Identiifcation Of A Novel Tumor Suppressor MicroRNA MiR-520e And Its Mechanism In Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is one of the most commonmalignant cancers worldwide. The chronic infection of hepatitis B virus(HBV) has been identified as a major risk factor in the development ofHCC. Hepatitis B virus X protein (HBx) plays a crucial role in thehepatocarcinogenesis. MicroRNAs (miRNAs) are small, non-codingRNA of approximately19-25nucleotides that negatively modulate geneexpression through mRNA cleavage and translational repression, servingas oncogenes or tumor suppressor genes. In the present study, we firstreport that microRNA-520e (miR-520e) is a novel tumor suppressormiRNA. The roles of miR-520e were systematically investigated,including three parts as follows:Part one: Effect of miR-520e on proliferation of hepatoma cellsTo investigate the roles of miR-520e in hepatocarcinogenesis, we analyzed the expression levels of miR-520e in human immortalized livercell lines (L-O2and Chang liver), hepatoma cell lines (HepG2, H7402and MHCC-97L),23paired clinical HCCs and adjacent nontumorousliver tissues. Our data showed that the expression levels of miR-520ewere significantly reduced in hepatoma cell lines and clinical HCCtissues relative to normal liver cells. To investigate the mechanism ofdownregulation of miR-520e, we examined the upstream region ofmiR-520e coding sequence in human genome using bioinformatics. Wenoted that there was a typical CpG island within the upstream region ofmiR-520e. Then, we tested the DNA methylation at the region bymethylation-specific PCR and bisulfitesequencing analysis. The resultsshowed that the CpG sites were highly methylated in hepatoma cell linesand clinical HCC tissues. Luciferase reporter gene assays revealed thatthe region containing CpG island hypermethylation was the core regionof miR-520e promoter. Thus, we conclude that the hypermethylation status of CpG islands is responsible for the downregulation of miR-520eexpression in hepatoma cells. Next, we sought to determine whethermiR-520e affects the growth of hepatoma cells. We observed that theintroduction of miR-520e dramatically suppressed the proliferation ofhepatoma cells (HepG2, H7402and MHCC-97L) by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,5-ethynyl-2-deoxyuridine (EdU) incorporation assay and colonyformation analysis. Moreover, the ectopic expression of miR-520einhibited the growth of hepatoma HepG2cells in nude mice. Inconclusion, miR-520e inhibits growth of hepatoma cells in vitro and invivo. To investigate the potential therapy application of miR-520e, weobserved that the direct injection with miR-520e into the tumor tissues intransplanted mice was able to efficiently inhibit the growth of tumor,suggesting that miR-520e has potential therapeutic value. Thus, weconclude that miR-520e is a novel tumor suppressor miRNA. Part two: The mechanism of miR-520e in suppression of hepatomacell growthTo explore the mechanisms underlying of miR-520e in suppression oftumor growth, we predicted the target genes of miR-520e in human byusing TargetScan and DIANA database. Bioinformatics showed thatNF-κB-inducing kinase (NIK) was one of target genes of miR-520e.Then, we confirmed that miR-520e directly bound to the3′untranslatedregion (3′UTR) of NIK, and silenced the expression of NIK at the levelsof mRNA and protein by luciferase reporter gene assay, RT-PCR,real-time PCR and Western blotting analysis. Our data revealed that theexpression of NIK was upregulated in hepatoma cells (HepG2, H7402and MHCC-97L) relative to hepatocytes (L-O2and Chang liver), as wellas in23cases of HCC tissues compared with adjacent nontumorous livertissues. The miR-520e level was inversely correlated with NIK expression in the hepatoma tissues. It suggests that miR-520e may inhibitthe growth of hepatoma cells through its target gene NIK. We found thatthe effect of anti-miR-520e on enhancement of cell proliferation waspartially attenuated by siRNA targeting human NIK mRNA (si NIK)when si NIK and anti-miR-520e were co-transfected into the Chang livercells, supporting that miR-520e inhibits the growth of hepatoma cellsthrough its target gene NIK. In addition, NIK significantly increased thephosphorylation level of ERK1/2(p-ERK1/2) and increased thetranscriptional activity and nuclear translocation of nuclear factor κB(NF-κB) in hepatoma cells. We conclude that miR-520e suppresses thegrowth of hepatoma cells by targeting NIK, involving theNIK-p-ERK1/2-NF-κB signaling pathway.Part three: Effect of miR-520e on HBx-enhanced growth ofhepatoma cellsTo further validate the role of miR-520e in tumor suppression, we investigated the significance of miR-520e in hepatocarcinogenesismediated by HBx. Our data showed that the expression of miR-520e wasdramatically reduced in HBx-transfected hepatoma HepG2-X cells andHBx-transfected immortalized liver L-O2-X cells. We found that theoverexpression of miR-520e inhibited the growth of HepG2-X andL-O2-X cells. Bioinformatics showed that the mitogen-activated proteinkinase kinase kinase2(MEKK2) was one of target genes of miR-520e.Then, we confirmed that miR-520e directly bound to the3′untranslatedregion (3′UTR) of MEKK2by luciferase reporter gene assay. RT-PCRand Western blotting analysis showed that miR-520e reduced theexpression of MEKK2at the levels of mRNA and protein. To testwhether MEKK2is involved in the supperession of miR-520e in cellproliferation, the ani-miR-520e and MEKK2siRNA were cotransfectedinto HepG2cells. We found that the knockdown of MEKK2blocked theenhanced proliferation mediated by anti-miR-520e. We conclude that HBx promotes the growth of hepatoma cells through downregualtingmiR-520e and miR-520e acts through target gene MEKK2.In summary, we demonstrate that miR-520e is a novel tumorsuppressor miRNA which acts through target gene NIK. In addition,miR-520e is involoved in the HBx-induced hepatocarcinogenesis throughtarget gene MEKK2. Our finding provides new insight into themechanism of hepatocarcinogenesis, indicating a therapeutic potential ofmiR-520e in the treatment of HCC.