以高通量定序方式解析微型核醣miR-148a於胃癌AGS細胞所扮演的角色

Abstract

微型核醣核酸為一種長約20~22鹼基小片段內生型核醣核酸，本身並不會轉錄出蛋白質，但會藉由結合標靶訊息核糖核酸抑制蛋白質的轉抑或是造成訊息核糖核酸的降解，進而調控基因表現。本實驗利用次世代定序RNA-seq研究微型核醣核酸148a對於胃癌細胞AGS的轉錄組造成的影響，進而探討微型核糖核酸148a可能尚未被發現的功能。從RNA-seq的資料中獲得了16,254個表現差異基因，利用基因表達量(RPKM)、基因表現量差異、錯誤發現率(FDR)來篩選顯著表現差異之基因，其中，有63個表現上升和48個表現下降的基因被挑選出來。藉由生物資訊工具，以基因本體(gene ontology)、基因功能性分析RNA-seq所挑選出的基因，得到前兩名結果分別為細胞死亡及細胞週期。在細胞集落形成測定法以及生長曲線監控實驗中，微型核糖核酸148a會抑制AGS細胞生長。利用4',6-二脒基-2-苯基吲哚(DAPI)染色在螢光顯微鏡的觀察下，並沒有發現DNA聚合、細胞凋亡情況發生；利用流式細胞儀，我們發現轉染微型核醣核酸148a的AGS細胞，在細胞週期G2/M族群分布增加。除此之外，磷酸化CDC2 (Thr-161)的蛋白質表現量下降，CDK4/6、Cyclin D1及磷酸化Rb的蛋白質表現量上升之結果，造成G1細胞周期活化進而使G2/M細胞週期累積有關。此實驗結果顯示，藉由RNA-seq揭露微型核糖核酸148a扮演調控細胞週期G1的新角色，可能提供了對於治療胃癌具有潛力的資訊。

MicroRNAs play an important role in various biological processes by post-transcriptionally regulating gene expression. To investigate the role of miR-148a in gastric cancer, the next generation RNA sequencing (RNA-seq) was applied to reveal the miR-148a-regulated gene expression profiles in gastric cancer cells. To uncover the role of miR-148a in gastric cancer, miR-148a was overexpressed in gastric cancer cell line AGS and followed by RNA-seq analysis. Using RNA-seq analysis, gene expression levels were estimated to identify differentially expressed genes in miR-148a-overexpressed cells. The biological processes and functional networks associated with the regulated genes were further analyzed by Ingenuity Pathway analysis (IPA). To validate the finding from RNA-seq, the expression level of the interested genes was measured by real-time reverse transcription PCR (qRT-PCR), the cell growth was monitored in real-time using the xCELLigence system, and the cell cycle analysis was examined by flow cytometry. We identified a total of 16,254 genes by comparing RNA-seq profiles of negative control (NTC) and miR-148a-overexpressed (Pre) AGS cells. Among them, 63 up-regulated and 48 down-regulated genes were differentially expressed in miR-148a-overexpressed cells. The IPA results showed that these miR-148a-regulated genes were significantly involved in the biological pathways including cell death and cell cycle. We demonstrated overexpression of miR-148a reduced the cell growth and alter the cell cycle G2/M phase population increased. This study illustrates the new role of miR-148a in controlling cell cycle by RNA-seq and provides information in gastric cancer therapy.