MiR-26a Inhibits Cell Proliferation, And Promotes Migration And Invasion In Hepatocellular Carcinoma

Background:miRNAs are small, noncoding RNAs(22-23nucleotides long), shown to regulate gene expression at translational and posttrans-lational levels. It regulates gene expression through interaction with3’untranslated regions (3’-UTRs) of mRNAs. Increasing evidence suggests that miRNAs are predicted to target over50%of all human protein-coding genes, enabling them to have numerous regulatory roles in many physiological and developmental processes, including organism development, cell proliferation, differentiation and apoptosis. miRNA expression seems to be altered in many human diseases, including cancer. They are often located in genomic unstable regions and therefore are typically downregulated in tumors; moreover, inhibiting some miRNAs biogenesis tends to enhance tumorigenesis. These type of miRNAs usually function as tumor suppressor genes. Of course, miRNAs can also act as oncogenes. For example, some miRNAs are overexpressed in cancer and seem to function as oncogenes themselves (miR-17-92, miR-155), a great number of miRNAs have been shown to be downregulated in cancer and have the potential to act as tumor suppressor genes (i.e., Let-7, miR-15/miR-16, miR-1/miR-206, miR-29, miR-124, miR-143/miR-145). Increasing researches suggest that silencing an oncogenic miRNA could allow reexpression of some tumor suppressor genes, while reexpressing a tumor suppressor miRNA could downregulate multiple oncogenes. miRNAs regulate a vast number of genes and pathways, so their final function depends on the cumulative effects of genes regulated by them. miRNAs are very complicated and some function of miRNAs in tumor is unknown.miR-26a located at3p21.3, composed of21nucleotides and it expresses in a large number of tissues. Numerous studies showed that miR-26a suppressed cell proliferation in nasopharyngeal carcinoma, breast cancer and liver cancer cells, but promoted cell proliferation in glioma. Interestingly, in leukemia, miR-26a inhibited acute myeloid leukemia cell growth, whereas stimulated T-cell acute lymphoblastic leukemia cell proliferation. These controversial results suggest that miR-26a might play different roles depending on tumor and tissue types. It is no doubt that the functions of miR-26a are extremely complicated and it is worth studying.Hepatocellular carcinoma (HCC) has the property of high degree of malignancy and metastasis in the early stage. In our experiment, HCC tissue specimens were obtained from hospital. qRT-PCR was used to detect the expression of miR-26a in human HCC tissues and adjacent non-tumor tissues. The results showed that the levels of miR-26a expression were significantly lower in human HCC tissues. Intriguingly, we found that the expression levels of miR-26a in HCC tissues with metastasis were significantly higher than HCC tissues without metastasis. Whether the results suggest that miR-26a enhances metastasis in HCC. The function of miR-26a in HCC metastasis was unclear. Therefore it promoted us to carry out this experiment.Methods:1. Detection of the expression profile of miR-26a in HCC cell lines and tissuesqRT-PCR was used to detect miR-26a expression in HCC-derived cell lines BEL-7402,7721, HepG2, Huh7, Hep3B, Sk-Hep-1, Plc and in Normal liver tissue miR-26a expression in43human HCC tissues and44adjacent non-tumor tissues was detected by qRT-PCR.2. Generation of HCC cell lines overexpressing miR-26a transgeneLentiviruses carrying miR-26a and enhancand green fluorescent protein (EGFP) genes were produced, followed by infecting HCC-derived cell line BEL-7402, HepG2and Sk-Hep-1using lentivirus supernatant. Finally, HCC cell lines overexpressing miR-26a transgene were confirmed to be generated by EGFP assay under inverted fluorescence microscope and qRT-PCR used to detect the expression of miR-26a transgene in BEL-7402, HepG2and Sk-Hep-1.3. The effects of miR-26a overexpression on HCC cell migration and invasion in vitroThe ability of cell migration was detected by wound-healing assay and transwell chamber assay, while the ability of cell invasion was detected by the matrigel invasion chamber assay.4. The effects of down-regulated expression of miR-26a in miR-26a-expressing HCC cells by miR-26a inhibitor on cell migration and invasionMiR-26a inhibitor was transfected into HCC cell lines BEL-7402-miR-26a and HepG2-miR-26a to down-regulate miR-26a expression. The