Function And Mechanism Study Of Long Noncoding RNAAC006159.3 In Cetuximab Resistance In Colorectal Cancer

Objective:To screen IncRNAs associated with cetuximab resistance in colorectal cancer and study the mechanism of its drug resistance,so as to provide a theoretical foundation for predicting and overcoming the resistance of cetuximab in colorectal cancer from the perspective of lncRNAMethods:(1)In order to filter lncRNAs that related to cetuximab resistance,lncRNA microarray was used to analyze the lncRNA expression profiles in colorectal cancer tissues and cell lines that were resistant to cetuximab in combination with bioinformatics analysis and molecular biology experiments.(2)To understand the relationship between the expression of lncRNA-AC006159.3 and cetuximab resistance,we detect the difference in the relative expression level of lncRNA-AC006159.3 in the cetuximab sensitive group and cetuximab resistance group.(3)The cell proliferation of wild type cells,cetuximab resistant cells and cetuximab resistant cells with lncRNA-AC006159.3 overexpressed were detected by CCK8 cell proliferation assay.So that to verify the relationship between lncRNA-AC006159.3 and cetuximab resistance.(4)Different cell lines(K-ras wild type cells,cetuximab resistant cells and cetuximab resistant cells with lncRNA-AC006159.3 overexpressed)were used to establish subcutaneous tumorigenesis model and liver metastasis model in nude mice in order to further study the relationship between lncRNA-AC006159.3 and cetuximab resistance.(5)Quantitative PCR was used to detect the expression level of lncRNA-AC0061 59.3 and c-Met in tissue samples to understand the relationship between the expression level of lncRNA-AC006159.3 and c-Met.we then observed the change of expression level of c-Met protein by regulating the expression of lncRNA-AC006159.3 in vitro(6)To verify targeted regulatory relationship between lncRNA-AC006159,3 and c-Met,we detected the changes of cell proliferation in cetuximab when interfering with lncRNA-AC006159.3 and c-Met gene expression,respectively.Results:(1)Using lncR:NA microarray,17 differential expressed lncRNAs which was related to cetuximab resistance of colon cancer was screened out.We found that lncRNA-AC006159.3 may play an important role in cetuximab resistance by using bioinformatics.(2)Results shown that the relative expression of lncRNA-AC006159.3 in cetuximab sensitive group was significantly higher than that of the cetuximab resistant group.The effect of cetuximab treatment was more significant in the group with lncRNA-AC006159.3 high expression.(3)The proliferation rates of wild-type cells,cetuximab resistant cells and cetuximab resistant cells with lncRNA-AC006159.3 overexpression were different.It was found that the overexpression of lncRNA-AC006159.3 could partly reverse the drug resistance of cetuximab(4)We found that tumor volume in the wild type group was the smallest,the volume of the drug resistant group was the largest,and the cetuximab resistant cells with lncRNA-AC006159.3 overexpression was in the middle.Moreover,we found that the number of metastatic nodules in the lncRNA-AC006159.3 overexpression group was significantly lower than that in the control group(5)There was a negative correlation between the expression level of c-Met and lncRNA-AC006159.3 in tumor tissue.The mRNA expression of c-Met was unregulated after the high expression of lncRNA-AC006159.3 was silenced at the cytological level.Overexpression of lncRNA-AC006159.3 significantly decreased the level of c-Met protein,while knockdown of lncRNA-AC006159.3 significantly increased the level of c-Met protein.(6)In the experiment of cell proliferation,interfering with the expression of lncRNA-AC006159.3 can reverse the inhibition of cetuximab on cell proliferation,while silencing the expression of c-Met gene can enhance the inhibition of cetuximab on cell proliferation.However,on the premise of knockdown c-Met using siRNA,there was no statistical difference in cell proliferation of the two groups with or without silencing lncRNA-AC006159.3.Conclusion:lncRNA-AC006159.3 is a negative regulator of cetuximab resistance.We preliminarily confirmed that lncRNA-AC006159.3 acts a significant role in cetuximab resistance in colorectal cancer by regulating c-Met gene.