miR-146a在HBV慢性感染中的機制研究及抑制肝癌作用
發(fā)布時間：2018-11-27 08:40
【摘要】：目的:研究miR-146a在HBV慢性感染中的免疫調節(jié)機制和對肝癌的作用。方法:1.提取HepG2.2.15肝癌細胞基因組DNA作為模板,PCR擴增miR-146a前體序列,雙酶切后連接到pmR-mCherry質粒,構建pmiR-146a真核表達載體,選用菌落PC.R、雙酶切初步鑒定,最終送公司測序鑒定。2.選用lip2000轉染pmiR-146a真核表達載體到HepG2.2.15細胞設為實驗組(lip2000+pmiR-146a),同時設空質粒組(lip2000+pmmR-mCherry)、空白組(lip2000)、knock down 組(簡稱 KD 組,lip2000+microFFTM has-miR-146a inhibitor)。細胞轉染24、48h后在熒光顯微鏡下觀察熒光蛋白的表達,qPCR檢測各組細胞miR-146a的表達量。3.細胞轉染后,分別于24、48h檢測各組細胞上清中HBsAg和HBeAg變化,轉染48h后Western blot檢測NF-κB蛋白表達量及ELISA法檢測細胞上清中TGF-β分泌量。4.細胞轉染后,分別于24、48h檢測各組細胞內c-Myc mRNA表達水平;48h后檢測c-Myc蛋白表達量;24、48、72h采用CCK-8法檢測細胞增殖情況。結果:1.經雙酶切、菌落PCR和DNA測序最終驗證,pre-miR-146a序列成功插入pmmR-mcherry載體中,表明pmiR-146a真核表達載體構建成功。2.細胞轉染24、48h后,熒光顯微鏡下觀察,實驗組和空質粒組可見強熒光,與非熒光條件下作對比,轉染效率在50%-600%之間;qPCR結果表明,與空白組相比,實驗組miR-146a表達量升高,KD組表達量下降,二者均具有統(tǒng)計學意義(P0.01),而空白組與空質粒組比較無統(tǒng)計學差異(P0.05)。3.細胞轉染后,化學發(fā)光法檢測細胞上清中HBeAg與HBsAg分泌量:與空白組比較,實驗組表達量均升高,KD組表達量下降,具有統(tǒng)計學意義(P0.05),而空白組與空質粒組比較無統(tǒng)計學差異(PO.05);Western blot檢測細胞內NF-κB表達量:與空白組比較,實驗組表達量下降,KD組表達量升高,具有統(tǒng)計學意義(P0.05),而空白組與空質粒組比較無統(tǒng)計學差異(PO.05);ELISA檢測細胞上清中TGF-β分泌量:與空白組對比,實驗組表達量升高,KD組表達量下降,具有統(tǒng)計學意義(P0.01),而空白組與空質粒組比較無統(tǒng)計學差異(P0.05)。4.細胞轉染后,qPCR檢測c-Myc mRNA表達量:與空白組比較,實驗組表達量下降,KD組表達量升高,具有統(tǒng)計學意義(P0.05),而空白組與空質粒組比較無統(tǒng)計學差異(PO.05);Western blot檢測細胞內c-Myc蛋白表達量:與空白組比較,實驗組表達量下降,KD組表達量升高,具有統(tǒng)計學意義(P0.05),而空白組與空質粒組比較無統(tǒng)計學差異(P0.05);CCK-8檢測細胞增殖:與空白組對比,實驗組細胞增殖能力降低,KD組細胞增殖能力升高,具有統(tǒng)計學意義(P0.01),而空白組與空質粒組比較無統(tǒng)計學差異(P0.05)。結論:miR-146a可在HBV慢性感染中促進病毒復制及表達,其作用機制是通過下調細胞內NF-κB的表達,同時上調TGF-β的表達,抑制細胞免疫應答,促進HBsAg、HBeAg的表達。miR-146a可以通過抑制c-Myc基因的表達,從而抑制肝癌細胞的增殖,起到抗腫瘤的作用。其可作為治療原發(fā)性肝癌的潛在靶點。
[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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62;R735.7
，
本文編號：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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62;R735.7
，
本文編號：2360081
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