Challenges in detection and treatment of multidrug resistant tuberculosis patients in Vietnam

Thuy Thi
Wertheim 6,1011

Thanh Hoang
Wertheim 6,1011

Nhung Viet Nguyen
Wertheim 6,1011

Ngoc Sy
Wertheim 6,1011

Dinh
Wertheim 6,1011

Hoa Binh Nguyen
Wertheim 6,1011

Frank Cobelens
Wertheim 6,1011

Guy Thwaites
Wertheim 6,1011

Huong Thien Nguyen
Wertheim 6,1011

Anh Thu Nguyen
Wertheim 6,1011

Pamela Wright
Wertheim 6,1011

Heiman F L
Wertheim 6,1011

Challenges in detection and treatment of multidrug resistant tuberculosis patients in Vietnam
10.1186/s12889-015-2338-5R E S E A R C H A R T I C L E Open Access
Background: Vietnam is ranked 14 th among 27 countries with high burden of multidrug-resistant tuberculosis (MDR-TB). In 2009, the Vietnamese government issued a policy on MDR-TB called Programmatic Management of Drug-resistant Tuberculosis (PMDT) to enhance and scale up diagnosis and treatment services for MDR-TB. Here we assess the PMDT performance in 2013 to determine the challenges to the successful identification and enrollment for treatment of MDR-TB in Vietnam. Methods: In 35 provinces implementing PMDT, we quantified the number of MDR-TB presumptive patients tested for MDR-TB by Xpert MTB/RIF and the number of MDR-TB patients started on second-line treatment. In addition, existing reports and documents related to MDR-TB policies and guidelines in Vietnam were reviewed, supplemented with focus group discussions and in-depth interviews with MDR-TB key staff members.Results: 5,668 (31.2 %) of estimated 18,165 MDR-TB presumptive cases were tested by Xpert MTB/RIF and second-line treatment was provided to 948 out of 5100 (18.7 %) of MDR-TB patients. Those tested for MDR-TB were 340/3224 (10.5 %) of TB-HIV co-infected patients and 290/2214 (13.1 %) of patients who remained sputum smear-positive after 2 and 3 months of category I TB regimen. Qualitative findings revealed the following challenges to detection and enrollment of MDR-TB in Vietnam: insufficient TB screening capacity at district hospitals where TB units were not available and poor communication and implementation of policy changes. Instructions for policy changes were not always received, and training was inconsistent between training courses. The private sector did not adequately report MDR-TB cases to the NTP. Conclusions: The proportion of MDR-TB patients diagnosed and enrolled for second-line treatment is less than 20 % of the estimated total. The low enrollment is largely due to the fact that many patients at risk are missed for MDR-TB screening. In order to detect more MDR-TB cases, Vietnam should intensify case finding of MDR-TB by a comprehensive strategy to screen for MDR-TB among new cases rather than targeting previously treated cases, in particular those with HIV co-infection and contacts of MDR-TB patients, and should engage the private sector in PMDT.

Background

Multidrug-resistant tuberculosis (MDR-TB) is a global health concern as treatment is prolonged, costly, and less effective compared to that of drug-susceptible TB. Globally, there are about 450,000 MDR-TB patients reported with an estimated 170,000 MDR-TB-related deaths annually. Regarding the TB and MDR-TB epidemiology in Vietnam, the country is ranked 12 nd among 22 high burden countries with TB, and 14 th among 27 countries with a high burden of MDR-TB. The incidence of TB in Vietnam is estimated at 144 per 100,000 population per year and the estimated prevalence is 209 cases per 100,000 population [1]. There are estimated to be about 5100 MDR-TB cases among notified TB cases per year. The proportion of TB cases with MDR-TB among new and retreatment cases is estimated to be 4 and 23 %, respectively, with around 6 % among TB cases co-infected with HIV [1]. A preliminary report from a TB drug resistance survey conducted in Vietnam from 2011-2012 showed that the estimated proportion of XDR-TB among MDR-TB was 5.6 %. Of the MDR-TB patients, 16.7 % showed resistance to fluoroquinolones (ofloxacin), 1.1 % to amikacin, 5.6 % to kanamycin, and 5.6 % to capreomycin (unpublished data).

In 2009, the Vietnamese government issued a policy on MDR-TB following WHO recommendations to enhance and scale up diagnosis and treatment services for MDR-TB [2]. It is based on the five essential control components that constitute the already implemented DOTS strategy for drug-susceptible TB. These components include: sustained political commitment, rational case finding, short-course treatment, an uninterrupted drugs supply, and standardized recording and reporting [3]. Since a pilot project in Ho Chi Minh City in 2009, diagnosis and treatment services for MDR-TB have become available in 35 of all 63 provinces by 2013. The programmatic management of drug-resistant tuberculosis (PMDT) is more complex than for susceptible TB as it requires greater human, financial and technical resources [3]. Between 2009 and 2011 the Vietnam PMDT program had a success rate, defined as MDR-TB patients who completed treatment and were cured, of approximately 75 %. This rate is high compared to many other countries reporting rates of 44-58 % [4,5]. Despite that the treatment outcome may depend on drug resistance patterns, this high success rate does reflect good compliance with the treatment regimen in Vietnam [6]. The current standard second-line regimen is kanamycin, capreomycin, levofloxacin, cycloserine, protionamide, and para-aminosalicyclic acid (PAS).

However, the number of MDR-TB patients detected and enrolled to second-line treatment is low compared to the expected number among notified TB cases, based on national drug susceptibility data [7]. This means that only a few of the MDR-TB patients get diagnosis and the recommended treatment. This can partly be explained by the fact that Vietnam did not have nation-wide PMDT coverage by 2013, and drug susceptibility testing was not yet done for all notified TB cases. However, PMDT was fully implemented in major cities by 2010, where just 30-50 % of the total estimated MDR-TB patients were enrolled for second-line treatment. This finding lead us to assess the proportion of MDR-TB patients enrolled out of the estimated number of MDR-TB in all PMDT areas of Vietnam in 2013 to determine the challenges to an efficient PMDT implementation and to provide recommendation to improve the PMDT enrollment.

PMDT includes five steps from diagnosis to treatment for MDR-TB patients:(1) identification of presumptive MDR-TB cases (individuals considered at high risk for MDR-TB) according to case definitions, (2) referring presumptive cases for diagnosis, (3) drug resistance testing, (4) obtaining informed consent from patients for treatment, and (5) enrollment of diagnosed MDR-TB patients to treatment. In this assessment, we used the data reported by the national TB control program to focus on three of these steps: identification of presumptive MDR-TB cases, drug resistance testing, and enrollment of diagnosed MDR-TB patients for treatment. Qualitative investigation was used for assessment of steps (2) and (4) and additionally to support the findings and their interpretation for the other steps.

