The Influence Of MiR-31 On Colorectal Cancer By Targeting RASA1

Objective:Colorectal Cancer (CRC) is a common gastrointestinal tumor which development and progression is regμlated by multiple factors. MicroRNA (miRNA) is a class of endogenous, about 22 non-coding single-stranded small RNA nucleotides, with a wide range of functions in human life activity. Especially in recent years, more and more researches focus on the role of miRNA in malignant tumors. With the study of miRNA deeper and deeper, we find that some miRNAs are related with the development and progression of CRC, which not only bring a new breakthrough for the study of the molecular mechanisms of CRC, but also suggest that miRNAs are new specific biomarkers for early diagnosis and prognosis of CRC. MiRNAs may act as the oncogene and anti-oncogene. The researchers on the mechanism of miRNAs regulation function find that the specific process of miRNAs regulating processes are through targeting some regulatory factor or signal pathways. The development and progression of CRC are regulated by many miRNAs, but the systematic study of its mechanism has not been reported. Thus, we investigated the expression of miR-31 in colorectal cancer cells and tumor tissues, and analysis the relationship of miR-31 expresion with clinicopathological parameters in colorectal cancer. Then we analyzed its effects on biological function on CRC cells like cell proliferation and migration, and we predicted target genes of miR-31 by bioinformatics software and explore its possible mechanism, to lay a theory foundation for clinical application of miR-31.Methods:The expression of miR-31 in colorectal cancer tissues and colorectal cancer cell lines were measured by RT-PCR. Total RNA Extraction Kit was used to extractd the total RNA of fresh frozen tissues and cell lines. PrimeScript RT kit was used to reverse transcriptase synthesis cDNA, and RT-PCR reactions were performed using ABI 7500 fluorescence quantitative PCR. The relationship of expression of miR-31 and clinical characteristics were analyzed. Then we also analyzed the relationship of expression of miR-31 and patients’ prognosis. Then we detected the cell proliferation and migration of colorectal cancer cell lines (DLD-1、SW480、 SW620、Caco-2、HCT116 and HT-29)transfected by miR-31 mimics using CKK8 and wound healing assay. The Cell Cycle Analysis of CRC cell lines were conducted by flow cytometry. We predicted the target protein of miR-31 by using TargetScan and miRanda database, then we verified it through dual luciferase experiment.The expression of RASA 1 colorectal cancer cell lines transfected by miR-31 mimics were detected by Western blotting.Results:The relative expression of miR-31 in colorectal cancer tissues was(3.69 ±0.22), which is significantly higher than normal tissues(P<0.05).And the relative expression of miR-31 in colorectal cancer cells lines (DLD-1、SW480、SW620、 Caco-2、HCT116) is also up-regulated compared to normal epithelial cells. Expression of miR-31 was significantly correlated with TNM stage and lymphatic metastasis (P<0.05).The patients with high miR-31 expression got poor prognosis. Overexpression of miR-31 in colorectal cancer cell lines could rrest the cells in S stage, which means the ratio of S stage increased and the ratio of G0/G1 decreased, with significant difference(P<0.05).We also find that overexpression of miR-31 in colorectal cancer cell lines could promote cell proliferation and migration. The bioinformatics prediction showed the target protein of miR-31 may be RASA1, Meanwhile, the expression of RASA1 in miR-31 mimics group was (0.21±0.05), which was significantly higher than that in miR-31 inhibition group(2.46±0.35).Conclusion:miR-31 is high expression in colorectal cancer, and may participate in the cell proliferation and migration of colorectal cancer cell lines, and the possible mechanism is targeting effect on RASA 1.