| Background Hepatocellular carcinoma (HCC) has a high prevalence in Southeast Asia and Africa, and the incidence of HCC has kept increasing in Europe and America. In China, HCC has become one of the main disease burdens as a result of the higher prevalence of hepatitis B virus (HBV) and hepatitis C virus infection. To date, early diagnosis and treatment of HCC are difficult as a consequence of a lack of reliable screening methods. Only 2/3 HCC patients can be diagnosed at the time of advanced carcinoma. Above all, HCC is resistant to most chemotherapeutic agents, its recurrence and metastasis are common in patients who have undergone surgery, with a postoperative 5-year survival rate of 30-40%. Therefore, better understanding of the pathogenesis of HCC is essential for the development of effective therapies and early diagnostic markers.The miR-26 family is composed of miR-26a-1, miR-26a-2 and miR-26b located on chromosomes 3,12 and 2, respectively. miR-26a is consistently down-regulated in a wide range of malignant tumors, such as hepatocellular carcinoma, nasopharyngeal carcinoma, lung cancer, and breast cancer. Therapeutic miR-26a delivery using adeno-associated virus (AAV) vector was able to inhibit cancer cell formation while inducing tumor-specific apoptosis and providing apparent protection from disease progression without side effects. As well, patients with reduced miR-26a expression in tumors had a significant improvement in overall survival after receiving adjuvant therapy with interferon (IFN)-α. Enhancer of zeste homologue 2 (EZH2) is the potential target gene for miR-26a, which methylates histone H3 lysine 27 (H3K27) and mediates transcriptional silencing. A previous study showed that silence of EZH2 gene in HCC cells is capable of reversing tumorigenicity in a nude mouse model, and demonstrated the potential therapeutic value of EZH2 inhibition in HCC. Recent studies have also reported the improved expression of EZH2 in HCC tissues, which was correlated with the aggressiveness or poor prognosis of HCCs.However, the mechanism underlining the sensitivity of HCCs to IFN-a is unclear; there is a need for applying molecular methods to elucidate the chain reactions induced by IFN-a therapy.Objective To discuss the expression of miR-26a and EZH2 in the occurrence, development, metastasis of hepatocarcinoma, and to demonstrate the mechanism. To identify whether EZH2 was the potential target gene for miR-26a. To research the mechanism of IFN-a treatment to HCC.The effect of miR-26a to hepatocarcinoma was discussed via mediating miR-26a, including the downstream target gene in order to supply data and thinking for theraptic miR-26a in the mechanism of hepatocarcinoma.Methods The expressions of miR-26a and EZH2 in hepatocyte, hepatocellular carcinoma cells, hepatocellular carcinoma and paracancerous tissues were detected. And pGL3-EZH2-3’UTR were established and transfection of miR-26a or inhibitor were also detected by luciferase to identify whether EZH2 was the direct target gene of miR-26a. Finally, using qRT-PCR, Western blot and Northern blot technology, detection of interferon alpha on the expression of miR-26a and EZH2 in HCC cells, and observe the effect of whether there is a dose-dependent.Then, we transfected the HepG2 cells with miR-26a mimics or a control and observed that miR-26a blocked HepG2 proliferation and invasion by decreasing EZH2 expression.Results1) miR-26a in six cell lines were detected, and compared with normal hepatocyte, miR-26a were decreased in hepatocellular carcinoma cells. miR-26a in paracancerous tissues were significantly higher than that in hepatocellular carcinoma tissues. The expression of EZH2 were significantly increased in SMMC-7721 and HepG-2 cells, and EZH2 in paracancerous tissues were significantly lower than that in hepatocellular carcinoma tissues.The expression of miR-26a and EZH2 was negative correlation in HCC cells.2) The relationship of miR-26 and EZH2 were analyzed by TargetScan software. And there were 7 basic group in complete complementary pairing between miR-26a and EZH2, also 4 basic group mutational site in EZH2-3’UTR. Transfection of miR-26a i could significantly decreased Luciferase activity of wild