MicroRNA-20a Is Invoved In Chememotherapy Resistance In Colorectal Adenocarcinoma

ObjectiveColorectal adencarcinoma is one of severe disease threatening our human being now, and chemotherapy is an important therapeutic approach. Unfortunately, chemoresistance of cancer cells to anti-tumor drugs is the main obstacle in chemotherapy treatment. So it is of great importance to find how the cancer cells obtain the ability to resist the anti-tumor drugs. miRNA, a kind of small noncoding RNA, is an important regulator that regulate growth, differentiation, proliferation and apoptosis by targeting on the corresponding3’UTR sequence of special target genes. We set out to see if miR-20a is related to chemotherapy resistance in colorectal adencarcinoma cell lines, SW620and SW480, and the possible mechanism.MethodsmiR-20a is one of the miRNAs that is higher in SW620than in SW480using miRNA profile array in our lab. Consist with higher level of miR-20a, SW620exhibit chemotherapy resistance to several chemotherapy drugs compared to SW480detected by MTT assay.20a-ASO and siR-20a was used to knock down and to over express endocellular miR-20a using Lipofectamine2000TM, then MTT assay was used to detect cell death rate after48h exposure to a serial concentrations of chemotherapy drugs commonly used in adencarcinoma chemotherapy. SW620was more sensitive to chemotherapy when miR-20a decreased, while overexpression of miR-20a in SW480resulted in decreased sensitivity to anti-tumor agents. Candidate target genes of miR-20a were predicted through bioinformatics using miRNA target prediction program, MiRanda, TargetScans and Pictar online. Then interested candidate target genes were confirmed directly, using the EGFP fluorescence reporter system.3’UTR of candidate target genes, obtained by PCR amplification, bearing miR-20a complementary binding sites or the mutant miR-20a binding sites, were cloned to the downstream of the stop codon of EGFP gene, using the BamH I and EcoR I multiple clone site of the pcDNA3B/EGFP vector to obtain the fluorescence reporter vectors,.Then either20a-ASO or siR-20a was contransfected with the fluorescence report vectors using Lipofectamine2000TM.60h post transfection, EGFP fluorescence was detected using fluorospectrophotometer under excitation light at488nm wavelength and emission light at507nm wavelength respectively, to detect the influence of miR-20a on EGFP expression.RT-PCR technique was used to detect mRNA level of candidate target genes after decreased and increased miR-20a level in SW620and SW480respectively.ResultsAfter20a-ASO decreased endocellular miR-20a level, SW620showed increased sensitivity to anti-tumor drugs, while miR-20a over expression through siR-20a transfection in SW480cell resulted in chemotherapy resistance to anti-tumor drugs. The above data confirmed that miR-20a was involved in the response of colorectal adencarcinoma to chemotherapy. The computational target prediction algorithm showed that bnip2, fastk and gab1were the predicted targets of miR-20a. The fluorescence report vectors, pcDNA3B/EGFP-Bnip23’UTR、pcDNA3B/EGFP-Fastk3’UTR、pcDNA3B/EGFP-Gabl3’UTR, bearing miR-20a complementary binding sites and the corresponding mutant vectors, were used in the following fluorescence reporter essay. The EGFP expression of EGFP increased greatly after20a-ASO decreased endocellular miR-20a, and its expression was not affected when the complementary miR-20a binding sequence mutant. The above data gave us direct certifications that miR-20a can bind directly to the3’UTR of bnip2, fastk and gab1, resulting in regulation of expression of these three genes, either decreasing mRNA level or inhibited corresponding protein expression.The endocellular mRNA level of bnip2, fastk, gab1increased when decreased miR-20a level in SW620, consistent with this, the mRNA level decreased when miR-20a level increased by siR-20a.ConclusionOur data gave strong certifications that miR-20a serves as a regulator of chemotherapy in colorectal adencarcinoma cell lines. And bnip2, fastk, gab1are target genes of miR-20a. And possibly, miR-20a regulated chemotherapy sensitivity of colorectal adencarcinoma cell line to anti-tumor drugs by targeting bnip2, fastk and gabl.