Regulatory effects of COL1A1 on apoptosis induced by radiation in cervical cancer cells

Abstract

Background: Cervical cancer is a common cancer of women in developing countries, and radiotherapy still remains its predominant therapeutic treatment. Collagen type I alpha 1 (COL1A1) has been shown to have a radioresistance effect in previous studies. However, such effect of COL1A1 has not yet been revealed in cervical cancer.

Methods: Expression of COL1A1 in cervical cancer tissues and normal tissues was assessed by qRT-PCR and immunohistochemistry. The effect of COL1A1 on radioresistance of human cervical cancer cell lines HeLa and CaSki was assessed using the colony formation assay. Apoptosis alterations were analyzed by flow cytometry. In addition, western blotting was used assessed the alterations of several critical apoptosis and signaling pathway related proteins.

Results: The expression of COL1A1 was significantly increased in cervical cancer tissues compared with normal tissues at the mRNA and protein level. Further, based on COL1A1 knock down and COL1A1 activation cell models, a negative correlation was observed between COL1A1 expression level and radiosensitivity. Moreover, the findings are further supported by apoptosis analysis that COL1A1 activation could inhibit the apoptosis of cervical cancer cells. Subsequently, a significantly decreased expression of p-AKT and Bcl-2, increased expression of Caspase-3 were observed in the LY294002 plus radiation group compared with radiation alone group, while these influences caused by LY294002 or X-ray radiation were reversed after COL1A1 activation.

Conclusions: To our knowledge, this is the only study to profile the mechanisms that COL1A1 plays a crucial role in cervical cells anti-apoptosis induced by radiation. Therefore, our identification of radioresistance-related COL1A1 in cervical cancer could be a starting point to explore the function of collagens, adding a new dimension to our understanding of the cervical cancer, assisting cancer biologists and clinical oncologists in novel therapeutic strategies.

Keywords: Apoptosis; COL1A1; Cervical cancer; Radioresistance.

Fig. 1

The expression of COL1A1 in cervical normal and cancer tissues. a COL1A1 was detected significantly increased in cervical cancer tissues (n = 20) compared with adjacent normal tissues (n = 20). b Representative tumor adjacent normal tissue, grade I–II, grade II, grade III cervical cancer tissues sections stained with an antibody against COL1A1, respectively. c Optical density measurements indicated that COL1A1 expression from normal cervical specimens to cervical cancer was enhanced gradually. Magnification, ×400. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01

Fig. 2

The expression of COL1A1 in Hela, CaSki, SiHa cells and cell models construction. a qRT-PCR analysis revealed that COL1A1 is differentially expressed in the cell lines. b Differentially expressed of COL1A1 was detected in three different cells by western blotting analysis. **P < 0.01 vs Hela, ^## P < 0.01 vs Caski

Fig. 3

The construction of over expression and interference models. a qRT-PCR analysis indicated that COL1A1 expression levels were increased significantly in CaSki and Hela cells with COL1A1 activation plasmids, while COL1A1 expression levels were decreased significantly in CaSki and Hela cells with COL1A1 shRNA plasmids. b Protein levels indicated the COL1A1-shRNA and COL1A1 Activation cell models were constructed successfully. *P < 0.05 vs control. Con, cells without plasmid. COL1A1 Activation, cells transfected with COL1A1 Activation plasmids. Activation NC, cells transfected with scramble control of activation plasmid. COL1A1 shRNA, cells transfected with COL1A1 shRNA plasmid. shRNA NC, cells transfected scramble control of shRNA plasmid

Fig. 4

The effects of COL1A1 in cervical cells after treating with radiation. a The expression of COL1A1 depended on the the increasing dose of radiation. *P < 0.005 for 4 Gy group vs 0 Gy group in Hela and CaSki cells. b Colony formation of different cells to X-ray radiation was detected. The images of the colonies were stained with 0.5% crystal violet in CaSki and Hela cells. c Survival fraction of different cells to a dose of X-ray radiation (0–10 Gy). **P < 0.01 vs 0 Gy

Fig. 5

The regulated effect of COL1A1 on apoptosis induced by radiation. In CaSki and HeLa cells, combination of COL1A1 shRNA transfection and radiations induced much more apoptosis than shRNA NC transfection or control cells. Whereas, combination of COL1A1 Activation and radiations group inhibiting apoptosis compared to Activation NC or control group. **P < 0.01 vs control

Fig. 6

Levels of apoptosis-related proteins are evaluated in cervical cancer cells. a Western blotting analysis of Caspase-3, Bax and Bcl-2 expression. The values indicate the COL1A1 may inhibit apoptosis of cervical cancer cells. b–d Western blotting analysis of individual signal events. The values indicate the COL1A1 may be involved in AKT pathway of apoptosis induced by radiation