KLF5和TNFRSF11a在宫颈癌变中的作用及机制研究
发布时间：2018-07-15 14:43
【摘要】：目的探讨转录因子Krüppel样因子5(Krüppel-like Factors 5,KLF5)和肿瘤坏死因子受体超家族成员11a(Tumor Necrosis Factor Receptor Superfamily 11a,TNFRSF11a)在人宫颈鳞癌组织中的表达并揭示其促进人宫颈癌细胞增殖、迁移和侵袭的分子与细胞生物学机制。方法收集就诊于河北省唐山市工人医院妇科门诊和住院部的汉族女性的正常宫颈组织和病变宫颈组织作为本次实验研究标本。严格按照宫颈组织的纳入标准和排除标准,从2014年9月到2016年3月期间,共收集160例。所有纳入实验的宫颈组织均由实验室研究人员与病理科医师共同确认病理类型和分级,包括:正常对照组47例、宫颈上皮内瘤变组(Cervical Intraepithelial Neoplasia,CIN)73例(其中CINⅠ27例,CINⅡ-Ⅲ46例)和宫颈鳞癌组(Cervical Squmaous Cell Carcinoma,CSCC)40例。利用mRNA表达谱芯片筛查3例对照宫颈组织和3例人宫颈鳞癌组织中细胞应答炎症反应相关基因的mRNA表达。通过实时定量反转录聚合酶链反应(Quantitative Real-time Polymerase Chain Reaction,q RT-PCR)对芯片筛查结果进行验证并分析KLF5和TNFRSF11a mRNA表达水平在不同病理人宫颈组织中的相关性。分析TNFRSF11a的mRNA表达与人宫颈鳞癌临床病理参数的关系。采用免疫双荧光染色法检测不同病理人宫颈组织中KLF5和TNFRSF11a的共表达。在人宫颈癌He La细胞中,采用脂质体转染特异性si RNA分别敲低KLF5和TNFRSF11a的表达,构建KLF5超表达腺病毒感染细胞过表达KLF5,应用q RTPCR和Western blot分别检测细胞内相关基因的mRNA和蛋白水平变化。采用CCK-8实验检测不同处理条件下He La细胞的增殖功能的改变。采用Transwell实验检测不同处理条件下He La细胞的迁移和侵袭功能的改变。采用双荧光素酶报告基因技术检测转录因子KLF5对TNFRSF11a基因启动子的转录活性并采用染色质免疫沉淀(Chromatin Immunoprecipitation,Ch IP)分析KLF5在TNFRSF11a基因启动子上的结合位点。结果1 mRNA表达谱芯片筛查结果显示,与人正常宫颈组织相比,宫颈鳞癌组织中细胞应答炎症反应的相关基因KLF5、Npnt、Socs5、TNFRSF11a、Cxcl13、Il6st、Cntfr、Plcb1表达均上调(P0.05)。2采用q RT-PCR实验对芯片检测结果进行验证,结果显示,宫颈鳞癌组织中KLF5、Npnt、Socs5、TNFRSF11a、Cxcl13、Il6st较正常组织表达均显著上调,差异有统计学意义(P0.05)。3免疫双荧光染色检测不同病理人宫颈组织中KLF5和TNFRSF11a共表达,结果显示,KLF5和TNFRSF11a共表达于宫颈组织,且与对照组比较,在CINⅠ组、CINⅡ-Ⅲ组和CSCC组中的表达水平逐渐升高,荧光强度增加,差异有统计学意义(P0.05)。4采用q RT-PCR实验检测不同病理人宫颈组织中KLF5和TNFRSF11a的mRNA水平,结果显示,与正常对照组相比,CINⅠ组、CINⅡ-Ⅲ组和CSCC组中KLF5和TNFRSF11a的mRNA表达水平均升高,且随病理程度的加重而升高,差异均有统计学意义(P0.05);与CINⅡ-Ⅲ组相比,CSCC组中KLF5和TNFRSF11a的mRNA表达水平增高,差异有统计学意义(P0.05)。5采用Spearman法分析不同病理人宫颈组织中KLF5与TNFRSF11a mRNA表达的相关性,在正常对照组和CINⅠ组中KLF5与TNFRSF11a的mRNA表达无明显相关性;在CINⅡ-Ⅲ组中KLF5与TNFRSF11a的mRNA表达呈正相关性(r=0.326,P=0.027);在CSCC组织中KLF5与TNFRSF11a的mRNA表达呈正相关(r=0.393,P=0.012)。6在He La细胞中分别过表达或敲低KLF5,Western blotting和q RT-PCR结果均显示,在基础水平和TNF-α诱导情况下,过表达或敲低KLF5均能够显著增加或降低TNFRSF11a的蛋白及mRNA水平(P0.05)。报告基因和Ch IP分析结果表明,KLF5通过直接与TNFRSF11a启动子结合而激活TNFRSF11a基因表达。7基因表达与细胞功能获得与缺失实验结果表明:过表达KLF5可促进He La细胞的增殖、迁移及侵袭(P0.05);采用si RNA抑制TNFRSF11a的表达能够抑制He La细胞的增殖、迁移及侵袭(P0.05);而敲低TNFRSF11a的表达同时过表达KLF5仍能够部分抑制KLF5介导He La细胞增殖、迁移及侵袭的功能(P0.05)。8临床参数相关分析结果表明:宫颈鳞癌组织中TNFRSF11a mRNA的表达与肿瘤的病理分化程度、肌层浸润深度、临床分期和淋巴结转移有关,差异有统计学意义(P0.05)。结论1人宫颈组织中KLF5和TNFRSF11a共表达促使宫颈鳞癌的发生、发展。2在宫颈癌细胞中KLF5可通过直接与TNFRSF11a基因启动子相互作用激活TNFRSF11a基因的转录。3转录因子KLF5促进宫颈癌细胞的增殖、迁移和侵袭部分依赖于TNFRSF11a基因的表达。
[Abstract]:Objective to investigate the expression of the transcription factor Kr u ppel like factor 5 (Kr u ppel-like Factors 5, KLF5) and the member of the tumor necrosis factor receptor superfamily 11a (Tumor Necrosis Factor Receptor Superfamily) in human cervical squamous cell carcinoma and to reveal the molecular and cellular biological machines that promote the proliferation, migration and invasion of human cervical cancer cells. Methods the normal cervical tissue and cervical tissue of the Han women in the gynecologic outpatient and inpatient department of Tangshan City workers' hospital in Hebei province were collected as the experimental specimens. According to the standard and exclusion criteria of cervical tissue, 160 cases were collected from September 2014 to March 2016. All the subjects were included in the laboratory. The cervical tissues were all confirmed by laboratory researchers and pathologists, including 47 cases of normal control, 73 cases of Cervical Intraepithelial Neoplasia (CIN), 73 cases of CIN I, CIN II - III, and 40 cases of cervical squamous cell carcinoma (Cervical Squmaous Cell Carcinoma, CSCC). MRNA expression of inflammatory response related genes in 3 cases of cervical tissue and 3 human cervical