Mechanism Of LncRNA EVADR On Cell Proliferation And EMT Occurrence In Colorectal Cancer Promoted By F.nucleatum

ObjectiveTo investigate the effet and potential mechanism of long non-coding RNA endogenous retroviral-associated adenocarcinoma(LncRNA EVADR)on the proliferation and epithelial to mesenchymal transition(EMT)of colorectal cancer cells caused by Fusobacterium nucleatum(F.nucleatum).Materials and Methods1.The pathological tissue samples of patients with colorectal cancer were collected and the changes of LncRNA bioinformatics in colorectal cancer tissue and adjacent cancer tissue were detected by high throughput gene chip analysis.qPCR technology further demonstrated the change of LncRNA EVADR,and qPCR detected the content of F.nucleatum.2.F.nucleatum respectively infected with HCT116,LOVO cells 0h,6h,12 h,or24h,and the two colon cancer cell infection models were established.qPCR method was used to detect the changes of LncRNA EVADR.qPCR and Westernblot methods were used to detect the changes of mRNA and protein levels of E-cadherin,respectively.3.Adenovirus was used to overexpresse the Lnc RNA EVADR level in HCT116,LOVO cell line.Four groups were estabulished including HCT116-NC group,HCT116-EVADR group,LOVO-NC group,LOVO-EVADR group.qRT-PCR was used to detect Lnc RNA EVADR expression.The changes of protein expression of E-cadherin,an EMT marker,were detected by Western blot method.The expression of EMT-related transcription factors Snail,Slug,ZEB1 and ZEB2 mRNA were detected by qRT-PCR.4.The proliferation ability of HCT116 and LOVO cells after Lnc RNA EVADR infection was detected by CCK-8 kit,and the migration and invasion ability of cell strain were detected by Transwell cell experiment.5.Effects of LncRNA EVADR adenovirus infection on the tumor formation,growth rate and liver metastasis of colorectal cancer cells were observed by subcutaneous tumorigenesis and caudal vein model in nude mice.Results1.F.nucleatum was higher in colorectal cancer tissue than in the paired colorectal cancer tissue.The expression of LncRNA detected by high-throughput gene microarray was significantly increased in colorectal cancer tissue by 5.23 times.2.The expression of LncRNA EVADR in the cells of HCT116 and LOVO showed a time-dependent increase with the increase of the F.nucleatum infected time.The expression of E-cadherin,a marker of EMT in colorectal cancer cells,decreased after F.nucleatum infected with 24 h.3.The expression level of LncRNA EVADR increased significantly after chronic virus infection with HCT116 and LOVO cells.The levels of protein expression of EMT marker E-cadherin in HCT 116-EVADR group,LOVO-EVADR group were significantly lower respectly compared with HCT 116-NC group,LOVO-NC.The expression of EMT-associated transcription factor markers Snail,Slug,ZEB1 and ZEB2 increased to different degrees.4.The results of CCK-8 showed that the proliferative ability of HCT 116-EVADR group,LOVO-EVADR group on day 2 to 5 was significantly higher than that of HCT116-NC group and LOVO-NC group respectively.The result of Transwell showed that the number of cells of HCT 116-EVADR group and LOVO-EVADR group passing through the transswell microporous filter was higher than that of HCT 116-NC group and LOVO-NC group.5.The tumor volume and weight were increased significantly in LncRNA EVADR overexpression nude mice.And the number of liver metastasis tumor nodules increased significantly after LncRNA EVADR overexpression in nude mice.Conclusion1.F.nucleatum content and LncRNA EVADR expression in colorectal cancer tissue samples was higher than matched colorectal cancer tissue samples.2.F.nucleatum infection can promote the expression of Lnc RNA EVADR in colorectal cancer cells and decrease the expression of E-cadherin,an EMT marker.3.Excessive expression of LncRNA EVADR can reduce the level of E-cadherin,which suggests that LncRNA EVADR promotes the occurrence of EMT in colorectalcancer cells.4.Excessive expression of LncRNA EVADR can promote the proliferation,migration and invasion of colorectal cancer cells.5.Expression of LncRNA EVADR can promote the proliferation and metastasis of colorectal cancer cells in nude mice6.F.nucleatum promotes the proliferation and transfer of colorectal cancer cells in nude mice by promoting the level of expression of LncRNA EVADR in colorectal cancer cells,facilitating the onset of EMT in colorectal cancer cells and promoting their proliferation,migration and invasion.