A Community of Strangers: The Dis-Embedding of Social Ties

Paolo Parigi email@example.com
Sociology Department
Stanford University
StanfordCaliforniaUnited States of America

Bogdan State
Sociology Department
Stanford University
StanfordCaliforniaUnited States of America

Diana Dakhlallah
Sociology Department
Stanford University
StanfordCaliforniaUnited States of America

Rense Corten
Sociology Department
Tilburg University
TilburgThe Netherlands

Karen Cook
Sociology Department
Stanford University
StanfordCaliforniaUnited States of America

A Community of Strangers: The Dis-Embedding of Social Ties
Received January 23, 2013; Accepted May 18, 2013; Published July 4, 2013Editor: Tobias Preis, University of Warwick, United Kingdom The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
In this paper we explore two contrasting perspectives on individuals' participation in associations. On the one hand, some have considered participation the byproduct of pre-existing friendship ties -the more friends one already has in the association, the more likely he or she is to participate. On the other hand, some have considered participation to be driven by the association's capacity to form new identities -the more new friends one meets in the association, the more likely he or she is to participate. We use detailed temporal data from an online association to adjudicate between these two mechanisms and explore their interplay. Our results show a significant impact of new friendship ties on participation, compared to a negligible impact of pre-existing friends, defined here as ties to other members formed outside of the organization's context. We relate this finding to the sociological literature on participation and we explore its implications in the discussion. Citation: Parigi P, State B, Dakhlallah D, Corten R, Cook K (2013) A Community of Strangers: The Dis-Embedding of Social Ties. PLoS ONE 8(7): e67388.

Introduction

Associations between individuals are considered fundamental to the lives of communities and significant for the proper functioning of democracies [1][2][3][4]. An active associational life has also been linked to the emergence of collective action and to an increased capacity for resolving social dilemmas [5,6]. However, the determinants of individual participation in associations are not fully understood [7,8]. Two competing explanations of participation are proposed: (a) participation is the byproduct of having multiple pre-existing friendship ties with individuals in the association [9,10] or, (b) participation is the result of the association's capacity to promote new identities among its members [11]. In the first view, the associational life of an individual benefits from embedding involvement in the association in her own social networks; for example, by having her friends join the association she is a member of or by simultaneously joining the same association with her friends. In the second view, associational life benefits from the association promoting a process that pushes in the opposite direction, i.e., toward dis-embedding engagement with the association from a member's pre-existing social networks; for example, by promoting ties to new friends through the association.

The difficulty in adjudicating between these two mechanisms resides not just in the fact that they are clearly interconnected, but also in the existing lack of detailed temporal data for determining the causal relationships between them and their varied effects on associational engagement. In the last decade or more, the Internet has emerged as a significant site for the promotion of associational life. An increasing number of associations have originated online and have become significant producers of meaning in the daily lives of their members. Associations born on the Internet typically collect temporal data on their members' social networks and these data have captured the attention of a number of social scientists [12]. We use social network data collected by one of these associations to determine the relative contribution of the proposed mechanisms to individuals' levels of participation.

Our results suggest that having many prior friends in the association decreases participation, while forming new friends through the association increases participation. We relate this finding to the sociological literature on the role of networks in the mobilization of individuals. In particular, we adopt Gould's argument that mobilization occurs along a continuum bounded on the one hand by established solidarities and on the other hand by new identities [7]. The role of pre-existing networks in explaining participation shifts from strong to weak along the continuum between these two poles. Our findings suggest that online associations may benefit more from promoting a new identity for their members rather than from embedding the association into the pre-existing social networks of their members. We discuss the main implications of this argument in the conclusion.

Following Friends or Building New Identity

Writing in the 1960s, Almond and Verba argued that what made Western democracies effective was active citizen participation in civic affairs. They developed what was called a ''psychocultural approach to the study of political phenomena'' [1]. According to this perspective, individual values and beliefs explain why some societies appeared more vibrant than others in creating associations and, consequently, why democracies differed in efficiency despite sharing similar institutions like universal suffrage, division of power, constitutions, free elections, and so forth. Similarly and more recently, the work of Robert Putnam [4] on Italian regional and local governments argued that regions in Northern Italy were better governed because of a longer tradition of civic associations compared to the regions in Southern Italy. Putnam explains the relationship between strong networks of citizen participation and positive institutional performance in terms of`social capital'' -the informal networks, norms of reciprocity and trust that are fostered among members of the same association.

While the two studies cited above stress the importance of macro level values and belief systems, several social scientists have articulated a structural perspective to explain the importance of associations and collective action more broadly [5,13]. From this perspective, social networks have emerged as a key factor in understanding modern associations [14]. Wellman, for instance, argued that contemporary associations and communities are better conceptualized as networks that are either locally or globally bounded rather than densely-knit, village-like groups [15]. Yet, within the literature that focuses on associations as networks tension, has emerged between two approaches. On the one hand, scholars have highlighted the role of social networks, and of friendship in particular, for explaining why people participate in associations. On the other hand, scholars have stressed the importance of identity processes in explaining the growth of associations. People join because the association provides them with a new identity and new circles of friends that they are interested in acquiring. With some notable exceptions, both approaches draw heavily from research on social movements.

The classic view of the importance of pre-existing network ties is perhaps articulated best in McAdam's Freedom Summer [16]. McAdam shows that activists who participated in the 1964 Mississippi summer camp were more likely to have friends already at the camp. McAdam and Fernandez analyzed the role of networks by studying the centrality of the people recruited to participate in the summer camp [17], further confirming the importance of pre-existing ties. The importance of network ties for participation received formal treatment by Centola and Macy [10]. Using a simulation model, Centola and Macy studied the diffusion of collective action with respect to different levels of risk. Innovation in mobilization tactics cascaded through the network when risk was minimal and thus adoption thresholds were low. The diffusion process stopped at local bridges, however, when the required threshold of adoption, i.e., the number of prior friends that have adopted, increased. In sum, this literature suggests that network ties matter for participation particularly when the risk involved in participating is perceived to be high.

In Peasants into Frenchmen [18], Eugene Weber articulated, on historical and institutional grounds, a view counter to the importance of pre-existing friendship ties for explaining participation. Weber describes the ways in which the French state facilitated the creation of a national identity among the locally identified members of the population by enabling contact among the soon-to-become`French'' individuals from the country's provinces. National universities, military service, corporations, and administrative bodies all facilitate the meeting of people from various parts of the state's territory. With contact comes the opportunity for the development of social relations, and the formation of such relations confirms one's loyalty to the nation. Membership in these institutions promotes new identities, which in turn influence participation. While Weber's argument focuses on institutions rather than associations, a similar argument about the importance of identity has been advanced by another set of social movement scholars interested in processes of identity formation and collective action [13,19]. Deborah Minkoff, for instance, argued that those in certain disadvantaged categories such as gays and lesbians, the elderly, and women, lacked access to the infrastructure that facilitates generation of ties between members. Mobilization of these groups created identities that then produced social ties [20]. Further reinforcing this argument and providing a more formalized approach to it, Kairam and co-authors used data from Ning online communities to show the existence of a non-network growth model for communities and, by extension, for associations [11]. In this case individuals join because they share a common interest with the community. The new relationships individuals form with members of the association subsequentily helped to promote a new identity.

