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中华实验和临床感染病杂志(电子版) ›› 2021, Vol. 15 ›› Issue (02) : 78 -85. doi: 10.3877/cma.j.issn.1674-1358.2021.02.002

所属专题： 文献；

论著

致癌相关环状RNA在慢性乙型肝炎病毒感染者的表达及临床意义
钱舒然1, 谭婧文1, 王萌婕1, 张思航1, 杨镇英1, 何越峰1, 李晖1,†()   
  1. 1. 650021　昆明市，云南大学附属医院/云南省第二人民医院肝病科；650500　昆明市，昆明医科大学
  • 收稿日期:2020-06-09 出版日期:2021-04-15
  • 通信作者: 李晖
  • 基金资助:
    云南省医疗卫生单位内设研究机构科研项目(No. 2018NS0004)

Expression and clinical significance of carcinoma-related circular RNA in patients with chronic hepatitis B virus infection

Shuran Qian1, Jingwen Tan1, Mengjie Wang1, Sihang Zhang1, Zhenying Yang1, Yuefeng He1, Hui Li1,†()   

  1. 1. Department of Hepatology, Affiliated Hospital of Yunnan University/The Second People’s Hospital of Yunnan Province, Kunming 650021, China; Kunming Medical University, Kunming 650500, China
  • Received:2020-06-09 Published:2021-04-15
  • Corresponding author: Hui Li
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引用本文：

钱舒然, 谭婧文, 王萌婕, 张思航, 杨镇英, 何越峰, 李晖. 致癌相关环状RNA在慢性乙型肝炎病毒感染者的表达及临床意义[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(02): 78-85.

Shuran Qian, Jingwen Tan, Mengjie Wang, Sihang Zhang, Zhenying Yang, Yuefeng He, Hui Li. Expression and clinical significance of carcinoma-related circular RNA in patients with chronic hepatitis B virus infection[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2021, 15(02): 78-85.

目的

探讨致癌相关环状RNA（circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1以及circ-MORC3）在慢性乙型肝炎病毒（HBV）感染者的表达变化及临床意义。

方法

选择云南大学附属医院/云南省第二人民医院2019年5月至12月住院和门诊收治的100例HBV感染者，其中慢性乙型肝炎患者50例（CHB组），HBV携带者50例（HBV携带组），另选择同期50例体检者为健康对照组。采用逆转录-实时荧光定量PCR（qRT-PCR）法检测入组研究对象外周血白细胞内circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1和circ-MORC3相对表达水平。采用Spearman相关分析检验circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1、circ-MORC3与乙型肝炎病毒表面抗原（HBsAg）浓度、HBV DNA水平的相关性，并应用ROC曲线评估上述环状RNA对于区分CHB患者和HBV携带者的诊断价值。

结果

CHB组、HBV携带者组和健康对照组患者中circ-ASXL1（H = 11.93、P = 0.003）、circ-BRIP1（H = 13.24、P = 0.001）、circ-CFLAR（H = 20.89、P < 0.001）、circ-LPAR1（H = 19.33、P < 0.001）和circ-MORC3（H = 14.81、P = 0.001）表达差异均具有统计学意义。与健康对照组相比，CHB组患者circ-ASXL1（Z =-3.26、P = 0.001）、circ-BRIP1（Z =-3.52、P < 0.001）、circ-CFLAR（Z =-4.23、P < 0.001）、circ-LPAR1（Z =-4.03、P < 0.001）和circ-MORC3（Z = -3.76、P < 0.001）表达水平显著升高，差异有统计学意义；而HBV携带者组的表达与健康对照组差异无统计学意义（P均> 0.05）。CHB患者和HBV携带者的HBsAg浓度与circ-ASXL1（r = 0.261、P = 0.009）、circ-BRIP1（r = 0.287、P = 0.004）、circ-CFLAR（r = 0.211、P = 0.037）、circ-LPAR1（r = 0.213、P = 0.035）和circ-MORC3（r = 0.237、P = 0.019）的表达水平均呈正相关，而HBV DNA水平与circ-ASXL1（r = 0.080、P = 0.430）、circ-BRIP1（r = 0.076、P = 0.454）、circ-CFLAR（r = 0.084、P = 0.406）、circ-LPAR1（r = 0.020、P = 0.846）和circ-MORC3（r = 0.070、P = 0.492）的表达水平无相关性。circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1和circ-MORC3区分CHB患者和健康人群的ROC曲线下面积分别为0.69（95%CI：0.59～0.79）、0.70（95%CI：0.60～0.81）、0.75（95%CI：0.65～0.84）、0.73（95%CI：0.64～0.83）和0.72（95%CI：0.62～0.82）。circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1和circ-MORC3区分CHB患者和HBV携带者的ROC曲线下面积分别为0.64（95%CI：0.53～0.75）、0.64（95% CI：0.53～0.75）、0.70（95%CI：0.60～0.80）、0.70（95%CI：0.60～0.80）和0.64（95%CI：0.50～0.75）。circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1和circ-MORC3区分HBV携带者和健康人群的ROC曲线下面积分别为0.57（95%CI：0.46～0.68）、0.58（95%CI：0.47～0.69）、0.57（95%CI：0.46～0.68）、0.56（95%CI：0.44～0.70）和0.59（95%CI：0.48～0.70）。

