Effect Of Dauricine On Apoptosis Of Colon Cancer Cells By Blocking Pre-miR-21 Shearing

Objective Screening miR-21 natural small molecule inhibitors and elucidating its mechanism of action against colon cancer.Methods(1)Mc-fold/Mc-Sym methods were used to construct the 3D structure of the stem-loop of pre-miR-21.The docking site of pre-miR-21 was used as a binding region for the traditional Chinese medicine.Natural screening of natural compounds in chemical databases.(2)MTT method was used to examine the inhibitory effect of candidate compounds on the proliferation of SW620 and HT29 colon cancer cell lines,and to determine miR-21 natural small molecule inhibitors with potential anti-colon cancer activity.(3)The effect of dauricine on migration of colon cancer cell lines SW620 and HT29 was tested by scratch assay.(4)Flow cytometry was used to detect the effect of dauricine on the cycle and apoptosis of colon cancer cell lines SW620 and HT29.(5)The effect of dauricine on the expression of miR-21 in SW620 and HT29 cell lines was detected by real-time fluorescence quantitative PCR.(6)The expression of PI3 K,PTEN,Akt,P-Akt,Bax,Bcl-2,caspase-3,cleaved-caspase-3,caspase-9 and cleaved-caspase-9 in SW620 and HT29 cell lines were detected by Western Blot.(7)In vivo nude mice tumor-bearing experiments,different doses of dauricine were compared in SW620 and HT29 cell lines in nude mice in vivo growth.(8)Real-time fluorescence quantitative PCR detected the effect of dauricine on the expression of miR-21 in tumor.(9)The expression of PI3 K,PTEN,Akt,P-Akt,Bax,Bcl-2,caspase-3,cleaved-caspase-3,caspase-9 and cleaved-caspase-9 in SW620 and HT29 cell lines were detected by Western Blot.Results(1)By molecular docking,14 candidate compounds that bind to the cleavage site of Dicer enzyme in the stem-loop of pre-miR-21 were obtained.(2)Using MTT assay,seven compounds with strong inhibition of the proliferation of SW620 and HT29 colon cancer cells were obtained,among which dauricine had the strongest inhibitory effect and showed a certain time-effect relationship and dose-effect relationship.(3)Dauricine can inhibit the migration of SW620 and HT29,induce the apoptosis of colon cancer cells through a G1 phase blocking effect,and show a dose-effect relationship.(4)Compared with the blank control group,dauricine can reduce the expression of miR-21 in colon cancer cells SW620 and HT29,and then weaken the inhibitory effect of miR-21 on its target gene PTEN,increase the expression of PTEN protein,thereby down-regulate PI3 K,AKT,p-AKT and Bcl-2 protein expression,up-regulation of Bax,Cleaved Caspase-3 and Cleaved Caspase-9 protein expression.(5)Dauricine can significantly inhibit the growth of tumors in nude mice bearing subcutaneous tumors of SW620 and HT29 colon cancer,and shows a dose-effect relationship.(6)Dauricine can reduce the expression of miR-21 in tumor tissues and increase the expression of PTEN,which in turn down-regulates the expression of PI3 K,AKT,p-AKT,and Bcl-2,and up-regulates the expression of Bax,Cleaved Caspase-3,and Cleaved Caspase-9.Conclusion Dalivacaine can induce apoptosis of colon cancer cells,and its mechanism may be related to inhibiting the cleavage of the stem-loop structure of pre-miR-21 by Dicer enzyme and regulating the PTEN/PI3K/AKT pathway.It may be used as a novel therapeutic agent for colon cancer.