To a certain extent the two mechanisms promoting participation are interrelated. Pre-existing friendship ties may be the first reason why one decides to begin to participate, but the capacity to form new friendships within the association may be the reason why one decides to continue participating. Roger Gould explored this interplay of forces directly in his analysis of the Paris Commune uprising in 1870. He argued that when mobilization occurs along the lines of pre-existing networks (e.g. the National Guard enlistment in Paris mirrored neighborhood boundaries) protest is more efficient but fails to produce extra levels of commitment to the organization or association beyond those already created by neighborhood ties [7]. In this case, the association does not have an independent identity. Conversely, when the association cuts across pre-existing networks, mobilization is harder to achieve unless it manages to create a total institution. In such a scenario, the association develops a strong identity of its own. In line with this reasoning, in a different case study, Peter Bearman shows that Confederate army battalions that had soldiers enlisted from the same region had more desertions during the Civil War than more regionally heterogeneous battalions (everything else being equal) [21]. The embedding of ties in pre-existing solidarities weakened the independence of the organization, in this case the Army battalion, leading to the quicker demise of mobilization during the final months of the war.

In light of this research we represent the role of pre-existing networks in participation along a continuum: at one end of the continuum, pre-existing ties are important and the association has a weak identity; at the other end, pre-existing networks are not relevant and the association has a strong identity.

Associations, Social Networks and the Internet

The question of whether the rise of the Internet has positive or negative effects on associational life and community formation has been the topic of heated debate. Early on, a`dystopian'' line of scholars feared that the use of Internet would erode communities and social relations. Internet use, they argued, would replace inperson interaction and long-distance online interaction would replace social interactions in local communities [22]. At the same time, a`utopian'' line of thought predicted that the Internet would enhance, or even radically transform, traditional forms of social capital [15]. Indeed, some early studies suggested that online interaction via e-mail and online discussion boards could strengthen local communities [9].

In the meantime, the nature of online interaction has evolved rapidly, most notably through the surprisingly swift rise of online social networks such as Facebook, MySpace and Twitter. While older forms of online social interaction such as e-mail seemed especially suited to support existing offline interaction structures, these newer online social networks allowed users not only to interact with their own contacts, but also to traverse the network by discovering the contacts of their own contacts [23], thereby making offline social structure potentially less relevant. Online social networks seem especially suited for the creation of new connections that bridge social contexts and thus may have an ameliorating effect on the cleavages found in modern societies, cleavages typically reinforced through certain patterns of offline interactions. Recent empirical research [24] suggests, for example, that while online networks are firmly rooted in existing offline social networks, they are positively associated with various forms of bridging social capital.

In a parallel debate, social movement scholars have emphasized the increasing importance of information and communication technology (ICT), and the Internet in particular, for social movements, arguing that social movements rely on networks to provide organizational structure and that ICT facilitates such networks [25]. Although much research on the relationship between social movements and online interaction is based on studies of older types of online interaction (such as e-mail, in contrast to modern online social media such as Facebook and Twitter), this literature initially predicted that ICT would be most valuable to social movements that are either large, well-established and centrally organized, or social movements that consist of informal and geographically dispersed networks [25]. These predictions seem largely at odds with recent developments such as the`Arab Spring,'' in which online social media (i.e., Facebook) appear to have been pivotal in coordinating local social movements [26]. In sum, scholarly work on the role of the Internet and ICT in contemporary societies has highlighted, on the one hand, how a type of social capital can be created in online interactions and, on the other hand, how ICT can be used to create global and local mobilization.

Regardless of perspective, whether the Internet promotes more participation or less and whether ICT favors global or local collective actions, associations that have their origins online have gone on to become significant producers of meaning in the daily lives of their members. We use social network data collected by one of these associations to determine the impact of the two distinct mechanisms identified above on individual levels of participation. The theoretical question we explore is thus: Do online associations primarily generate participation by tapping into pre-existing networks of their members or by promoting a new identity independent of these networks?

Data and Methods

For our analysis we use a unique dataset collected by CouchSurfing.org-the world's largest and fasted growing social travel network-whose stated mission is to ''to make the world a smaller and friendlier place, one life-changing experience at a time.''' After negotiating access to the data with CouchSurfing and signing a Non-Disclosure Agreement with them, we submitted our research protocol to the Internal Review Board (IRB) of Stanford University that determined that it posed no more than minimal risk (`Notice of Exempt Review,'' received in April 2012). All the data we used in our analysis were anonymized. Members of CouchSurfing engage not only in hospitality exchanges, visiting each other often in disparate corners of the world, but also in organizing interest groups and social gatherings for members that live in the same area. Local chapters of CouchSurfing resemble leisure associations whose goal is to facilitate interactions among their members. While some interactions occur offline, it is by logging into their personal webpages that members learn about gatherings and about the local initiatives of the various interest groups. In other words, on CouchSurfing the online platform plays a key role in facilitating the offline meeting of like-minded people. For this reason, we use logins as an indicator of the overall level of individual participation with the life of the association.

Compared to traditional social networking websites, Couch-Surfing's online platform introduced a number of innovations that arguably resulted in more credible data being collected about social ties. Rather than simply allowing users to add`friends'' or buddies,'' CouchSurfing's platform asks probing questions about new social connections -whether the tie is one's best friend, a good friend, or a mere acquaintance, how much trust the friends have in each other, as well as the context in which the two individuals met. Even among the numerous online platforms that collect social network data, few gather network data at this level of detail. As a consequence, CouchSurfing has attracted a good deal of attention from the scientific community and from mainstream media [27][28][29]. We contend that data collected through such a strategy are likely to reflect meaningful social ties.

The period of our analysis spans the time from September 2003 to December 2010. We created a random sample of 10,000 American users, whose monthly logins were counted and recorded over their career in the association. Among American users, 43% are females, 75% are 29 years old or younger and, on average, have hosted or surfed, i.e., visited somebody, for 4.38 days and 4.51 days respectively. Figure 1 shows the information recorded by CouchSurfing for each tie a member reports with another member. We made the page completely anonymous by eliminating the names and pictures of all tie recipients. We used the information from the circled question (bottom left corner) to capture the extent to which CouchSurfing facilitated the formation of a friendship tie. In the example, the focal member reported that the tie predated membership in CouchSurfing. We called this type of relationship an external tie; we labeled a tie formed because of CouchSurfing an associational tie. Thus and within the scope of our argument, external ties capture pre-existing friendships formed outside of the organization while associational ties capture new friendships formed within the organization. Of the ten thousand members in our random sample, 2,941 of them acquired either or both of these types of ties during the period of analysis.