Methods

By 2013, 35 provinces had implemented the PMDT and participated in the MDR-TB enrollment assessment. Thirty-one provinces were selected by the NTP to implement PMDT based on their prior MDR-TB case-load and case management capacity. The remaining four provinces with a low MDR-TB case-load were selected as they had a high HIV prevalence. All 35 provinces were provided access to diagnostic equipment for intensified case finding using the Xpert MTB/RIF (Xpert) assay, a within-cartridge real-time PCR assay that detects M. tuberculosis as well as mutations in the rpoB gene conferring resistance to rifampicin in clinical specimens [8]. The selected provinces are required to provide PMDT treatment services in case MDR-TB patients are diagnosed. For the assessment, we reviewed all existing reports and documents related to MDR-TB policies and guidance since 2009 through December 2013.

A presumptive MDR-TB case requiring testing was defined as belonging to at least one of the risk groups as defined in the NTP guidelines (see Additional file 1). In order to calculate the proportion of those screened for MDR-TB, we divided the number of tested presumptive MDR-TB cases by the number of presumptive MDR-TB cases estimated using annual data reported by each province to the NTP (step1). All PMDT provinces provide quarterly reports to the NTP of the number of Xpert MTB/RIF tests done and the number of MDR-TB cases detected. We used the 2013 Xpert MTB/RIF data for assessing the number of presumptive cases screened per risk category (step 3).

For national estimation of MDR-TB, we used the number of all notified TB cases per province and applied the result of the most recent national DRS in Vietnam (year 2011-2012) which showed prevalence of MDR-TB among new and previously treated cases of 4.0 % and 23.0 %, respectively [1]. This allowed us to estimate the expected number of incident MDR-TB cases in all provinces irrespective of detection by the NTP. We used the standard errors from the MDR-TB prevalence as estimated in the DRS to calculate confidence intervals for the proportion of MDR-TB patients enrolled for secondline treatment in this study. The proportion with 95 % confidence intervals (95 % CI) was estimated for the number of MDR-TB patients enrolled for second-line treatment among (i) the estimated number of MDR-TB cases in the whole country and (ii) the estimated number of MDR-TB cases in the 35 PMDT provinces (step5).

This review was followed by focus group discussions (FGD) in face-to-face workshops, which encompassed five separate one-day sessions with approximately 30 key provincial TB staff members (for all PMDT steps including steps 2 and 4). FGD were used to discuss the PMDT program, challenges to implementation and potential solutions. Five groups were invited from eight different geographic areas (see Additional file 1). TB staff included provincial program managers, program officers with expected knowledge of the local overall situation and staff responsible for implementation of PMDT in their province. Guidance was provided for FGD facilitators who had previous FGD discussion experience and knowledge of the NTP. Discussions were continued until saturation was reached before facilitators moved to a new topic. FGD summaries were checked and agreed upon by all participants to be finalized for content analysis later on. The sessions were not audio recorded and transcribed due to lack of funds.

Furthermore, in-depth interviews to supplement the focus group discussions was conducted by the same group of interviewers with eighty TB health staff at different levels of the NTP, including 8 central staff from PMDT team, 56 provincial staff from variety of departments, and 16 district and commune level health staff (for all steps). The interview guide was developed and piloted before the interviews and included questions regarding: structure of the TB health unit at specific levels in the health system, training provided to staff, their knowledge and awareness about MDR-TB risk groups, their role and job description, and challenges encountered in PMDT implementation. The in-depth interview also allowed the interviewee to reflect on findings from the FGD. These interviews were conducted during monitoring visits in 2014 in eight randomly selected provinces among the 35 PMDT provinces from three zones: four provinces were randomly selected from the southern zone as there exists the highest disease burden, two from the north and two from the centre (see Additional file 1). The interviews were conducted privately. During the interview and FGDs, detailed notes were taken and used subsequently to identify key themes. In the analysis, similarities and differences between the various interviews and FGDs were looked for and summarized to identify key findings. The interviews were not recorded.

This study was approved by the research and ethics committee of the National Lung Hospital in Hanoi. Informed consent was obtained from participants in workshops and in-depth interviews.

Results

The estimated number of MDR-TB cases in Viet Nam

Based on the notification report of the NTP in 2013 and the results of the recent national DRS, we estimated that in 2013 there were 5065 (95 % CI: 3355-6700) MDR-TB patients among 102,196 notified TB cases, resulting in a proportion of 5.0 % MDR-TB among notified cases for the year 2013 (95 % CI: 3.3-6.6 %). These cases were mainly concentrated in the South-East, Mekong Delta and Red River Delta regions. The PMDT program already covered provinces with an expected high number of MDR-TB cases ( Fig. 1 and Table 1). We estimated that the majority 3982/5065 (78.6 %) of the national MDR-TB case-load originates in the provinces participating in the PMDT ( Table 1).

Identification of MDR-TB presumptive cases to be tested (step 1)

In 2013, there were an estimated 18,165 cases identified as presumptive MDR-TB nationally, with the majority (n = 14,998) in the 35 PMDT provinces (82.6 %). There was no data available on the number of MDR-TB presumptive cases identified by the health staff of PMDT areas who were referred for screening.

Testing practices per risk category by Xpert MTB/RIF and detection of MDR-TB (step 3)

A total of 32 Xpert MTB/RIF instruments were distributed among the 35 PMDT provinces. The estimated number of MDR-TB cases to be detected per machine per year for these instruments varied by region, ranging from 9 to 338 MDR-TB cases (see Additional file 1). The instrument's capacity is expected to be sufficient as one instrument with four modules is able to test approximately 3000 MDR-TB presumptive cases to detect around 500 rifampicin resistant TB cases per year (see Additional file 1).

The proportion of MDR-TB presumptive cases tested by Xpert MTB/RIF was 31.2 % (5668/18,165) for the whole country and 37.8 % (5668/14,998) in the 35 PMDT provinces (Table 2). In PMDT provinces, the highest proportion of MDR-TB presumptive cases tested was in the North-West (43/46; 93.9 %) and the Southern Central Coast (668/897; 74.5 %) regions. The lowest proportions were in the Central Highlands (0/69; 0 %) and the Mekong Delta (516/3935; 13.1 %; Table 2) regions. In the North-West region, all provinces together only had 43/214 (20.1 %) of presumptive cases tested while that percentage was 43/46 (93.9 %) in the PMDT provinces. Among 5668 MDR-TB presumptive cases tested, 997 cases were detected as rifampicin resistant (17.6 %).

The proportions of HIV infected TB cases and new TB non-converters tested by Xpert MTB/RIF were 10.5 % (340/3224) and 13.1 % (290/2214), respectively (Table 3). Retreatment patients who showed no sputum conversion after 3 months of the category II regimen were tested frequently: 85.7 % (413/482). We estimated that 249/402 (62.0 %) of symptomatic household contacts of MDR-TB patients were tested. Among 249 MDR-TB symptomatic household contacts screened within 1 year after MDR-TB patients diagnosed, 77 TB patients were detected (30.9 %) including 9 TB patients resistant to rifampicin (3.6 %). We could not calculate the testing rate among failure cases since no data were available for the number of people tested. However, this rate was estimated to be at least 68.5 % because one failure case could receive up to two tests.

