IEX-1在宫颈癌中的表达及其与HPV感染的相关性
发布时间：2018-09-10 16:31
【摘要】：背景和目的子宫颈癌是女性生殖系统最常见的恶性肿瘤之一,全球范围内宫颈癌发病率在女性癌症中占第三,仅次于肺癌和胃癌,约有90%的宫颈癌死亡病例发生在发展中国,严重威胁着女性生命健康。宫颈癌最主要的两种类型是鳞状细胞癌(squamous cell carcinoma,SCC)和腺癌(adenocarcinoma,ADCA),宫颈鳞癌约占80%左右,近年来宫颈腺癌的发病率不断增加。持续的高危型人乳头瘤病毒(high risk human papilloma virus,HR-HPV)感染是宫颈癌最主要的致癌因子,其中HPV16和HPV18是最常见的HR-HPV型别。早期蛋白E6和E7是HR-HPV的主要癌蛋白,E6可泛素化水解P53,导致P53依赖的细胞凋亡和/或衰老的损失,而E7结合p RB从而导致细胞周期紊乱。因此,HR-HPV感染可导致细胞的恶性转化和肿瘤的发展。1993年Charles等在小鼠成纤维细胞中发现了即刻早期反应基因(Immediate early response gene X-1,IEX-1),作为即刻早期基因(immediate early gene,IEG)一员,IEX-1可以在多种刺激条件下短暂快速地表达。这些刺激因子包括生长因子、细胞因子、电离辐射、病毒感染和其他类型的细胞应激因素。IEX-1参与细胞的代谢、增殖、凋亡、分化及细胞周期调节等多种生物学效应。IEX-1在调节细胞周期和凋亡过程中发挥着复杂的作用。虽然目前研究已经证实在多种人类恶性肿瘤中IEX-1表达失调,但在宫颈癌中IEX-1的研究较少,IEX-1在宫颈癌中的表达情况如何,IEX-1表达与宫颈HPV感染是否相关尚不清楚。本研究将通过免疫组化法分析IEX-1在宫颈癌组织、宫颈上皮内瘤变(Cervical Intraepithelial Neoplasia,CIN)组织及正常宫颈组织中的表达情况,分析IEX-1的表达与宫颈癌临床病理关系,预测IEX-1在宫颈癌发生发展中的作用。并通过实时荧光定量PRC(RT-q PCR)法及Western Blot法分析不同HPV感染状态的宫颈癌细胞中IEX-1的表达差异。RNA干扰(RNA interference,RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA,ds RNA)诱发的、同源m RNA高效特异性降解的现象。RNAi法具有高度的序列专一性,通过瞬时转染si RNA导入细胞可以特异地使特定基因沉默。我们通过设计针对HPV16 E6的si RNA,转染Si Ha细胞,特异性沉默Si Ha细胞中E6的表达,检测沉默E6前后Si Ha细胞中IEX-1的表达,以探索IEX-1的表达与HPV感染的相关性及其可能涉及的机制。本课题将为明确宫颈癌变过程中IEX-1的作用及研究其与HPV的相互作用提供理论依据。材料与方法1.人宫颈癌细胞株Si Ha(HPV16+)、He La(HPV18+)、C-33a(HPV-)均由华中科技大学惠赠。收集2010年1月到2014年10月在郑州大学第一附属医院病理科存档蜡块标本132份(其中手术标本124例,活检标本8例)。其中正常宫颈29例,年龄32-61岁;CIN 46例(Ⅰ级19例、Ⅱ级17例、Ⅲ级10例),年龄29-58岁;宫颈癌57例(鳞癌35例、腺癌22例),年龄27-65岁。宫颈癌根据FIGO临床病例分期:Ⅰ期23例,Ⅱ期26例,Ⅲ期6例,Ⅳ期2例。分化等级:高分化13例,中分化18例,低分化26例。2.免疫组化SP法检测IEX-1在正常宫颈组织、CIN组织及宫颈癌组织中的表达情况,并分析其与临床病理特征的关系。3.以RT-q PCR法检测Si Ha、He La、C-33a宫颈癌细胞系中IEX-1表达,Western Blot法检测Si Ha、He La、C-33a宫颈癌细胞系中IEX-1蛋白质表达。4.RT-q PCR和Western Blot法检测E6特异性si RNA干扰Si Ha细胞前后Si Ha细胞中E6及IEX-1的m RNA及蛋白表达水平。5.统计学处理:采用SPSS17.0软件统计分析,定性资料比较采用卡方检验,定量资料比较采用单因素方差分析,以P0.05为差异有统计学意义。两两比较采用Bonferroni法检验矫正检验系数。结果1.免疫组化法显示:IEX-1在正常宫颈组织、CIN组织和宫颈癌组织中的表达依次减少,三者之间差异有统计学意义(c2=75.905,P=0.0000.017)。IEX-1在宫颈癌组织中的表达与患者年龄、病理类型及FIGO分期均无关(均P0.05)。IEX-1的表达与宫颈癌分化程度及肌层浸润深度相关。分化程度越低,IEX-1蛋白阳性表达率越低(c2=6.642,P=0.0090.05);深肌层浸润者IEX-1蛋白阳性表达率明显低于仅浅肌层浸润者(c2=5.076,P=0.0250.05)。2.RT-q PCR法分别检测Si Ha细胞、He La细胞和C-33a细胞中IEX-1 m RNA表达水平显示:IEX-1 m RNA在C-33a细胞中表达(21.652±9.020)高于Si Ha细胞(1.006±0.027)和He La细胞(1.742±0.854)(PSi Ha-C-33a=0.0030.05,PHe La-C-33a=0.0030.05)。Si Ha细胞和He La细胞间IEX-1m RNA表达无明显差异(PSi Ha-He La=0.8680.05)。3.Western Blot法分别检测Si Ha细胞、He La细胞和C-33a细胞中IEX-1蛋白的表达水平显示:IEX-1蛋白在C-33a细胞(18.017±1.351)中表达高于Si Ha细胞(1.028±0.039)和He La细胞(2.004±0.363)(PSi Ha-C-33a0.001,PHe La-C-33a0.001)。Si Ha细胞和He La细胞间IEX-1蛋白表达无明显差异(PSi Ha-He La=0.1520.05)。4.将Si Ha细胞分为干扰组、阴性对照组和空白对照组,干扰组转染入E6特异性Si RNA核苷酸序列,阴性对照组转入无义RNA核苷酸序列,空白对照组使用RNase-free water代替。转染后用RT-q PCR法分别检测干扰组、阴性对照组和空白对照组E6 m RNA和IEX-1 m RNA表达水平显示:E6 m RNA在干扰组(0.218±0.106)表达水平明显低于阴性对照组(1.031±0.072)及空白对照组(1.003±0.015)(P干扰-阴性对照0.001,P干扰-空白对照0.001),E6 m RNA表达水平在阴性对照组和空白对照组差异无统计学意义(P阴性对照-空白对照=0.2070.05)。IEX-1m RNA在干扰组(20.083±0.672)表达水平明显高于阴性对照组(1.613±0.408)及空白对照组(1.018±0.032)(P干扰-阴性对照0.001,P干扰-空白对照0.001),IEX-1 m RNA表达水平在阴性对照组和空白对照组差异无统计学意义(P阴性对照-空白对照=0.3040.05)5.转染后用Western Blot法分别检测干扰组、阴性对照组和空白对照组E6、IEX-1蛋白表达水平显示:E6蛋白在干扰组(0.184±0.274)表达水平均明显低于阴性对照组(0.692±0.133)及空白对照组(0.783±0.097)(P干扰-阴性对照0.001,P干扰-空白对照0.001),E6蛋白表达水平在阴性对照组和空白对照组间差异无统计学意义(P阴性对照-空白对照=0.1840.05)。IEX-1蛋白在干扰组(0.672±0.135)表达水平均明显高于阴性对照组(0.137±0.071)及空白对照组(0.081±0.009)(P干扰-阴性对照=0.0010.05,P干扰-空白对照0.001),IEX-1蛋白表达水平在阴性对照组和空白对照组间差异无统计学意义(P阴性对照-空白对照=0.0940.05)结论1.IEX-1在宫颈癌组织中表达减低,其表达水平与宫颈癌恶性程度呈负相关,宫颈上皮细胞中IEX-1的表达减低或缺失可能参与了宫颈癌的发生与发展。2.IEX-1在宫颈癌细胞中的表达水平与HPV感染与否呈负相关,与感染的HPV型别无关,IEX-1与HPV在宫颈癌变过程中可能发挥相反作用。3.E6特异性Si RNA可沉默宫颈癌细胞系中E6蛋白的表达。宫颈癌细胞系中HPV E6沉默后,IEX-1表达上调,HPV可能通过E6癌蛋白下调了IEX-1的表达。
