黄鉴涛,胡海浩,马嘉霖,战盈瑾,闫秀英,简纪常

• 1:广东海洋大学水产学院/广东省水产动物病害防控与健康养殖重点实验室/水产经济动物病害控制广东普通高校重点实验室

摘要(Abstract)：

【目的】鉴定草鱼（Ctenopharyngodon idella）miR-218(cid-miR-218)家族成员，分析cid-miR-218在鱼淋巴囊肿病毒中国株（LCDV-cn）感染草鱼卵巢细胞系（Grass carp ovary cells,GCO）过程中的表达特性，探索cid-miR-218-5p对LCDV-cn感染的调控作用。【方法】从LCDV-cn感染的GCO细胞中，通过茎环RT-PCR获取cid-miR-218家族成员，并进行测序验证和生物信息学分析；用定量PCR分析cid-miR-218家族在LCDV-cn感染GCO过程中的表达；用生物信息学方法预测cid-miR-218家族的靶基因，并利用双荧光素酶报告基因系统验证cid-miR-218-5p的靶基因；通过转染cid-miR-218-5p mimic和cid-miR-218-5p inhibitor上调和下调cid-miR-218-5p的表达，用定量PCR分析cid-miR-218-5p、靶基因il-10和LCDV-cn mcp基因的表达情况；应用GO和KEGG数据库，对cid-miR-218家族可能参与调控的信号通路进行富集分析。【结果】在LCDV-cn感染GCO过程中，鉴定到cid-miR-218-5p、cid-miR-218a、cid-miR-218b、cid-miR-218b-1和cid-miR-218b-2等5个cid-miR-218家族成员，其中cid-miR-218-5p的表达呈先升后降再上升的变化趋势，且差异表达最显著（P <0.05）；经生物信息学分析预测，cid-miR-218家族成员有较多共同的潜在靶基因，il-10是共有的靶基因。经验证，pmirGLO-il-10重组质粒与家族成员代表cid-miR-218-5p结合后，其荧光素酶活性降低（P <0.05），证明il-10是cid-miR-218-5p的靶基因。cid-miR-218-5p过表达后，il-10与LCDV-cn mcp表达均显著下降（P <0.05），而抑制cid-miR-218-5p表达后，il-10与LCDV-cn mcp表达则显著上升（P <0.05）。预测表明cid-miR-218家族参与调控病毒感染致瘤相关信号通路。【结论】在LCDV-cn感染GCO过程中，cid-miR-218家族中cid-miR-218-5p起重要调控作用；cid-miR-218-5p负调控其靶基因il-10的表达，并负调控LCDV-cn mcp基因的表达；cid-miR-218-5p和IL-10对鱼淋巴囊肿病诊断和防控有潜在的临床应用价值。

关键词(KeyWords)： 草鱼卵巢细胞系;cid-miR-218家族;鱼淋巴囊肿病毒中国株;靶基因;白介素10

Abstract:

Keywords:

