Expression profile and promoter analysis of HEPIS

Introduction

The human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10 (SARS-CoV nsp-10; HEPIS) gene is a novel gene that was initially discovered by Hong et al (1) in 2008 from a cDNA library of human embryo lung tissues. The HEPIS protein is able to interact with SARS-CoV nsp-10 (1). SARS-CoV nsp-10 is produced by the coronavirus main protease, which cleaves polyproteins pp1a-pp1ab during infection; this protein is able to function as a viral transcriptase (2). The HEPIS protein consists of 147 amino acids and has several casein kinase II phosphorylation sites (1). In a previous study, HEPIS was demonstrated to interact specifically with the TATA sequence of the heat shock protein 70 promoter, suggesting that HEPIS may be associated with gene transcriptional regulation (1). However, the expression profile and promoter activity of HEPIS are yet to be elucidated.

Changes in the expression of specific gene products are regulated by a wide range of mechanisms, including transcriptional and translational regulation (3). Octamer transcription factor-1 (OCT-1), nuclear factor κB (NF-κB) and activator protein 1 (AP-1) are important transcription factors that serve roles in cancer cell proliferation, survival, transformation, invasion, metastasis, angiogenesis and chemotherapy/radiotherapy resistance (4). OCTs are a class of transcription factor that bind to the ‘ATTTGCAT’ sequence of the gene promoter (5). OCT-1 (also termed POU2F1) is a ubiquitously expressed transcription factor containing a POU domain with a homeobox subdomain (6). OCT-1 serves an important regulatory role in cellular transcription via binding to a specific promoter octamer sequence on the target genes (7). Furthermore, OCT-1 binds to cofactors that interact with the POU DNA-binding domain to either positively or negatively regulate a variety of genes (8). Previous studies have reported that OCT-1 affects the occurrence and development of several cancers, including breast cancer (9), LNCaP prostate cancer (10), esophageal squamous cell carcinoma (11) and colorectal cancer (12). NF-κB is a dimeric transcription factor that belongs to the Rel/NF-κB family and is formed by hetero- or homodimerization (13). NF-κB is known to serve a vital role in the regulation of inflammation, immunity, cell proliferation and apoptosis (13–16). AP-1, which is a dimeric transcriptional activator composed of Jun, Fos, activating transcription factor and musculoaponeurotic fibrosarcoma protein subunits (17,18), serves important roles in the regulation of cellular proliferation, transformation, differentiation and apoptosis via binding to a common AP-1-binding site in the target gene promoter (19,20).

In the present study, in situ RNA hybridization and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect the HEPIS gene expression profile in several organ tissues and breast cancer cell lines. The promoter activity of the HEPIS gene was also investigated. The first step was to identify the core HEPIS promoter to enable subsequent determination of the important transcription factors. The promoter region and transcription factor binding sites of the HEPIS gene were predicted by bioinformatics analysis. The AP-1, NF-κB and OCT-1 binding sites of the HEPIS promoter region were identified using site-directed mutagenesis, dual luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays, respectively.

Materials and methods

RNA in situ hybridization

A DNA microarray containing samples from 72 cases of tumor and normal tissue was obtained from Shaanxi Chaoying Biotechnology Co., Ltd. (cat. no. BCN721; Xian, China). The samples were from the following 12 organs: Esophagus, stomach, colon, rectum, liver, lung, kidney, breast, uterine cervix, ovary, prostate and pancreas; with 3 cores positive for cancer and 3 cores of adjacent normal tissue from each organ and one cancer tissue core and one adjacent normal tissue core per case. The following sense and antisense probes matching the HEPIS core responding sequence were used: Antisense, digoxigenin (DIG)-TCT GCC CAT ATG TCA GGA TTG GAA ATA ATG GAT −3′ and sense, DIG-ATC CAT TAT TTC CAA TCC TGA CAT ATG GGC AGA-3′. All probes were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Hybridization procedures were performed as previously described (21). Staining was scored using a 0–3+ scale. 0, no staining; 1+, 2+ and 3+ indicate increased intensity of the staining. Sub-regions excluding necrosis, macrophages and infiltrated neutrophils and lymphocytes were selected and scored. The intensity score for an array spot is the mean of all its sub-regions.

