LncRNA SNHG6 Promotes Chemoresistance Through ULK1-Induced Autophagy By Sponging MiR-26a-5p In Colorectal Cancer Cells

Background and purposeColorectal cancer（CRC）is one of the most common malignant tumors^[1].Chemotherapy resistance is the main reason for the low cure rate and high mortality rate of CRC^[2-4].Therefore,it is important to reveal the mechanism of chemotherapy resistance and explore new targets for treatment of CRC.Long non-coding RNA（lncRNA）can regulate the occurrence and development of tumor in many ways^[5-6].As a resut,the purpose of this study was to explore the mechanism of lncRNA to regulate tumor chemical resistance.Methods1.The exprssion level of lncRNA SNHG6 and the correlation analysis between the expression of lncRNA SNHG6 and clinical prognosis of patient.1)To find the differentially expressed lncRNA in CRC by consulting the literature and analyzing GSE9348;2)To analyze the expression of lncRNA SNHG6 in CRC by bioinformatics;3)To verify the expression of lncRNA SNHG6 in CRC tissue and cell line by qRT-PCR;4)To analyze the correlation between lncRNA SNHG6 expression and clinical prognosis of patient by the data of TCGA,GSE17538,GSE14333 and GSE39582.2.1ncRNA SNHG6 promotes the proliferation,migration and invasion of CRC cells.1)Proliferation in vitro:lncRNA SNHG6 interference lentivirus and overexpression plasmid were transfected into CRC cells respectively.The expression efficiency was verified by qRT-PCR and CCK8 assay was carried out;2)Proliferation in vivo:subcutaneous tumorigenesis in nude mice was built by using lncRNA SNHG6 knock-down CRC cells;3)Migration and invasion in vitro:transwell chamber migration and invasion test as well as scratch test were done.3.1ncRNA SNHG6 increases the resistance of CRC cells to 5-fluorouracil.1)To detect the IC50 of 5-fluorouracil:CCK-8 was used to detect the number of CRC cells in groups treated with different concentration of 5-fluorouracil for 48 hours,and the IC50 of each group was calculated;2)Apoptosis:flow cytometry was used to detect the apoptosis rate of cells treated with different concentrations of 5-fluorouracil for 48 hours;3)5-fluorouracil resistance in vivo experiment:subcutaneous tumor test in nude mice was carried out with lncRNA SNHG6 knock-down CRC cells,and then the nudes were divided into groups for abdominal injection of 5-fluorouracil or PBS.Finally,the tumor growth inhibition rate of each group was calculated.4.Study on the mechanism of lncRNA SNHG6 in increasing the resistance of CRC cells to 5-fluorouracil.1)Nuclear-cytoplasmic separation experiment:the distribution of lncRNA SNHG6 in CRC cells was explored;2)To search the targeted microRNA（miRNA）of lncRNA SNHG6:to predict the miRNA that could bind to lncRNA SNHG6 by bioinformatics database,which was then verified by qRT-PCR and double luciferase reporter gene experiment;3)To search the targeted mRNA of miR-26a-5p:to predict the mRNA that could bind to miR-26a-5p by bioinformatics database,which was then verified by qRT-PCR and double luciferase reporter gene experiment;4)QRT-PCR was used to verify the correlation between lncRNA SNHG6 and ULK1 in CRC specimens;5)Detection of autophagy:the occurrence of autophagy was detected by western blot,electron microscope and autophagy double-labeled adenovirus transfection experiment.5.lncRNA SNHG6/miR-26a-5p/ULKl axis induces drug resistance in CRC by inducing autophagy.1)LncRNA SNHG6/miR-26a-5p/ULK1 axis was verified by western blot and qRT-PCR;2)LncRNA SNHG6 indirectly upregulated ULK1 depending on miR-26a-5p was further verified by western Blot,qRT-PCR and apoptosis experiments after lncRNA SNHG6 knockdown and overexpression of colorectal cancer cell lines were transferred with inhibitors and mimics of miR-26a-5p,respectively;3)After adding autophagy inhibitor 3MA to lncRNA SNHG6 knockdown CRC cells and control group,western Blot,IC50 and apoptosis experiments were carried out to further verify that lncRNA SNHG6 reduced the sensitivity of CRC cells to 5-fluorouracil depending on autophagy.Results1.LncRNA SNHG6 was highly expressed in CRC tissues and cell lines,and was related to the poor prognosis of patients;2.LncRNA SNHG6 could promote the proliferation,migration and invasion of CRC cells;3.LncRNA SNHG6 could reduce the sensitivity of CRC cells to 5-fluorouracil;4.LncRNA SNHG6 was mainly distributed in the cytoplasm of CRC cell lines and could bind to miR-26a-5p.It was further found that ULK1 was the target gene of miR-26a-5p and positively correlated with lncRNA SNHG6.LncRNA SNHG6 could promote autophagy in CRC cell lines;5.LncRNA SNHG6 up-regulating the expression of ULK1 depended on miR-26a-5p,and reducing the sensitivity of CRC cells to 5-fluorouracil depended on autophagy.ConclusionThis study found that lncRNA SNHG6 was highly expressed in CRC tissues and cells,and was related to poor clinical prognosis of patients;LncRNA SNHG6 could adsorb miR-26a-5p and then up-regulated ULK1 to induce autophagy and reduced the drug sensitivity of CRC cells to 5-fluorouracil.