Qualitative investigations (all steps)

This section reveals the challenges to a successful diagnosis and enrollment for treatment of MDR-TB (Table 4). It was noted that TB units (i.e. small departments operating under the NTP that identify MDR-TB presumptive, provide TB diagnostic and first-line treatment services by specifically trained staff) are located mainly in district health centers. Although there is a policy change ongoing to have TB units also in (district) hospitals this had not yet been implemented in all districts. As a result, several district hospitals, where many MDR-TB presumptive cases present did not have TB diagnostic facilities and hospital staff had not been trained and instructed to screen patients for TB and MDR-TB.

Provincial and district staff reported that they were overall not well informed about PMDT procedures. The first guideline for PMDT was issued in 2009 [2] and has not been updated for any of the multiple changes since then. The changes in policies over time had been updated through separate instruction letters from the central level and though yearly PMDT training courses without documenting these in a clear set of guidelines.

A total of 17 instruction letters to inform about changes have been sent out from central level to provinces between 2009 to late 2013. However, regularly these update letters were not received by the provincial PMDT officer or the information was not disseminated to the relevant staff at lower levels. A further challenge was regular health care staff turnover or change in health care staff 's responsibilities over time. As a consequence, it is Standard operating procedures (SOPs) for PMDT were developed and released in 2012, three years since the MDR-TB guideline was released. The SOPs lacked clear instructions for steps that need to be taken [9]. No standard PMDT training modules are available as the training and presentations were prepared and updated by different lecturers, based on their interpretation of the available guidelines and SOPs. Inconsistencies across training courses were mentioned by trained health staff, which generated uncertainty. In addition, the majority of district level staff was not trained due to lack of funding. In 2013, the majority of district staff with an assigned responsibility to identify presumptive MDR-TB had not been trained to identify risk groups. Supervision visits conducted between April and July 2014 revealed that, although all districts under PMDT had been trained, their focus was on retreatment cases, while nonconverters among new cases and HIV-positive TB patients were generally neglected.

Many TB units were unable to send sputum for testing by Xpert MTB/RIF as no appropriate financial compensation mechanisms were in place for consumable procurement and sputum transportation fees. Partly this was due to a delay in an award from the Global Fund. In addition it was not straightforward to send specimens by mail as no agreements with the postal services were in place, and sputum referral using health staff or public transportation were used instead. This may explain why in some regions with Xpert MTB/RIF capacity, like the Mekong delta region, the proportion tested and enrolled was limited. Finally, there had been stock outs of Xpert MTB/RIF test kits due to delays in cartridge forecast  reporting. Although challenges were mentioned for sending sputum, there was always a confirmation and communication between referring sites and laboratory after samples had been sent. A small number of referred samples were rejected by the laboratory for being considered low-quality or low-volume, and requested to recollect and re-send. For MDR-TB patients that were diagnosed, health staff mentioned that informed consent was obtained for all of these patients before starting treatment. There were issues regarding enrolling MDR-TB patients for treatment due to lack of hospitalization capacity. It was reported that poor links existed between the public and private sector, as patients cannot be referred from the private to the public sector to continue their treatment. Furthermore, there are no management systems in place to screen and treat MDR-TB patients in prison (see Table 4 for more details).

Discussion

We estimated that only one third of MDR-TB presumptive cases are screened by Xpert MTB/RIF in Vietnam. However, it is encouraging that 95 % of the patients who were tested positive for MDR-TB were enrolled for second-line treatment. The low enrollment rate is mainly attributable to low number of presumptive MDR-TB cases identified and subsequently tested, and not due to poor enrollment of patients after diagnosis. There were some inconsistencies between the percentage of MDR-TB presumptive cases tested in 35 PMDT provinces (Table 2) and the enrollment rate of MDR-TB (Table 1). While there were relative high testing proportion in the North-East, North-West, Northern Central and Southern Central Coast regions, the enrollment proportion for treatment among number estimated were low, varying from 0 % to 27.3 %. In other regions, the variations in enrollment rates were in line with the variations in percentage of MDR-TB presumptive cases tested. The reasons behind these inconsistencies were limited capacity for hospitalization and other issues as revealed by our qualitative assessment.

This study showed that access to Xpert MTB/RIF testing was overall sufficient for Vietnam. However, regions with a higher number of operational Xpert MTB/RIF instruments had a higher enrollment proportion compared to regions with fewer instruments, except for the Mekong River Delta (Fig. 3). While the burden of MDR-TB in Mekong Delta region was high, with Xpert MTB/RIF instruments widely available, the enrollment proportion in this region was very low: 7.5 % (Table 1, Fig. 3). The reason behind the low enrollment proportion in Mekong Delta region is a weak system for transferring specimens for Xpert MTB/RIF testing as revealed by our qualitative study. In addition, procurement of test kits needs improvement to avoid stock outs, and redistribution of Xpert MTB/RIF instruments from the Red River Delta with 9 instruments and a low workload to the Northern Central region with just one instrument and a high workload may improve access to testing for risk categories. Testing coverage was nil in the Central Highlands region. Although this area has a relatively low number of estimated MDR-TB cases, facilitating a good specimen referral system for testing could increase the coverage. Main challenges for effective MDR-TB diagnosis and treatment and potential solutions proposed by our study team for under enrollment are listed in Table 4.

New TB patients whose sputum had not converted after 2 or 3 months of treatment and TB patients with HIV, both considered risk groups for MDR-TB, were largely neglected for screening and had the lowest proportions of MDR-TB screening done while they account for the majority of presumptive MDR-TB cases [2,3]. Since the proportion of detected rifampicin-resistant patients among these groups in 2013 was 11.3 % [10], it is likely that a considerable number of MDR-TB remain undiagnosed due to inadequate screening among these groups. Importantly, undetected MDR-TB/HIV patients who have high mortality rates [11,12] would lose the opportunity for adequate treatment. Moreover, TB-HIV patients in 2013 are underreported due to limited collaboration between the TB and HIV control programs [13].

The qualitative assessment revealed that many patients refuse treatment in units far from home. Other patients reported problems getting health insurance reimbursement in case they were referred to another province. Lack of collaboration between treatment centers and adjacent provinces for referring patients for treatment also contributed to drop outs. Another contributing factor to treatment delays and poor enrollment was the irregular delivery and stock outs of MDR-TB treatment drugs due to procurement delays. Occasionally, patients insisted to be enrolled, but they had to wait for a long time to start treatment.

