Li Chunjing, Cao Yu, Zhang Li, Li Jierong, Wang Jianfeng, Zhou Yanfen, Wei Huiling, Guo Mingjuan, Liu Liang, Liu Chunxiao, Zhang Shilin, Liu Guoqing
Affiliated Foshan Maternal and Child Healthcare Hospital, Southern Medical University, Foshan, China.
Department of Urology, Zhujiang Hospital of Southern Medical University, Guangzhou, China.
Front Mol Biosci. 2021 Mar 9;8:638995. doi: 10.3389/fmolb.2021.638995. eCollection 2021.
CRISPR-CasRx technology provides a new and powerful method for studying cellular RNA in human cancer. Herein, the pattern of expression of long noncoding RNA 00341 (LINC00341) as well as its biological function in bladder cancer were studied using CRISPR-CasRx. qRT-PCR was employed to quantify the levels of expression of LINC00341 in tumor tissues along with the matched non-tumor tissues. sgRNA targeting LINC00341 or the sgRNA negative control were transiently transfected into the T24 as well as 5,637 human bladder cancer cell lines. CCK-8, ELISA as well as wound healing methods were employed to explore cell proliferation, apoptosis and migration, respectively. The tumorigenicity experiment in nude mice also performed to detect cell proliferation. The expression of p21, Bax as well as E-cadherin were assayed using western blot. The results demonstrated that LINC00341 was overexpressed in bladder cancer in contrast with the healthy tissues. The LINC00341 expression level in high-grade tumors was higher in contrast with that in low-grade tumors. The expression of linc00341 was higher relative to that of non-invasive tumors. In T24 as well as 5637-cell lines harboring LINC00341-sgRNA, inhibition of cell proliferation ( and ), elevated apoptosis rate and diminished migration ability. Moreover, silencing LINC00341 upregulated the expressions of p21, Bax as well as E-cadherin. Knockout of these genes could eliminate the phenotypic changes caused by sgRNA targeting LINC00341. Our data demonstrate that LINC00341 has a carcinogenic role in human bladder cancer.
CRISPR-CasRx技术为研究人类癌症中的细胞RNA提供了一种全新且强大的方法。在此，我们利用CRISPR-CasRx技术研究了长链非编码RNA 00341（LINC00341）在膀胱癌中的表达模式及其生物学功能。采用qRT-PCR对肿瘤组织及配对的非肿瘤组织中LINC00341的表达水平进行定量。将靶向LINC00341的sgRNA或sgRNA阴性对照瞬时转染至T24和5637人膀胱癌细胞系中。分别采用CCK-8、ELISA和伤口愈合方法来探究细胞增殖、凋亡和迁移情况。还进行了裸鼠成瘤实验以检测细胞增殖。通过蛋白质免疫印迹法检测p21、Bax和E-钙黏蛋白的表达。结果表明，与健康组织相比，LINC00341在膀胱癌中高表达。与低级别肿瘤相比，高级别肿瘤中LINC00341的表达水平更高。相对于非侵袭性肿瘤，linc00341的表达更高。在携带LINC00341-sgRNA的T24和5637细胞系中，细胞增殖受到抑制（以及），凋亡率升高，迁移能力降低。此外，沉默LINC00341可上调p21、Bax和E-钙黏蛋白的表达。敲除这些基因可消除靶向LINC00341的sgRNA所引起的表型变化。我们的数据表明，LINC00341在人类膀胱癌中具有致癌作用。
