基于生物信息学对SLC25A21下游靶基因的筛选及验证

doi:10.16288/j.yczz.24-230

遗传 ›› 2024, Vol. 46 ›› Issue (12): 1055-1065.doi: 10.16288/j.yczz.24-230

基于生物信息学对SLC25A21下游靶基因的筛选及验证

陈瑶(), 温馨, 袁芳媛, 彭钞灵, 王翠喆, 张君(), 孟平平()

石河子大学医学院，新疆地方与民族高发病教育部重点实验室，石河子 832000

收稿日期:2024-08-25 修回日期:2024-10-25 出版日期:2024-12-20 发布日期:2024-11-15
通讯作者: 张君，博士，教授，研究方向：肥胖、炎症及代谢相关疾病中关键信号转导分子的调控机制和干预方式基础研究。E-mail: zhangjunyc@163.com;
孟平平，硕士，讲师，研究方向：代谢相关性疾病方面的研究。E-mail: mengvictoria@126.com
作者简介:陈瑶，硕士研究生，专业方向：生物化学与分子生物学。E-mail: 1694094508@qq.com
基金资助:
国家自然科学基金项目(82260162);新疆维吾尔自治区“天山英才”项目(2023TSYCCX0116);新疆维吾尔自治区“天山英才”项目(2023TSYCQNTJ0032);新疆生产建设兵团指导性科技计划项目(2022ZD001);新疆生产建设兵团指导性科技计划项目(2023ZD037)

Screening and validation of downstream target genes of SLC25A21 based on bioinformatics

Yao Chen(), Xin Wen, Fangyuan Yuan, Chaoling Peng, Cuizhe Wang, Jun Zhang(), Pingping Meng()

Key laboratory of Xinjiang Endemic and Ethnic Diseases, School of Medicine, Shihezi Uniiversity, Shihezi 832000, China

Received:2024-08-25 Revised:2024-10-25 Published:2024-12-20 Online:2024-11-15
Supported by:
National Natural Science Foundation Project of China(82260162);Tianshan Talent Project of Xinjiang Autonomous Region(2023TSYCCX0116);Tianshan Talent Project of Xinjiang Autonomous Region(2023TSYCQNTJ0032);Guiding Science and Technology Planning Project of Xinjiang Production and Construction Corps(2022ZD001);Guiding Science and Technology Planning Project of Xinjiang Production and Construction Corps(2023ZD037)

摘要/Abstract

摘要：

溶质载体家族25成员21 (solute carrier 25 member 21, SLC25A21)是氧代二羧酸盐载体，其主要功能是通过反向交换机制将胞浆内的2-氧代己二酸转运至线粒体。前期研究发现，在过表达SLC25A21的3T3-L1细胞中葡萄糖消耗能力显著增加。为进一步挖掘SLC25A21下游关键代谢基因，本研究在3T3-L1细胞中上调SLC25A21表达，利用高通量测序结合生物信息学分析获得差异表达基因，并通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction，qRT-PCR)验证关键基因表达。结果显示：(1)过表达SLC25A21的脂肪细胞中有26个上调基因，66个下调基因；(2)基因本体论(gene ontology, GO)分析表明，差异表达基因的生物学功能主要涉及脂质合成和代谢过程；京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes，KEGG)和基因集富集分析(gene set enrichment analysis, GSEA)表明，差异表达基因主要富集在鞘脂代谢、胰岛素和胰高血糖素样肽1分泌和合成等信号通路；(3) CytoHubba筛选出GRB2、SOS1、SHC1、CBL、HRAS、SOS2、EGFR、MET、PLCG2和KRAS等10个评分最高的关键基因，主要参与了细胞的糖、脂代谢过程；(4)在脂肪细胞中过表达SLC25A21，qRT-PCR验证结果表明除KRAS表达无明显变化外，其余基因的mRNA表达水平均出现相应的增高。本研究结果将为今后深入研究SLC25A21在糖、脂代谢过程中的作用及机制提供理论依据。

关键词: SLC25A21, 生物信息学, 差异表达基因, 筛选及验证

Abstract:

Solute carrier 25 member 21 (SLC25A21) serves as an oxodicarboxylate carrier, which mainly conveys 2-oxoadipate from the cytoplasm to the mitochondria via a reverse exchange mechanism. Previous studies have indicated that the capacity for glucose consumption is significantly enhanced in 3T3-L1 cells overexpressing SLC25A21. In this study, we upregulate SLC25A21 in 3T3-L1 cells to further probe into the downstream key metabolic genes of SLC25A21. Through high-throughput sequencing combined with bioinformatics analysis, differentially expressed genes are obtained, and the expression of key genes is verified by qRT-PCR. The results demonstrat that: (1) There are 26 up-regulated genes and 66 down-regulated genes in the adipocytes overexpressing SLC25A21; (2) GO (gene ontology) analysis indicates that the biological functions of differentially expressed genes are predominantly involved in lipid synthesis and metabolism, and KEGG (Kyoto encyclopedia of genes and genomes) and GSEA (gene set enrichment analysis) analyses reveal that differentially expressed genes are mainly concentrated in sphingolipid metabolism, secretion and synthesis of insulin and glucagon-like peptide 1; (3) By means of cytoHubba, 10 key genes with the highest scores, such as GRB2, SOS1, SHC1, CBL, HRAS, SOS2, EGFR, MET, PLCG2 and KRAS, were screened out and they are mainly involved in the sugar and lipid metabolism processes of cells; (4) SLC25A21 is overexpressed in adipocytes, and the qRT-PCR verification results show that the mRNA expression levels of other genes increased correspondingly, except for KRAS expression, which exhibits no significant change. These results provide a theoretical basis for further investigations on the role and mechanism of SLC25A21 in the process of glucose and lipid metabolism.

