lncRNA316686对NGFR的调控机制研究

The cause of dopamine agonist-resistant prolactinomas is primarily due to a decrease in D2R and NGFR on tumor cell surface. Although studies have shown that NGF prompts D2R expression by activating NF-κB, the mechanisms underlying the regulation of NGFR expression remain unclear. Recent studies show that long non-coding RNAs (lncRNA) are essential regulators for a number of biological processes and diseases. Our previous studies showed that lncRNA316686 might involve in regulating NGFR expression by screening of lncRNA and mRNA common expressive profile microarray and that there was a significant differential NGFR and lncRNA316686 expression in dopamine-sensitive and -resistant prolactinomas by RT-PCR analysis. In this proposed study, we will further confirm whether NGFR is regulated by lncRNA316686 and then identify the molecular mechanisms underlying the regulation of NGFR expression in prolactinoma cell line. Next, we will determine whether targeted interventions can reverse the resistance to dopamine agonist in vitro and in vivo. We expect that lncRNA316686 can control NGFR expression level, which may, at least in part, diminish the resistance to dopamine agonists in prolactinoma. Meanwhile, lncRNA316686 can be used as a molecular biomarker for prolactinoma's sensitivity to dopamine agonist therapy and thus guide the clinical pharmacotherapy. If successful, the findings from this study will give us insight into the molecular mechanism of dopamine agonists-resistant prolactinomas and provide us a new avenue for treatment of dopamine-resistant prolactinomas.

垂体泌乳素腺瘤细胞表面D2R和NGFR数量的减少被认为是肿瘤耐药的主要原因，NGF通过与其受体NGFR结合激活NF-κB来促使D2R表达，但对NGFR的调控机制未明。长链非编码RNA（lncRNA）是最近发现的在多种生物学过程以及疾病中起重要调控作用的非编码RNA。本课题前期采用lncRNA和mRNA芯片共表达谱分析筛选并用RT-PCR方法验证泌乳素腺瘤药物治疗中NGFR相关的lncRNA316686。本课题将进一步在细胞水平验证lncRNA316686对NGFR的调控及机制，同时体外和体内靶向干预观察逆转耐药效果，以期在lncRNA水平调控NGFR表达，从而逆转耐药泌乳素腺瘤对药物治疗的反应性，同时lncRNA316686可以作为泌乳素腺瘤药物治疗敏感性的生物学分子指标从而指导临床用药。如果实验成功，将为临床解决耐药泌乳素腺瘤的治疗难题奠定实验基础。

长链非编码RNA在肿瘤中的功能得到越来越多的重视和研究，但是在垂体瘤中的研究相对很少，在耐药中的机制研究尚未见相关报道。本课题通过对3例药物治疗敏感的垂体泌乳素瘤标本和药物治疗耐药的垂体泌乳素瘤标本进行芯片分析，找到差异性表达的长链非编码RNA-H19，通过体内和体外实验证明H19在垂体瘤的耐药中扮演者重要作用。结果显示：(1)干预及过表达GH3细胞的H19水平，联合溴隐亭和卡麦角林治疗后细胞存活率明显下降。说明H19增加了GH3细胞对于药物治疗的敏感性。（2）经转染H19的GH3细胞的裸鼠皮下移植瘤的H19表达水平升高，联合卡麦角林的治疗可明显抑制皮下移植瘤生长。体内实验再次证明H19增加了GH3细胞对于药物治疗的敏感性。（3）调节H19的表达水平后，通过WB和rtPCR技术发现H19在蛋白水平调节了Atg7的表达水平，并通过RNA免疫共沉淀实验证明H19与Atg7蛋白存在结合。（4）通过对Atg7的蛋白降解途径的研究发现，H19通过与Atg7结合调节了Atg7的泛素化降解途径。