Effect Of Oridonin/miR-200b-3p-mediated EMT In Human Pancreatic Cancer Cells

Objective To research the effects of oridonin via a miRNA-dependent mechanism,miRNA expression profiling of oridonin-treated BxPC-3 human pancreatic cancer cells was performed using a miRNA microarray assay.Further study the relationship among oridonin,miR-200b-3p and pancreatic cancer cells on EMT to look for the molecular mechanism or signaling pathways on the migration in pancreatic cancer cells.Exploring the anti-cancer.mechanism of oridonin/miR-200b-3p on pancreatic cancer cells provide not only the possible anti-cancer accesses but also the clinical data for treatment.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.MicroRNA expression profiling of oridonin-treated BxPC-3 cells was performed using a miRNA microarray assay,and miRNA target prediction,Gene Ontology and KEGG pathway analysis were performed to investigate possible pathways involved.3.MicroRNA expression profiling was validated by using real-time PCR.miR-200b-3p was selected for the further research and its expression was verified in PANC-1 cells,whose target gene is predicted.4.BxPC-3 and PANC-1 cells were treated with oridonin or transfected by miR-200b-3p mimics or inhibitor respectively.Those cells were used for Western Blot assay to verified the effects of oridonin or miR-200b-3p on EMT.5.BxPC-3 and PANC-1 cells were treated with oridonin or transfected by miR-200b-3p Lentiviral Vector or Lentiviral Vector NC respectively.Those cells were used for Transwell assay to verified the effects of oridonin or miR-200b-3p on migration.6.The effects of oridonin on cell adhesion molecules ICAM-1 and VCAM-1 in BxPC-3 and PANC-1 cells were measured by ELISA.7.The effects of oridonin on cytoskeleton in PANC-1 cells were observed by immunofluorescence staining8.The effects of oridonin/miR-200b-3p on pancreatic cancer in vivo were observed by tumorigenesis assay in nude mice.Results 1.CCK-8 array showed that oridonin inhibited the proliferation of BxPC-3 and PANC-1 cells in a manner as dose-dependent,the IC50 of oridonin in BxPC-3 and PANC-1 cells were 87.8 μM and 55.8 μM respectively(24 hours).2.Compared with control group,microarray analysis of miRNA expression in BxPC-3 cells treated with oridonin showed that 49 miRNAs were significantly down-regulated,whereas 56 were significantly up-regulated by oridonin(signal reading>500,p≤0.01,|Log2-valueb|≥1).3.The RT-PCR results showed that miR-409-3p was upregulated 2.04 times,miR-103a-3p was downregulated 1.85 times,miR-200b-3p was downregulated 2.22 times and miR-107 was downregulated 2.13 times in the oridonin treatment group compared with the control,which correlated well with the microarray results.ZEB1 was a predicted target gene for miR-200b-3p.The miR-200b-3p was downregulated 2.39 times in the oridonin treatment group compared with the control in PANC-1 cells.4.Compared with control group,results showed that oridonin could down-regulate the expressions of ZEB1,E-cadherin,N-cadherin and fibronectin significantly(P<0.05);miR-200b-3p could up-regulate the expressions of E-cadherin and down-regulate the expressions of ZEB1,N-cadherin and fibronectin significantly(P<0.05)in BxPC-3 and PANC-1 cells by Western Blot.5.The Transwell array showed that oridonin or overexpression of miR-200b-3p inhibit cell migration in BxPC-3 and PANC-1 cells compared with the control.6.The ELISA analysis showed that the cell adhesion molecules ICAM-1 and VCAM-1 were decreased by oridonin in BxPC-3 and PANC-1 cells compared with the control.7.The cytoskeleton in PANC-1 cells were altered by oridonin compared with the control.8.The tumorigenesis assay in nude mice showed that oridonin or overexpression of miR-200b-3p inhibit the tumor growth.Conclusion 1.Oridonin could inhibit the proliferation and migration of BxPC-3 and PANC-1 cells,down-regulat the expressions of miR-200b-3p.2.miR-200b-3p could inhibit the EMT of BxPC-3 and PANC-1 cells by targeted ZEB1.3.Oridonin had a certain effect on the migration in BxPC-3 and PANC-1 cells,but not thoughⅢ-EMT by miR-200-3p/ZEB1 axis,may be related to other miRNAs,Ⅱ-EMT,tumor microenvironment or altering the cytoskeleton.4.Oridonin/miR-200b-3p inhibit the pancreatic cancer growth in nude mice.