环状RNA ciRS-7靶向miR-219a-5p影响微小隐孢子虫体外增殖的机制

doi:10.11843/j.issn.0366-6964.2022.11.024

摘要/Abstract

摘要： 旨在探究环状RNA ciRS-7靶向miR-219a-5p影响微小隐孢子虫（Cryptosporidium parvum）在HCT-8细胞中增殖的作用机制。以C.parvum感染HCT-8细胞为研究模型，利用Western blot检测HCT-8细胞中的自噬蛋白LC3B-II和p62的表达情况；利用qRT-PCR和双荧光素酶报告基因试验检测和验证ciRS-7与miR-219a-5p的靶向关系，利用qRT-PCR检测HCT-8细胞中C.parvum的荷虫量（C.parvum hsp70基因的mRNA水平）。结果显示：C.parvum感染诱导HCT-8细胞中LC3B-II和p62的蛋白质表达水平在感染后8、12、24、36和48 h显著上调；过表达ciRS-7显著抑制了C.parvum感染细胞中LC3B-II的蛋白质表达水平，但升高了p62的蛋白质表达水平，而干扰ciRS-7表达的作用相反；C.parvum感染诱导HCT-8细胞中miR-219a-5p的显著下调表达，且ciRS-7可靶向调节miR-219a-5p的表达；miR-219a-5p mimics显著增加了C.parvum感染细胞中LC3B-II的蛋白质表达水平，但降低了p62的蛋白质表达水平，而miR-219a-5p inhibitor的作用相反；miR-219a-5p mimics可逆转过表达ciRS-7对C.parvum感染细胞中LC3B-II蛋白质表达的抑制和p62蛋白质表达的促进作用；miR-219a-5p mimics显著抑制了感染细胞中C.parvum hsp70基因的mRNA表达水平，而miR-219a-5p inhibitor的作用相反；miR-219a-5p mimics逆转了过表达ciRS-7对感染细胞中C.parvum hsp70基因的mRNA表达水平的上调作用。ciRS-7通过靶向miR-219a-5p抑制C.parvum感染诱发的HCT-8细胞自噬，进而促进C.parvum在HCT-8细胞中的增殖。

关键词: 微小隐孢子虫, circRNA, ciRS-7/miR-219a-5p轴, 自噬, 增殖

Abstract: The present study was carried out to investigate the impact of circRNA ciRS-7 on the propagation of Cryptosporidium parvum in HCT-8 cells via targeting miR-219a-5p. Using C. parvum-infecting HCT-8 cells as the model, we detected the expression of the autophagy associated-proteins of LC3B-II and p62 in HCT-8 cells by using Western bolt assays. The targeting relationship between ciRS-7 and miR-219a-5p were detected and verified by using qRT-PCR and dual luciferase reporter assays, and the infection burden of C. parvum (the mRNA level of C. parvum hsp70 gene) in HCT-8 cells was determined by using qRT-PCR. C. parvum infection induced significantly up-regulation of the protein levels of LC3B-II and p62 in HCT-8 cells at 8 h, 12 h, 24 h, 36 h and 48 h post-infection. Overexpression of ciRS-7 markedly inhibited the protein level of LC3B-II but promoted the protein level of p62 in C. parvum-infecting cells, whereas interfering the expression of ciRS-7 obtained the opposite results. C. parvum infection induced significant down-regulation of miR-219a-5p in HCT-8 cells, and ciRS-7 could target to regulate the expression of miR-219a-5p. miR-219a-5p mimics significantly increased the protein level of LC3B-II but decreased the protein level of p62 in C. parvum-infecting cells, while miR-219a-5p inhibitor had the opposite effects, and the inhibition effect on the protein level of LC3B-II and the promoted effect on the protein level of p62 by overexpression of ciRS-7 in C. parvum-infecting cells were reversed by miR-219a-5p mimics. miR-219a-5p mimics significantly inhibited the mRNA level of C. parvum hsp70 in infected cells, while miR-219a-5p inhibitor had the opposite effect. The upregulated effect of overexpression of ciRS-7 on the mRNA level of C. parvum hsp70 gene in infected cells was attenuated by miR-219a-5p mimics. The results revealed that ciRS-7 promoted the propagation of C. parvum in HCT-8 cells by targeting miR-219a-5p to inhibit C. parvum-induced cell autophagy.

