SNHG6/miR-101-3p Axis Induces Epithelial-mesenchymal Transition In Esophageal Squamous Carcinoma Cells Via Promoting ZEB1 Expression

Part one The expression of lncRNA SNHG6 in esophageal squamous cell cellsObjective:To detect the expression of long non-coding RNA?lncRNA?small nuclear RNA host gene 6?SNHG6?in esophageal squamous cell carcinoma?ESCC?tissues and cells,and to explore its effects on the cell proliferation,migration and invasion in ESCC cells.Methods:1. The specimens of ESCC tissues and the adjacent tissues were collected from 36 patients who underwent surgery at the Fourth Hospital of Hebei Medical University from February to September 2019.None of these ESCC patients received any anti-tumor treatment before surgery.Specimens of ESCC and the adjacent tissues were removed immediately after surgery and stored in-80°C refrigerator for RNA extraction.All the patients signed informed consent before surgery.2.Quantitative real-time polymerase chain reaction?qRT-PCR?was used to detect the SNHG6 expression in ESCC tissues.3.Quantitative polymerase chain reaction?qPCR?was used to detect the SNHG6 expression in ESCC cell lines?TE1,Yes-2,EC9706 and Kyse150?.The TE1 and EC9706 cells with higher SNHG6 expression were selected for subsequent functional experiments.4.SNHG6-siRNA?si-SNHG6-1,si-SNHG6-2,si-SNHG6-3?was transfected into TE1 and EC9706 cells to construct SNHG6 low-expression ESCC cells by lipofectamine ^TM2000.siRNA-NC was transfected as a control group?Control?.qRT-PCR method was used to detect the transfection efficiency.Two knock-down sequences with the higher transfection efficiency were selected for subsequent experiments.5.Colony formation assay,wound healing assay and transwell invasion assay were used to detect the effect on the abilities of cell proliferation,migration and invasion of ESCC before and after SNHG6 knockdown.Results:1. qRT-PCR results showed that SNHG6 was highly expressed in ESCC tissues compared with the adjacent tissues?P<0.05?.2. qPCR results showed that SNHG6 was highly expressed in ESCC cells.TE1 and EC9706 cell lines were selected for subsequent functional experiments.3.The expression of SNHG6 m RNA was significantly reduced by transfecting SNHG6-siRNA in ESCC cells?P<0.05?,which demonstrated that the cell lines with low expression of SNHG6 were successfully constructed.The si-SNHG6-1 and si-SNHG6-2 sequences were selected for subsequent functional experiments because of their lower knockdown efficiency.4.After SNHG6 knockdown,the proliferation ability of ESCC cells was significantly reduced compared with the control group?P<0.01?.5.After SNHG6 knockdown,the migration ability of ESCC cells was significantly reduced compared with the control group?P<0.05?.6.After SNHG6 knockdown,the invasive ability of ESCC cells was significantly reduced compared with the control group?P<0.01?.Conclusions:1.LncRNA SNHG6 is highly expressed in esophageal squamous cell carcinoma tissues.2.LncRNA SNHG6 promotes the abilities of proliferation,migration and invasion of ESCC cells.Part two SNHG6/miR-101-3p axis promotes epithelial-mesenchymal upregulating ZEB1 expressionObjective:The software was used to predict the downstream target genes and target proteins of lncRNA SNHG6 and to verify them by molecular biology experiments.To explore the mechanism of SNHG6 affecting the epithelial-mesenchymal transition?EMT?in ESCC.Methods:1.Bioinformatics software were used to predict the lncRNA SNHG6target micro RNA?miRNA?.2.Liposome transfection method was used to treat 293T cells by lipofectamine ^TM2000 and for the following experiments.Cells were divided into four groups?miR-NC+SNHG6-WT,miR-101-3p mimic+SNHG6-WT,miR-101-3p mimic+SNHG6-MUT,miR-NC+SNHG6-MUT?.Dual luciferase reporter assay and qRT-PCR were used to verified preliminarily the correlation between SNHG6 and miR-101-3p.3. qRT-PCR was used to detect the expression of miR-101-3p in ESCC tissues.4. Liposome transfection method was used to transfect miR-101-3p mimic into ESCC TE1 and EC9706 cells to construct ESCC cell lines that overexpressed miR-101-3p