INTS11通过介导CDK2和CYCLIND1的转录促进牛成肌细胞增殖

doi:10.11843/j.issn.0366-6964.2024.07.013

摘要/Abstract

摘要：

旨在验证牛INTS11基因在成肌细胞中的功能作用，通过生物信息学分析探讨INTS11的CDS区序列及其编码蛋白的特征，并使用不同分子试验技术验证其在牛成肌细胞增殖过程中发挥的作用。本研究对牛INTS11基因序列与其他物种进行同源性对比并构建生物进化树，对其编码蛋白进行理化性质和功能结构分析以及亚细胞定位。并以体外分离培养的3月龄健康胎牛成肌细胞为试验材料，克隆成肌细胞中INTS11的全部CDS区序列，构建INTS11的过表达载体并设计基因的干扰序列，通过CCK8、Edu、RT-qPCR等技术探究其对成肌细胞增殖的影响。生物信息分析结果表明，CDS区序列全长为1 800 bp，具有MBL-foldmetallo-hydro和β-CASP基序两个功能结构域，属于非跨膜蛋白，且细胞定位主要分布于细胞质。在成肌细胞中转染pcDNA3.1-INTS11质粒，结果显示在72~96 h时试验组的成肌细胞比对照组的增殖速度显著增加(P＜0.05)；Edu试验发现阳性细胞数目显著增加(P＜0.05)；RT-qPCR结果表明增殖标志因子CDK2、CYCLIND1的mRNA表达水平与对照组相比显著上升(P＜0.05)。转入干扰序列后，细胞增殖速度显著下降(P＜0.05)，同时增殖标志因子CDK2、cyclin D1的mRNA表达水平与对照组相比显著下降(P＜0.05)。本研究预测了INTS11在家养动物中的保守性, 并且INTS11蛋白具有两个功能结构域，发现其通过介导CDK2、CYCLIND1的mRNA转录促进牛成肌细胞增殖，其结果完善了调控骨骼肌的基因网络，为后序探讨调控骨骼肌生长机制提供理论支持。

关键词: INTS11, 成肌细胞, 细胞增殖, 过表达, 干扰

Abstract:

The study aimed to verify the functional role of bovine INTS11 gene in myogenic cells, explore the sequence of the CDS region of INTS11 and the characteristics of its encoded protein by bioinformatics analysis, and verify its role in the proliferation process of bovine myogenic cells using different molecular test techniques. The bovine INTS11 gene sequence was compared with other species. This involved the construction of a phylogenetic tree and the examination of the physicochemical properties, functional structure, and subcellular localization of the encoded protein. The test material comprised 3-month-old healthy fetal bovine myoblasts isolated and cultured in vitro. The sequences of the coding sequence (CDS) region of INTS11 in myoblasts were cloned. An overexpression vector of INTS11 was constructed, and interfering sequences of the gene were designed. This was done in order to investigate its effect on the proliferation of myoblasts by CCK8, Edu, RT-qPCR and other techniques. Bioinformatic analysis showed that INTS11 had a CDS region of 1 800 bp, two functional domains (MBL-foldmetallo-hydro and β-CASP motif), which belonged to the non-transmembrane proteins and the cellular localization was mainly distributed in the cytoplasm. Transfection of pcDNA3.1-INTS11 plasmid showed that the proliferation rate of myoblasts in the test group was significantly increased compared with that in the control group at 72-96 h (P<0.05); Edu assay revealed a significant increase in the number of positive cells (P<0.05); and the RT-qPCR results showed that the mRNA expression levels of proliferation marker factors CDK2 and CYCLIND1 were significantly increased (P<0.05) compared with that in the control group. After transferring the interference sequence, the cell proliferation rate was significantly decreased (P<0.05), while the mRNA expression levels of proliferation marker factors CDK2 and CYCLIND1 were significantly decreased (P<0.05) compared with the control group. In this study, the conservation of INTS11 was predicted in the domesticated animals, and that INTS11 protein has two functional structural domains, and it promotes the proliferation of bovine myoblasts by mediating the transcription of CDK2 and CYCLIND1. This research has perfected the gene network regulating skeletal muscle growth, provided theoretical support for further exploration of the regulating mechanisms of skeletal muscle growth in the post-sequence.

Key words: INTS11, myoblast, cell proliferation, overexpression, interference

中图分类号:

S823.2

王子岩, 王亚慧, 吴天弋, 高晨, 杜振伟, 葛菲, 张晓贝, 赵文轩, 张路培, 高会江, 董焕声, 李俊雅. INTS11通过介导CDK2和CYCLIND1的转录促进牛成肌细胞增殖[J]. 畜牧兽医学报, 2024, 55(7): 2927-2939.

Ziyan WANG, Yahui WANG, Tianyi WU, Chen GAO, Zhenwei DU, Fei GE, Xiaobei ZHANG, Wenxuan ZHAO, Lupei ZHANG, Huijiang GAO, Huansheng DONG, Junya LI. INTS11 Promotes the Proliferation of Bovine Myoblasts by Mediating the Transcription of CDK2 and CYCLIND1[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 2927-2939.

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基因Gene	上游引物(5′→3′) Forward primer sequence	下游引物(5′→3′) Reverse primer sequence
INTS11	AGGATGCCCGAGATCAGGGT	TGTTCTCTACACAGATGAGCGATG
INTS11-m	ccaagctggctagttaagcttATGCCCGAGATCAGGGTCA	agtggatccgagctcggtaccTCAGCTGGCCTGGGGCA

基因Gene	上游引物(5′→3′) Forward primer sequence	下游引物(5′→3′) Reverse primer sequence
INTS11	AGATCAGGGTCACGCCCTT	GCAGTCCAGAAAGTCGGTCA
CDK2	TCTTTGCTGAGATGGTGACCC	TAACTCCTGGCCAAACCACC
CYCLIND1	GACGAGCTGCTGCACATGGA	TGCTTGTTCTCCTCGGCCAC
GAPDH	CGAGCTGCTGCACATG	GGCGACGATGTCCACTTTG

SOPMA	数量 Number	百分比/% Percentage
α-螺旋Alpha helix(Hh)	219	36.56
延伸链Extended strand(Ee)	121	20.20
β-转角Beta turn(Tt)	33	5.51
无规则卷曲Random coil(Cc)	226	37.73

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