在HTR-8细胞中BCL6参与复发性流产的可变剪接调控

Involvement of BCL6 in variable splicing regulation of recurrent abortion in HTR-8 cells

目的构建慢病毒过表达B细胞淋巴瘤6(BCL6)的HTR-8稳定细胞模型进行高通量测序,分析其下游的可变剪接基因及其通路。方法BCL6过表达的慢病毒感染HTR-8细胞1~2周获得稳定细胞系后采用Illumina Novaseq6000高通量测序平台执行双端测序,并借助分层索引(HISAT2)工具对各实验样本的转录本剪接事件进行了比对分析。采用学生t检验(t-test)方法比较每个基因的同一剪接类型在不同细胞样本中的可变剪接水平变化,筛选出差异表达的可变剪接的基因(DASG)和差异表达基因(DEG)进行基因本体(GO)和京都基因与基因组百科全书(KEGG)分析富集分析。结果测序结果显示过表达BCL6的HTR-8细胞在可变剪接事件的比例均高于对照组,其中内含子保留发生频率明显增加。测序共筛选出880个DASG基因,670个DEG基因,其中共表达32个基因。GO富集显示生物功能富集结果为蛋白质结合,主要存在于细胞溶质中,KEGG通路富集在淀粉和蔗糖代谢。结论过表达BCL6基因作用于单纯疱疹病毒I型感染和淀粉蔗糖代谢通路增加可变剪接的位点数目进一步引起复发性流产的发生,BCL6可能是复发性流产的潜在生物学标志物。

Objective To construct a lentiviral overexpression of B cell lymphoma 6(BCL6)gene in the HTR-8 stable cell model for high-throughput sequencing to analyze potential variable splicing sites and pathways.Methods Lentiviral infection of HTR-8 cells for 1-2 weeks to obtain a stable cell line was followed by bipartite sequencing using the Illumina Novaseq 6000 sequencing platform,and hierarchical indexing for spliced alignment of transcripts(HISAT2)was used to analyze the splicing sites of each sample.The student t-test(t-test)was utilized to filter out differentially expressed genes(DEG)and differentially alternative splicing genes(DASG)by comparing the change in variable splicing level of the same splicing type of each gene in various cell samples.DASG and DEG were improved for Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)study.Results Sequencing results showed that HTR-8 cells overexpressing the BCL6 gene had a higher proportion of variable splicing events than controls in all cases,in which intron retention occurred with significantly increased frequency.Sequencing screened a total of 880 DASG genes,and 670 DEG genes,of which a total of 32 genes were expressed,and GO enrichment showed that the biological functions were mainly protein binding,which existed with the cytosol.KEGG pathway was enriched in starch and sucrose metabolism.Conclusion Overexpression of the BCL6 gene acts on herpes simplex virus type I infection and starch-sucrose metabolism pathway to increase the number of variable splicing sites to cause the occurrence of recurrent miscarriage further,BCL6 may be a potential biomarker for recurrent miscarriage.