长链非编码RNA在PEDV感染Vero-E6细胞中对自噬的调控作用

doi:10.16431/j.cnki.1671-7236.2022.05.035

Abstract

Abstract: 【Objective】 The purpose of the experiment was to study the regulation of long noncoding RNA (lncRNA) on autophagy in the process of Porcine epidemic diarrhea virus (PEDV) infecting African green monkey kidney cells (Vero-E6),so as to provide a new idea for the development of new drug targets against PEDV.【Method】 According to the human published lncRNA-HOTAIR gene sequence (accession No.:NR_047517.1) in GenBank,the monkey homologous sequence of lncRNA-HOTAIR (lncRNA-M) was screened by LongMan online tool,and the secondary structure and nucleocytoplasmic distribution were predicted by RNAfold and LncLocator online prediction tools.The Vero-E6 cell model infected by PEDV was established.The cells were collected at 0,6,12,24,36 and 48 h,and Real-time quantitative PCR was used to detect microtubule associated protein 1 light chain 3 (LC3),in order to determine the occurrence of autophagy at different time points after virus infected cells.The expression levels of lncRNA-M in Vero-E6 cell model infected by PEDV at 0,6,12,24,36 and 48 h were detected by Real-time quantitative PCR.Three lncRNA-M interference fragments siRNA-1,siRNA-2 and siRNA-3 were designed,synthesized and transfected into Vero-E6 cells respectively.When the confluence of Vero-E6 cells reached 90%,the cells were infected with PEDV,and after infection for 48 h,the expression of lncRNA-M was detected by Real-time quantitative PCR,and the interference fragment with the highest interference efficiency was screened.After transfecting the interference fragment with the highest interference efficiency into Vero-E6 cells,and the cells were infected with PEDV,the expression level of LC3 protein at 24 and 48 h after infection was detected by Western blotting to verify the occurrence of autophagy.【Result】 The homologous sequence of monkey lncRNA-HOTAIR was successfully screened as lncRNA 0.1,and the RNA minimum free energy secondary structure of lncRNA 0.1 was generated according to the prediction results of RNAfold.The prediction results of Lnlocator showed that the distribution of lncRNA 0.1 in nucleus and cytoplasm were 41.88% and 37.78%,respectively.48 h after PEDV infected Vero-E6 cells,the cells shrank and clustered,and the cell membrane fused to form syncytia body.1.0% agarose gel electrophoresis showed that the target band appeared at 1 326 bp,indicating that the PEDV infection Vero-E6 cell model was successfully established.In Vero-E6 cell model infected by PEDV,Western blotting results showed that the expression of LC3Ⅱ was increased in the experimental group at 6,12,24,36 and 48 h,the ratio of LC3Ⅱ /LC3Ⅰ was the highest at 48 h and was extremely significantly higher than that of control group (P<0.01).Real-time quantitative PCR results showed that the mRNA expression of LC3 was the highest at 48 h and was extremely significantly higher than that of control group (P<0.01),autophagy was activated.The expression of lncRNA 0.1 showed an upward trend,and the expression was the highest at 48 h,and the expression of lncRNA 0.1 at 48 h test group was extremely significantly higher than that in control group (P<0.01).Real-time quantitative PCR showed that siRNA-2 had the best interference effect,and the interference efficiency was 80%.After interfering with lncRNA 0.1,Western blotting results showed that the expression of LC3 protein was decreased and autophagy was inhibited in 24 and 48 h interference groups.【Conclusion】 PEDV infection could induce Vero-E6 autophagy,and lncRNA 0.1 played a role in promoting autophagy during infection.

Key words: Porcine epidemic diarrhea virus(PEDV); autophagy; long noncoding RNA; LC3 protein

CLC Number:

S852.65⁺9.2

WANG Zihang, DENG Xingmei, QIU Runhui, ZHU Dexin, LI Jia, TAO Tingting, ZHU Jiale, SUN Zhihua, ZHANG Hui. Regulation of Long Noncoding RNAs in Autophagy Induced by PEDV Infected Vero-E6 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1942-1950.

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Regulation of Long Noncoding RNAs in Autophagy Induced by PEDV Infected Vero-E6 Cells

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