This study also revealed poor links between the public and private sector. Due to absence of a referral mechanism between the two systems, MDR-TB presumptive were not referred from private sector to the NTP for diagnosis, and patients who withdrew from MDR-TB treatment in 3

Absence of a sound referral system for sending sputum samples to a laboratory.

A sound national referral system should be set up with a shipping agency who can do this safely.

4 Current Vietnam policy is to require patient to be hospitalized at the start of treatment. However, there is insufficient hospitalization capacity and patients may refuse to be referred to another treatment centre in another province either due to distance from hometown or additional costs without getting health insurance reimbursement.

The NTP policy needs consider the adoption for ambulatory treatment with community based care as also recommended by WHO [3]. Health insurance need to support MDR-TB patients in reimbursing costs in case there is a need for referral to another province for PMDT treatment.

5

Temporary MDR-TB drug stock-out due to procurement and distribution delay resulted in patients either not enrolled for treatment or a delay in treatment.

Improve drug procurement and distribution system.  [14]. Also MDR-TB patients in prisons pose a challenge as second-line treatment is not available, and no procedures for management of MDR-TB patients after discharge from prison are in place.

The current PMDT policy is that each district should have a TB unit. The Ministry of Health has as a policy to locate TB units in general hospitals, or in district health centers if a general hospital is not present [15]. This policy is aimed at strengthening the utilization of diagnosis and treatment services for TB units in the district hospital, where most patients seek health care. However, our study found that not all district hospitals have a TB unit with TB screening capacity. Health staff lack proper training and MDR-TB screening skills and do not have access to updated guidelines/SOPs to support them in identifying patients. This results in confusion and limited confidence among staff to implement the PMDT properly. Inconsistencies across training courses also contributed to confusion around the PMDT policies. Clear guidelines, instructions and training for health staff is needed to improve this.

Our study has several limitations. To estimate the MDR-TB enrollment proportion we used two different data sets: the 2013 NTP report and the DRS conducted in 2011-2012. For the number of MDR-TB cases detected, we only used laboratory reports from Xpert MTB/RIF testing and we did not include reports from other tests such as line-probe assays or phenotypic drug susceptibility testing. As the number of tests done by these other methods is small, less than 5 % with considerable overlap with Xpert MTB/RIF testing, we decided to exclude these.

Furthermore, as we do not know the prevalence of rifampicin resistance for each MDR-TB risk group we could not assess the positive predictive value (PPV) of Xpert MTB/RIF separately among each category of presumptive MDR-TB patients. However, for most categories, the estimated prevalence of rifampicin resistance is relative high (around one-fourth, given the 23 % prevalence among previously treated patients in the DRS). In addition, recent studies from South Africa [16] and Brazil [17] suggest a PPV on the order of 90 % even with relatively low prevalence of rifampicin resistance. Therefore we do not expect that the PPV in our study will have been much below 90 %, and thus did not affect the results to considerable extent.

A final limitation is that we did not involve the private sector in our study. However, our qualitative assessment suggests poor management of MDR-TB in the private sector and a lack of a good referral system between the private and public sector. This situation causes patients to be under screened, poorly treated, and underreported to the NTP. Better links need to be established between the private and public sector.

Conclusion

The proportion of MDR-TB patients enrolled for second-line treatment among the total estimated number of MDR-TB cases in Vietnam in 2013 was 18.7 % (948/5065). The low enrollment was considered due to under-screening of MDR-TB presumptive cases, especially for HIV infected and new TB non-converters. Multiple reasons exist for under-screening, including: poor communication and implementation of policy changes, lack of involvement of general district hospitals, and limited resources. However, Vietnam has achieved a high proportion of enrollment to second-line treatment among of detected MDR-TB cases and a high treatment success rate, which can be considered an opportunity to mobilize resources and expand the program to detect and treat more MDR-TB cases. Vietnam should expand the intensified case finding of MDR TB by a comprehensive strategy with more focus on new detected cases, in particular those with HIV co-infection and contacts of MDR-TB patients. The capacity to treat new MDR-TB cases will need to be increased in case the screening is improved. The private sector needs to be engaged in the PMDT.

Additional file

rifampicin-resistant cases detected, 948 (95.1 %) were enrolled for MDR-TB treatment, accounting for just 18.7 % (95 % CI: 14.1-28.3 %) of the estimated 5065 MDR-TB cases in the whole of Vietnam (Fig. 2). The region with the highest proportion of MDR-TB patient enrolled for second-line treatment was the South-East region with 495/1546 (32.0 %; 95 % CI: 24.3-47.8 %), followed by the Red River Delta with 234/859 (27.3 %; 95 % CI: 20.5-41.4 %). The lowest enrollments were in the Mekong Delta region: 81/1288 (6.3 %; 95 % CI:

Fig. 1
1Estimated number of MDR-TB patients among notified TB cases in Viet Nam, 2013 4.8-9.5 %). No enrollments were reported in PMDT for North-East, North-West and Central Highlands (Table 1, Fig. 3). Also in the 35 PMDT provinces, the enrollment proportion of MDR-TB cases was low: 948/ 3982 (23.8 %; 95 % CI:18.0-35.9 %). The highest proportion was seen in the PMDT provinces in the Red River Delta with 234/627(37.3 %; 95 % CI: 28.2-56.5 %), and South-East region: 495/1408 (35.2 %; 95 % CI: 26.7-52.4 %)

Fig. 2
2PMDT performance for case detection and enrollment of MDR-TB in Vietnam

Fig. 3
3The enrollment proportion into the second-line treatment program of MDR-TB cases among the estimated number of notifiable MDR-TB cases in 35 PMDT provinces in 2013. Data are presented per socio-economic region

Table 2
2Percentage of presumptive MDR-TB patients tested by regions Actual enrollment and estimated total number of MDR-TB cases nationwide and in 35 PMDT provinces (the numbers in brackets are for lower and upper CI)Ser. No
Region
Number of
presumptive
MDR-TB
patients tested

All provinces
35 PMDT provinces

Estimated Number of
presumptive
MDR-TB patients

% tested
Estimated Number of
presumptive MDR-TB patients

% tested

1
Red River Delta
801
2,519
31.8 %
2,035
39.4 %

2
North-East
263
1,154
22.8 %
627
42.0 %

3
North-West
43
214
20.1 %
46 a
93.9 %

4
Northern Central
460
1,144
40.2 %
886
51.9 %

5
Southern Central Coast
668
1,117
59.8 %
897
74.5 %

6
Central Highland
0
290
0.0 %
69
0.0 %

7
South -East
2,917
6,991
41.7 %
6,503
44.9 %

8
Mekong Delta
516
4,736
10.9 %
3,935
13.1 %

Total
5,668
18,165
31.2 %
14,998
37.8 %

a

one PMDT province in North -West

Table 3
3Presumptive MDR-TB patients estimated and tested by risk category in the whole country and in 35 PMDT provinces of Vietnam 2013Presumptive category
Estimate number for whole
country a

