The Screening For Oxaliplatin Resistant MicroRNAs And Research On Their Mechanisms In Colon Cancer Cell

Objective:To investigate the mechanisms of small RNA (microRNA, miRNA) and the molecular mechanisms that play a role in colon cancer cells OXA resistant, looking for the resistance-associated regulation molecules, to provide the experimental basis for an effective reversal of tumor resistant.Methods:1:The establishment of drug oxaliplatin (oxaliplatin, OXA) colon cancer cell lines using OXA concentration gradient induced method2:MiRNA microarray and real-time quantitative PCR technology were used to detect differentially expressed miRNA in resistant and parental colon cancer cell lines.3:The miRNA-137that was low expression in the resistant cell lines was selected as the target molecule of this study. And using the MTT assay, the influence of miRNA-137on colon cancer cell line SW480and HCT-15resistant to OXA was investigated.4:The candidate target genes YBX1of miRNA-137was screened using the bioinformatics method and using fluorescent reporter vector experiment to test the direction regulation of miRNA-137on YBX1expression. Real-time PCR and Western blot experiments were used to detect target gene YBX1mRNA and protein expression levels when miRNA-137expression was enhanced in colon cancer cells. The sensitivity of colon cancer cells to OXA was detected when the target gene YBX1was inhibited using RNA interference techniques,5:The relationship between OXA resistance and hsa-miR-137, YBX1expression in the clinical colon cancer specimens was further validated by real-time PCR technology and immunohistochemistry.Results:1:The drug-resistant colon cancer cell lines were established using the OXA whose concentration is from0.2to2.0μM2:The hsa-miR-93, hsa-miR-191, hsa-miR-137, hsa-miR-491-3p, hsa-miR-96, hsa-miR-21, hsa-miR-22, hsa-miR-192were significantly differentially expressed between the parental SW480, HCT-15and resistant SW480/OXA,HCT-15/OXA cell lines. The MTT assay in colon cancer cells showed that miR-93, miR-137and miR-21can regulate the chemosensitivity of colon cancer cells to OX A3:The fluorescent reporter vectors experiments showed that miRNA-137can target YBX1gene3’untranslated region and negatively regulate its expression, the mRNA and protein expression levels of YBX1gene were significantly increased when miRNA-137expression was decreased in colon cancer cells,4:The drug resistance of colon cancer cells to OXA was significantly decreased when the expression of YBX1was inhibited in colon cancer cells.5:Quantitative real-time PCR results showed that the expression level of miR-137was negatively correlated with OXA resistant ability in clinical colon cancer tissues. Western blot and immunohistochemistry results showed that the expression level of YBX1was positively correlated with OXA resistant ability in clinical colon cancer tissues.Conclusion:1:Abbrent expression of many miRNAs contributed to OXA resistance in colon cancer cells2:miR-137can negatively regulate its target gene YBX1expression in colon cancer cells,3:MiR-137regulates the sensitivity of colon cancer cells to chemotherapeutic drugs OXA through target YBX14:The OXA resistance in clinical colon cancer tissue is related with miR-137and its target gene YBX1expression...