miR-146a在HBV慢性感染中的机制研究及抑制肝癌作用
发布时间：2018-11-27 08:40
【摘要】：目的:研究miR-146a在HBV慢性感染中的免疫调节机制和对肝癌的作用。方法:1.提取HepG2.2.15肝癌细胞基因组DNA作为模板,PCR扩增miR-146a前体序列,双酶切后连接到pmR-mCherry质粒,构建pmiR-146a真核表达载体,选用菌落PC.R、双酶切初步鉴定,最终送公司测序鉴定。2.选用lip2000转染pmiR-146a真核表达载体到HepG2.2.15细胞设为实验组(lip2000+pmiR-146a),同时设空质粒组(lip2000+pmmR-mCherry)、空白组(lip2000)、knock down 组(简称 KD 组,lip2000+microFFTM has-miR-146a inhibitor)。细胞转染24、48h后在荧光显微镜下观察荧光蛋白的表达,qPCR检测各组细胞miR-146a的表达量。3.细胞转染后,分别于24、48h检测各组细胞上清中HBsAg和HBeAg变化,转染48h后Western blot检测NF-κB蛋白表达量及ELISA法检测细胞上清中TGF-β分泌量。4.细胞转染后,分别于24、48h检测各组细胞内c-Myc mRNA表达水平;48h后检测c-Myc蛋白表达量;24、48、72h采用CCK-8法检测细胞增殖情况。结果:1.经双酶切、菌落PCR和DNA测序最终验证,pre-miR-146a序列成功插入pmmR-mcherry载体中,表明pmiR-146a真核表达载体构建成功。2.细胞转染24、48h后,荧光显微镜下观察,实验组和空质粒组可见强荧光,与非荧光条件下作对比,转染效率在50%-600%之间;qPCR结果表明,与空白组相比,实验组miR-146a表达量升高,KD组表达量下降,二者均具有统计学意义(P0.01),而空白组与空质粒组比较无统计学差异(P0.05)。3.细胞转染后,化学发光法检测细胞上清中HBeAg与HBsAg分泌量:与空白组比较,实验组表达量均升高,KD组表达量下降,具有统计学意义(P0.05),而空白组与空质粒组比较无统计学差异(PO.05);Western blot检测细胞内NF-κB表达量:与空白组比较,实验组表达量下降,KD组表达量升高,具有统计学意义(P0.05),而空白组与空质粒组比较无统计学差异(PO.05);ELISA检测细胞上清中TGF-β分泌量:与空白组对比,实验组表达量升高,KD组表达量下降,具有统计学意义(P0.01),而空白组与空质粒组比较无统计学差异(P0.05)。4.细胞转染后,qPCR检测c-Myc mRNA表达量:与空白组比较,实验组表达量下降,KD组表达量升高,具有统计学意义(P0.05),而空白组与空质粒组比较无统计学差异(PO.05);Western blot检测细胞内c-Myc蛋白表达量:与空白组比较,实验组表达量下降,KD组表达量升高,具有统计学意义(P0.05),而空白组与空质粒组比较无统计学差异(P0.05);CCK-8检测细胞增殖:与空白组对比,实验组细胞增殖能力降低,KD组细胞增殖能力升高,具有统计学意义(P0.01),而空白组与空质粒组比较无统计学差异(P0.05)。结论:miR-146a可在HBV慢性感染中促进病毒复制及表达,其作用机制是通过下调细胞内NF-κB的表达,同时上调TGF-β的表达,抑制细胞免疫应答,促进HBsAg、HBeAg的表达。miR-146a可以通过抑制c-Myc基因的表达,从而抑制肝癌细胞的增殖,起到抗肿瘤的作用。其可作为治疗原发性肝癌的潜在靶点。
[Abstract]:Objective: to study the immunomodulatory mechanism of miR-146a in chronic HBV infection and its effect on hepatocellular carcinoma. Methods: 1. Genomic DNA of HepG2.2.15 hepatoma cells was extracted as template. MiR-146a precursor sequence was amplified by PCR and ligated to pmR-mCherry plasmid after double enzyme digestion. The eukaryotic expression vector of pmiR-146a was constructed and identified by double enzyme digestion of colony PC.R,. Finally sent to the company sequencing identification. 2. Lip2000 transfection of pmiR-146a eukaryotic expression vector into HepG2.2.15 cells was selected as experimental group (lip2000 pmiR-146a), empty plasmid group (lip2000 pmmR-mCherry) and blank group (lip2000), knock down group, KD group, lip2000 microFFTM has-miR-146a inhibitor). Group). The expression of fluorescent protein was observed under fluorescence microscope 48 h after transfection, and the expression of miR-146a was detected by qPCR. After transfection, the changes of HBsAg and HBeAg in the supernatant of each group were detected at 24 h after transfection, the expression of NF- 魏 B protein by Western blot and the secretion of TGF- 尾 in the supernatant by ELISA assay at 48 h after transfection. After transfection, the expression level of c-Myc mRNA was detected at 24: 48h, the expression of c-Myc protein was detected after 48h, and the proliferation of cells was detected by CCK-8 assay for 72 hours. Results: 1. After double enzyme digestion, the colony PCR and DNA sequencing proved that the pre-miR-146a sequence was successfully inserted into the pmmR-mcherry vector, indicating that the pmiR-146a eukaryotic expression vector was successfully constructed. 