Estimate number for PMDT
areas only

Number of Xpert tests
performed

% tested in PMDT
areas

Retreatment
Failure b
593
519
711
ND

Relapse
7,059
5,673
2,641
46.6 %

Defaulters
472
376
129
34.3 %

Other
739
662
210
31.7 %

Non-converters at 2 and
3 months

New
2,713
2,214
290
13.1 %

Retreatment 586
482
413
85.7 %

MDR contacts
402
402
249
62.0 %

TB/HIV
3,828
3,224
340
10.5 %

>1 month using TB drugs
1,773
1,446
685
47.4 %

Total MDR-TB suspects
18,165
14,998
5,668
37.8 %

a

The denominator is the number of presumptive by categories that need to be tested

b

Failure is defined as sputum smear positive at 5 months or later during treatment, so one failure case could receive one or 2 tests (at 5 and/or 7 months). ND:
not done as failure cases can be tested on multiple occasions

Table 4
4Obstacles to enrolling MDR-TB patients for treatment and solutions for an effective PMDTSer. No Obstacle

6TB units in many districts remain located in health centers that focus on prevention, and are separate from the general hospitals, which is discrepant from MoH policy.Enforce policy to locate TB units in the district general hospitals. Training should be provided to appropriate staff. Poor links between the NTP and public sector and no management system for MDR-TB patients in prison. There is no mechanism to refer MDR-TB presumptives or MDR-TB patients from the private sector to the NTP to be diagnosed or for treatment. The private sectors often do not notify MDR-TB cases to the NTP.Establish the collaboration between the private sector and PMDT. Ensure private sector adheres to treatment guidelines for TB and MDR-TB. Provide diagnosis and treatment service for MDR-TB patients in prison. the private sector for financial or other reasons were unable to enroll to continue treatment in the public PMDT. Currently, only limited data for MDR-TB patients in the private sector are available, where default rates are high, up to 75 %, implying poor MDR-TB management in the private sector that may lead to drug resistance amplification7

AcknowledgmentsThis work was approved by the NTP to be conducted and financially supported by the Wellcome Trust Major Overseas Program, Vietnam. The sponsor had no role in the study design, data collection, analysis or decision to submit for publication.Competing interestsThe authors declare that they have no competing interests.Authors' contributions NVN, HTTT, HW, NTA conceptualized and designed the study. HTTT was responsible for data collection, analysis and interpretation of data. HTTT wrote the first draft of the paper with critical advice provided by NVN, HW, NTA, PW, NTH, NBH and FC. All authors reviewed and approved the manuscript as submitted.Authors' information Not applicable.
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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. A Trajman, B Durovnil, V Saraceni, M Cordeiro, F Cobelens, S Van Den Hoff, Eur Respir J. 446High positive predictive value of Xpert in a low rifampicin resistance prevalence setting. Submit your manuscript at www.biomedcentral.com/submitTrajman A, Durovnil B, Saraceni V, Cordeiro M, Cobelens F, van den Hoff S. High positive predictive value of Xpert in a low rifampicin resistance prevalence setting. Eur Respir J. 2014;44(6):1711-3. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


Oncogenic miRNA-182-5p Targets Smad4 and RECK in Human Bladder Cancer
2012

H Hirata
K Ueno
V Shahryari
Y Tanaka
Z L Tabatabai
Oncogenic miRNA-182-5p Targets Smad4 and RECK in Human Bladder Cancer

PLoS ONE
711510562012Received September 14, 2012; Accepted October 29, 2012;This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. 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. *
Onco-miR-182-5p has been reported to be over-expressed in bladder cancer (BC) tissues however a detailed functional analysis of miR-182-5p has not been carried out in BC. Therefore the purpose of this study was to: 1. conduct a functional analysis of miR-182-5p in bladder cancer, 2. assess its usefulness as a tumor marker, 3. identify miR-182-5p target genes in BC. Initially we found that miR-182-5p expression was significantly higher in bladder cancer compared to normal tissues and high miR-182-5p expression was associated with shorter overall survival in BC patients. To study the functional significance of miR-182-5p, we over-expressed miR-182-5p with miR-182-5p precursor and observed that cell proliferation, migration and invasion abilities were increased in BC cells. However cell apoptosis was inhibited by miR-182-5p. We also identified Smad4 and RECK as potential target genes of miR-182-5p using several algorithms. 39UTR luciferase activity of these target genes was significantly decreased and protein expression of these target genes was significantly up-regulated in miR-182-5p inhibitor transfected bladder cancer cells. MiR-182-5p also increased nuclear beta-catenin expression and while Smad4 repressed nuclear beta-catenin expression. In conclusion, our data suggests that miR-182-5p plays an important role as an oncogene by knocking down RECK and Smad4, resulting in activation of the Wnt-beta-catenin signaling pathway in bladder cancer.

Introduction

Bladder cancer (BC) is the third leading cause of death among urological tumors and the most common histological type of bladder cancer is urothelial carcinoma (UC), formerly known as transitional cell carcinoma (TCC) [1]. Approximately 75% of patients are ''non-muscle invasive UC (pTa, pTis, pT1) and have a 5-year survival rate of between 88-98% [2]. The common treatment for these patients is endoscopic resection [1,3]. Patients with muscle invasive UC are usually treated with radical cystectomy or chemo radiotherapy [1,4]. However half of muscle invasive UC patients develop subsequent metastatic disease after the first aggressive treatment [1,5]. Previous studies have identified several potential molecular biomarkers for bladder cancer [6,7]. Inactivation of tumor suppressor genes TP53 and Rb and Ras oncogene activation have been regarded as important key players in bladder cancer carcinogenesis [6].

Activation of Wnt-beta-catenin signaling has also been studied and reported to be associated with cancer progression and poor prognosis in bladder cancer [8,9]. Transforming growth factor beta (TGF-beta) plays a crucial role in embryonic development and pathogenesis of several diseases and cancer [10]. Evidence of crosstalk between TGF-beta and other signaling pathways including Wnt signaling have been reported [10]. Smad4 is a central intracellular signal transduction component of TGF-beta and recent studies have shown that Smad4 cooperates with betacatenin in several caners [11][12][13][14].

RECK is crucial repressor of matrix metalloproteinases (MMPs) and previous studies have shown that RECK expression is significantly lower in bladder cancer tissues compared to normal urothelial tissues [15][16][17].

So far many microRNAs have been identified and reported to be important in several cancers [18]. MicroRNAs (miRNAs) are small non-coding RNAs, approximately 22 nucleotides in length, that are capable of regulating gene expression at both the transcription and translation levels [19]. MiRNAs bind to the 39UTR of target mRNA and repress translation from mRNA to protein or induce mRNA cleavage and thereby regulate the expression of target genes [20].

