| [Background and Objective]Hepatocellular carcinoma (HCC) is one the most common malignant tumors with an increasing incidence in the worldwide and the second leading cause of cancer-related mortality in China with an increasing mortality. Despite the improved treatment, the improvement in survival rates for HCC is still very unsatisfactory over the decades. Among so many factors affecting the survival of HCC, the metastasis and recurrence is almost always the harbinger of eventual cancer mortality for HCC patients. Therefore, elucidating the molecular mechanism for invasion and metastasis, clarifying the initiating factor of invasion and metastasis, screening and identification molecules that could be used for early diagnosis of invasion and metastasis or used for new targets to be intervened to prevent or inhibit the invasion and metastasis in HCC, will has important theoretical and clinical significance in HCC treatment. Recently, more and more reports have demonstrated that miRNAs which play important roles in many biological processes by post-transcriptionaly regulating gene expression via mRNAs degradation or translation blockade, are aberrantly expressed in almost all human cancer types including HCC, functions as oncogenes and tumor suppressors. Basic research and clinical observation showed miRNAs are involved in all aspects of cancer including tumorigenesis, progression and treatment outcome. miRNAs may be involved in cancer invasion and metastasis via influencing cancer cell motility, angiogenesis and cancer microenvironment. Several miRNAs have been reported involved in HCC. However, the role and mechanisms of miRNAs in HCC invasion and metastasis is still far from full understanding. Here, we investigated the expression pattern of miR-217in HCC cell lines and tissue specimens. We also explored the role and the mechanisms of miR-217in HCC invasion using bioinformatics analysis combined with experimental confirmation. After the completion of this project, the signal network responsible for invasion and metastasis in HCC will be improved and miR-217may be provided as new biomarkers for metastasis prognosis or reversion.[Methods]1. The low invasion potential HCC cell lines (Huh7, HepG2, SMMC7721and MHCC-97L) and high invasion potential cell lines (MHCC-97H) were routinely cultured and the invasion potential was measured by Trans well assay. miRNAs and mRNAs were isolated and purified with miRNA isolation system. cDNA was generated with the miScript Ⅱ RT Kit and the quantitative real-time PCR was done to determine the expression of miR-217and U6was used as endogenous control;2. The expression of miR-217in MHCC-97H cells was restored by miR-217mimics transfection using lipofectamin2000and the change of invasion potential was measured by trans well assay;3. The function inhibition in Huh7and MHCC-97L cells was performed by miR-217inhibitor transfection and the change of invasion potential was measured by Trans well assay;4. A total of48clinical HCC tissue samples and normal liver tissue samples were collected and divided into "non-metastasis" group and "metastasis" group of which the specimens were obtained from HCC patients with one or more phenotypes such as intrahepatic metastasis, vascular invasion, portal vein tumor thrombus or pulmonary metastasis. miRNAs were isolated and purified with miRNA isolation system. cDNA was generated with the miScript Ⅱ RT Kit and the quantitative real-time PCR was done to determine the expression of miR-217and U6was used as endogenous control5. We used the public database-TargetScan (http://www.targetscan.org) to predict potential target of miR-217;6. mRNAs in HCC cells with or without miR-21interference was isolated and cDNA was generated. The expression of E2F3was determined in mRNA levels with qTR-PCR and in protein level with western blot;7. The luciferase reporter vector with the putative E2F33’UTR target site for miR-217downstream of the luciferase gene (pMir-E2F3-Wt, as wide type) and mutant version with a deletion of7bp in the seed region was constructed (pMir-E2F3-Mut, as mutant type). Cells were seeded in96well-plates and co-transfected with pMir-Report luciferase vector, pRL-TK Renilla luciferase vector and miR-217mimics.48h later the luciferase activities were determined using a Dual-Luciferase Reporter Assay System (Promega) where the Renilla luciferase activity was used as internal control;8. The specific siRNAs targeting E2F3were designed and transfected into MHCC-97H cells, and the efficiency was determined by western blot. The change of invasion potential after E2F3knockdwon was measured by Trans well assay;9. The Immunohistochemistry assay was used to measure the protein expression of E2F3in HCC tissue samples and normal liver tissue samples.[Results]1. MCHH-97H cells were more invasive than Huh7, HepG2, SMMC7721and MHCC-97L cells. The expression of miR-217in MHCC-97H cells was much lower than that in Huh7, HepG2, SMMC7721and MHCC-97L cells;2. Restored miR-217expression with mimics could efficiently inhibit invasion of MHCC-97H cells (miR-217mimics group,8±1.28cells per field; control group,52±4.54cells per field, p<0.05);3. miR-217function inhibition with specific inhibitor significantly increased invasion of both Huh7(control group,2±0.20cells per field, miR-217inhibitor group,32±2.52cells per field; p<0.05)(Fig.1D) and MHCC-97L cells (control group,3±0.25cells per field, miR-217inhibitor group,37±2.85cells per field; p<0.05);4. miR-217was significantly downregulated in the "metastasis" group tissues (n-=21) normalized to normal liver tissues with approximately90.48%of the cases displaying>50%reduction compared to the "non-metastasis" group with approximately only 14.81%of the cases displaying>50%reduction (p<0.05),5. E2F3with critically conserved binding site was selected for further molecular and functional confirmation, There was no significant different expression of E2F3at mRNA level among selected HCC cell lines, but the protein expression of E2F3in MHCC-97H cells with endogenous low expression of miR-217was much higher compared to other selected HCC cell lines. E2F3protein in MHCC-97H cells significantly decreased after ectopic miR-217expression with miR-217mimics. However, miR-217inhibitor upregulated E2F3protein level in Huh7and MHCC-97L cells;6. The luciferase activity of pMir-E2F3-Wt in MHCC-97H cells was about48.46%and42.24%more than that in Huh7and MHCC-97L cells respectively, without difference on luciferase activity of pMir-E2F3-Mut. Moreover, the lucifarase reporter assay performed in HEK293T cells showed miR-217reduced the luciferase activity of the vector with the wild-type E2F33’UTR by about54.82%, but the mutant version abrogated the repressive ability of miR-217;7. Compared to the control, si-2#markedly decreased E2F3protein expression. E2F3knockdown significantly inhibit MHCC-97H cells invasion (control group,58±5cells per field; si-E2F3group,18±3cells per field, p<0.05). In the selected48clinical HCC tissues, high E2F3protein expression was detected in "metastasis" group, compared to "non-metastasis" group and normal liver tissue.[Conclusions]1. miR-217expression is downregulated in highly invasive HCC cell lines and metastatic HCC tissue specimens;2. Restored miR-217expression inhibits HCC cells invasion and miR-217inhibition enhances HCC cells invasion;3. miR-217directly targets E2F3expression in HCC cells;4. E2F3knockdown inhibits HCC cells invasion;5. miR-217expression is negatively related with HCC invasion and metastasis and is with the potential to be a marker for early diagnosis and a target for intervention for HCC invasion and metastasis.There are totally14figures,5tables, and101references. |
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