| Objective Screening several lncRNA differentially expressed in human pancreatic cancer cells after treatment of oridonin.Studying the effect of oridonin on the expression of lncRNA gene in pancreatic cancer cells.Investigating the effect of oridonin and siRNA AFAP1-AS1 on the apoptosis,cycle and EMT of pancreatic cancer cells.Investigating the molecular mechanism of oridonin anti-tumor with siRNA AFAP1-AS 1 and providing possible pathways and theoretical basis for the treatment of pancreatic cancer.Methods 1.BxPC-3 and PANC-1 cells were cultivated sterilely and the IC50 of oridonin in BxPC-3 and PANC-1 cells were obtained by CCK-8 array(for 24h).2.The expression of lncRNA,which was overexpressed in pancreatic cancer,was detected by Real-time Quantitative PCR after treatment with oridonin.The expression of lncRNA AFAP1-AS1 was significantly down-regulated in BxPC-3 and PANC-1 cell treated with oridonin.lncRNA AFAP1-AS 1 was selected as the study object.3.The BxPC-3 and PANC-1 cells were transfected with interferon RNA(siAFAP1-AS1)and its negative control(siNC).Four experimental groups were set up:siNC,siAFAPl-AS1,siNCori,siAFAP1-ASlori.The apoptosis and cycle of pancreatic cancer cells were detected by flow cytometry.Apoptosis of pancreatic cancer cells was also detected by Hoechst 33258 staining.The effects of oridonin and lncRNA AFAP1-AS1 on the migration of pancreatic cancer cells were detected by Transwell migration assay and RTCA.Western blot was used to detect the expression of related proteins in pancreatic cancer cells.4.The pancreatic cancer BxPC-3 and PANC-1 cells were transfected with the lentiviral vector targeting AFAP1-AS1 to construct the cell lines and control cell lines with stable knockdown of AFAP1-AS1.The effects of oridonin and shAFAP1-ASl on pancreatic cancer in vivo were observed by tumorigenesis assay in nude miceResults 1.CCK-8 results showed that oridonin could inhibit the proliferation of BxPC-3 and PANC-1 cells in a dose-dependent manner with IC50 of 96.109 μM and 97.324 μM.2.The results of Q-PCR showed that lncRNA AFAP1-AS1 was significantly down-regulated in the pancreatic cancer BxPC-3 and PANC-1 cells treated with oridonin in lncRNA overexpressed in pancreatic cancer.3.SiAFAP1-AS1 and its negative control siNC were transiently transfected successfully.The fluorescence results showed that the transfection efficiency was>85%.The results of Q-PCR showed that the expression of lncRNA AFAP1-AS1 in siAFAP 1-AS1 group was significantly lower than that in siNC group,the difference was statistically significant.4.Hoechst fluorescence staining showed that both siNC and siAFAP1-AS1 showed homogeneous and weak fluorescence.While the cells of siNCori and siAFAP1-ASlori were densely dense and strongly blue,and the strong blue fluorescence cells of siAFAP1-ASlori group were more than siNCori group.5.The results of flow cytometry showed that AFAP1-AS1 had little effect on the cell cycle and apoptosis of pancreatic cancer BxPC-3 and PANC-1 cells,and the apoptotic rate and G2/M phase cells were significantly increased in siAFAP1-ASlori group compared with siNCori group.6.Western Blot showed that the expression of Bcl-2,CDK1 and Bax in BxPC-3 and PANC-1 cells were not significantly affected by knockdown of AFAP1-AS1,and the expression of Bcl-2 and CDK1 was significantly down-regulated and Bax was significantly up-regulated in siAFAP 1-AS lori group compared with siNCori group.7.Transwell migration assay and RTCA showed that knockdown of AFAP1-AS1 significantly inhibited the migration of BxPC-3 and PANC-1 cells,and the inhibitory effect of siAFAP1-AS1ori group on cell migration was more obvious.8.Western Blot showed that the expression of Fibronectin,ZEB1,N-cadherin,MMP9,MMP2,Slug and Snail was down-regulated and the expression of E-cadherin was up-regulated in BxPC-3 and PANC-1 cells after knockdown of AFAP1-AS1.The expression of Fibronectin,ZEB1,N-cadherin,MMP9,MMP2,Slug and Snail in the siAFAP1-AS1ori group was significantly lower and the expression of E-cadherin was significantly higher than that in the siNCori group.9.Animal experiments show that knockdown of AFAP1-AS1 followed by oridonin treatment can significantly inhibit tumor growth.Conclusion 1.Oridonin could inhibit the proliferation of BxPC-3 and PANC-1 cells and down-regulat the expressions of lncRNA AFAP1-AS 1.2.Knockdown of AFAP1-AS1 had little effect on cell cycle and apoptosis of pancreatic cancer.Knockdown of AFAP1-AS1 enhanced the ability of oridonin to induce apoptosis and cell cycle arrest in pancreatic cancer cells BxPC-3 and PANC-1.3.Knockdown of AFAP1-AS1 and oridonin treatment could inhibit the EMT of pancreatic cancer cells BxPC-3 and PANC-1,and the combination of both could enhance the inhibitory effect.4.knockdown of AFAP1-AS1 and oridonin treatment can inhibit the growth of pancreatic tumors and the combination of both can enhance the inhibition in nude mice. |
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