These data allowed us to test the impact of pre-existing relationships, i.e., external ties, on participation (logins) controlling for the individual propensity to form new friendships within the association (associational ties). The model that we used for our estimates, a panel vector autocorrelation model (PVAR), took into account the possibility of feedback loops among all the variables in the model [30]. In particular, the PVAR model controlled for the possibility that having many pre-existing friends joining the association reinforced the likelihood of forming new ties, that is, of developing a new identity. More formally the model is specified as:
L t D t P t 0 B @ 1 C A~P J j~1 b t{j 1,1 b t{j 1,2 b t{j 1,3 b t{j 2,1 b t{j 2,2 b t{j 2,3 b t{j 3,1 b t{j 3,2 b t{j 3,3 0 B B @ 1 C C A L t{j D t{j P t{j 0 B @ 1 C Az e L,t e D,t e P,t 0 B @ 1 C Að1Þ
The variables in the model are L, D and P that respectively represent logins, nodal degrees (total number of friends, however acquired) and proportion of associational ties at time t. Each variable is time-demeaned to take into account any secular trends. We controlled for heteroskedasticity by dividing each variable by its time dependent standard deviation. We addressed autocorrelation of the individual observations by subtracting the forward mean, which corresponds to the mean of all future observations for each individual (Helmert transformation). Time was measured in month intervals. In the formula above, betas represent the estimated coefficients of the effects of each variable on the other variables in the model and J is the order of the model. The Lagrange multiplier test indicated that using three-month lagged variables did not create serial correlation. Furthermore, using a lag of three months maximized the Akaike Information Criterion (AIC) and improved the fit of the model. The estimates were obtained using the software Stata and the panel VAR procedure developed by Love and Zicchino [30].

We also considered how the PVAR system reacted to external shocks (Impulse-Response Function, or IRF). Unexpected shocks are defined as the impact of a one standard deviation increase of the error terms (e) on the variables in the system. Since the PVAR model does not differentiate between endogenous and exogenous factors, analyzing the reaction of logins to a shock in one of the other variables estimated in the model allowed us to isolate the explanatory role of nodal degree (D) and proportion of associational ties (P) on participation.

We focused in particular on the role of associational ties, i.e., on the impact of a shock in e P,t on logins L t for tzs time periods. We set s to be equal to five so that the IRF estimated the impact of the shock for a six month period. We used Monte Carlo simulation with 500 repetitions to generate 95% error bands. This made possible estimation of the causal role of associational ties on participation within the constraints of the model.

Results

Before estimating the PVAR model we explore the possibility that the propensity to form new friendships and, consequently, of participating more in the life of the association is strongly influenced by the size of the local chapter. For each member in our sample, we identified the metropolitan area of his or her residency. We matched these areas with the combined statistical areas (CSAs) as defined by the U.S. Census Bureau. Figure 2 (top panel) suggests that the greater the size of the local chapter, the more new social ties members acquire through CouchSurfing. The mean ratio between the two types of ties for small chapters (less than a 1,000 members) is.22 while for larger chapters (more than 5,000 members) is almost.60. Yet, a closer examination reveals that the relationship between the type of ties and the size of the chapter is rather weak. The bottom panel of Figure 2 shows the fit of a quadratic function, producing a R 2~: 33. Not surprisingly, chapters in larger CSAs appear to have higher ratios than chapters in smaller CSAs. Thus, CouchSurfing members located in New York City had a greater proportion of associational ties compared to CouchSurfing members in Lake Charles, LA. More members create more opportunities for interactions among previously unknown members, making it easier for two strangers to meet.

While the aggregate analysis of the type of ties by the size of the chapters does not reveal a strong association between the two, we examine the extent to which opportunities to meet strangers within the association are structured by the overall number of friends one has (Figure 3). In the figure, people with 15 friends (top line) have a   Table 1 consistently higher proportion of associational ties than people with smaller circles of friends-5 and 10 friends-and, at the same time, participate more in the life of the association. Independent of the size of the local chapter therefore, the formation of new friendship ties, i.e., of associational ties, appears strongly impacted by the individual general propensity to form friendships. Figure 3 reinforces our confidence in focusing the analysis on individual level determinants of participation rather than at the aggregate level of local chapters. Table 1 reports the z-score estimates of the PVAR model while Figure 4 shows the graphical interpretation of the same model. We focus the discussion on the latter. Figure 4 suggests that while having many friends in the association has a two-month lagged impact on decreasing logins (negative coefficient in Table 1 forD t,2 on L t ), every increase in the proportion of friends met through CouchSurfing offsets this decrease in logins by almost one percentage point (a lagged effect of one month). After two months, associational ties lose their positive impact on engagement and behave more like external ties, i.e., they negatively affect logins. Yet, the magnitude of this latter effect is considerably smaller (see Table 1). Furthermore, the model indicates that the proportion of associational ties does not correlate with the total number of friends, and vice-versa. These findings suggest the existence of a separation between one's pre-existing networks of friends and the new circle of friends met via membership in the online association. Such a separation is strongly reinforced by one's participation in the association (compare the magnitudes of the effects of L t{1 on D t and P t ).

We also explore the impact on logins of a shock in associational ties and track this effect in time for six months. Such a shock rapidly increases logins in the first month, producing an increase of 4.3% in logins and remains largely constant at this level for the next five time periods. This suggests that the positive impact of a dis-embedding process on associational engagement is stronger at the beginning of one's membership in the association rather than later. Further, the consistency of the effect in time suggests that the dis-embedding process has a lasting impact on participation.

Conclusions

Recently we have witnessed rapid growth in both associational life and the mobilization of collective action facilitated by the Internet. It is not hard to imagine a proximate future in which many human associations will be overwhelmingly made up of online communities. Individual social networks have been thought to produce positive effects on associational life by encouraging participation. Our analysis compares the effects of two competing mechanisms underlying individual levels of participation. We discovered that pre-existing social networks do not always promote associational life. In fact, while forming new social ties in the association does increase engagement, interestingly, having one's prior friends join the association you belong to actually decreases your overall engagement with the organization.