In this study, we found that miR-182-5p was significantly higher in bladder cancer tissues compared to normal urothelial tissues and high miR-182-5p expression was significantly associated with shorter overall survival. So far there have been no reports about the function of miR-182-5p in bladder cancer. Thus we focused on miR-182-5p, performed functional analyses, identified several target genes of miR-182-5p using several algorithms and identified Smad4 and RECK as target genes. Finally, we over-expressed these target genes (Smad4 and RECK) in bladder cancer cells to examine the mechanism of miR-182-5p function.

Results

miRNA-182-5p Expression is Significantly Higher in Bladder Cancer Tissues and Associated with Shorter Overall Survival

We compared miRNA-182-5p expression levels in bladder cancer tissues (n = 18) and normal urothelial tissues (n = 6) by realtime PCR. The miR-182-5p expression was significantly higher in bladder cancer tissues (Fig 1A). We investigated the association of miR-182-5p and several clinical parameters as shown in Figure 1-B and observed that high miR-182-5p expression was significantly associated with shorter overall survival.

Regarding several other clinical parameters, no significant relationship was observed. We divided the 18 bladder cancer patients into two categories based on the median value and Kaplan Meier plots showed that overall survival was shorter in the high miR-182-5p expressing group (p value = 0.0349, Log-rank test) (Figure 1-C).

miRNA-182-5p Expression is Significantly Increased in Bladder Cancer Cell Lines

We compared miR-182-5p expression in several bladder cancer cell lines and its expression in T24 and UM-UC-3 cells was in the range of that in bladder cancer tissues. Thus we used these two bladder cancer cell lines for further experiments in this study ( Figure 1A).

To confirm the function of miR-182-5p, we transfected miR-182-5p precursor into bladder cancer cell lines (T24 and UM-UC-3). At 24 hours after transfection of miR-NC or miR-182-5p precursor into bladder cancer cells, the miR-182-5p expression level was verified by real time PCR (fold change; 4545, 5920, respectively Fig. 2-A). Then several functional analyses were performed. We observed significantly increased cell proliferation ( Fig. 2  RECK mRNA has one while Smad4 has two potential complimentary binding site with miR-182-5p within its 39 UTR ( Fig. 3-A). Based on these results, we performed 39UTR luciferase assays and found that the relative luciferase activities with these sites were significantly decreased in miR-182-5p transfected bladder cancer cells (T24, UM-UC-3) (Fig. 3-B). These results suggest that RECK and Smad4 mRNAs are potential target genes of miR-182-5p. Western analysis confirmed that RECK and Smad4 protein expression was significantly increased in miR-182-5p inhibitor transfected cells ( Fig. 3-C).

Effects of Over-expression of RECK and Smad4 on Bladder Cancer Cell (T24) Function

To look at the function of RECK and Smad4, we overexpressed RECK and Smad4 in T24 cells which was confirmed by measuring mRNA (Fig. 4-A) and protein expression levels ( Fig. 4-B). Then we performed several functional analyses. As shown in Figure

Effect of miR-182-5p and Smad4 on Beta-catenin Expression in the Nuclear Fraction

As shown in Figure 4-G, beta-catenin expression in the nuclear fraction was significantly increased in miR-182-5p transfected T24 cells. In contrast, beta-catenin expression in the nuclear fraction was significantly decreased in Smad4 transfected T24 cells.  [21][22][23][24][25][26][27][28][29]. Two miRNAs (miR-21 and miR-129) have been identified and confirmed as oncogenes in bladder cancer [30,31].

Discussion

MiR-182-5p has also been reported to be an oncogene in several cancers [32][33][34][35][36][37]. Similar to our results, one report found that miR-182-5p expression was significantly higher in bladder cancer tissues compared to normal urothelium, however functional analysis was not performed in this study [38]. Thus we investigated the relationship between miR-182-5p expression and clinical parameters including pathological stages and patient outcomes and found that miR-182-5p expression was correlated to shorter overall survival after operation in bladder cancer patients. These results suggest that miR-182-5p may be a new and useful diagnostic biomarker in bladder cancer.

Our next aim was to determine whether miR-182-5p functions as a bladder cancer oncogene. Of three bladder cancer cell lines (T24, UM-UC-3, J82), the expression of miR-182-5p in two cell lines (T24 and UM-UC-3) was in the range of expression observed in bladder cancer tissues. Thus we used these two bladder cancer cell lines (T24 and UM-UC-3) for functional analysis experiments. We found that over-expression of miR-182-5p significantly promoted bladder cancer cell viability, migration and invasion and inhibited apoptosis. We used several algorithms to search for potential miR-182-5p target genes since microRNAs exert their effects by regulating target gene expression. We identified RECK and Smad4 as potential target genes by 39UTR luciferase assay and Western analyses.

MMP plays an important role in cancer invasion and metastasis and MMP-2 elevation in bladder cancer tissues has been reported to be correlated with tumor stage and poor prognosis in bladder cancer patients [39,40]. RECK is a crucial MMP-2 repressor and RECK expression was previously reported to be down regulated in bladder cancer tissues compared to normal urothelium [16,17]. In addition, DNA methylation has been identified as a RECK silencing mechanism [41]. Recently miR-21 was identified as a regulator of RECK gene expression in several cancers [42], but to date there has been no report showing direct regulation of RECK by miR-182-5p.

We also investigated the function of RECK by over expressing it in a bladder cancer cell line (T24). As shown, RECK inhibited cell proliferation, migration and invasion abilities in bladder cancer cells and the number of apoptotic cells was increased by RECK transfection.

Smad4 is an important signal transduction component of TGFbeta and recent studies show that Smad4 functions by cooperating with beta-catenin in several cancers.

Wnt-beta catenin signaling is crucial for embryogenesis and tumourigenesis [43]. In cancer cells, the Wnt pathway is usually activated causing unphosphorylated beta-catenin to accumulate in the cytoplasm and moves to the nucleus, where it binds to TCF/ LEF and transcriptionally regulates Wnt target genes promoting tumorigenesis [43]. In bladder cancer, deregulated Wnt-betacatenin signaling plays an important role in progression and metastasis. Thus we looked to see whether beta-catenin expression was altered by either miRNA-182 or Smad4 transfection. As we observed, miR-182-5p increased nuclear beta-catenin expression while Smad4 decreased nuclear beta-catenin expression. As far as we know, there have been no reports about miR-182 and Wntbeta-catenin signaling and our results suggest that onco-miR-182-5p may be involved in the regulation of Wnt-beta-catenin related genes.