[Abstract]:BACKGROUND AND OBJECTIVE Cervical cancer is one of the most common malignant tumors in the female reproductive system. The incidence of cervical cancer is the third in female cancers worldwide, next only to lung cancer and gastric cancer. About 90% of cervical cancer deaths occur in developing countries, which seriously threaten women's life and health. Squamous cell carcinoma (SCC) and adenocarcinoma (ADCA), cervical squamous cell carcinoma (SCC) account for about 80%, the incidence of cervical adenocarcinoma in recent years is increasing. Persistent high-risk human papilloma virus (HR-HPV) infection is the most important carcinogen of cervical cancer, HPV16 and HPV18 are the most common HR-HPV. Type. E6 and E7 are the major cancer proteins of HR-HPV. E6 can hydrolyze P53 by ubiquitination, leading to loss of apoptosis and/or senescence in P53-dependent cells. E7 binding to P RB leads to cell cycle disorders. Therefore, HR-HPV infection can lead to malignant transformation and tumor development. Charles et al. found in mouse fibroblasts in 1993 Immediate early response gene X-1 (IEX-1), as a member of immediate early gene (IEG), can be expressed briefly and rapidly under a variety of stimulation conditions. These stimulating factors include growth factors, cytokines, ionizing radiation, virus infection and other types of cell stress factors. IEX-1 plays a complex role in regulating cell cycle and apoptosis. Although the expression of IEX-1 has been proved to be out of balance in many human malignant tumors, there is little research on IEX-1 in cervical cancer and IEX-1 in cervical cancer. The expression of IEX-1 in cervical cancer, cervical intraepithelial neoplasia (CIN) and normal cervical tissues was analyzed by immunohistochemistry. The relationship between IEX-1 expression and clinical pathology of cervical cancer was analyzed. To determine the role of IEX-1 in the development of cervical cancer, real-time fluorescence quantitative PRC (RT-q PCR) and Western Blot were used to analyze the expression of IEX-1 in cervical cancer cells with different HPV infection status. Homologous m RNA degrades efficiently and specifically. RNAi method has high sequence specificity. Specific gene silencing can be achieved by transient transfection of Si RNA into cells. Material and Methods 1. Human cervical cancer cell lines Si Ha (HPV16 +), He La (HPV18 +), C-33a (HPV -) were given by Huazhong University of Science and Technology. From January 2010 to October 2014, 132 cervical specimens (124 surgical specimens and 8 biopsy specimens) were collected from the Department of Pathology, First Affiliated Hospital of Zhengzhou University. Among them, 29 cases were normal cervix, aged 32-61 years; 46 cases of CIN (19 cases of grade I, 17 cases of grade II, 10 cases of grade III), aged 29-58 