LncRNA SNHG9敲除对胶质瘤细胞增殖影响及其作用机制的生物信息学探讨

Effects of LncRNA SNHG9 knockout on glioma cell proliferation and bioinformatic exploration of its mechanism

目的利用CRISPR-Cas9结合流式单细胞分选构建lncRNA SNHG9基因敲除的U251细胞株,检测基因敲除对细胞增殖能力的影响。利用生物信息学分析探讨SNHG9基因发挥功能的可能机制。方法在SNHG9基因上、下游分别设计一个Cas9作用靶点,将两对单链向导RNA的引物模板退火连接px330-EGFP载体,构建成功的目的质粒共转染U251细胞后,通过流式单细胞分选在96孔板中培养,通过PCR扩增和测序验证筛选得到SNHG9敲除成功的单克隆细胞株并扩大培养。RTCA方法检测SNHG9敲除后细胞增殖能力的改变。利用在线工具分析lncRNA SNHG9的表达情况,查找共表达基因并进行富集分析。结果构建成功用于SNHG9敲除的质粒。培养共存活了8个克隆,且得到1株SNHG9成功敲除的细胞株。敲除细胞的增殖能力明显降低(P<0.05)。分析显示SNHG9在胶质母细胞瘤(GBM)组织中高表达,富集分析显示共表达基因主要与线粒体的功能相关。结论成功建立了SNHG9敲除的克隆细胞株且能抑制U251细胞的增殖,生物信息学分析显示SNHG9可能参与线粒体的功能调控。

Objective To establish SNHG9-knocked out U251 cells by combination of CRISPR/Cas9 and single cell sorting through flow cytometry,and test the effect of SNHG9 deletion on cell proliferation.And to explore the potential mechanism that SNHG9 involved by bioinformatic analysis.Methods Two targets were separately designed for Cas9 at the genomic sites upstream and downstream of SNHG9 gene,the two pairs of primers that guide the synthesis of sgRNA were annealed and cloned into px330-EGFP,and U251 cell were co-transfected with the two successfully constructed plasmids followed by single cell sorting into 96-well plates.Cell clones were raised and the genotypes were identified Gby PCR and sequencing,SNHG9-deleted clone cells were selected for subsequent experiments.Effect of SNHG9 deletion on cell proliferation was assessed by RTCA.Expression of lncRNA SNHG9,its co-expressed genes and their enrichment analysis were performed by online tools.Results Effective targets were designed and plasmid of SNHG9 knockout was successfully constructed.8 clones survived after single cell sorting and culture,from which 1 clone with SNHG9 totally deleted was obtained.The proliferation capacity of U251 cells significantly decreased after SNHG9 knocking-out(P<0.05).SNHG9 was overexpressed glioblastoma multiforme(GBM)samples when compared with normal tissues,the co-expressed genes most significantly enriched in mitochondrion related functions.Conclusion The SNHG9-deleted U251 clone was successfully established;SNHG9 knocking-out suppressed the proliferation of U251 cells;bioinformatic analysis showed that SNHG9 might involve in the regulation of mitochondrial function.