An alignment analysis was performed to compare the sequences of the transcript variants available in GenBank and the mRNA B isoforms of the ST3GAL4 gene reported by Kitagawa et al in 1996 (11). The gene bank accession numbers for the ST3GAL4 isoforms are: V1 isoform: NM_006278; V2 isoform: NM_001254757; V3 isoform: NM_001254758 and V4 isoform: NM_001254759.

The human keratinocyte cell line HaCaT was obtained from the Centro de Investigación Biomédica de Occidente (Guadalajara, Mexico), and human cervical cell lines (SiHa, HeLa, C33A) were obtained from the Centro de Investigación Biomédica de Oriente (Puebla, Mexico). The cells were grown in monolayer culture in p25 cm² flasks and were maintained at 37°C in an atmosphere of CO₂ in Dulbecco's modified Eagle's medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with 10 mmol/l HEPES, which was supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA). The culture medium was replaced every two days. Subconfluent adherent cells were harvested using a mixture of trypsin (0.025%) and EDTA (0.02%); (Sigma-Aldrich; Merck KGaA) and were washed with phosphate buffered saline.

Cervical specimens were obtained at the Centro Médico Nacional sXXI (Mexico City, Mexico), between January 2013 and January 2014, from female patients (aged 18–65 years). Samples of squamous cell carcinoma of the cervix were obtained from women that underwent radical hysterectomy. Samples exhibiting premalignant lesions (low and high grade squamous intraepithelial lesions) were obtained from women who were treated by cervical conisation. Normal tissue was obtained from women undergoing hysterectomy for uterine miomatosis. Women with a clinical history of other cancer types, as well as samples with degraded RNA, were excluded from the present study. All data, including age and pathological results, were prospectively recorded. The present study was approved by the Ethics Committee of Instituto Mexicano del Seguro Social (Mexico City, Mexico; Number R-2012-785-061). All patients provided informed consent according to the guidelines of the Human Ethics Committee. In total, 8 normal samples, 7 samples of premalignant lesions and 8 samples of cervical cancer were included.

Total RNA was extracted from cell lines using a Nucleo Spin RNA II kit (Macherey-Nagel GmbH, Düren, Germany) according to the manufacturer's protocols.

Biopsies were maintained in RNAlater solution (Qiagen, Inc., Valencia, CA, USA), and 25 mg of tissue was disrupted with the TissueLyser II (Qiagen, Inc.) for 2 min. Subsequently, RNA was extracted with an RNeasy Plus Mini kit (Qiagen, Inc.) according to the manufacturer's protocols.

First-strand cDNA synthesis was performed with a RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, US) using the following concentrations: 1X of reaction buffer, 1 U/µl of Ribolock RNAse Inhibitor, 1 mM of dNTP mix, 10 U/µl of RevertAid H Minus M-MuLV Reverse Transcriptase, 5 µM of random primers and 1 µg of total RNA, in a final volume of 20 µl, according to the manufacturer's protocols. The reaction was incubated for 5 min at 25°C followed by incubation for 60 min at 42°C.

RT-sqPCR was performed on a 1000 Touch thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a final volume of 20 µl using the following: 10 µl of 2X PCR Master Mix (Promega Corporation, Madison, WI, USA), 1 µl of 10 µM forward and reverse primer, and 2 µl of cDNA. The primer sequences were as follows: V1 transcript forward 5′-gac tgt gct gga ggt gac ag-3′, reverse 5′-acc atg ttt ctc agc agg ca-3′; V2 forward 5′-gaa ccg tgc tgc ccc gcc cc-3′; reverse 5′-ggg act tgc tgac cat gtt t-3′; V3 forward 5′-ttt gta gtg ttt ccc gcc ca-3′, reverse 5′-gct gac cat gtt tct cag ca-3′. A positive control containing primers for hypoxanthine-guanine phosphoribosyltransferase (HPRT) (forward 5′-ccc tgg cgt cgt gat tag tga-3′; reverse 5′-agc aag acg ttc agt cct gtc c-3′) and a negative control (with no template) were added into each assay. The program cycle was: 95°C for 2 min, 40 cycles of 95°C for 30 sec, 56°C for 1 min and 72°C for 1 min. A total of 40 cycles were performed to ensure for the successful detection of RNA variants with a low copy number. The presence of the variants was confirmed, by performing the reactions twice. The amplification product was assessed on an agarose gel for low molecular weight fragments at a 2% concentration. The gels were stained with ethidium bromide (1 mg/ml), observed in an ultraviolet transilluminator, and then photographed using a digital camera (ELP110HS; Canon, Inc., Tokyo, Japan). To confirm that the amplified product corresponded to the variant transcript, direct sequencing of the PCR products was performed using the Sanger method in the Laboratory of Biodiversity and Genomics, CINVESTAV (Irapuato, Mexico).