by lipofectamine ^TM2000,and transfected miR-NC as a negative control group.qRT-PCR was used to detect transfection efficiency.5.Colony formation assay,wound healing assay,transwell invasion assay were used to detect the effect on the abilities of proliferation,migration,invasion of ESCC before and after miR-101-3p overexpression.6.Liposome transfection method was used to treat 293T cells by lipofectamine ^TM2000 and for the following experiments,cells were divided into four groups?miR-NC+ZEB1-WT,miR-101-3p mimic+ZEB1-WT,miR-101-3p mimic+ZEB1-MUT,miR-NC+ZEB1-MUT?.Dual luciferase reporter assay and qRT-PCR were used to verify preliminarily the correlation between miR-101-3p and ZEB1.7.Liposome transfection method was used to treat ESCC TE1 and EC9706 cells in vitro with lipofectamine ^TM2000.The cells were divided into SNHG6 knockdown control group?si-NC?,SNHG6 knockdown group?si-SNHG6-1?,SNHG6 knockdown and miR-101-3p knockdown control group?si-SNHG6-1+miR-NC inhibitor?,SNHG6 and miR-101-3p knockdown group?si-SNHG6-1+miR-101-3p inhibitor?,SNHG6 knockdown control and miR-101-3p overexpression control group?si-NC+miR-NC mimic?,SNHG6knockdown control and miR-101-3p overexpression group?si-NC+miR-101-3p mimic?.Western blot was used to detect the protein expression levels of ZEB1 and epithelial-mesenchymal transition?EMT?markers?E-cadherin,N-cadherin,Twist and Snail?in ESCC cells.Results:1.Bioinformatics software predicts that lncRNA SNHG6 has specific binding sites to miR-101-3p,suggesting that miR-101-3p may be the target miRNA of SNHG6.Dual luciferase reporter assay and qRT-PCR were used to verified preliminarily the correlation between SNHG6 and miRNA in ESCC cells.Dual luciferase reporter assay showed that the luciferase activity in the miR-101-3p mimic+SNHG6-WT group was significantly reduced?P<0.05?,while there were no statistical difference in the luciferase activity between miR-NC+SNHG6-WT,miR-101-3p mimic+SNHG6-MUT and miR-NC+SNHG6-MUT?P>0.05?.qRT-PCR results showed that the expression of miR-101-3p in ESCC cells in the si-SNHG6-1 and si-SNHG6-2 groups was significantly higher than that in the control group?P<0.05?,indicating that miR-101-3p is the target miRNA of SNHG6,and the expression of SNHG6 is negatively correlated with miR-101-3p.2.qRT-PCR results showed that the expression of miR-101-3p in esophageal cancer tissue was significantly lower than that in adjacent tissues?P<0.05?.3.qRT-PCR results showed that the expression of miR-101-3p in ESCC was significantly increased after miR-101-3p mimic transfection?P<0.05?,and the cell lines with miR-101-3p overexpression was successfully constructed.4.After miR-101-3p overexpression,the proliferation ability of ESCC cells was significantly reduced compared with the miR-NC group?P<0.01?.5. After miR-101-3p overexpression,the migration ability of ESCC cells was significantly reduced compared with the miR-NC group?P<0.05?.6. After miR-101-3p overexpression,the invasive ability of ESCC cells was significantly reduced compared with the miR-NC group?P<0.01?.7. Bioinformatics software predicts that miR-101-3p has specific binding sites to ZEB1,suggesting that ZEB1 may be the target m RNA of miR-101-3p.Dual luciferase reporter assay and qRT-PCR were used to verified preliminarily the correlation between miR-101-3p and ZEB1 in ESCC cells.Dual luciferase reporter assay showed that no statistical difference in the luciferase activity between miR-NC+ZEB1-WT,miR-101-3p mimic+ZEB1-MUT,miR-NC+ZEB1-MUT.The luciferase activity in the miR-101-3p mimic+ZEB1-WT group was significantly reduced?P<0.05?.qRT-PCR results showed that the expression of ZEB1 in ESCC cells in the miR-101-3p mimic was significantly lower than that in the miR-NC group?P<0.05?,indicating that ZEB1 is the target miRNA of miR-101-3p,and the expression of the two is negatively correlated.8. Western blot results showed that compared with the si-NC group,the expression of ZEB1 in the si-SNHG6-1 group was significantly reduced,the expression of EMT-associated protein E-cadherin was increased,and N-cadherin,Snail,Twist