Silencing the Expression of Cyclin G1 Enhances the Radiosensitivity of Hepatocellular Carcinoma In Vitro and In Vivo by Inducing Apoptosis

Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 is a novel member of the cyclin family, and it is abnormally expressed in HCC. In this study we investigated the role of cyclin G1 in the radiotherapy of HCC cells. The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells and Huh7 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and Western blot analysis. The proliferation was analyzed using MTT assay, and the radiosensitivity of HCC cells was detected using colony formation assay and a xenograft tumor model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by Western blot analysis, and caspase-3 was detected using fluorimetry. The expression of cyclin G1 mRNA and protein in HepG2/Huh7-cyclin G1-siRNA cells was found to be significantly decreased compared to that in HepG2/Huh7 cells. Silencing the expression of cyclin G1 inhibited the proliferation of HCC cells and enhanced radiosensitivity in HCC cells in vitro and in vivo. Knockdown of cyclin G1 expression significantly decreased Bcl-2 expression, and increased Bax expression and caspase-3 activity in HCC cells. Silencing of cyclin G1 expression enhances the radiosensitivity of HCC cells in vitro and in vivo. The mechanism for this may be related to the regulation of apoptosis-related proteins.


INTRODUCTION
Hepatocellular carcinoma (HCC) is the fifth most common cancer in men and the seventh most common in women. Patients with HCC mostly receive palliative treatments because few patients have the opportunity for curative resection (1). Radiotherapy, such as three-dimensional conformal radiotherapy (3-DCRT), intensity modulated radiotherapy (IMRT), image-guided radiotherapy (IGRT) or stereotactic body radiation therapy (SBRT), plays an important role in the treatment of HCC (2)(3)(4); however, there are also many adverse effects associated with radiotherapy (5). Radiotherapy is limited by the risk of radiation-induced hepatic toxicity (6,7). HCC is usually accompanied by cirrhosis of the liver with poor hepatic function, which makes it difficult to treat with high-dose radiotherapy (8). Low-dose radiotherapy always requires cancer cells to be highly radiosensitive to achieve curative effects. Currently, the precise mechanism modulating the radiosensitivity of HCC cells remains poorly understood (9).
Cyclin G1 was originally identified as a novel member of the cyclin family (10). Many published studies have shown that cyclin G1 is abnormally expressed in many malignant cancers, such as HCC (11), cervical carcinoma (12) and lung carcinoma (13), and also contributes to the recurrence and chemoresistance of hepatoma cells (11). Targeting cyclin G1 exerts anticancer effects in many cancers (14)(15)(16). Kimura et al. reported that cyclin G1 is involved in cell cycle arrest (G 2 /M block) induced by radiation and that radiosensitivity is significantly enhanced in cyclin G1 genedeleted mice and embryonic fibroblasts (17). Cyclin G1 is also one of the target genes of the transcription factor p53 (18,19). The p53 tumor suppressor gene is involved in the tumorigenesis, development and progression of many cancers and is closely related to the radiosensitivity of cancer cells (20,21). Therefore, we hypothesized that cyclin G1 is related to the radiosensitivity of HCC cells.
In the current study, we investigated the role of cyclin G1 in the radiosensitivity of HCC cells. We evaluated the effects of silencing the expression of cyclin G1 on the proliferation and radiosensitivity of HCC cells. The results of this study suggest that silencing of cyclin G1 expression promotes proliferation and enhances the radiosensitivity of HCC cells in vitro and in vivo, and that the mechanism is possibly related to the regulation of apoptosis.

Ethics Approval and Consent to Participate
The experimental protocol established here was approved by the Ethics Committee of the Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, China.

Cell Culture and siRNA Transfection
HepG2 cells and Huh7 cells were obtained from the School of Medical Sciences, Zhengzhou University, and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClonee Laboratories, Logan, UT) with 10% fetal bovine serum (FBS) (HyClone), 100 lg/ml penicillin and 0.25 lg/ml streptomycin at 378C and 5% CO 2 .