To prove that the efficiencies of the ST3Gal 4 gene and HPRT amplification were similar and optimal, RT-qPCR was performed to validate the endogenous gene and to quantify the V1 transcription in cell lines and biopsies. Each reaction was performed in a final volume of 10 µl with the concentrations: 5 µl of 2X Maxima SYBR Green/Rox qPCR Master Mix (Thermo Fisher Scientific, Inc.), 1 µl of 10 mM forward and reverse primers for the V1 isoform of the ST3GAL4 gene or HPRT, and cDNA from SiHa, HeLa, and C33A cell lines that had been previously synthesized, using the aforementioned procedure. A standard curve was determined with the concentrations: 2, 1, 0.5, 0.25, 0.125 and 0.0625 ng/µl. The reactions were performed in triplicate using a StepOne Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 30 sec at 60°C, and 70°C for 30 sec.

Relative quantification was performed using the comparative CT method with the formula: 2^−ΔΔCq (13). The qPCR reaction was performed with the StepOneReal-Time PCR System (Applied Biosytems; Thermo Fisher Scientific, Inc.). The final reaction volume of 10 µl included 1 µl of cDNA template (0.5 ng final concentration in cell lines and 12 ng in biopsies), 5 µl of 2X Maxima SYBR Green/Rox qPCR Master Mix (Thermo Fisher Scientific, Inc.), 0.5 µl of forward and reverse primers (0.5 µM final concentration) and 3 µl of RNase free water. RT-qPCR was performed under the following conditions: 1 cycle at 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 60°C for 1 min. Triplicates of each sample were analysed, and negative controls with no cDNA template were included in each assay. Transcript levels were normalized against HPRT expression.

Statistical analysis was performed using the Graph Pad program (version 7; GraphPad Software, Inc., La Jolla, CA, USA). A one-way analysis of variance followed by Tukey's post-test was performed for multiple comparisons. Data are presented as mean ± standard error deviation. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated in triplicate.

Since isoforms A1 and A2 are reported exclusively in testicle, ovary and placenta these isoforms were excluded from subsequent analysis. First, an alignment was performed among isoforms B1, B2, B3 and B4 reported by Kitagawa et al (11) with those transcript variants reported in GenBank to determine their correspondence and their sequence identity. Table I indicates the B isoforms whose sequence corresponded to the current nomenclature of transcript variants reported in GenBank and the protein isoform coded. Analysis demonstrated that B1 isoform corresponds to V1 variant, BX to V2 and B3 to V4. The B2 isoform sequence was not available in the GenBank data base, and the V3 transcript was not reported by Kitagawa et al (11) (Access date April 29, 2016).

Next, the presence of the transcripts variants V1, V2 and V3 was evaluated in cervical cell lines. The RT-PCR analysis demonstrated that V1 and V2 transcripts were present in all the cervical cell lines, SiHa, HeLa, and C33A, and in the human keratinocyte cell line HaCaT; V3 transcript was present in SiHa, HeLa and HaCaT cell lines, however not in C33A (Fig. 1).

The expression level of the V1 transcript was determined in the cervical cell lines SiHa, HeLa and C33A. The ΔΔCq analysis demonstrated that SiHa cells exhibited the highest expression (Fig. 2) compared with HeLa, C33A and HaCaT. In every case, significant differences were identified.

Next, the V1 transcript levels in biopsies were analysed to determine whether the V1 transcript modifies its expression in relation to cervical neoplasia grade. Relative quantification was performed by RT-qPCR using 8 samples of normal tissue, 7 samples of squamous intraepithelial lesions (low and high grade) and 8 samples of cervical cancer. Premalignant lesions and cervical cancer demonstrated a decreased level of V1 transcript compared with normal tissue (Fig. 3). In every case, significant differences were identified.