Fig 2: SEMA3C promotes cervical cancer growth both in vitro and in vivo. (A) Western blotting analyses of SEMA3C expression in cervical cancer cell lines. (B) SEMA3C protein levels transfected with SEMA3C siRNA were determined by western blotting analysis. (C) Proliferation of silenced SEMA3C cells was evaluated by CCK-8 assays. Data represent the mean (±SD) of three independent experiments, each performed in silenced SEMA3C HeLa and SiHa cells, compared with the control. Error bars indicate S.D. (**p < 0.01; ***p < 0.001). (D) Silencing SEMA3C inhibited HeLa and SiHa cell colony formation. Data represent the mean (±SD) of three independent experiments, each performed in silenced SEMA3C HeLa and SiHa cells, compared with the control. Error bars indicate S.D. (**p < 0.01; ***p < 0.001). (E) SiHa shSEMA3C or control cells were subcutaneously injected into nude mice for xenograft assay. Tumor growth curve and average weight in each group are shown. Data represent the mean (±SD) of five mice. Error bars indicate S.D. (**p < 0.01; ***p < 0.001).

Fig 3: SEMA3C activates the MAPK signaling pathway in cervical cancer cells. (A) SiHa cells transfected with siSEMA3C and control cells were subjected to western blotting assay. (B) Activation of ERK (p44/42) in SiHa cells treated with varying concentrations of recombinant SEMA3C protein for 10 min. (C,D) GSEA results showed that “HALLMARK_KRAS_SIGNALING_UP” and “KEGG_MAPK_SIGNALING_PATHWAY” were differentially enriched in the SEMA3C high-expression cervical cancer patients in the TCGA cohort. (E) SiHa cells transfected with siSEMA3C and control cells were subjected to western blotting assay. The effect of SEMA3C silencing on receptor tyrosine kinase pathway signaling is shown by western blotting assay with phospho-specific antibodies against EGFR, HER2, MET, and SRC. (F) Activation of kinases in SiHa cells treated with varying concentrations of recombinant SEMA3C protein for 10 min.

Fig 4: SEMA3C predicts poor prognosis of cervical cancer patients. (A) Real-time PCR analysis of SEMA3C expression in 12 pairs of cervical cancer tissues and adjacent normal tissues. (B) Western blot assay of SEMA3C expression in cervical cancer tissues (T) and paired adjacent normal tissues (N) from 12 patients. (C) Immunohistochemical staining and scoring of SEMA3C expression was performed in 87 human cervical cancer samples. Representative views were shown. (D) Kaplan–Meier analysis results for overall survival correlation with SEMA3C expression assessed by immunohistochemical staining in our cohort are presented. The patients were divided into two groups according to the median value. (E) Kaplan–Meier analysis results for overall survival correlation with SEMA3C expression assessed by sequencing in the TCGA cohort are presented. High SEMA3C group, n = 201; low SEMA3C group, n = 103. The patients were divided into two groups according to the cutoff representing the value that yields maximal difference with regard to survival at the lowest log-rank P-value calculated by R software. The dotted lines indicate 95% confidence intervals. T, tumor tissue; N, adjacent normal tissue.