Cell Proliferation Assay
Cells were seeded into 96-well plates at 1.5 3 10 3 cells and 2 3 10 3 cells per well and incubated overnight. The cells were cultured for five days at 378C. 3-(4,5-Dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed at different time points: 24, 48, 72, 96 and 120 h. Then, 20 ll MTT solution (5 mg/ml) was added to each well and incubated for an additional 4 h at 378C. Then, the MTT solution was aspirated, and 100 ll DMSO was added to dissolve the formazan crystals. The number of cells was counted using a microplate reader at a wavelength of 490 nm.

Colony Formation Assays
Cells were digested, and single-cell suspensions were prepared. Then, cells were plated in six-well plates at appropriate dilutions (200, 200, 400, 400 and 800 cells/well for the 0, 2, 4, 6 and 8 Gy irradiated groups, respectively) and allowed to attach overnight. The cells were irradiated with different doses using an ONCOR linear accelerator (Siemens, Munich, Germany) at a dose rate of 300 cGy/min, cultured for another 14 days, fixed with methanol and stained with 0.05% crystal violet (Sigma-Aldricht, St. Louis MO). The number of colonies consisting of 50 or more cells was counted, and the surviving fraction was calculated. Dosesurvival curves were plotted with a single-hit multitarget model using GraphPad 5 software (San Diego, CA), and the values of D 0 , D q and SER Dq were calculated. The experiment was performed three times.

Xenograft Transplantation and Irradiation
To develop xenograft tumors, HepG2 cells, HepG2-cyclin G1-siRNA negative control cells and Hep-cyclin G1-siRNA cells (1.0 3 10 5 cells) were subcutaneously implanted into BALB/c nude mice (n ¼ 5). When xenograft tumors grew to a mean volume of approximately 50 mm 3 , tumors were 20 Gy irradiated in five fractions (4 Gy per fraction, once every two days). Each animal was earmarked and followed individually throughout the experiment. Tumor volume (mm 3 ) was calculated using the following formula: V (mm 3 ) ¼ length (mm) 3 width (mm) 2 /2. The body weights of the mice were also recorded every two days during the experiment. All tumors were observed for 21 days, and the mice were then sacrificed and tumors were weighed.

Caspase-3 Assay
Caspase-3 activity was determined using a caspase-3 kit (Sigma-Aldrich) 48 h postirradiation according to the manufacturer's instructions. Briefly, 25 ll of 13 lysis buffer was added into the well, and incubated on ice for 15-20 min, then 200 ll of 13 assay buffer containing substrate was added and mixed well, then 200 ll was transferred to a fluorimeter multi-well plate, fluorescence was read in a kinetic mode every 10 min for 40-60 min at room temperature (fluorimeter was set at: excitation of 360 nm, emission of 460 nm, slit width of 5 nm). The results were calculated using a 7amino-4-methylcoumarin (AMC) standard curve.

Statistical Analysis
Data are expressed as the mean 6 standard deviation (SD) and were analyzed using SPSSt version 10.0 software (IBMt, Armonk, NY). Differences between groups were determined using analysis of variance (ANOVA) or Student's t test. P , 0.05 was considered statistically significant.

Silencing the Expression of Cyclin G1 Inhibits HCC Cell Proliferation
The expression of cyclin G1 mRNA in HCC cells (HepG2 and Huh7) was analyzed by q-PCR. We selected three siRNAs to silence the expression of cyclin G1 and detected it at different time points to avoid false positive results. As shown in Fig. 1A, the expression of cyclin G1 mRNA in HepG2/Huh7-cyclin G1-siRNA cells was significantly lower than that in HepG2/Huh7 cells and HepG2/Huh7 cyclin G1-siRNA negative control cells, and siRNA-3 had the best silence effect. Therefore, we selected siRNA-3 to silence the expression of cyclinG1 in subsequent experiments. Meanwhile, there was no significant difference between HepG2/Huh7 cells and HepG2/ Huh7-cyclin G1-siRNA negative control cells. Cyclin G1 protein expression was also decreased after transduction with cyclin G1-siRNA, which was confirmed by Western blot analysis (Fig. 1B). The MTT assay showed that cyclin G1-siRNA significantly inhibited the proliferation of HepG2 and Huh7 cells (Fig. 1C).

