Gene expression changes associated with knockdown of lncRNA PVT1 in ovarian cancer cell line SK-OV3. Gene expression changes associated with knockdown of lncRNA PVT1 in ovarian cancer cell line SK-OV3

lncRNA PVT1 is an emerging lncRNA of significance in cancer due to alterations in both the RNA and genomic locus in multiple cancers and its established relationship to the oncogene MYC. Several recent studies have documented potential important roles for the lncRNA in ovarian cancer. Herein RNA sequencing was performed to determine the impact of PVT1 on global gene expression by performing RNA sequencing in SK-OV3 cells after silencing PVT1 (siPVT1) of cells grown upon transient knockdown of the lncRNA PVT1. SK-OV3 cells were cultured to 50% confluence in 6 well plates. Pooled siRNA’s to human PVT1 or non targeting control siRNA’s from Dharmacon were used to transfect SK-OV3 cells for 48 hrs in full serum media carefully maintaining cell confluence to not exceed approximately 80%. This was followed by RNA extraction and verification of knockdown using primers to PVT1 followed by sequencing. We find that 450 protein coding genes were differentially expressed between control (siControl) and siPVT1 cells with 50 additional found to be non-protein coding. The top 50 differentially expressed genes include 12 that were downregulated by siPVT1 and 32 that were upregulated. Several pathways associated with metabolic and stress processes, ribosome biogenesis and ncRNA processing were altered based on GO pathway analysis. Additional pathways included pathways associated with cell motility and differentiation. Overall design: mRNA sequencing of human ovarian cancer (SKOV3) cells, control and PVT1 knockdown with siRNA Pooled siRNA’s to human PVT1 or scrambled control siRNA’s from Dharmacon were used to transfect SK-OV3 cells for 48 hrs followed by RNA extraction.

长链非编码RNA（long non-coding RNA, lncRNA）PVT1是一类在癌症中具有重要研究价值的新兴lncRNA，这是因为在多种癌症中，其RNA转录本与基因组位点均存在异常改变，且该分子与致癌基因MYC已被证实存在紧密关联。近年来多项研究已证实，该lncRNA在卵巢癌中可能发挥关键调控作用。本研究通过RNA测序技术，探究瞬时敲低PVT1后对SK-OV3细胞全局基因表达的影响：将SK-OV3细胞接种于6孔板中，培养至细胞汇合度达50%后，使用Dharmacon公司合成的人源PVT1混合小干扰RNA（pooled siRNA）或非靶向对照siRNA进行转染，在完全血清培养基中培养48小时，期间严格维持细胞汇合度不超过约80%。转染完成后，提取细胞总RNA，并利用针对PVT1的引物验证敲低效率，随后进行RNA测序。本研究发现，对照组（siControl）与siPVT1组细胞间共有450个蛋白编码基因存在差异表达，另有50个非蛋白编码基因出现表达差异。排名前50的差异表达基因中，12个在siPVT1处理后被下调，32个被上调。基于基因本体（Gene Ontology, GO）通路分析，代谢与应激过程、核糖体生物发生以及非编码RNA加工等相关通路发生显著改变，此外还包括细胞运动与分化相关通路。实验整体设计：对人卵巢癌SKOV3细胞进行mRNA测序，分为对照组与PVT1基因敲低组。使用Dharmacon公司合成的人源PVT1 pooled siRNA或乱序对照siRNA转染SK-OV3细胞，培养48小时后提取总RNA并进行测序。