2. After transfection for 48 h, the transfection efficiency was between 50% and 600% in the experimental group and the blank plasmid group. QPCR results showed that compared with the blank group, the expression of miR-146a increased in the experimental group, and decreased in the KD group (P0.01), but there was no significant difference between the blank group and the blank plasmid group (P0.05). After transfection, chemiluminescence assay was used to detect the secretion of HBeAg and HBsAg in the supernatant. Compared with the blank group, the expression of HBeAg and HBsAg in the experimental group was higher than that in the control group, and the expression level in the KD group was decreased (P0.05). There was no significant difference (PO.05) between blank group and blank plasmid group. The expression of NF- 魏 B was detected by Western blot: compared with the blank group, the expression of NF- 魏 B decreased in the experimental group and increased in the KD group (P0.05), but there was no significant difference between the blank group and the blank plasmid group (PO.05). ELISA detection of TGF- 尾 secretion in the supernatant: compared with the blank group, the expression of TGF- 尾 increased in the experimental group, decreased in the KD group (P0.01), but there was no significant difference between the blank group and the blank plasmid group (P0.05). After transfection, qPCR was used to detect the expression of c-Myc mRNA: compared with the blank group, the expression of c-Myc mRNA in the experimental group was decreased, and the expression in the KD group was increased (P0.05), but there was no significant difference between the blank group and the blank plasmid group (PO.05). Western blot detection of intracellular c-Myc protein expression: compared with the blank group, the expression of the experimental group decreased, the expression of KD group increased, with statistical significance (P0.05), but there was no significant difference between the blank group and the blank plasmid group (P0.05). CCK-8 detection of cell proliferation: compared with the blank group, the experimental group cell proliferation ability decreased, KD group cell proliferation ability increased, with statistical significance (P0.01), but blank group and empty plasmid group had no statistical difference (P0.05). Conclusion: miR-146a can promote viral replication and expression in chronic HBV infection by down-regulating the expression of NF- 魏 B, up-regulating the expression of TGF- 尾, inhibiting cellular immune response and promoting HBsAg,. The expression of HBeAg. MiR-146a can inhibit the proliferation of hepatoma cells by inhibiting the expression of c-Myc gene. It can be used as a potential target for the treatment of primary liver cancer.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62;R735.7
，
本文编号：2360081
[Abstract]:Objective: to study the immunomodulatory mechanism of miR-146a in chronic HBV infection and its effect on hepatocellular carcinoma. Methods: 1. Genomic DNA of HepG2.2.15 hepatoma cells was extracted as template. MiR-146a precursor sequence was amplified by PCR and ligated to pmR-mCherry plasmid after double enzyme digestion. The eukaryotic expression vector of pmiR-146a was constructed and identified by double enzyme digestion of colony PC.R,. Finally sent to the company sequencing identification. 