The Potential Function And Mechanism Of MicroRNA-26a In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide.High tumor recurrence and metastasis has been one major reason that contributes to the poor prognosis of HCC patients. Tumor angiogenesis was confirmed to be associated with the invasion and metastasis of HCC.Therefore it is urgently needed to explore the molecular mchanisms of underlying HCC metastasis and tumor angiogenesis, and provide potential effective therapeutic targets to improve HCC patients survival.MicroRNAs (miRNAs) are a class of highly conserved short RNAs that inhibit translation or induce mRNA degradation in general by binding to the3’-untranslated region (3’-UTR) of target mRNAs. MicroRNAs regulate diverse cellular processes including growth, proliferation, differentiation, and apoptosis. Numerous reports have shown that miRNA dysfunction is involved in the development and progression of various human cancers. Many studies have demonstrated that some miRNAs are crucial for tumorigenicity, proliferation, apoptosis, angiogenesis, invasion, and metastasis of hepatocellular carcinoma. Therefore, miRNAs could serve as therapeutic targets in HCC.Emerging data shows that miRNA-26a (miR-26a) serve as a potential tumor suppressor or exhibit oncogenic properties in several distinct cancer types.The real reason of the dual effects of miR-26a is not clear yet, it might be in part due to organ-specific actions and the different cellular contexts of tumors.We previously reported that patients whose tumors had low miR-26a expression had a shorter overall survival (OS) but a better response to adjuvant interferon therapy versus patients whose tumors had high expression of miR-26a. However, the potential mechanisms by which miR-26a affects the malignant phenotype of HCC cells remain largely unknown.In this study, we investigated the prognosic value of miR-26a and potential function of miR-26a in the development and progression of HCC. We further explored the underlying mechanisms by which miR-26a inhibits HCC metastasis and tumor angiogenesis.PART ONEmiR-26a Expression in HCC and its Effects on HCC PrognosisPurpose:The aim of this study was to elucidate the relationship between miR-26a and recurrence and metastasis of HCC, and the prediction of miR-26a for HCC prognosis by detecting the expression levels of miR-26a in tumor tissues, the plasma of HCC patients and HCC cell lines.Methods:The expression levels of miR-26a in130HCC with40paried adjacent non-tumorous liver tissue,62patients plasma and HCC cell lines with different metastatic potentials was assessed by real-time quantitative PCR using Taqman probes. Next,we analyzed miR-26a expression in HCC tissues and corresponding adjacent non-tumor tissues, recurrent and non-recurrent HCC tissues, metastatic and non-metastatic HCC tissues, and HCC cell lines with different metastatic potentials. The curves of overall survival(OS) and time to recurrence (TTR) of miR-26a in HCC tissues and plasma were drew by Kaplan-Meier method, and comparisons between groups were performed by log-rank test. Univariate and multivariate survival analysis determined whether the expression of miR-26a was used as an independent predictor for HCC prognosis by Cox proportional hazards model.Results:(1) miR-26a levels in HCC were negatively related with tumor metastasis and recurrence. miR-26a expression levels in HCC tissues were significantly lower than their corresponding non-tumor tissues (P<0.01); miR-26a expression levels were significantly lower in metastatic HCC tissues than non-metastatic HCC tissues (P <0.01), and in recurrent HCC tissues than non-recurrent HCC tissues (P<0.01).(2) Plasma miR-26a levels were negatively correlated with liver cancer metastasis and recurrence. Plasma miR-26a levels was significantly lower in metastatic HCC tissues compared with non-metastatic HCC tissues (P<0.01), and in recurrent HCC tissues in comprision to non-recurrent HCC tissues (P<0.01). miR-26a level in HCC cell lines was negative related with their metastatic potentials. miR-26a level in HCC cell lines compared with normal hepatic cell were significantly down-regulated (P<0.01). miR-26a levels in HCC cell lines with high metastatic potentials shower lower miR-26a level than cell lines with low metastatic potentials (P<0.01). High miR-26a expression in HCC tissues or plasma showed a longer survival time and longer time to recurrence than low miR-26a expression