The Research Of MicroRNA-30a-5p On The Proliferation And Migration Of The Lung Cancer Cells

Lung cancer is the leading incidence and the highest mortality cancer worldwide, of which 80% belongs to non-small cell lung cancer (NSCLC). Although early diagnosis technology, surgery, chemotherapy and biological target therapy in lung cancer in clinic have made great progress, the 5-year-survival rate of NSCLC is still very low. To increase the survival rate and to reduce the recurrence rate, it is not enough to explore the clinical investigation. Further research about etiology, especially about the cancer related genes should be strengthened, and the identification of new molecule for diagnosis and prognostic evaluation becomes urgent problems. The finding and in-depth research of miRNAs offer this possibility.MicroRNAs () are a kind of endogenous single-chain small noncoding RNAs which have length of about 22 nucleotides. miRNA, which can hybridize to the 3’UTR region of target mRNA sequence, regulate target gene expression both at the level if mRNA degradation and translation, then affect the life activities. Disorders of important process, like cellular differentiation, proliferation, apoptosis, may be caused by the dysfunction of miRNAs. MiRNAs can act as tumor-suppressor or oncogene in tumor progression. MiR-30a-5p, as one of the miRNAs, is closed related to many kinds of tumors, but the role of miR-30a-5p in lung cancer is not clear.In this study, the purpose is to reveal the differential expression and the tumor-suppressive role of miR-30a-5p in lung cancer. As further clarify the mechanism that the inhibitory effect of miR-30a-5p on the proliferation and migration of lung cancer cells, our study will provide an important theoretical basis for the pathogenesis of lung cancer. Our finding that miR-30a-5p inhibits LDHA expression will also help develop a potential therapeutic target for lung cancer therapy.Objective:To investigate the different expression of miR-30a-5p in lung cancer and its clinical significance, and to further study the biological effect and possible mechanism of miR-30a-5p effect on lung cancer cell proliferation and migration, which may provide the important theory basis to clarify the pathogenesis of lung cancer, and contribute to find a new target for diagnosis and treatment of lung cancer.Method:By using real-time quantitative PCR, the expression of miR-30a-5p in lung cancer tissues and the corresponding adjacent normal tissues were detected. The statistic correlation analysis for the expression of miR-30a-5p and the clinical factors were performed, and so did between the low expression of miR-30a-5p and clinical prognosis. The expression of miR-30a-5p were also detected in human lung cancer cell lines A549, HTB182, H460 and the normal human embryo lung cell line MRC-5. Furthermore, using bioinformatical softwares (RNAhybrid, Findtar3 and MiRanda) to predict the candidate target gene of miR-30a-5p. Using double luciferase reporter gene assay to verify the target gene. By real-time quantitative PCR and western blot, the effect of miR-30a-5p regulating the target gene at the level of mRNA ad protein were further investigated. Furthermore, the cell proliferation assay, cell colony formation assay, cell cycle assay, cell apoptosis assay, cell wound scratch assay, and cell migration assay were performed to investigate the proliferation and migration of cancer cells transfected with miR-30a-5p mimics or siR-LDHA in vitro. In addition, The lung cancer cell line, which were stably overexpressed with miR-30a-5p, was constructed, then the tumor xenograft was also established in mice and the tumor volume was calculated. Immunohistochemistry assays were used to detect cell proliferation antigen PCNA, LDHA expression and apoptosis in the transplanted tumor.Results:We found that the expression of miR-30a-5p decreased in both clinical lung cancer specimens and lung cancer cell lines. According to the statistic correlation analysis between differential expression of miR-30a-5p and clinical factors (age, gender, tumor size, tumor differentiation, tumor TNM stage, metastasis) in lung cancer, the low expression of miR-30a-5p had significant correlation with clinical factors including TNM stage and metastasis (P<0.05) but no correlation with clinical factors including age, gender, tumor size, tumor differentiation (P> 0.05). Compared to non-metastasis lung cancer, lower expression of miR-30a-5p was identified in metastasis lung cancer tissue (P <0.05). Kaplan-Merier curve showed that the lower expression of miR-30a-5p was positive associated with a poor prognosis (P<0.05). furthermore, we found that lactate dehydrogenase A (LDHA) was a target of miR-30a-5p. Western blotting showed that miR-30a-5p could inhibit the expression of LDHA protein in A549 and H460 cell lines. Dual luciferase report assay confirmed that miR-30a-5p was able to bind to the 3 ’UTR region of LDHA mRNA. We found that the proliferation and migration of lung cancer cells were suppressed after transfecting with miR-30a-5p mimics or siR-LDHA in vitro. The results of flow cytometry assays showed that the loss-of LDHA protein, which was mediated by miR-30a-5p, could induce cell cycle arrest in G0/G1 phase but not induce apoptosis in vitro. We also found that miR-30a-5p could inhibit the growth of transplanted lung tumor in mice. The results of immunochemistry assays showed that miR-30a-5p could inhibit the expression of both PCNA and LDHA proteins but not induce the apoptosis in vivo.Conclusion:MiR-30a-5p expression decreases in both lung cancer cell lines and lung cancer tumor tissues, and the low expression is positive correlated with TNM stage, metastasis, poor prognosis in lung cancer. Lactate dehydrogenase A (LDHA) is the direct target of miR-30a-5p in lung cancer. The decreased expression of LDHA protein, which is regulated by miR-30a-5p, induces the cell cycle arrest in G1 phase and suppresses the proliferation of lung cancer cells in vitro; also suppresses the growth of lung cancer cells in vivo. The decreased expression of LDHA protein mediated by miR-30a-5p also suppresses the migration of lung cancer cells in vitro. Taken together, these data strongly suggest that miR-30a-5p plays an important suppressive role in lung cancer.