The Study On MiR-496-induced Epithelial-mesenchymal Transition By Targeting RASSF6 In Colorectal Cancer

Background:Colorectal cancer(CRC)is a malignant tumor with high incidence worldwide,which seriously endangers people's health.Similar to most solid tumors,the molecular pathogenesis of CRC is complicated,and inactivation of important tumor suppressor genes such as APC and TP53 is an important basis for its pathogenesis.Therefore,it is of great significance to study and clarify the expression and regulation of tumor suppressor genes to correctly understand the pathogenesis of CRC.Recently,we used whole exome sequencing and functional experiments to report for the first time that the expression of the tumor suppressor gene RASSF6 is lost or down-regulated to participate in the occurrence and development of CRC.Micro RNA,as a short-sequence non-coding RNA that regulates m RNA expression levels,has been paid more and more attention.We previously used bioinformatics methods to determine the CRC new oncogenic miRNA(miR-496)upstream of RASSF6.As far as we know,systematic analysis of the targeted regulation of miRNAs upstream of RASSF6 and the molecular mechanism of miR-496 in the occurrence and development of CRC have not been reported.Therefore,the completion of this study will help us to further understand the RASSF6 expression regulatory network during the occurrence and development of CRC,and to clarify the mechanism of action of the new oncogene miR-496,which has important theoretical significance.Research methods:1.Use micro RNA.org online prediction software,Pun Med database,TCGA database to analyze the relevant data and information of CRC clinical samples to determine the new oncogenic miRNA of CRC: miR-496.2.Analyze the expression of miR-496 and RASSF6 in CRC clinical tissue samples and their adjacent cancer tissue samples from the m RNA level using Q-PCR.3.The detection relationship between miR-496 and RASSF6 3'-UTR was detected using dual luciferase reporting system detection experiments.4.Based on the cell(SW480 and Lo Vo)level,Western blot was used to verify the effect of miR-496 on RASSF6 expression from the protein level.5.Select SW480 and Lo Vo cell lines and transfect them with miR-496 mimics or inhibitor,respectively.The effects of miR-496 on the proliferation and migration ability of CRC cells were compared by CCK8,clone formation experiments,cell scratching experiments,and Transwell migration experiments.SW480 cell line was selected,and Western blot was used to test whether the protein expression of migration and EMT-related marker molecules had the same trend as above.6.In SW480 and Lo Vo cell lines,the Rescue test using Transwell was performed to verify the ability of miR-496 to directly target RASSF6 to inhibit the migration of colorectal cancer cells.The SW480 cell line was selected,and the Rescue test was performed using Western blot to detect whether the protein expression of migration and EMT-related marker molecules had a trend consistent with the above.7.Select the SW480 cell line,transfect it with miR-496 mimics or inhibitor,and use Western blot to detect whether the Wnt signaling pathway is activated or inhibited.Simultaneously perform rescue tests to verify whether the activation or inhibition of the Wnt signaling pathway induced by miR-496 mimics or inhibitor is related to RASSF6.Results:1.Bioinformatics analysis to determine the novel oncogenic miRNA of CRC: miR-496;the double luciferase reporting system detection experiments showed that RASSF6 is the target gene of miR-496;SW480 and Lo Vo cell lines were transfected with miR-496mimics/inhibitor and the expression of RASSF6 protein changed accordingly.2.In clinical samples,the expression of miR-496 in colorectal cancer tissues was significantly higher than in adjacent normal tissues,and the expression of RASSF6 in colorectal cancer tissues was lower than in adjacent normal tissues,and there was a significant negative correlation between the two in colorectal cancer tissue samples.3.After SW480 and Lo Vo cell lines were transfected with miR-496 mimics,there was no difference in cell proliferation rate and number of colony formation,and cell migration ability was enhanced.After transfection of miR-496 inhibitor,there was no difference in cell proliferation rate and number of colony formation,and cell migration ability was reduced.4.In the Rescue test,it was determined by Transwell experiment analysis that knockdown of RASSF6 promoted the migration ability of Lo Vo and SW480 cells,and inhibition of miR-496 could reduce these effects.On the contrary,overexpression of RASSF6 reduced the migration ability of Lo Vo and SW480 cells,and miR-496 overexpression can attenuatethese effects.5.In SW480 cell line,after transfection of miR-496 mimics/inhibitor,the protein expression of Wnt signaling markers,migration markers,and EMT markers changed.The results of the Rescue test showed that inhibition of miR-496 could attenuate the promotion effects of RASSF6 low expression on CRC migration,EMT and Wnt signaling;overexpression of miR-496 could attenuate the inhibitory effects of RASSF6 high expression on CRC migration,EMT and Wnt signaling.Conclusion:MiR-496 targets RASSF6 and inhibits RASSF6 expression,and promotes CRC cell migration and epithelial-mesenchymal transition(EMT)by activating Wnt signaling pathway.This suggests that miR-496 acts as an oncogene in CRC.