In light of sociological work on the role of pre-existing networks on the mobilization of collective action, our main finding suggests that CouchSurfing succeeded in forming a strong identity among its members, thereby reducing the importance of embedding participation within pre-existing networks to foster participation. This finding is also consistent with Centola and Macy's analysis [10] if we assume that joining a local CouchSurfing chapter is a low risk activity. Whether our finding is the byproduct of the nature of the specific association we targeted or is instead a characteristic of online associations, we cannot directly determine from our analysis. It is nevertheless useful to entertain the possibility that a potential effect of online associations would be that of promoting new identities that are independent of preexisting ones.

Regardless of the precise scenario, we believe the study of online platforms for social networking such as the one provided by CouchSurfing can provide important insights into the world of all associations and the nature of their broader social, political and economic consequences. While the limited availability of data has restricted the scope of the debates about links between personal networks and levels of associational engagement in the case of primarily face-to-face associations, new data collection capabilities can give us important new insights into the complex nature of the interaction between networks and associational life. Further investigation is required to explore the similarities and potential differences between membership in online associations and those that are primarily face-to-face as well as the consequences of such membership for participation in civic activities.

Figure 1 .
1The information members enter after a friendship tie is reported on the website. doi:10.1371/journal.pone.0067388.g001

Figure 2 .
2Ratio of ties by size of local chapters (top panel) and by (log) number of members (bottom panel). Each dot represents the total proportion of the two types of ties in a metropolitan area of the United States (CSA) as of December 2010 (N = 83.). We excluded CSAs where more than 90% of the members had no ties. In the top panel diamonds show the group means with 95% confidence intervals (One Way ANOVA, F ratio = 21.22, pv:001). The bottom panel shows a quadratic fit to the data with 95% confidence interval. R 2~: 33. doi:10.1371/journal.pone.0067388.g002

Figure 3 .
3Total logins by number of associational ties. doi:10.1371/journal.pone.0067388.g003

Figure 4 .
4Graphical representation of the PVAR model with a three-lag period. L = number of logins (logs); D = number of ties; P = proportion of associational ties. Arrows represent statistically significant effects (at the 95% confidence level or more). Magnitude of the effects reported in

. doi:10.1371/journal.pone.0067388.g004

Table 1 .
1PVAR Estimates.Source: CouchSurfing US dataset. Legend: *pv:05, ** pv:01, ***pv:001. N = 67,183. Each variable is time-demeaned to take into account any secular trends. We controlled for heteroskedasticity by dividing each variable by its time dependent standard deviation. We addressed autocorrelation of individual observations by subtracting the forward mean, which corresponds to the mean of all future observations for each individual (Helmert transformation). The reported coefficients are z-scores. doi:10.1371/journal.pone.0067388.t001Dependent
Variable
(Unlagged)

Ln
(logins): L
Degree: D

%
Assoc.
Ties: P

Lag (months) Coef.
(S.E.)
t-statistic
Coef.
(S.E.)
t-statistic
Coef.
(S.E.)
t-statistic

Ln (logins)
One
0.475***
(0.007)
69.659
0.095***
(0.011)
8.448
0.531***
(0.043)
12.282

Two
0.075***
(0.006)
13.296
0.002
(0.007)
0.359
20.066*
(0.033)
22.028

Three
0.055***
(0.005)
11.998
0.004
(0.006)
0.620
0.002
(0.025)
0.085

Degree
One
0.007
(0.015)
0.454
1.010***
(0.062)
16.231
20.034
(0.036)
20.946

Two
20.034**
(0.013)
22.605
20.094
(0.051)
21.845
0.014
(0.036)
0.376

Three
0.016
(0.008)
1.923
0.020
(0.034)
0.592
0.023
(0.024)
0.967

% Assoc. Ties One
0.009***
(0.002)
6.295
20.002
(0.002)
21.320
0.878***
(0.010)
86.283

Two
20.003**
(0.001)
22.670
20.001
(0.002)
20.398
20.008
(0.007)
21.051

Three
0.000
(0.001)
0.533
20.001
(0.001)
20.724
20.010*
(0.004)
22.442

PLOS ONE | www.plosone.org
July 2013 | Volume 8 | Issue 7 | e67388
AcknowledgmentsWe thank Ravi Jain and Piero Landolfi for useful comments on earlier drafts of this paper.Author ContributionsConceived and designed the experiments: PP BS DD RC KC. Analyzed the data: BS DD PP. Wrote the paper: PP RC KC BS DD.
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Hsa_circ_0051079 functions as an oncogene by regulating miR-26a-5p/TGF-β1 in osteosarcoma

Zuojun Zhang
Ming Zhao
Guojie Wang
Hsa_circ_0051079 functions as an oncogene by regulating miR-26a-5p/TGF-β1 in osteosarcoma
10.1186/s13578-019-0355-2Zhang et al. Cell Biosci (2019) 9:94 RESEARCHHsa_circ_0051079miR-26a-5pTGF-β1Osteosarcoma
Background: Osteosarcoma is a most common bone malignant tumor which threatens children and adolescents. Circular RNAs (circRNAs) fundamentally play essential roles in the progress and development of human cancers by sponging with microRNAs (miRNAs). However, the role of circRNAs in osteosarcoma is not clear. The aim of the study was to investigate the roles and molecular mechanism of circRNAs in osteosarcoma.Results:The data from qRT-PCR showed that circ_0051079 expression was higher in osteosarcoma cells and tissues compared to their normal controls. Meanwhile, bioinformatic analysis indicated that circ_0051079 was a sponge of miR-26a-5p, which was verified by luciferase activity assay. Subsequently, TGF-β1 was verified as a putative target mRNA of miR-26a-5p by luciferase assay. Cellular function assays were conducted and the findings revealed that circ_0051079/miR-26a-5p/TGF-β1 regulated osteosarcoma proliferation and metastasis.Conclusion:The study demonstrated that circ_0051079 could act as an oncogene via regulating miR-26a-5p/TGF-β1 and a potential biomarker for osteosarcoma diagnose.