Cell culture

MDA-MB-231, MCF-7, T-47D, ZR-75-30 and 293T cells (China Center for Type Culture Collection, Wuhan, China) were maintained in high-glucose Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and incubated at 37°C with 5% CO2. 293T cells were seeded at a density of 15×104 cells/well in 6-well plates for quantitative ChIP assays. 293T cells were seeded at a density of 5×104 cells/well in 24-well plates for luciferase assays.

RT-qPCR

Total RNA was extracted from MDA-MB-231, MCF-7, T-47D and ZR-75-30 breast cancer cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The extracted total RNA (0.5 µg per sample) was then used to synthesize first-strand cDNA using a GoScript™ Reverse Transcription System kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. The primers used for PCR were as follows: HEPIS, forward, 5′-ATGTGGCTCAGTTTGTCCTC-3′ and reverse, 5′-AGCAAGATTTCCTCCAGGTC-3′; GAPDH, forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. GAPDH was used as an internal control. qPCR was performed using a SYBR Master Mixture (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol using the following cycling conditions: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The expression of HEPIS was analyzed as previously described (22).

Plasmid construction

The promoter sequence of the HEPIS gene (pHEPIS) was obtained by PCR from MCF-7 cell genomic DNA using the following primers: pHEPIS-F1.7k (−1334), forward 5′-ATCCTCGAGCATCACAAGTAGGGCAGCAT-3′; pHEPIS-F1.6k (−1060), forward 5′-ATCCTCGAGGAGTCTTCAAAGGGAGTG-3′; pHEPIS-F1.4k (−1203), forward 5′-ATCCTCGAGTCCTGGTATGCCAAGAAA-3′; pHEPIS-F1.3k (−899), forward 5′-ATCCTCGAGCAAGCTGATAGCCACCAA-3′; pHEPIS-F1.1k (−759), forward 5′-ATCCTCGAGAGGTTGGCAGGCCGGATAT-3′; pHEPIS-F0.6k (−279), forward 5′-ATCCTCGAGCGAAGAGGAGGGAGGTAG-3′; pHEPIS-R (+373), reverse 5′-AGTAAGCTTACTTCGCACCTTCGGCTA-3′. PCR was performed using pyrobest DNA polymerase (Takara Biotechnology Co., Ltd.). The PCR amplification reaction system conditions and PCR products were purified as previously described (23). Purified products were cloned into the XhoI (CTCGAG) and HindIII (AAGCTT) restriction enzyme sites of the pGL3-basic vector (Promega Corporation) using T4 DNA ligase (Sangon Biotech Co., Ltd.) according to the manufacturer's protocol.

In the present study, the transcription factor database TRANSFAC (www.cbrc.jp/research/db/TFSEARCH.html) was used for the search, and several AP-1, NF-κB and OCT-1 transcription factor-binding sites were predicted within the HEPIS promoter region. Site-directed mutageneses of the OCT-1 (−1236/−1223, negative numbers indicate that it is upstream of the transcription initiation site), NF-κB (−1186/−1176) and C-JUN (−856/−846) binding sites in the HEPIS promoter were performed using a Quick Change Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) according to manufacturer's protocol, using the following primers: pHEPIS-OCT-1-M, forward 5′-TTATAGGTGTCAAATTCATCATCACCATCAAAACTGCGTGCTTCTGCACTGAAACA-3′ and reverse 5′-TGTTTCAGTGCAGAAGCACGCAGTTTTGATGGTGATGATGAATTTGACACCTATAA-3′; pHEPIS-NF-κB-M, forward 5′-GAGTCTTCAAAGGGAGTGGAATTACCTGGATCTTCTGTTG-3′ and reverse 5′-CAACAGAAGATCCAGGTAATTCCAACTCCCTTTGAAGACTC-3′; pHEPIS-C-JUN-M, forward 5′-AATAACAAATTCATCATTGTTAGTTTGTAGCAGGATTGCACTGGAGACAGAGATTCC-3′ and reverse 5′-GGAATCTCTGTCTCCAGTGCAATCCTGCTACAAACTAACAATGATGAATTTGTTATT-3′. Underlined base pairs indicate mutation sites.