in group (P<0.01). Univariate and multivariate analysis showed that miR-26a is an independent predictor for the prognosis of HCC patients. Conclusions:miR-26a is downregulated in HCC,and negatively correlated with tumor metastasis and recurrence. miR-26a is an independent predictor for the prognosis of HCC patients.PART TWOmiR-26a Suppresses Tumor Growth and Metastasis of Human Hepatocellular Carcinnoma by Targeting to Inhibit IL-6-stat3PathwayPurpose:This study was to further explore the molecular mechanism by which miR-26a regulated tumor invasion and metastasis using in vitro and in vivo function tests.Methods:Using in vitro proliferation, scratches and invasion tests,cell cycle and apoptosis determinations and nude mice experiments validated the functions of miR-26a in HCC. To further explore the molecular mechanism by which miR-26a inhibited HCC,we investgated target genes of miR-26a by dual fluorescent reporter system analysis and we further verified downstream IL-6-stat3pathway inhibiting tumor growth and metastasis of HCC by Western-blot analysis. Univariate and multivariate survival analysis determined whether miR-26a or IL-6or combination of miR-26a and IL-6was used as an independent predictor for HCC prognosis by Cox proportional hazards model.Results:(1) miR-26a overexpression suppressed in vitro cell proliferation and invasion, induced cell cycle arrest, promoted apoptosis, and restrained in vivo tumor growth and metastasis. Conversely, down-regulation of miR-26a inhibited apoptosis and promoted proliferation, invasion, and cell cycle transition to S phase.(2)We found that miR-26a inhibits tumor growth and metastasis in part by suppressing IL-6. The mRNA levels of miR-26a inversely correlated with IL-6levels in HCC tissues. Up-regulation of miR-26a significantly reduced IL-6levels in HCC cells, whereas down-regulation of miR-26a increased IL-6levels. Overexpression of miR-26a decreased the luciferase reporter activity of wild-type3’UTR but not mutant3’UTR of IL-6. More importantly, the effects of miR-26a modulation on cell proliferation, apoptosis, and invasion of HCC cells were accompanied by changes in IL-6levels and activities. Introduction of IL-6abrogated the effects induced by miR-26a and downregulation of IL-6produced the similar effects to miR-26a.(3) miR-26a play important roles by inhibiting IL-6-stat3signaling pathway including some target genes, including Bcl-2, Mcl-1, cyclin D1and MMP2. miR-26a overexpression down-regulated in vitro and in vivo the expression of target genes, including Bcl-2, Mcl-1, cyclin D1and MMP2, but the effects was abrogated by introduction of IL-6. Downregulation of IL-6produced the similar effects to miR-26a.(4) The patients with high miR-26a levels, low IL-6levels or high miR-26a low IL-6had better overall survival and lower possibilities of tumor recurrence than the patients with low miR-26a levels, high IL-6levels or low miR-26a high IL-6, respectively. miR-26a, IL-6, combination of miR-26a and IL-6were conformed to be independent prognostic indicators. Combination of miR-26a and IL-6showed the better prognostic value than miR-26a or IL-6alone.Conclusion:miR-26a inhibited in vitro and in vivo HCC growth, invasion and metastasis through inhibition of IL-6-stat3signaling pathway.PART THREEmiR-26a suppresses Angiogenesis of Human Hepatocellular Carcinnoma by Targeting to Inhibit HGF-cMet PathwayPurpose:To validate and elaborate the molecular mechanism of miR-26a in the regulation of HCC angiogenesis by analyzing the effects of miR-26a on VEGF expression of HCC cell lines and proliferation,migration and tube formation of endothelial cell in vitro and in vivo.Methods:We detected miR-26a, VEGF and MVD (microvessel density) levels in120 HCC tissues using PCR and immunohistochemical staining to confirm the correlation between miR-26a levels and VEGF levels or miR-26a levels and MVD levels. We verified miR-26a function in the regulation of HCC angiogenesis by analyzing the effects of miR-26a on VEGF expression of HCC cell lines and proliferation,migration and tube formation of Human Umbilical Vein Endothelial Cells(HUVECs) in vitro and in vivo. To further explore the molecular mechanism by which miR-26a inhibited HCC angiogenesis, we investgated target genes of miR-26a by dual fluorescent reporter system analysis and downstream HGF-cMet signal pathways inhibiting tumor angiogenesis were further verified by Western-blot analysis. Univariate and multivariate survival analysis determined whether miR-26a, HGF, VEGF, MVD, or combination of miR-26a and HGF was used as an independent predictor for HCC prognosis by Cox proportional hazards model.Results:(1) miR-26a levels significantly negatively correlated with VEGF or MVD levels in HCC tissues.VEGF and MVD levels was significantly higher in vascular invasive HCC tissue than non-vascular invasive HCC tissue(P<0.01), and higher in metastatic HCC tissues than non-metastatic HCC tissue(.P<0.01). Spearman correlation analysis showed that miR-26a expression significantly negatively correlated with VEGF or MVD levels(P<0.001).