type EZH2-3’UTR, no effect on mutant sequence of EZH2-3’UTR.However, Transfection of miR-26a inhibitor could significantly increased Luciferase activity of wild type EZH2-3’UTR, no effect on mutant sequence of EZH2-3’UTR.In hepatocytes BNL CL.2 and hepatocellular carcinoma cells Hepal-6, over expression of miR-26a would significantly suppress EZH2 mRNA, while inhibition of miR-26a would increase EZH2 mRNA. miR-26a could act on EZH2 and also inhibit the expression of EZH2.3) HepG2 cells were sampled at the time point of 0,12,24 and 48 hours after IFN-a treatment. Then the copies of miR-26a and the house-keeping gene U6 were subsequently measured by RT-qPCR and northern blot. The relative miR-26a level to U6 didn’t increase significantly at 12 hours post-treatment; the level rose to a higher level at the point of 24 hours and maintained this level until 48 hours after IFN-a intervention.The EZH2 mRNA transcription level after IFN-a intervention was also evaluated, EZH2 mRNA level decreased significantly at 24 hours and 48 hours post-IFN-a treatment. In contrast with 102 IU of IFN-α,103 IU of IFN-a suppressed the EZH2 mRNA level to a lower level. Next, we checked the EZH2 protein expression in the treated HCC cells, IFN-a led to a dose-dependent decrease in EZH2 protein levels, specially, at 103 IU dosages of IFN-a, EZH2 expression was decreased by approximately 34% compared with the negative control.These results showed that IFN- alpha inhibied the expression of EZH2 through up-regulation expression of miR-26α in HCC cells.To confirm the association between miR-26a and EZH2, the HepG2 cells were transfected with miR-26a mimic or miR control at the same amount, separately. Next, we checked the EZH2 mRNA level to β-actin in HepG2 cells every 24 hours after transfection; a significant difference was observed between the miR-26a mimic group and control group, and EZH2 mRNA level was stable during 72 hours post- transfection. Finally, we evaluated the EZH2 expression level by western blot at different time points post-transfection; dramatic difference was clearly disclosed between the control group and miR-26a group during the 3 time intervals. These results demonstrated that up-regulated miR-26a can inhibit the EZH2 expression in the transcription level.To explore the effect of miR-26a on cell proliferation, HepG2 cells were transiently transfected with miR-26a mimics and miR control, respectively.The results of MTT assay displayed that miR-26a mimics inhibited cell proliferation in Hep G2 cells by 33% (P< 0.05) by 48 hours and 50% (P<0.05) at 72 hours post-transfection, separately. To investigate the effects of miR-26a on HCC cell invasion and migration, we conducted cell migration and invasion assays on HepG2 cells. It was shown that upregulated expression of miR-26a significantly suppressed the migratory and invasive abilities of HepG2 cells 48 hours after transfection. The number of migrated cells per field in miR-26a mimics group was decreased by 36% compared with the control group. Also, the number of invasive cells in the miR-26a group declined by 37.5% and 62.5% compared with the control group, respectively, when the HepG2 cells were transfected with mimics at concentration of 25nM and 50nM separately. These results suggested the inhibitory role of miR-26a in HepG2 cells metastasis with timeliness and dose dependence.Conclusion miR-26a were decreased in hepatocellular carcinoma cells,while the expression of EZH2 were significantly increased in cells. EZH2 was the direct target gene of miR-26a. The expression of miR-26a and EZH2 was negative correlation in HCC cells.IFN- alpha suppressed the expression of EZH2 through up-regulation expression of miR-26a in HCC cells.Reintroduction of miR-26a mimics into the tumor may provide an alternative therapy by reducing expression of target gene EZH2. IFN-a-induced miR-26a inhibited migration and proliferation of HepG2 in vitro and the inhibitory effects were partially mediated by EZH2. Although miRNA-related therapies still remain immature, our findings on IFN-a-induced inhibition on HCC suggest the miR-26a delivery may be a promising therapy for the treatment of HCC. |
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