Chinese Journal of Tissue Engineering Research ›› 2022, Vol. 26 ›› Issue (30): 4773-4779.doi: 10.12307/2022.756
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Lin Bo, Chen Xinyu, Jin Qiu, Zhu Zhiman, Zhao Wenhui
Received:
2021-09-17
Accepted:
2021-11-03
Online:
2022-10-28
Published:
2022-03-29
Contact:
Zhao Wenhui, PhD, Associate professor, Basic Medical Department, Jiangsu Vocational College of Nursing, Huaian 223005, Jiangsu Province, China
About author:
Lin Bo, Master, Lecturer, Basic Medical Department, Jiangsu Vocational College of Nursing, Huaian 223005, Jiangsu Province, China
Supported by:
CLC Number:
Lin Bo, Chen Xinyu, Jin Qiu, Zhu Zhiman, Zhao Wenhui. Effects of miR-126-3p from adipose-derived mesenchymal stem cell released exosomes on human umbilical vein endothelial cell glucolipotoxicity[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(30): 4773-4779.
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2.5 MiR-126-3p的靶基因分析 利用生物信息学软件预测miR-126-3p靶基因。通过4种软件分析预测结果的交集作为共同靶基因,共有11种基因(DIP2C、PTPN9、SPRED1、CRK、RNF165、KANK2、PIK3R2、ITGA6、PLXNB2、ADAM9和TSC1)符合,占总量0.2%,见图5A。采用RT-qPCR分析11种共同靶基因在miR-126-3p过表达细胞模型中有差异表达的靶基因,结果表明,只有CRK mRNA相对表达水平降低,差异有显著性意义(P < 0.001),见图5B。MiR-126-3p能够与野生型CRK的3’非编码区结合,见图5C。双荧光素酶基因报告实验结果表明,与对照组相比,miR-126-3p能够负向调控野生型CRK基因的荧光素酶活性(P < 0.001),而对突变型没有影响,见图5D,并对靶基因CRK相关信号通路进行预测,最终选取AKT信号通路进行后续研究。"
2.6 预测并验证 miR-126-3p作用机制 为验证miR-126-3p改善HUVECs糖脂毒性的可能作用机制,构建miR-126-3p靶基因CRK过表达转染HUVECs糖脂毒性细胞模型,Western blot检测凋亡相关蛋白,结果表明,CRK过表达能够显著提高细胞凋亡相关蛋白Cleaved caspase 3,9的表达量,表明CRK基因大量异常表达可促进细胞凋亡,见图6A。p-AKT蛋白磷酸化水平显著提高(P < 0.01),证实AKT途径能够被CRK激活,见图6B。使用AKT途径抑制剂RG7440与miR-126-3p共处理糖脂毒性HUVECs,结果表明,与对照组相比,miR-126-3p可显著降低Cleaved caspase 3,9蛋白的表达量(P < 0.001),但与miR-126-3p过表达组相比,RG7440与miR-126-3p共处理对Cleaved caspase 3,9的表达水平没有明显作用,见图6C。与外泌体组相比,RG7440与外泌体共处理能够显著提高细胞凋亡的能力(P < 0.01),见图6D。"
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