| Objective(s): By mining and analyzing cervical cancer data from The Cancer Genome Atlas(TCGA)to find differentially expressed genes,we screened out bovine pox virus-associated kinase 1(VRK1)and found that VRK1 was significantly more highly expressed in cervical cancer tissues compared with normal cervical tissues.We further examined the m RNA and protein expression levels of VRK1 in cervical cancer cell lines and human immortalized keratin-forming cells,cervical cancer tissues and normal tissue specimens adjacent to cancer,and investigated the effects of VRK1 on cell proliferation,migration and invasion in cervical cancer cell lines,as well as the regulation of VRK1 on cell cycle and apoptosis pathways,and finally explored the potential of VRK1 to promote the development of cervical cancer molecular mechanisms.Methods:1.The differentially expressed genes(DEGs)in cervical cancer and adjacent normal tissues were obtained from TCGA database by bioinformatics method,and the genes VRK1 involved in the regulation of cell cycle and apoptosis pathway were screened,and the correlation between VRK1 and prognosis was furth er analyzed.2.The m RNA and protein expression levels of VRK1 were measured in various human cervical cancer cell lines(He La,Si Ha,MS751,CASKI,C33A)and human immortalized keratin-forming cells(Hacat)using RT-q PCR and Western-Blotting,respectively.3.Freshly excised specimens from cervical cancer patients who underwent surgical treatment at our Hospital between 01/2022 and 06/2022 were collected,and the m RNA and protein expression levels of VRK1 were detected by RT-q PCR,Western-Blotting and IHC,respectively.4.Combining the expression of VRK1 in cervical cancer cell lines,cervical cancer cell lines and control cell lines with exogenous overexpression of VRK1 and interference with endogenous VRK1 expression were constructed using lentiviral stab le expression vectors,and transfection efficiency was detected with the help of fluorescence microscopy observation,RT-q PCR and Western-Blotting.5.In the above constructed successful cell models,CCK-8 assay and clone formation assay were used to detec t the proliferation ability of cells,Transwell assay was used to detect the migration and invasion ability of cells,flow cytometric cell cycle assay was used to detect the change of cell cycle,and flow apoptosis assay was used to detect the apoptosis of cells.6.Exploring the potential molecular mechanism of VRK1 for cervical carcinogenesis and development with the help of transcriptome sequencing technology.Results:1.Analysis of data from cervical cancer tissues and normal tissues adjacent to cancer in the TCGA database revealed that the m RNA expression of VRK1 was significantly upregulated in cervical cancer tissues and the difference was statistically significant.High VRK1 expression was significantly associated with worse overall survival.2.The m RNA and protein expression levels of VRK1 were significantly upregulated in some cervical cancer cell lines compared to immortalized keratinocytes,and the differences were statistically significant.3.A total of 72 postoperative fresh tissue specimens of cervical cancer patients meeting the inclusion criteria were collected,and the m RNA and protein expression levels of VRK1 were significantly upregulated in cervical cancer tissues compared with normal tissues adjacent to the cancer,and the differences were statistically significant.4.Based on the expression of VRK1 in cervical cancer cell lines,we successfully constructed exogenous overexpression of VRK1 in cervical cancer cell lines He La-VRK1,Si Ha-VRK1 and control cells He La-GFP and Si Ha-GFP;and at the same time,we successfully constructed the cervical cancer cell lines MS751-sh VRK1-2,MS751-sh VRK1-3,CASKI-sh VRK1-2,CASKIsh VRK1-3 and their control cells MS751-sh Control and CASKIsh Control,which interfered with the expression of endogenous VRK1.The cell transfection efficiency was verified by fluorescence microscopy,RTq PCR and Western-Blotting.5.CCK-8 assay and clone formation assay revealed that the proliferation ability of He La-VRK1 and Si Ha-VRK1 cells was significantly enhanced compared to He La-GFP and Si Ha-GFP,while the cell viability of MS751-sh VRK1-2,MS751-sh VRK1-3,CASKI-sh VRK1-2 and CASKI-sh VRK1-3cells was significantly weaker compared to MS751-sh Control and CASKIsh Control.Transwell assay revealed that the migration and invasion ability of He La-VRK1 and Si Ha-VRK1 cells were significantly enhanced compared with He La-GFP and Si Ha-GFP,while the migration and invasion ability of MS751-sh VRK1-2,MS751-sh VRK1-3 and CASKI-sh VRK1-2,CASKI-sh VRK1-3 cells was significantly reduced compared with MS751-sh Control and CASKI-sh Control.Flow cytometric cell cycle assays revealed that more He La-VRK1,Si Ha-VRK1 cells entered S phase and G2/M phase compared to He La-GFP,Si Ha-GFP,while more MS751-sh VRK12,MS751-sh VRK1-3 and CASKI-sh VRK1-2 and CASKI-sh VRK1-3 cells entered G0-phase compared to MS751-sh Control and CASKI-sh Control.The flow apoptosis assay revealed that He La-VRK1 and Si Ha-VRK1 cells had suppressed apoptosis compared with He La-GFP and Si Ha-GFP cells,while MS751-sh VRK1-2,MS751-sh VRK1-3,CASKI-sh VRK1-2 and CASKI-sh VRK13 cells had increased apoptosis compared with MS751-sh Control and CASKI-sh Control cells.6.Transcriptome sequencing technology combined with bioinformatics analysis revealed that endothelin-1(EDN1)may be involved as a key target gene in the regulation of VRK1 on cervical cancer cell proliferation,migration and invasion,as well as cell cycle and apoptosis.Conclusion(s):1.Based on the analysis of TCGA database,it was found that there was differential expression of VRK1 in cervical cancer tissue and normal cervical tissue,and VRK1 showed significantly high expression in cervical cancer tissue,which was closely related to the poor prognosis of patients with cervical cancer.2.The m RNA and protein expression levels of VRK1 in some cervical cancer cell lines and cervical cancer tissues were significantly higher than those in immortable keratinocytes and paracancer normal tissues.3.VRK1 promotes cervical cancer cells proliferation,migration and invasion in vitro,and promotes cervical cancer cell to enter the proliferation cycle and inhibits cell apoptosis. |
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