Transfection and dual luciferase reporter assay

293T cells were cotransfected with 1 µg pGL3-basic vector, pHEPIS-1.7K, pHEPIS-1.6K, pHEPIS-1.4K, pHEPIS-1.3K, pHEPIS-1.1K, pHEPIS-0.6K, pHEPIS-1.7K-M-OCT-1, pHEPIS-1.6K-M-NF-κB, pHEPIS-1.3k-M-C-JUN or pHEPIS-1.7K-3M and 0.2 µg pRL-TK (Promega Corporation) plasmid DNA/well in 24-well plates using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The luciferase activity of the extracts was assessed 24 h following transfection using a Betascope analyzer Infinite M200, (Tecan Group Ltd., Männedorf, Switzerland) and analyzed as previously described (23). The pRL-TK plasmid containing the Renilla luciferase gene was used as an internal control.

ChIP assays

ChIP assays were performed according to the manufacturer's protocol using a Millipore ChIP assay kit (EMD Millipore, Billerica, MA, USA). The following primary antibodies were used: Rabbit polyclonal antibodies against NF-κB p65 (cat. no. ab7970, 1:200), OCT-1 (cat. no. ab66132, 1:200; both Abcam, Cambridge, UK) and C-JUN (cat. no. sc-1694, 1:100), and anti-rabbit normal immunoglobulin G (cat. no. sc-2345, 1:100; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as an negative control. The above antibodies were used per chromatin sample and rotated overnight at 4°C. Protein A/G Agarose/Salmon Sperm DNA Secondary antibody (1:400; cat. nos. 16-157 and 16-201; EMD Millipore) was added per sample for 1 h at 4°C with rotation. The amount of each specific DNA fragment in the immunoprecipitates was determined using PCR reactions with the following primers: OCT-1, forward 5′-ATGTAATCCAGTAGCCTGTC-3′ and reverse 5′-CTCCCTTTGAAGACTCTGA-3′; NF-κB, forward 5′-TTCAGAGTCTTCAAAGGGAG-3′ and reverse 5′-GCATACCAGGAGACAATAAAC-3′; C-JUN, forward 5′-GCCACCAACAATAACAAA-3′ and reverse 5′-AGGAGGACATTCACTTGC-3′. The PCR was performed using a PCR Master Mix (Sangon Biotech, Shanghai, China) according to the manufacturer's protocol using the following cycling conditions: 95°C for 30 sec, followed by 30 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 10 sec; 72°C for 5 min.

Statistical analysis

Statistical analysis was performed using SPSS 9.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Student's t-test and one-way analysis of variance followed by a Dunnett's test were used to analyze data. P<0.05 was considered to indicate a statistically significant difference.

Results

HEPIS expression profile in tissues and breast cancer cells

The HEPIS expression profile was detected by RNA in situ hybridization in a tissue microarray. HEPIS expression in esophageal squamous cell carcinoma and rectal adenocarcinoma tissues was the opposite of that in normal esophageal and rectal tissues (Table I; Fig. 1); HEPIS expression was positive in esophageal squamous cell carcinoma and negative in normal esophageal tissue, whereas it was positive in normal rectal tissue and negative in rectal adenocarcinoma. HEPIS was positively expressed in tumor and normal tissues from the stomach, liver, colon, prostate, lung, uterine cervix and pancreas (Table I). The expression of HEPIS was positive in some tumor and normal tissues of the kidneys and ovaries and negative in others. HEPIS was positively expressed in nonspecific infiltrating duct carcinoma of the breast and partial positive expression was observed in normal breast tissue. HEPIS expression levels in four human breast cancer cell lines was examined using RT-qPCR (Fig. 2). The expression of HEPIS was significantly increased in the osteolytic breast cancer T-47D cell line compared with ZR-75-30, MDA-MB-231 and MCF-7 cells. HEPIS mRNA levels in T-47D cells were ~8-fold higher compared with MCF-7 cells (P<0.01), and in ZR-75-30, MDA-MB-231 cells were ~1.8-fold higher compared with MCF-7 cells (P<0.05). These results suggest that HEPIS is expressed at different levels in various organs and breast cancer cell lines.

Figure 1. RNA in situ hybridization demonstrating HEPIS expression in clear cell carcinoma of the kidney, squamous cell carcinoma of the uterine cervix, and hepatocellular carcinoma. Magnification, ×200. HEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10; anti-p, antisense probe used to detect HEPIS mRNA expression; sense-p, sense probe used as a negative control.

