Study Of Mechanism Of The Effect Of Emodin On The Inhibition EMT Of Pancreatic Cancer By Elevated MiRNA-1271

Pancreatic carcinoma is a high malignant digestive system tumor with a high mortality rate,nearly 60% of pancreatic cancer patients died of liver metastases.As none of the existing treatments against pancreatic carcinoma including surgeries and chemoradiotherapy,are ideal,current studies on pancreatic carcinoma focus on how to effectively cure pancreatic carcinoma and inhibit its metastasis.Extensive studies have shown that multiple mi RNAs are closely related to tumorigenesis and tumor progression.miRNA-1271 has an extensive tumor-suppression effect by inhibiting EMT in tumor cells and induce tumor cell apoptosis.However,reports on its influence and regulatory effect on pancreatic cancer cells are rare.Traditional Chinese Medicine has been a crucial part of the comprehensive treatment against pancreatic carcinoma.Studies have shown that emodin,an active ingredient found in many herbs,has a therapeutic action against various malignant tumors including pancreatic carcinoma,but studies on the mechanism through which emodin inhibit pancreatic metastasis are yet to be reported.Proceeding with the EMT regulatory mechanism of pancreatic carcinoma,this experiment was intended to study the inhibiting effect of miRNA-1271 and emodin against invasion and metastasis of pancreatic carcinoma.The experiment was carried out in three parts.Part 1 Objective: To verify the influence of miRNA-1271 on EMT in pancreatic tumor cells.Methods: miRNA-1271 mimics,mi RNA-1271 inhibitor and negative control were respectively transfected to pancreatic tumor cells and a blank control group was set up.Protein variations of EMT-related phenotypic markers(E-cadherin,ZEB1 and TWIST1)in pancreatic tumor cells were measured through Western-blot;mRNA level of miRNA-1271,E-cadherin,ZEB1 and TWIST1 in pancreatic tumor cells from experimental groups were measured through RT-PCR;Transwell cell model was used to detect the invasive ability of pancreatic tumor cells in experimental groups.Results: In the experimental group where mi RNA-1271 mimics group,mRNA expression level of miRNA-1271 and E-cadherin was elevated compared to that of other experimental groups(P<0.05),while there was no significant difference in mRNA levels of ZEB1 and TWIST1 among all experimental groups(P>0.05).Protein expression level of E-cadherin in miRNA-1271 mimics group was higher while protein expression levels of ZEB1 and TWIST1 were lower than those of other experimental groups(P<0.05).In the experimental group where miRNA-1271 inhibitor was transfected,protein expression level of E-cadherin was the lowest among all experimental groups while the protein expression levels of ZEB1 and TWIST1 were significantly higher than those in other experimental groups(P<0.05).In Transwell cell model experiment,invasive ability of pancreatic tumor cells from miRNA-1271 mimics group significantly decreased compared to the results of other experimental groups(P<0.05).Conclusion: Results have shown that miRNA-1271 can significantly inhibit EMT in pancreatic tumor cells.Part 2 Objective: To observe the variation of mi RNA-1271 level and of EMT phenotypes in processed cells by processing pancreatic tumor cells with emodin of different concentration.Methods: Pancreatic tumor cells were processed with emodin of high and low concentration,then a control group was set up.Protein variations of EMT-related phenotypic markers(E-cadherin,ZEB1 and TWIST1)in pancreatic tumor cells were measured through Western-blot;mRNA level of mi RNA-1271,E-cadherin,ZEB1 and TWIST1 found in pancreatic tumor cells in experimental groups were measured through RT-PCR;Transwell cell model was used to measure the invasive ability of pancreatic tumor cells in both experimental groups.Results: In experimental groups where pancreatic tumor cells were processed with emodin,mRNA expression level of miRNA-1271 and E-cadherin was elevated compared to the results of the control group,while there was no difference in mRNA levels of ZEB1 and TWIST1 among experimental groups(P<0.05).Protein expression level of E-cadherin in emodin groups was higher while protein expression levels of ZEB1 and TWIST1 was lower than those of the control group(P<0.05).In Transwell cell model experiment,invasive ability of pancreatic tumor cells in emodin groups had significantly decreased compared to that of the control group(P<0.05).In the experimental group where high-concentration emodin were used,a stronger inhibiting effect against EMT in pancreatic tumor cells was demonstrated compared to the other experimental group(P<0.05).Conclusion: Experimental results have shown that emodin inhibits EMT in pancreatic tumor cells by raising the level of miRNA-1271 in them.Part 3 Objective: To verify the inhibiting effect of emodin against liver metastasis of pancreatic cancer in vivo by setting up a liver metastatic model with an implantation of pancreatic tumor tissue into the spleens of nude mice.Methods: Animal models of pancreatic cancer with liver metastasis were set up by injection of pancreatic tumor cells into spleens of 45 nude mice.Three groups of 15 nude mice were set up: high-dose emodin group(daily gavage of emodin,50mg/Kg),low-dose emodin group(daily gavage of emodin,20mg/Kg)and a control group.The mice were sacrificed in 6 weeks and the quantity of tumor nodes of hepatic metastasis,growth inhibition rate of liver metastasis and inhibition ratio against metastasis in all nude mice were measured.m RNA level of miRNA-1271,E-cadherin,ZEB1 and TWIST1 found in liver metastasis tissue in experimental groups were measured through RT-PCR.Protein expression levels of E-cadherin,ZEB1 and Twist1 in tumor tissue of liver metastasis was measured through Western-blot and IHC methods.Results: Both high-dose emodin group and low-dose emodin group excelled the control group in indicators including the quantity of tumor nodes of hepatic metastasis,growth inhibition rate of liver metastasis and inhibition ratio against metastasis in nude mice(P<0.05).Results of Western-blot and IHC tests showed a significantly elevated protein expression level of E-cadherin and a significant lowered protein expression level of ZEB1 and Twist1 in tumor tissue of liver metastasis compared to those of the control group(P<0.05).mRNA expression level of miRNA-1271 and E-cadherin in emodin groups was elevated compared to the results of control group,and there was no significant difference in mRNA levels of ZEB1 and TWIST1 among experimental groups(P>0.05).Conclusion: In vivo experiments have verified the inhibiting effect of emodin against liver metastasis of pancreatic cancer by inhibiting EMT in pancreatic tumor cells.