Screening Of Wnt Signaling Pathway Related Genes Regulated By LI-cadherin In Colorectal Cancer

Background:Colorectal Cancer(CRC)is one of the most common malignant tumors in digestive tract.The cancer incidence rate of CRC ranks third in the global,and the mortality ranks fourth.The main reason for the high mortality rate is closely related to the recurrence and metastasis of postoperative tumors.Some studies have shown that the abnormal expression of liver-intestine cadherin(LI-cadherin,or Cadherin-17,CDH17)in colorectal cancer is associated with cell invasion and metastasis,but the specific mechanism remains largely unknown.Previous studies have found that Wnt signaling pathway is involved in the invasion and metastasis of various human tumors Therefore,it is important to clarify whether LI-cadherin is involved in the regulation of Wnt signaling pathway-related gene expression,to reveal the molecular mechanism of invasion and metastasis of colorectal cancer cells,and to explore the knot.New targets for the diagnosis and prevention of rectal cancer are of great significance.Objective:To screen the possible molecular mechanisms of LI-cadherin being involved in invasion and metastasis of colon cancer by screening for Wnt signaling pathway related genes in colon cancer induced by LI-cadherin expressionMethods:1.The colon cancer cell line was cultured,LI-cadherin-shRNA lentiviral silencing expression and LI-cadherin lentivirus negative control vectors were constructed,and the lentivirus was packaged and infected to LoVo and COLO205 cells respectively.RT-qPCR and Western blot were used to detected LI-cadherin gene silencing efficiecy at mRNA and protein levels.The stable transfection cell lines of LI-cadherin knockout gene2.The total RNA of the experimental group and control group was extracted and everse transcripted for first chain cDNA.Wnt Signaling Pathway PCR Array was used to screen Wnt signaling pathway related genes regulated by LI-cadherin.Finally,the result was further verified by RT-qPCR,Western blot and immunofluorescence methods.Results:1.LI-cadherin-shRNA lentiviral silencing expression vector and LI-cadherin lentivirus negative control vector were successfully constructed,and the colon cancer LoVo and COLO205 cell lines stably silenced by LI-cadherin gene after lentivirus infection were obtained2.Wnt Signaling Pathway PCR Array was used to screen out the Wnt signaling pathway genes that LI-cadherin regulated in colon cancer LoVo and COLO205 cells The results showed that The mRNA transcriptional level of 40 WNT signaling pathway related genes was changed significantly after the silencing LI-cadherin gene3.The result of Wnt Signaling Pathway PCR Array was further validated and detected by RT-qPCR,Western blot and immunofluorescence method to clarify the effect of silencing LI-cadherin gene on Wnt signaling pathway.It was showed that the expression of 18 Wnt signaling pathway related genes was significantly reducedConclusion:The expression of three branch Wnt signaling pathway-related genes was affected by silencing LI-cadherin gene in colon cancer LoVo and COLO0205 cells:Seven classical Wnt/β-catenin pathway related genes were significantly changed:APC,AXIN2,GSK3β,FZD1,FZD3,FZD7,TCF7;One Wnt/Ca2+pathway related gene was changed:WNT5A;Six Wnt/PCP pathways related genes were changed:VANGL2,WNT4,WNT5A,WNT5B and WNT9B.In addition,four downstream target genes of Wnt signaling pathway regulated by LI-cadherin were changed:MYC,CCND1,MMP7 and PPARD.It shows that all of the three pathways of Wnt signaling pathway are regulated by LI-cadherin.