Figure 2. HEPIS mRNA levels in MDA-MB-231, MCF-7, T-47D and ZR-75-30 breast cancer cells. *P<0.05 and **P<0.01 vs. MCF-7. HEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10.

Table I. HEPIS expression in multiple organ cancer and normal tissue.

Table I.

HEPIS expression in multiple organ cancer and normal tissue.

Organ	Pathology diagnosis	Tissues/samples (n)	HEPIS mRNA-positive tumors, n (+/++/+++)	HEPIS mRNA-negative tumors, n
Esophagus	Squamous cell carcinoma	3	3 (0/3/0)	0
	Normal tissue	3	0	3
Stomach	Adenocarcinoma	3	3 (1/2/0)	0
	Normal tissue	3	3 (0/1/2)	0
Colon	Adenocarcinoma	3	3 (0/2/1)	0
	Normal tissue	3	3 (0/0/3)	0
Rectum	Adenocarcinoma	3	0	3
	Normal tissue	3	3 (0/3/0)	0
Liver	Hepatocellular carcinoma	3	3 (0/2/1)	0
	Normal tissue	3	3 (0/0/3)	0
Lung	Squamous cell carcinoma	3	3 (0/3/0)	0
	Normal tissue	3	3 (3/0/0)	0
Kidney	Clear cell carcinoma	3	2 (2/0/0)	1
	Normal tissue	3	1 (1/0/0)	2
Breast	Non-specific infiltrating duct carcinoma	3	3 (2/1/0)	0
	Normal tissue	3	2 (1/1/0)	1
Uterine cervix	Squamous cell carcinoma	3	3 (3/0/0)	0
	Normal tissue	3	3 (3/0/0)	0
Ovary	Serous cystadenocarcinoma	3	1 (1/0/0)	2
	Normal tissue	3	1 (1/0/0)	2
Prostate	Adenocarcinoma	3	3 (1/1/1)	0
	Normal tissue	3	3 (2/1/0)	0
Pancreas	Duct adenocarcinoma	3	3 (3/0/0)	0
	Normal tissue	3	3 (0/3/0)	0

[i] HEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10.

Cloning and activity of the human HEPIS promoter

To understand the mechanism by which HEPIS gene transcripts are expressed, dual luciferase reporter assays were used to detect HEPIS promoter activity. A total of six different truncated lengths of the HEPIS promoter regulatory sequences were amplified and the PCR products were cloned into the pGL3-basic vector (Fig. 3A). Dual luciferase reporter assay analysis of the six recombined plasmids revealed that the −1334/+373, −1203/+373, −1060/+373, and −899/+373 bp reporter gene fragments exhibited higher activity levels compared with pGL3-basic (P<0.01); and the −759/+373 bp and −279/+373 bp reporter gene fragments exhibited higher activity levels compared with pGL3-basic (P<0.05; Fig. 3B).

Figure 3. Cloning and activity of pHEPIS. (A) Six different truncated pHEPISs were cloned into a pGL3-basic LUC expression vector. These plasmids were designated as pHEPIS-1.7K, pHEPIS-1.6K, pHEPIS-1.4K, pHEPIS-1.3K, pHEPIS-1.1K and pHEPIS-0.6K. (B) Dual LUC activity assays of six pHEPIS constructs. Six recombinant vectors containing pHEPISs of different lengths and pRL-TK were cotransfected into 293T cells. *P<0.05, **P<0.01 vs. pGL3-basic. HEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10; pHEPIS, HEPIS promoter; LUC, luciferase.