years; 57 cases of cervical cancer (35 cases of squamous cell carcinoma, 22 cases of adenocarcinoma), aged 27-65 years. According to FIGO clinical staging, 23 cases of cervical cancer were stage I, 26 cases of stage II, 6 cases of stage III and 2 cases of stage IV. Differentiation grade: 13 cases were well-differentiated, 18 cases were moderately differentiated and 26 cases were poorly differentiated. The expression of IEX-1 in Si Ha, He La, C-33a cervical cancer cell lines was detected by Western Blot method, and the expression of IEX-1 protein in Si Ha, He La, C-33a cervical cancer cell lines was detected by Western Blot method. Statistical analysis showed that the expression of IEX-1 in normal cervical tissues, CIN tissues and cervical cancer tissues decreased in turn. The expression of IEX-1 in cervical cancer tissues was not correlated with age, pathological type and FIGO stage (all P 0.05). The expression of IEX-1 was correlated with the degree of differentiation and the depth of myometrial invasion. The lower the degree of differentiation, the lower the positive expression rate of IEX-1 protein (c 2 = 6.642, P = 0.0090.05). The positive expression rate of IEX-1 protein in deep myometrial infiltrators was significantly lower than that in superficial myometrial infiltrators only (c2 = 5.076, P = 0.0250.05). 2. Detection of IEX-1 m RNA expression in Si Ha cells, He La cells and C-33a cells by RT-q PCR showed that the expression of IEX-1 m RNA in C-33a cells (21.652 + 9.020) was higher than that in Si Ha cells (1.006 + 0.027) and He La cells (1.742 + 0.027). 854 (PSi Ha-C-33a = 0.0030.05, PHe La-C-33a = 0.0030.05). There was no significant difference in IEX-1m RNA expression between Si Ha cells and He La cells (PSi Ha-He La = 0.8680.05). 3. Western Blot assay of IEX-1 protein expression in Si Ha cells, He La cells and C-33a cells showed that IEX-1 protein expression in C-33a cells was higher than that in Si Ha cells (18.017 + 1.351). There was no significant difference in IEX-1 protein expression between Si Ha cells and He La cells (PSi Ha-C-33a 0.001, PHe La-C-33a 0.001). 4. Si Ha cells were divided into interference group, negative control group and blank control group. The interference group was transfected into E6-specific Si RNA sequence, negative pair. After transfection, the expression levels of E6 m RNA and IEX-1 m RNA in the negative control group and blank control group were detected by RT-q PCR respectively. The expression levels of E6 m RNA and IEX-1 m RNA in the interference group (0.218 +0.106) were significantly lower than those in the negative control group (1.031 +0.072) and blank control group (1.031 +0.072). There was no significant difference in the expression of E6 m RNA between the negative control group and the blank control group (P-negative control = 0.2070.05). The expression of IEX-1m RNA in the interference group (20.083 + 0.672) was significantly higher than that in the negative control group (1.613 + 0.408) and the blank control group (0.001). There was no significant difference in the expression of IEX-1 m RNA between negative control group and blank control group (P-negative control = 0.3040.05). 