tRF-Pro-CGG对小...物学行为的影响及其分子机制_符庆胜.pdf

241欢迎关注本刊公众号中国癌症杂志2023年第33卷第3期 CHINA ONCOLOGY 2023 Vol.33 No.3论 著tRF-Pro-CGG对小鼠胰腺癌细胞生物学行为的影响及其分子机制符庆胜1，2，金雷2，张旭东2，徐荧晨1，朱春富2，秦锡虎2，吴宝强1，21.蚌埠医学院研究生院，安徽 蚌埠 233030；2.常州市第二人民医院肝胆胰外科，江苏 常州 213003摘要 背景与目的：tRNA衍生的片段（tRNA-derived fragments，tRF）是一类长度为14 30 nt的小分子非编码RNA，其影响着恶性肿瘤的发展进程。本研究旨在探讨tRF-Pro-CGG对小鼠胰腺癌细胞生物学行为的影响及其可能的分子机制。方法：采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测tRF-Pro-CGG在小鼠胰腺癌细胞系pan02、LTPA，人胰腺癌细胞系Capan-2和正常胰腺细胞HPDE6-C7中的表达水平。通过慢病毒转染技术过表达pan02细胞及敲低LTPA细胞中tRF-Pro-CGG的表达，采用RTFQ-PCR和蛋白质印迹法（Western blot）检测过表达和敲低效果。采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）检测细胞增殖情况。采用transwell实验检测细胞迁移和侵袭能力。采用动物模型检测tRF-Pro-CGG对胰腺癌裸鼠移植瘤生长和转移的影响。采用H-E染色观察移植瘤的组织病理学结构。采用Western blot检测移植瘤组织中Ki-67增殖指数、转移相关蛋白E-钙黏蛋白（E-cadherin）、波形蛋白（vimentin）的表达及磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）/蛋白激酶B（protein kinase B，AKT）信号转导通路蛋白的表达及磷酸化情况。结果：小鼠胰腺癌细胞系pan02中tRF-Pro-CGG表达最低，在pan02细胞中转染tRF-Pro-CGG mimics后，tRF-Pro-CGG的mRNA和蛋白表达水平均显著升高（P0.01），细胞增殖能力显著降低（P0.01），细胞迁移（P0.001）和侵袭能力（P0.001）显著降低，裸鼠移植瘤体积（P0.01）和重量（P0.001）均显著降低，裸鼠移植瘤组织中出现明显坏死和凋亡细胞；裸鼠移植瘤组织中Ki-67增殖指数和vimentin的表达显著降低（P0.001），而E-cadherin的表达显著升高（P0.001），PI3K、P-PI3K、AKT和P-AKT的表达显著降低（P0.001），胰腺癌肝转移例数差异无统计学意义（P0.05）。小鼠胰腺癌细胞系LTPA中tRF-Pro-CGG表达最高，在LTPA细胞中转染tRF-Pro-CGG inhibitor后，tRF-Pro-CGG的mRNA和蛋白表达水平显著降低（P0.01），细胞增殖能力显著升高（P0.01），细胞迁移（P0.001）和侵袭能力（P0.001）显著升高，裸鼠移植瘤体积（P0.01）和重量（P0.01）均显著升高，裸鼠移植瘤组织中出现少量的坏死及凋亡细胞；裸鼠移植瘤组织中Ki-67和vimentin的表达显著升高（P0.001），而E-cadherin的表达显著降低（P0.001），PI3K、P-PI3K、AKT和P-AKT的表达显著升高（P0.001），胰腺癌肝转移例数差异无统计学意义（P0.05）。结论：过表达tRF-Pro-CGG可以抑制小鼠胰腺癌细胞增殖、迁移和侵袭，抑制胰腺癌裸鼠移植瘤的生长，下调Ki-67增殖指数、vimentin的表达水平和PI3K/AKT磷酸化水平。tRF-Pro-CGG可能通过调节PI3K/AKT信号转导通路抑制胰腺癌的发生、发展。关键词 胰腺癌；tRNA衍生的片段-Pro-CGG；增殖；迁移；侵袭；裸鼠移植瘤模型中图分类号：R735.9 文献标志码：ADOI:10.19401/ki.1007-3639.2023.03.007Effect of tRF-Pro-CGG on the biological behavior of mouse pancreatic cancer cells and its molecular mechanism FU Qingsheng1,2,JIN Lei2,ZHANG Xudong2,XU Yingchen1,ZHU Chunfu2,QIN Xihu2,WU Baoqiang1,2(1.School of Graduate,Bengbu Medical College,Bengbu 233030,Anhui Province,China;2.Department of Hepatobiliary and Pancreatic Surgery,Changzhou No.2 Peoples Hospital,Changzhou 213003,Jiangsu Province,China)Correspondence to:WU Baoqiang,E-mail:.基金项目：常州市卫健委科技项目（ZD202023）。第一作者：符庆胜（ORCID:0000-0002-0764-6854），硕士，住院医师。通信作者：吴宝强（ORCID:0000-0001-5811-5212），博士，主任医师，E-mail:。242符庆胜，等 tRF-Pro-CGG对小鼠胰腺癌细胞生物学行为的影响及其分子机制Abstract Background and purpose:tRNA-derived fragments(tRF)are a kind of short non-coding RNA(14-30 nt)that influences the course of cancer.This study aimed to investigate the molecular pathways that might underlie the effects of tRF-Pro-CGG on the biological behavior of mouse pancreatic cancer cells.Methods:Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was used to assess the expression of tRF-Pro-CGG in mouse pancreatic cancer cell lines pan02 and LTPA,human pancreatic cancer cell line Capan-2,and normal pancreatic cells HPDE6-C7.tRF-Pro-CGG overexpression in pan02 cells and LTPA cell suppression were achieved through lentiviral transfection,and RTFQ-PCR and Western blot were used to determine overexpression and knockdown effects.Cell counting kit-8(CCK-8)was used to detect cell proliferation.Transwell assays were used to detect cell migration and invasion ability.The effect of tRF-Pro-CGG on the growth and metastasis of pancreatic cancer transplantation tumors in nude mice model was investigated.H-E staining was used to observe the histopathological structure of transplantation tumors.Western blot was used to detect the expression and phosphorylation of proliferation-related protein Ki-67 and metastasis-related proteins.Western blot was used to assess the expressions of cadherin,vimentin,phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)pathway protein and phosphorylation in transplanted tumor tissues.Results:tRF-Pro-CGG expression was lowest in the mouse pancreatic cancer cell line pan02.Both mRNA and protein expression levels of tRF-Pro-CGG were significantly increased(P0.01)after transfection of tRF-Pro-CGG mimics in pan02 cells,and cell proliferation ability(P0.01),cell migration(P0.001)and invasion ability(P0.001)were significantly reduced.A significant decrease in the volume(P0.01)and weight(P0.001)of transplanted tumors in nude mice was observed,and significant necrotic and apoptotic cells in transplanted tumor were identified.In transplanted tumor tissues of nude mice,the Ki-67 proliferatien index and expression of vimentin were significantly decreased(P0.001),while E-cadherin was increased(P0.001).The expressions of PI3K,P-PI3K,AKT and P-AKT were significantly decreased(P0.001).There was no significant difference in the number of liver metastases from pancreatic cancer(P0.05).The mouse pancreatic cancer cell line LTPA had the greatest level of tRF-Pro-CGG expression.The mRNA and protein expression levels of tRF-Pro-CGG were significantly reduced (P0.01)after transfection of tRF-Pro-CGG inhibitor in LTPA cells.The proliferation ability of cells was significantly increased(P