mdv1-miR-M4-5p对MDCC-MSB1细胞增殖和凋亡的影响

doi:10.11843/j.issn.0366-6964.2024.08.039

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (8): 3678-3687.doi: 10.11843/j.issn.0366-6964.2024.08.039

mdv1-miR-M4-5p对MDCC-MSB1细胞增殖和凋亡的影响

余祖华¹^,²(), 高梦茹¹^,², 何雷¹^,², 魏颖¹^,², 陈建¹^,², 陈松彪¹^,², 丁轲¹^,²^,³^,*()

1. 河南科技大学动物科技学院/功能微生物与畜禽健康实验室/动物疫病与公共卫生重点实验室, 洛阳 471023
2. 洛阳市活载体生物材料与动物疫病防控重点实验室, 洛阳 471023
3. 河南科技大学学院动物科技学院, 新乡 453003

收稿日期:2023-11-08 出版日期:2024-08-23 发布日期:2024-08-28
通讯作者: 丁轲 E-mail:yzhd05@163.com;keding19@163.com
作者简介:余祖华(1977-)女，河南商城人，副教授，博士，主要从事动物疫病病原与分子免疫学研究，E-mail: yzhd05@163.com
基金资助:
国家自然科学基金(U1504308);国家自然科学基金(31702207)

Effects of mdv1-miR-M4-5p Encoded by MDV on Proliferation and Apoptosis of MDCC-MSB1 Cells

Zuhua YU¹^,²(), Mengru GAO¹^,², Lei HE¹^,², Ying WEI¹^,², Jian CHEN¹^,², Songbiao CHEN¹^,², Ke DING¹^,²^,³^,*()

1. Laboratory of Functional Microbiology and Animal Health/The Key Laboratory of Animal Disease and Public Health, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
2. Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control, Luoyang 471023, China
3. College of Animal Science and Technology, Henan UniversityInstitute of Science and Technology, Xinxiang 453003, China

Received:2023-11-08 Online:2024-08-23 Published:2024-08-28
Contact: Ke DING E-mail:yzhd05@163.com;keding19@163.com

摘要/Abstract

摘要：

旨在研究马立克病病毒(Marek's disease virus, MDV)编码的mdv1-miR-M4-5p对MDCC-MSB1细胞增殖和凋亡的影响，将mdv1-miR-M4-5p模拟物、抑制物及其阴性对照转染MDCC-MSB1细胞后48 h，采用实时荧光定量PCR(qRT-PCR)检测细胞中mdv1-miR-M4-5p、TGF-β1、Smad2以及caspase-9、caspase-3、cyt-c、cyclinD1、Bcl-2等增殖和凋亡相关分子的转录；CCK-8检测细胞增殖，流式细胞术检测细胞周期和凋亡。结果显示：与对照组相比，mdv1-miR-M4-5p模拟物转染显著上调MDCC-MSB1细胞中mdv1-miR-M4-5p、cyclinD1、Bcl-2的转录水平，下调TGF-β1、Smad2、cyt-c、caspase-9和caspase-3的表达转录，促进细胞的增殖，减少G1期细胞，增加S和G2细胞，降低细胞凋亡率。与对照组相比，mdv1-miR-M4-5p抑制物转染后MDCC-MSB1细胞的增殖、凋亡及其相关分子转录的结果与mdv1-miR-M4-5p模拟物转染后的结果相反。综上，Mdv1-miR-M4-5p可促进MDCC-MSB1细胞增殖，抑制其凋亡，这可能是通过抑制TGF-β1/Smad2信号通路来实现的。

关键词: mdv1-miR-M4-5p, MDV, 增殖, 凋亡, TGF-β1/Smad2

Abstract:

