Novel Perspective of Regulating P53/Bcl2/Caspase-3 via In vitro Targeted AFP Gene Knocks Out in HepG2 Cells Using CRISPR/Cas9 Editing Tool.

Abstract

Introduction: Hepatocellular carcinoma (HCC) is a major health burden worldwide, with a persistent need for molecular target drugs. Alpha-fetoprotein (AFP) is a major concern during HCC, as it has an incompletely solved action. CRISPR/Cas9 is a gene editing tool that aids in cancer treatment research; thus, this study evaluated the effect of in vitro knockout of AFP on HCC using CRISPR/Cas9 technique.

Methods: Two sgRNAs targeting specific sites in AFP exon 2 were separately cloned to the mammalian expression vector pSpCas9 (BB)-2a-GFP (PX458). HepG2 cells were transfected with CRISPR constructs I and II, and a pool of the two constructs (M) for 6 -, 24- and 39 hours using liopfectamine3000. AFP editing was evaluated regarding genomic DNA sequence, RNA, and protein expression levels. In addition, the effect of AFP knocking out on HepG2 viability, and apoptotic genes mRNA and protein expression levels were evaluated using crystal violet assay, real-time PCR, and western blot analysis respectively.

Results: The results revealed efficient delivery of the AFP/CRISPR constructs to HepG2 cells. Insertion and deletion mutations introduced to the AFP genomic sequence were analyzed using TIDE software analysis and the Expasy translation tool. The viability of the HepG2 cells was reduced 39 hours post-transfection with significant modulation in the expression of the apoptotic markers P53, BAX, Bcl2, and caspase-3.

Conclusion: This study succeeded in developing AFP/CRISPR constructs that could disrupt the AFP genomic sequence, reduce its expression, and restore the activity of cell-specific apoptotic factors, demonstrating the potential inhibitory effect of AFP downregulation on HCC progression.