hepatitis b virus ( hbv ) infection is a significant public health problem with approximately 350 million infections worldwide . hbv causes acute and chronic hepatitis and is one of the major causes of cirrhosis and hepatocellular carcinoma ( hcc ) [ 2 , 3 ] . hbv is not directly cytopathogenic for infected hepatocytes and the pathogenesis of liver diseases results from complicated interactions between the hbv replication and host immune responses [ 4 , 5 ] . thus , exploring the mechanisms of immune responses in chronic hepatitis b ( chb ) patients has been the focus of intense research . proinflammatory cytokines such as tnf- , mcp-1 , il-1a , and il-6 play a central role in the induction of immune responses in the pathogenesis of various infectious diseases [ 610 ] . cytokines released by immune cells in response to virus infections including hbv , respiratory syncytial virus ( rsv ) , and cytomegalovirus ( cmv ) exert the main functions by recruiting inflammatory cells , constraining virus replication and spread , and inducing adaptive immunity [ 610 ] . however , when the production of chemokines in the context of viral infection is continuous , it may become harmful to the host . for example , numerous proinflammatory mediators produced and released in association with human rsv challenge , including mcp-1 and il-6 , may promote a viral pathology . of interest , it has been recently shown that interleukin- ( il- ) 8 , an important inflammatory cytokine , might be involved in the pathogenesis of hbv infectious diseases , such as chb . il-8 is a cxc chemokine able to elicit granulocytes , nk cells , and t cell chemotaxis at the inflammatory site . clinical studies showed that as the severity of liver inflammation got higher , il-8 levels increased gradually [ 12 , 13 ] . this is probably because il-8 is closely related to natural killer ( nk ) cells that activate a variety of immune cells to release inflammatory mediators , leading to repeated inflammation and damage of liver function . in addition , il-8 can reduce hbv sensitivity to ifn - a , thereby counteracting its antiviral action . previous data had shown that the hbv - encoded regulatory hbx protein is able to transactivate the il-8 promoter , thereby upregulating its expression . however , recent studies showed that the epigenetic mechanisms are also involved in the regulation of il-8 gene expression . epigenetic modifications refer to alterations in gene expression without modification of the dna sequence , by mechanisms involved with dna methylation , histone modifications , and mirna regulation . variation in methylation status of the il-8 gene promoters in human cell models investigating periodontitis appears to predispose some subjects to chronic inflammation . increasing histone acetylation in human intestinal epithelial cells ( iec ) was able to potentiate the cellular response to lipopolysaccharide ( lps ) as measured by il-8 protein production . a recent study showed that aza - tsa ( inhibiting dna methyltransferase and histone diacetyl transferase ) treatment significantly alters lps - induced il-8 production . mirnas are small endogenous noncoding rnas that modulate the expression of genes at the posttranscriptional level by binding to the 3 untranslated region ( 3utr ) of their target mrnas . martinez - nunez et al . demonstrated that a network of micrornas ( mir-18a , mir-27a , mir-128 , and mir-155 ) modulates il-8 expression in the asthmatic bronchial epithelium . furthermore , dalbeth et al . reported that mir-146a reduced msu crystal - induced il-8 gene expression . mirnas are demonstrated to be involved in the pathogenesis of many diseases , such as viral infection and cancer . the hcmv - encoded mirna , hcmv - mir - us25 - 1 - 5p , could inhibit viral replication during viral infection . several mirnas ( e.g. , mir-135b , mir-200 , and mir-101 ) were found to be involved in the development of many cancers , such as colorectal cancer , breast cancer , and glioma [ 2325 ] . chen et al . reported that mir-197 is decreased in peripheral blood mononuclear cells ( pbmcs ) from chb patients and it is implicated in the pathogenesis of chb by targeting il-18 , a key regulator in inflammation and immunity . recently , zhang et al . examined the mirna expression profiles in 327 hcc patients , including 327 tumor and 43 adjacent nontumor tissues , and they built a unique 7-mirna prognosis marker that could significantly predict overall survival ( os ) of hcc patients . mir-582 - 5p was demonstrated to inhibit proliferation of hepatocellular carcinoma by targeting cdk1 and akt3 . during viral infection , virus associated molecular patterns were recognized by toll - like receptors ( tlrs ) , followed by recruitment of their distinct adaptor proteins , sequentially activating signalling cascades to induce cytokine production . mirnas participate in the process of hbv infection mainly by regulating tlrs and cytokine signaling pathway [ 31 , 32 ] . repression of the let-7 family relieves il-6 and il-10 mrnas from negative posttranscriptional control in the tlr4 signaling pathway . mir146a impairs the ifn - induced anti - hbv immune response by downregulating stat1 in hepatocytes . thus , it is important to investigate the role of mirnas in immune response during hbv infection . our group took advantage of mirna expression profile to identify differentially expressed mirnas in pbmcs from chb patients and healthy controls ( data not shown ) , among which mir-106a was found to be significantly downregulated . in the present study , we verified the expression of mir-106a by real - time pcr in chb patients and investigated its possible role . our results provide a new concept to understand the role of mir-106a in immune responses induced by hbv infection . blood samples were collected from 56 patients with chb with abnormal liver function as well as 40 healthy people . patients with chb were selected from the clinic or hospitalization unit at the department of infection of the fifth affiliated hospital of sun yat - sen university during january 2012 and may 2015 . the standards of diagnosis complied with the standards for diagnoses of chb prevention and treatment guidelines recommended by liver disease of the chinese medical association . all patients with a history and clinical features of drug - induced liver injury , alcoholic hepatitis , and steatohepatitis as well as those treated with nucleotide or nucleotide - analog antiviral or immunomodulatory drugs were excluded from this study . this study was approved by the human ethics committee of sun yat - sen university . the activities of alt were calculated spectrophotometrically in serum , using beckman coulter kits by autoanalyzer ( unicel d  c 800 synchron , brea , california , usa ) . pbmcs were isolated from heparinized blood by density gradient centrifugation with a lymphocyte separation medium . total pbmcs rna was isolated using the trizol reagents as instructed ( invitrogen , san diego , ca , usa ) , and rna concentrations were determined with a nanodrop instrument ( nanodrop technologies , wilmington , de ) . the expression of mir-106a was determined by miscript sybr green pcr kit ( qiagen , germany ) . the 20  l pcr reactions consisted of 10  l of 2  quantitect sybr green pcr master mix , 2  l of 10  miscript universal primer , 2  l of 10miscript primer assay , 1  l of rna , and 2  l of rnase - free water . mammalian u6b small nuclear rna in the pbmcs was used to normalize the mirna expression level . the mir-106a and u6b primers were purchased from qiagen . for quantification of il-8 mrna , 1  g of extracted rna for each sample was treated with rq1 rnase - free dnase ( promega , madison , wi ) for 1  h at 37c and used as a template for cdna synthesis using reverse - transcribe kit ( promega co. , madison , wi , usa ) in accordance with the manufacturer 's protocol . the relative il-8 expression level was determined by using the following specific primers 5- actccaaacctttccacc -3 and 5- aacttctccacaaccctc -3   and normalized to glyceraldehyde phosphate dehydrogenase ( gapdh ) . the pcr amplification was performed with a volume of 20  l containing 10  l sybr qpcr mix ( takara , dalian , china ) . the relative expression of mir-106a and il-8 ( defined as fold change ) was calculated by the 2 ( ct = ct  ct ; ct = ct  ct ) . the isolated pbmcs were cultured in rpmi 1640 medium supplemented with 10% fetal calf serum ( fcs ) and penicillin / streptomycin in u - bottom 24-well plates at 37c with 5% carbon dioxide . the human embryonic kidney ( hek ) the hsa - mir-106a mimics , hsa - mir-106a inhibitor , and unrelated sequence positive control ( control mimics ) and negative control ( control inhibitors ) were purchased from genecopoeia ( jiangsu , china ) . pbmcs were transfected using lipofectamine 2000 reagents according to the manufacturer 's instructions ( invitrogen ) . then , the cells were subjected to analysis of mir-106a and il-8 mrna expression as