CREPT在口腔鳞癌组织中的表达及功能研究
发布时间：2018-08-04 16:26
【摘要】：目的:初次研究CREPT基因在口腔鳞状细胞癌(Oral squamous cell carcinoma, OSCC)组织样本及正常组织样本中的表达情况,对CREPT基因在OSCC中起到的作用及其临床意义进行分析。初步研究分析CREPT shRNA转染SCC25和CAL27细胞系后,对鳞癌细胞系生物学功能的影响及机制。通过小鼠成瘤实验,研究CAL27细胞经慢病毒携带CREPT shRNA转染后,裸鼠肿瘤生长情况的变化。为探索口腔鳞状细胞癌发病机制、预后以及治疗方法提供新的线索及研究靶点。方法:采用免疫组化技术(Immunohistochemistry, IHC),蛋白印迹法(Western blot, WB)和实时定量聚合酶链反应法(Quantitative Real time Polymerase Chain Reaction, qRT-PCR)检测分析,在OSCC组织样本和正常组织样本中,CREPT的表达情况;利用慢病毒携带shCREPT序列,敲低CREPT表达,建立SCC25con、SCC25shCREPT和CAL27con、CAL27shCREPT稳定转染细胞系,进行细胞生长检测实验,单细胞克隆实验,划痕实验,流式细胞术检测细胞凋亡实验,验证CREPT在SCC25和CAL27细胞中的功能;小鼠成瘤实验检测抑制CREPT表达后,CAL27细胞在BALB/c裸鼠体内成瘤情况。结果:第一部分:CREPT在口腔鳞状细胞癌组织中的表达情况1、对SCC25与CAL27细胞系检测发现,CREPT的基因与蛋白高表达。2、对新鲜口腔鳞状细胞癌组织检测发现,CREPT在肿瘤组织中的表达高于正常组织。3、对OSCC肿瘤石蜡组织样本检测发现,CREPT呈阴性至强阳性表达;在正常口腔黏膜石蜡组织样本当中,CREPT表达呈阴性至阳性表达;肿瘤组与正常组组间差异显著;4、临床资料统计结果显示:CREPT阳性表达与肿瘤大小及淋巴转移具有显著相关性。第二部分:CREPT对口腔鳞状细胞癌细胞系体外生物学行为的影响1、下调CREPT的表达,SCC25和CAL27细胞的增殖能力能受到抑制;2、下调CREPT的表达,SCC25和CAL27细胞的单细胞克隆形成能力受到抑制;3、下调CREPT的表达,SCC25和CAL27细胞的迁移能力受到抑制;4、下调CREPT的表达,SCC25和CAL27细胞凋亡水平增高;5、下调CREPT的表达,SCC25和CAL27细胞的cyclin D1、 c-Myc的表达降低。第三部分:CREPT对口腔鳞状细胞癌细胞系体内生物学行为的影响1、CREPTshRNA转染CAL27细胞后,裸鼠体内实验显示,肿瘤的生长受到抑制,2、CREPTshRNA转染CAL27细胞后,形成瘤体的CREPT、cyclinD1与c-Myc表达降低。结论:1、CREPT在OSCC细胞系及肿瘤组织中表达升高,其阳性表达与OSCC的T、N分期正相关。2、下调CREPT表达可以抑制SCC25和CAL27细胞增殖能力、细胞迁移能力,促进细胞系凋亡的发生,抑制cyclin D1、c-Myc的表达。3、下调CREPT表达可以抑制CAL27细胞在裸鼠体内肿瘤的生长。
[Abstract]:Objective: to investigate the expression of CREPT gene in oral squamous cell carcinoma (OSCC) (Oral squamous cell carcinoma, OSCC) tissues and normal tissues for the first time, and to analyze the role of CREPT gene in OSCC and its clinical significance. The effect and mechanism of CREPT shRNA transfection on the biological function of SCC25 and CAL27 cell lines were studied. The changes of tumor growth in nude mice after CAL27 cells were transfected with lentivirus carrying CREPT shRNA were studied by mouse tumorigenesis test. To explore the pathogenesis, prognosis and treatment of oral squamous cell carcinoma (OSCC). Methods: the expression of crept in OSCC tissue samples and normal tissue samples was detected by immunohistochemical (Immunohistochemistry, IHC), Western blot (Western blot, WB) and real-time quantitative polymerase chain reaction (Quantitative Real time Polymerase Chain Reaction, qRT-PCR), and the shCREPT sequence was carried by lentivirus. The stable transfection cell lines of SCC25shCREPT and CAL27cong CAL27shCREPT were established. Cell growth assay, single cell clone assay, scratch assay, flow cytometry were used to detect the apoptosis of SCC25 and CAL27 cells, and the function of CREPT in SCC25 and CAL27 cells was verified. Tumorigenesis in BALB/c nude mice was detected by murine tumorigenesis assay after inhibiting the expression of CREPT. Results: the first part was the expression of crept in oral squamous cell carcinoma (1). The gene and protein expression of crept in SCC25 and CAL27 cell line was found to be higher than that in oral squamous cell carcinoma, and in the tissue of fresh oral squamous cell carcinoma, crept was found in tumor tissue. The expression of CREPT in paraffin tissue of OSCC was negative and strong. In the paraffin tissues of normal oral mucosa, the expression of crept was negative to positive, and the difference between tumor group and normal group was significant (4). The clinical data showed that the positive expression of crept was significantly correlated with tumor size and lymphatic metastasis. The second part: the effect of crept on the biological behavior of oral squamous cell carcinoma cell line in vitro. 1. Down-regulating the expression of CREPT and the proliferation ability of SCC25 and CAL27 cells can be inhibited, and down-regulating the expression of CREPT and the single cell clone formation ability of CAL27 cells. The down-regulation of CREPT expression and the migration ability of CAL27 cells were inhibited. The expression of CREPT was down-regulated and the apoptotic level of SCC25 and CAL27 cells was increased. The expression of CREPT and CAL27 cells was down-regulated. The cyclin D1 and c-Myc expression of SCC25 and CAL27 cells were decreased. Effect of: crept on the biological behavior of oral squamous cell carcinoma cell lines in vivo. 1 after transfection of CREPTshRNA into CAL27 cells, the results of nude mice experiments showed that the expression of cyclinD1 and c-Myc in the tumor formation decreased after the tumor growth was inhibited and CREPTshRNA was transfected into CAL27 cells. Conclusion the positive expression of crept in OSCC cell line and tumor tissue is increased, and its positive expression is positively correlated with the OSCC stage. Down-regulation of CREPT expression can inhibit the proliferation and migration of SCC25 and CAL27 cells, and promote the apoptosis of the cell line. [WT5 "HZ] [WT5" BZ] [WT5 "BZ] [WT5" BZ] [WT5BZ] Inhibition of the expression of cyclin D1Tc-Myc. 3 and down-regulation of CREPT expression could inhibit the growth of CAL27 cells in nude mice.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.8
