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中华肺部疾病杂志(电子版) ›› 2020, Vol. 13 ›› Issue (04) : 461 -465. doi: 10.3877/cma.j.issn.1674-6902.2020.04.006

论著

靶向核壳蛋白基因的siRNA对流感病毒的预防和治疗作用
史亮1, 尹申慧2, 任浩2, 高荣2, 马壮1,†(), 孙文武1, 曹建平1   
  1. 1. 10016　沈阳，北部战区总医院呼吸与危重症医学科
    2. 200433　上海，海军（第二）军医大学海军医学系
  • 收稿日期:2020-03-19 出版日期:2020-08-25
  • 通信作者: 马壮
  • 基金资助:
    中国人民解放军十二五科研基金项目(CSY14C005)

Prophylactic and therapeutic effects of small interfering RNA targeting nucleocapsid protein gene on influenza A virus

Liang Shi1, Shenhui Yin2, Hao Ren2, Rong Gao2, Zhuang Ma1,†(), Wenwu Sun1, Jianping Cao1   

  1. 1. Department of Respiratory and Critical Care Medicine, General Hospital of Northern Theater Command, Shenyang 110016, China
    2. Department of Naval Medicine, Naval Medical University, Shanghai 200433, China
  • Received:2020-03-19 Published:2020-08-25
  • Corresponding author: Zhuang Ma
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引用本文：

史亮, 尹申慧, 任浩, 高荣, 马壮, 孙文武, 曹建平. 靶向核壳蛋白基因的siRNA对流感病毒的预防和治疗作用[J]. 中华肺部疾病杂志(电子版), 2020, 13(04): 461-465.

Liang Shi, Shenhui Yin, Hao Ren, Rong Gao, Zhuang Ma, Wenwu Sun, Jianping Cao. Prophylactic and therapeutic effects of small interfering RNA targeting nucleocapsid protein gene on influenza A virus[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2020, 13(04): 461-465.

目的

探讨靶向核壳蛋白(nucleoprotein, NP)的siRNA对甲型流感病毒(influenza virus A, IAV)的预防及治疗作用。

方法

在对IAV测序的基础上设计对NP保守区特异的小干扰RNA(small interfering RNA, siRNA)，并观察在犬肾细胞(Madin-Darby canine kidney, MDCK)中先转染了siRNA然后以IAV感染，或先以IAV感染，然后转染siRNA时MDCK细胞中IAV载量的变化。

结果

1．转染了siRNA的MDCK细胞再进行IAV感染，不同剂量siRNA组均较转染剂对照组IVA病毒载量显著降低(20 pmol siRNA组，P<0.05；40 pmol siRNA组，P<0.05；80 pmol siRNA组，P<0.01)；2.当细胞在转染siRNA之前感染IAV，siRNA组的病毒载量显著低于对照组(P<0.01)，也低于转染试剂组(P<0.05)；3.免疫细胞化学结果显示：无论MDCK细胞预先转染siRNA还是感染了IVA后再进行siRNA转染，siRNA组的NP蛋白表达均明显低于对照组(P<0.01)。

结论

靶向NP的siRNA通过干扰IVA的NP蛋白合成，从而对IVA的生长产生抑制作用；无论MDCK细胞感染IVA前后应用靶向NP的siRNA转染均可以降低IVA的产生，说明靶向NP的siRNA对于IVA感染具有预防及治疗作用。

关键词: 流感病毒, RNA干扰, NP, 转染, qRT-PCR, 免疫细胞化学
Objective

To explore the prophylactic and therapeutic effects of small interfering RNA (siRNA) targeting nucleocapsid protein (NP) gene on influenza A virus (IAV).

Methods

On the basis of sequencing IAV, the siRNA targeting NP segment of IAV was synthesized. The changes of IAV loads in the Madin-Darby canine kidney (MDCK) were observed after siRNA transfection and IAV infection or after IAV infection and siRNA transfection.

Results

The virus load was lower or much lower in the siRNA-transfected cells than the transfection agent controls (P<0.05 for 20 pmol siRNA group and 40 pmol siRNA group, and P<0.01 for 80 pmol siRNA group). When the cells were infected with IAV prior to siRNA transfection, the virus load was significantly lower in the siRNA group than the controls (P<0.01) and lower in the siRNA group than the transfection agent group (P<0.05). The immunohistochemical results showed that the expression of NP protein was significantly lower in the siRNA group than the controls (P<0.01), no matter the MDCK cells were firstly transfected with siRNA or infected with IAV.

Conclusion

The production of IAV can be inhibited by siRNAs targeting NP in a dose-dependent manner and can also be inhibited by siRNA targeting NP after the cells were infected with IAV. The inhibition of IAV by siRNA targeting NP is resulted from the interference with NP protein synthesis.