squamous carcinoma tissues was screened by spectral chip. The results of chip screening were verified by real-time quantitative reverse transcription polymerase chain reaction (Quantitative Real-time Polymerase Chain Reaction, Q RT-PCR) and the expression level of KLF5 and TNFRSF11a mRNA was analyzed. The relationship between the mRNA expression of TNFRSF11a and the clinicopathological parameters of human cervical squamous cell carcinoma was analyzed. The co expression of KLF5 and TNFRSF11a in different histopathological cervical tissues was detected by immunofluorescence staining. In He La cells of human cervical cancer, the specific Si RNA was transfected by liposomes to knock low KLF5, respectively. And the expression of TNFRSF11a, KLF5 overexpression of adenovirus infected cells overexpressed KLF5, Q RTPCR and Western blot were used to detect the changes of mRNA and protein levels of intracellular related genes, and CCK-8 test was used to detect the proliferation of He La cells under different treatment conditions. Change of cell migration and invasion function. The transcriptional activity of transcription factor KLF5 to TNFRSF11a gene promoter was detected by double luciferase reporter gene technique and the binding site of KLF5 on the promoter of TNFRSF11a gene was analyzed by chromatin immunoprecipitation (Chromatin Immunoprecipitation, Ch IP). Results 1 mRNA expression spectrum chip screening was used. Compared with normal cervical tissue, the expression of KLF5, Npnt, Socs5, TNFRSF11a, Cxcl13, Il6st, Cntfr, Plcb1 in cervical squamous cell carcinoma tissues were up regulated (P0.05).2 (P0.05).2 using Q RT-PCR test to verify the results of the chip detection. The expression of Il6st was significantly higher than that of normal tissue. The difference was statistically significant (P0.05).3 immunofluorescence staining was used to detect the co expression of KLF5 and TNFRSF11a in different histopathological cervical tissues. The results showed that KLF5 and TNFRSF11a were co expressed in cervical tissue, and compared with the control group, the expression level in the CIN II group and CSCC group was gradually increased in CIN I group. High fluorescence intensity increased, and the difference was statistically significant (P0.05).4 using Q RT-PCR test to detect mRNA levels of KLF5 and TNFRSF11a in different pathological human cervix tissues. The results showed that the levels of KLF5 and TNFRSF11a in the CIN I group, CIN II - III and CSCC groups were all higher than those in the normal control group, and increased with the aggravation of the pathological degree. The difference was statistically significant (P0.05). Compared with group CIN II - III, the mRNA expression level of KLF5 and TNFRSF11a increased in group CSCC, and the difference was statistically significant (P0.05).5 used Spearman method to analyze the correlation between KLF5 and TNFRSF11a mRNA expression in different histopathological cervical tissues. There was no significant correlation between the expression of mRNA and TNFRSF11a in CIN II - III group (r=0.326, P=0.027), and KLF5 in CSCC tissues was positively correlated with the expression of TNFRSF11a (r=0.393, P=0.012). Under the condition of overexpression or knock down KLF5, the protein and mRNA levels of TNFRSF11a were significantly increased or reduced (P0.05). The results of the reporter gene and Ch IP analysis showed that KLF5 activates the expression of the TNFRSF11a gene expression.7 gene and the cell function acquisition and deletion experiments by binding to the TNFRSF11a promoter directly. The proliferation, migration