| As one of the most common malignant tumors developing in the female reproductive tract,endometrial cancer(EC)is second only to cervical cancer in the incidence of female reproductive tract malignancies in China.Over the past few years,the incidence of endometrial cancer has increased with the aging population.Most patients with endometrial cancer are diagnosed in early stages and can be treated through surgery and adjuvant therapy,with good prognosis achieved.Though about75% of patients with early endometrial cancer can survive for 5 years,such treatments as surgery,radiotherapy and chemotherapy are still ineffective for the patients with advanced,recurrent or metastatic endometrial cancer.Besides,the prognosis is still unsatisfactory.Therefore,it is a pressing need to find new prognostic markers and therapeutic targets.Objective1.To analyze and sort out the endometrial cancer-related data in The Cancer Genome Atlas(TCGA)public database to find the differentially expressed gene CKMT1A(creatine kinase mitochondrial 1A,CKMT1A)related to staging and prognosis.2.To collect clinical tissue samples for detecting the expression level of CKMT1A in different tissues and the significance of clinical expression.3.To explore the mechanism of CKMT1A in the occurrence and development of endometrial cancer at the cellular level,thus providing a new theoretical basis for further understanding the pathogenesis of endometrial cancer and finding new prognostic markers and therapeutic targets.Methods1.The transcriptome data on normal endometrial and endometrial cancer as well as the corresponding clinical data were collected from the Cancer Genome Atlas(TCGA)database,with the R language applied to standardize them.According to the database sample FIGO stage information,the differential gene expression in endometrial tissues of different stages was compared,based on which a Venn diagram was drawn to further clarify the differential genes related to stage and prognosis.2.GO,Kegg,GSEA function enrichment analysis and PPI network drawing were conducted to determine the relevant functions.Both TCGA data and human protein atlas data were applied to verify the association between the screened differential genes and the prognosis in endometrial cancer.3.SP method immunohistochemical and q RT-PCR were performed to detect the CKMT1A protein expression and the relative expression of CKMT1A m RNA for 39 pairs of endometrial cancer tissues,based on which the correlation between CKMT1A and the clinical characteristics of endometrial cancer was analyzed.In addition,western blot was applied to detect the protein expression of CKMT1A in different stages of endometrial cancer.4.The endometrial cancer cell Ishikawa was cultured,and the cell transfection protocol was designed for three groups,including the targeted knockdown CKMT1A group,the negative control group transfected with NC,and the untransfected blank control group.Western blot and q RT-PCR were performed to obtain the knockdown efficiency.5.CCK-8 was used and scratch test was conducted to explore the effect of CKMT1A on the proliferation and migration of endometrial cancer Ishikawa cells.Results1.Compared with stage Ⅰ endometrial cancer tissues,there were 54 highly expressed genes found in stage Ⅱ endometrial cancer tissues.Compared to stage Ⅲ endometrial cancer tissues,there were 58 genes highly expressed in stage Ⅳ endometrial cancer tumor tissues.According to the Venn diagram,the gene related to stage and prognosis was screened out as CKMT1A.As revealed by GO,Kegg and GSEA enrichment analysis,CKMT1A is closely related to metabolic processes.2.The relative expression level of CKMT1A in 164 endometrial cancer tissues and 11 normal endometrial tissues included in the TCGA database reached6.535±0.1698 and 4.259±0.3378,respectively.While the expression of CKMT1A m RNA in endometrial cancer tissues was higher than in normal endometrium,and the difference was statistically significant(P<0.001).A significant difference was also observed in stage(P<0.01),pathological type(P<0.05),as well as the degree of differentiation(P<0.05)and prognosis(P<0.05)in endometrial cancer.3.As confirmed by the human protein atlas database,CKMT1A protein was highly expressed in the endometrial cancer tissues relative to normal endometrial tissue,and the difference showed statistical significance(P<0.01).Additionally,the higher the expression of CKMT1A,the worse the prognosis for patients(P<0.05).4.The relative expression level of CKMT1A in stages Ⅰ,Ⅱ,Ⅲ,and Ⅳ among 39 clinical endometrial cancer tissue specimens reached 0.0013±0.0002232,0.006808±0.001042,0.01643±0.001035,0.06064±0.01521,respectively.Besides,the higher the stage,the higher the relative expression of CKMT1A,with the difference found to be statistically significant(P<0.0001).The expression of CKMT1A in endometrial cancer was associated with the degree of differentiation,and the difference showed statistical significance(P<0.01).According to the results of Kaplan-Meier survival analysis,the higher the expression of CKMT1A,the worse the prognosis(P<0.01).5.The positive protein rate of CKMT1A in endometrial cancer tissue reached87.2%,which is significantly higher than in normal endometrial tissue(46.1%).The difference between the two was observed to be statistically significant(P<0.0001).In endometrial cancer tissue,FIGO Ⅲ+Ⅳ stage CKMT1A immunohistochemical score was clearly higher as compared to Ⅰ+Ⅱ stage,and the difference was statistically significant(P<0.0001).Also,the higher the immunohistochemical score,the worse the prognosis for patients(P< 0.01).6.According to western blot results,the expression of CKMT1A gradually increased with FIGO staging.7.In the Ishikawa cell experiment,the relative expression of CKMT1A m RNA in the blank control group,negative control group,si-CKMT1A-1 experimental group,si-CKMT1A-2 experimental group and si-CKMT1A-3 experimental group reached1.952±0.0908,1.847±0.0326,1.705±0.04213,0.4886±0.03853 and 1.924±0.03064,respectively.The relative expression of CKMT1A in the si-CKMT1A-2 experimental group was significantly lower than in the blank control group and the negative control group,while the difference was statistically significant(P<0.0001),indicating the effectiveness of si-CKMT1A-2 in knocking down the expression of CKMT1A in Ishikawa cells.In the Ishikawa cell experiment,the level of CKMT1A protein expression in the si-CKMT1A-2 experimental group was considerably lower than in the blank control group and the negative control group,suggesting the effectiveness of si-CKMT1A-2 in knocking down the expression of CKMT1A in Ishikawa cells.9.According to the results of cell proliferation-toxicity(CCK-8)experiment,the cell proliferation of the si-CKMT1A-2 experimental group was significantly reduced when compared with the blank control and negative control cells,while the difference was statistically significant(P<0.0001).10.As revealed by the scratch test,the cell migration ability of the si-CKMT1A-2experimental group was significantly weakened as compared to the blank control group and the negative control group,while the difference was statistically significant(P<0.0001).Conclusion1.CKMT1A is highly expressed in endometrial cancer tissues,and the expression of CKMT1A is associated with FIGO staging and tissue differentiation.2.The ability of Ishikawa cells with low CKMT1A expression to proliferate and migrate is significantly weakened,suggesting the possibility that CKMT1A plays a role in the occurrence and development of endometrial cancer.Also,it is indicated that CKMT1A may be suitable as therapeutic target for endometrial cancer. |
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