above . and the protein expression levels of il-8 were measured in supernatant by elisa ( r&d systems , minneapolis , mn , usa ) . to generate luciferase reporter constructs , the 3-utr regions of il-8 containing the predicted target sites of mir-106a were cloned into pmir - report mirna expression reporter vector ( yinrun , china ) by using synthesized fragments ( 5- ctagttgttgtgaggacatgtggaagcactttaagttttttcatcataa -3 and 5- agctttatgatgaaaaaacttaaagtgcttccacatgtcctcacaacaa -3 ) . the corresponding mutant plasmid was also constructed with the fragments ( 5- ctagttgttgtgaggacatgtggaacccgcaaaagttttttcatcata a -3 and 5- agctttatgatgaaaaaacttttgcgggttccacatgtcctcacaacaa -3 ) . for luciferase reporter assays , pmir - il-8 - 3-utr ( wt or mut ) plasmid was cotransfected with mir-106a mimics into the hek293 cells as previously described [ 36 , 37 ] . , the cells were collected and determined by using a dual - luciferase reporter assay system ( promega ) . all statistical analyses were conducted with the statistical product and service solutions ( spss ) version 16.0 ( spss , chicago , il , usa ) . the differences of mir-106a and il-8 expressions were assessed by using unpaired student't test . for determination of correlation between different variables to identify the mirnas potentially associated with hbv infection , the expression levels of mir-106a from 56 chb patients and 40 healthy controls were determined by qrt - pcr . we observed that the mir-106a expression level was decreased in pbmcs from chb patients compared with that in healthy controls ( figure 1(a ) ) . serum alanine aminotransferase ( alt ) and tbil have been widely recognized as an important marker of liver disease . here , we analyzed the relationship between mir-106a expression , serum alt , and tbil level in chb patients . the results showed that mir-106a expression was inversely proportional to serum alt level ( r = 0.693 , p = 0.000 ) and tbil level ( r = 0.545 , p = 0.000 ) ( figures 1(b ) and 1(c ) ) . to evaluate whether il-8 protein and mrna were increased in chb , the levels of il-8 protein and mrna in chb patients and healthy individuals were analyzed . as expected , il-8 protein and mrna were significantly higher in chb patients than that of health controls ( figure 2(a ) ) . and the analysis results revealed that there was positive correlation between serum il-8 and alt levels ( r = 0.569 , p = 0.000 ) ( figure 2(b ) ) . il-8 mrna in pbmcs was also positively correlated with serum alt level ( r = 0.645 , p = 0.000 ) ( figure 2(b ) ) . in addition , serum tbil levels were found to be significantly correlated with il-8 mrna level ( r = 0.498 , p = 0.000 ) and serum il-8 protein level ( r = 0.476 , p = 0.000 ) ( figure 2(c ) ) . using the targetscan 6.0 , we found that il-8 was one of the possible target genes for mir-106a . to demonstrate it , hek293 cells were cotransfected with the il-8 3-utr construct ( wt ) or its mutant ( mut ) and mir-106a mimics or mir-106a inhibitor , followed by a measurement with the luciferase reporter assay ( figure 3(a ) ) . as expected , mir-106a mimics decreased il-8 translation , whereas mir-106a inhibitor increased the activity of il-8 translation ( figure 3(a ) ) . however , the luciferase activity of the il-8 mutant was not influenced by mir-106a mimics or mir-106a inhibitor ( figure 3(a ) ) . next , we determined the effect of mir-106a on il-8 mrna and protein expression levels by qrt - pcr and elisa . we firstly determined the efficiency of the transfection of mir-106a mimics and inhibitor . as is shown in figure 2(b ) , transfection of mir-106a mimics into pbmcs can significantly increase its expression , while mir-106a inhibitor downregulated its expression level . il-8 mrna and protein levels were significantly downregulated in healthy pbmcs transfected with mir-106a mimics compared with the cells transfected with control mimics , whereas the expressions of il-8 were significantly upregulated in cells transfected with mir-106a inhibitors compared with cells transfected with control inhibitors ( figures 3(c ) and 3(d ) ) . next , we assessed the relationship between mir-106a and il-8 mrna levels in pbmcs of chb patients . results showed that mir-106a was negatively correlated with il-8 mrna levels in pbmcs from chb patients ( r = 0.700 , p = 0.000 ) ( figure 4 ) . mirnas play an important role in hbv infection and hbv - related disease , such as hcc , and changes in mirna expression patterns are important indicators of these