CRISPR-Cas13-Mediated Knockdown of lncRNA-GACAT3 Inhibited Cell Proliferation and Motility, and Induced Apoptosis by Increasing p21, Bax, and E-Cadherin Expression in Bladder Cancer

Abstract

The current study is to investigate the expression pattern and biological function of long non-coding RNA Focally gastric cancer-associated transcript3 (GACAT3) in bladder cancer. Real-time quantitative qPCR was used to detect the expression level of GACAT-3 in tumor tissues and paired normal tissues. Human bladder cancer T24 and 5637 cell lines were transiently transfected with specific CRISPR-Cas13 or negative control CRISPR-Cas13. Cell migration, proliferation, and apoptosis were measured by using wound healing assay CCK-8 assay and Caspase-3 ELISA assay, respectively. The expression changes of p21, Bax, and E-cadherin after knockdown of GACAT3 were detected by using Western blot. The results demonstrated that GACAT3 was up-regulated in bladder cancer tissues than that in the paired normal tissues. Inhibition of cell proliferation, increased apoptosis, and decreased motility were observed in T24 and 5637 cell lines transfected by CRISPR-Cas13 targeting GACAT3. Downregulation of GACAT3 increased p21, Bax, and E-cadherin expression and silencing these genes could eliminate the phenotypic changes induced by knockdown of GACAT3. A ceRNA mechanism for GACAT3 was also revealed. By using CRISPR-Cas13 biotechnology, we suggested that GACAT3 may be a novel target for diagnosis and treatment of bladder cancer.

LncRNA GACAT3: A Promising Biomarker and Therapeutic Target in Human Cancers

“…In CRISPR-Cas13-transfected bladder cancer T24 and 5637 cell lines, GACAT3 promotes cell proliferation, suppresses apoptosis, and enhances cell migration by downregulating p21, BAX, and E-Cadherin protein levels, respectively. Mechanistically, GACAT3 acts as a ceRNA by interacting with miRNA-497 in the cytoplasm ( Zhang et al, 2020a ). These observations indicate that GACAT3 -targeted therapy is a promising strategy for bladder cancer.…”

“…A recent breakthrough in cancer research has been the discovery and application of lncRNA in comprehensive tumor therapy ( Bhan et al, 2017 ; Peng et al, 2017 ; Wang et al, 2019b ). An abundance of evidence has demonstrated that GACAT3 is a novel oncogenic lncRNA that functions as a ceRNA to modulate the occurrence and development of various tumors ( Feng et al, 2018 ; Zhong et al, 2018 ; Zhang et al, 2020a ).…”

From Junk to Function: LncRNAs in CNS Health and Disease

“…Recent progress in genome-editing techniques, such as CRISPR-based methods including CRISPR-interference (CRISPRi) and CRISPR-activation (CRISPRa), has brought the exciting possibility of transcriptionally silence or activate lncRNA expressing loci, and clearly demonstrate that these tools will be crucial for a better understanding of lncRNA biology ( Jinek et al, 2012 ; Cong et al, 2013 ; Mali et al, 2013 ; Qi et al, 2013 ; Gilbert et al, 2014 ; Thakore et al, 2015 ; Zhu et al, 2016 ; Abudayyeh et al, 2017 ; Liu S. J. et al, 2017 ; Chen et al, 2019 ; Phelan and Staudt, 2020 ; Wolter et al, 2020 ; Xu D. et al, 2020 ; Zhang et al, 2021 ). One way to achieve such transcriptional modulation is to use a dead-Cas9 approach.…”

Reprogramming the anti-tumor immune response via CRISPR genetic and epigenetic editing

“…Significant attention has been directed toward applying CRISPR-Cas13 (RNA-targeting) technology as a diagnostic in the detection of low-frequency cancer somatic mutations 144 and in RNA editing. 85 , 145 Additionally, the CRISPR-Cas13 system has demonstrated highly efficient and specific knockdown of oncogenic mutant drivers, 146 gene fusions, 88 and ncRNA 147 transcripts in vitro with minimal off-targets, highlighting its potential for use as an RNA-based therapeutic tool. More recently, the potential of Cas13-based gene suppression in cancer immunotherapy has drawn attention due to its growing success in pre-clinical studies, as explored below.…”