In our study, Smad4 overexpression decreased bladder cancer cell proliferation, migration and invasion ability. Apoptosis was also increased with Smad4 overexpression in bladder cancer cells. Since loss of Smad4 has been reported to play a causal role in initiating squamous cell carcinomas of the skin, upper digestive tract as well as adenocarcinoma of the gastrointestinal tract [44], our results may indicate that Smad4 plays an important role in bladder cancer. Since we focused only on Smad4 in the Wntsignaling cascade, it is possible that other genes may be directly or indirectly regulated by miR-182-5p. Additional experiments will be needed to elucidate the exact role of miR-182-5p in Wnt-beta catenin signaling. Taken together, this study shows that miR-182-5p exerts its oncogenic effects in bladder cancer cells by downregulating RECK and Smad4. This is the first report to also document that miR-182-5p expression is significantly increased in bladder cancer tissues where it functions as an oncogene by inhibiting RECK and Smad4 expression and may be potentially useful as a prognostic biomarker. These results also suggest that miR-182-5p may have therapeutic potential for the treatment of bladder cancer.

Materials and Methods

Ethics Statement

Formalin-fixed, paraffin-embedded (FFPE) bladder cancer samples were obtained from the San Francisco Veterans Affairs (VA) Medical Center. Written informed consent was obtained from all patients and the study was approved by the UCSF Committee on Human Research (Approval number: H9058-35751-01).

Clinical Samples

RNA and Protein Extraction

RNA (microRNA and total RNA) was extracted from formalinfixed, paraffin-embedded (FFPE) human bladder cancer and noncancerous normal bladder tissues (urothelial cells) using a miRNeasy FFPE kit (Qiagen) after laser micro-dissection based on pathologists reviews. Total RNA was also extracted from bladder cancer cell lines using a miRNeasy mini kit (QIAGEN). Cells were lysed with RIPA buffer (Thermo Scientific, Rockford, IL) containing protease inhibitors (Sigma, St. Louis, MO). Protein quantification was done using a BCA protein assay kit (Thermo Scientific, Rockford, IL). The NE-PER Nuclear and Cytoplasmic Extraction Reagent was used to extract nuclear and cytoplasmic

MicroRNA Transfection (pre-miR Precursor and miR Inhibitor)

Pre-miR TM miRNA precursors [negative control (miR-NC) or hsa-miR-182-5p, Ambion] were transiently transfected into bladder cancer cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Anti-miR TM miRNA inhibitor [negative control (inh-NC) or miR-182-5p inhibitor (miR-182 inhibitor), Ambion] were transiently transfected into bladder cancer cells by siPORT NeoFX Transfection Agent (Ambion) according to the manufacturer's instructions. After transfection, cells were incubated at 37uC for 48 hours until assessment.

Cell Viability, Cell Invasion, Wound Healing Assay

Cell viability was measured 3 days after transfection with MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega). Data are the mean 6 S.D. of 6 independent experiments. Cell invasion assays were performed with the CytoSelect 24-well cell invasion assay kit (Cell BioLab, San Diego, CA) according to the manufacturer's instructions. Transfected cells were re-suspended in culture medium without FBS and placed in the upper chamber in triplicate. After 48 hours incubation at 37 o C (5% CO2), cells migrating through the membrane were stained. The results were expressed as invaded cells quantified at OD 560nm. The wound healing process begins with tissue matrix remodeling, migration, and eventual closing of the wound area. Therefore this assay is frequently used for assessment of cancer cell migration. Wound healing assay was performed with the CytoSelect 24-well wound healing assay kit according to the manufacturer's instructions. To generate a wound field, transfected cells were cultured until they formed a monolayer around the insert. After removing the insert, a 0.9 mm open wound field was generated and cells were allowed to migrate from either side of the gap. Wound closure was monitored and the percent closure was measured. [Percent closure rate (%) = migrated cell surface area/ total surface area x100)].

Apoptosis Analyses

Cells (48 hours after transfection) were washed twice with 1xPBS and trypsinized. After inactivating trypsin in complete medium, the cells were re-suspended in ice-cold 1x binding buffer (70 ml). Annexin V-FITC solution (10 ml) and 7-AAD viability dye (20 ml) were added to 70 ml of the cell suspensions. After incubation for 15 minutes in the dark, 400 ml of ice-cold 1x binding buffer was added. The apoptotic distribution of the cells in each sample was then determined using a FACS (Cell Lab QUANTA SC, Beckman Coulter, Fullerton, CA). Data are the mean 6 S.D. of four independent experiments.

Plasmid Construction and 39UTR-Luciferase Assay

We constructed individual plasmids for each binding site in the 39UTR of mRNA from potential target genes based on microRNA.org information. Then we confirmed miR-182-5p binding to the target genes mRNA 39UTR by luciferase assay with miR-182-5p precursor. PmirGLO Dual-Luciferase miRNA Target Expression Vector was used to perform 39UTR luciferase assay (Promega, Madison, WI, USA). The primer sequences used for plasmid inserts are shown in Table 1. In a total volume of 20 ml, 5 ml each of 100 mM forward primer and reverse primer, 2 ml of 10x annealing buffer (100 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA) and 8 ml water were added to a 200 ml PCR tube and incubated at 95uC for 5 minutes then placed at room temperature for 1 hr. The oligonucleotides were ligated into the PmeI-XbaI site of pmirGLO Dual-Luciferase miRNA Target Expression Vector. Colony direct PCR was performed for insert recognition using REDTaq (Sigma, St. Louis, MO, USA). The primers used for PCR were as follows: forward primer, 59cgtgctggaacacggtaaaa-39; reverse primer, 59-gcagccaactcagcttcctt-39. PCR parameters for cycling were as follows: 94uC for 3 minutes, 30 cycles of PCR at 94uC for 30 seconds, 55uC for 30 seconds and 72uC for 30 seconds, 72uC for 10 minutes and 4uC for 10 minutes. The PCR product was digested with NotI (TaKaRa/ Fisher Scientific, Pittsburgh, PA, USA). The sizes of vectors containing inserts were about 200 bp and 100 bp by electrophoresis since the NotI recognition sequence was incorporated into the primers. For miR-182-5p precursor transfection, bladder cancer cells were co-transfected with miR-NC and pmirGLO or miR-182-5p and pmirGLO Dual-Luciferase miRNA Target Expression Vectors using Lipofectamine 2000 (Invitrogen). Luciferase activity was assessed using the Dual-LuciferaseH Reporter Assay System (Promega) (48 hours after their transfection).

Overexpression Plasmid of Target Genes (RECK, Smad4) and Functional Analyses

In order to construct target gene (RECK, Smad4) over expressing plasmids, the genes were amplified with total RNA from human adult normal kidney tissues (catalog#: R1234142-50, Biochain Institute, Newark, CA) and RWPE-1 by transcriptionpolymerase chain reaction (RT-PCR). The sequences of primers for cloning are shown in Table 1. Polymerase chain reaction products were cloned into the pTargeT-Mammalian Expression Vector System (Promega, Madison, WI). Then pCMV6-RECK or pCMV6-Smad4 was obtained by subcloning a NheI-XhoI fragment from pTargeT-RECK/Smad4 into the NheI-XhoI site of pCMV6-Entry Vector.

