Effect Of Long Non-coding RNA ANO1-AS1 On Migration And Invasion Of Esophageal Squamous Cell Carcinoma Cells

Objective:To study the effect of long non-codingRNA（lncRNA）ANO1-AS1 on migration and invasion of ESCC cells and its possible molecular mechanism.Methods:（1）Routine culture of esophageal cancer cell lines（EC109,TE）,to detect optimal MOI values according to viral titers,transfection of ANO1-AS1 knockdown lentivirus according to MOI,ANO1-AS1knockdown virus was transfected into esophageal cancer cell lines.The ANO1-AS1 expression silencing group was named LV-sh-ANO1-AS1,the negative control group was named LV-Con and untreated cells were blank control group.（2）The expression levels of ANO1-AS1 and ANO1 in esophageal cancer cells were detected by real-time PCR.（3）The scratch test and Transwell^TM migration test were used to detect the migration ability of cells.（4）The Transwell^TM invasion test was used to detect the invasion ability of cells.（5）The expressions of ANO1,matrix metalloproteinases（MMPs）family proteins MMP2,MMP9,epithelial mesenchymal transitions（EMT）related proteins and ERK/MAPK signal pathway related proteins were detected by Western blot.（6）Predicted target genes of lncRNA ANO1-AS1 by mi Rcode and Lnc Base Predicted v.2 website.Results:（1）After transfection of lentivirus according to the lentivirus transfection manual and screening with puromycin three times,the transfection rate of all groups of cells in both esophageal cancer cell lines was above 80%when observed under fluorescent inverted microscope.（2）q RT-PCR showed that after transfection with ANO1-AS1 knockdown virus,the expression levels of lncRNA ANO1-AS1 and mRNA ANO1 in LV-sh-ANO1-AS1 group were lower than those in LV-Con control group and blank group（P<0.05）.（3）The results of cell scratch test showed that the healing rate of cell scratch in LV-sh-ANO1-AS1 group was slower than that in LV-Con group and blank group（P<0.05）.（4）The results of Transwell experiment showed that the migration and invasion ability of LV-sh-ANO1-AS1 group were reduced compared with LV-Con group and blank group（P<0.05）.（5）Western blot showed that the expression levels of MMP2,MMP9,Vimentin,p-ERK1/2 and ANO1 protein were decreased（P<0.05）,and the expression level of E-cadherin protein was increased in LV-sh-ANO1-AS1 group（P<0.05）.（6）The mi Rcode and Lnc Base Predicted v.2 websites predict the target genes of lncRNA ANO1-AS1,and the two were intersected to find mi R-424-5p as a possible downstream target gene of lncRNA ANO1-AS1.Conclusion:（1）Inhibition of lncRNA ANO1-AS1 expression could inhibit the migration and invasion ability of esophageal squamous carcinoma cells EC109,TE（2）The study suggests that lncRNA ANO1-AS1may promote the migration and invasion of esophageal squamous cell carcinoma cells EC109 and TE by regulating ERK/MAPK signaling pathway.