结论

circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1和circ-MORC3可能与CHB的致病调控相关，可作为生物学标记物区分CHB患者、HBV携带者和健康人群。

关键词: 肝炎, 乙型, 慢性, 环状RNA, 免疫调控
Objective

To investigate the differential expression of circular RNA, such as circ-ASXL1, circ-BRIP1, circ-CFLAR, circ-LPAR1 and circ-MORC3 in patients with chronic hepatitis B virus (HBV) infection and their clinical significance.

Methods

Total of 100 patients with HBV infection in Affiliated Hospital of Yunnan University/The Second People’s Hospital of Yunnan Province from May to December 2019 were selected, including 50 patients of chronic hepatitis B (CHB), 50 HBV carriers and 50 healthy controls. Expressions of circ-ASXL1, circ-BRIP1, circ-CFLAR, circ-LPAR1 and circ-MORC3 in peripheral blood leukocytes of all subjects were detected by fluorescence real-time quantitative RT-PCR (qRT-PCR). The correlation between HBV surface antigen (HBsAg) concentration and HBV DNA level and expressions of circ-ASXL1, circ-BRIP1, circ-CFLAR, circ-LPAR1 and circ-MORC3 were analyzed by Spearman correlation analysis. The diagnostic value of these circular RNA in distinguishing between CHB patients and HBV carriers were evaluated by receiver operating characteristic (ROC) curve.

Results

The expressions of circ-ASXL1 (H = 11.93, P = 0.003), circ-BRIP1 (H = 13.24, P = 0.001), circ-CFLAR (H = 20.89, P < 0.001), circ-LPAR1 (H = 19.33, P < 0.001) and circ-MORC3 (H = 14.81, P = 0.001) between patients of CHB group, HBV carriers group and control group were significantly different. The expressions of circ-ASXL1 (Z =-3.26, P = 0.001), circ-BRIP1 (Z =-3.52, P < 0.001), circ-CFLAR (Z =-4.23, P < 0.001), circ-LPAR1 (Z =-4.03, P < 0.001) and circ-MORC3 (Z =-3.76, P < 0.001) in CHB group were significantly higher than those in control group, while there were no significant difference between HBV carriers group and control group (all P > 0.05). The HBsAg concentration had significantly positive relationship with expression levels of circ-ASXL1 (r = 0.261, P = 0.009), circ-BRIP1 (r = 0.287, P = 0.004), circ-CFLAR (r = 0.211, P = 0.037), circ-LPAR1 (r = 0.213, P = 0.035), circ-MORC3 (r = 0.237, P = 0.019) in CHB group and HBV carriers group, but there were no significant correlation between HBV DNA level and expression levels of circ-ASXL1 (r = 0.080, P = 0.430), circ-BRIP1 (r = 0.076, P = 0.454), circ-CFLAR (r = 0.084, P = 0.406), circ-LPAR1 (r = 0.020, P = 0.846), circ-MORC3 (r = 0.070, P = 0.492). The area under the ROC curve of these circRNAs for distinguishing patients of CHB from control were 0.69 (95%CI: 0.59-0.79) for circ-ASXL1; 0.70 (95%CI: 0.60-0.81) for circ-BRIP1; 0.75 (95%CI: 0.65-0.84) for circ-CFLAR, 0.73 (95%CI: 0.64-0.83) for circ-LPAR1; 0.72 (95%CI: 0.62-0.82) for circ-MORC3. The area under the ROC curve of these circRNAs for distinguishing patients of CHB from HBV carriers were 0.64 (95%CI: 0.53-0.75) for circ-ASXL1, 0.64 (95%CI: 0.53-0.75) for circ-BRIP1, 0.70 (95%CI: 0.60-0.80) for circ-CFLAR, 0.70 (95%CI: 0.60-0.80) for circ-LPAR1, 0.64 (95%CI: 0.50-0.75) for circ-MORC3. The area under the ROC curve of these circRNAs for distinguishing patients of HBV carriers from control were 0.57 (95%CI: 0.46-0.68) for circ-ASXL1, 0.58 (95%CI: 0.47-0.69) for circ-BRIP1, 0.57 (95%CI: 0.46-0.680) for circ-CFLAR, 0.56 (95%CI: 0.44-0.70) for circ-LPAR1, 0.59 (95%CI: 0.48-0.70) for circ-MORC3.