Initially we transfected pCMV6-empty and pCMV6-RECK or -Smad4 into bladder cancer cells and RNA and protein were extracted. Overexpression of RECK or Smad4 was confirmed by real time RT-PCR and Western Blot analysis and functional analyses were performed.

Quantitative Real-time RT-PCR

Quantitative real-time RT-PCR was performed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacture's protocol (Applied Biosystems Inc., Foster City, CA, USA). The TaqMan probes and primers were purchased from Applied Biosystems. Human GAPDH and RNU48 were used as an endogenous control. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems).

Western Analysis

Total cell protein (15-20 mg) was used for Western blotting. Samples were resolved in 4-20% Precise Protein Gels (Thermo Scientific, Rockford, IL) and transferred to PVDF membranes (Amersham Biosciences, Fairfield, CT). The membranes were immersed in 0.3% skim milk in TBS containing 0.1% Tween 20 for 1 hour and probed overnight at 4 4uC with primary polyclonal and monoclonal antibody against Smad4 (#9515), RECK (#3433), beta-catenin (#9562), CREB (#9197) and beta-tubulin (#2128) from Cell Signaling Technology, Beverly, MA. Blots were washed in TBS containing 0.1% Tween20 and labeled with miR-182-5p and Bladder Cancer horseradish peroxidase (HRP)-conjugated secondary anti-mouse or anti-rabbit antibody (Cell Signaling Technology, Beverly, MA). Proteins were enhanced by chemiluminescence (Amersham ECL plus Western Blotting detection system, Fairfield, CT) for visualization. The protein expression levels were expressed relative to beta-tubulin or CREB levels.

Statistical Analysis

All statistical analyses were performed using StatView (version 5; SAS Institute Inc., NC). A p-value of ,0.05 was regarded as statistically significant.

-B), invasion (Fig. 2-C) and migration (Fig. 2-D) in miRNA-182-5p transfected cells compared to miR-NC transfected cells. In addition, miR-182-5p significantly decreased cell apoptosis (Fig. 2-E).

Figure 1 .
1miR-182-5p expression and association with clinical parameters in bladder cancer tissues. A. miR-182-5p expression in clinical samples and bladder cancer cell lines, B. Association of miR-182-5p with clinic-pathological parameters, C. Kaplan Meier plots of overall survival. doi:10.1371/journal.pone.

4, cell viability (Fig. 4-C), invasion (Fig. 4-D) and migration (Fig. 4-E) were significantly inhibited in RECK and Smad4 transfected T24 bladder cancer cells. As shown inFigure 4-F, the percentage of apoptotic cells was significantly increased in RECK or Smad4 transfected cells.

A number of microRNAs have been identified as tumor suppressor or oncogenes based on their expression level in bladder cancer tissues and/or functional analysis. However, many miRNA studies have focused on tumor suppressor miRNAs including miR-125b, -133a, -143, -145, -200 family (200a, 200b, 200c, 141, 429), -203, -205, -218, -449a, -493 and -517a

Figure 2 .
2Effect of miR-182-5p over-expression on bladder cancer cell function (T24, UM-UC-3). Two bladder cancer cell lines (T24 and UM-UC-3) were transiently transfected with either miR-182-5p precursor or control (miR-NC). A. Relative miR-182-5p expression, B. Cell viability assay, C. Invasion assay, D. Wound healing assay (24 hours), E. Flow cytometric analysis of apoptosis in miR-NC or miR-182-5p transfected BC cells. doi:10.1371/journal.pone.

Figure 3 .
3miR-182-5p binds to the 39 UTR of RECK and Smad4 mRNAs and down-regulates expression. A. RECK and Smad4 39UTR position and complementary miR-182-5p sequences. B. 39UTR Luciferase assay (miR-NC and miR-182-5p precursor), C. RECK, Smad4 and beta-tubulin protein expression in miR-NC inhibitor or miR-182-5p inhibitor transfected bladder cancer cells (T24, UM-UC-3). doi:10.1371/journal.pone.

A total of 18 male patients with pathologically confirmed bladder cancer were enrolled in this study (Veterans Affairs Medical Center at San Francisco).Cell CultureThree bladder cancer cell lines (J82; ATCC number: HTB-1, T24; ATCC number: HTB-4, UM-UC-3; ATCC number: CRL-1749) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The J82 and UM-UC-3 cell lines were cultured in MEM Eagle's with Earle's BSS supplemented with 10% fetal bovine serum. The T24 cell line was cultured in McCoy's 5A medium with 10% fetal bovine serum.

Figure 4 .
4Effect of RECK and Smad4 over-expression on bladder cancer cell (T24) function. A. At 24 hours after transfection of either pCMV6-empty, pCMV6-RECK or pCMV6-Smad4 into bladder cancer cells (T24), RECK and Smad4 expression levels were verified by real time RT-PCR (fold change; 24744, 8563, respectively) and Western analysis (B) C. Cell viability assay, D. Invasion assay, E. Wound healing assay, F. Flow cytometric analysis of apoptosis in empty, RECK and Smad4 transfected T24 cells. Data are the mean 6 S.D. of four independent experiments. G. beta-catenin expression in nuclear fraction, CREB was used as control. doi:10.1371/journal.pone.

Table 1 .
1Primer sequences used for plasmid construction. doi:10.1371/journal.pone.0051056.t001 miR-182-5p and Bladder Cancer PLOS ONE | www.plosone.orgname
sequence

RECK NheI cloning forward primer
5' GCTAGCggccaagctgggtccgagcatcccg
3'

RECK XhoI cloning reverse primer
5' CTCGAGcaactacaaaccagcagtcctgaat
3'

Smad4 NheI cloning forward primer
5' GCTAGCttgcttcagaaattggagacatatt
3'

Smad4 XhoI cloning reverse primer
5' CTCGAGattttgtagtccaccatcctgataa
3'

RECK-S
5' AAACTAGCGGCCGCTAGTgcTGCTACTTATATAATTGCCAAaT
3'

RECK-AS
5' CTAGAtTTGGCAATTATATAAGTAGCAgcACTAGCGGCCGCTAGTTT
3'

Smad4-1S
5' AAACTAGCGGCCGCTAGTaactTCAGTTTTATTTTTGCCAAgT
3'

Smad4-1AS
5' CTAGAcTTGGCAAAAATAAAACTGAagttACTAGCGGCCGCTAGTTT
3'

Smad4-2S
5' AAACTAGCGGCCGCTAGTttgctcccTTCGTTATTGCCAAcT
3'

Smad4-2AS
5' CTAGAgTTGGCAATAACGAAgggagcaaACTAGCGGCCGCTAGTTT
3'

November 2012 | Volume 7 | Issue 11 | e51056
AcknowledgmentsWe thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript.Author Contributions
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