Abstract
Background: CRISPR/Cas9 system is a prokaryotic adaptive immune response system that uses noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. Hepatocyte growth factor (HGF) is a well-known growth factor that plays a crucial role in cell growth and organ development. According to recent studies, it has been reported that HGF promoted growth of hepatocellular carcinoma (HCC) cells. Here, we investigated the apoptotic effects in HCC cells. Methods: Crispr-HGF plasmid was constructed using GeneArt CRISPR Nuclease Vector. pMex-HGF plasmid that targets HGF overexpressing gene were designed with pMex-neo plasmid. We performed real time-polymerase chain reaction to measure the expression of HGF mRNA. We performed cell counting assay and colony formation assay to evaluate cell proliferation. We also carried out migration assay and invasion assay to reveal the inhibitory effects of Crispr-HGF in HCC cells. Furthermore, we performed cell cycle analysis to detect transfection of Crispr-HGF induced cell cycle arrest. Collectively, we performed annexin V/PI staining assay and Western blot assay. Results: In Crispr-HGF-transfected group, the mRNA expression levels of HGF were markedly downregulated compared to pMex-HGF-transfected group. Moreover, Crispr-HGF inhibited cell viability in HCC cells. We detected that wound area and invaded cells were suppressed in Crispr-HGF-transfected cells. The results showed that transfection of Crispr-HGF induced cell cycle arrest and apoptosis in HCC cells. Expression of the phosphorylation of mitogen activated protein kinases and c-Met protein was regulated in Crispr-HGF-transfected group. Interestingly, we found that the expression of HGF protein in conditioned media significantly decreased in Crispr-HGF-transfected group. Conclusions: Taken together, we found that inhibition of HGF through transfection of Crispr-HGF suppressed cell proliferation and induced apoptotic effects in HCC Huh7 and Hep3B cells.
Highlights
Hepatocellular carcinoma (HCC) is known to be the most common disease in some areas of Asia and Africa [1]
The results showed that the expression levels of c-Met, p38, Akt, extracellular signal-regulated kinases (Erk), and Jun amino-terminal kinases (Jnk) proteins were maintained regardless of Hepatocyte growth factor (HGF) dose in both cell lines
The expression levels of phosphorylated c-Met, p38, Akt, Erk, and Jnk increased as HGF concentration increased in Huh7 cells
Summary
Hepatocellular carcinoma (HCC) is known to be the most common disease in some areas of Asia and Africa [1]. HCC is being concentrated as a medical issue in the world Many treatments such as chemotherapy and radiofrequency ablation for HCC have been developed. These therapies have some drawbacks, such as detrimental effects of radiation therapy on normal liver tissue [4]. We carried out migration assay and invasion assay to reveal the inhibitory effects of Crispr-HGF in HCC cells. We performed cell cycle analysis to detect transfection of Crispr-HGF induced cell cycle arrest. We detected that wound area and invaded cells were suppressed in Crispr-HGF-transfected cells. The results showed that transfection of Crispr-HGF induced cell cycle arrest and apoptosis in HCC cells. Conclusions: Taken together, we found that inhibition of HGF through transfection of Crispr-HGF suppressed cell proliferation and induced apoptotic effects in HCC Huh and Hep3B cells
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