CRISPR-CAS9 HSF4-targeted for clear cell renal cell carcinoma proliferation capacity, colony and tumorspheres formation / Mei Juan Wong

Mei , Juan Wong (2022) CRISPR-CAS9 HSF4-targeted for clear cell renal cell carcinoma proliferation capacity, colony and tumorspheres formation / Mei Juan Wong. Masters thesis, Universiti Malaya.

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Abstract

Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer, accounts for about 75% of all kidney cancer cases. Although anti-angiogenic drugs targeting the receptor tyrosine kinases and mTOR inhibitors were clinically approved for ccRCC, but the current available drugs are still having low efficacy and most of the patients can rapidly develop therapeutic resistance. Prior analysis of TCGA RNA-Seq data on the downstream analysis of KLF6-driven transcriptional programs in ccRCC revealed that HSF4 is among the genes those are significantly upregulated in ccRCC as compared to normal kidney tissues and high expression of HSF4 in ccRCC patients is associated with poor prognosis. To interrogate the HSF4 functional relevance in supporting kidney cancer cells growth and cellular fitness, we targeted HSF4 gene in ccRCC cell line (786-M1A) by using CRISPR-Cas9 (CRISPRko) system and carried out in-vitro functional assays to determine the phenotypic effects of targeting HSF4 in ccRCC cells. A point mutation of insertion was introduced into the HSF4-targeted 786-M1A cells and cause frameshift mutation and reduction of HSF4 protein synthesis. The in-vitro functional assays showed to decrease cell activities in terms of proliferation rate, colony formation, and ability to form tumorspheres in HSF4-targeted 786-M1A cells as compared with the non-targeted 786-M1A cells. The results indicated the HSF4 gene displayed functional role in maintain 786-M1A cells growth and cellular fitness. The further analysis will be focused on the analysis of the downstream pathways that are regulated by HSF4 gene. These findings are believed to be able to contribute to the foundation of the understanding on the molecular drivers in ccRCC pathogenesis and the development of effective therapeutic strategy for ccRCC patients.

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