MiR-21Promotes Migration And Invasion By The MiR-21-PDCD4-AP-1Feedback Loop In Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors of digestive tract diagnosed in Asian countries, and is the second leading cause of cancer mortality in China, only next to gastric carcinoma. HCC has high malignancy and poor prognosis and the5-year survival rate after operation has been reported to range from only36-50%. Like many other cancers, distant metastasis is the main contributor to poor prognosis. Migration and invasion of HCC is a multi-phase developmental process that involves changes in multi-gene signatures. Analysis of the HCC genome and proteome has demonstrated that focusing on molecular heterogeneity within HCC maybe available approach to identify and develop novel therapeutics.MicroRNAs (miRNAs) are a class of small non-coding RNAs that are18-25nucleotides in length and negatively regu late gene expression by binding to the3’-untranslated region (3’-UTR) of specific target messenger mRNAs (mRNAs), which in turn causes mRNA degradation or translational repression. Aberrant miRNA expression has been linked to human diseases, including cancer. As an oncogene, microRNA-21(miR-21) was found to be overexpressed in a variety of malig nancies, and be implicated in multiple malignancy-related processes, including cell proliferation, apoptosis, invasion and metastasis. Tumor suppressor PDCD4is one of target gene with significant biological function of miR-21. Although the miR-21-PDCD4axis has been addressed in other cancer types, the involvement of miR-21in the migration/invasion of HCC and its underlying mechanism is not yet known.This article intended to study "The relationship between miR-21and hepatocellular carcinoma". Inspired by the phenomenon that "miR-21was significantly up-regulated in malig nancies compared with non-neoplastic tissues", we assumed that whether miR-21as a oncogene or potential cancer therapeutic target in hepatocellular carcinoma? Accordingly, the design of this experiment was as follows:1.To investigate the expression levels of miR-21and PDCD4in paired HCC tumor tissues and adjacent non-tumor liver tissues, and normal human hepatic cells (LO2) and four HCC cancer cell lines with different invasive potential, and exploring the relationships between the expression level of miR-21and PDCD4and tumor invasion and metastasis.2. To explore the effects of miR-21and PDCD4on migration/invasion activities in HCC cells, and expression of downstream signaling pathway molecules, and to investigate the molecular mechanism of modulating tumor migration and invasion involved in miR-21-PDCD4axis.Part I The tissues and cells expression and significance of miR-21and PDCD4in HCCObjectiveTo explore the expression pattern of miR-21and PDCD4in HCC tumor tissues and HCC cell lines with different invasive potential.MethodsThe expression of miR-21and PDCD4protein in paired HCC tumor tissues and adjacent non-tumor liver tissues, normal human hepatic cells (LO2) and four HCC cell lines (HepG2, Bel-7402, MHCC97-H, Huh-7) with different invasive potential were examined respectively by real-time PCR and Western blot, and to analyze the relationship among miR-21, PDCD4and clinicopathologic characteristics of the HCC.Results1.miR-21was significantly up-regulated in HCC tissues, as compared with that detected in matched non-tumor tissues, miR-21expression was significantly higher in the invasive/metastatic group and the patients with stage III-IV disease. miR-21was highly up-regulated in HCC cell lines, as compared with human normal hepatic L02cells, and the miR-21levels were gradually up-regulated in the HCC cell lines with increasing invasive capacity (MHCC97H>Be17402>HepG2>Huh7).2. In comparison with the non-tumor counterparts, lower levels of PDCD4were detected in tumor tissues. Furthermore, PDCD4protein was down-regulated in HCC samples with invasion/metastasis and in patients with stage III-IV disease. The levels of PDCD4protein in the4HCC cell lines were much lower than that in human normal hepatic cells, and PDCD4expression was negatively correlated with the cell invasive capacity.3. we observed a significant inverse correlation between PDCD4protein expression levels and miR-21levels in both HCC tissues and cells.Conclusion1. miR-21expression has been shown to be greatly up-regulated in both human HCC tissues and cells compared to matched non-tumor tissues and normal hepatic cells, and to be significantly higher in the invasive/metastatic group and the patients with stage III-IV disease. miR-21expression was positively correlated with the cell invasive capacity.2. Lower levels of PDCD4protein were detected in tumor tissues, HCC cells, invasive/metastatic group and the patients with stage III-IV disease. Its down-regulation is associated with invasive potential of HCC cells.3. PDCD4protein expression was negatively