Key words: SLC25A21, bioinformatics, differentially expressed gene, screening and verification

陈瑶, 温馨, 袁芳媛, 彭钞灵, 王翠喆, 张君, 孟平平. 基于生物信息学对SLC25A21下游靶基因的筛选及验证[J]. 遗传, 2024, 46(12): 1055-1065.

Yao Chen, Xin Wen, Fangyuan Yuan, Chaoling Peng, Cuizhe Wang, Jun Zhang, Pingping Meng. Screening and validation of downstream target genes of SLC25A21 based on bioinformatics[J]. Hereditas(Beijing), 2024, 46(12): 1055-1065.

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基因	上游引物序列(5′→3′)	下游引物序列(5′→3′)
GRB2	GCAAAATCCCCAGAGCCAAG	GCAAAATCCCCAGAGCCAAG
SOS1	GAGACATCCCACACCTCTGC	ATGCTGTGCTTTCCGTCTCA
SHC1	TTCGCCAAGACAACTGAGCA	TGCCCGAAAGCAGAAGGTAG
CBL	CAGGCAGGGAGTTAGCACAA	TCACGGCAAGCCTACAGAAC
HRAS	ACCGGAAACAGGTGGTCATT	TCCCGCATGGCACTATACTC
KRAS	GCGCCTTGACGATACAGCTAA	TACACAAAGAAAGCCCTCCCC
SOS2	GGCGTTAGAAAAAGGCGAGC	AACCCAGTGGCGAAAGACAT
EGFR	GCAATGTTCCCATCGCTGTC	CAGGTGTCTTTGCATGTGGC
PLCG2	GATGAGGCTTCGATACCCCG	CTGCGCTTGGCTTTGTAGTC
MET	CCCAGCCCAAACTACCTCTG	ACCAGCTTTGGGAGGCTAAC

样本	原始reads 数目(M)	原始测序量(Gb)	过滤后的 reads数目(M)	过滤后的测序量(Gb)	有效碱基百分比(%)	Q30(%)	GC(%)
NC1	48.63	7.16	46.9	6.91	96.45	93.26	50.74
NC2	49.27	7.26	47.57	7.01	96.54	93.34	50.59
NC3	49.27	7.23	47.35	6.95	96.1	93.31	50.75
OE1	49.87	7.32	47.93	7.04	96.11	93.17	50.74
OE2	48.81	7.2	47.11	6.95	96.52	93.1	50.63
OE3	48.29	7.14	46.79	6.92	96.9	93.32	50.28

样本	Total reads	Total mapped reads	Multiple mapped	Uniquely mapped	Read	Non-splice reads	Splice reads	Reads mapped in proper pairs
NC1	46899752	45533180 (97.09%)	2265197 (4.83%)	43267983 (92.26%)	21654638 (46.17%)	24015475 (51.21%)	19252508 (41.05%)	42694074 (91.03%)
NC2	47565180	46131900 (96.99%)	2324947 (4.89%)	43806953 (92.10%)	21919628 (46.08%)	24346626 (51.19%)	19460327 (40.91%)	43211796 (90.85%)
NC3	47350582	45976707 (97.10%)	2344644 (4.95%)	43632063 (92.15%)	21837383 (46.12%)	24004218 (50.69%)	19627845 (41.45%)	43059252 (90.94%)
OE1	47933810	46408234 (96.82%)	2373324 (4.95%)	44034910 (91.87%)	22039724 (45.98%)	24351292 (50.80%)	19683618 (41.06%)	43439056 (90.62%)
OE2	47107546	45678757 (96.97%)	2316938 (4.92%)	43361819 (92.05%)	21709112 (46.08%)	23931808 (50.80%)	19430011 (41.25%)	42764390 (90.78%)
OE3	46791584	45401464 (97.03%)	2315495 (4.95%)	43085969 (92.08%)	21568015 (46.09%)	24044762 (51.39%)	19041207 (40.69%)	42485994 (90.80%)

基于生物信息学对SLC25A21下游靶基因的筛选及验证

Screening and validation of downstream target genes of SLC25A21 based on bioinformatics

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