Mutations at transcription factor binding sites and luciferase activity analysis

To investigate whether these putative response elements regulate the transcription of HEPIS, the OCT-1 (5′-CTATTTGCTTCTG-3′, −1236/−1223 bp), NF-κB (5′-GGAATCCCCT-3′, −1186/−1176bp), and C-JUN (5′-TTGAGTCAGG-3′, −856/−846bp) response elements on the human HEPIS promoter were mutated to generate pHEPIS-1.7K-M-OCT-1, pHEPIS-1.6K-M-NF-κB and pHEPIS-1.3K-M-C-JUN, which were constructed individually (Fig. 4A). The dual luciferase assay results demonstrated that the luciferase activities of pHEPIS-1.7K-M-OCT-1, pHEPIS-1.6K-M-NF-κB and pHEPIS-1.3k-M-C-JUN were significantly decreased compared with the activities of pHEPIS-1.7K, pHEPIS-1.6K and pHEPIS-1.3K, respectively (P<0.05; Fig. 4B), suggesting that C-JUN, OCT-1 and NF-κB activate the reporter. Furthermore, the OCT-1, NF-κB and C-JUN binding elements of the HEPIS promoter were simultaneously mutated to generate pHEPIS-1.7K-3M. When all three sites were mutated, the pHEPIS-1.7K-3M promoter activity was significantly decreased compared with the pHEPIS-1.7K (P<0.05; Fig. 4B); however, the level of suppression with the three mutations did not exceed the combined level of suppression by the individual point mutations, which suggests that the three mutations act jointly. Taken together, these results suggest that the OCT-1, NF-κB and C-JUN sites serve an important role in inhibiting the transcriptional activity of HEPIS.

Figure 4. Mutation of transcription factor binding sites and LUC assay analysis of human pHEPIS. (A) The OCT-1, NF-κB and C-JUN elements were mutated individually on pHEPISs and designated as pHEPIS-1.7K-M-OCT-1, pHEPIS-1.6K-M-NF-κB and pHEPIS-1.3k-M-C-JUN. All three binding elements were mutated on the pHEPIS to generate pHEPIS-1.7K-3M. (B) Dual LUC activity assays of mutated pHEPIS constructs. A total of six recombinant vectors containing mutated pHEPIS fragments and pRL-TK were cotransfected into 293T cells. *P<0.05. LUC, luciferase; pHEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10 promoter; OCT-1, octamer-binding transcription factor 1; NF-κB, nuclear factor-κB.

Identification of transcription factors in the HEPIS promoter

Identifying the transcription factor binding sites within the HEPIS promoter region is important for determining the mechanism of HEPIS gene transcription. To determine whether OCT-1, NF-κB and C-JUN were able to bind to the endogenous HEPIS promoter, a ChIP assay was performed to investigate transcription factor binding. The results indicated that OCT-1, NF-κB and C-JUN bind to the endogenous HEPIS promoter in 293T cells, which suggests that they may serve an important role in regulating HEPIS expression (Fig. 5).

Figure 5. Binding of OCT-1, NF-κB and C-JUN to the endogenous pHEPIS promoter was analyzed using a chromatin immunoprecipitation assay in 293T cells. An amplified pHEPIS fragment with the OCT-1, NF-κB and C-JUN elements is presented. The amount of DNA in the input confirms equal loading of chromatin. ‘+’ indicates the positive control in which the template of genomic DNA fragments from 293T cells was used. OCT-1, octamer-binding transcription factor 1; NF-κB, nuclear factor-κB; pHEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10 promoter.

Analysis of HEPIS promoters in multiple species

Table II lists the putative HEPIS promoter among various species with the same sequence length. The HEPIS promoter is conserved among vertebrates (Fig. 6). The sequence of the Homo sapiens (human) HEPIS promoter shares the highest homology (100%) with that of Pan troglodytes (chimpanzees). The HEPIS promoters of Rattus norvegicus (Norway rats), Mus musculus (house mice), Bos taurus (cattle), chimpanzees, humans, Canis lupus familiaris (dogs) and Macaca mulatta (Rhesus monkeys) were analyzed using TRANSFAC and several AP-1, C-JUN, C-Fos, NF-κB and OCT-1 transcription factor binding sites were predicted within the promoter region (Table II).

Figure 6. Homology tree of the HEPIS promoter (2.0kb upstream from the 5′-end of the HEPIS gene). HEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10.

Table II. Analysis of HEPIS promoters in multiple species.

Table II.

Analysis of HEPIS promoters in multiple species.