correlated with miR-21in both HCC tissues and cells.4. The expression of miR-21and PDCD4protein was modest in HepG2cells, and this cell line was selected for further experiment. Part II Effects of miR-21-PDCD4-AP-1feedback loop on migration and invasion of HCC cell line HepG2ObjectiveResearch on the effects of expression miR-21and PDCD4on the cell migration and invasion ability in HepG2cells and on the expression of relative downstream signaling pathway molecules, and to investigate the molecular mechanism of modulating tumor migration and invasion involved in miR-21-PDCD4-AP-1feedback loop.Methods1.To explore the positive regulation pathway firstly. The experiments were consists of two groups:single transfection group (MOCK, NC, miR-21inhibitor, PDCD4siRNA) and combined transfection group (inhibitor NC+siRNA NC, miR-21inhibitor+siRNA NC, miR-21inhibitor+PDCD4siRNA)(1) to purchase chemically synthesized miR-21inhibitor, PDCD4siRNA and respective negative control.(2) The effect of miR-21and PDCD4on migration of HepG2cells was investigated using a Transwell migration assay to evaluate cell migration. The significance of PDCD4in the process was analyzed.(3) Matrigel invasion assay in Transwell culture chambers to determine the effect of miR-21and PDCD4on in vitro HepG2invasion. The role of PDCD4was also assessed.(4) The expression of PDCD4and downstream molecules (c-Jun、 p-c-Jun、MMP-2、MMP-9) in each group were examined respectively by real-time PCR and Western blot.2. To explore the reverse regulation pathway secondly. The experiments were divided into three groups:MOCK, NC, c-Jun siRNA. The expression of miR-21were tested by real-time PCR.Results1. single transfection group:(1) Transwell migration assay:the average migration cells of each group was as follows:MOCK group,(75.3±2.5)/HP; NC group,(71.3±2.1)/HP; miR-21inhibitor group,(39.7±2.5)/HP; PDCD4 siRNA group,(101±4.0)/HP. The number of migrated cells in miR-21inhibitor group was much lower compared with MOCK and NC group (P<0.05), while that in PDCD4siRNA group was much more that control.(2) Transwell invasion assay:the average invasion cells of each group was as follows:MOCK group,(58.3±1.5)/HP; NC group,(56.7±2.1)/HP; miR-21inhibitor group,(33.7±2.5)/HP; PDCD4siRNA group,(73±3.6)/HP. The number of HepG2cells that passed through Matrigel in miR-21inhibitor group was much lower compared with MOCK and NC group (P<0.05), while that in PDCD4siRNA group was much more that control.(3) As confirmed by Western blot and real time PCR, the expression of PDCD4protein and mRNA was significantly more in miR-21inhibitor group compared with MOCK and NC(P<0.05).(4) In miR-21inhibitor group the expression of p-c-Jun protein exhibited a notable decrease compared with MOCK and NC(P<0.05), while there was no significant difference of c-Jun protein among three groups.(5) Compared with MOCK and NC, the expression of MMP-2mRNA and MMP-9mRNA showed obviously decreased in miR-21inhibitor group (P<0.05).(6) Compared with MOCK and NC, the expression of p-c-Jun protein, MMP-2mRNA and MMP-9mRNA showed markedly increased in PDCD4siRNA group (P<0.05), but c-Jun protein expression were unaffected in these three groups.2.combined transfection group:(1) Transwell migration assay:the average migrated cells in (miR-21inhibitor+PDCD4siRNA) group were obviously more than (miR-21inhibitor+siRNA NC) group [(83.7±3.1)/HP vs (57.8±3.4)/HP](P<0.05).There were also significant difference in the two group in Transwell invasion assay[(69.3±2.5)/HP vs (32.0±2.7)/HP](P<0.05).(2) Compared with (miR-21inhibitor+siRNA NC) group, the expression of p-c-Jun protein, MMP-2mRNA and MMP-9mRNA showed markedly increased in (miR-21inhibitor+PDCD4siRNA) group (P<0.05), but c-Jun protein expression were unchanged in the two groups.3. The miR-21expression in c-Jun siRNA group was significantly lower than MOCK and NC(P<0.05). There was no significant difference between MOCK and NC group(P>0.05).Conclusion1. In HepG2cells, down-regulated miR-21expression potently inhibits tumor cell migration and invasion, while knockdown of PDCD4in HepG2cells signifi cantly promoted their migration and invasion.2. miR-21inhibits target PDCD4expression at transcriptional and translational level, promoting downstream signaling pathway molecules c-Jun phosphorylation and AP-1activity, increasing transcriptional expression of MMPs. PDCD4plays a critical role in mediating miR-21dependent biological functions in HCC.3. In HepG2cell line, AP-1could directly activate miR-21transcription.4. miR-21acts as both target and regulator of AP-1. A positive feedback loop of miR-21-PDCD4-AP-1maintains the miR-21-mediated biological effects of the HCC malignant phenotype. Our data suggest that molecular introduction strategies interfering with the miR-21-PDCD4-AP-1feedback loop might provide strong rationale for preventing invasion/metastasis in HCC in the future.