32 cervical cancer tissue specimens and paracancerous tissue specimens were obtained from patients who underwent surgical at the Gynecology and Obstetrics Department of the Third Affiliated Hospital of Zhengzhou University (Zhengzhou, China) from January 2017 to October 2017. All patients were not receiving radiotherapy or chemotherapy before the surgical. All patients involved in the study were informed, in addition to being provided with informed consent documentation, which they subsequently signed. Experiments were approved by the Ethics Committee of clinical trials of the Third Affiliated Hospital of Zhengzhou University. SiHa (cervical squamous cell carcinoma) and HeLa (cervical adenocarcinoma) cell lines were bought from the Shanghai Institute for Biology Sciences of the Chinese Academy of Sciences. The cell lines were cultured in DMEM medium with 10% (v/v) prenatal bovine serum, which is not heat-activated (all from Gibco^®; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 mg/ml streptomycin, and 100 U/ml penicillin (Sangon Co., Ltd, Shanghai, China) at 37°C in a humidified incubator with 5% CO₂.

Tissues were fixed in formalin and embedded in paraffin wax. The paraffin-embedded samples were cut into 4-mm-thick, then were deparaffinized and rehydrated in xylene and serially diluted alcohol solutions. Slides were heated by microwaving in 0.01 M sodium citrate solution for 15 min to retrieve antigen. Subsequently, the slides were incubated with ASRGL1 rabbit polyclonal antibody (1:100; Abcam, Cambridge, UK) at 4°C overnight. Then, each slice was incubated with second antibody for 30 min. Finally, all slides were stained with diaminobenzidine followed by the hematoxylin was applied to counterstain. As a negative control (NC), the primary antibody was replaced with PBS. All the images were analyzed by conventional optical microscopy.

The cellular RNA from SiHa cells was extracted by TRIzol (Invitrogen, Shanghai, China). Subsequently, 2 µg mRNA was reverse transcribed into cDNA. The primers were designed as follows: ASRGL1 forward, 5′-CGAGTTCAACGCAGGTTGTG-3′ and reverse, 5′-GGGATTTGCTATACACTGGACTG-3′; GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and 5′-CACCCTGTTGCTGTAGCCAAA-3′. qPCR was performed using the SYBR-Green kit (Takara Biotechnology Co., Ltd., Dalian, China) and the ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative expression as determined using the 2^−∆∆Cq method as described previously (19). The RT-qPCR reaction was as follows: 95°C for a period of 15 sec, followed by 45 cycles including denaturation at 95°C for 5 sec, annealing at 45–62°C for 30 sec. The PCR amplification of GAPDH and ASRGL1 provided an amplicon of 121 and 127 bp, respectively.

GeneChem Co., Ltd. (Shanghai, China) made the blueprint of the mutually complementary DNA sequence (5′-CAGTCCAGTGTATAGCAAA-3′) of ASRGL1. The knockdown efficiencies of these oligonucleotides were tested by incorporating into the lentivirus-based psc14173 (GeneChem Co., Ltd.) via AgeI/EcoRI sites. The lentivirus particles functioned as described previously (20). With respect to the lentivirus infection, SiHa cells were seeded in 6-well plates. Then, ASRGL1-shRNA-lentivirus or NC lentivirus was added according to the multiplicity of infection (MOI). The cells were examined using fluorescence microscope (Olympus, Tokyo, Japan) after three days.

After several washes with cold phosphate-buffered saline (PBS), the cells were solubilized in precooled 2X lysis buffer (100 mM Tris, pH 6.8, 10% glycerol, 4% SDS, 2% 2-ME, 10 mM EDTA) for 30 min on ice. Then, the supernatants were collected after the lysates were centrifuged at 14,000 × g, 4°C for 10 min. An equivalent amount of protein is separated by 12% SDS-PAGE and transferred on PVDF membrane (Millipore, Bedford, MA). Then, the membrane was blocked in 5% fat-free milk, in TBST buffer, followed by probing with the anti-ASRGL1 (1:1,000), Bcl-2-associated X protein (anti-Bax; 1:1,000), anti-B-cell lymphoma 2 (Bcl-2; 1:1,000), anti-cyclin dependent kinase (CDK2; 1:1,000), anti-cyclin A2 (1:1,000; all Abcam) antibodies at 4°C overnight. GAPDH (1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) is an endogenous control. After 3 washes in TBST, the membrane was incubated with the HRP-conjugated goat anti-mouse and goat anti-rabbit secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Finally, it was detected using ECL reagent (ECL-Plus kit; Amersham, Piscataway, NJ, USA).

SiHa cells in the logarithmic stage were digested and resuspended; 2,000 cells/well were seeded into 96-well plates. Each experiment was performed three times independently. Since day 2, Cellomics ArrayScan VT1 Readers (Cellomics, Inc., Pittsburgh, PA, USA) calculated the cell number once a day at an interval of five days. By adjusting the analysis settings of input parameters, the number of cells with green fluorescence in each scan orifice were calculated accurately. Finally, the cell proliferation curve was plotted.

