KLF5和TNFRSF11a在宮頸癌變中的作用及機制研究
發(fā)布時間：2018-07-15 14:43
【摘要】：目的探討轉(zhuǎn)錄因子Krüppel樣因子5(Krüppel-like Factors 5,KLF5)和腫瘤壞死因子受體超家族成員11a(Tumor Necrosis Factor Receptor Superfamily 11a,TNFRSF11a)在人宮頸鱗癌組織中的表達并揭示其促進人宮頸癌細(xì)胞增殖、遷移和侵襲的分子與細(xì)胞生物學(xué)機制。方法收集就診于河北省唐山市工人醫(yī)院婦科門診和住院部的漢族女性的正常宮頸組織和病變宮頸組織作為本次實驗研究標(biāo)本。嚴(yán)格按照宮頸組織的納入標(biāo)準(zhǔn)和排除標(biāo)準(zhǔn),從2014年9月到2016年3月期間,共收集160例。所有納入實驗的宮頸組織均由實驗室研究人員與病理科醫(yī)師共同確認(rèn)病理類型和分級,包括:正常對照組47例、宮頸上皮內(nèi)瘤變組(Cervical Intraepithelial Neoplasia,CIN)73例(其中CINⅠ27例,CINⅡ-Ⅲ46例)和宮頸鱗癌組(Cervical Squmaous Cell Carcinoma,CSCC)40例。利用mRNA表達譜芯片篩查3例對照宮頸組織和3例人宮頸鱗癌組織中細(xì)胞應(yīng)答炎癥反應(yīng)相關(guān)基因的mRNA表達。通過實時定量反轉(zhuǎn)錄聚合酶鏈反應(yīng)(Quantitative Real-time Polymerase Chain Reaction,q RT-PCR)對芯片篩查結(jié)果進行驗證并分析KLF5和TNFRSF11a mRNA表達水平在不同病理人宮頸組織中的相關(guān)性。分析TNFRSF11a的mRNA表達與人宮頸鱗癌臨床病理參數(shù)的關(guān)系。采用免疫雙熒光染色法檢測不同病理人宮頸組織中KLF5和TNFRSF11a的共表達。在人宮頸癌He La細(xì)胞中,采用脂質(zhì)體轉(zhuǎn)染特異性si RNA分別敲低KLF5和TNFRSF11a的表達,構(gòu)建KLF5超表達腺病毒感染細(xì)胞過表達KLF5,應(yīng)用q RTPCR和Western blot分別檢測細(xì)胞內(nèi)相關(guān)基因的mRNA和蛋白水平變化。采用CCK-8實驗檢測不同處理條件下He La細(xì)胞的增殖功能的改變。采用Transwell實驗檢測不同處理條件下He La細(xì)胞的遷移和侵襲功能的改變。采用雙熒光素酶報告基因技術(shù)檢測轉(zhuǎn)錄因子KLF5對TNFRSF11a基因啟動子的轉(zhuǎn)錄活性并采用染色質(zhì)免疫沉淀(Chromatin Immunoprecipitation,Ch IP)分析KLF5在TNFRSF11a基因啟動子上的結(jié)合位點。結(jié)果1 mRNA表達譜芯片篩查結(jié)果顯示,與人正常宮頸組織相比,宮頸鱗癌組織中細(xì)胞應(yīng)答炎癥反應(yīng)的相關(guān)基因KLF5、Npnt、Socs5、TNFRSF11a、Cxcl13、Il6st、Cntfr、Plcb1表達均上調(diào)(P0.05)。2采用q RT-PCR實驗對芯片檢測結(jié)果進行驗證,結(jié)果顯示,宮頸鱗癌組織中KLF5、Npnt、Socs5、TNFRSF11a、Cxcl13、Il6st較正常組織表達均顯著上調(diào),差異有統(tǒng)計學(xué)意義(P0.05)。3免疫雙熒光染色檢測不同病理人宮頸組織中KLF5和TNFRSF11a共表達,結(jié)果顯示,KLF5和TNFRSF11a共表達于宮頸組織,且與對照組比較,在CINⅠ組、CINⅡ-Ⅲ組和CSCC組中的表達水平逐漸升高,熒光強度增加,差異有統(tǒng)計學(xué)意義(P0.05)。4采用q RT-PCR實驗檢測不同病理人宮頸組織中KLF5和TNFRSF11a的mRNA水平,結(jié)果顯示,與正常對照組相比,CINⅠ組、CINⅡ-Ⅲ組和CSCC組中KLF5和TNFRSF11a的mRNA表達水平均升高,且隨病理程度的加重而升高,差異均有統(tǒng)計學(xué)意義(P0.05);與CINⅡ-Ⅲ組相比,CSCC組中KLF5和TNFRSF11a的mRNA表達水平增高,差異有統(tǒng)計學(xué)意義(P0.05)。5采用Spearman法分析不同病理人宮頸組織中KLF5與TNFRSF11a mRNA表達的相關(guān)性,在正常對照組和CINⅠ組中KLF5與TNFRSF11a的mRNA表達無明顯相關(guān)性;在CINⅡ-Ⅲ組中KLF5與TNFRSF11a的mRNA表達呈正相關(guān)性(r=0.326,P=0.027);在CSCC組織中KLF5與TNFRSF11a的mRNA表達呈正相關(guān)(r=0.393,P=0.012)。6在He La細(xì)胞中分別過表達或敲低KLF5,Western blotting和q RT-PCR結(jié)果均顯示,在基礎(chǔ)水平和TNF-α誘導(dǎo)情況下,過表達或敲低KLF5均能夠顯著增加或降低TNFRSF11a的蛋白及mRNA水平(P0.05)。報告基因和Ch IP分析結(jié)果表明,KLF5通過直接與TNFRSF11a啟動子結(jié)合而激活TNFRSF11a基因表達。7基因表達與細(xì)胞功能獲得與缺失實驗結(jié)果表明:過表達KLF5可促進He La細(xì)胞的增殖、遷移及侵襲(P0.05);采用si RNA抑制TNFRSF11a的表達能夠抑制He La細(xì)胞的增殖、遷移及侵襲(P0.05);而敲低TNFRSF11a的表達同時過表達KLF5仍能夠部分抑制KLF5介導(dǎo)He La細(xì)胞增殖、遷移及侵襲的功能(P0.05)。8臨床參數(shù)相關(guān)分析結(jié)果表明:宮頸鱗癌組織中TNFRSF11a mRNA的表達與腫瘤的病理分化程度、肌層浸潤深度、臨床分期和淋巴結(jié)轉(zhuǎn)移有關(guān),差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論1人宮頸組織中KLF5和TNFRSF11a共表達促使宮頸鱗癌的發(fā)生、發(fā)展。2在宮頸癌細(xì)胞中KLF5可通過直接與TNFRSF11a基因啟動子相互作用激活TNFRSF11a基因的轉(zhuǎn)錄。3轉(zhuǎn)錄因子KLF5促進宮頸癌細(xì)胞的增殖、遷移和侵襲部分依賴于TNFRSF11a基因的表達。
[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.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33
本文編號：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.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33
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