Key words: Influenza virus, RNA interference, Nucleocapsid protein, Transfection, qRT-PCR, Immunohistochemistry
图1 NP特异的siRNA对MDCK细胞中IAV的产生的影响；注：**：P<0.01,和IAV对照相比；#：P<0.05，和转染液对照相比；##：P<0.01，和转染液对照相比
图2 NP特异的siRNA对已感染IAV的MDCK细胞中IAV的产生的影响；注：**：P<0.01，和IAV对照相比；*：P<0.05，和转染试剂对照相比
图3 预先转染siRNA对MDCK感染IAV后NP蛋白合成的影响；注：A：转染了NP特异性siRNA的MDCK细胞的免疫荧光图像；(A)对照组细胞的荧光图像；(B)转染了siRNA细胞的荧光图像；(C)对照组中以Hoechst 33342染色的细胞核；(D)转染了siRNA的细胞中以Hoechst 33342染色的细胞核；B：对照组和转染了siRNA细胞的荧光强度；以Image J软件分析两组细胞NP蛋白的荧光强度。**，P<0.01和对照组相比，(n＝30)
图4 siRNA对预先感染IAV的MDCK细胞NP蛋白合成的影响；注：A：转染了NP特异性siRNA的MDCK细胞的免疫荧光图像；(A)对照组细胞的荧光图像；(B)转染了siRNA细胞的荧光图像；(C)对照组中以Hoechst 33342染色的细胞核；(D)转染了siRNA的细胞中以Hoechst 33342染色的细胞核；B：对照组和转染了siRNA细胞的荧光强度；以Image J软件分析两组细胞NP蛋白的荧光强度。**，P<0.01，和对照组相比，(n＝30)
1
任成山，钱桂生. 甲型H1N1流感的流行特点及防控对策[J]. 中华医学杂志，2009, 89(46): 3241-3243.
2
张曦木，张雪倩，冯　聪，等. 流行性感冒合并肺炎临床特征分析[J/CD]. 中华肺部疾病杂志(电子版), 2020, 13(1): 63-68.
3
WHO. Influenza (seasonal) fact sheet No 211. [(accessed on 31 March 2012)]. Available online:

URL    
4
Tamura D, Mitamura K, Yamazaki M, et al. Oseltamivir-resistant influenza A viruses circulating in Japan[J]. J Clin Microbiol, 2009, 47(5): 1424-1427.
5
Bright RA, Shay DK, Shu B, et al. Adamantane resistance among influenza A viruses isolated early during the 2005-2006 influenza season in the United States[J]. JAMA, 2006, 295: 891-894.
6
Govorkova EA, Beranovich T, Seiler P, et al. Antiviral resistance among highly pathogenic influenza A(H5N1) viruses isolated worldwide in 2002-2012 shows need for continued monitoring[J]. Antiviral Res, 2013, 98(2): 297-304.
7
Kiso M, Kubo S, Ozawa M, et al. Efficacy of the new neuraminidase inhibitor CS-8958 against H5N1 influenza viruses[J]. PLoS Pathog, 2010, 6(2): e1000786.
8
Tran TH, Nguyen TL, Nguyen TD, et al. Avian influenza A (H5N1) in 10 patients in Vietnam[J]. N Engl J Med, 2004, 350: 1179-1188.
9
Yen HL, Monto AS, Webster RG, et al. Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/1203/04 influenza virus in mice[J]. J Infect Dis, 2005, 192(2): 665-672.
10
Rappuoli R, Dormitzer PR. Influenza: options to improve pandemic preparation[J]. Science, 2012, 336: 1531-1533.
11
Fedson DS. What treating Ebola means for pandemic influenza[J]. J Public Health Policy, 2018, 39(3): 268-282.
12
Lenny BJ, Sonnberg S, Danner AF, et al. Evaluation of multivalent H2 influenza pandemic vaccines in mice[J]. Vaccine, 2017, 35(10): 1455-1463.
13
Beigel John H, Farrar Jeremy, Han Aye Maung, et al. Avian influenza A (H5N1) infection in humans[J]. N Engl J Med, 2005, 353(13): 1374-1385.
14
Uyeki TM, Peiris M. Novel avian influenza A virus infections of Humans[J]. Infect Dis Clin North Am, 2019, 33(4): 907-932.
15
Vaucheret H, Beclin C, Faqard M. Post-transcriptional gene silencing in plants[J]. J Cell Sci, 2001, 114: 3083-3091.
16
Sharp PA. RNA interference-2001[J]. Genes Dev, 2001, 15: 485-490.
17
Brantl S. Antisense-RNA regulation and RNA interference[J]．Biochim Biophys Acta, 2002, 1575(1-3): 15-25.
18
Ge Qing, McManus Michael T, Nguyen Tam, et al. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription[J]. Proc Natl Acad Sci USA, 2003, 100(5): 2718-2723.
19
Ge Q, Filip L, Bai A, et al. Inhibition of influenza virus production in virus-infected mice by RNA interference[J]. Proc Natl Acad Sci USA, 2004, 101: 8676-8681.
20
Zhou HB, Jin ML, Yu ZJ, et al. Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice[J]. Antiviral Res, 2007, 76(2): 186-193.
21
Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elengans[J]. Nature, 1998, 391(6669): 806-811.
22
Montgomery MK, Xu S, Fire A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegant[J]. Proc Natl Acad Sci USA, 1998, 95(26): 15502-15507.
23
Huang F, Hua X, Yang S, et al. Effective inhibition of hepatitis E virus replication in A549 cells and piglets by RNA interference (RNAi) targeting RNA-dependent RNA polymerase[J]. Antiviral Res, 2009, 83: 274-281.
24
Li WY, Yang XF, Jiang Y, et al. Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene[J]. Arch Virol, 2011, 156(11): 1979-1987.
25
Kumar A, Panda SK, Durgapal H, et al. Inhibition of Hepatitis E virus replication using short hairpin RNA(shRNA)[J]. Antiviral Res, 2010, 85(3): 541-550.
26
Kaushik N, Subramani C, Anang S, et al. Zinc salts block Hepatitis E virus replication by inhibiting the activity of viral RNA-dependent RNA polymerase[J]. J Virol, 2017, 91(2): e00754-e00717.
27
DeVincenzo J, Lambkin-Williams R, Wilkinson T, et al. A randomized，double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus[J]. Proc Natl Acad Sci USA, 2010, 107(19): 8800-8805.
28
Sui HY, Zhao GY, Huang JD, et al. Small interfering RNA targeting M2 gene induces effective and long term inhibition of influenza A virus replication[J]. 2009, 4(5): e5671.
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