5. Western Blot method was used to detect the expression of E6 and IEX-1 protein in interference group, negative control group and blank control group respectively. The level of E6 protein expression in the interference group (0.184+0.274) was significantly lower than that in the negative control group (0.692+0.133) and the blank control group (0.783+0.097) (P interference-negative control 0.001, P interference-blank control 0.001), and there was no significant difference between the negative control group and the blank control group (P negative control-blank control = 0.001). 1840.05). The expression level of IEX-1 protein in the interference group (0.672.135) was significantly higher than that in the negative control group (0.137.071) and the blank control group (0.081.009) (P interference-negative control = 0.0010.05, P interference-blank control 0.001). There was no significant difference in the expression level of IEX-1 protein between the negative control group and blank control group (P negative control-blank control-blank control). Conclusion 1. The expression of IEX-1 in cervical cancer tissues decreased, and its expression level was negatively correlated with the malignant degree of cervical cancer. The decrease or deletion of IEX-1 expression in cervical epithelial cells may be involved in the occurrence and development of cervical cancer. 2. The expression level of IEX-1 in cervical cancer cells was negatively correlated with HPV infection and H infection. IEX-1 and HPV may play an opposite role in cervical carcinogenesis. 3. E6-specific Si RNA can silence the expression of E6 protein in cervical cancer cell lines. After HPV E6 silencing, the expression of IEX-1 is up-regulated, and HPV may down-regulate the expression of IEX-1 through E6 oncoprotein.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.33
[Abstract]:BACKGROUND AND OBJECTIVE Cervical cancer is one of the most common malignant tumors in the female reproductive system. The incidence of cervical cancer is the third in female cancers worldwide, next only to lung cancer and gastric cancer. About 90% of cervical cancer deaths occur in developing countries, which seriously threaten women's life and health. Squamous cell carcinoma (SCC) and adenocarcinoma (ADCA), cervical squamous cell carcinoma (SCC) account for about 80%, the incidence of cervical adenocarcinoma in recent years is increasing. Persistent high-risk human papilloma virus (HR-HPV) infection is the most important carcinogen of cervical cancer, HPV16 and HPV18 are the most common HR-HPV. Type. E6 and E7 are the major cancer proteins of HR-HPV. E6 can hydrolyze P53 by ubiquitination, leading to loss of apoptosis and/or senescence in P53-dependent cells. E7 binding to P RB leads to cell cycle disorders. Therefore, HR-HPV infection can lead to malignant transformation and tumor development. Charles et al. found in mouse fibroblasts in 1993 Immediate early response gene X-1 (IEX-1), as a member of immediate early gene (IEG), can be expressed briefly and rapidly under a variety of stimulation conditions. These stimulating factors include growth factors, cytokines, ionizing radiation, virus infection and other types of cell stress factors. IEX-1 plays a complex role in regulating cell cycle and apoptosis. Although