Background

Osteosarcoma is regarded as the most common devastating malignant bone cancer in children and adolescents [1,2] and results in high morbidity and mortality [3]. Presently, the main therapies for osteosarcoma are surgery, adjuvant chemotherapy and radiotherapy, which increase the survival rates, but those conventional therapies still limit its efficacy [4,5]. The overall survival rate of osteosarcoma patients remains dismal due to the metastases and relapse [6]. So, it is urgent for scientists to discover in depth the molecular mechanisms underlying osteosarcoma progression and identify some relevant biomarkers for diagnosis and prognosis for patients. Circular RNAs (circRNAs) are novel of endogenous non-coding RNAs which were firstly found in 1976 and considered as the byproducts of splicing errors with a low expression level [7,8]. Recently, with the development of high-throughput sequencing and novel computational approaches, circRNAs are identified to play critical roles in regulating gene expression at transcriptional and posttranscriptional levels [8]. Many studies have reported the functions of circRNAs including mediating alternative splicing [9], interacting with proteins [10], and acting as microRNAs (miRNAs) sponges [11,12]. Specially, circR-NAs can serve as competing endogenous RNA (ceRNA) to inhibit miRNAs activity and further regulate the down-stream gene expression. So, many circRNAs were reported to take part in the cancer progression and function as biomarkers for the cancer diagnosis and prognosis [13]. Increasing evidence has demonstrated that circR-NAs were revealed in many malignant tumors to regulate cell proliferation, migration, invasion and apoptosis. For instance, hsa_circ_0008039 exerted oncogenic roles in breast cancer [14]. Hsa_circ_0000673 exerted tumorsuppressing effects and served as a promising diagnosis

Open Access

Cell & Bioscience *Correspondence: firstname.lastname@example.com 2 Luoyang Orthopedic Hospital of Henan Province, Orthopedic Hospital of Henan Province, 82 Qiming South Road, Chanhe District, Luoyang 122400, Henan, China Full list of author information is available at the end of the article biomarker and a therapeutic target in gastric cancer [15]. circ_0001649 could inhibit lung cancer cells growth and metastasis [16]. However, whether and how circRNAs control the development of osteosarcoma is unclear.

In the current study, it was found circ_0051079 was upregulated in osteosarcoma by circRNA array and the data of cellular functions verified that circ_0051079 played an important role in osteosarcoma development and could function as an oncogene. The investigation in exploring the molecular mechanism identified that circ_0051079 sponged with miR-26a-5p to increase TGF-β1 expression. The study revealed that circ_0051079 could function as an oncogene in osteosarcoma.

Results

CircRNA expression in osteosarcoma tissues

To explore the different circRNA profiles between osteosarcoma tissues and adjacent tissues, unsupervised hierarchical clustering was performed to visualize the differential circRNAs. The heatmap showed the top fifteen most increased and decreased circRNAs in osteosarcoma tissues and the matched non-tumor tissues by circRNAs Arraystar Chip (Fig. 1a). Two significant up-regulated and two down-regulated circRNAs were selected to validate in 45 osteosarcoma tumor tissues and adjacent non-tumor tissues by real-time quantitative RT-PCR. The levels of circ_0051079 and circ_0011038 expression were significantly higher in osteosarcoma tissues than their adjacent normal tissues (Fig. 1b, c). The two down-regulated circRNAs including circ_0070372 and circ_0035114 were lower in osteosarcoma tissues than in their adjacent normal tissues (Fig. 1d, e). The circ_0051079 levels were changed significantly and might be an oncogene, so it was chosen for the further investigation.

Circ_0051079 was upregulated in osteosarcoma and correlated with poor survival ability

Circ_0051079 was confirmed as a significant up-regulated circRNA and selected to explore its function in osteosarcoma. Firstly, we detected the circ_0051079 expression level in serum samples from osteosarcoma patients (n = 32) and healthy individuals (n = 32). The data showed that the expression of circ_0051079 upregulated greatly in the blood samples from osteosarcoma patients compared to the healthy individuals ( Fig. 2a). At the same time, we found that there was a negative relationship between the overall survival and circ_0051079 expression levels. Patients with high circ_0051079 levels have a shorter survival time than the patients with low circ_0051079 levels (Fig. 2b).  Figure 2c showed that the circ_0051079 expression was significantly up-regulated in HOS, KH-OS, MG63, 143B, U2OS, SaoS2 cells compared to hFOB cells. Remarkably, the expression levels of circ_0051079 in the growth medium were also higher in HOS, KH-OS, MG63,143B, U2OS, SaoS2 cells compared to hFOB cells (Fig. 2d). Therefore, the results demonstrated that circ_0051079 was up-regulated in osteosarcoma and had a poor correlation with the overall survival.

Circ_0051079 negatively regulated miR-26a-5p in osteosarcoma cells

CircRNAs regulate the target gene expression by sponging with miRNAs. To further explore the underlying mechanism of circ_0051079 in osteosarcoma, targetscan and miRanda were used to search the potential targets of circ_0051079. Bioinformatics analysis predicted that circ_0051079 was the potential sponge of miR-26a-5p (Fig. 3a). Luciferase reporter assay was conducted to determine the reliability. The result illustrated that upregulation of miR-26a-5p notably decreased the luciferase activity of circ_0051079 wild-type, but had no effect on circ_0051079 mutant-type (Fig. 3b, c). Moreover, circ_0051079 shRNAs were transfected to osteosarcoma cells (U2OS and SaoS2) and miR-26a-5p expression was detected by qRT-PCR. We found that down-regulation of circ_0051079 decreased the expression of miR-26a-5p in the osteosarcoma cells (Fig. 3d). However, overexpression of miR-26a-5p does not alter the expression level of circ_0051079 (Fig. 3e). Furthermore, the miR-26a-5p expression was detected in osteosarcoma tissues. The result revealed that miR-26a-5p was down-regulated in 45 primary osteosarcoma tissues in contrast to paired adjacent normal tissues (Fig. 3f ). These results proved that circ_0051079 might bind to miR-26a-5p in osteosarcoma cells.

Circ_0051079 played an oncogenic role in osteosarcoma cells via miR-26a-5p

To deeply study the function of circ_0051079 in osteosarcoma cells, circ_0051079 shRNA and/or miR-26a-5p was introduced into osteosarcoma cells (U2OS and SaoS2). MTT assay was used to observe the proliferation of U2OS and SaoS2. The data showed that cell proliferation was dramatically inhibited by knockingdown of circ_0051079 or up-regulation of miR-26a-5p in U2OS and SaoS2 cells respectively (Fig. 4a, b). The result of colony formation assay indicated that the colonies remarkably decreased in U2OS and SaoS2 cells with suppressing circ_0051079 expression and/or overexpressing miR-26a-5p expression (Fig. 4c, d). Similarly, we detected the migration and invasion ability in U2OS and SaoS2 cells transfected with circ_0051079 shRNA or miR-26a-5p mimics. Wound-healing assay indicated that circ_0051079 inhibition and/or miR-26a-5p over-expression could effectively decrease the migration ability in U2OS and SaoS2 cells (Fig. 4e, f and Additional file 1: Figure S2). At the same time, the invasion assay also proved that the silence of circ_0051079 and/or up-regulation of miR-26a-5p could inhibit the invasion ability of U2OS and SaoS2 cells (Fig. 4g, h and Additional file 1: Figure S2). Furthermore, the in vivo data indicated that inhibiting circ_0051079 expression led to tumor growth suppression (Fig. 4i, j). Thus, these experiments in vitro and in vivo demonstrated that circ_0051079 silencing inhibited the proliferation, migration and invasion by regulating miR-26a-5p, suggesting the oncogenic role of circ_0051079 in osteosarcoma cells via miR-26a-5p.

