Study On The Mechanism Of Aloperine Inhibits Colorectal Cancer Cell Proliferation And Metastasis Progress Via Regulating MiR-296-5p/STAT3 Axis

Objective:To observe the effect of Aloperine(ALO)on the biological behavior of colorectal cancer(CRC)cells,and to study the effect of ALO on the expression of mir-296-5p and STAT3 in colorectal cancer,so as to further explore the anti CRC effect of ALO and its potential mechanism.Methods:1.CRC cells were processed by different concentrations of ALO,and subsequently the cell proliferation was detected by MTT assay.2.qRT-PCR was used to detect the expression of mir-296-5p in CRC cells treated with different concentrations of ALO.3.Target genes based on miRNA sequences were predicted by targetscan v7.2,and the predicted results were verified by dual luciferase reporter assay.4.miR-296-5p mimics,STAT3 overexpression plasmid,mir-296-5p inhibitor and si STAT3 were transfected or co-transfected at room temperature and treated with ALO.qRT-PCR was used to detect the expression of mir-296-5p in HCT116 cells and SW480 cells after different transfection schemes.Western blot was used to detect the expression of STAT3 in HCT116 cells and SW480 cells after different transfection schemes.5.The proliferation of HCT116 and SW480 cells treated with ALO,which transfected or co-transfrcted was detected by cell clone formation assay,the apoptosis was detected by flow cytometry,and the apoptosis related proteins were detected by Western blot.6.Wound healing assay was used to detect the migration of HCT116 and SW480 cells after ALO treatment,and Transwell invasion test was used to test the invasion of HCT116 and SW480 cells.7.Western blot was used to detect the expression of EMT related proteins N-cadherin(n-cad)and E-cadherin(E-cad).Results:1.Using MTT assay,the results showed that ALO had different degrees of inhibition on the proliferation of different colorectal cancer cells,and the inhibition was dose-dependent.The inhibitory effect on SW480 cells was the strongest,but the inhibitory effect on HCT116 cells was the weakest.The IC50 of SW480 cells was 0.611 mmol/L,while that of HCT116 cells was 1.141 mmol/L.2.By starbasev3.0 analysis,miR-296-5p was compared between colorectal adenocarcinoma and normal samples,and the low expression of mir-296-5p in colorectal adenocarcinoma was confirmed.qRT-PCR was used to verify that the expression of mir-296-5p in colorectal cancer cell lines(SW480,HCT116,HT29,SW620)was lower than that in human intestinal epithelial cell line(HIEC),and the expression of miR-296-5p was the highest in SW480 cells and the lowest in HCT116 cells.SW480 cells and HCT116 cells were selected as representatives.After 24 hours of treatment with ALO,the expression of mir-296-5p was detected.The results showed that ALO could up regulate the expression of miR-296-5p in SW480 cells and HCT116 cells in a dose-dependent manner.3.Targetscan v7.2 was used to predict the target gene of miR-296-5p.The results showed that STAT3 was the target gene and the target site was located in 3'-UTR.The 3'UTR luciferase expression vector of wild-type(STAT3-WT)and mutant(STAT3-MUT,)gene target STAT3 was constructed.HCT116 cells with the lowest expression of miR-296-5p were co-transfected with miR-296-5p mimic,and SW480 cells with the highest expression of mir-296-5p were co-transfected with miR-296-5p inhibitor.Dual-luciferase reporter assay showed that compared with the cells co-transfected with STAT3-MUT,the luciferase activity of HCT116 cells co-transfected with miR-296-5p mimic and STAT3-WT was significantly inhibited,while the luciferase activity of SW480 cells co-transfected with miR-296-5p inhibitor and STAT3-WT was enhanced,which proved that STAT3 was the target gene of mir-296-5p.4.MiR-296-5p mimics and ALO inhibited the expression of STAT3 in colorectal cancer cells,but miR-296-5p inhibitor enhanced the expression of STAT3.Up-regulation of mir-296-5p and ALO inhibited the expression of STAT3.5.Up-regulation of mir-296-5p and ALO inhibited the proliferation and bcl-2 expression of colorectal cancer cells,but promoted apoptosis and Bax expression,and these effects were reversed by overexpression of STAT3.The results showed that ALO regulated the proliferation and apoptosis of colorectal cancer cells by regulating miR-296-5p/STAT3 pathway.6.Up-regulation of miR-296-5p and ALO can inhibit the migration and invasion of colorectal cancer cells,and can be reversed by overexpression of STAT3.These results indicate that ALO regulates the migration and invasion of colorectal cancer cells by regulating mir-296-5p/STAT3 pathway.7.Up-regulation of miR-296-5p and ALO inhibits the expression of N-cadherin in CRC cells,but promotes the expression of E-cadherin.These effects can be reversed by overexpression of STAT3.The results show that alo regulates the expression of EMT related proteins by regulating mir-296-5p/STAT3 pathway.Conclusion:ALO inhibited CRC cell proliferation in a dose-dependent manner.MiR-296-5p was low-expressed in CRC tissues and cells,and ALO promoted miR-296-5p expression.STAT3 was targeted by miR-296-5p.Up-regulation of miR-296-5p and ALO treatment both suppressed STAT3 expression.ALO can regulate the proliferation,apoptosis,migration,invasion and EMT of CRC by regulating miR-296-5p/STAT3 pathway.