Key words: Cryptosporidium parvum, circRNA, ciRS-7/miR-219a-5p axis, autophagy, propagation

中图分类号:

S852.723

尹艳玲, 黄爽, 姚倩, 吴江平, 郭浩晨, 杨新, 宋军科, 赵光辉. 环状RNA ciRS-7靶向miR-219a-5p影响微小隐孢子虫体外增殖的机制[J]. 畜牧兽医学报, 2022, 53(11): 3989-3999.

YIN Yanling, HUANG Shuang, YAO Qian, WU Jiangping, GUO Haochen, YANG Xin, SONG Junke, ZHAO Guanghui. The Mechanisms of Circular RNA ciRS-7 Affecting the Propagation of Cryptosporidium parvum in HCT-8 Cells via Targeting miR-219a-5p[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3989-3999.

参考文献 37

[1]	CHEN X M, KEITHLY J S, PAYA C V, et al.Cryptosporidiosis[J].N Engl J Med, 2002, 346(22):1723-1731.
[2]	LIU A Q, GONG B Y, LIU X H, et al.A retrospective epidemiological analysis of human Cryptosporidium infection in China during the past three decades (1987-2018)[J].PLoS Negl Trop Dis, 2020, 14(3):e0008146.
[3]	KARABEY M, CAN H, ÖNER T Ö, et al.Cryptosporidium spp.during chemotherapy:a cross-sectional study of 94 patients with malignant solid tumor[J].Ann Saudi Med, 2021, 41(5):293-298.
[4]	KOTLOFF K L, NATARO J P, BLACKWELDER W C, et al.Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS):a prospective, case-control study[J].Lancet, 2013, 382(9888):209-222.
[5]	CHECKLEY W, WHITE A C Jr, JAGANATH D, et al.A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium[J].Lancet Infect Dis, 2015, 15(1):85-94.
[6]	SOW S O, MUHSEN K, NASRIN D, et al.The burden of Cryptosporidium diarrheal disease among children
[7]	ABUBAKAR I, ALIYU S H, ARUMUGAM C, et al.Prevention and treatment of cryptosporidiosis in immunocompromised patients[J].Cochrane Database Syst Rev, 2007(1):CD004932.
[8]	LOVE M S, CHOY R K M.Emerging treatment options for cryptosporidiosis[J].Curr Opin Infect Dis, 2021, 34(5):455-462.
[9]	JECK W R, SHARPLESS N E.Detecting and characterizing circular RNAs[J].Nat Biotechnol, 2014, 32(5):453-461.
[10]	ZHOU W Y, CAI Z R, LIU J, et al.Circular RNA:metabolism, functions and interactions with proteins[J].Mol Cancer, 2020, 19(1):172.
[11]	YUE B L, WANG J, SONG C C, et al.Biogenesis and ceRNA role of circular RNAs in skeletal muscle myogenesis[J].Int J Biochem Cell Biol, 2019, 117:105621.
[12]	PATOP I L, WVST S, KADENER S.Past, present, and future of circRNAs[J].EMBO J, 2019, 38(16):e100836.
[13]	YANG R C, XU B J, YANG B, et al.Circular RNA transcriptomic analysis of primary human brain microvascular endothelial cells infected with meningitic Escherichia coli[J].Mol Ther Nucleic Acids, 2018, 13:651-664.
[14]	ZHANG Y, ZHANG H, AN M H, et al.Crosstalk in competing endogenous RNA networks reveals new circular RNAs involved in the pathogenesis of early HIV infection[J].J Transl Med, 2018, 16(1):332.
[15]	YU H L, MI C H, WANG Q, et al.Comprehensive analyses of circRNA expression profiles and function prediction in chicken cecums after Eimeria tenella infection[J].Front Cell Infect Microbiol, 2021, 11:628667.
[16]	YIN Y L, LIU T L, YAO Q, et al.Circular RNA ciRS-7 affects the propagation of Cryptosporidium parvum in HCT-8 cells by sponging miR-1270 to activate the NF-κB signaling pathway[J].Parasit Vectors, 2021, 14(1):238.
[17]	MAHURKAR-JOSHI S, RANKIN C R, VIDRLOCK E J, et al.The colonic mucosal microRNAs, microRNA-219a-5p, and microRNA-338-3p are downregulated in irritable bowel syndrome and are associated with barrier function and MAPK signaling[J].Gastroenterology, 2021, 160(7):2409-2422.e19.
[18]	XIAO Y, ZHANG S H, LI Q, et al.miR-219a-5p ameliorates hepatic ischemia/reperfusion injury via impairing TP53BP2[J].Dig Dis Sci, 2019, 64(8):2177-2186.