(2) miR-26a significantly suppressed supernatant VEGF expression of HCCLM3and MHCC97H cells(P<0.01) and inhibited proliferation, migration and tube formation of HUVECs; reduced miR-26a promoted supernatant VEGF expression of HCC cells (P<0.01) and increased proliferation, migration and tube formation of HUVECs. In vivo experiments suggested that tumor MVD number and VEGF protein levels significantly reduced in miR-26a overexpression group compared with the control(P <0.01).(3) HGF was conformed to be a downstream target of miR-26a mediating VEGF expression of HCC and proliferation, migration and tube formation of HUVECs. The mRNA levels of miR-26a inversely correlated with HGF levels in HCC tissues. Up-regulation of miR-26a significantly reduced HGF levels in HCC cells, whereas down-regulation of miR-26a increased HGF levels. Overexpression of miR-26a decreased the luciferase reporter activity of wild-type3’UTR but not mutant3’UTR of HGF. More importantly, the effects of miR-26a modulation on VEGF expression of HCC and proliferation, migration and tube formation of HUVECs were accompanied by changes in HGF levels and activities. Introduction of HGF abrogated the effects induced by miR-26a and downregulation of HGF produced the similar effects to miR-26a.(4) miR-26a downregulated VEGF expression by inhibiting the HGF-cMet,and both downstream the PI3K/Akt/mTOR/S6K signaling pathway and HIF-1α-VEGF signaling pathway, in turn, inhibited VEGFR2signaling pathway in endothelial cell. Introduction of HGF abrogated the effects induced by miR-26a and downregulation of HGF produced the similar effects to miR-26a.(5) The patients with high miR-26a levels, low HGF levels, high miR-26a low HGF, low VEGF levels or low MVD levels had better overall survival and lower possibilities of tumor recurrence than the patients with low miR-26a levels, high HGF levels, low miR-26a high HGF, high VEGF levels or high MVD levels, respectively. miR-26a, HGF, VEGF, MVD, combination of miR-26a and HGF were conformed to be independent prognostic indicators. MVD showed best prognostic value, combination of miR-26a and HGF followed, but better than miR-26a or HGF alone.Conclusion:miR-26a expression levels was significantly negatively correlated with VEGF or MVD levels. miR-26a could significantly inhibit the expression of VEGF of HCC cells and proliferation, migration and tube formation of HUVECs by directly inhibiting HGF-cMet signaling pathway, and therefore can be used as a clinical potential therapeutic target.PART FOURTherapeutic upregulation of miR-26a by combination therapy of interferon-alb and5-FU induced apoptosis of HCC cells partly through the mitochondrial pathwayPurpose:To explore the michanism of miR-26a upregulation and combination therapy of interferon-alb and5-FU induced the apoptosis of HCC cells by investgating the effects of miR-26a on in vitro and in vivo HCC cell apoptosis.Methods:We examined miR-26a levels, tumor cell apoptosis and the apoptotic pathway-related protein levels in58HCC tissues to confirm the correlation between miR-26a and tumor cell apoptosis or miR-26a and the apoptotic pathway-related proteins. Then in vitro experiments validated the effect of miR-26a upregulation on HCC cell apoptosis and the apoptosis-related proteins expressions.Whether the cell apoptosis was induced partly through the mitochondrial apoptotic pathway was verified by determining the loss of mitochondrial membrane potential and cytochrome c release. In vitro and in vivo experiments of combinated intervention of IFN-alb and5-FU were performed to analyze miR-26a levels,tumor cell apoptosis, apoptosis related proteins expressions,the loss of mitochondrial membrane potential and cytochrome C release in order to further verify whether conbination of IFN-alb and5-FU induced mitochondrial apoptosis of