the expression of IEX-1 has been proved to be out of balance in many human malignant tumors, there is little research on IEX-1 in cervical cancer and IEX-1 in cervical cancer. The expression of IEX-1 in cervical cancer, cervical intraepithelial neoplasia (CIN) and normal cervical tissues was analyzed by immunohistochemistry. The relationship between IEX-1 expression and clinical pathology of cervical cancer was analyzed. To determine the role of IEX-1 in the development of cervical cancer, real-time fluorescence quantitative PRC (RT-q PCR) and Western Blot were used to analyze the expression of IEX-1 in cervical cancer cells with different HPV infection status. Homologous m RNA degrades efficiently and specifically. RNAi method has high sequence specificity. Specific gene silencing can be achieved by transient transfection of Si RNA into cells. Material and Methods 1. Human cervical cancer cell lines Si Ha (HPV16 +), He La (HPV18 +), C-33a (HPV -) were given by Huazhong University of Science and Technology. From January 2010 to October 2014, 132 cervical specimens (124 surgical specimens and 8 biopsy specimens) were collected from the Department of Pathology, First Affiliated Hospital of Zhengzhou University. Among them, 29 cases were normal cervix, aged 32-61 years; 46 cases of CIN (19 cases of grade I, 17 cases of grade II, 10 cases of grade III), aged 29-58 years; 57 cases of cervical cancer (35 cases of squamous cell carcinoma, 22 cases of adenocarcinoma), aged 27-65 years. According to FIGO clinical staging, 23 cases of cervical cancer were stage I, 26 cases of stage II, 6 cases of stage III and 2 cases of stage IV. Differentiation grade: 13 cases were well-differentiated, 18 cases were moderately differentiated and 26 cases were poorly differentiated. The expression of IEX-1 in Si Ha, He La, C-33a cervical cancer cell lines was detected by Western Blot method, and the expression of IEX-1 protein in Si Ha, He La, C-33a cervical cancer cell lines was detected by Western Blot method. Statistical analysis showed that the expression of IEX-1 in normal cervical tissues, CIN tissues and cervical cancer tissues decreased in turn. The expression of IEX-1 in cervical cancer tissues was not correlated with age, pathological type and FIGO stage (all P 0.05). The expression of IEX-1 was correlated with the degree of differentiation and the depth of myometrial invasion. The lower the degree of differentiation, the lower the positive expression rate of IEX-1 protein (c 2 = 6.642, P = 0.0090.05). The positive expression rate of IEX-1 protein in deep myometrial infiltrators was significantly lower than that in superficial myometrial infiltrators only (c2 = 5.076, P = 0.0250.05). 2. Detection of IEX-1 m RNA expression in Si Ha cells, He La cells and C-33a cells by RT-q PCR showed that the expression of IEX-1 m RNA in C-33a cells (21.652 + 9.020) was higher than that in Si Ha cells (1.006 + 0.027) and He La cells (1.742 + 0.027). 854 (PSi Ha-C-33a = 0.0030.05, PHe La-C-33a = 0.0030.05). There was no significant difference in IEX-1m RNA expression between Si Ha cells and He La cells (PSi Ha-He La = 0.8680.05). 