Expression profile and promoter analysis of HEPIS

Abstract

Human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10 (HEPIS) is a novel transcriptional repressor, the expression profile and promoter activity of which have not been well studied. In the present study, in situ hybridization of RNA was used to study differential HEPIS expression levels in different types of cancer and normal tissues. A total of six truncated lengths of the HEPIS promoter regulatory sequences were cloned into the pGL3-basic vector, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and dual luciferase reporter assays were performed. The results of RT-qPCR demonstrated that HEPIS expression levels differed across four breast cancer cell lines. The results of the dual luciferase reporter assays revealed that the activities of the reporter gene fragments spanning -1334/+373, -1203/+373, -1060/+373 and -899/+373 bp were higher compared with the reporter gene fragments spanning -759/+373 and -279/+373 bp. A search of the transcription factor database TRANSFAC identified numerous octamer transcription factor-1 (OCT-1), nuclear factor (NF)-κB and C-JUN transcription factor binding sites located on the HEPIS promoter (pHEPIS). Furthermore, the results revealed that mutations of the OCT-1 (-1236/-1223 bp), NF-κB (-1186/-1176 bp) and C-JUN (-856/-846 bp) sites on the human pHEPIS resulted in a decrease in luciferase activity. A chromatin immunoprecipitation assay revealed that OCT-1, NF-κB and C-JUN bound to pHEPIS in a site-dependent manner at the basal state. The TRANSFAC database was used to analyze the pHEPIS of multiple species and several activator protein-1, NF-κB and OCT-1 transcription factor binding sites were predicted. In conclusion, the results of the present study suggest that HEPIS is expressed at different levels in multiple organs and breast cancer cell lines. Furthermore, these findings indicate that OCT-1, NF-κB and C-JUN transcription factors are associated with transcriptional regulation of the HEPIS gene.

Keywords: core promoter; expression profile; human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10; transcriptional regulation.

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.

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.

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.

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.

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.