The Study On The Mechanism Of VRK1 In Promoting Proliferation And Migration Of HCC Cell Sthrough Regulating The CHD1L/Snail Axis

BackgroundGlobal health is challenged by liver cancer,whose incidence rates ranked sixth and mortality rates ranked fourth among malignant tumors.The most common form of liver cancer is hepatocellular carcinoma(HCC),which accounts for～90%of liver cancer.Most HCC patients are diagnosed at an advanced stage and cannot benefit from surgery.The preferred treatment for advanced HCC patients is targeted therapy.The first-line targeted therapeutic drugs,sorafenib and lenvatinib,can extend survival of advanced HCC patients.But some patients are resistant or fail to respond to sorafenib and lenvatinib.It is necessary to understand the mechanisms of liver cancer development and find new therapeutic targets.Vaccinia-related kinase 1(VRK1)is a Ser-Thr chromatin kinase in the nucleus and is involved in many cellular processes,including cell cycle regulation,DNA damage repair,nuclear envelope assembly,transcription,and so on.VRK1 is overexpressed in many types of tumors and is associated with poor prognosis.It is reported that VRK1 promotes proliferation of liver cancer by regulating cell cycle progression.However,the function and mechanism of VRK1 in liver cancer migration and progression remains poorly understood.We investigated the effects of VRK1 on cell proliferation and migration using HCC cell lines with knockdown of VRK1.Validating the effect of VRK1 on HCC biological functions in a mouse xenograft tumor model.Mechanistically,we screened the downstream target genes and cellular signaling pathways regulated by VRK1 using RNA sequencing.The interacting proteins of VRK1 were explored by immunoprecipitation combined with mass spectrometry.Clarifying the mechanism that VRK1 promotes HCC progression by searching for the above key molecules.Purpose1.To investigate the effects of VRK1 on the proliferation and migration in HCC cells.2.To reveal downstream target genes and signaling pathways regulated by VRK1 in HCC.3.To clarify the mechanism of VRK1 regulating downstream target genes and promoting HCC progression.Methods1.Analyze the expression of VRK1 in tumor tissues of HCC patients and the relationship between VRK1 and the survival or tumor stage of HCC patients by TCGA.2.Study the effects of VRK1 on the proliferation and migration in HCC cells using CCK8,colony formation assay and Transwell assay.3.Detect the effect of VRK1 on tumor growth in vivo using a mouse xenograft tumor model.4.Screen downstream target genes and cellular signaling pathways regulated by VRK1 using RNA-seq and GSEA.5.Overexpression Snail after knockdown of VRK1,to study whether VRK1 promoted HCC cell proliferation and migration through its target protein,Snail,using EdU and Transwell assay.6.Reveal the interacting proteins of VRK1 by immunoprecipitation combined with mass spectrometry.7.Using Western blotting and qRT-PCR to study whether VRK1 regulates Snail(SNAI1)through its interacting protein,CHD1L.8.Detect whether VRK1 phosphorylates CHD1L using Western blotting.9.Investigate whether VRK1 regulates Snail expression through phosphorylation of CHD1L at S122 site using CHD1L phosphorylated inactivated mutant(CHD1L S122A).Results1.High expression of VRK1 in HCC is significantly associated with poor prognosisAnalysis of TCGA showed that VRK1 was highly expressed in the tumor tissues of HCC patients,and the survival of HCC patients with high VRK1 expression was short.The expression of VRK1 in tumor tissues of HCC patients increased with tumor stage,and there was a correlation between VRK1 and the malignancy of tumors in HCC patients.2.Knockdown of VRK1 inhibits the proliferation and migration of HCC cellsCompared with the control,knockdown of VRK1 significantly inhibited the proliferation,clone formation and migration ability of Huh7·and·HepG2·cells.3.Knockdown of VRK1 inhibits tumor growth in vivoCompared with the control group,the tumor in the knockdown VRK1 group were both reduced in volume and weight,indicating that knockdown VRK1 inhibited tumor growth in vivo.4.VRK1 regulates SNAI1 expressionRNA sequencing showed that there were 325 differential genes between the control and knockdown VRK1 group of which 151 genes were down-regulated and 171 genes were up-regulated in the knockdown VRK1 group.GSEA analysis showed that the differential genes were significantly enriched in the epithelial-mesenchymal transition pathway.The expression of SNAI1(encode Snail),a marker of mesenchyme cells,was significantly down-regulated in the knockdown VRK1 group.The results of qRT-PCR and Western blotting confirmed that VRK1 regulates the mRNA and protein expression of SNAI1.5.VRK1 promotes the proliferation and migration of HCC cells through SnailThe effect of overexpression Snail after knockdown of VRK1 on the proliferation and migration ability of HCC cells was examined in a rescue assay which showed that VRK1·promotes the proliferation and migration of hepatocellular carcinoma cells through Snail.6.VRK1 interacts with CHD1LImmunoprecipitation combined with mass spectrometry analysis revealed that·175 proteins were enriched in the VRK1 overexpression group compared to the control group.CHD1L is the protein with the most bound peptides.The interaction between VRK1 and CHD1L was confirmed by immunoprecipitation assay.7.VRK1·regulates Snail expression through CHD1LThe protein and mRNA expression of Snail was elevated in the CHD1L overexpression group after knockdown of VRK1 compared to the VRK1 knockdown group,indicating that VRK1 regulates Snail expression through CHD1L.8.VRK1 phosphorylates CHD1L at S122 siteCompared with the control group,the phosphorylation level of CHDL was reduced in the knockdown VRK1 group,indicating that VRK1 could phosphorylate CHD1L.The phosphorylation level of inactivating mutation of the CHD1L(CHD1L S122A)cannot be affected by knockdown of VRK1,indicating that VRK1 phosphorylates CHD1L at S122 site.9.VRK1·regulates Snail expression through phosphorylation of CHD1L at S122 siteCompared with the VRK1 knockdown group,the expression of Snail increased in CHD1L rescued group,but did not change in CHD1L S122A rescued group,indicating that VRK1 regulated the expression of Snail through phosphorylation CHD1L at the S122 site.Conclusion1.VRK1 is highly expressed in hepatocellular carcinoma tissues and associated with poor prognosis,VRK1 promotes the proliferation,migration and tumor growth in HCC;2.VRK1 interacts with CHD1L and phosphorylates CHD1L at S122 site;3.VRK1 regulates Snail expression through CHD1L phosphorylation to promote proliferation and migration of liver cancer cells.