| Breast cancer is the most common cancer in women and is a major cause of cancer-related morbidity and mortality.The neuropeptide substance P(SP) is an important member of the tachykinin receptor family, exert its biological action through transmembrane G-protein coupled receptors, neurokinin-1(NK1R) in human breast cancer development and progression. NK1 R has two isoforms: full-length NK1R(NK1R-FL) and truncated NK1R(NK1R-Tr), the latter lacking the cytoplasmic terminal 96-amino acid residues of the full-length protein. We have previously shown that expression of NK1R-FL inversely correlates with the progression of human breast cancer and we have identified three candidate miR-206 target sites within 3’-untranslated region(3’-UTR) of the NK1R-FL gene from bioinformatics databases searches. MicroRNAs(miRNAs) are a novel class of small non-coding RNAs(~22 bp) that regulate gene expression by directly binding to 3’-UTR of target mRNAs, causing translational inhibition or mRNA degradation. Recent studies demonstrated that serum miRNAs are involved in multiple pathological processes, for example tumor occurrence, development, invasion and metastasis.Our study aimed to explore the relationship of the expression of miR-206 and NK1R-FL in the human breast cancer, then further validated at a cellular level. At last we discuss the influence of the miR-206/NK1R-FL pathway for the biological behavior of breast cancer invasion and metastasis and proliferation. Methods:1. The tissues level82 clinical cases of breast cancer and paired adjacent normal tissues were collected, and total RNA of those paired tissues were extracted, then the expression of miR-206 and NK1R-FL were detected thereafter by qRT-PCR, the expression of NK1 R and NK1R-FL were detected by immunohistochemistry. NK1R-FL and miR-206 expression levels of breast cancer cell line MDA-MB-231, MCF-7, T47 D, SK-BR-3 and nontumorigenic breast epithelial cell line HBL-100 were detected by qRT-PCR. The correlation of miR-206 and NK1R-FL mRNA expression levels was tested by Pearson’s correlation.2. Validation of target prediction and control each other: whether is there target site and regulation function of miR-206.By using bioinformatics tools, we found that those are three candidate miR-206 target sites within 3’-untranslated region(3’-UTR) of the NK1R-FL gene and then further validated by dual-luciferase assay, The expression levels of NK1R-FL were determined after the overexpression of miR-206 in breast cancer cell line SK-BR-3 and nontumorigenic breast epithelial cell line HBL-100, or knockdown of miR-206 in breast cancer cell line MDA-MB-231, the expression of NK1R-FL was detected by Western Blot and qRT-PCR.3. SP-NK1 R signaling pathway analysis: To vetify the influence of the miR-206 for the Ca2+ influx and pathway by targeting NK1R-FL.We research the influence of miR-206-NK1R-FL pathway for breast cancer by detecting the change of the concentration of Ins(1,4,5)P3, to explore the influence of miR-206-NK1R-FL pathway for breast cancer cells ERK1/2 pathway by detecteding the change of the expression level of p-ERK1/2 in non-transfected group(NC) and miR-206 mimic- transfected group.4. Biological behavior research: To vetify the influence of the miR-206/NK1R-FL pathway for the biological behavior of breast cancer invasion and metastasis and proliferation.The expression levels of NK1R-FL were determined after the overexpression of miR-206 in breast cancer cell line SK-BR-3 and nontumorigenic breast epithelial cell line HBL-100 or knockdown of mi R-206 in breast cancer cell line MDA-MB-231. And the CCK8 assays, transwell, and colony formation assays were performed to verify the functional infiuence of miR-206 for migration, invasion, proliferation and colony formation in breast cancer cells. Results:1. The analysis of NK1R-FL and miR-206 expression in breast cancer tissuesCompared with adjacent normal tissues, the expression of NK1R-FL protein showed clear decrease in breast cancer tissues and the expression level of miR-206 was higher, NK1R-FL and mi R-206 positivity were significantly associated with advanced histological grade, advanced TNM stage and lymph node metastasis. In MDA-MB-231, T47 D, and MCF-7 breast cancer cell lines, miR-206 expression was sinificantly upregulated when compared with control HBL-100 cells and MDA-MB-231, T47 D, and MCF-7 cells expressed only low levels of NK1R-FL. 2. Validation of target prediction and control each other:Using microRNA.org, miRanda, Target-Scan, and Pictar databases, we identified three candidate miR-206 target sequences within the 3’-UTR of NK1R-FL and then further validated that miR-206 regulates NK1R-FL expression by directly binding the 3’-UTR of NK1R-FL mRNA using dual-luciferase assay. The expression levels of NK1R-FL were down-regulated after the overexpression of miR-206 in breast cancer cell line SK-BR-3 and nontumorigenic breast epithelial cell line HBL-100, the expression levels of NK1R-FL were up-regulated after the knockdown of miR-206 in breast cancer cell line MDA-MB-2313. SP-NK1 R signaling pathway analysis:MiR-206 causes the reduce of the intracellular levels of Ins(1,4,5) P3 by inhibiting the NK1R-FL expression and the duration of the Ca2+ influx was longer, the time of activation of the intracellular ERK1/2 was longer.4. Biological behavior research:MiR-206 can directly bind to the 3’-UTR of NK1R-FL mRNA and play the role of transcriptional regulation, causing the downregulation of NK1R-FL expression levels and thereby promote tumor invasion and metastasis, proliferation and colony formation.Conclusion:In the present study, mi R-206 was found to be up-regulated while the expression of NK1R-FL was inhibited in tumor tissues of breast cancer. Studies shown that miR-206 inhibited NK1R-FL expression by directly binding with 3’-UTR of mRNA and influence downstream signaling pathway, thus promoting tumor invasion and metastasis, proliferation and colony formation. |
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