MiR-26a-5p targeted TGFB1 in osteosarcoma cells

Bioinformatics analysis was applied to predict the binding site between miR-26a-5p and TGF-β1 (Fig. 5a). Subsequently, luciferase reporter assay confirmed that TGF-β1 was targeted by miR-26a-5p. Over-expression of miR-26a-5p effectively inhibited the luciferase activity of TGF-β1-WT (wild-type), but made no difference to TGF-β1-MUT (mutant-type) in both U2OS and SaoS2 cells (Fig. 5b, c). Then, we built a miR-26a-5p overexpression cell models by transfecting miR-26a-5p overexpression vector. The qRT-PCR and western blotting result found that miR-26a-5p overexpression significantly suppressed TGF-β1 mRNA and protein levels (Fig. 5d, e). In osteosarcoma tissues, TGF-β1 mRNA level was higher than adjacent tissues (Fig. 5f ). In summary, we confirmed miR-26a-5p targeted TGF-β1 in osteosarcoma cells.

Circ_0051079 promoted osteosarcoma progression by miR-26a-5p/TGF-β1

To clarify the underlying mechanism of circ_0051079 in osteosarcoma, the overexpressed circ_0051079 Circ_0051079 played an oncogenic role in osteosarcoma cells via miR-26a-5p. a, b Cell proliferation ofU2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26-5p mimics for 24 h and then MTT assay was used to evaluate the cell proliferation of U2OS and SaoS2 cells. c, d Cell proliferation of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26-5p mimics for 24 h and then cell colony formation assay was used to evaluate the cell proliferation of U2OS and SaoS2 cells. e, f The migratory ability of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26a-5p mimics for 24 h and cell migration was analyzed. g, h The invasion ability of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26a-5p mimics for 24 h and cell migration was analyzed. i, j The growth curve of U2OS and SaoS2 cells in vivo. U2OS and SaoS2 cells with circ_0051079 shRNA or miR-26a-5p mimics were injected to the nude mice and the tumor size was recorded and analyzed every 7 days. **p < 0.01 vector was transfected to U2OS and SaoS2 cells. TGF-β1 mRNA level was up-regulated along with circ_0051079 overexpression (Fig. 6a). MTT assay showed that circ_0051079 remarkably promoted osteosarcoma cells proliferation, however, this effect was significantly abrogated by co-transfection with miR-26a-5p mimics or TGF-β1 siRNAs (Fig. 6b, c). Consistently, the enhanced cell invasive and migrant capacity of both cell lines induced by circ_0051079 was abolished by co-transfection with miR-26a-5p mimics or TGF-β1 siRNA (Fig. 6d-g). In addition, when the cells were transfected with circ_0051079 shRNA, the TGF-β1 signaling pathway was inhibited (Additional file 1: Figure S2). The relationship from the clinic data analysis showed that there was a positive relationship between circ_0051079 and TGF-β1 expression in osteosarcoma (Additional file 1: Figure S3). Overall, these results showed that circ_0051079 promoted osteosarcoma progression by sponging miR-26a-5p which suppressed TGF-β1 expression.

Discussion

In clinic, there are great obstacles to treat osteosarcoma. It is necessary to find new target molecules for the early diagnosis and therapy of osteosarcoma. To better learn the roles of circRNAs in osteosarcoma, circRNA array was performed to find the different circRNAs in osteosarcoma. Circ_0051079 was verified as a significant up-regulated circRNA in osteosarcoma patients and associated with a short survival time. Circ_0001564 was found to regulate osteosarcoma proliferation and apoptosis [17]. The investigation of the underlying mechanism of circ_0051079 in osteosarcoma showed that it was sponged with miR-26a-5p and regulated osteosarcoma cell growth and metastasis by targeting TGF-β expression.

MiRNAs are attached to non-coding RNA and responsible to cellular and physiological processes by targeting protein-coding genes at post-transcriptional levels [18,19]. MiR-26a-5p, locating on the human chromosome 3p22 [20], was the targeted downstream gene The luciferase activity of TGF-β1 3′UTR. U2OS and SaoS2 cells were transfected with plasmids containing wild type reporter for TGF-β1, or mutated reporter for TGF-β1 or miR-26a-5p and then luciferase activity was analyzed by dual luciferase system. d, e TGF-β1 mRNA and protein levels in U2OS and SaoS2 cells with miR-26a-5p up-regulation. U2OS and SaoS2 cells were transfected with miR-26a-5p and total RNA or protein was extracted for real time RT-PCR or Western blotting respectively. f Total RNA was extracted from osteosarcoma tissues for TGF-β1 mRNA detection by real time RT-PCR. The adjacent tissues were the controls. **p < 0.01 which predicted by target scan and functions in the development, differentiation, and cycle regulation for many cancers. For example, Xu et al. demonstrated that miR-26a-5p potentiated lung cancer cell metastasis via JAK2/STAT3 pathway by targeting ITGb8 [19]. Wang et al. found that miR-26a-5p regulated hepatoblastoma development by restraining the LIN28B-RAN-AURKA pathway [21]. Milena Rizzo et al. demonstrated that the re-expression of miR-26a-5p in DU-145 prostate tumor cells reduced the proliferation [22]. In the present study, miR-26a-5p was proved to the target bindings of circ_0051079 by dual-luciferase activity assay. Then, a series of experiments in vitro indicated that miR-26a-5p over-expression could inhibit osteosarcoma cells proliferation, migration and invasion. So, it was suggested that miR-26a-5p could function as anosteosarcoma suppressor. Furthermore, we found that TGF-β1 was a direct target gene of miR-26a-5p and involved in osteosarcoma progression and development.

TGF-β is a multifunctional growth factor involved in cell proliferation, differentiation, and extracellular matrix protein synthesis [23]. TGF-β1 encodes a secreted ligand of TGF-β that could bind various TGF-β receptors leading to recruitment and activation of SMAD family transcription factors that regulate gene expression. According to many reports, TGF-β1 plays a critical role in tumorigenesis as well as metastasis within tumor progression and development [24][25][26][27][28][29]. TGF-β1 extremely exerted essential role in many aspects of cancers [24][25][26][27][28][29], but the specific function and mechanism of TGF-β1 was not elucidated in osteosarcoma. In current study, we confirmed that miR-26a-5p could interact with 3′-untranslated regions (3′-UTR) of TGF-β1 mRNAs through luciferase activity assay. Meanwhile, the mRNA level and protein level of TGF-β1 were found to be higher in osteosarcoma cell lines and tissues compared with normal one. Thus, we confirmed that circ_0051079 promoted osteosarcoma progression by sponging miR-26a-5p and upregulation of TGF-β1.