Conclusions

circ-ASXL1, circ-BRIP1, circ-CFLAR, circ-LPAR1 and circ-MORC3 may be involved in the pathogenic regulation of CHB and could be used as biological markers to distinguish patients of CHB, HBV carriers and healthy controls.

Key words: Chronic hepatitis B, Circular ribonucleic acid, Immunomodulation
表1 circ-ASXL1、circ-BRIP1、circ-CFLAR、circ-LPAR1和circ-MORC3引物序列
引物 引物序列（5'→3'）
circ-ASXL1 上游：GCATGCCTCAATGCTATGCT
  下游：GGGGAAGTCCCACTTCTCAT
circ-BRIP1 上游：TCTGTGTGCCAGACTGTGAG
  下游：ACACCAAGTTCTGACGAAAAGG
circ-CFLAR 上游：TGGACTTCATAGCCCAGGGA
  下游：GAGCAAGCCCCTAGGAATCTG
circ-LPAR1 上游：AGGTGTCAGTCCTGAGAAGTCA
  下游：GTAGTTCTGGGGCGTGTTCA
circ-MORC3 上游：CATCCTACGTGGACAGAAAGTGAA
  下游：CTGTTCCGTGGAAAACAGAGAAT
β-actin 上游：CCTGTACGCCAACACAGTGC
  下游：ATACTCCTGCTTGCTGATCC
表2 5种环状RNA在各组研究对象白细胞中的表达[M（P25，P75）]
组别 例数 circ-ASXL1 circ-BRIP1 circ-CFLAR circ-LPAR1 circ-MORC3
CHB组 50 1.77（0.80，5.27） 2.12（0.85，6.29） 2.81（1.01，5.20） 3.17（0.99，5.89） 2.21（0.92，4.89）
HBV携带者组 50 1.08（0.64，2.21） 1.22（0.66，2.48） 1.07（0.72，2.17） 1.20（0.65，2.06） 1.28（0.67，2.43）
健康对照组 50 1.00（0.56，1.47） 0.97（0.48，1.79） 0.99（0.56，1.56） 1.03（0.59，1.77） 0.96（0.48，1.64）
H值   11.93 13.24 20.89 19.33 14.81
P值   0.003 0.001 < 0.001 < 0.001 0.001
图1 5种circRNA与HBsAg浓度相关性分析
图2 5种circRNA与HBV DNA水平相关性分析
表3 5种环状RNA与HBV DNA、HBsAg相关性分析
指标 circ-ASXL1 circ-BRIP1 circ-CFLAR circ-LPAR1 circ-MORC3
r值 P值 r值 P值 r值 P值 r值 P值 r值 P值
HBV DNA 0.080 0.430 0.076 0.454 0.084 0.406 0.020 0.846 0.070 0.492
HBsAg 0.261 0.009 0.287 0.004 0.211 0.037 0.213 0.035 0.237 0.019
图3 5种circRNA区分CHB患者和健康人群的ROC曲线
表4 5种环状RNA的诊断效能
组别 circ-ASXL1 circ-BRIP1 circ-CFLAR circ-LPAR1 circ-MORC3
CHB组vs.健康对照组          
  AUC（95%CI） 0.69（0.59～0.79） 0.70（0.60～0.81） 0.75（0.65～0.84） 0.73（0.64～0.83） 0.72（0.62～0.82）
  灵敏度（%） 48.0 44.0 58.0 48.0 52.0
  特异度（%） 90.0 94.0 88.0 88.0 94.0
  阳性预测值（%） 82.8 88.0 82.9 88.9 80.8
  阴性预测值（%） 63.4 62.7 67.7 64.4 60.8
CHB组vs. HBV携带者组          
  AUC（95%CI） 0.64（0.53～0.75） 0.64（0.53～0.75） 0.70（0.60～0.80） 0.70（0.60～0.80） 0.64（0.50～0.75）
  灵敏度（%） 44.0 48.0 58.0 50.0 40.0
  特异度（%） 86.0 76.0 78.0 90.0 86.0
  阳性预测值（%） 74.2 66.7 72.5 83.3 70.1
  阴性预测值（%） 60.9 59.4 65.8 64.2 60.0
HBV携带者组vs.健康对照组          
  AUC（95%CI） 0.57（0.46～0.68） 0.58（0.47～0.69） 0.57（0.46～0.68） 0.56（0.46～0.70） 0.59（0.48～0.70）
  灵敏度（%） 38.0 40.0 78.0 68.0 32.0
  特异度（%） 82.0 78.0 40.0 48.0 88.0
  阳性预测值（%） 70.4 64.5 56.5 57.6 76.2
  阴性预测值（%） 57.5 56.5 64.5 61.0 57.0
图4 5种circRNA区分CHB患者和HBV携带者的ROC曲线
图5 5种circRNA区分HBV携带者和健康人群的ROC曲线
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