人多形性腺瘤中与cyclin D1共表达LncRNA的初步筛选及鉴定

Preliminary screening and validation of cyclin D1 co-expressed long non-coding RNAs in human pleomorphic adenoma

目的:在人多形性腺瘤中筛选出与差异表达基因细胞周期蛋白D1 (cyclin D1)表达相关的长链非编码RNA(LncRNA),并进行初步验证。方法:利用实时定量PCR、Western免疫印迹及基因芯片等方法,验证人多形性腺瘤组织中cyclin D1的表达,通过计算Pearson相关系数,筛选出与cyclin D1共表达的LncRNA。随机挑选其中的3个LncRNA(GSE61474TCONS00180431、ENST00000603829和T253381),利用实时荧光定量PCR鉴定其在人多形性腺瘤组织中的表达是否存在差异。采用SPSS19.0软件包对数据进行统计学分析。结果:Cyclin D1在人多形性腺瘤中的表达显著增高(P<0.05),共筛选出与cyclin D1共表达的9个LncRNA,其中上调6个,下调3个。实时荧光定量PCR结果显示,所挑选的3个LncRNA在肿瘤组织中的表达趋势与基因芯片结果一致,但后续扩大样本验证结果显示,仅ENST00000603829的表达差异具有统计学意义(P<0.05)。结论:本研究成功筛选及验证人多形性腺瘤中cyclin D1相关LncRNA,提示这些LncRNA可能与cyclin D1相互作用从而在人多形性腺瘤的发生、发展中起到重要的调节作用。

PURPOSE: To screen out and validate the differentially expressed long non-coding RNAs(LncRNAs) that co-expressed with cyclin D1 in human pleomorphic adenoma. METHODS: The expression of cyclin D1 in human pleomorphic adenoma was first validated through real time-PCR, Western blot and micro-array analysis. The differentially expressed LncRNAs from the micro-array results that co-expressed with cyclin D1 were screened out via calculating the Pearson’s correlation coefficient(PCC), and then the expression of three randomly selected lncRNAs(GSE61474TCONS00180431, ENST00000603829 and T253381) were validated with real time PCR. Statistical analysis was completed with SPSS 19.0 software package. RESULTS: The expression of cyclin D1 was significantly up-regulated in human pleomorphic adenoma than the controls(P<0.05). A total of 9 LncRNAs that co-expressed with cyclin D1 were screened out, among which, 6 were up-regulated and 3 were down-regulated. Real-time PCR results demonstrated that same expression trends of the three selected LncRNAs were shown with micro-array results. However, only ENST00000603829 expressed significantly in the subsequent enlarged tumor samples(P <0.05). CONCLUSIONS: The differentially expressed LncRNAs that co-expressed with cyclin D1 were screened out and validated in human pleomorphic adenoma. These LncRNAs could play important roles in pathogenesis and development of pleomorphic adenoma, possibly through interacting with cyclin D1.