miR-210及其靶基因转录因子E2F3与喉鳞状细胞癌的相关研究

发布时间：2018-07-04 09:02

本文选题：喉肿瘤 + 鳞状细胞癌　；参考：《山西医科大学》2012年硕士论文

【摘要】：目的: 探讨miR-210及其靶基因E2F3在喉鳞状细胞癌细胞系中的表达，并证明两者直接调控关系。同时探讨miR-210及E2F3的表达与喉鳞癌临床病理参数的关系，为喉鳞状细胞癌的诊断和预后评估寻找辅助生物学指标奠定基础。方法: 1.通过qRT-PCR方法测定两种喉癌细胞株中miR-210及其下游靶基因E2F3的表达水平，并通过双荧光检测验证miR-210与E2F3之间的直接调控关系；2.通过qRT-PCR方法检测20例喉鳞状细胞癌和癌旁正常切缘新鲜样本中miR-210和E2F3 mRNA水平，分析二者在喉鳞状细胞癌中表达的相关性以及二者与喉鳞状细胞癌患者各项临床病理学参数的关系；3.采用免疫组化技术检测喉鳞状细胞癌组织及癌旁正常切缘组织石蜡标本中E2F3蛋白表达，探讨E2F3在喉鳞状细胞癌发生发展过程中可能的作用；4.生存率统计采用寿命表法和Kaplan-Meier法，其差异性检验用Log-rank法；5.采用Cox回归模型分析E2F3及临床病理因素在喉鳞癌患者预后中的价值。所有数据均采用SPSS 16.0软件包进行统计学分析。结果: 1.检测两种喉癌细胞株Hep-2和TU177中miR-210和E2F3水平，结果显示：在miR-210相对高表达的Hep-2细胞株中E2F3表达水平较miR-210相对低表达的TU177细胞株中低，通过双荧光检测证实：miR-210+E2F3细胞株组较miR-210+mutE2F3细胞株组的荧光强度明显减弱，且差异有统计学意义(P＜0.05)；miR-210+E2F3细胞株组较NC组的荧光强度明显减弱，且差异有统计学意义(P＜0.05)； 2.统计学方法分析20例喉鳞状细胞癌和癌旁正常切缘新鲜样本中miR-210和E2F3的水平，结果显示：miR-210在喉鳞状细胞癌组织和癌旁正常切缘组织中表达水平的均数±标准差分别为15.488±9.371和4.437±3.039，二者差异有统计学意义(P=0.000)；E2F3mRNA在喉鳞状细胞癌组织和癌旁正常切缘组织中表达水平的均数±标准差分别为6.683±5.599和2.728±1.431，，二者差异有统计学意义(P=0.008)；喉鳞状细胞癌新鲜组织的miR-210和E2F3 mRNA表达水平之间呈线性负相关(rs=-0.645, P=0.002)； 3.免疫组化检测E2F3蛋白在喉鳞状细胞癌与癌旁正常切缘中的表达率，结果显示：两组中E2F3蛋白阳性表达率分别为90.44% (123/136)和27.85% (22/79)，差异具有显著统计学差异(P＜0.001)，E2F3蛋白胞核中高表达与喉鳞状细胞癌患者年龄、临床分期、组织分化和肿瘤分型有关；胞浆中高表达只于肿瘤分化有关(P＜0.05)； 4.单因素分析显示：临床分期、淋巴结有无转移、T分期、E2F3蛋白核表达和肿瘤分型是喉鳞状细胞癌患者生存期的影响因素（P＜0.05）； 5.多因素分析显示：T分期、淋巴结有无转移和E2F3蛋白核表达是影响喉鳞状细胞癌患者预后的危险因素（P＜0.05）。结论: 1. miR-210与靶基因E2F3之间存在直接调控关系； 2. E2F3蛋白核表达可能参与喉鳞状细胞癌发生发展过程； 3. T分期、淋巴结有无转移和E2F3蛋白核表达是喉鳞状细胞癌患者预后影响因素，检测E2F3蛋白核表达可为判断喉鳞状细胞癌患者预后评估提供帮助。
[Abstract]:Purpose :
To investigate the expression of miR - 210 and its target gene E2F3 in laryngeal squamous cell carcinoma cell lines and to demonstrate their direct control relationship . Meanwhile , the relationship between expression of miR - 210 and E2F3 and the clinical pathological parameters of laryngeal squamous cell carcinoma was discussed , which laid the foundation for the diagnosis and prognosis evaluation of laryngeal squamous cell carcinoma .
Method :
1 . The expression level of miR - 210 and its downstream target gene E2F3 was determined by qRT - PCR , and the direct control relationship between miR - 210 and E2F3 was verified by double fluorescence detection ;
2 . The levels of miR - 210 and E2F3 mRNA were detected by qRT - PCR in the fresh samples of laryngeal squamous cell carcinoma and adjacent normal margins , and the correlation between the expression of both in laryngeal squamous cell carcinoma and the clinical pathological parameters of laryngeal squamous cell carcinoma were analyzed .
3 . The expression of E2F3 protein in paraffin specimens of laryngeal squamous cell carcinoma and normal margin tissue was detected by immunohistochemistry , and the possible role of E2F3 in the development of laryngeal squamous cell carcinoma was discussed .
4 . Life table method and Kaplan - Meier method were used for survival statistics . Log - rank method was used for differential test .
5 . Cox regression model was used to analyze the value of E2F3 and clinicopathological factors in the prognosis of laryngeal squamous cell carcinoma . All the data were analyzed by SPSS 16.0 software package .
Results :
1 . The levels of miR - 210 and E2F3 in the two types of laryngeal cancer cell lines were tested . The results showed that the expression level of E2F3 was lower than that of the low - expressed TU177 cell line of miR - 210 . The results showed that the fluorescence intensity of the miR - 210 + E2F3 cell line group was significantly lower than that of the miR - 210 + mutE2F3 cell line group , and the difference was statistically significant ( P < 0.05 ) .
Compared with NC group , the fluorescence intensity of the miR - 210 + E2F3 cell line group was significantly decreased , and the difference was statistically significant ( P < 0.05 ) .

