Based On The Effect Of MiR-21 On The EMT Of Pancreatic Stellate Cells, We Explored The Mechanism Of Cinocobalamin Improving The Microenvironment Of Pancreatic Cancer

ObjectivesTo determine whether cinobufacin can interfere with the EMT process of activated pancreatic stellate cells through miR-21 regulation of TGB?/Smad signaling pathways,thereby affecting the pancreatic cancer microenvironment and exploring its specific mechanisms.MethodsCultured human pancreatic stellate cells(PSC).after 48 h of combined treatment with different concentrations of toadine,gemcitabine and two drugs,the inhibition rate of PSC was detected by CCK-8 method.the PSC activation model was prepared by TGF? stimulation to observe cell morphology within the living cell dynamic imaging system.toadine was used as the drug intervention and named as TGF?1 group,toadolin TGF?1 group,toadolin group,toadolin group,to miR-21 high expression group,to miR-21 inhibitor group,to idling control group and to blank control group.Transfection of miR-21 mimics?miR-21 inhibitor?mimics NC.with Lipo 2000 reagent qRT-PCR?western blot were used to determine the gene and protein expression levels of miR-21?TGF?/Smad pathway molecules and EMT molecules in each group.To explore the specific mechanism of the miR-21 regulation of the EMT phenotype of pancreatic stellate cells in the microenvironment of pancreatic cancerResults(1)CCK-8 method was used to detect the inhibition rate of PSC in each group after treatment for 48 hours.The 50%inhibitory concentration(IC50)of-cinobufagin on PSC was 28.405 mg/ml,and the IC50 of gemcitabine on PSC was 1.24 ?g/ml.The IC50 of cinobufacin combined with gemcitabine on PSC was 4.71 mg/ml+gemcitabine 1 ?g/ml.The inhibitory effect of cinobufacin combined with gemcitabine on PSC was concentration-dependent.The higher the concentration of cinobufacin,the more obvious the inhibition;(2)After 48 hours of TGF?1 treatment,the morphology of the PSC changed significantly compared with that before dosing.The volume of static PSC,cells without TGF?1 stimulation is small?showing typical round-like and polygonal,and the volume of PSC,cells after TGF?1 stimulation increases,showing the morphology of long spindle-shaped myofibroblasts,indicating that PSC activation occurs;(3)The relative expression of miR-21 in each group was detected by qRT-PCR method.The relative expression of miR-21 decreased with the increase of dr?g concentration at 15 mg/ml?30 mg/ml?60 mg/ml,and the inhibition of-miR-21 was most obvious at 60 mg/ml;(4)The miR-21 expression was increased after activation with TGF-?1 stimulation PSC,and the relative expression of miR-21 decreased after the addition of cinobufacin in the group.The expression of PSC,miR-21 after transfection was significantly increased and the expression of miR-21 after transfection was significantly inhibited;(5)The expression levels of EMT related genes and proteins were detected by qRT-PCR and WB methods.the expression of epithelial marker E-cadherin was downregulated after TGF-?1 activation and miR-21 mimic transfection,while the expression of interstitial marker N-cadherin increased;the results were opposite after use of miR-21 inhibitor.The combined use of vasopressin increased E-cadherin in activated PSC and decreased N-cadherin expression;(6)The gene and protein expression levels of key molecules in TGF-?/Smad pathways were detected by qRT-PCR and WB methods.the gene and protein expression of PSC,Smad2/3 were significantly increased after TGF-?1 activation and transfection;the gene and protein expression of Smad2/3 was decreased after treatment with miR-21 inhibitors.