基金项目(Foundation): 广东省科技特派员专项（GDKTP2021029800）;; 国家自然科学基金（31602199）

作者(Author): 黄鉴涛,胡海浩,马嘉霖,战盈瑾,闫秀英,简纪常

参考文献(References)：

• [1]金爱琴,倪红兵,孙宝兰,等. microRNA-218在急性淋巴细胞白血病细胞CCRF-CEM中的作用及机制研究[J].中国免疫学杂志, 2015, 31(1):103-108.
• [2]王涛,马思聪,戚星星,等. microRNA-218下调肝细胞癌中的E2F2基因表达抑制细胞增殖的研究[J].肝脏, 2016,21(3):179-182.
• [3] LI L, YU H, REN Q. miR-218-5p suppresses the progression of retinoblastoma through targeting NACC1 and inhibiting the AKT/mTOR signaling pathway[J]. Cancer Management and Research, 2020, 12:6959-6967.
• [4]齐会娟. miR-218、ROBO1在宫颈癌组织中的表达及与临床病理特征的关系[D].唐山:华北理工大学, 2021.
• [5] LI Z X, QIAN R J, ZHANG J D, et al. miR-218-5p targets LHFPL3 to regulate proliferation, migration, and epithelialmesenchymal transitions of human glioma cells[J]. Bioscience Reports, 2019, 39(3):BSR20180879.
• [6]王园园,吕小红. miR-218-5p通过靶向抑制TDP1表达促进鱼藤酮诱导的胃癌细胞凋亡[J].肿瘤防治研究, 2022,49(1):12-17.
• [7] ZHANG L, ZHANG L, PAN Y, et al. Downregulation of miR-218 by porcine reproductive and respiratory syndrome virus facilitates viral replication via inhibition of type I interferon responses[J]. The Journal of Biological Chemistry,2021, 296:100683.
• [8] QIU Y L, GENG X X, BAN J D, et al. micro RNA-218 inhibits type I interferon production and facilitates virus immune evasion via targeting RIG-I[J]. Biotechnology and Applied Biochemistry, 2020, 67(3):396-403.
• [9] LI Y, LIU J, YUAN C, et al. High-risk human papillomavirus reduces the expression of microRNA-218 in women with cervical intraepithelial neoplasia[J]. The Journal of International Medical Research, 2010, 38(5):1730-1736.
• [10]路尧. miR-218-5p/CDCP1信号轴介导KSHV vIRF1诱导内皮细胞迁移侵袭的研究[D].南京:南京医科大学, 2015.
• [11]闫秀英,吴灶和,简纪常,等.淋巴囊肿病毒中国分离株基因组序列和结构分析[J].广东海洋大学学报, 2011, 31(1):1-6.
• [12] CHENG H B, XIE C, ZHANG R Y, et al. Xeroderma pigmentosum complementation group F polymorphisms influence risk of glioma[J]. Asian Pacific Journal of Cancer Prevention, 2013, 14(7):4083-4087.
• [13] LUO W W, LIANG X, HUANG S Q, et al. Molecular cloning, expression analysis and mi RNA prediction of vascular endothelial growth factor A(VEGFAa and VEGFAb)in pond loach Misgurnus anguillicaudatus, an air-breathing fish[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 2016, 202:39-47.
• [14]胡海浩,黄鉴涛,马嘉霖,等. cid-miR-146a在LCDV-cn感染中的差异表达及其调控作用[J].广东海洋大学学报, 2022, 42(4):31-41.
• [15]周宏专,苏霞,林路路,等.犬细小病毒感染F81细胞microRNA荧光定量PCR内参基因的筛选[J].畜牧与兽医, 2023, 55(3):96-103.
• [16]李迎娣,孙志宾,马爱军,等.双锯鱼(Amphiprion)胚胎不同发育阶段和成鱼不同组织中内参基因的筛选及应用[J].海洋与湖沼, 2022, 53(1):150-160.
• [17] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4):402-408.
• [18] CHEN G, WANG Q H, WANG K Y. microRNA-218-5p affects lung adenocarcinoma progression through targeting endoplasmic reticulum oxidoreductase 1 alpha[J]. Bioengineered, 2022, 13(4):10061-10070.
• [19]王江波,梁皓,陆允平. miR-218-5p靶向EGLN3抑制肺腺癌增殖、迁移和侵袭[J].中国比较医学杂志, 2022, 32(11):57-67.
• [20] PENG P, CHEN T, WANG Q, et al. Decreased miR-218-5p levels as a serum biomarker in bone metastasis of prostate cancer[J]. Oncology Research and Treatment, 2019, 42(4):165-181.
• [21]朱耀魁,程妮,胡颖,等.微RNA在宫颈癌发生发展中作用及其与HPV相互关系[J].生理科学进展, 2012, 43(4):251-256.
• [22] OCADIZ-DELGADO R, LIZCANO-MENESES S, TREJOVAZQUEZ J A, et al. Circulating miR-15b, miR-34a and miR-218 as promising novel early low-invasive biomarkers of cervical carcinogenesis[J]. APMIS, 2021, 129(2):70-79.
• [23] LI W, WANG Q X, FENG Q, et al. Oncogenic KSHVencoded interferon regulatory factor upregulates HMGB2and CMPK1 expression to promote cell invasion by disrupting a complex lncRNA-OIP5-AS1/miR-218-5p network[J]. PLoS Pathogens, 2019, 15(1):e1007578.
• [24] CHAROSTAD J, NAKHAIE M, AZARAN A, et al. Mi RNA-218 is frequently downregulated in malignant breast tumors:a footprint of epstein-barr virus infection[J]. Iranian Journal of Pathology, 2021, 16(4):376-385.
• [25]于晓妮,于玲燕,朱高香,等.高危型人乳头瘤病毒感染与宫颈癌中miR-218表达的关系[J].中国微生态学杂志, 2020, 32(3):325-329.
• [26] OFT M. IL-10:master switch from tumor-promoting inflammation to antitumor immunity[J]. Cancer Immunology Research, 2014, 2(3):194-199.
• [27] MAIMON A, LEVI-YAHID V, BEN-MEIR K, et al.Myeloid cell-derived PROS1 inhibits tumor metastasis by regulating inflammatory and immune responses via IL-10[J].Journal of Clinical Investigation, 2021:131(10):e126089.
• [28] BERMúDEZ-MORALES V H, GUTIéRREZ L X,ALCOCER-GONZáLEZ J M, et al. Correlation between IL-10 gene expression and HPV infection in cervical cancer:a mechanism for immune response escape[J]. Cancer Investigation, 2008, 26(10):1037-1043.
• [29] LONG D L, SONG H L, QU P P. Cytokines profiles in cervical mucosa in patients with cervical high-risk human papillomavirus infection[J]. The Journal of Infection in Developing Countries, 2021, 15(5):719-725.
• [30] MIN Z H, PU X W, GU Z R. Correlative analysis of the expression of IL-10 and Ki-67 in human cervical cancer and cervical intraepithelial neoplasias and human papillomavirus infection[J]. Oncology Letters, 2018, 16(6):7189-7194.
• [31] YEH W W, CHEN Y Q, YANG W S, et al. SUMO modification of histone demethylase KDM4A in Kaposi’s sarcomaassociated herpesvirus-induced primary effusion lymphoma[J].Journal of Virology, 2022, 96(16):e0075522.
• [32] CARO-VEGAS C, SELLERS S, HOST K M, et al. Runaway Kaposi Sarcoma-associated herpesvirus replication correlates with systemic IL-10 levels[J]. Virology, 2020, 539:18-25.
• [33] WU Y X, TIAN S J, CHEN Y J, et al. miR-218 inhibits gastric tumorigenesis through regulating Bmi-1/Akt signaling pathway[J]. Pathology-Research and Practice,2019, 215(2):243-250.
• [34]曾文兴,万颖,敖继红.不同情况淋巴瘤患者血清LDH、β2-MG、IL-2R及IL-10水平变化及临床意义[J].检验医学与临床, 2022, 19(24):3377-3381.
• [35] RALLIS K S, CORRIGAN A E, DADAH H, et al. Cytokinebased cancer immunotherapy:challenges and opportunities for IL-10[J]. Anticancer Research, 2021, 41(7):3247-3252.
• [36]陈静妮,代礼平,雷燕,等.草鱼NCCRP-1和IL-10的表达、抗体制备及组织病理变化[J].广东海洋大学学报,2021, 41(5):10-18.