The human cervical cancer HeLa cell line (Institute for Regenerative Medicine, ZhuJiang Hospital of Southern Medical University, Guangzhou, China), GenBank (National Center for Biotechnology Information, Bethesda, MD USA), Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA), fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA), Ex Taq, T4 DNA ligase, pMD18-T vector, restriction enzymes SacI and HindIII (all from Takara Bio, Inc., Otsu, Japan), primer synthesis (Sangon Biotech Co., Ltd., Shanghai, China), pGL3-Basic vector, PGL3-control vector, PRL-TK vector, dual-luciferase reporter assay kit (all Promega Corporation, Madison, WI, USA), Escherichia coli DH5α (Institute for Regenerative Medicine, ZhuJiang Hospital of Southern Medical University), genomic DNA extraction kit, agarose gel extraction kit, plasmid minipreparation medium kit (DP304; Tiangen Biotech Co., Ltd., Beijing, China), Lipofectamine^™ 2000 (Thermo Fisher Scientific Inc.), and electrophoresis-grade agarose (12% separation gel and 5% spacer gel) were used.

HeLa cells were cultured in high glucose DMEM containing 10% fetal bovine serum. The cells were maintained at 37°C, in a saturated humidity and 5% CO₂ incubator, and were passaged every 2–3 days. The cells at the logarithmic phase were used in all the experiments. The TIANamp Genomic DNA kit (DP304) was used to extract the genomic DNA of HeLa cells, and all procedures were conducted according to the manufacturer's protocol (Tiangen Biotech Co., Ltd.).

The primer was designed according to the 5′end of Hes1 gene from GenBanksp16 (ncbi.nlm.nih.gov/nuccore/NT_005612.17?from=100428715&to=100434890&report=genbank). The Hes1 gene sequence was analyzed using BIO-XM^™ (Biomax Informatics AG, Planegg, Germany) and the −747-+66 segment at the 5 end was amplified. The following primers were used: Upstream, 5-CGA GCT CAGCGGGAACTTTAGATGTG-3 and downstream, 5-CCCAAG CTTGTTGACACTGGCTGGGGTA-3. The underlined parts indicate the enzyme sites-SacI and HindIII. The genomic DNA of HeLa cells was used as a template, and polymerase chain reaction (PCR) was used to amplify the Hes1 promoter segment. The Hes1 promoter sequences are listed in Table I.

The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 56.2°C for 30 sec, elongation at 72°C for 45 sec, there were 35 cycles in total, followed by elongation at 72°C for 10 min. Following PCR, 5 µl product was added in 1.5% agarose gel for electrophoresis. Subsequently, the gel extraction kit was used for the purification of the PCR product. The procedures were conducted according to the manufacturer's protocol [DNA amplification and extraction kit (cat. no. ER103; Tiangen Biotech Co., Ltd.)].

The collected PCR product (Hes1 gene) was transfected by Lipofectamine^™ 2000 into the pMD18-T vector, which was transformed and amplified to extract the plasmids. Subsequently, restriction enzymes for SacI and HindIII dual-enzyme digestion were used and the sequences were detected, which was termed the pMD18-T-Hes1-promoter.

The recombinant T vector with the correct sequence and pGL3-Basic plasmid received SacI and HindIII dual-enzyme digestion and separated by agarose electrophoresis, the gel extraction kit was used to collect the target segment, and then T4 DNA ligase was used to transform, amplify and extract the plasmids. The product received dual-enzyme digestion and sequence detection, which was termed the pGL3-Hes1-promoter.

The HeLa cells were inoculated in 24-well plate at 4 h before transfection, 0.5 ml complete medium and 0.5×10⁵ cells were added in each well and the transfection was conducted when the cells reached 70–80% confluency. The procedures were done according to the manufacturer's instructions for Lipofectamine 2000. The transfected plasmids included negative control plasmid pGL3-Basic, positive control plasmid pGL3-Control containing the SV40 enhancer/promoter, recombinant plasmid pGL3-Hes1-promoter and internal control PRL-TK plasmid. A total of 48 h after transfection, the Dual-luciferase reporter assay kit from Promega was used to detect the activity of the transfected plasmid, and there were 3 repeated wells for every group and the experiments were repeated 3 times.

The data were analyzed by SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance and the Q test were used to analyze the data. The data were presented as mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The genomic DNA of HeLa cell was used as a template and the promoter segment of the Hes1 gene was amplified by PCR, and the electrophoresis results indicated that there was a specific band at 813 base pairs (bp), as presented in Fig. 1A.

The PCR production of the Hes1 promoter was purified and connected to the pMD18-T vector to establish the recombinant vector pMD18-T-Hes1-promoter. Subsequent to transformation the plasmids were extracted for SacI and HindIII dual enzyme digestion. The electrophoresis results demonstrated that there were two specific bands at 813 and 2,700 bp, as presented in Fig. 1B.

DNA sequence analysis demonstrated that compared with the Hes1 gene promoter sequence from GenBank, the 5′ and 3′ ends of the cloned target segment of recombinant T cloning vector were located upstream −747 bp and downstream +66 bp of ATG, the size was 813 bp as presented in Fig. 2.

The electrophoresis of recombinant expression vector pGL3-Hes1-promoter after SacI and HindIII dual-enzyme digestion demonstrated that there were specific bands at 813 and 4,300 bp, as presented in Fig. 3.

DNA sequencing results demonstrated that compared with the Hes1 promoter sequence from GenBank, the 5′ and 3′ end of the target segment sequence of cloned expression vector pGL3-Hes1-promoter located at upstream −747 bp and downstream +66 bp of ATM, the size was 813 bp, as presented in Fig. 4.

At 48 h after HeLa cell transfection by recombinant expression plasmid pGL3-Hes1-promoter, pGL3-Basic and pGL3-Control, the luciferase expression was detected. The ratio of first and second fluorescence was defined as the relative luciferase activity. The relative luciferase of pGL3-Basic was 1.076, of pGL3-Control was 100.47 and of pGL3-Hes1-promoter was 34.44, there was significant difference (P<0.05; Table II).