		Transcriptional factor binding sites, n
Species	Context of HEPIS promoter (−2.0k)	OCT-1	NF-κB	AP-1	C-JUN	C-Fos
Rattus norvegicus (Norway rat)	Chr3: 91195981-91197981 [-]	11	0	6	2	0
Mus musculus (house mouse)	Chr2: 101629105–101631105 [-]	13	11	6	2	1
Homo Sapiens (human)	Chr11: 36592229–36594229 [+]	14	5	5	2	0
Bos Taurus (cattle)	Chr15: 67842229–67844229 [+]	17	3	9	4	1
Pan troglodytes (chimpanzee)	Chr11: 36583771–36585771 [+]	14	6	6	2	0
Canis lupus familiaris (dog)	Chr18: 31618122–31620122 [-]	15	3	7	4	3
Macaca mulatta (rhesus monkey)	Chr14: 29326253–29328253 [-]	14	4	5	2	0

[i] HEPIS, human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10; OCT-1, octamer-binding transcription factor 1; NF-κB, nuclear factor-κB; AP-1, activator protein 1.

Discussion

It has previously been reported that HEPIS is able to inhibit the proliferation of HeLa cells and may serve as an anti-oncoprotein (1). HEPIS is also able to inhibit the expression of the chloramphenicol acetyltransferase gene and may function as a factor of transcriptional repression (1). The aim of the present study was to determine the expression profile of the HEPIS gene and further elucidate the mechanism by which HEPIS transcriptional levels differ. RNA in situ hybridization (RISH) is a method of identifying the mRNA transcriptional expression pattern within the cytoplasm by hybridizing the sequence of interest to a labeled probe (24). Probes include radioactive probes and non-radioactive probes and RISH experiments performed with non-radioactive probes have several advantages over the radioactive procedures, including signal resolution, safety, shelf-life and cost (25). Due to the limited availability of the HEPIS antibody, the expression of the HEPIS gene in cancer and adjacent normal tissues in 12 organs was assessed using RNA in situ hybridization with a specific digoxigenin-labelled probe. However, due to the limited number of specimens available, it was necessary to further increase the number of specimens analyzed in order to obtain accurate results. HEPIS expression levels in four human breast cancer cell lines was examined using RT-qPCR, however, HEPIS expression in other types of cell lines remains unknown. Determining the differential expression of HEPIS allows analysis of its function in a variety of diseases.

Promoters control gene transcription. They may be located upstream of the gene transcription start site and can be very long (26). The binding of transcription factors to a promoter is an important mechanism by which gene expression is controlled (26). Investigating HEPIS promoter activity revealed that the luciferase activity varied between pHEPIS-1.3k and pHEPIS-1.1k, suggesting that transcriptional regulation occurs at the-899/−759 bp region of the promoter. Furthermore, the results suggest that mutations of C-JUN (TTGAGTCAGG, −856/−846 bp), OCT-1 (CTATTTGCTTCTG, −1236/−1223 bp) and NF-κB (GGAATCCCCT, −1186/−1176 bp) result in a marked reduction in luciferase activity, which indicated that C-JUN, OCT-1 and NF-κB are activators. However, no significant changes in luciferase activity were observed following truncation of the −1334/1203 bp and −1203/−1060 bp regions. These results suggest that the −1334/1203 bp and −1203/−1060 bp regions also contain repressor-binding sites. The findings of the present study indicate that the apparent changes in transcriptional activity of the HEPIS gene may result from complex interactions of different transcription factors with the promoter. The association of HEPIS gene and the above transcription factors maybe widespread and therefore, further study is required.

In the present study, sequence analysis identified numerous transcription factor-binding sites within the HEPIS promoter sequence. Of these, OCT-1 NF-κB and C-JUN were ubiquitously expressed; these have previously been reported to serve a variety of roles in the progression of numerous cancers (5,9,16,17,19,20). The results of the ChIP assay indicated that OCT-1, NF-κB and C-JUN are able to bind to the endogenous HEPIS promoter in 293T cells. Several AP-1, C-JUN, C-Fos, NF-κB and OCT-1 transcription factor-binding sites were predicted within the putative HEPIS promoter in various species.

In conclusion, the results of this present research revealed that HEPIS has different expression levels in multiple types of cancer and normal tissues, and four breast cancer cell lines; and the OCT-1, NF-κB and C-JUN transcription factors are associated with transcriptional regulation of the HEPIS gene. These findings provide further insight into the expression profile and the mechanism of HEPIS gene transcriptional regulation.

Acknowledgements

The present study was supported by the General Higher Education Young Talents Program of Hebei Province (grant no. BJ2014027) and the National Natural Science Foundation of China (grant no. 81302323).

References