2 . The levels of miR - 210 and E2F3 in the fresh samples of laryngeal squamous cell carcinoma and adjacent normal margins were analyzed by statistical methods . The results showed that the mean 卤 standard deviation of miR - 210 was 15.488 卤 9.371 and 4.437 卤 3.39 , respectively , in the tissues of laryngeal squamous cell carcinoma and adjacent normal margin tissues ( P = 0.000 ) .
The mean 卤 SD of E2F3mRNA was 6.683 卤 5.599 and 2.728 卤 1.431 in normal margin of laryngeal squamous cell carcinoma ( P = 0.008 ) .
There was a linear negative correlation between miR - 210 and E2F3 mRNA expression levels in fresh tissue of laryngeal squamous cell carcinoma ( rs = - 0.645 , P = 0.002 ) ;

3 . The expression rate of E2F3 protein in laryngeal squamous cell carcinoma and normal margin of carcinoma was detected by immunohistochemistry . The results showed that the positive rates of E2F3 protein were 90.44 % ( 123 / 136 ) and 27.85 % ( 22 / 79 ) in both groups , and the difference was statistically significant ( P < 0.001 ) .
High expression in cytoplasm was only related to tumor differentiation ( P < 0.05 ) .

4 . Single factor analysis showed that clinical stage , lymph node metastasis , T stage , E2F3 protein core expression and tumor typing were the influencing factors of survival time of laryngeal squamous cell carcinoma ( P < 0.05 ) ;

5 . Multi - factor analysis showed that T staging , lymph node metastasis and E2F3 protein expression were the risk factors affecting the prognosis of patients with laryngeal squamous cell carcinoma ( P < 0.05 ) .
Conclusion :
1.There is a direct regulatory relationship between miR - 210 and the target gene E2F3 ;

2 . The expression of E2F3 protein may be involved in the development of laryngeal squamous cell carcinoma .

3.T staging , lymph node metastasis and the expression of E2F3 protein were the prognostic factors in laryngeal squamous cell carcinoma .
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.65

【参考文献】