diseases . results from recent research have shown that the mirna expressions of pbmc , such as hsa - mirplus - e1063 , hsa - mirplus - e1236 , ebv - mir - bhrf1 - 2 , hsa - mirplus - a1098 , hsa - mir-1267 , and hsa - mirplus - e1163 , were associated with the severity of hbv - induced liver disease and therapeutic outcome of ifn- therapy in chb patients . have demonstrated significantly different expression profiles of mirna molecules during ia phases of chb , including hsa - mir-548ah-5p , hsa - mir-4804 - 3p , hsa - mir-483 - 3p , hsa - mir-3607 - 3p , and hsa - mir-44475 . mir-548ah- 5p can influence the molecular function of ifn- through targeting ifn-r1 and inhibiting its expression . our group also used mirna expression profile to identify differentially expressed mirnas in pbmcs from chb patients and healthy controls ( data not shown ) . the microarray results showed that mir-106a was significantly downregulated in chb patients . in the current study , we further verified the expression of mir-106a in pbmcs of chb patients by real - time pcr assays and demonstrated that mir-106a is downregulated in chb patients compared with that of healthy controls . furthermore , clinical analysis revealed that it is negatively correlated with serum alt and tbil in chb patients . thus , our results firstly revealed that the abnormal expression of mir-106a in pbmcs may have an important role in the damage of liver function during chronic hbv infection . the pathogenesis is largely mediated by immune responses following hbv infection [ 4 , 5 ] . il-8 , a member of the cysteine - x - cysteine ( cxc ) chemokine subfamily , is an important mediator of inflammatory responses to many viruses and bacteria , as it recruits granulocytes , nk cells , and t cells at the inflammatory sites [ 40 , 41 ] . recent evidence has suggested that this chemokine may be involved in immune pathogenesis of hbv infection and in the resistance of hbv to ifn-. moreover , it is strictly correlated with malignant features of tumors , a frequent event in chronic hbv . by using computational methods , we predicted that mir-106a is highly likely to interact with the 3utr of il-8 . to confirm whether il-8 is a direct target of mir-106a , we subcloned il-8 3utr containing the target sequences into a luciferase reporter vector , and the corresponding pmir-3-utr - mut was also constructed . our results showed that exogenous expression of mir-106a could significantly repress il-8 expression at both the mrna and protein levels , whereas mir-106a inhibitor had the opposite effects . further statistical analysis showed that mir-106a is negatively related to il-8 expression in pbmcs from chb patients . these data revealed that mir-106a may be an effective strategy for regulating the expression of il-8 . in summary , our study firstly provides the evidence that mir-106a is downregulated in pbmcs of chb patients , and mir-106a expression levels were negatively associated with the severity of hbv - induced liver disease . in addition , our data demonstrated that mir-106a downregulates il-8 expression by targeting its 3utr . we believe that our study may help to develop a better understanding of clinical characterization and pathogenesis of chb .
aims . this study aimed to investigate mir-106a expression in peripheral blood mononuclear cells ( pbmcs ) of chronic hepatitis b ( chb ) patients and to analyze the function of mir-106a . 
 materials and methods . 
 mir-106a expression levels in pbmcs from 40 healthy controls and 56 chb patients were analyzed by quantitative real - time polymerase chain reaction ( qrt - pcr ) . 
 the luciferase activity assays were used to determine whether mir-106a binds to 3utr of il-8 . 
 mir-106a mimics and inhibitors were transfected into healthy pbmcs . 
 il-8 mrna and protein levels were detected and determined by qrt - pcr and elisa , respectively . 
 results . 
 the qrt - pcr results suggested that the pbmc mir-106a levels were decreased in chb patients . 
 il-8 was augmented in chb patients and was inversely correlated with mir-106a levels . 
 the luciferase activity assays indicated that il-8 is a target of mir-106a . 
 exogenous expression of mir-106a could significantly repress il-8 expression at both mrna and protein levels in pbmcs , whereas mir-106a inhibitor had the opposite effects . 
 conclusions . 
 this study suggested that mir-106a is downregulated in pbmcs of chb patients and that mir-106a may play an important role in chb by targeting il-8 .