The purpose of this experiment was to study the effects of mdv1-miR-M4-5p analog encoded by Marek's disease virus (MDV) on proliferation and apoptosis of MDCC-MSB1 cells. In this study, mdv1-miR-M4-5p mimics, inhibitors and their negative controls were transfected into MSB1 cells for 48 h. Real-time quantitative fluorescent PCR (qRT-PCR) was used to detect the transcription of mdv1-miR-M4-5p, TGF-β1, Smad2, caspase-9, caspase-3, cyt-c, cyclinD1, Bcl-2 et al other molecules related to proliferation and apoptosis in MDCC-MSB1 cells. Cell proliferation was detected by CCK-8 and cell cycle and apoptosis were detected by flow cytometry. The results showed that compared with the control group, the transcription levels of mdv1-miR-M4-5p, cyclinD1 and Bcl-2 in MDCC-MSB1 cells transfected with mdv1-miR-M4-5p mimics was significantly up-regulated. The transcription of TGF-β1, Smad2, cyt-c, caspase-9 and caspase-3 was down-regulated, the proliferation of MDCC-MSB1 cells was promoted, the the number of G1 phase cells were decreased, the S and G2 cells were increased, and the apoptosis rate was decreased. Compared with the control group, the results of the proliferation and apoptosis of MDCC-MSB1 cells and the expression of related molecules transfected with mdv1-miR-M4-5p inhibitor were opposite to those transfected with mdv1-miR-M4-5p mimics. The results showed that Mdv1-miR-M4-5p could promote the proliferation and inhibit the apoptosis of MDCC-MSB1 cells. This may be conducted by inhibiting the TGF-β1/Smad2 signaling pathway.

Key words: mdv1-miR-M4-5p, MDV, proliferation, apoptosis, TGF-β1/Smad2

中图分类号:

余祖华, 高梦茹, 何雷, 魏颖, 陈建, 陈松彪, 丁轲. mdv1-miR-M4-5p对MDCC-MSB1细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2024, 55(8): 3678-3687.

Zuhua YU, Mengru GAO, Lei HE, Ying WEI, Jian CHEN, Songbiao CHEN, Ke DING. Effects of mdv1-miR-M4-5p Encoded by MDV on Proliferation and Apoptosis of MDCC-MSB1 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3678-3687.

图/表 5

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名称 Name	引物序列(5′→3′) Sequence	用途 Application
U6 RT prime	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGATACA	反转录
U6 forward	CGCTTCGGCAGCACATATAC	Real-time PCR
miR-M4-5p RT prime	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAAGGGTT	反转录
miR-M4-5p forward	TGCGCTTAATGCTGTATCGGAAC	Real-time PCR
miR-M4-5p/U6 reverse	CCAGTGCAGGGTCCGAGGTATT	Real-time PCR
GAPDH forward	CCAGAACATCATCCCAGCGT	Real-time PCR
GAPDH reverse	CAGGTCAGGTCAACAACAGA	Real-time PCR
TGF-β1 forward	GTATTGGGCCAAAGAGCTGC	Real-time PCR
TGF-β1 reverse	ATTGCCGTAACCCTGGTACA	Real-time PCR
Smad2 forward	GTGGCAGATGAGGGTGATTT	Real-time PCR
Smad2 reverse	TGCAGCCACAGGACGAAAC	Real-time PCR
Caspase-3 forward	TGTGGACTCTGGAATTCTGC	Real-time PCR
Caspase-3 reverse	TGCCTGAACGAGATGACAGT	Real-time PCR
Caspase-9 forward	CCGAAGGGAGCAAGCACG	Real-time PCR
Caspase-9 reverse	AGGTTGGACTGGGATGGAC	Real-time PCR
Cyt-c forward	AGGCAAGCACAAGACTGGA	Real-time PCR
Cyt-c reverse	CTGACTATCACCAAGAACCACC	Real-time PCR
Bcl-2 forward	TACCAGAGGGACTTCGC	Real-time PCR
Bcl-2 reverse	TTGACCCCATCACGGA	Real-time PCR
cyclinD1 forward	AGGTTGGACTGGGATGGAC	Real-time PCR
cyclinD1 reverse	TGCAGCCACAGGACGAAACAG	Real-time PCR

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mdv1-miR-M4-5p对MDCC-MSB1细胞增殖和凋亡的影响

Effects of mdv1-miR-M4-5p Encoded by MDV on Proliferation and Apoptosis of MDCC-MSB1 Cells

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