| Objective: Colon cancer is the third most common malignancy in the world and the fourth leading cause of cancer-related deaths worldwide.However,the detailed mechanism of colon cancer development is currently not fully understood.Therefore,exploring the pathogenesis of colon cancer is still necessary for its early diagnosis and treatment.NFX1-type zinc finger containing protein 1 antisense RNA 1(ZFAS1)is a lncRNA located on chromosome 20q13.13.The lncRNA ZFAS1 is upregulated in colon cancer,and its high expression is associated with TNM staging,lymphatic infiltration,and poor prognosis in colon cancer patients,which can be used as a prognostic biomarker and potential therapeutic target for colon cancer.The the specific mechanism of its participation in regulating the development of colon cancer needs to be further analyzed.In summary,this study aims to investigate the impact of lncRNA ZFAS1 on the progression of colon cancer.In addition,lncRNA often acts as a competitive endogenous RNA(ce RNA)to regulate the expression of specific micro RNAs(miRNAs)and their downstream target genes.In this study,we also screened the miR-1271-5p/METTL1/ZEB1 ce RNA axis that related to lncRNA ZFAS1,and further validated the impact and mechanism of lncRNA ZFAS1 on colon cancer progression through the miR-1271-5p/METTL1/ZEB1 axis.This study will provide a theoretical basis for lncRNA ZFAS1 as a potential target for the diagnosis and treatment of colon cancer.Methods: Section 1: The highly expressed lncRNAs in colon cancer were screened by GSE126092 dataset and bioinformatics online database.The expression of lncRNA ZFAS1 in colon cancer tissues and cells was detected by q RT-PCR.The knockdown lncRNA ZFAS1 lentivirus were transfected into the colon cancer cells,and the transfection efficiency was verified by q RT-PCR.CCK-8,Ed U and plate clonal formation assay were carried out to analysis the effect of knockdown lncRNA ZFAS1 on the proliferation of colon cancer cells.Flow cytometry was used to detect the effect of knockdown lncRNA ZFAS1 on the apoptosis of colon cancer cells.Transwell migration and invasion assay were used to verify the effects of knockdown lncRNA ZFAS1 on migration and invasion of colon cancer cells.Western blot and immunofluorescence were performed to analysis the effect of knockdown lncRNA ZFAS1 on EMT-related protein expression in colon cancer cells The knockdown lncRNA ZFAS1 colon cancer cells were injected subcutaneously into the nude mice to observe the size,volume and weight of the transplanted tumors.The expression of proliferative marker Ki-67 was detected by immunohistochemistry,and the expression of EMT-related proteins in the transplanted tumors was detected by western blot and immunofluorescence.miR-1271-5p targeting binding to lncRNA ZFAS1 was predicted by bioinformatics and validated by double luciferase reporter gene experiments and AGO2-RIP.Then q RT-PCR was performed to detect the effect of knockdown lncRNA ZFAS1 on the expression of miR-1271-5p in colon cancer cells,as well as the expression of which in colon cancer tissues.At last,the correlation between lncRNA ZFAS1 and miR-1271-5p expression in colon cancer tissues was analyzed by Pearson correlation analysis.Colon cancer cells were transfected with miR-1271-5p mimetic or inhibitor,and the transfection efficiency was verified by q RT-PCR.The effects of miR-1271-5p mimetic or inhibitor on the proliferation,migration and invasion of colon cancer cells and the expression of EMT-related proteins were detected by CCK-8,Transwell and Western blot.The miR-1271-5p inhibitor was transfected with colon cancer cells that knocked down lncRNA ZFAS1 and verified the transfection efficiency by q RT-PCR CCK-8,Ed U and plate clonal formation assays were performed to detect the effect of transfected miR-1271-5p inhibitors on the proliferation of colon cancer cells with knockdown lncRNA ZFAS1.Flow cytometry was used to detect the effect of transfected miR-1271-5p inhibitors on the apoptosis of colon cancer cells with knockdown lncRNA ZFAS1.Transwell migration assay and invasion assay were used to detect the effect of transfection of miR-1271-5p inhibitors on the migration and invasion of colon cancer cells with knockout lncRNA ZFAS1.Western blot and immunofluorescence were used to detect the effect of miR-1271-5p inhibitors on the expression of EMT-related proteins in colon cancer cells with lncRNA ZFAS1 knockdown.After the antagomiR-1271-5p was injected into colon cancer cells to inhibit the expression of miR-1271-5p in tumorbearing mice with knockout lncRNA ZFAS1,the size,volume and weight of the transplanted tumor was observed,and the expression of Ki-67,a proliferative marker in the transplanted tumor,was detected by immunohistochemistry.Emt-related proteins were detected by western blot and immunofluorescence.Section 2: METTL1 and ZEB1 m RNAs that bind to miR-1271-5p were predicted by bioinformatics,and verified by dual luciferase reporter and AGO2-RIP assays.The effects of miR-1271-5p mimicry and inhibitors on the expression of METTL1 and ZEB1 in colon cancer cells were detected