Next Generation Sequencing from Tumor Tissues And Tumor-adjacent Tissues of Renal Cell Carcinoma. Next Generation Sequencing from Tumor Tissues And Tumor-adjacent Tissues of Renal Cell Carcinoma

Tumor-adjacent tissues are very important for tumor research. This study aimed to identify hub genes in tumor-adjacent tissues of mice transplanted with 786-o renal cell carcinoma cells. 786-o cells were implanted in nude mice raised to 1 month of age, and normal tissues, tumor tissues, and adjacent tissues were obtained for RNA sequencing. All tissues were sequenced for transcriptome genes. This was the first systematic exploration of the hub genes in the tumor-adjacent tissues of mice transplanted with 786-o renal cell carcinoma cells, which provides a genetic basis for further in-depth study on the underlying regulatory mechanism of renal cell carcinoma. Overall design: Ten 4-week-old male BALB/c nude mice were obtained from the Animal Center of Shanghai Tenth People’s Hospital (Shanghai, China), every five mice were kept in a cage, kept under specific pathogen-free conditions at 21±2°C and 55±10% humidity under a 12 h light/dark cycle, and fed normal chow with ad libitum access to water. All mice were treated humanely. The 786-o cells were cultured in a 10 cm dish (DMEM medium, 10% fetal bovine serum, PS double antibody). When the cell density was 70-80%, the cells were collected, mixed into a cell suspension with 100 µl sterile phosphate-buffered saline solution, and injected into the right back of a nude mouse at approximately the midpoint of the spinal column and right lower limb. Before injection, 0.5 cm of tissue was taken from the tail of each nude mouse. The mice were fed normally, and the growth of transplanted tumors was observed every day. Seven of the ten mice transplanted tumors successfully. One month later, three mice with tumor diameter of 1-1.5 cm were selected for the next study. Mice were euthanized in a carbon dioxide box, and the tumor and tumor-adjacent tissues (more than 1 cm from tumor tissue capsule) were removed. All tissues were stored in liquid nitrogen and sent to Shanghai Majorbio company for RNA-seq analysis. Different tissues of each mouse were self-compared, three mice were repeated three times.

癌旁组织在肿瘤研究中具有至关重要的研究价值。本研究旨在筛选移植786-O肾细胞癌细胞的裸鼠癌旁组织中的枢纽基因（hub gene）。将786-O细胞接种于饲养至1月龄的裸鼠体内，收集正常组织、瘤体组织及癌旁组织进行RNA测序（RNA sequencing），对所有组织的转录组基因进行测序分析。本研究是首次针对移植786-O肾细胞癌细胞的裸鼠癌旁组织中的枢纽基因开展系统性探究，可为后续深入解析肾细胞癌的潜在调控机制提供遗传学依据。

整体实验设计：从中国上海第十人民医院动物中心购入10只4周龄雄性BALB/c裸鼠，每5只饲养于同一笼具中，饲养环境为特定无病原体（specific pathogen-free, SPF）级：温度控制在21±2℃，湿度为55±10%，光照周期为12小时明暗交替，饲喂正常饲料并自由饮水。所有小鼠均以人道方式进行饲养与实验处理。

将786-O细胞培养于10cm培养皿中，采用DMEM培养基、10%胎牛血清及青霉素-链霉素双抗（PS双抗）进行培养。当细胞融合度达70%-80%时，收集细胞并以100μl无菌磷酸盐缓冲液（phosphate-buffered saline, PBS）重悬为细胞悬液，将其注射至裸鼠脊柱中点与右下肢之间的右侧背部区域。注射前，从每只裸鼠尾部截取0.5cm组织样本。随后正常饲养小鼠，每日观察移植瘤的生长情况。10只裸鼠中共有7只成瘤，饲养1个月后，选取3只瘤体直径为1-1.5cm的裸鼠开展后续实验。采用二氧化碳窒息法处死裸鼠，摘取瘤体及癌旁组织（距肿瘤包膜1cm以上区域）。所有组织样本均置于液氮中保存，并送至上海美吉生物医药（Majorbio）进行RNA测序（RNA-seq）分析。每只裸鼠的不同组织样本均进行自身对照，共设置3次生物学重复。