摘要
乙型肝炎病毒(Hepatitis B virus,HBV)感染是威胁人类健康,可能会直接或间接地导致多种肝外病变,并且通过某些机制引起的肾炎。为了探讨miR-146b-5p在乙型肝炎病毒相关性肾炎诊断中的应用价值及其在乙型肝炎病毒相关性肾小球肾炎(Hepatitis B virus-associated glomerulonephritis,HBV-GN)发生中的机制研究,本研究选取在医院通过肾穿刺活组织检查明确诊断为HBV-GN患者及人肾小管上皮细胞HK-2为研究对象,通过CCK-8实验分析细胞活性,通过流式细胞术和Annexin V-FITC/PI双染检测细胞凋亡并计算凋亡率,通过qRT-PCR检测miR-146b-5p和丙酮酸脱氢酶B(Pyruvate dehydrogenase beta,PDHB)mRNA表达情况,通过Western Blot检测凋亡相关蛋白(Bcl-2、Bax、cleaved-caspase3)和PDHB蛋白的表达。结果显示,与对照组患者相比较,HBV-GN患者组织中miR-146b-5p表达量增加。在细胞中转染正常对照(Non-specific control,NC)抑制剂、miR-146b-5p抑制剂后,与NC组相比较,转染miR-146b-5p inhibitor能够促进细胞增殖,抑制细胞凋亡,且凋亡相关蛋白的差异具有统计学意义(P<0.05)。通过生物信息学分析显示PDHB是miR-146b-5p靶基因,并通过双荧光素酶报告实验证实miR-146b-5p可以直接靶向PDHB;通过qRT-PCR和Western Blot证实miR-146b-5p能够靶向抑制PDHB表达,从而得出结论:miR-146b-5p可通过靶向PDHB在乙型肝炎病毒相关性肾炎中发挥抑制细胞增殖并促进细胞凋亡的作用。本研究首次揭示了miR-146b-5p在乙型肝炎病毒相关性肾炎中的临床意义及致病的机制。HBV感染的HK-2细胞与乙型肝炎病毒相关性肾炎临床样本中的miR-146b-5p表达量显著升高。细胞中miR-146b-5p抑制能够促进细胞增殖,抑制细胞凋亡。通过对靶基因的分析及双荧光素报告实验验证,得出miR-146b-5p通过靶向PDHB发挥其调节功能,促进病变细胞凋亡,进而导致HBV-GN。
Hepatitis B virus(HBV)infection is a threat to human health,and may directly or indirectly cause various extrahepatic diseases and nephritis.In order to explore the application value of mRNA(miR)-146 b-5 p in the diagnosis of HBV-related nephritis,and study the mechanism of action of miR-146 b-5 p in the occurrence of HBV-associated glomerulonephritis(HBV-GN).Patients diagnosed with HBV-GN confirmed by renal biopsy in the hospital and human renal tubular epithelial cells(HK-2)were selected as the research objects.Cell activity was analyzed by cell counting kit-8 experiments;flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide were used.Double-staining was employed to detect apoptosis and calculate the apoptosis index.Expression of miR-146 b-5 p and pyruvate dehydrogenase beta(PDHB)mRNA was measured by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).Levels of apoptosis-related proteins(Bcl-2,Bax,cleaved-caspase3)and PDHB protein were measured by Western Blot.Results showed that:compared with the control group,miR-146 b-5 p expression was increased in the tissues of HBV-GN patients.After transfection of an NC inhibitor and miR-146 b-5 p inhibitor in cells,compared with the NC group,transfection of the miR-146 b-5 p inhibitor could promote cell proliferation and inhibit apoptosis,and differences in levels of apoptosis-related proteins were significant.Bioinformatics analysis showed that PDHB was a target gene of miR-146 b-5 p,and that miR-146 b-5 p could be targeted directly by PDHB according to dualluciferase assays;qRT-PCR and Western Blot confirmed that miR-146 b-5 p could target PDHB expression.Then we revealed,for the first time,the clinical importance and pathogenic mechanism of miR-146 b-5 p in HBV-GN.miR-146 b-5 p expression in HBV-infected HK-2 cells and clinical samples from HBV-GN patients was increased significantly.Inhibition of miR-146 b-5 p expression in cells can promote cell proliferation and inhibit apoptosis.Based on analyses of target genes and duallucife
作者
张玉娟
钱峻
丁晓兰
ZHANG Yujuan;QIAN Jun;DING Xiaolan(Changzhou Second People's Hospital Affiliated to Nanjing Medical University,Changzhou 213003,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2020年第6期1101-1108,共8页
Chinese Journal of Virology