Cells were infected with ASRGL1 shRNA lentivirus or control and were cultured to 80% confluency. Subsequently, the cells were digested, resuspended, and centrifuged at 1,300 rpm for 5 min, washed in chilled PBS, and fixed in 75% alcohol for 1 h, followed by staining with propidium iodide (PI; 50 µg/ml, Sigma-Aldrich^®, St. Louis, MO, USA) in the presence of RNase A (100 µg/ml; Fermentas^®, Shanghai, China). The cell cycle was analyzed using BD FACSCalibur flow cytometer (FCM; BD Biosciences, San Diego, CA, USA).

Cells were seeded into 6-well plates and transfected with ASRGL1-shRNA or NC lentivirus for 72 h. Then, the cells were digested, resuspended, centrifuged at 1,300 rpm for 5 min, washed in cold PBS and 1X binding buffer, followed by resuspension in 1 ml 1X staining buffer, and 5 ml Annexin V-APC (eBioscience, San Diego, CA, USA) into 100 ml cell suspension. The reaction was incubated in the dark for 15 min. The cells were analyzed by FCM.

The cell cultures were analyzed in triplicate using the SPSS version 21.0 software (IBM SPSS, Corp., Armonk, NY, USA). The original data were presented as mean ± standard deviation (SD). The discrepancies in ASRGL1 knockdown and control cells were comparison using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The ASRGL1 proteins were mainly located in the cytoplasm and was found to be overexpressed in cervical tissues from patients with cervical cancer when compared with the paracancerous tissue specimens, as analyzed by immunohistochemistry (Fig. 1) (P<0.05).

The efficiency of ASRGL1 in SiHa cervical cancer cells was examined by infecting the cells with ASRGL1 shRNA lentivirus and NC shRNA lentivirus-expressing GFP, respectively. Then, we examined the fluorescence level of GFP protein to determine the infection efficiency. As seen in Fig. 2A, 72 h after infection, >80% cells were expressing GFP as assessed by fluorescence microscopy. The mRNA and protein expression level of ASRGL1 after shRNA infection was evaluated by quantitative PCR and western blot analysis, respectively. As observed in Fig. 2B, the ASRGL1 mRNA level was decreased by almost 90% in cell lines infected with ASRGL1 shRNA lentivirus as compared to the NC group. Furthermore, Fig. 2C showed the downregulation of ASRGL1 protein expression in SiHa cells infected with ASRGL1 shRNA lentivirus. Fig. 2D, the quantitative analysis of western blotting brands.

In order to evaluate the function of ASRGL1 in the proliferation of cervical cancer cells, GFP-expressing cells were infected with ASRGL1 shRNA lentivirus and NC shRNA lentivirus, respectively using Cellomics ArrayScan VT1 Readers continuously for 5 days. As shown in Fig. 3A, the cells transfected with NC shRNA lentivirus were significantly more as compared to the ASRGL1-shRNA-transfected cells on day 5 (P<0.05). Fig. 3B further confirmed that knockdown of ASRGL1 had a dramatic impact on curbing the SiHa cells' number.

FCM was used to analyze whether the knockdown of ASRGL1 will affect the cell cycle progression in SiHa cells. As illustrated in Fig. 4A, the ASRGL1-shRNA-transfected cells were distributed in G1 phase (37.71±0.86%, P<0.01); S phase (51.95±0.68%, P<0.01), and G2/M (10.34±1.31%, P<0.01). However, the NC-shRNA-transfected cells were distributed in G1 phase (66.62±0.27%, P<0.01); S phase (15.30±0.26%, P<0.01), and G2/M (18.08±0.19%, P<0.01). Furthermore, the ratios of S stage cells in ASRGL1-shRNA transfected group were significantly larger than those in the negative shRNA control group (P<0.01). Additionally, the levels of CDK2 and cyclin A2 were significantly reduced in the ASRGL1 siRNA-transfected cells (Fig. 4B). Cyclin A2 appeared in the late Go, can combine with CDK2 and promote the synthesis of DNA in S phase. Consequently, the knockdown of ASRGL1 might capture the cell cycle during the S stage in SiHa cells.

FCM was employed to analyze whether the knockdown of ASRGL1 affected the cell apoptosis in SiHa cells (Fig. 5A). The rate of apoptosis in the ASRGL1-shRNA exhibited a dramatic increase as compared to the negative shRNA control group (ASRGL1-shRNA 11.483±0.55% vs. NC-shRNA 4.36±0.15%, P<0.05). Hence, silencing the expression of ASRGL1 in SiHa cells induces apoptosis. Subsequently, we measured the expression level of cell apoptosis-associated proteins Bax and Bcl-2 after the knockdown of ASRGL1 (Fig. 5B). The proapoptotic protein, Bax, was significantly enhanced, whereas the expression of anti-apoptosis Bcl-2 protein was reduced compared with the control.