by q RT-PCR and western blot.RIP experiments with m7 G antibodies detected the effect of overexpression METTL1 on ZEB1 m RNA m7 G modification.The effect of overexpression of METTL1 on the expression of ZEB1 in colon cancer cells was detected by q RT-PCR and western blot.q RT-PCR and western blot assays were also used to detect the effect of miR-1271-5p inhibitor transfected on the expression of METTL1 and ZEB1 in colon cancer cells with lncRNA ZFAS1 knockdown,as well as the effect of METTL1 overexpression on ZEB1 expression of lncRNA ZFAS1 knockout colon cancer cells.The effect of overexpression of ZEB1 on the proliferation of colon cancer cells with knockdown lncRNA ZFAS1 was detected by CCK-8,Ed U and plate clonal formation assays.And flow cytometry was performed to analysis the effect of overexpression of ZEB1 on apoptosis of lncRNA ZFAS1 knockout colon cancer cells.The effect of overexpression of ZEB1 on the migration and invasion of lncRNA ZFAS1 knockdown colon cancer cells was examined by Transwell migration and invasion assays.Western blot and immunofluorescence were used to detect the effect of overexpression of ZEB1 on the expression of EMT-related proteins in colon cancer cells with lncRNA ZFAS1 knockdown.The effect of overexpression of ZEB1 on the tumor growth of colon cancer cells with knockdown lncRNA ZFAS1 was detected by establishing a nude mouse transplanted tumor model.The size,volume and weight of the transplanted tumor were monitored.The expression of Ki-67,a proliferative marker in the transplanted tumor,was detected by immunohistochemistry.Emt-related proteins were detected by western blot and immunofluorescence.Results: Section 1: LncRNA ZFAS1 is highly expressed in both colon cancer tissues and cells.Knocking down of lncRNA ZFAS1 reduced the expression of lncRNA ZFAS1 in colon cancer cells,inhibited cell proliferation,migration,invasion and promoted apoptosis,and downregulated the expression of N-cadherin,Vimentin,Snail and Slug proteins in cells.The knockdown of lncRNA ZFAS1 also decreased the tumor volume and mass of colon cancer cells,down-regulated the expression of Ki-67 and decreased the expressions of N-cadherin,Vimentin,Snail and Slug proteins.lncRNA ZFAS1 binds with miR-1271-5p,and has potential binding sites.Knockdown of lncRNA ZFAS1 increased the expression of miR-1271-5p in colon cancer cells.miR-1271-5p was low expressed in colon cancer tissues and cells,and which is negatively correlated with the lncRNA ZFAS1 in colon cancer tissues.Transfection of miR-1271-5p mimetic increased the expression of miR-1271-5p in colon cancer cells,inhibited cell proliferation,migration and invasion,and down-regulated the expression of N-cadherin,Vimentin,Snail and Slug.On the contrary,transfection of miR-1271-5p inhibitor decreased the expression of miR-1271-5p in colon cancer cells,promoted cell proliferation,migration and invasion,and up-regulated the expression of N-cadherin,Vimentin,Snail and Slug.Transfection with miR-1271-5p inhibitors decreased the expression of miR-1271-5p in colon cancer knockdown lncRNA ZFAS1,promoted cell proliferation,migration,invasion,inhibited cell apoptosis,and up-regulated the expression of N-cadherin,Vimentin,Snail and Slug proteins.Transfection of antagomiR-1271-5p increased the tumor volume and mass of colon cancer cell transplanted tumors with knockdown lncRNA ZFAS1,up-regulated the expression of Ki-67,and increased the expression of N-cadherin,Vimentin,Snail and Slug proteins in transplanted tumors.Section 2: The miR-1271-5p was targeted binding to METTL1 and ZEB1 m RNAs,and the 3’-UTR of METTL1 and ZEB1 m RNAs contained potential binding sites of miR-1271-5p.Transfection with miR-1271-5p mimicers decreased the expression of METTL1 and ZEB1 m RNAs in colon cancer cells,while transfection with miR-1271-5p inhibitors increased METTL1 and ZEB1 m RNAs expression.Overexpression of METTL1 increased ZEB1 m RNA and m7 G modification,and increased the expression of ZEB1 protein in colon cancer cells,but showed non effects on the ZEB1 m RNA expression.Transfection with miR-1271-5p inhibitor increased the expression of METTL1 and ZEB1 in colon cancer cells with knockdown lncRNA ZFAS1.Overexpression of ZEB1 decreased the expression of miR-1271-5p in colon cancer with knockdown lncRNA ZFAS1,promoted cell proliferation,migration and invasion,inhibited cell apoptosis,and upregulated the expression of N-cadherin,Vimentin,Snail and Slug proteins.Overexpression of ZEB1 also increased the tumor volume and weight of colon cancer cell transplant tumors with knockdown lncRNA ZFAS1,upregulated the expression of Ki-67,and increased the expressions of N-cadherin,Vimentin,Snail and Slug proteins.Conclusion: LncRNA ZFAS1 is highly expressed in colon cancer,and it promotes the proliferation,migration,invasion and EMT of colon cancer cells in vitro and increases tumor growth in vivo,and inhibits cell apoptosis by up-regulating the expression of target genes of METTL1 and ZEB1 by sponging miR-1271-5p.Mettl1-mediated m7 G modification promotes the translation and up-regulates the expression of ZEB1. |