and invasion (P0.05) of a cells, Si RNA inhibition of TNFRSF11a expression can inhibit the proliferation, migration and invasion (P0.05) of He La cells, while low TNFRSF11a expression and overexpression of KLF5 can partly inhibit the KLF5 mediated proliferation, migration and invasion of He cells. The expression of TNFRSF11a mRNA in the tissue of cervical squamous cell carcinoma is related to the degree of pathological differentiation of the tumor, the depth of myometrium infiltration, the clinical stage and lymph node metastasis, and the difference is statistically significant (P0.05). Conclusion the co expression of KLF5 and TNFRSF11a in 1 cervical tissues promotes the occurrence of squamous cell carcinoma of the cervix, and the development of.2 in cervical cancer cells can be achieved by direct and TNFRSF1 in the cervical cancer cells. The 1A gene promoter interaction activates the transcriptional.3 transcription factor KLF5 of the TNFRSF11a gene to promote the proliferation of cervical cancer cells, and the migration and invasion depends on the expression of the TNFRSF11a gene.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
本文编号：2124419
[Abstract]:Objective to investigate the expression of the transcription factor Kr u ppel like factor 5 (Kr u ppel-like Factors 5, KLF5) and the member of the tumor necrosis factor receptor superfamily 11a (Tumor Necrosis Factor Receptor Superfamily) in human cervical squamous cell carcinoma and to reveal the molecular and cellular biological machines that promote the proliferation, migration and invasion of human cervical cancer cells. Methods the normal cervical tissue and cervical tissue of the Han women in the gynecologic outpatient and inpatient department of Tangshan City workers' hospital in Hebei province were collected as the experimental specimens. According to the standard and exclusion criteria of cervical tissue, 160 cases were collected from September 2014 to March 2016. All the subjects were included in the laboratory. The cervical tissues were all confirmed by laboratory researchers and pathologists, including 47 cases of normal control, 73 cases of Cervical Intraepithelial Neoplasia (CIN), 73 cases of CIN I, CIN II - III, and 40 cases of cervical squamous cell carcinoma (Cervical Squmaous Cell Carcinoma, CSCC). MRNA expression of inflammatory response related genes in 3 cases of cervical tissue and 3 human cervical squamous carcinoma tissues was screened by spectral chip. The results of chip screening were verified by real-time quantitative reverse transcription polymerase chain reaction (Quantitative Real-time Polymerase Chain Reaction, Q RT-PCR) and the expression level of KLF5 and TNFRSF11a mRNA was analyzed. The relationship between the mRNA expression of TNFRSF11a and the clinicopathological parameters of human cervical squamous cell carcinoma was analyzed. The co expression of KLF5 and TNFRSF11a in different histopathological cervical tissues was detected by immunofluorescence staining. In He La cells of human cervical cancer, the specific Si RNA was transfected by liposomes to knock low KLF5, respectively. And the expression of TNFRSF11a, KLF5 overexpression of adenovirus infected cells overexpressed KLF5, Q RTPCR and Western blot were used to detect the changes of mRNA and protein levels of intracellular related genes, and CCK-8 test was used to detect the proliferation of He La cells under different treatment conditions. Change of cell migration and invasion function. The transcriptional activity of transcription factor KLF5 to TNFRSF11a gene promoter was detected by double luciferase