Abstract:

Objective To explore the correlation between IκB kinase-interacting protein （IKBIP） expression in tumor cells within cervical cancer tissues （TCCCT） and the clinical pathological characteristics and prognosis of patients， as well as its impact on the biological behaviors of cervical cancer （CC） cells HeLa and SiHa， and to elucidate its potential mechanism. Methods GENT2 and Kaplan-Meier plotter databases were utilized to analyze the differential expression of IKBIP mRNA in CC and normal cervical tissues， as well as its correlation with the clinical prognosis of CC patients. The Hallmark reference gene set was chosen for pathway enrichment analysis using the Gene Set Enrichment Analysis（GSEA） software. Immunohistochemistry method was used to detect the IKBIP protein expression in TCCCT and epithelial cells in normal cervical tissue （ECNCT）， and analyze the correlations between its expression level and clinicopathological characteristics and prognosis of CC patients. Furthermore， univariate and multivariate Cox regression analyses were performed to identify the risk factors impacting the prognosis of CC patients. The stably transfected cells of CC （HeLa cells and SiHa cells） with IKBIP knockdown were established for the experiment， which were divided into sh-NC group and sh-IKBIP group. The expression of IKBIP protein in various groups was assessed using Western blotting method. The cell proliferation activity and the percentage of 5-ethynyl-2'-deoxyuridine （EdU） positive cells were measured using cell counting kit-8 （CCK-8） and EdU methods， while Transwell chamber assay was employed to determine the numbers of migration and invasion cells in various groups. Additionally， the expression levels of E-cadherin， N-cadherin， and Snail proteins in the cells in various groups were analyzed by Western blotting method. Results The GENT2 database analysis revealed that the expression level of IKBIP mRNA in CC tissue was higher than that in normal cervical tissue （P<0.001）. The GSEA enrichment analysis identified the epithelial-mesenchymal transition （EMT） pathway as the top-ranked pathway in IKBIP high-expression group. The immunohistochemistry results indicated the positive expression rate of IKBIP protein in TCCCT was higher than that in ECNCT （50.5% vs 8.0%）， and its over-expression was associated with FIGO stage （2018） and differentiation grade of tumor （P<0.05）. The univariate and multivariate Cox regression analyses suggested that lymph node metastasis （LNM） and high expression of IKBIP were the risk factors affecting the overall survival （OS） and progression-free survival （PFS） of CC patients （P<0.05）. The Western blotting method results showed that compared with sh-NC group， the expression level of IKBIP protein in the cells in sh-IKBIP group was decreased （P<0.05）. The CCK-8 and EdU assay results showed that compared with sh-NC group， the proliferation activity and the percentage of EdU positive cells in sh-IKBIP group were decreased （P<0.05）. The Transwell chamber assay results showed that compared with sh-NC group， the numbers of migration and invasion cells in sh-IKBIP group were significantly decreased （P<0.05）. Additionally， the Western blotting method results indicated that compared with sh-NC group， the expression level of E-cadherin protein in sh-IKBIP group in CC cells was increased （P<0.05）， while the expression levels of N-cadherin and Snail protein were decreased （P<0.05）. Conclusion The expression of IKBIP protein is significantly up-regulated in HeLa and SiHa cells derived from CC， and it is closely correlated with poor prognosis in CC patients. Suppression of IKBIP protein expression can effectively inhibit the proliferation， invasion and migration capabilities as well as EMT process of CC cells.

Key words: I kappa B kinase-interacting protein, Cervical neoplasms, Cell proliferation, Cell invasion, Epithelial-mesenchymal transition