人多形性腺瘤中与cyclin D1共表达LncRNA的初步筛选及鉴定
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摘要
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(GSE61474_TCONS_00180431、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(GSE61474_TCONS_00180431, 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.
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(GSE61474_TCONS_00180431, 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.
引文
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[21]Fan S,Yang Z,Ke Z,et al.Downregulation of the long noncoding RNA TUG1 is associated with cell proliferation,migration,and invasion in breast cancer[J].Biomed Pharmacother,2017,95:1636-1643.
[2]Eveson JW PA,Gnepp DR,El-Naggar AK.Tumours of the salivary glands[M].3rd Ed.Lyon:IARC Press,2005.
[3]Hu YH,Zhang CY,Xia RH,et al.Prognostic factors of carcinoma ex pleomorphic adenoma of the salivary glands,with emphasis on the widely invasive carcinoma:a clinicopathologic analysis of 361 cases in a Chinese population[J].Oral Surg Oral Med Oral Pathol Oral Radiol,2016,122(5):598-608.
[4]夏亮,汪洋,胡宇华,等.唾液腺恶性多形性腺瘤HER2基因扩增与临床病理特征及预后的关系[J].中国口腔颌面外科杂志,2018,16(1):12-19.
[5]Villegas SL,Darb-Esfahani S,von Minckwitz G,et al.Expression of Cyclin D1 protein in residual tumor after neoadjuvant chemotherapy for breast cancer[J].Breast Cancer Res Treat,2018,168(1):179-187.
[6]Khabaz MN,Abdelrahman AS,Butt NS,et al.Cyclin D1 is significantly associated with stage of tumor and predicts poor survival in endometrial carcinoma patients[J].Ann Diagn Pathol,2017,30(1):47-51.
[7]Jour G,West K,Ghali V,et al.Differential expression of p16(INK4A)and cyclin D1 in benign and malignant salivary gland tumors:a study of 44 Cases[J].Head Neck Pathol,2013,7(3):224-231.
[8]尚文军,王洋,刘丽敏,等.ERK通路抑制剂苯乙酸钠对转基因小鼠唾液腺肿瘤的作用[J].中国口腔颌面外科杂志,2013,11(5):359-364.
[9]Wang Y,Shang W,Lei X,et al.Opposing functions of PLAG1 in pleomorphic adenoma:a microarray analysis of PLAG1 transgenic mice[J].Biotechnol Lett,2013,35(9):1377-1385.
[10]Zheng Q,Lin Z,Xu J,et al.Long noncoding RNA MEG3suppresses liver cancer cells growth through inhibiting betacatenin by activating PKM2 and inactivating PTEN[J].Cell Death Dis,2018,9(3):253-271.
[11]Mitra R,Chen X,Greenawalt EJ,et al.Decoding critical long non-coding RNA in ovarian cancer epithelial-to-mesenchymal transition[J].Nat Commun,2017,8(1):1604-1615.
[12]Gupta RA,Shah N,Wang KC,et al.Long non-coding RNAHOTAIR reprograms chromatin state to promote cancer metastasis[J].Nature,2010,464(7291):1071-1076.
[13]徐万林,刘丽敏,卢浩,等.PLAG1转基因小鼠中差异表达长链非编码RNA的筛选分析[J].中国口腔颌面外科杂志,2019,17(1):20-25.
[14]Ponting CP,Oliver PL,Reik W.Evolution and functions of long noncoding RNAs[J].Cell,2009,136(4):629-641.
[15]Chen Z,Lin S,Li JL,et al.CRTC1-MAML2 fusion-induced lncRNA LINC00473 expression maintains the growth and survival of human mucoepidermoid carcinoma cells[J].Oncogene,2018,37(14):1885-1895.
[16]何巍,何伟,曹选平,等.细胞周期素D1在唾液腺腺样囊性癌中的表达及意义[J].华西口腔医学杂志,2005,23(3):191-193.
[17]Liu T,Zhu E,Wang L,et al.Abnormal expression of Rb pathway-related proteins in salivary gland acinic cell carcinoma[J].Hum Pathol,2005,36(9):962-970.
[18]Liu W,Zhu E,Wang R,et al.Cyclin D1 gene polymorphism,A870G,is associated with an increased risk of salivary gland tumors in the Chinese population[J].Cancer Epidemiol,2011,35(4):e12-17.
[19]Zhou JJ,Cheng D,He XY,et al.Knockdown of Hotair suppresses proliferation and cell cycle progression in hepatocellular carcinoma cell by downregulating CCND1 expression[J].Mol Med Rep,2017,16(4):4980-4986.
[20]Yu T,Shan TD,Li JY,et al.Knockdown of linc-UFC1suppresses proliferation and induces apoptosis of colorectal cancer[J].Cell Death Dis,2016,7(5):e2228-2236.
[21]Fan S,Yang Z,Ke Z,et al.Downregulation of the long noncoding RNA TUG1 is associated with cell proliferation,migration,and invasion in breast cancer[J].Biomed Pharmacother,2017,95:1636-1643.