3. Western Blot assay of IEX-1 protein expression in Si Ha cells, He La cells and C-33a cells showed that IEX-1 protein expression in C-33a cells was higher than that in Si Ha cells (18.017 + 1.351). There was no significant difference in IEX-1 protein expression between Si Ha cells and He La cells (PSi Ha-C-33a 0.001, PHe La-C-33a 0.001). 4. Si Ha cells were divided into interference group, negative control group and blank control group. The interference group was transfected into E6-specific Si RNA sequence, negative pair. After transfection, the expression levels of E6 m RNA and IEX-1 m RNA in the negative control group and blank control group were detected by RT-q PCR respectively. The expression levels of E6 m RNA and IEX-1 m RNA in the interference group (0.218 +0.106) were significantly lower than those in the negative control group (1.031 +0.072) and blank control group (1.031 +0.072). There was no significant difference in the expression of E6 m RNA between the negative control group and the blank control group (P-negative control = 0.2070.05). The expression of IEX-1m RNA in the interference group (20.083 + 0.672) was significantly higher than that in the negative control group (1.613 + 0.408) and the blank control group (0.001). There was no significant difference in the expression of IEX-1 m RNA between negative control group and blank control group (P-negative control = 0.3040.05). 5. Western Blot method was used to detect the expression of E6 and IEX-1 protein in interference group, negative control group and blank control group respectively. The level of E6 protein expression in the interference group (0.184+0.274) was significantly lower than that in the negative control group (0.692+0.133) and the blank control group (0.783+0.097) (P interference-negative control 0.001, P interference-blank control 0.001), and there was no significant difference between the negative control group and the blank control group (P negative control-blank control = 0.001). 1840.05). The expression level of IEX-1 protein in the interference group (0.672.135) was significantly higher than that in the negative control group (0.137.071) and the blank control group (0.081.009) (P interference-negative control = 0.0010.05, P interference-blank control 0.001). There was no significant difference in the expression level of IEX-1 protein between the negative control group and blank control group (P negative control-blank control-blank control). Conclusion 1. The expression of IEX-1 in cervical cancer tissues decreased, and its expression level was negatively correlated with the malignant degree of cervical cancer. The decrease or deletion of IEX-1 expression in cervical epithelial cells may be involved in the occurrence and development of cervical cancer. 2. The expression level of IEX-1 in cervical cancer cells was negatively correlated with HPV infection and H infection. IEX-1 and HPV may play an opposite role in cervical carcinogenesis. 3. E6-specific Si RNA can silence the expression of E6 protein in cervical cancer cell lines. After HPV E6 silencing, the expression of IEX-1 is up-regulated, and HPV may down-regulate the expression of IEX-1 through E6 oncoprotein.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.33
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