Genome-wide small RNA sequencing analysis of KSHV long-term-infected endothelial (LTC) and parental uninfected TIVE cells [miRNA-seq]

Purpose: To characterize genome-wide microRNA (miRNA) expression profiles in the context of Kaposi's sarcoma-associated herpesvirus (KSHV)-induced oncogenesis using isogenic cell lines for a Kaposi's sarcoma (KS) xenograft model: KSHV-infected human endothelial cells (LTC) that form tumors with properties closely resembling KS lesions in nude mice and uninfected parental (TIVE) cells. Methods: miRNA expression profiles of LTC and TIVE cells were generated by deep sequencing using Illumina HiSeq 2500. The mappable reads were aligned to the human genome and miRBase using Bowtie. Results: We show, through global cellular miRNA transcriptome analysis, that KSHV infection has a profound impact on the host miRNA expression landscape, up-regulating multiple miRNAs with oncogenic roles while down-regulating many tumor suppressive miRNAs, thereby contributing to KS oncogenesis. In particular, we identify miR-127-3p as one of the most significantly down-regulated miRNAs in LTC and restoring this miRNA inhibits KSHV-induced transformation, proliferation and tumorigenesis. Conclusions: This study provides a detailed analysis of the cellular miRNA transcriptome in LTC and TIVE cells using small RNA sequencing technology. We identify significant dysregulation of cellular miRNAs with important oncogenic or tumor-suppressive functions that may be physiologically relevant to KSHV oncogenesis. Moreover, our results identify a previously unrecognized tumor suppressor function for miR-127-3p in KS, indicating that its restoration offers potential as a therapeutic intervention for KS. Overall design: miRNA expression profiles of LTC and TIVE cells were generated by deep sequencing using Illumina HiSeq 2500.

研究目的：本研究旨在利用卡波西肉瘤（Kaposi's sarcoma, KS）异种移植模型的同基因细胞系，解析卡波西肉瘤相关疱疹病毒（Kaposi's sarcoma-associated herpesvirus, KSHV）诱导肿瘤发生过程中的全基因组微小RNA（microRNA, miRNA）表达谱，该细胞系包含感染KSHV且可在裸鼠体内形成特性与KS病灶高度相似的肿瘤的人内皮细胞（LTC），以及未感染的亲代TIVE细胞。
研究方法：采用Illumina HiSeq 2500平台开展高通量测序，获取LTC与TIVE细胞的miRNA表达谱。通过Bowtie将可比对的测序reads比对至人类基因组及miRBase数据库。
研究结果：通过全细胞miRNA转录组分析可知，KSHV感染对宿主miRNA表达谱具有显著影响：可上调多种具有致癌功能的miRNA，同时下调大量抑癌miRNA，进而参与KS的肿瘤发生过程。尤为关键的是，本研究鉴定出miR-127-3p是LTC细胞中下调幅度最显著的miRNA之一；恢复该miRNA的表达能够抑制KSHV诱导的细胞转化、增殖及肿瘤发生。
研究结论：本研究借助小RNA测序技术，对LTC与TIVE细胞的细胞miRNA转录组进行了详细分析。我们鉴定出一批与KSHV肿瘤发生存在潜在生理相关性的、存在显著表达失调的兼具重要致癌或抑癌功能的细胞miRNA。此外，本研究发现miR-127-3p在KS中具有此前未被报道的抑癌功能，提示恢复其表达可作为KS的潜在治疗干预手段。
整体实验设计：采用Illumina HiSeq 2500平台开展高通量测序，获取LTC与TIVE细胞的miRNA表达谱。