| Background and ObjectiveHepatocelluar carcinoma (HCC) is one of the most common malignancies with high mortality rates worldwide. Metastasis and recurrence has become one major obstacle for further improving the long-term survival of HCC patients. Therefore, it is critical to elucidate the mechanisms involved in HCC tumorigenesis, progress and metastasis. Aurora-A, an important member of Aurora kinase family, is a novel serine / threonine kinase promoting mitotic spindle assembly by regulating centrosome duplication and separation. The overexpression of Aurora-A has been found in a variety of human malignancies, including HCC. In our previous studies, we have shown that high Aurora-A mRNA level was significantly correlated with tumor stage, more frequent lymph node or hematogenous metastasis, and poor prognosis of HCC patients. Also,downregulation of Aurora-A could lead to growth inhibition and apoptosis induction in HCC cells both in vitro and in vivo, suggesting the potential value of Aurora-A as a therapeutic target.MicroRNAs (MiRNA) are a class of small noncoding RNAs that function as key regulators of gene expression at the post-transcriptional level. Increasing evidence has revealed that miRNAs play important roles in various human biological and pathological processes, including cell differentiation, development, apoptosis, morphogenesis and metastasis. This study aimed to explore the roles of microRNAs in regulation of Aurora-A expression. In the present study, we screened and identified a set of miRNAs that directly targeted and regulated Aurora-A expression by bioinformatics analysis and luciferase activity assay. Next, we comprehensively explore the expression, clinicalpathological significance and biological roles of miR-129-3p in HCC and the possible molecular mechanisms, with the aim of better elucidating the mechanisms of Aurora-A dysregulation and laying a foundation for formulating novel therapeutic target for treatment of HCC.Materials, Methods and ResultsPart I Screening and Identification of MicroRNAs that Negatively Regulate the Aurora-A Expression in HCCMaterials and Methods1. Four different online miRNA databases (TargetScanHuman 6.0, PicTar 5.0, MicroRNA.org and microRNA Targets) were employed for prediction of miRNAs that directly target the 3’-UTR of Aurora-A.2. Synthesize the mimics of the 60 miRNA predicted in step 1 and transfect the HEK293T cells that contains the 3’-UTR of Aurora-A gene. Luciferase reporter assay was performed to further confirm the results.3. Synthesize the mimics of the 8 miRNA indentified in step 2. Western blot was performed to determine their effect on Aurora-A expression.Results1. A total of 60 miRNAs were predicted by online miRNA databases.2. 8 miRNAs could significantly decrease the fluorescence intensity, including let-7a, miR-124,miR-129-3p, miR-140-5p, miR-153-3p, miR-363-3p, miR-885-3p and miR-92a-3p.3. 6 miRNAs could significantly regulate the Aurora-A expression, including miR-129-3p,miR-140-5p, miR-153-3p, miR-363-3p, miR-885-3p and miR-92a-3p.Part II The Analysis of Expression and Clinical Significance of miR-129-3p in HCCMaterials and Methods1. MiR-129-3p was selected for further study. Real-time RT-PCR analysis was performed to detect the expression of miR-129-3p in a panel of human HCC cell lines with different metastatic potentials (HepG2, BEL-7402, HCCLM3 and MHCC97-H).2. qRT-PCR was performed to dectect the miR-129-3p expression while qRT-PCR and western blot were performed to dectect Aurora-A expression in 20 of 88 primary HCC and corresponding nontumor liver tissues obtained from HCC patients. Next,immunohistochemistry stain of Aurora-A was applied to all the 88 HCC tissues and corresponding nontumor liver tissues. The correlation between Aurora-A positive rate and miR-129-3p was analyzed.3. The correlations between miR-129-3p expression levels and the clinicopathological factors as well as prognosis were determined in the whole 88 HCC tissues.Results1. MiR-129-3p expression