环状非编码RNA hsa_circ_001842对肾癌细胞增殖、凋亡、侵袭和成瘤能力的影响

Effects of circular non-coding RNA hsa_circ_001842 on proliferation，apoptosis，invasion and tumorigenicity of renal cancer cells

摘要:

目的：探究环状非编码RNA hsa_circ_001842作为癌基因在肾细胞癌（renal cell carcinoma，RCC）患者肾癌组织及细胞中的表达情况，以及其对肾癌细胞增殖、凋亡、侵袭与成瘤能力的影响。方法：收集2018年9月至2019年10月绵阳市中心医院泌尿外科进行手术切除的肾癌标本及癌旁组织标本各64例。利用qRT-PCR检测所有RCC组织与癌旁正常组织中hsa_circ_001842的表达情况。将Caki-1肾癌细胞分别转染hsa_circ_001842基因过表达质粒（ox-circ_001842）、沉默质粒（sh-circ_001842）和空质粒（ox-NC和sh-NC），采用细胞克隆形成实验、流式细胞术、细胞划痕实验和细胞侵袭实验，检测hsa_circ_001842对肾癌细胞增殖凋亡和迁移侵袭能力的影响。使用Western blot检测各组肾癌细胞上皮间充质转化（epithelial mesenchymal transition，EMT）相关蛋白与MMP-9蛋白表达水平。将转染sh-NC和sh-circ_001842的Caki-1肾癌细胞分别接种于裸鼠皮下，每7天测量一次肿瘤体积及体质量，4周后处死各组小鼠，并使用免疫组化技术和qRT-PCR检测肿瘤组织中EMT相关蛋白的表达情况。结果：RCC患者肾癌组织中hsa_circ_001842的表达量明显高于癌旁正常组织（P<0.05）。体外实验表明，与ox-NC组和sh-circ_001842组相比，ox-circ_001842组和sh-NC组Caki-1肾癌细胞的集落形成率、划痕愈合比率、细胞侵袭能力和E-cadherin蛋白的表达均明显增加（P<0.05），而细胞凋亡率、N-cadherin和MMP-9蛋白的表达明显降低（P<0.05）。裸鼠成瘤实验表明，与sh-NC组相比，sh-circ_001842组裸鼠形成的肿瘤体积和体质量均小于sh-NC组形成的肿瘤（P<0.05），并且E-cadherin蛋白表达水平明显上升，N-cadherin蛋白表达水平明显下降（P<0.05）。结论：hsa_circ_001842在肾癌组织及细胞中表达上调，抑制hsa_circ_001842的表达可以抑制癌细胞的增殖、迁移、侵袭和成瘤能力。

Abstract:

Objective：To investigate the expression of circular non-coding RNA hsa_circ_001842 as an oncogene in renal cancer tissues and renal cells of patients with renal cell carcinoma（RCC），and to investigate its effects on the proliferation，apoptosis，invasion and tumorigenicity. Methods：A total of 64 cases of renal cancer specimens and adjacent tissue specimens resected from September 2018 to October 2019 in Mianyang Central Hospital were collected. The qRT-PCR was used to detect the expression of all RCC tissues and adjacent normal tissues. Caki-1 kidney cancer cells were divided into ox-NC group（transfected with over-express－ing empty plasmid），ox-circ_001842 group（transfected with circ_001842 over-expression plasmid），sh-NC group（transfected with silent empty plasmid），and sh-circ_001842 group（trans－fected with circ_001842 silencing plasmid）. Cell clone formation experiment，flow cytometry，cell scratch test and cell invasion experiment were used to detect the effect of hsa_circ_001842 on the proliferation，apoptosis，migration and invasion of RCCs. Western blot was used to detect the expression of epithelial mesenchymal transition（EMT）-related protein and MMP-9 protein in RCCs. Caki-1 cells transfected with sh-NC and sh-circ_001842 were inoculated subcutaneously in nude mice，and tumor volume and weight were measured every 7 days. Mice in each group were executed after 4 weeks and immunohistochemical techniques and qRT-PCR were used to detect the expression of EMT-related proteins in tumor tissues. Results：The expression of hsa_circ_001842 in renal cancer tissues of renal cell carcinoma patients was significantly higher than that in normal adjacent tissues（P<0.05）. Compared with the ox-NC group and the sh-circ_001842 group，the Caki-1 renal cell’s colony formation rate，wound healing ratio，cell invasion ability and E-cadherin protein in the ox-circ_001842 group and the sh-NC group were significantly increased（P<0.05），while the apoptosis rate，N-cadherin and MMP-9 protein expression were significantly reduced（P<0.05）. Nude tumor formation experiments showed that the tumor volume and weight in the sh-circ_001842 group were smaller than those in the sh-NC group（P<0.05），and the expression level of E-cadherin protein increased significantly，while expression level of N-cadherin protein was decreased significantly（P<0.05）. Conclusion：Expression of hsa_circ_001842 in renal cancer tissues and cells is up-regulated，inhibiting the proliferation，migration，invasion and tumorigenicity by inhibiting the expression of hsa_circ_001842.