[19]	ZHU X T, CHEN L, LIN J H.miR-219a-5p represses migration and invasion of osteosarcoma cells via targeting EYA2[J].Artif Cells Nanomed Biotechnol, 2018, 46(S3):S1004-S1010.
[20]	LI Y M, ZHANG J Z, PAN S, et al.CircRNA CDR1as knockdown inhibits progression of non-small-cell lung cancer by regulating miR-219a-5p/SOX5 axis[J].Thorac Cancer, 2020, 11(3):537-548.
[21]	LIU T L, FAN X C, LI Y H, et al.Expression profiles of mRNA and lncRNA in HCT-8 cells infected with Cryptosporidium parvum IId subtype[J].Front Microbiol, 2018, 9:1409.
[22]	LIU T L, FAN X C, WANG Y, et al.Micro-RNA expression profile of chicken small intestines during Eimeria necatrix infection[J].Poult Sci, 2020, 99(5):2444-2451.
[23]	曹大龙.双荧光素酶报告基因检测miR-185与AKT1靶标关系[D].蚌埠:蚌埠医学院, 2015.CAO D L.Research on target relationship between miR-185 and AKT1 by dual luciferase report gene assay[D].Bengbu:Bengbu Medical College, 2015.(in Chinese)
[24]	SUBAUSTE C S.Recent advances in the roles of autophagy and autophagy proteins in host cells during Toxoplasma gondii infection and potential therapeutic implications[J].Front Cell Dev Biol, 2021, 9:673813.
[25]	SCHMUCKLI-MAURER J, REBER V, WACKER R, et al.Inverted recruitment of autophagy proteins to the Plasmodium berghei parasitophorous vacuole membrane[J].PLoS One, 2017, 12(8):e0183797.
[26]	MUNIZ-FELICIANO L, VAN GROL J, PORTILLO J A C, et al.Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite[J].PLoS Pathog, 2013, 9(12):e1003809.
[27]	REAL E, RODRIGUES L, CABAL G G, et al.Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes[J].Nat Microbiol, 2018, 3(1):17-25.
[28]	ONIZUKA Y, TAKAHASHI C, UEMATSU A, et al.Inhibition of autolysosome formation in host autophagy by Trypanosoma cruzi infection[J].Acta Trop, 2017, 170:57-62.
[29]	THOMAS S A, NANDAN D, KASS J, et al.Countervailing, time-dependent effects on host autophagy promotes intracellular survival of Leishmania[J].J Biol Chem, 2018, 293(7):2617-2630.
[30]	蒋衡.隐孢子虫通过microRNA-MAPK信号通路调控细胞自噬的机制[D].长春:吉林大学, 2020.JIANG H.Mechanism of cell autophagy regulated by Cryptosporidium parvum via microRNA-MAPK signaling pathway[D].Changchun:Jilin University, 2020.(in Chinese)
[31]	PRIYAMVADA S, JAYAWARDENA D, BHALALA J, et al.Cryptosporidium parvum infection induces autophagy in intestinal epithelial cells[J].Cell Microbiol, 2021, 23(4):e13298.
[32]	ZHOU Z X, ZHANG Y F, GAO J N, et al.Circular RNAs act as regulators of autophagy in cancer[J].Mol Ther Oncolytics, 2021, 21:242-254.
[33]	MENG L J, LIU S H, DING P A, et al.Circular RNA ciRS-7 inhibits autophagy of ESCC cells by functioning as miR-1299 sponge to target EGFR signaling[J].J Cell Biochem, 2020, 121(2):1039-1049.
[34]	ZHOU X D, LI J, ZHOU Y S, et al.Down-regulated ciRS-7/up-regulated miR-7 axis aggravated cartilage degradation and autophagy defection by PI3K/AKT/mTOR activation mediated by IL-17A in osteoarthritis[J].Aging (Albany NY), 2020, 12(20):20163-20183.
[35]	CHEN X M, SPLINTER P L, O'HARA S P, et al.A cellular micro-RNA, let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection[J].J Biol Chem, 2007, 282(39):28929-28938.
[36]	LI J, JIN K H, LI M, et al.A host cell long noncoding RNA NR_033736 regulates type I interferon-mediated gene transcription and modulates intestinal epithelial anti-Cryptosporidium defense[J].PLoS Pathog, 2021, 17(1):e1009241.
[37]	HE W, LI J, GONG A Y, et al.Cryptosporidial infection suppresses intestinal epithelial cell MAPK signaling impairing host anti-parasitic defense[J].Microorganisms, 2021, 9(1):151.