influences of miR-26a downregulation on migration ability and invasion were evaluated by transwell chamber assay and the matrigel invasion chamber assay, respectively. qRT-PCR was used to detect the expression of metastasis-related genes in miR-26a-expressing HCC cells with and without transfected with miR-26a inhibitor.5. The effects of miR-26a overexpression in HCC cells on cell proliferation in vitro and in vivoThe ability of cell proliferation was detected in vitro by CCK8aasay, cell cycle and colony formation assay; The ability of cell proliferation was detected in vivo by nude mice tunor growth assay.Results:1. Expression profile of miR-26a in HCC cell lines and tissues qRT-PCR was used to detect the expression of miR-26a in HCC cell lines and human HCC tissues. The results indicated that the levels of miR-26a expression were significantly lower in HCC cell lines (such as BEL-7402,7721, HepG2, Huh7, Hep3B, Sk-Hep-1and Plc) than in normal liver tissue366N (figure1-1A). qRT-PCR was used to detect the expression of miR-26a in41human HCC tissues and44adjacent non-tumor tissues. The results showed that the levels of miR-26a expression were significantly lower in human HCC tissues(figure1-1B). Interestingly, we found that miR-26a expression levels in HCC tissues with metastasis were significantly higher than HCC tissues without metastasis(figure1-1C).2. Generation of HCC cell lines stably overexpressing miR-26a transgeneHCC cell lines (i.e., BEL-7402, HepG2and Sk-Hep-1) stably overexpressing miR-26a transgene were confirmed to be successfully generated by EGFP assay under inverted fluorescence microscope(figure2-3) and qRT-PCR used to detect the expression of miR-26a transgene in BEL-7402, HepG2and Sk-Hep-1(Table2-2,figure2-4).3. The effects of miR-26a overexpression on HCC cell migration and invasion in vitro3.1The ability of migrationThe results of wound-healing assay and transwell chamber assay showed that miR-26a overexpression in BEL-7402and HepG2cells enhanced cell migration in vitro(Table2-3, figure2-5A,B).3.2The ability of invasionThe matrigel invasion chamber assay indicated that miR-26a overexpression in BEL-7402and HepG2cells enhanced cell invasion in vitro(Table2-4, figure2-5C).4. The down-regulated miR-26a expression in miR-26a-expressing HCC cells inhibited cell migration and invasion4.1miR-26a inhibitor down-regulated miR-26a expression in miR-26a-expressing HCC cellsThe results of qRT-PCR showed that the efficiency of down-regulated miR-26a was more than95%(Table2-5,figure2-6).4.2The ability of migrationTranswell chamber assay showed that down-regulated miR-26a in miR-26a-expressing HCC cells inhibited cell migration in vitro (Table2-6,figure2-7A).4.3The ability of invasionThe results of the matrigel invasion chamber assay indicated that down-regulated miR-26a in miR-26a-expressing HCC cells inhibited cell invasion in vitro(Table2-7,figure2-7B).4.4The change of metastasis-related genesThe results of qRT-PCR indicated that miR-26a overexpression in BEL-7402and HepG2cells up-regulated the expression of metastasis-related genes(i.e., MMP1, MMP2, MMP9, MMP10), while the down-regulated expression of miR-26a caused the down-regulation of metastasis-related gene expression (figure2-8).5. The effects of miR-26a overexpression in HCC cells on cell proliferation in vitro and in vivo5.1miR-26a overexpression in HCC cells suppressed cell proliferation in vitroThe results from CCk8assay, colony formation assay and cell cycle strongly supported that miR-26a overexpression suppressed cell growth in miR-26a-expressing BEL-7402and HepG2cells(Table3-1,2,3and figure3-1A,C,D), whereas the data derived from CCk8assay and cell cycle indicated that the down-regulated expression of miR-26a in miR-26a-xpressing HCC cells by miR-26a inhibitor led to the enhanced proliferation(Table3-4,5and figure3-1B,E).5.1miR-26a overexpression in HCC cells inhibited cell proliferation in vivoThe results of nude mice tumor growth assay showed that miR-26a overexpression suppressed cell growth in vivo (figure3-2A,B,C). BrdU and p21-stained sections of transplanted tumors formed by miR-26a-expressing HepG2cells showed that there were fewer BrdU-positive cells and more p21-positive cells in miR-26a overexpression group than that in control group (Table3-6,figure3-2D).Conclusion:Taken together, these findings demonstrate that miR-26a can inhibit cell proliferation, but promotes migration and invasion in hepatocellular carcinoma.