Conclusions

Circ_0051079 was up-regulated in osteosarcoma and correlated with poor survival time. Circ_0051079 was verified as a sponge of miR-26a-5p. Furthermore, miR-26a-5p targeted TGF-β1 to involve in the regulation of osteosarcoma. In brief, circ_0051079/miR-26a-5p/TGF-β1 regulated osteosarcoma progression and development. Our findings revealed that circ_0051079 could act as an oncogene and a potential biomarker for osteosarcoma.

Methods

Osteosarcoma samples

Total 45 osteosarcoma tissues and adjacent normal tissues were collected from Luoyang Orthopedic Hospital of Henan Province (Zhengzhou, China) between 2013 and 2016. The samples from the healthy individuals were from the healthy peoples without any diseases. The blood samples from osteosarcoma patients and healthy individuals were collected for use. All samples were instantly snapfrozen and stored at − 80 °C until use. Informed contents were written and obtained from all patients (Table 1).

Cell culture

Six human osteosarcoma cell lines (HOS, KH-OS, MG63, 143B, U2OS and SaoS2) and one normal osteoblast cell line (hFOB1.19) were all purchased from American Type Culture Collection (ATCC). They were cultured in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, Gaithers-burg, MD, USA) growth medium with 10% fetal bovine serum (Gibco), 50 μg/ml of penicillin and 50 μg/ ml of streptomycin at 37 ℃ containing a humidified atmosphere of 5% CO 2 .

Cell transfection

Lipofectamine 2000 (Invitrogen) was used to transfect recombinant vectors or control vectors into U2OS and SaoS2 cells according to the manufacturer's instruction. The recombinant vectors included circ_0051079, circ_0051079 shRNA, miR-26a-5p, miR-26a-5p mimics or TGF-β1 siRNA (Santa Cruz). All the vectors were constructed by Hanheng Biotech (Shanghai, China). The sequences were covered by a patent. About 2 × 10 5 cells were planted in six well plates. The recombinant vector was transfected after 24 h. The efficiency of knockdown and up-regulation was analyzed using qRT-PCR.

RNA extraction and real-time PCR analysis

Microarray analysis

After being obtained from surgical specimens, samples were immediately frozen using liquid nitrogen. Sample preparation and microarray hybridization were performed according to the protocols of Arraystar (Rockville, MD, USA). CircRNAs were enriched through removing linear RNAs with Rnase R (Epicentre, Madison, WI, USA), and then amplified and labelled using Arraystar Super RNA Labeling Kit (Arraystar). mRNAs were purified using rRNA removal kit (Arraystar). Subsequently, Arraystar Human circRNA Array (8 × 15K) and LncPath Human Cancer Array were used for hybridization, and then scanned by the Agilent Scanner G2505C (Jamul, CA, USA). circRNAs and mRNAs demonstrating fold-changes of ≥ 2 and p-values of less than 0.05 were regarded as significantly differentially expressed.

ceRNA analysis and target prediction

We constructed a hsa_circRNA_100290-miRNA-target gene network using Cytoscape to visualize their interactions based on our circRNA microarray data and mRNA microarray data. In the network, we predicted the cir-cRNA/miRNA interaction with miRNA target prediction software (Arraystar's home-made) established from Tar-getScan and miRanda. The triple network was based on the theory of ceRNA that the circRNA shared the same miRNA with mRNA or other non-coding RNAs in one triplet.

Cell proliferation

MTT (Sigma, USA) was performed for the cell proliferation analysis based on the manufacturer's protocol. The transfected osteosarcoma cells were seeded in 96-well plates with 5 × 10 3 cells in every well. The second day, 20 μl MTT solution (5 mg/ml) was added to cells per well incubated at 37 °C for 4 h. Then, abandon supernatant and add 150 μl DMSO (dimethylsulfoxide) to dissolve sediment. Lastly, the absorbance was measured at 570 nm through microplate reader (BioTek). All experiments were repeated at least three times independently.

Nude mouse experiments

To study tumor growth, 5-to 6-week-old female, athymic nude BALB/c mice (Vital River Laboratory Animal Technology Co. Ltd., China) were injected with 2 × 106 U2OS and SaoS2 cells in 1 ml PBS. The mice were then divided into a circ_0051079 shRNA group and a control group with six mice in each group. At the day 7, 14, 21, 28, 35 following tumor cell injection, tumor size was measured and tumor growth was analyzed.

Colony formation assay

The transfected osteosarcoma cells, about 1 × 10 3 , were planted in 6-well plate and cultured with DMEM completed growth medium at 37 °C for 14 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 30 min. Then, wash cells and take photos. Colonies containing > 50 cells were manually counted. Each experiment was conducted in triplicate for more than three independent experiments.

Wound healing assays

Osteosarcoma cells were seeded in six-well plates and cultured to 90% confluence under same culture condition. A plastic pipette tip was used to scratch the cell monolayer and generate wounds. Phosphate buffered saline (PBS) was used to rinse and remove the floating cells. The wounds distance was measured with Image pro plus 6.0 software (Media Cybernetics Inc., MD, USA) at 48 h. The data were representative of three individual experiments carried out in triplicate.

Transwell assays

About 1 × 10 4 transfected cells were added into the transwell chambers (Corning, USA) which coated with Matrigel (BD Bioscience, MA, USA) in upper surface. Growth medium without fetal bovine serum was added in the upper chamber and 600 μl medium containing 10% fetal bovine serum was added into the low chamber. After 24 h, the upper cells invade to bottom surface. The cells under the membrane were fixed and strained using 0.1% crystal violet. Three randomly selected fields were captured and calculate the number of cells that had success fully invaded and transmigrated the Matrigel with Image J software.

Dual luciferase reporter assay

The binding sites were identified by JASPAR (http:// jaspa r.gener eg.net/) within circ_0051079, miR-26a-5p and TGF-β1. The constructed luciferase reporter plasmid pLG4.0 (Promega, Madison, WI, USA) or empty vector was transfected into cells. The luciferase activity was measured using the dual-luciferase reporter kit (Promega, Madison, WI, USA) after transduction 48 h. The relative firefly luciferase activity was normalized to Renilla luciferase activity.

Western blotting

The total protein of the osteosarcoma tissues and cells was harvested with cold RIPA (radio immunoprecipitation assay buffer). Bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA) was performed to qualify the protein concentration. Then, equal protein samples were loaded into 10% SDS-PAGEand transferred onto a PVDF membrane, which was incubated in the primary antibodies and then the secondary antibodies. Antibodies to TGF-β1 and GAPDH used in this study were purchased from ABCAM.

Statistical analysis

Data from three independent experiments were performed using the SPSS 15.0 statistical software (SPSS Incorporation, Chicago, IL) and emerged as mean ± standard deviation (SD). Difference between two groups was assessed using Student's t test or one-way ANOVA. Kaplan-Meier method was used to analyze the survival curves for patients with high or low expression of circ_0051079. p < 0.05 was considered to be statistically significant.

