The Tumor-promoting Effects of FAM92A1-289 in Cervical Carcinoma Cells

Materials and Methods

Cells and animals. The human cervical carcinoma cell line (HeLa cells) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin-streptomycin solution (all from Invitrogen, Shanghai, China) and incubated at 37°C in a humidified atmosphere with 5% CO₂. Subculture was performed once newly grown cells reached subconfluence. All cells used in the present study were limited in 3-5 passages. Nude mice (female, 4-6 weeks of age) were purchased from the Model Animal Research Center at Nanjing University (Nanjing, China) and housed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocols of the present study were approved by the Animal Care Committee at Hubei University of Medicine (Shiyan, China). The mice were subject to xenotransplantation of HeLa cells followed by series of imaging detection under anesthesia. Anesthesia was performed using intraperitoneal injection of pentobarbital (90 mg/kg; Shanghai Chemical Reagent, Shanghai, China).

RNA isolation from HeLa cells. Total RNA was isolated from Hela cells using TRIzol reagent (Invitrogen) as per manufacturer's instruction. RNA integrity was confirmed by electrophoresis on a 1% agarose gel. Hela cell-derived cDNA was used as the template for PCNA amplification, which was involved in the construction of expression vector.

The construction of expression vectors. The fragments of GFP-FAM92A1-289 and PCNA were amplified by PCR. The PCR template for the GFP-FAM92A1-289 was pEGFP-FAM92A1-289 plasmid that was constructed in our previous study (5). The PCR primers for this fragment were 5’-CGGAATTCATGA TGAGGCGCACC-3’ (forward, containing an ECoRI site) and 5’-CCGCTCGAGTTACTTGTACAGCTCGT-3’ (reverse, containing an XhoI site). Hela cell-derived cDNA was used as the template for PCNA amplification and the primers for amplifying the complete coding region of PCNA were 5’-CGCCTCGAGATGTTCGAGGCGCGCCTGGT-3’ (forward, containing an XhoI site) and 5’-CGGGGATCCCTAAGATCCTTCTTCATCCT-3’ (reverse, containing a BamHI site). The primers used in this study were designed based on the sequences of human FAM92A1-289 and PCNA in GenBank database (accession numbers: DQ327716 and NM182649) and synthesized by Sangon Biotech (Shanghai, China). The PCR amplification was conducted at 95°C for 5 min, then for 30 cycles of 95°C of 30 sec each, 55°C for 30 sec, 72°C for 1 min and, lastly, with a final extension cycle of 72°C for 10 min. The amplified DNA fragments were identified by 1% agarose gel with ethidium bromide staining.

The amplified fragments of GFP-FAM92A1-289 and PCNA were inserted into the expression vector CMV-SP6-TALEN (SIDANSAI) with puromycin-resistance gene and the pcDNA3.1(−) vector. Briefly, the target fragments were purified with universal DNA purification/recovery kit (Tiangen, Beijing, China). Then, both the amplified DNA fragments and the vector were doubly digested with endonucleases, correspondingly. The target fragments and the target vector backbone were extracted from the gel using universal DNA purification/recovery kit and then they were ligated overnight at 16°C. The ligation products were transfected into competent E. coli cells DH5α and screened with LB plate for 16 h at 37°C. The obtained recombinant plasmids were extracted with a DNA mini-extraction kit (Sigma, St. Louis, MO, USA) and confirmed by DNA sequencing and restriction enzyme digestion (Takara, Dalian, China).

Optimal puromycin concentration for the screening of HeLa cell line. Since the CMV-SP6-TALEN vector contained a puromycin-resistance gene, the optimal puromycin concentration was determined for screening purpose. The HeLa cells in logarithmic phase were cultured in 24-well plates with the same density of 5×10⁴ cells/well. When the cells reached 80% confluency, puromycin was added into the culture medium for screening. The concentrations of puromycin solutions were set as 0.25, 0.5, 0.75, 1, 1.5, 2 and 3 μg/ml. Triplicate tests were performed for each concentration. After 9 days of culture, all Hela cells cultured with 1.5, 2 and 3 μg/ml puromycin solution died. Therefore, the concentration of 1.5 μg/ml was considered as optimal puromycin concentration for screening HeLa cells.

Screening of stably transfected cell line and target protein identification. HeLa cells were cultured till 80% confluency and then transfected with the CMV-SP6-TALEN-GFP-289 vector using Lipofectamine™ 2000 transfection reagent (Invitrogen) as per manufacturer's instruction. After 24 h of incubation, the transfection medium was replaced with fresh medium containing 1.5μg/ml puromycin to initiate positive HeLa cell screening procedure. The same culture medium was replaced every third day for 9 days. On day 10, the HeLa cells transfected with CMV-SP6-TALEN-GFP-289 (HeLa/GFP-289) showed good growth and were observed under a microscope to confirm the presence of green fluorescence.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify the expression of target protein, i.e. GFP-FAM92A1-289. Briefly, HeLa/GFP-289, HeLa /PEGFP-N1 (positive control) and wild type HeLa cells (negative control) were cultured in 6-well plates. Cells of each group were collected in 1.5ml microcentrifuge tubes, washed twice with ice-cold PBS and lysed in 100μl cell lysis buffer (Beyotime, Jiangsu, China) containing 1 mM phenymethylsulfonyl fluoride (PMSF). Samples were chilled on ice for 30 min and then centrifuged at 10,000 x g at 4°C for 5 min. The supernatant was re-suspended in 6X sample buffer and boiled for 5 minutes.