Silencing of Cyclin G1 Expression Enhances the Radiosensitivity of HepG2 and Huh7 Cells In Vitro
Colony formation assays showed that radiation exposure caused a dose-dependent cytotoxic effect on HepG2 and Huh7 cells. Transduction with cyclin G1-siRNA sensitized HepG2 and Huh7 cells and successfully enhanced the effects of radiation (Fig. 2). The radiosensitization effects of cyclin G1-siRNA in HepG2 and Huh7 cells are summarized in Table 1.

Silencing of Cyclin G1 Expression Enhances the Radiosensitivity of HCC In Vivo
To detect the effect of cyclin G1 on the radiosensitivity of HepG2 tumor xenografts in vivo, a xenograft analysis was performed. The data showed that silencing of cyclin G1 expression in HepG2 cells inhibited the growth of tumor xenografts compared to the control group. After irradiation, the tumor volume of tumor xenografts was significantly inhibited in the HepG2-cyclin G1-siRNA group compared to the HepG2 group, HepG2-cyclin G1-siRNA negative control group and HepG2 with irradiated group (Fig. 3A  and B). There was no significant difference in the body weight of the mice (Fig. 3C). The change in tumor weight illustrated an inhibitory effect similar to that seen with the change in tumor volume (Fig. 3D).

Silencing of Cyclin G1 Expression Decreases Bcl-2 Expression and Increases Bax Expression and Caspase-3 Activity
Bcl-2 and Bax proteins were detected by Western blot. As shown in Fig. 4, the expression of Bcl-2 decreased, and the expression of Bax and the activity of caspase-3 increased significantly in the HepG2/Huh7-cyclin G1-siRNA group compared to the control group, and the change in the HepG2/Huh7-cyclin G1-siRNA combined with X-ray irradiated group was the most obvious.

DISCUSSION
While radiotherapy can lead to local control of HCC in patients, the dose is always limited by radiation-induced toxicity to the liver and adjacent luminal gastrointestinal organs. The application of radiosensitizers may increase the   (23), thus targeting cyclin G1 enhanced radiosensitivity, which is consistent with our results. Cyclin G1 plays an important role in the proliferation of many cancers, such as osteogenic sarcoma (15), ovarian cancer (16), breast cancer (24), pancreatic ductal adenocarcinoma (25) and papillary thyroid carcinoma (26). In our study, we found that silencing the expression of cyclin G1 inhibited the proliferation of HepG2 cells and xenograft tumor growth in vivo. According to the literature, the effect of cyclin G1 on proliferation is probably related to p53 or MDM2 (27). In cyclin G1-deficient tissue, p53 levels increase, and the loss of cyclin G1 protects against N-diethylnitrosamine (DEN)induced hepatocarcinogenesis (28).
Numerous published studies have shown that the radiosensitivity of tumor cells is associated with apoptosis (29) and that induction of apoptosis can enhance the radiosensitivity of cancer cells. Loss of cyclin G1 can induce apoptosis in uterine leiomyoma cells (30). Bcl-2, Bax and caspase-3 play important roles in apoptosis (31). Caspase-3 may induce apoptosis in HCC cells (32). We found that X-ray irradiation and silencing of cyclin G1 expression can inhibit Bcl-2 expression, promote Bax expression, and increase caspase-3 activity. These results indicate that X-ray irradiation and cyclin G1 silencing can induce apoptosis, and that cyclin G1-siRNA enhanced the effects of radiation.

CONCLUSION
Silencing of cyclin G1 expression can enhance the radiosensitivity of HepG2 and Huh7 cells. The mechanism may be related to the regulation of apoptosis-related proteins. This study provides a new potential pathway to increase the radiosensitivity of HCC cells.

SUPPLEMENTARY INFORMATION
Western blot data.