| The latest GLOB OCAN data revealed that gastric cancer(GC)has the fifth incidence rate and the fourth mortality rate in the world,posing a major threat to human health.Although chemotherapy can prolong survival and improve the quality of life,the median survival of advanced GC is still difficult to exceed one year,and the 5-year survival rate was less than 10%.Therefore,it is especially necessary and urgent to devote to the research on the pathogenesis of GC,to explore new breakthrough points for GC treatment,and to improve the survival of GC patients.The development of GC was a multi-stage and multifactorial process.Recent studies have revealed that mutations in key proteins of the chromatin remodeling complex can affect normal gene expression and participate in tumor development.With the development of high-throughput genomics technologies,bioinformatics has been increasingly used to screen for disease-specific biomarkers for research.In the first part of this study,the results of weighted gene co-expression network analysis and differentially expressed gene analysis were integrated to screen chromatin remodeling-related genes implicated in the development of GC.The results suggested that Centromere protein K(CENPK)is a novel strategic gene for GC prognosis.Public database analysis indicated that CENPK expression was upregulated in GC,and overall survival(OS)was shorter in GC patients with high CENPK expression,and multivariate COX regression suggested that high CENPK expression could be an independent poor prognostic factor for GC.We subsequently validated this phenomenon.qRT-PCR and Western blot assays demonstrated that CENPK expression was significantly upregulated in the GC cell line.Immunohistochemical staining of 101 GC patients’ tissue microarrays also suggested that CENPK was overexpressed in GC tissues,and multivariate logistic results indicated that overexpression of CENPK was positively correlated with tumor T-stage,vascular endothelial growth factor receptor(VEGFR)and Ki-67 levels.Histological samples alsodemonstrated worse OS in CENPK-positive GC patients,which could be an independent factor for poor GC prognosis.In the second part,the biological functions of CENPK in GC were further explored.We performed CENPK knockdown in GC cell lines AGS and SGC-7901,and overexpressed CENPK in HGC-27 and MGC-803 cells.Functional assays demonstrated that knockdown of CENPK inhibited proliferation,migration,and invasive ability of GC cells,induced G1-phase arrest,and promoted apoptosis in vitro.Overexpression of CENPK promoted the proliferation,migration,and invasive ability of GC cells,induced S-phase arrest,and inhibited apoptosis.Additionally,the experiment of constructing GC nude mice subcutaneous transplantation tumor model by tail vein injection of CENPK knockdown AGS cells indicated that knockdown of CENPK could inhibit the growth of nude mice subcutaneous tumors in vivo,and the expression level of CENPK in the knockdown group of nude mice tumors was also significantly lower than that in the control group.The liver metastasis model of GC was established by injecting CENPK-overexpressed MGC-803 cells into the spleen of nude mice,and the overexpression of CENPK promoted the liver metastasis of GC cells.All of the above results suggest that CENPK may be an important new oncogene involved in the progression of GC.In the third part,we focus on exploring the molecular mechanism of CENPK promoting GC proliferation and metastasis based on the previous work.(1)First,the results of enrichment analysis based on the TCGA database revealed that the E2Fs transcription factor family was significantly enriched.Correlation analysis suggested that E2F1,E2F2,and E2F7 were significantly correlated with CENPK.qRT-PCR examined the changes of the above transcription factors in knockdown CENPK and control GC cells,and the results indicated that the expression of E2F2 was significantly reduced,which was consistent with the results of bio informatics analysis,suggesting that E2F2 may be regulated by CENPK as a key target gene.The mRNA data from public databases showed that E2F2 expression was significantly upregulated in GC tissues and patients with high E2F2 expression had a worse prognosis.Meanwhile,protein expression data of E2F2 in microarrays from 101 GC tissues suggested that E2F2 was also highly expressed in GC.Multivariate logistic results demonstrated that the expression level of E2F2 was positively correlated with VEGFR expression,and survival analysis suggested that E2F2-positive GC patients had shorter OS.CENPK was significantly and positively correlated with E2F2 expression,and the correlation analysis results suggested a correlation coefficient r=0.36,p<0.0001.We then further investigated the functional role of E2F2 in CENPKmediated GC progression.Interfering with E2F2 reversed the proliferation,migration,and invasive ability of GC cells promoted by CENPK overexpression and restored the decrease in the proportion of G1-phase cells and the increase in S-phase and G2/Mphase caused by CENPK overexpression.Similarly,the apparent inhibition of apoptosis in GC cells by overexpression of CENPK was partially reversed by siE2F2.All of the above results suggest that CENPK may promote GC proliferation and metastasis by regulating E2F2.(2)Secondly,enrichment analysis based on TCGA gastric cancer database suggested that PI3K/AKT signaling pathway was enriched.pAKT and PI3K expression were significantly decreased after CENPK knockdown by western blot assay.PI3K inhibitor LY294002 reversed the proliferation,migration,and invasive ability of GC cells promoted by CENPK overexpression.Overexpression of CENPK decreased the proportion of the G1-phase and increased S-phase and the addition of LY294002 increased the proportion of the G1-phase and decreased Sphase.The addition of LY294002 partially reversed the inhibition of apoptosis caused by the overexpression of CENPK.Expressions of p-AKT and E2F2 in the subcutaneous tumor tissues of GC with CENPK knockdown were significantly lower than those of the control group.The above results suggest that CENPK can play a role in promoting GC proliferation and metastasis through the E2F2-PI3K/AKT axis,which provides a theoretical basis for finding new targets for GC diagnosis and treatment.In the fourth part,based on our previous findings,both CENPK and CENPKregulated downstream target gene E2F2 were significantly and positively correlated with the expression of VEGFR.Results of GSEA enrichment analysis of CENPK also suggested that the angiogenic pathway was also significantly enriched.Blocking the signal transduction pathway of VEGFR and thus inhibiting tumor angiogenesis is an important anti-tumor pathway in GC.Apatinib is a commonly used drug for advanced GC in clinical practice.Apatinib can highly selectively inhibit the activity of VEGFR2 tyrosine kinase to exert anti-tumor effects.In this section,we first returned to the clinic and analyzed the correlation between CENPK expression and the efficacy of GC patients treated with apatinib-based regimens.Thirty-eight patients with advanced GC treated in first-line with apatinib-based regimens were retrospectively collected.The expression of CENPK in the paraffin tissues of these patients was identified using immunohistochemistry.The results revealed that most of the GC patients with effective treatment with apatinib-based regimens had low expression of CENPK,and further survival analysis revealed that the CENPK-negative group had longer progression-free survival and OS than the CENPK-positive group.Furthermore,we performed in vitro functional assays using apatinib-treated CENPK knockdown and control GC cells.The results illustrated that the ability of apatinib to inhibit GC growth and metastasis was more pronounced after the knockdown of CENPK.The above results tentatively suggest a correlation between CENPK and the efficacy of gastric cancer treated with apatinib,which still needs further validation in large samples and in-depth molecular mechanism studies.In summary,this study screened CENPK as a new key gene for GC prognosis,starting from high-throughput bioinformatics analysis and histological validation,and clarified that CENPK is a pro-GC growth and metastasis gene and that GC patients with low CENPK expression have better efficacy when receiving apatinib-based regimens.The above findings provide a new research direction for the pathogenesis of GC and the drug sensitivity study of apatinib. |
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