Protein samples (30 μg) were electrophoresed in SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature followed by an overnight incubation with primary antibody against GFP (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. After washing three times with Tris-buffered saline, 0.1% Tween 20 (TBST), the membrane was incubated with alkaline phosphatase-conjugated secondary antibody (1:500; Beyotime) for 1 h at room temperature. The membrane was washed three times with TBST and imaged with a gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Scratch assay. The scratch assay was used to detect the effects of FAM92A1-289 on cell migration capability. HeLa/GFP-289 and wild type HeLa cells were cultured in 6-well plates (1×10⁵ cells/well). When the cells reached complete confluency, a line was scratched in the middle of the wells using a 200-μl pipette tip. The plates were washed with PBS for three times. Cells were then incubated with fresh serum-free DMEM medium at 37°C with 5% CO₂ for 48 h. As described in a previous study (10), the width of scratched line was measured and the percentage of narrow down was compared at 0 h, 24 h and 48 h, respectively. The experiment was performed in triplicates.

MTT assay. Cell viability was evaluated using the MTT assay as previously described (11). HeLa cells and HeLa/GFP-289 cells were seeded into 96-well microplate at a density of 2.5×10³ cells/well. Five replicates were set for each type of the cells. On days 1, 3, 5 and 7 after incubation, 20 μl methylthiazol tetrazolium ((MTT), 5 mg/ml; Beyotime) was added to each well followed by 4 h incubation. Then, 100 μl formazan solution was added to dissolve the crystal violet. The optical absorbance at wavelength 570 nm (A570) was recorded with a Vmax Microplate reader (Thermo Scientific, Waltham, MA, USA). The value of A570 is proportional to the number of viable cells in each well.

Colony forming ability assay. The HeLa cells and HeLa/GFP-289 cells in good growth condition were counted and 1×10³ cells were plated into a 12-well plate pre-coated with 1% gelatin. The cells were cultured with serum-free DMEM/F12 medium (containing 100 U/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml epidermal growth factor (EGF), 10 ng/ml basic fibroblast growth factor (bFGF)) at 37°C for 10-14 days. Three replicates were set for each type of the cells. The culture medium was replaced according to the cell growth rate and the color changes of the culture medium. The cultures were photographed regularly under the microscope to observe colony formation.

Xenograft test. Since the In Vivo Imaging System (IVIS) is preferentially sensitive to bioluminescence, luciferase gene-transfected HeLa cells (HeLa-luc cells) were used for the xenograft test, including HeLa-luc cells and FAM92A1-289-transfected HeLa-luc cells. The cells were harvested when they reached logarithmic phase of growth. Then, 1×10⁷ of these cells were subcutaneously injected from the right of the inguinal region of nude female mouse (5 mice each group). All the xenografted animals were imaged every week using a small animal image system (IVIS; Caliper Life Sciences, Hopkinton, MA, USA) until the mice died. Each test was conducted 10 minutes after an anesthetized mouse received 200 μl D-luciferin solution (0.15 mg/ml, Caliper Life Sciences) intraperitoneal injection. The growth rate and size of tumors were analyzed using the radiance value, which is proportional to the number of bioluminescence-producing cells (10).

Co-immunoprecipitation assay. Because HeLa cells express PCNA, it is an ideal cell model to study protein interaction between FAM92A1-289 and PCNA. However, the content of endogenous PCNA is too low to perform co-immunoprecipitation assay; therefore, pcDNA3.1-PCNA plasmid was transfected into HeLa cells to enhance the expression of PCNA. After pcDNA3.1-PCNA was transfected into HeLa/GFP-289 cells, these cells could overexpress both GFP-FAM92A1-289 and PCNA simultaneously. The protein sample extracted from the double transfected HeLa cells was used for co-immunoprecipitation assay. Sufficient amount of PCNA antibody was added into 200 μg total protein sample and gently rotated at 4°C overnight. To pull down PCNA antibodies, protein sample was incubated with 40 μl protein A/G Agarose beads (Beyotime) for an additional 3 h period at 4°C. The precipitate was washed for five times with ice-cold radioimmunoprecipitation assay (RIPA) buffer and re-suspended in 1X sample buffer followed by boiling for 5 minutes to dissociate the immunocomplex from the beads. The supernatant was collected by centrifugation and subjected to Western blot. The protein of co-immunoprecipitation was detected by antibodies against GFP and PCNA in order to observe whether GFP-289 and PCNA coexisted in the precipitation.

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Figure 1.

Gel electrophoresis of FAM92A1-289 gene PCR products. The PCR products of GFP-FAM92A1-289 gene (A) and PCNA gene (B) were isolated by 1% agarose gel. M: DNA ladders.

Statistical analysis. Numerical data were presented as the mean±deviation (SD). χ² test was used to compare the rate of two samples. Statistical analyses were performed with the t-test by using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) and p<0.05 was considered statistically significant.