Research On Inhibition Of Endothlial-mesenchymal Transition By Targeting RUNX3

Aim1. To observe TGF-β1 induce endothelial mesenchymal transition and its correlation with the change of Runx3.2. To observe the effect on endothelial mesenchymal transition by the target of Runx3. 3. Investingating the effect and related mechanisms on mesenchymal transition induced by Runx3. Methods1.Vascular endothelial cells were divided into seven different groups: ①Control groups: endothelial cells were cultured in common nutrient solution six days. ②1 ng/ml groups: endothelial cells were cultured in nutrient solution containing 1ng/ml TGF-β1. ③5ng/ml groups : endothelial cells were cultured in nutrient solution containing 5ng/ml TGF-β1. ④10ng/ml groups:endothelial cells were cultured in nutrient solution containing 10ng/ml TGF-β1. ⑤10ng/ml+Runx3-siRNA virus groups:endothelial cells were treated with virus transfecion for 72 hours, then cultured in nutrient solution containing 10 ng/ml TGF-β1.2. The expressions of Runx3 mRNA, CD31 mRNA, α-SMAmRNA and SNAILmRNA were detected by the method of qRT-PCR in endothelial cells of each group. The expressions of Runx3, p-AKT, SNAIL, eNOS were detected by the method of Western blot in endothelial cells of each group and compared. The expression of VEGF was detected by the method of ELISA simultaneously.Results1. The lentivirus vector of Runx3-siRNA were built successfully. The virus transfection efficiency was strongest when infected 72 hours, MOI was 30.2. The expressions of Runx3 mRNA, CD31 mRNA, α-SMAmRNA and SNAILmRNA were detected by the method of qRT-PCR in endothelial cells of each group. The results were showed that the expression of CD31 mRNA down-regulated(P<0.05), α-SMAmRNA up-regulated(P<0.05), SNAILmRNA up-regulated(P<0.05), Runx3 mRNA up-regulated(P<0.01)in endothelial cells in containing 1ng/ml TGF-β1 groups compared with control groups. The expression of CD31 mRNA down-regulated(P<0.05), α-SMAmRNA up-regulated(P<0.05), SNAILmRNA up-regulated(P<0.05), Runx3 mRNA up-regulated(P<0.01) in endothelial cells in containing 5ng/ml TGF-β1 groups compared with control groups. The expression of CD31 mRNA down-regulated( P<0.05), α-SMAmRNA up-regulated( P<0.05), SNAILmRNA up-regulated( P<0.01), Runx3 mRNA up-regulated(P<0.01) in endothelial cells in containing 10ng/ml TGF-β1 groups compared with control groups.The expressions of CD31 mRNA up-regulated(P<0.05), α-SMAmRNA down-regulated(P<0.05), SNAILmRNA down-regulated(P<0.05), Runx3 mRNA down-regulated(P<0.05) in endothelial cells in containing Runx3-siRNA virus accordingly groups compared with containing 10 ng/ml TGF-β1 groups.3. The expression of Runx3, eNOS, SNAIL, p-AKT were detected by the method of Western blot in endothelial cells of each group. The results showed that the expressions of Runx3 up-regulated(P<0.05), SNAIL up-regulated(P<0.05), eNOS down-regulated(P<0.05), p-AKT up-regulated(P<0.05) in endothelial cells in containing 1ng/ml TGF-β1 groups compared with control groups. The expressions of Runx3 up-regulated(P<0.01),SNAIL up-regulated(P<0.05), eNOS down-regulated(P<0.05), p-AKT up-regulated(P<0.05) in endothelial cells in containing 5ng/ml TGF-β1 groups compared with control groups. The expressions of Runx3 up-regulated(P<0.01), SNAIL up-regulated(P<0.01), eNOS down-regulated(P<0.05), p-AKT up-regulated(P<0.05) in endothelial cells in containing 10ng/ml TGF-β1 groups compared with control groups.The expression of Runx3 down-regulated(P<0.01), SNAIL down-regulated(P<0.01), eNOS up-regulated(P<0.01), p-AKT up-regulated(P<0.01) in endothelial cells in containing Runx3- siRNA virus accordingly groups compared with containing 10ng/ml TGF-β1 groups.4. The expression of VEGF was detected by the method of ELISA simultaneously. The results were showed that the expressions of VEGF up-regulated(P<0.05) in endothelial cells in containing 1ng/ml TGF-β1 groups compared with control groups. The expressions of VEGF up-regulated(P<0.05) in endothelial cells in containing 5ng/ml TGF-β1 groups compared with control groups. The expressions of VEGF up-regulated(P<0.05) in endothelial cells in containing 10ng/ml TGF-β1 groups compared with control groups. The expressions of VEGF up-regulated(P<0.05) in endothelial cells in containing Runx3-siRNA virus accordingly groups compared with containing 10ng/ml TGF-β1 groups.. Conclusion1. TGF-β1 can induce endothelial mesenchymal transition, Runx3 was involved in the process.2. Runx3 down-regulation can relief endothelial mesenchymal transition.3. Runx3 down-regulation can reduce the expression of SNAIL, increase the expression of eNOS and VEGF, activate AKT, may participate in the mechanism of relief endothelial mesenchymal transition.