探討經由外泌體傳播之類致癌基因長鏈非編碼核糖核酸促使膀胱癌細胞上皮間質轉化之機制
黃晟碩1、何嘉益2、于承平3、于大雄4
1國防醫學院病理及寄生蟲研究所，2國防醫學院生命科學研究所，3三軍總醫院病理部，4三軍總醫院外科部泌尿外科
The Study of Mechanisms of Exosome-transmitted Like Oncogene Long non-Coding RNA Promotes Epithelial-Mesenchymal Transition in Bladder Cancer Cells
Cheng-Shuo Huang1 , Jar-Yi Ho2, Cheng-Ping Yu3 , Dah-Shyong Yu4
1Graduate Institute of Pathology and Parasitology, National Defense Medical Center, 2Graduate Institute of Life Science, National Defense Medical Center, 3Department of Pathology, Tri-Service General Hospital, 4Division of Urology, Department of Surgery, Tri-Service General Hospital
Purpose. This study was aimed to evaluate how high-grade bladder cancer cells derived exosomes aggravated cellular functions of low- grade bladder cancer cells, including cell proliferation, migration, and invasiveness. And this study also tried to characterize which exosome–transmitted long non-coding (lnc) RNAs promoted bladder cancer progression and to realize their regulatory mechanisms.
Materials and Methods. High-grade urothelial carcinoma (UC) cell lines, T24 and J82 and low-grade UC cell line TSGH-8301 were employed in this study. Exosomes were extracted from the conditioned medium using the ultracentrifuge method. The qualitative and quantitative of exosomes were analyzed by Nanoparticle Tracking Analysis (NTA), western blots, and transmission electron microscope (TEM). The biological effects were evaluated in TSGH-8301 cells after treated with 0.2 μm-filtered conditioned medium or exosomes derived from T24 or J82 cells, including cell proliferation activity using MTT assay and migration/invasion activity using wound-healing and transwell assays. Exosome-transmitted lncRNA candidates were screened with bioinformatic pipelines (the lnCAR database) and validated with RT-q PCR in bladder cancer cells and exosomes. Two novel lncRNAs, LINC00960 and LINC02470, were selected for investigation of their molecular regulation in tumor cells with western blots.
Results. The average exosome diameters were similar among TSGH-8301, T24, and J82 (116.8 nm, 118.5 nm, and 124.2 nm, respectively). Conditioned media and exosomes of high-grade bladder cancer cells (T24 and J82) enhanced cell proliferation, migration, and invasion of low-grade bladder cancer cell (TSGH-8301). Two lncRNA candidates, LINC00960 and LINC02470, were significantly higher in T24 and J82 cells and exosomes than TSGH-8301. In molecular regulation, T24 or J82 derived exosome induced epithelial-mesenchymal transition (EMT) via upregulated NOTCH1, HES1 signaling and SLUG/SNAIL/TWIST/ZEB2 transcription factors in TSGH8301.
Conclusion. Our findings indicate that exosomes-transmitted LINC00960 and LINC02470 of high-grade bladder cancer cells can aggravate low-grade bladder cancer cells and promote epithelial-mesenchymal transition via upregulating NOTCH1, HES1 signaling, and SLUG/SNAIL/TWIST/ZEB2 transcription factors.