decreased progressively from the normal human hepatocyte cell line(HH), to low metastatic HCC cell lines (HepG2 and BEL-7402), and finally to highly metastatic HCC cell lines (HCCLM3 and MHCC97-H).2. It was observed that the relative levels of Aurora-A mRNA and protein were significantly upregulted, while the miR-129-3p expression was significantly downregulated in 20 cases of HCC tissues in comparison with corresponding nontumor liver tissues. By linear regression analysis, it was found that there was a significant inverse association between the expression of miR-129-3p and that of Aurora-A mRNA (r = -0.221; p=0.0001). Compared with corresponding nontumor liver tissues, the immunoreactivity of Aurora-A was markedly increased in the 88 HCC tissues. The stronger immunoreactivity of Aurora-A in HCC tissues was significantly correlated with lower miR-129-3p expression (p<0.01).3. In 88 cases of HCC tissues, miR-129-3p expression level was associated with clinicopathological characteristics including lymph node metastasis, vascular invasion, TNM stage and recurrence. The Kaplan-Meier survival analysis indicated that the reduced expression of miR-129-3p was significantly correlated with shorter DFS (p<0.001) and OS(p<0.001) of HCC patients.Part Ⅲ The Impacts of MiR-129-3p on Biological Behaviors of HCC Cells Materials and Methods1. A miR-129-3p expression plasmid vector (pLMP/miR-129-3p) was constructed and introduced into highly metastatic HCC cell lines (HCCLM3 and MHCC97-H) through stable transfection, whereas miR-129-3p inhibitor (anti-miR-129-3p) was introduced into low metastatic HCC cell lines (HepG2 and BEL-7402) through transient transfection. Wound healing assays and transwell assays with or without Matrigel were performed to detect the in vitro cell migration and invasion abilities.2. The stable HCCLM3/miR-129-3p or MHCC97-H/miR-129-3p cells were transplanted into the left hepatic lobe of nude mice to establish the orthotopic HCC animal models. After 10 weeks, mice were sacrificed, and their livers and lungs were dissected and prepared for histological analysis. The intrahepatic and lung metastasis status was determined compared with the control groups (HCCLM3/miR-NC or MHCC97-H/miR-NC group). Establish the orthotopic HCC animal models in another group of nude mice and feed them until all die.Compare the survival time of tumor-bearing mice with the control groups.3. Immunofluorescence and Western blotting assays were performed to analyze the effect of miR-129-3p on the expression of epithelial and mesenchymal molecular makers in HCC cells.Results1. Significant suppression of the in vitro migration and invasion was observed in both HCCLM3/miR-129-3p and MHCC97-H/miR-129-3p cells (p<0.05).Conversely,downregulation of miR-129-3p could lead to the decreased migration and invasion of HepG2 and BEL-7402 cells.2. In the orthotopic HCC animal model whose tumors derive from the HCCLM3/miR-129-3p or MHCC97H/miR-129-3p cells, the incidence of both intrahepatic and lung metastasis and the number of both intrahepatic and lung metastatic nodules were notably reduced compared with the control groups (HCCLM3/miR-NC or MHCC97-H/miR-NC group) , while the survival time of tumor-bearing mice was significantly increased. Therefore, miR-129-3p could reduce the in vivo metastatic capacities of HCC cells.3. Upregulation of miR-129-3p could lead to increased expression of epithelial markers(E-cadherin and β-catenin) and decreased expression of mesenchymal markers (N-cadherin and Vimentin) in HCCLM3 and MHCC97-H cells. In contrast, in miR-129-3p-downregulated HepG2 and BEL-7402 cells, the expression of epithelial markers was markedly decreased and the expression of mesenchymal markers was significantly increased.Part IV The Analysis of Molecular Mechanisms of miR-129-3p in Regulating the Motility and Metastasis of HCCMaterials and Methods1. HCCLM3 and MHCC97-H cells were stably transfected with pSil/shAurora-A vector, and then, wound healing assays and transwell assays with