microRNA-206和microRNA-22对乳腺癌细胞神经激肽受体-1的表达调控研究

Substance P (SP) is an important tachykinin family member, exert its biological action through its preferential ligands neurokinin-1 receptor (NK1R). Two naturally occurring NK1R mediate the effects of SP: a full-length receptor (NK1R-FL) and a truncated (NK1R-Tr). SP coupled NK1R can act directly or indirectly on cancer cells modulating their responses. Study on breast cancer tissues and cell lines showed that NK1R-FL was reduced,but NK1R-Tr increased gradually with malignancy advance in breast cancer. To explore the potential role of NK1R-FL and NK1R-Tr in breast cancer malignant phenotypes, we studied the effect with ectopically overexpression of NK1R-FL and NK1R-Tr in MDA-MB-231 and HBL-100 cells, respectively. Cell growth curve, cell cycle distribution, soft-agar colony formation, in vitro adhesion assay, invasion and migration assays, athymic mice in vivo models tests indicated that NK1R-FL wan relate to invasion and migration negatively,but NK1R-Tr promoted migration of breast cancer.These results indicate the property of NK1R-Tr as a membrane receptor oncogene in human breast cancer.. As microRNAs (miRNAs) can regulate gene expression and they can function as tumor suppressors or oncogenes, we predicted that they may also have an important role in modulating NK1R expression in breast cancer. Several target prediction algorithms were used to predict miRNAs to target NK1R-FL and NK1R-Tr. As the results, bioinformatics analyses revealed that NK1R-FL 3'UTR contained putative miR-206 binding sites, NK1R-Tr 3'UTR contained putative miR-22 binding sites. Because miRNAs generally regulate gene expression by binding to the 3'-UTRs of their target genes, we predicted that miR-206 represses NK1R-FL expression levels, but miR-22 represses NK1R-Tr expression levels. Test on breast cancer tissues showed that higher expression of NK1R-FL corresponded to lower expression of miR-206, but lower expression of NK1R-Tr corresponded to higher expression of miR-22. Furthermore, ERα contained putative miR-206 and miR-22 target sites within its respective 3′-UTRs, and miR-206 expression is down-regulated in estrogen receptor alpha-positive human breast cancer, miR-22 could act as a tumor suppressor in breast cancer. It is known that ERα had important role in development and/or progression of breast cancer, ERα can direct interaction of estrogen with NK1R,and also can crosstalk between ERα and GPCR cell signals. For these reasons, study on interaction among miR-206/ miR-22, ERα and NK1R-FL/ NK1R-Tr by methods such as reporter gene assay and chromatin immunoprecipitation assay in this project, will help us to explore regulation relationship among miR-206/ miR-22, ERα and NK1R-FL/ NK1R-Tr, and to provide evidence that miR-206 and miR-22 regulate expression of NK1R-FL and NK1R-Tr to influence invasion and migration of breast cancer cells.

神经激肽受体-1（NK1R）是速激肽P物质的偏嗜性受体，前期研究显示其全长型变异体NK1R-FL与乳腺癌细胞的侵袭转移呈负相关；截断型变异体NK1R-Tr随着乳腺癌的进展表达不断升高，促进了乳腺癌细胞的远处转移并与乳腺癌细胞在骨髓微环境中的静息和复发相关。研究发现:miR-206和miR-22的种子区分别与NK1R-FL和NK1R-Tr的3'UTR区特异性结合，从而可能参与对二者的表达调控；miR-206和miR-22与乳腺癌细胞ERα的表达调控相关；ERα也可能通过基因和非基因组途径与NK1R相互作用。因此本课题拟通过miR-206/22对乳腺癌细胞NK1R-FL/Tr表达调控的研究，利用报告基因测定、CHIP等方法，探讨miR-206/22是通过直接调控NK1R-FL/Tr表达，还是通过调控ERα并经ERα与NK1R-FL/Tr的相互作用，影响了乳腺癌细胞的侵袭和转移。

神经激肽受体-1（NK1R）是速激肽P物质的偏嗜性受体，其全长型变异体NK1R-FL与乳腺癌细胞的侵袭转移呈负相关；截断型变异体NK1R-Tr随着乳腺癌的进展表达不断升高。miR-206和miR-22的种子区分别与NK1R-FL、NK1R-Tr和ER的3‘UTR区特异性结合，从而可能参与对三者的表达调控。本课题旨在探讨miR-206和miR-22是否参与了ERα与NK1R之间的相互作用，并因此影响了乳腺癌细胞生长、增殖、侵袭和转移等生物学行为。免疫组化法和RT PCR分析显示乳腺癌患者组织NK1R-FL和miRNA-22的表达降低、NK1R-Tr和miRNA-206升高与乳腺癌患者组织的分型、分期、组织分级、淋巴结转移、ER和PR阴性、 Ki-67阳性、HER2阳性等显著相关。HBL-100和SK-BR-3细胞高表达miR-22, 其表达水平显著高于MDA-MB-231、T-47D和MCF-7细胞；MDA-MB-231、MCF-7、T47D、SK-BR-3细胞miR-206的表达均显著高于HBL-100细胞。荧光素酶报告基因研究证实在乳腺癌细胞系中，miRNA-206和miRNA-22可以在蛋白和mRNA水平分别靶向下调NK1R-FL和 NK1R-Tr的表达。染色质免疫共沉淀研究和ERα沉默实验显示ERα可以通过NK1R基因启动子上游ERE序列正向调控NK1R的表达。在乳腺癌细胞系MDA-MB-231、MCF-7、T47D、SK-BR-3及乳腺非肿瘤源性上皮细胞HBL-100内转染pre-22、pre-206， anti-206和anti-22，免疫印迹和RT PCR结果显示SP刺激下，转染了miRNA-206的乳腺癌细胞中的1，4，5-IP3水平增加，并且ERK信号通路的激活延迟。在转染了miRNA-206的乳腺癌细胞中，miRNA-206可以通过下调NK1R-FL的表达，使细胞的增殖、侵袭和转移能力升高；在转染了miRNA-22的乳腺癌细胞中，miRNA-22可以通过下调NK1R-Tr的表达，使细胞的增殖、侵袭和转移能力降低。miRNA-206作为促癌因子，可以通过降低乳腺癌细胞中NK1R-FL的表达，从而促进了乳腺癌细胞的恶性转化。miRNA-22作为抑癌因子，通过ERα间接降低NK1R-Tr的表达，使乳腺癌细胞的增殖、侵袭、转移能力下降。