Lee Han Ki, Lim Heui Min, Park See-Hyoung, Nam Myeong Jin
Department of Biological Science, Gachon University, Seongnam 13120, Korea.
Department of Bio and Chemical Engineering, Hongik University, Sejong 30016, Korea.
J Pers Med. 2021 Sep 29;11(10):983. doi: 10.3390/jpm11100983.
: 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. 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. 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. 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.
CRISPR/Cas9系统是一种原核生物适应性免疫反应系统，它利用非编码RNA引导Cas9核酸酶诱导位点特异性DNA切割。肝细胞生长因子（HGF）是一种著名的生长因子，在细胞生长和器官发育中起关键作用。根据最近的研究，有报道称HGF促进了肝细胞癌（HCC）细胞的生长。在此，我们研究了其对HCC细胞的凋亡作用。使用GeneArt CRISPR核酸酶载体构建了Crispr-HGF质粒。用pMex-neo质粒设计了靶向HGF过表达基因的pMex-HGF质粒。我们进行实时聚合酶链反应以测量HGF mRNA的表达。我们进行细胞计数测定和集落形成测定以评估细胞增殖。我们还进行了迁移测定和侵袭测定以揭示Crispr-HGF对HCC细胞的抑制作用。此外，我们进行细胞周期分析以检测Crispr-HGF转染诱导的细胞周期停滞。总体而言，我们进行了膜联蛋白V/PI染色测定和蛋白质印迹测定。在Crispr-HGF转染组中，与pMex-HGF转染组相比，HGF的mRNA表达水平明显下调。此外，Crispr-HGF抑制了HCC细胞的活力。我们检测到Crispr-HGF转染的细胞中伤口面积和侵袭细胞受到抑制。结果表明，Crispr-HGF转染诱导了HCC细胞的细胞周期停滞和凋亡。在Crispr-HGF转染组中，丝裂原活化蛋白激酶和c-Met蛋白的磷酸化表达受到调节。有趣的是，我们发现Crispr-HGF转染组中条件培养基中HGF蛋白的表达明显降低。综上所述，我们发现通过转染Crispr-HGF抑制HGF可抑制HCC Huh7和Hep3B细胞的增殖并诱导凋亡作用。