reporter gene technique and the binding site of KLF5 on the promoter of TNFRSF11a gene was analyzed by chromatin immunoprecipitation (Chromatin Immunoprecipitation, Ch IP). Results 1 mRNA expression spectrum chip screening was used. Compared with normal cervical tissue, the expression of KLF5, Npnt, Socs5, TNFRSF11a, Cxcl13, Il6st, Cntfr, Plcb1 in cervical squamous cell carcinoma tissues were up regulated (P0.05).2 (P0.05).2 using Q RT-PCR test to verify the results of the chip detection. The expression of Il6st was significantly higher than that of normal tissue. The difference was statistically significant (P0.05).3 immunofluorescence staining was used to detect the co expression of KLF5 and TNFRSF11a in different histopathological cervical tissues. The results showed that KLF5 and TNFRSF11a were co expressed in cervical tissue, and compared with the control group, the expression level in the CIN II group and CSCC group was gradually increased in CIN I group. High fluorescence intensity increased, and the difference was statistically significant (P0.05).4 using Q RT-PCR test to detect mRNA levels of KLF5 and TNFRSF11a in different pathological human cervix tissues. The results showed that the levels of KLF5 and TNFRSF11a in the CIN I group, CIN II - III and CSCC groups were all higher than those in the normal control group, and increased with the aggravation of the pathological degree. The difference was statistically significant (P0.05). Compared with group CIN II - III, the mRNA expression level of KLF5 and TNFRSF11a increased in group CSCC, and the difference was statistically significant (P0.05).5 used Spearman method to analyze the correlation between KLF5 and TNFRSF11a mRNA expression in different histopathological cervical tissues. There was no significant correlation between the expression of mRNA and TNFRSF11a in CIN II - III group (r=0.326, P=0.027), and KLF5 in CSCC tissues was positively correlated with the expression of TNFRSF11a (r=0.393, P=0.012). Under the condition of overexpression or knock down KLF5, the protein and mRNA levels of TNFRSF11a were significantly increased or reduced (P0.05). The results of the reporter gene and Ch IP analysis showed that KLF5 activates the expression of the TNFRSF11a gene expression.7 gene and the cell function acquisition and deletion experiments by binding to the TNFRSF11a promoter directly. The proliferation, migration and invasion (P0.05) of a cells, Si RNA inhibition of TNFRSF11a expression can inhibit the proliferation, migration and invasion (P0.05) of He La cells, while low TNFRSF11a expression and overexpression of KLF5 can partly inhibit the KLF5 mediated proliferation, migration and invasion of He cells. The expression of TNFRSF11a mRNA in the tissue of cervical squamous cell carcinoma is related to the degree of pathological differentiation of the tumor, the depth of myometrium infiltration, the clinical stage and lymph node metastasis, and the difference is statistically significant (P0.05). Conclusion the co expression of KLF5 and TNFRSF11a in 1 cervical tissues promotes the occurrence of squamous cell carcinoma of the cervix, and the development of.2 in cervical cancer cells can be achieved by direct and TNFRSF1 in the cervical cancer cells. The 1A gene promoter interaction activates the transcriptional.3 transcription factor KLF5 of the TNFRSF11a gene to promote the proliferation of cervical cancer cells, and the migration and invasion depends on the expression of the TNFRSF11a gene.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
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