or without Matrigel were performed to detect the in vitro cell migration and invasion abilities. Immunofluorescence and Western blotting assays were performed to analyze the effect of Aurora-A knockdown on the expression of epithelial and mesenchymal molecular makers in HCC cells.2. The stable HCCLM3/shAurotra-A or MHCC97-H/shAurora-A cells were transplanted into the left hepatic lobe of nude mice to establish the orthotopic HCC animal models. After 10 weeks, mice were sacrificed, and their livers and lungs were dissected and prepared for histological analysis. The intrahepatic and lung metastasis status was determined compared with the control groups (HCCLM3/shcontrol or MHCC97-H/shcontrol). Establish the orthotopic HCC animal models in another group of nude mice and feed them until all die.Compare the survival time of tumor-bearing mice with the control groups.3. A plasmid vector carrying WT Aurora-A (pMD-Auro) or the control plasmid vector pMD-NC was introduced into HCCLM3/miR-129-3p cells through stable transfection. Western blot assay was performed to detect the expression of Aurora-A. Wound healing assays and transwell assays with or without Matrigel were performed to detect the in vitro cell migration and invasion abilities. Immunofluorescence and Western blotting assays were performed to analyze the effect of Aurora-A restoration on the miR-129-3p-induced expression change of epithelial and mesenchymal molecular makers in HCC cells.4. The stable HCCLM3/miR-129-3p/pMD-Auro cells were transplanted into the left hepatic lobe of nude mice to establish the orthotopic HCC animal models. After 10 weeks, mice were sacrificed, and their livers and lungs were dissected and prepared for histological analysis.The intrahepatic and lung metastasis status was determined compared with the control group(HCCLM3/miR-129-3p/pMD-NC). Establish the orthotopic HCC animal models in another group of nude mice and feed them until all die. Compare the survival time of tumor-bearing mice with the control groups.5. Western blot assay was performed to detect the expression levels of Aurora-A,phosphorylated Akt (p-Akt), p38 MAPK (p-p38 MAPK), the total Akt and p38 MAPK,pro-MMP-2 and active MMP-2 in HCCLM3/miR-129-3p and HCCLM3/miR-NC cells.Moreover, a selective inhibitor of p38 MAPK (SB202190) or Akt (LY294002) was added into the anti-miR-129-3p-transfected HepG2, and then, the same molecular markers were detected by Western blot assays in these HCC cell lines.6. A selective inhibitor of p38 MAPK (SB202190) or / and Akt (LY294002) was added alone or in combination into HCCLM3 and MHCC97-H cells, or the anti-miR-129-3p-transfected HepG2 and BEL-7402 cells, and then, wound healing assays and transwell assays with or without Matrigel were performed to detect the in vitro cell migration and invasion abilities.7. A selective inhibitor of p38 MAPK (SB202190) and Akt (LY294002) were added in combination into the anti-miR-129-3p-transfected HepG2, and then, western blot was performed to analyze the expression of epithelial and mesenchymal molecular makers in HCC cells.Results1. Aurora-A knockdown in both HCCLM3 and MHCC97-H cells could significantly inhibit the in vitro cell migration and invasion (P<0.05), and at the same time, increase the expression of epithelial markers (E-cadherin and β-catenin) and decrease the expression of mesenchymal markers (N-cadherin and Vimentin).2. In the orthotopic HCC animal model whose tumors derive from the HCCLM3/shAurotra-A or MHCC97-H/shAurora-A cells, the incidence of both intrahepatic and lung metastasis and the number of both intrahepatic and lung metastatic nodules were notably reduced compared with the control groups (HCCLM3/shcontrol or MHCC97-H/shcontrol, while the survival time of tumor-bearing mice was significantly increased. Thus, suppression of Aurora-A could mimic the effects of miR-129-3p overexpression on invasion and metastasis of HCC cells.3. Exotic Aurora-A expression could partially rescue the decreased expression of Aurora-A