本文编号：2164443
[Abstract]:Objective: to investigate the expression of CREPT gene in oral squamous cell carcinoma (OSCC) (Oral squamous cell carcinoma, OSCC) tissues and normal tissues for the first time, and to analyze the role of CREPT gene in OSCC and its clinical significance. The effect and mechanism of CREPT shRNA transfection on the biological function of SCC25 and CAL27 cell lines were studied. The changes of tumor growth in nude mice after CAL27 cells were transfected with lentivirus carrying CREPT shRNA were studied by mouse tumorigenesis test. To explore the pathogenesis, prognosis and treatment of oral squamous cell carcinoma (OSCC). Methods: the expression of crept in OSCC tissue samples and normal tissue samples was detected by immunohistochemical (Immunohistochemistry, IHC), Western blot (Western blot, WB) and real-time quantitative polymerase chain reaction (Quantitative Real time Polymerase Chain Reaction, qRT-PCR), and the shCREPT sequence was carried by lentivirus. The stable transfection cell lines of SCC25shCREPT and CAL27cong CAL27shCREPT were established. Cell growth assay, single cell clone assay, scratch assay, flow cytometry were used to detect the apoptosis of SCC25 and CAL27 cells, and the function of CREPT in SCC25 and CAL27 cells was verified. Tumorigenesis in BALB/c nude mice was detected by murine tumorigenesis assay after inhibiting the expression of CREPT. Results: the first part was the expression of crept in oral squamous cell carcinoma (1). The gene and protein expression of crept in SCC25 and CAL27 cell line was found to be higher than that in oral squamous cell carcinoma, and in the tissue of fresh oral squamous cell carcinoma, crept was found in tumor tissue. The expression of CREPT in paraffin tissue of OSCC was negative and strong. In the paraffin tissues of normal oral mucosa, the expression of crept was negative to positive, and the difference between tumor group and normal group was significant (4). The clinical data showed that the positive expression of crept was significantly correlated with tumor size and lymphatic metastasis. The second part: the effect of crept on the biological behavior of oral squamous cell carcinoma cell line in vitro. 1. Down-regulating the expression of CREPT and the proliferation ability of SCC25 and CAL27 cells can be inhibited, and down-regulating the expression of CREPT and the single cell clone formation ability of CAL27 cells. The down-regulation of CREPT expression and the migration ability of CAL27 cells were inhibited. The expression of CREPT was down-regulated and the apoptotic level of SCC25 and CAL27 cells was increased. The expression of CREPT and CAL27 cells was down-regulated. The cyclin D1 and c-Myc expression of SCC25 and CAL27 cells were decreased. Effect of: crept on the biological behavior of oral squamous cell carcinoma cell lines in vivo. 1 after transfection of CREPTshRNA into CAL27 cells, the results of nude mice experiments showed that the expression of cyclinD1 and c-Myc in the tumor formation decreased after the tumor growth was inhibited and CREPTshRNA was transfected into CAL27 cells. Conclusion the positive expression of crept in OSCC cell line and tumor tissue is increased, and its positive expression is positively correlated with the OSCC stage. Down-regulation of CREPT expression can inhibit the proliferation and migration of SCC25 and CAL27 cells, and promote the apoptosis of the cell line. [WT5 "HZ] [WT5" BZ] [WT5 "BZ] [WT5" BZ] [WT5BZ] Inhibition of the expression of cyclin D1Tc-Myc. 3 and down-regulation of CREPT expression could inhibit the growth of CAL27 cells in nude mice.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.8
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