Chi-miR-182对NTS和TES基因及子宫内膜上皮细胞调控作用的研究

发布时间：2018-03-04 22:28

本文选题：奶山羊　切入点：子宫内膜上皮细胞　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：本研究以奶山羊子宫内膜上皮细胞(EEC)为研究对象,构建双荧光素酶报告载体验证miR-182与NTS和TES的靶向关系,用Real-time PCR、Western Blot等方法研究miR-182对基因NTS、TES和Caspase-8、p38表达量的影响。构建pcDNA3.1(+)过表达载体,研究NTS和TES的相互作用、对Caspase-8和p38蛋白表达量以及对子宫内膜上皮细胞凋亡的影响。主要研究结果如下:1.验证mi R-182与NTS和TES的靶向调节关系生物信息学预测发现奶山羊NTS基因3'UTR存在mi R-182靶位点,TES基因CDS区存在miR-182靶位点。构建双荧光素酶报告载体,分别与miR-182模拟物、Negative control(NC)共转染到293T细胞中。结果表明,与对照组NC相比,miR-182模拟物显著抑制了荧光素酶活性,表明miR-182通过与NTS-3'UTR相互作用导致荧光素酶活性降低(P0.05),与TES基因CDS区相互作用导致荧光素酶活性降低(P0.05)。据此,本研究初步鉴定NTS和TES为miR-182的靶基因。2.验证mi R-182对NTS、TES、Caspase-8、p38表达量的影响采用Real-time PCR和Western Blot技术检测NTS和TES基因的表达水平。结果表明,与对照组相比,miR-182在mRNA和蛋白水平均显著抑制靶基因NTS和TES的表达(P0.05)。采用Western Blot技术发现mi R-182显著增加了Caspase-8的蛋白表达量(P0.05),减少了p38的蛋白表达量(P0.05)。3.检测NTS和TES对Caspase-8、p38表达量的影响构建pcDNA3.1(+)-NTS和pcDNA3.1(+)-TES过表达载体并转染EEC,采用Western Blot技术检测Caspase-8和p38表达量,并与对照组相比。结果显示过表达NTS和TES显著降低了Caspase-8蛋白表达量(P0.05),增加了p38的蛋白表达量(P0.05)。这一结果与miR-182对Caspase-8、p38表达量的影响结果相反。4.验证NTS和TES的相互作用和对EEC凋亡的影响将pcDNA3.1(+)-NTS和pcDNA3.1(+)-TES过表达载体转染EEC,通过Real-time PCR和Western Blot技术检测NTS和TES基因的表达水平。结果发现过表达NTS显著抑制了TES mRNA和蛋白水平表达量(P0.05)。而过表达TES基因只减少了NTS蛋白的表达量(P0.05),对NTS mRNA没有显著影响(P0.05)。用流式细胞仪检测pcDNA3.1(+)-NTS和pcDNA3.1(+)-TES对EEC凋亡的影响,结果表明NTS和TES均显著抑制了EEC凋亡(P0.05)。以上结果表明,NTS和TES为miR-182的靶基因,mi R-182可能通过下调NTS和TES,上调Caspase-8而影响奶山羊子宫内膜上皮细胞的凋亡。
[Abstract]:In this study, a double luciferase report vector was constructed to study the targeting relationship between miR-182 and NTS and TES in dairy goat endometrial epithelial cells (EECs). The effect of miR-182 on the expression of NTSnTES and Caspase-8 p38 was studied by Real-time PCR Western Blot. PcDNA3.1 () overexpression vector was constructed to study the interaction between NTS and TES. The main results are as follows: 1. To verify the targeted regulatory relationship between miR-182 and NTS and TES. Bioinformatics prediction found that the 3UTR of NTS gene of dairy goats exists mi R-182. There is a miR-182 target site in the CDS region of the tes gene. A double luciferase reporter vector was constructed. The results showed that the mimics of nmmiR-182 significantly inhibited luciferase activity in 293T cells compared with the control group (NC). The results showed that the activity of luciferase decreased by interaction of miR-182 with NTS-3'UTR, and the activity of luciferase decreased by interaction with CDS region of TES gene. In this study, NTS and TES were identified as target genes of miR-182. The effect of miR-182 on the expression of Caspase-8 p38 was detected by Real-time PCR and Western Blot techniques. Compared with the control group, pmiR-182 significantly inhibited the expression of target gene NTS and TES at both mRNA and protein levels. Using Western Blot technique, it was found that miR-182 significantly increased the protein expression of Caspase-8 and decreased the protein expression of p38. Detection of NTS and TES on Caspase-8p38 was carried out. PcDNA3.1 (Western Blot technique was used to detect the expression of Caspase-8 and p38). The results showed that overexpression of NTS and TES significantly decreased the expression of Caspase-8 protein, increased the expression of p38 protein and increased the expression of p38 protein. This result was contrary to the effect of miR-182 on the expression of Caspase-8 p38. 4. The interaction between NTS and TES was verified. Real-time PCR and Western Blot were used to detect the expression level of NTS and TES genes. The results showed that overexpression of NTS significantly inhibited the expression of TES mRNA and protein levels (P0.05). The expression of TES gene only reduced the expression of NTS protein (P0.05), but had no significant effect on NTS mRNA. Flow cytometry was used to detect the effect of pcDNA3.1 (pnts) and pcDNA3.1 (P0.05) on the apoptosis of EEC. The results showed that both NTS and TES inhibited EEC apoptosis significantly (P 0.05). The results indicated that the target genes of miR-182, TES and TES, might affect the apoptosis of endometrial epithelial cells of dairy goats by down-regulating NTS and tes and up-regulating Caspase-8.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

【相似文献】