| Objective:Currently,the role of tumour immune infiltration is a study hotspot in the carcinogenesis and progression of cervical cancer.This project aims to explore the immune infiltration-related markers in cervical cancer and their clinicopathological application value through bioinformatics combined with clinical sample analysis.Methods:This study includes two parts:bioinformatics and clinical sample analysis.In the first part,the expression profile from GSE63514 and corresponding information of for sample with cervical cancer were obtained from the high-throughput gene expression database(GEO)using the bioinformatics method.The single sample gene set enrichment analysis(ss GSEA)algorithm was used to analyze the difference in immune infiltration levels in normal cervical tissues,cervical intraepithelial neoplasia(CIN)tissues and cancer tissues.Limma R package was used to screen differential genes in different tissues.Pearson correlation was used to analyze the correlation between the intersection differential genes and immune infiltration.Weighted gene co-expression network analysis(WGCNA)was used to screen out key genes associated with tumour immune infiltration.The expression was verified in the Cancer Genome Atlas(TCGA)database.The second part is based on the analysis of clinical samples.The paraffin-embedding tissues of cancer and peri-cancer tissues from 69patients with cervical cancer were collected.Immunohistochemical technique was used to detect the expression of immune infiltration-related genes in cancer and para-cancer tissues.The clinicopathological application value of immune infiltration-related genes was analyzed.Results:(1)The data in the first stage were from GSE63514,a gene expression profile by high-throughput sequence in the normal cervix tissues(n=24),CIN tissues(n=76)and cervical cancer tissues(n=28).Compared with the control,76 CIN-related genes and 497cervical cancer-related different genes were screened,and 72 genes were intersected.Functional enrichment analyses showed that these 72 intersection genes mediated cell division,cell cycle,mitotic cell cycle,microtubule movement,regulation of cyclin-dependent serine/threonine kinase activity,chromosome separation and other biological processes mainly through cell components such as the nucleus,cytoplasm,cytoplasm,chromosomes,intermediates,mitotic spindles,and centromere.By ss GSEA algorithm analysis,the results showed that there were significant differences in the levels of immune infiltration of activated CD4~+T cells,natural killer T cells,follicular helper T cells and neutrophils in normal control tissues,CIN tissues and cervical cancer tissues(P<0.05).Based on the WGCNA method and cyto Hubba analysis,three key markers related to tumor immune infiltration were selected,namely KI67,SPAG5 and KIF15.The expression levels of these markers in tissue samples with cervical cancer were significantly higher than those in control samples through the analysis of TCGA data(P<0.05).(2)The results of the second stage showed that the immunohistochemical expression levels of hub genes-related proteins Ki67,SPAG5,and KIF15 in cervical cancer tissue were significantly higher than those in para-carcinoma normal tissue(P<0.05).According to ROC analysis,the optimal truncation values for predicting cancer and normal samples were calculated,which were 1.5,2.5 and 3.5,respectively.According to the optimal cut-off value,69 patients were divided into high and low-expression groups.The proportion of stage III tumors in the high expression group(34.55%)was higher than that in the low expression group(0%),P<0.05.The proportion of histological grading with low differentiation in the high SPAG5 expression group(85.25%)was higher than that in the low SPAG5 expression group(50%),P<0.05.The rate of cervical invasion depth(9.25±5.10),vascular cancer thrombus(44.64%),lymph node metastasis(35.71%)and advanced cancer(33.93%)in the high KIF15 expression group was higher than that in the low KIF15 expression group(3.93±3.10),(7.69%),(7.69%),(0%),P<0.05.There was a significant difference between the high Ki67 and SPAG5 co-expression group and the low SPAG5 co-expression group in vascular thrombus and tumour staging(P<0.05).There were significant differences in cervical invasion depth,vascular thrombus,lymph node metastasis and tumor staging between the high-expression group of KIF15 and Ki67 and the low-expression group(P<0.05).There were statistically significant differences between the high expression group of KIF15 and SPAG5 and the low expression group in vascular cancer thrombus,degree of differentiation,and tumor stage(P<0.05).There was a significant difference between the high expression group of KIF15,Ki67 and SPAG5 and the low expression group in vascular thrombus,lymph node metastasis and tumor staging(P<0.05).COX regression model analysis showed that high expression of KIF15 was a risk factor for poor prognosis of cervical cancer patients RR=1.935(95%CI=1.091-3.433),P=0.024.Conclusion:The results of this study indicate that the levels of immune infiltration in Activated CD4+T cells and Natural killer T cells in cervical cancer tissue increase,while the levels of immune infiltration in T follicular helper cells and Neutral decrease.This abnormality was closely related to the increased expressions of Ki67,SPAG5 and KIF15 in cancer tissue,which were closely related to the pathological characteristics of vascular cancer thrombus,degree of differentiation,and tumor stage.These results suggested that the increased expressions of Ki67,SPAG5 and KIF15 genes in cervical cancer tumor tissue were correlated with tumor immune invasion,which may be important markers for predicting the evolution of cervical cancer. |
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