ABSTRACT
Objective To analyze the regulation of estrogen receptor α (ERα) on truncated neurokinin-1 receptor (NK1R-Tr), and the influence of this regulation on cell proliferation in estrogen receptor-positive breast cancer cell lines. Methods The chromatin immune coprecipitation (CHIP) was used to observe the transcriptional regulation function of ERαon NK1R-Tr in breast cancer cells. Luciferase reporter gene assay was used to verify whether ERα played a positive regulatory role in the expression of NK1R-Tr. Western blot assay and real-time-PCR were used to detect the expression of ERα and NK1R-Tr in breast cancer cells, MCF-7 and T47D, as well as the expression of NK1R-Tr protein and mRNA level. NK1R-Tr levels were also detected after using estradiol (E2, ERα agonist) and small interfering RNA (knock out ERα). CCK-8 and clone formation experimen were used to detect the proliferation ability of breast cancer cells after knocking out NK1R-Tr with small interfering RNAs. Results CHIP test and Luciferase reporter gene assay proved that ERα can positively regulate the expression of NK1R-Tr via the ERα sequences in the upstream of the NK1R-Tr gene promoter. The expression of NK1R-Tr at both protein level and mRNA level dropped in the estrogen receptor-positive breast cancer cell line MCF-7 upon knocking out ERα. After knocking out NK1R-Tr, the proliferation ability of estrogen receptor-positive breast cancer cells was lower than that of the control group. Conclusion The ERα positively regulates the expression of NK1R-Tr, resulting in the increased cell proliferation in estrogen positive breast cancer cells.
ABSTRACT
Objective:To determine the expression of the full-length (NK1R-FL) and truncated (NK1R-Tr) neurokinin-1 receptor (NK1R) and the neurokinin-2 receptor (NK2R) in breast cancer tissues and cell lines, as well as to study the effects of the NK1R and NK2R antagonists on the growth of breast cancer cells. Methods:Immunohistochemistry and Western blot assays were used to detect NK1R, NK1R-FL, and NK2R expression in clinical samples of primary breast cancer tissue, benign lesions, and normal breast tissue, as well as in different breast cancer cell lines. Cell proliferation and soft agar growth tests were performed on cells treated with the NK1R and NK2R antagonists to study the ectopic overexpression of NK1R-FL and NK1R-Tr in breast cancer cell lines. Results:Total NK1R expression was detected in the breast cancer tissues, benign lesions, and normal breast tissues. Compared with the normal breast epithe-lia and benign breast lesions, the expression levels of NK1R-FL and NK2R decreased in the carcinoma. These changes were also relat-ed to the carcinoma type, histological grade, lymph node metastasis, HER2 and Ki-67 expression, and estrogen and progesterone recep-tors in breast cancer. The expression levels of NK1R-FL and NK2R were high in the HBL-100 breast cell lines of para-neoplastic tis-sues, but NK1R-Tr expression was low. The MDA-MB-231, T-47D, and MCF-7 cells only expressed NK1R-Tr. NK1R-Tr or NK1R-FL overexpression caused the decreased inhibition rate or increased levels of the NK1R and NK2R antagonists in the breast cancer cells. Conclusion:NK1R-FL and NK2R are co-expressed in normal cells. NK1R-Tr is highly expressed in breast cancer cells and exerts nega-tive feedback to regulate NK1R-FL and NK2R expression in all cells, especially cancer cells.