Rab12 Promotes Radioresistance of HPV-Positive Cervical Cancer Cells by Increasing G2/M Arrest

Abstract

Background: HPV-positive (HPV+) cervical cancer cells are more radioresistant compared with HPV-negative (HPV-) cervical cancer cells, but the underlying mechanism is not fully illuminated. Our previous mass spectrometry data showed that Ras-associated binding protein Rab12 was up-regulated by HPV, and this study is to investigate the role of Rab12 in the radioresistance of HPV-positive cervical cancer cells.

Methods: CCK-8 assay, colony formation assay, flow cytometry, and Western blot were performed to determine cell proliferation, apoptosis, cell cycle distribution, and protein expressions. DNA damage and repair levels were measured by comet assays and detection of γ-H2AX, XRCC4, and pBRCA1 protein expressions.

Results: Rab12 mRNA and protein expressions were up-regulated in cervical cancer tissues and HPV+ cervical cancer cells. Knockdown of Rab12 enhanced radiosensitivity while overexpression of Rab12 promotes radioresistance. Knockdown of Rab12 alleviated G2/M arrest by decreasing p-Cdc2(Tyr15) after radiation, which was a result of the reduction of p-Cdc25C(Ser216). Rab12 knockdown caused more DNA double-strand breaks (DSBs) and inhibited DNA homologous recombination repair (HRR) after radiation. Instead, overexpression of Rab12 enhanced radioresistance by increasing G2/M arrest, which provided more time for DNA HRR.

Conclusions: Rab12 may serve as a potential therapeutic target to improve clinical treatment outcome of cervical cancer.

Keywords: Cdc2; G2/M arrest; HPV; Rab12; cervical cancer; radiation.

Figure 1

Rab12 was highly expressed in cervical cancer and oncoproteins E6 and E7 promoted the expression of Rab12. (A) Real-time PCR showed that Rab12 mRNA level was higher in clinical cervical cancer tissues (n=63) compared with non-cancer cervix tissues (n=46). (B) Kaplan-Meier curves for overall survival of 60 patients with cervical cancer according to Rab12 expression (Log-Rank test, P > 0.05). (C) Rab12 protein expression was analyzed by Western blot in HaCaT and cervical cancer cell lines HeLa, SiHa, Caski, C33A. (D, E) Western blot analysis of Rab12 proteins in HaCaT cells overexpressing 16E7 and Rab12, p53, E2F1 proteins in RPE1 cells overexpressing 16E6, 16E7; GAPDH was used as the loading control. (F–H) Protein levels of Rab12, p53, E2F1 in HeLa, SiHa, Caski cells after transfection with siRNA targeting 18E6, 18E6E7, or 16E6, 16E6E7 for 48 h; Comparable Western blots were observed in three independent experiments. Data are presented as mean ± SD from three independent experiments, **p < 0.01, ***p < 0.001.

Figure 2

Radiation promoted the expression of Rab12 at both mRNA and protein levels. (A) Western blot analysis of Rab12 expression in SiHa cells at different radiation doses (24 h). (B) Rab12 expression in SiHa cells at different times after radiation (6Gy). (C) Effects of radiation (6Gy, 24 h) on Rab12 expression in SiHa cells were determined by real-time PCR and Western blot analysis. (D) Immunofluorescent staining and comet assay showed the presence of nuclear debris and DNA breaks following exposure to 6Gy radiation. Comparable Western blots were observed in three independent experiments. Data are presented as mean ± SD from three independent experiments, ***p < 0.001.

Figure 3

Rab12 affected radiosensitivity of cervical cancer cells. (A) Western blot analysis of Rab12 expression after transfection of shRab12 lentivirus. (B, C) The effects of radiation on cell proliferation and viability of SiHa-ShRab12 and control cells were detected by the colony formation and CCK-8 assays. (D) Western blot analysis of Rab12 expression after transfection of pENTER-Rab12, adenovirus vector overexpressing Rab12. (E, F) The effects of radiation on cell proliferation and viability of C33A cells overexpression Rab12 were detected by the colony formation and CCK-8 assays. Data presented as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Rab12 exerted no effects on apoptosis of cervical cancer cells after radiation. (A, C) Flow cytometry was used to detect the effect of knockdown or overexpression Rab12 on apoptosis 24 h after radiation at 6 Gy. (B, D) The expression of apoptosis related proteins PARP, Caspase3, and Cleaved-Caspase3 were detected by Western blot analysis 24 h after radiation at 6 Gy. Data are presented as mean ± SD from three independent experiments.

Figure 5

Rab12 induced more G2/M arrest by regulating cell cycle-related proteins after radiation. (A, C) Flow cytometry analysis of the effect of Rab12 knockdown and overexpression on cell cycle distribution 24 h after radiation at 6 Gy (stained with PI), G1, G2 phases are indicated; (B, D) Cell cycle related proteins before and after radiation in Rab12 knockdown and overexpression cells were detected by Western blot. Comparable Western blots were observed in three independent experiments. Data are presented as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Rab12 alleviated DNA damage after radiation. (A, D) Double-strand breaks (DSBs) were analyzed using the neutral comet assay and immunofluorescent staining of γ-H2AX. Immunofluorescence showed that γ-H2AX was located in the nuclear of SiHa and C33A cells (red). There were more γ-H2AX foci in SiHa-ShRab12 cells than control cells, and γ-H2AX foci in C33A-pENTER-Rab12 cells decreased after exposure to radiation than control cells (6Gy, 24 h). DAPI (blue): nuclear counterstain. (B, E) The comet assay showed changes in tails in SiHa and C33A cells with Rab12 knockdown or overexpression after radiation. DNA was stained with DAPI (blue). The cells were visualized by fluorescence microcopy. (C, F) DSB repair proteins pBRCA1 and XRCC4 in Rab12 knockdown and overexpression cells after irradiation were detected by Western blot. Data are presented as mean ± SD from three independent experiments, *p < 0.5, **p < 0.01, ***p < 0.001.