Fig. 1
1Deregulated circRNAs in osteosarcoma tumor tissues. a The heat map of significant circRNAs in osteosarcoma tissues and the matched non-tumor tissues. circRNAs were analyzed by circRNAs Arraystar Chip. The samples were from 5 osteosarcoma patients. b-e Relative expression of the four circRNAs from 45 osteosarcoma tumor tissues and adjacent non-tumor tissues listed in (a) measured by RT-qPCR. **p < 0.01 Subsequently, the circ_0051079 expression was performed in six osteosarcoma cell lines (HOS, KH-OS, MG63, 143B, U2OS, SaoS2) and one normal osteoblastic cell line (hFOB).

Fig. 2
2Circ_0051079 was upregulated in osteosarcoma patients and correlated with poor survival. a The expression level of circ_0051079 was detected in serum samples from osteosarcoma patients (n = 32) and healthy individuals (n = 32). b Kaplan-Meier curves for overall survival. There were 105 patients including 63 males and 42 females. The average age was 36.2 years old. c The expression level of circ_0051079 in six osteosarcoma cell lines and one normal osteoblastic cell line hFOB. d The expression level of circ_0051079 in the growth medium from six osteosarcoma cell lines and one normal osteoblastic cell line hFOB. **p < 0.01

Fig. 3
3Circ_0051079 negatively regulated miR-26a-5p in osteosarcoma cells. a The putative sequences of miR-26-5p and circ_0051079 binding site. b, c Luciferase activity of circ_0051079 in osteosarcoma cells. Cells were transfected with miR-26a-5p and the plasmids with the circ_0051079 reporter and then luciferase activity was analyzed by dual-luciferase system. d MiR-26a-5p expression in osteosarcoma cells. The cells were transfected with circ_0051079 shRNA for 48 h and the levels of it was detected by real time RT-PCR. e Circ_0051079 expression in osteosarcoma cells. The cells were transfected with miR-26a-5p mimics for 48 h and the levels of it was detected by real time RT-PCR. f MiR-26a-5p expression in osteosarcoma tissues. Total RNA from 32 primary osteosarcoma tissues was extracted for miR-26a-5p examination by real time RT-PCR. **p < 0.01

Fig. 4
4Fig. 4 Circ_0051079 played an oncogenic role in osteosarcoma cells via miR-26a-5p. a, b Cell proliferation ofU2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26-5p mimics for 24 h and then MTT assay was used to evaluate the cell proliferation of U2OS and SaoS2 cells. c, d Cell proliferation of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26-5p mimics for 24 h and then cell colony formation assay was used to evaluate the cell proliferation of U2OS and SaoS2 cells. e, f The migratory ability of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26a-5p mimics for 24 h and cell migration was analyzed. g, h The invasion ability of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26a-5p mimics for 24 h and cell migration was analyzed. i, j The growth curve of U2OS and SaoS2 cells in vivo. U2OS and SaoS2 cells with circ_0051079 shRNA or miR-26a-5p mimics were injected to the nude mice and the tumor size was recorded and analyzed every 7 days. **p < 0.01

Fig. 5
5MiR-26a-5p suppressed osteosarcoma cell proliferation and invasion by targeting TGFB1 expression. a The putative sequences of miR-26a-5p and TGF-β1 with a binding site. b, c

Fig. 6
6Circ_0051079 promoted osteosarcoma cellproliferation and metastasis through miR-26a-5p/TGF-β1. a TGFB1 mRNA and protein levels in U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 plasmids and circ_0051079 levels were assayed by real time RT-PCR. b, c Cell proliferation in U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079, miR-26a-5p mimics or TGF-β1 shRNAs for 24 h and then cell proliferation was assayed by MTT. d, e Cell migration in U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079, miR-26a-5p mimics or TGF-β1 shRNAs for 24 h and then cell migration was assayed by transwell chamber. f, g Cell invasion in U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079, miR-26a-5p mimics or TGF-1 shRNAs for 24 h and then cell invasion was assayed by transwell system. **p < 0.01Zhang et al. Cell Biosci  (2019) 9:94

Table 1
1The information of patients with osteosarcomaCharacteristics
Numbers

Gender

Male
27

Female
18

Age (years)

< 40
32
≥ 40
13

Tumor size (cm)

< 6
29
≥ 6
16

WHO grade

I-II
31

III
14

AcknowledgementsThe study was supported by Luoyang Orthopedic Hospital of Henan Province (Orthopedic Hospital of Henan Province).1Supplementary informationSupplementary information accompanies this paper at https ://doi. org/10.1186/s1357 8-019-0355-2.Additional file 1:Figure S1. a The microscopic images of migration assays. The migratory ability of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26a-5p mimics for 24 h and cell wound healing assay was performed and the photos were taken under a microscope. b The invasion ability of U2OS and SaoS2 cells. U2OS and SaoS2 cells were transfected with circ_0051079 shRNA or miR-26a-5p mimics for 24 h and the photos of invaded cells were taken under a microscope.Figure S2. The effects of Hsa_circ_0051079 knockdown or overexpression on the protein expression of TGFB1 and the activation of TGFB signaling. The total protein from U2OS and SaoS2 cells were extracted for western blotting.Figure S3. The correlation between Hsa_circ_0051079 and TGF-β1 in patients.Authors' contributionsZZ, the corresponding author, designed and conducted this study and was a major contributor in writing the manuscript. MZ and GW consulted literatures and performed the experiment conduction and data analysis. All authors read and approved the final manuscript.FundingNone.Availability of data and materialsThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Ethics approval and consent to participateWritten informed consent was obtained from all patients prior to participation.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Author details
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Transforming growth factor-β1/Smad7 in intestinal immunity, inflammation, and cancer. E Troncone, I Marafini, C Stolfi, G Monteleone, Front Immunol. 91407Troncone E, Marafini I, Stolfi C, Monteleone G. Transforming growth factor-β1/Smad7 in intestinal immunity, inflammation, and cancer. Front Immunol. 2018;9:1407.

In situ expressions of protein 16 (p16(CDKN2A)) and transforming growth factor β-1 in patients with cervical intraepithelial neoplasia and cervical cancer. M E Viloria, J Bravo, Y Carrero, J A Mosquera, Eur J Obstet Gynecol Reprod Biol. 228Viloria ME, Bravo J, Carrero Y, Mosquera JA. In situ expressions of protein 16 (p16(CDKN2A)) and transforming growth factor β-1 in patients with cervical intraepithelial neoplasia and cervical cancer. Eur J Obstet Gynecol Reprod Biol. 2018;228:303-7.

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