mRNA and protein in HCCLM3/miR-129-3p cells. The migratory and invasive capacity of HCCLM3/miR-129-3p cells was partially rescued after transfection of pMD-Aurora-A.Reexpression of Aurora-A could also induce EMT in HCCLM3/miR-129-3p cells, which was correlated with downregulation of epithelial markers (E-cadherin and β-catenin), and upregulation of mesenchymal markers (N-cadherin and Vimentin).4. In the orthotopic HCC animal model whose tumors derive from the HCCLM3/miR-129-3p/pMD-Auro cells, the incidence of both intrahepatic and lung metastasis and the number of both intrahepatic and lung metastatic nodules were notably reduced compared with the control group (HCCLM3/miR-129-3p/pMD-NC), while the survival time of tumor-bearing mice was significantly increased. It was suggested that reexpression of Aurora-A could partially rescue miR-129-3p-mediated EMT, cell invasion and metastasis in HCC cells.5. In HCCLM3/miR-129-3p cells, the phosphorylation of Akt and p38 MAPK were significantly downregulated compared with HCCLM3/miR-NC cells, with downregulation of MMP-2. However, ectopic overexpression of Aurora-A could partially rescue the downregulated levels of pAkt, p-p38 MAPK and MMP-2 proteins in HCCLM3/miR-129-3p cells. Moreover, transfection with anti-miR-129-3p combined with SB202190 or LY294002 treatment could partially rescue the effects of anti-miR-129-3p on the activation of p38 MAPK and Akt signaling pathways in HepG2 and BEL-7402 cells. It was indicated that miR-129-3p could regulate the activation of p38 MAPK and Akt signaling and the expression of MMP-2 though targeting Aurora-A.6. SB202190 or / and LY294002 alone or combined treatment significantly reduced the capacities of mobility, migration and invasion of HCCLM3 and MHCC97-H cells. In contrary, transfection with anti-miR-129-3p combined with SB202190 or / and LY294002 treatment could partially rescue the effects of anti-miR-129-3p on migration, invasion in HepG2 and BEL-7402 cells.7. Transfection with anti-miR-129-3p combined with SB202190 and LY294002 treatment could partially rescue the effects of anti-miR-129-3p on the EMT process in HepG2 and BEL-7402 cells. It was suggested that miR-129-3p could inhibit HCC migration and invasion, at least partially by inactivation of PI3K/Akt and p38 MAPK signaling pathways, which eventually induced the reversal of EMT phenotype and downregulation of MMP-2 in HCC cells.Conclusions1. Our data indicated that 6 miRNA including miR-129-3p, miR-140-5p, miR-153-3p,miR-363-3p,miR-885-3p and miR,92a-3p could directly target the 3’-UTR of Aurora-A mRNA and regulate its expression.2. MiR-129-3p was significantly downregulated in HCC tissues, with upregulation of Aurora-A expression. The upregulation of Aurora-A was correlated with lymph node metastasis in HCC patients, while the downregulation of miR-129-3p was correlated with early recurrence,tumor metastasis and poor survival in HCC patients.3. MiR-129-3p could inhibit invasion and metastasis as well as the EMT process of HCC cells in vitro and in vivo.4. miR-129-3p could inhibit invasion and metastasis and reverse EMT of HCC cejls, at least in part by inactivating PI3K/Akt and p38 MAPK signaling pathways through targeting Aurora-A.5. To our knowledge, this study is the first to identify the miRNAs that directly target Aurora-A and regulate its expression. We have showed the dysregulation of miR-129-3p/Aurora-A axis in hepatocellular carcinoma and its association with the clinicalpathological factors and prognosis, the impacts of miR-129-3p on the invasion, metastasis and EMT process of HCC cells, and moreover, the molecular mechanisms of miR-129-3p in regulating the above biological behaviors. This study uncovers the mechanisms of Aurora-A dysregulation in hepatocellular carcinoma at the post-transcriptional level, indicating the posiblity of targeting miR-129-3p/Aurora-A axis to prevent and reverse the hepatocellular carcinoma metastasis in clinic. |
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