2. Lip2000 transfection of pmiR-146a eukaryotic expression vector into HepG2.2.15 cells was selected as experimental group (lip2000 pmiR-146a), empty plasmid group (lip2000 pmmR-mCherry) and blank group (lip2000), knock down group, KD group, lip2000 microFFTM has-miR-146a inhibitor). Group). The expression of fluorescent protein was observed under fluorescence microscope 48 h after transfection, and the expression of miR-146a was detected by qPCR. After transfection, the changes of HBsAg and HBeAg in the supernatant of each group were detected at 24 h after transfection, the expression of NF- 魏 B protein by Western blot and the secretion of TGF- 尾 in the supernatant by ELISA assay at 48 h after transfection. After transfection, the expression level of c-Myc mRNA was detected at 24: 48h, the expression of c-Myc protein was detected after 48h, and the proliferation of cells was detected by CCK-8 assay for 72 hours. Results: 1. After double enzyme digestion, the colony PCR and DNA sequencing proved that the pre-miR-146a sequence was successfully inserted into the pmmR-mcherry vector, indicating that the pmiR-146a eukaryotic expression vector was successfully constructed. 2. After transfection for 48 h, the transfection efficiency was between 50% and 600% in the experimental group and the blank plasmid group. QPCR results showed that compared with the blank group, the expression of miR-146a increased in the experimental group, and decreased in the KD group (P0.01), but there was no significant difference between the blank group and the blank plasmid group (P0.05). After transfection, chemiluminescence assay was used to detect the secretion of HBeAg and HBsAg in the supernatant. Compared with the blank group, the expression of HBeAg and HBsAg in the experimental group was higher than that in the control group, and the expression level in the KD group was decreased (P0.05). There was no significant difference (PO.05) between blank group and blank plasmid group. The expression of NF- 魏 B was detected by Western blot: compared with the blank group, the expression of NF- 魏 B decreased in the experimental group and increased in the KD group (P0.05), but there was no significant difference between the blank group and the blank plasmid group (PO.05). ELISA detection of TGF- 尾 secretion in the supernatant: compared with the blank group, the expression of TGF- 尾 increased in the experimental group, decreased in the KD group (P0.01), but there was no significant difference between the blank group and the blank plasmid group (P0.05). After transfection, qPCR was used to detect the expression of c-Myc mRNA: compared with the blank group, the expression of c-Myc mRNA in the experimental group was decreased, and the expression in the KD group was increased (P0.05), but there was no significant difference between the blank group and the blank plasmid group (PO.05). Western blot detection of intracellular c-Myc protein expression: compared with the blank group, the expression of the experimental group decreased, the expression of KD group increased, with statistical significance (P0.05), but there was no significant difference between the blank group and the blank plasmid group (P0.05). CCK-8 detection of cell proliferation: compared with the blank group, the experimental group cell proliferation ability decreased, KD group cell proliferation ability increased, with statistical significance (P0.01), but blank group and empty plasmid group had no statistical difference (P0.05). Conclusion: miR-146a can promote viral replication and expression in chronic HBV infection by down-regulating the expression of NF- 魏 B, up-regulating the expression of TGF- 尾, inhibiting cellular immune response and promoting HBsAg,. The expression of HBeAg. MiR-146a can inhibit the proliferation of hepatoma cells by inhibiting the expression of c-Myc gene. It can be used as a potential target for the treatment of primary liver cancer.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62;R735.7
，
本文编号：2360081
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