Researchers from universities such as South China University of Technology, Jinan University, and Huazhong University of Science and Technology published a study titled CRISPR Activation Screening in a Mouse Model for Drivers of Hepatocellular Carcinoma Growth and Metastasis** in iScience, which was used in the studyCRISPR-activated (CRISPRA) SAM2 lentiviral library screens genome-wide growth and metastasis drivers for hepatocellular carcinoma, which provides a roadmap for in vivo screening of functional genes related to liver tumor invasion and metastasis.
Primary liver cancer is the sixth most common malignancy in the world, of which hepatocellular carcinoma (HCC) is the most common primary liver cancer, accounting for 90% of all cases. In this study, the investigators by:crispraSystematic phenotypic measurements were performed on gain-of-function screening during the growth and metastasis of primary liver tumors, and candidate targets Xage1B, PLK4, LMO1, and MyADML2 were identified to promote cell proliferation and invasion
In 8 separate replicate experiments of infection, the researchers used:crispr/cas9 sam2 pooled libraryHEPG2 cells were transduced, 43106 cells were cultured in vitro for 1 week and then transplanted into the livers of immunocompromised BLABC Nu Nu mice to construct orthotopic tumor models, and the mouse models were screened for functional gain transfer. The results showed that the body weight and survival rate of SAM2 library transduced mice were lower than those of the control group, and the SAM2 library transduced and untransduced HEPG2 cells formed tumors at the injection site under macrovisual observation. In addition, the expression levels of VEGF, Vimentin and MMP9 proteins in the liver of SAM2-transduced mice were significantly increased, and the expression levels of VEGF and MMP9 proteins in the lungs of SAM2-transduced mice were higher. These results suggest that SAM2 library transduction enhances the ability of HEPG2 cells to form metastases in the lung.
Fig.1 Growth and metastasis of tumors in HEPG2 cells transplanted from the SAM2 library.
The researchers took the sgRNA sequences in the liver and lung genomes for PCR amplification, calculated the coverage of sgRNA in cells through high-throughput sequencing, and compared the normalized sgRNA abundance differences between lung and liver. The results showed that the abundance of sgRNAs in the H gene was higher in the lung than in the liver, which was a specific gene for lung metastasisType C genes may promote tumor growth and lung metastasis;The L-type gene sgRNAs are less abundant than those of the liver and may primarily promote tumor growth rather than metastasis. These genes were further analyzed by KEGG pathway analysis, and the SgRNAs commonly expressed differentially expressed H genes were enriched in cells such as cell adhesion molecules (CAMS) and NoT, and the C-type genes were related to metabolism, PI3KAKT, actin cytoskeletal regulation, MAPK and FOXO signaling pathways, and L-type genes suggested that they were involved in VEGF, tight junctions, cell cycle, and abnormal transcriptional regulation in tumor pathways.
Fig.2 sgRNA library screening identifies pathways affecting liver and lung tumor growth and metastasis.
The researchers performed a Kaplan-Meier analysis of 421 samples and found that 385 genes of TCGA intersected 385 genes in group C, 177 genes in group L, and 18 genes in group H out of 6,765 genes in TCGA. The volcano plot shows differential analysis of 580 intersecting HCC genes, representing a comparison of 371 cancer samples and 50 paracancerous samples and 50 pairs of matched HCC and paracancerous comparisons. The enrichment genes were identified in primary liver tumors and lung metastases, and seven genes (polr2l, xage1b, hoxd1, plk4, c17orf99, lmo1 and myadml2) affecting tumor growth and metastasis were screened.
Fig.3 CRISPRA screening of sgRNAs enriched in the genome for liver tumor growth and metastasis
Researchers use lentivirus to deliver CRISPRApolr2l, xage1b, hoxd1, plk4, c17orf99, lmo1, and myadml2 and controls infected with hepatoma cell lines hepg2 and huh7 to verify the effect of candidate genes screened out by the crispra library on hcc cell phenotype. q-PCR confirmed that all sgRNA sequences were effective in upregulating the expression of seven candidate genes in HEPG2 and HUH7 cells. CCK8 assay showed that overexpression of the seven candidate genes could significantly promote HCC cell proliferation and increase the survival rate of hepatocellular carcinoma cells. The cells with high expression of seven candidate genes were subjected to cell invasion experiments, and the number of HEPG2 cells invaded by all tested genes was significantly increased compared with the control group, and the overexpression of LMO1, MyADML2, PLK4 and XAGE1B significantly promoted the invasion of HUH7 cells, indicating that these genes accelerated the invasion of HCC cells.
Fig.4 Overexpression of candidate genes promoted the proliferation and invasion of hepatoma cells.
The researchers used WB to detect protein expression levels, screened out the siRNAs with the best inhibitory effect, and used CCK8 and Transwell to detect cell proliferation and migration. The results showed that down-regulation of LMO1, MyADML2, PLK4 and XAGE1B significantly inhibited the proliferation of HCC cells at 24h, 48h and 72h, and the Transwell assay clearly showed that the invasive cells of LMO1, MyADML2, PLK4 and XAGE1B-siRNA groups were significantly reduced at 48h compared with the SICs group of HEPG2 and HUH7 cells.
Fig.5 Inhibition of candidate gene expression inhibited the proliferation and invasion of hepatocellular carcinoma cells.
The researchers used the cancer genealist RNA sequencing data to understand the expression profiles of these candidate genes in HCC and to evaluate the expression levels of these candidate genes in the TCGA database. The results showed that HCC patients with high expression of LMO1, MyADML2, PLK4 and XAGE1B had lower overall survival and worse expression of HCC, and univariate analysis showed that LMO1, MYADML2, PLK4 and XAGE1B were valuable factors for the survival of HCC patients. Immunohistochemistry was used to detect the expression of MyADML2 protein in 80 HCC tissues, and it was found that MyADML2 was mainly located on the cytoplasm and cell membrane. Kaplan-Meier analysis showed that HCC patients with high MyADML2 protein levels had worse overall survival than HCC patients with low MyADML2 protein levels. The results showed that the high expression of LMO1, MyADML2, PLK4 and XAGE1B not only led to the increase in the incidence of lung metastasis, but also affected the prognosis of liver cancer, and MyADML2 was the most important prognostic factor.
Fig.6 Candidate genes were analyzed using cancer gene profiles and immunohistochemistry.
The researchers analyzed the proportionality of 15 immune cell types in the TCGA dataset and found that PLK4, LMO1 and MyADML2 were consistent with M0 macrophage infiltration levels, and PLK4 was consistent with dendritic cell infiltration levels. In addition, PLK4 and MyADML2 were negatively correlated with the infiltration levels of monocytes, **B cells, and M2 macrophages, suggesting that dendritic cells, macrophages, and B cells play an important role in promoting HCC proliferation and invasion.
Fig.7 Analysis of immune cell composition of hepatocellular carcinoma.
In summary, the researchers used CRISPRA library screening technology to provide a roadmap for in vivo screening of functional genes associated with liver tumor invasion and metastasis, and the high expression of the screened LMO1, MyADML2, PLK4 and XAGE1B genes not only means a higher probability of lung metastasis, but also affects the prognosis of liver cancer, and these findings provide new potential biological targets for the clinical diagnosis, ** and prognostic evaluation of liver cancer.
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