expression were decreased?all P<0.05?,indicating that knocking down SNHG6 can significantly reduce ZEB1 expression and reverse EMT transformation.Compared with the si-NC+miR-NC mimic group,ZEB1 expression in the si-NC+miR-101-3p mimic group was significantly reduced,E-cadherin expression was increased,and N-cadherin,Snail,and Twist expression were reduced?all P<0.05?,indicating that contrary to the effect of SNHG6,overexpression of miR-101-3p can reduce ZEB1expression and reverse EMT transformation.In order to verify whether SNHG6 exerts biological functions through the miR-101-3p/ZEB1 axis,this study knocked down both SNHG6 and miR-101-3p in ESCC cells,set a negative control group,and then observe the changes of downstream related protein expression levels.Western blot results showed that compared with si-SNHG6-1+miR-NC inhibitor,the expression of ZEB1 in si-SNHG6-1+miR-101-3p inhibitor group was significantly increased,and the expression of E-cadherin was decreased.N-cadherin,Snail,Twist expression were increased?all P<0.05?.It shows that knocking down miR-101-3p expression can rescue ZEB1 down-regulation and EMT reversal caused by SNHG6 knockdown.The above results indicate that lncRNA SNHG6 up-regulates ZEB1 expression through the SNHG6/miR-101-3p axis,induces ESCC epithelial-mesenchymal transition,and thus participates in the genesis and development of ESCC as a pro-oncogene.Conclusions:lncRNA SNHG6 induces EMT of ESCC through the miR-101-3p/ZEB1axis which plays a carcinogenic role in the occurrence and development of ESCC.Part three SNHG6/miR-101-3p/ZEB1 promotes the development of esophageal squamous cell carcinoma in vivoObjective:To explore the carcinogenic effect of SNHG6/miR-101-3p/ZEB1 axis in nude mice,and to lay a theoretical foundation for the reversal treatment of ESCC.Methods:1.Cell lines stably knockdown of SNHG6 gene were constructed by transfecting with sh-RNA construct containing desired vector.2.Two groups of EC9706 cells were injected subcutaneously into the4-week-old female BALB/c nude mice to construct a nude mouse xenograft model.3.The nude mice were weighed every 3 days Tumor volume of nude mice was measured with vernier calipers in vitro every 1 week,and then the tumor growth curve was drawn After 3 weeks,all nude mice were sacrificed by cervical dislocation and the primary tumor was removed and weighed.4.qRT-PCR was used to detect the expression of SNHG6 and miR-101-3p in tumor tissues of mice.5.Western blot was used to detect the expression of ZEB1 and EMT-related proteins in tumor tissues of mice.Results:1.The tumor growth curve showed that the growth rate of the transplanted tumor in the sh-SNHG6 group was significantly lower than that in the sh-NC group?P<0.01?.The tumor size and weight of the sh-SNHG6group were significantly smaller and lighter than those of the sh-NC group?P<0.05?.Knockdown of SNHG6 expression in vivo can significantly inhibit the growth of xenografts in nude mice and reduce the malignant degree of ESCC.2.qRT-PCR results showed that the expression of SNHG6 in the sh-SNHG6 group was significantly lower than that in the sh-NC group?P<0.05?The expression of miR-101-3p in the sh-SNHG6 group was significantly higher than that in the sh-NC group?P<0.05?.Knockdown of SNHG6 in nude mice can significantly increase the expression of miR-101-3p.3.Western blot results showed that compared with the sh-NC group,the expression of ZEB1 in the sh-SNHG6 group was reduced,the expression of E-cadherin was increased,and the expression of N-cadherin,Snail,and Twist were reduced?all P<0.05?.This is consistent with the biological function of SNHG6 verified by in vitro experiments through miR-101-3p/ZEB1 axis.Knockdown of SNHG6 expression in vivo significantly inhibits ZEB1expression in transplanted tumors and reverses EMT transformation.Conclusions:1.Knockdown of lncRNA SNHG6 expression significantly inhibits tumor growth in vivo.2.LncRNA SNHG6 exerts carcinogenesis in nude mice through miR-101-3p/ZEB1 axis,inducing EMT and promoting the development of esophageal squamous cell carcinoma.