Application of hsa-miRNA-155-5p for preparing medicine for inhibiting human enterovirus 71

A human enterovirus, inhibition technology, applied in the direction of antiviral agents, to achieve the effect of reducing endocytosis and inhibiting replication

Inactive Publication Date: 2018-07-17
ZHENJIANG NO 1 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, miR-155 has not been reported

Method used

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  • Application of hsa-miRNA-155-5p for preparing medicine for inhibiting human enterovirus 71
  • Application of hsa-miRNA-155-5p for preparing medicine for inhibiting human enterovirus 71
  • Application of hsa-miRNA-155-5p for preparing medicine for inhibiting human enterovirus 71

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: EV71 infection up-regulates the expression of intracellular miR-155

[0018] Neuroblastoma SK-N-SH cells were cultured overnight. When the confluence reached 70-80%, the culture medium was aspirated, washed with PBS and discarded; added EV71 virus solution with MOI=10, and incubated at 37°C for 1.5 hours ; Discard the virus liquid, discard after washing with PBS once; add DMEM culture medium containing 2% fetal bovine serum, after 36 hours, collect and collect SK-N-SH cells uninfected and infected with EV71, extract total RNA, and quantify To detect the expression level of miR-155, see figure 1 .

[0019] The result is as figure 1 Shown: Fluorescence quantitative results show that compared with cells not infected with virus (control), the expression of miR-155 in SK-N-SH cells infected with EV71 (EV71 infection) is significantly increased, and the results are statistically significant.

Embodiment 2

[0020] Example 2: miR-155 mimics the ability to inhibit the replication of EV71 in vitro

[0021] (1) Increased intracellular miR-155 levels after miR-155 mimic transfection

[0022] miRNA mimic is a double-stranded RNA synthesized by chemical methods, which can simulate the high-level expression of endogenous mature miRNA in cells, so as to enhance the regulation of endogenous miRNA and conduct gain-of-function research.

[0023] Both miR-155 mimic and miRNA mimic negative control (miR-mimic-NC) were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. After miR-155 mimic and negative control miRNA (miR-mimic-NC) were transfected into SK-N-SH cells, the level of intracellular miR-155 was detected by fluorescent quantitative PCR.

[0024] (2) miR-155 mimic inhibits the replication of EV71 after transfection

[0025] After transfection with miR-155 mimic, the virus titer, VP1 protein and viral nucleic acid of EV71 were detected. Compared with transfected miR-mimic-NC, the ...

Embodiment 3

[0027] Example 3: Verification of the target gene of miR-155—picalm gene

[0028] The picalm gene was predicted to be the target gene of miR-155 by Targetscan, MiRBase and other miRNA biological prediction software, and this targeting relationship was verified by the detection of the dual luciferase reporter system. In order to verify the direct binding between miR-155 and PICALM, a sequence containing the predicted binding site in the 3'UTR of the PICALM gene (sequence information from Genebank, accession number NM_007166.3) was inserted into the psi-CHECKTM-2 double fluorescein Then, miR-155 mimic and the reporter gene carrier were co-transfected into 293T cells, and the targeting effect of miR-155 on the picalm gene was reflected by detecting the fluorescent signal.

[0029] The result is as image 3 Shown: there is a sequence complementary relationship between miR-155 and picalm gene ( image 3 A); if image 3 As shown in B, compared with the miR-155 mimic NC of the con...

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Abstract

The invention discloses application of hsa-miRNA-155-5p (miR-155 for short) for preparing medicine for inhibiting human enterovirus 71 (EV71 for short). The molecular biological technology is used forimproving expression of miR-155 in cells after the cells are infected with EV71. The miR-155 can assemble protein PICALM through targeting phosphatidylinositol clathrin, and then copying of the EV71is inhibited. The application provides the theoretical and experiment basis for diagnosing and treating diseases such as hand-foot-and-mouth diseases caused by the EV71, and can be applied to development of the medicine for treating EV71 infection.

Description

technical field [0001] The invention relates to the application of hsa-miRNA-155-5p (abbreviated as miR-155) in the preparation of medicines for inhibiting human enterovirus 71 (abbreviated as EV71), which belongs to the fields of virology, molecular biology and biomedicine. Background technique [0002] Enterovirus 71 (EV71) is a member of the Enterovirus genus of the Picornaviridae family and is one of the most important pathogens causing hand, foot and mouth disease. Since it was isolated for the first time in 1969, the EV71 virus epidemic has broken out in many countries and regions on a large scale, including China, Japan, Malaysia, and Singapore. In China, from 2009 to 2016, there were about 2 million infections in China every year, ranking first among Class C infectious diseases. Due to the lack of effective anti-EV71 drugs, patients with severe hand, foot and mouth disease die every year. [0003] microRNA (miRNA) is a single-stranded non-coding small molecule RNA ...

Claims

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Application Information

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IPC IPC(8): A61K31/7105A61P31/14
CPCA61K31/7105
Inventor 茅凌翔吴静沈俐顾佳奇邹欣然许化溪王胜军
Owner ZHENJIANG NO 1 PEOPLES HOSPITAL
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