1.Role of miR-486-5p in apoptosis of human bone marrow mesenchymal stem cells induced by hydrogen peroxide
Ming HU ; Jiao LI ; Ningning LIU ; Zhenjun HUANG ; Chonghai WU ; Yun ZHONG ; Shiming LIU
Chinese Journal of Pathophysiology 2015;(3):524-529
AIM:To investigate the role of microRNA-486-5p (miR-486-5p) in the apoptosis of human bone marrow mesenchymal stem cells (hMSCs) induced by hydrogen peroxide (H2O2).METHODS: The hMSCs were cul-tured in vitro and exposed to serum-free medium and H2O2(10 mmol/L).The changes of miR-486-5p expression in oxida-tive stress-related apoptosis of hMSCs were measured by real-time PCR.The hMSCs were transfected with miR-486-5p mimic or inhibitor at concentration of 30 nmol/L by Lipofectamine RNAiMAX.The effect of miR-486-5p on H2 O2-induced decrease in cell viability was evaluated by MTT assay.Hoechst 33342 staining and flow cytometry were applied to determine the role of miR-486-5p in the apoptosis of hMSCs.The protein expression was evaluated by Western blotting.Caspase-3 ac-tivity was determined using a caspase-3 activity kit.RESULTS:Compared with control group, the expression of miR-486-5p significantly decreased after treated with H2O2(P<0.05).In addition, over-expression of miR-486-5p in the hMSCs reduced the cell viability, accelerated apoptosis, down-regulated Bcl-2/Bax ratio, caspase-3 enzyme precursor content and phosphorylation of Akt, and activated caspase-3 activity.Conversely, down-regulation of miR-486-5p significantly inhibited H2 O2-induced cell apoptosis and the caspase-3 activity, increased cell viability and up-regulated Bcl-2/Bax ratio and phos-phorylation level of Akt.CONCLUSION:Over-expression of miR-486-5p promotes H2 O2-induced hMSCs apoptosis, and repression of miR-486-5p protects hMSCs from H2 O2-induced cellular apoptosis, which may be mediated by regulating Akt signaling pathway.
2.Analysis of epidemiological and pathogenic characteristics of a mumps outbreak in a school in Xining, Qinghai province
Zhifang ZHANG ; Ning XU ; Jing WU ; Chonghai LI ; Xiaotong WANG ; Aili CUI ; Lixia FAN
Chinese Journal of Experimental and Clinical Virology 2020;34(4):401-405
Objective:To investigate the epidemiological and pathogenic characteristics of a mumps outbreak in a primary school in Xining, Qinghai province.Methods:The epidemiological investigation was carried out to analyze the epidemiological distribution of mumps cases in the outbreak. Serum and throat swab samples were collected from 9 suspected mumps cases for laboratory testing. The throat swab samples detected positive for nucleic acid of mumps virus were subjected to virus isolation. Then the SH gene was amplified by RT-PCR for positive virus isolates, and the genotypes of mumps virus were identified and gene characteristics were analyzed.Results:A total of 13 cases were reported in this outbreak. The age of cases was mainly 7-11 years old, and the cases were mainly concentrated at 8 years old (69.23%. 9/13). The male to female ratio is 1.6: 1. None of the 13 cases had a history of mumps vaccination. And there was an obvious in-class clustering in this mumps outbreak. Of the 9 suspected mumps cases, 8 were double positive for mumps specific IgM antibody and viral nucleic acid. Two positive mumps virus isolates were obtained and identified by genotyping as F genotype, and the SH gene sequence of the two mumps virus isolates had 100% homology.Conclusions:This outbreak is caused by genotype F mumps virus. MuV immunization activities were recommended to conduct among unvaccinated students in primary and secondary schools.
3.Genetic characterizations of an imported monkeypox virus in Qinghai province
Youju LEI ; Shengcang ZHAO ; Zhijian TANG ; Wenling WANG ; Changcheng WU ; Chonghai LI ; Roujian LU ; Xiaotong WANG ; Lifang HE ; Mengqi YIN
Chinese Journal of Experimental and Clinical Virology 2023;37(5):518-523
Objective:Based on targeted amplicon technology combined with high-throughput sequencing technology and bioinformatic analysis technology, to understand the characteristics of the whole genome of the monkeypox virus and its variation, and to construct a method for the analysis of monkeypox virus variation and molecular traceability of the case in Qinghai province, and to provide technical support for the prevention and control of monkeypox epidemic in the future.Methods:The extracted viral DNA was used as a template, and the genome of monkeypox virus was specifically amplified by Ion AmpliSeq Monkeypox Panel with the number of amplicons 1 609 and the length of 125 bp-275 bp, and the sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and sequenced by Ion Torrent GeneStudio S5. The sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and the monkeypox virus genome was sequenced using Ion Torrent GeneStudio S5 sequencer. Monkeypox virus was analyzed for genomic profiling and mutation site analysis using the online analysis tool Nextclade. The genomic sequence of the case virus in this study was compared with some sequences in the GIASID monkeypox virus database and a phylogenetic tree was constructed to analyze the potential origin of the case virus.Results:The Ct values of monkeypox virus genes in the rash swab and oropharyngeal swab samples were 32.13 and 36.91, respectively. The rash swab sample had a reads number match of 99.99% and a genome coverage of 99.45% after whole-genome sequencing of monkeypox virus, and the sequences belonged to the IIb (West African branch) B. 1.3 type. The analysis of nucleotide mutation sites and phylogenetic tree showed that the sequences were in the same branch with four monkeypox virus genome sequences recently submitted by China and Japan in the GISAID monkeypox virus database, and had the closest evolutionary relationship with the sequence EPI_ISL_18059184 (sampled on 2023-07-03) submitted by Yunnan, China, which shared 82 single-nucleotide mutation sites, among which the sequence from Yunnan was only present in all of the shared 82 single-nucleotide mutation sites. The sequence in this study has 2 additional nucleotide mutation sites on top of the shared 82 single nucleotide mutation sites. The sequence submitted by Japan, EPI_ISL_17692269 (sampled on 2023-04-28), is more closely related in evolution, sharing 78 single nucleotide mutation sites, with 7 single nucleotide mutation site differences, and the Japanese sequence shares 78 single nucleotide mutation sites. The Japanese sequence shared 78 mutation sites with one additional nucleotide mutation site (G57786A), while the present sequence had six additional nucleotide mutation sites (G13563A, C21062T, G101241A, C142797T, G152866A, T169721A).Conclusions:The whole genome sequence of monkeypox virus of 197 084 bp was successfully obtained from a sample with low viral load, and the average. We constructed a method for sequencing and analyzing the whole genome of monkeypox virus.