HCC cells by upregulating miR-26a.Results:(1) miR-26a levels significantly positively correlated with tumor cells apoptosis in HCC tissues. Apoptotic cells in high miR-26a group were significantly more than low miR-26a group(P<0.01). We found that miR-26a levels was significantly associated with the apoptosis of tumor cells (r=0.5479, P<0.001). Anti-apoptotic proteins Bcl-2and Bcl-xL level was significantly lower in high miR-26a group than low miR-26a group (P<0.01); while the pro-apoptotic proteins Bax, cleaved the level caspase3and cleaved caspase9significantly increased in high miR-26a group compared with low miR-26a group (P<0.01). miR-26a levels were significantly inversely correlated with protein expression levels of Bcl-2and Bcl-xL (r=-0.6259, P<0.001and r=-0.7452, P<0.001), while positively correlated to protein levels of Bax, cleaved caspase3and cleaved caspase9in HCC tissues(r=0.5778, P<0.001; r=0.5398, P<0.001and r=0.4663, P<0.001).(2) In vitro experiments confirmed that miR-26a induced apoptosis of HCC cells part through the mitochondrial pathway. miR-26a induced apoptosis of HCC cells, inhibited the expression of Bcl-2and Bcl-xL, increased expression levels of of Bax, caspase3and caspase9and the loss of mitochondrial membrane potential, and to promote the release of cytochrome c from the mitochondria to the cytoplasm.(3) miR-26a was up-regulated by conbination of IFN-alb and5-FU. In vitro and in vivo experiments of combinated intervention of IFN-alb and5-FU confirmed that miR-26a mediated combination therapy of IFN-alb and5-FU inducing apoptosis partly through the mitochondrial apoptotic pathway and miR-26a also increased the sensitivity of conbination of IFN-alb and5-FU which induced apoptosis of HCC cells.Conclusion:Therapeutic up-regulation of miR-26a induces cells apoptosis partly through the mitochondrial pathway in human hepatocellular carcinoma by combinated intervention of IFN-alb and5-FU and miR-26a sensitizes HCC cells to apoptosis induced by combination of IFN-α1b and5-FU. The Conclusion of the Full Text1.miR-26a is downregulated in HCC,and negatively correlated with tumor metastasis and recurrence. miR-26a is an independent predictor for the prognosis of HCC patients.2. miR-26a inhibited in vitro and in vivo HCC growth, invasion and metastasis through inhibition of IL-6-stat3signaling pathway.3. miR-26a expression levels was significantly negatively correlated with VEGF or MVD levels in HCC. miR-26a could significantly inhibit the expression of VEGF and proliferation, migration and tube formation of HUVECs by directly inhibiting HGF-cMet signaling pathway, and therefore can be used as a clinical potential therapeutic target.4. Therapeutic up-regulation of miR-26a induces cells apoptosis partly through the mitochondrial pathway in human hepatocellular carcinoma by combinated intervention of IFN-alb and5-FU and miR-26a sensitizes HCC cells to apoptosis induced by combination of IFN-alb and5-FU.The Innovative Point and Significance of the Study1.We firstly demonstrated that miR-26a was downregulated in HCC,and negatively correlated with tumor metastasis and recurrence.2.1t was firstly reported that miR-26a inhibited in vitro and in vivo HCC growth, invasion and metastasis through inhibition of IL-6-stat3signaling pathway.3. For the first time, we discovered that miR-26a inhibited angiogenesis of human hepatocellular carcinnoma by targeting to inhibit HGF-cMet pathway.4. We firstly confirmed that therapeutic up-regulation of miR-26a induces cells apoptosis partly through the mitochondrial pathway in human hepatocellular carcinoma by combinated intervention of IFN-alb and5-FU and miR-26a sensitizes HCC cells to apoptosis induced by combination of IFN-alb and5-FU. Potential Value of this Study1.We can predict the prognosis of HCC patients by detecting miR-26a expression levels in HCC tissues.2.Up-regulating miR-26a is conformed to inhibit IL-6-stat3signaling pathway and HGF-cMet signaling pathways which involves multi-targets to regulate growth, invasion, metastasis and angiogenesis in HCC. Therefore, miR-26a has a very important clinical application prospects for potential molecular targeted drugs to inhibit HCC metastasis and tumor angiogenesis and to improve HCC patients survival.3. Combined intervention of IFN-alb and5-FU induced apoptosis of HCC cells by up-regulating miR-26a which provide theoretical basis for combination therapy of IFN-alb and5-FU to inhibit HCC.