【作者】 王乐；

【导师】 谢勇；

【作者基本信息】 南昌大学 ， 临床医学/内科学/消化系病， 2023， 博士

【摘要】 研究背景及目的:胰腺癌不断增长的发病率和居高不下的病死率使其成为公共卫生领域的重大挑战。胰腺癌发生发展的机制是当前的研究热点,这一领域的研究有助于寻找用于诊断的标志物和新的治疗靶点。FOXP1作为转录因子,可通过影响不同的下游分子的转录,在不同的肿瘤起促癌或抑癌作用。FOXP1还可在多种肿瘤和正常细胞中影响细胞增殖。此外,生物信息学预测显示FOXP1有数以千计的潜在靶基因,其中许多调控着细胞增殖与凋亡,并在肿瘤的发生发展起重要作用。虽然FOXP1在胰腺癌中的作用尚不明确,但是细胞的增殖与凋亡异常在胰腺癌发生发展中起关键作用。因此,我们推测,FOXP1可能通过影响细胞增殖相关生物学行为在胰腺癌的发生发展中起作用,但是它在胰腺癌中起抑癌作用还是促癌作用目前尚不清楚,在胰腺癌中它的下游效应分子也不清楚。转录因子IRF1在多种细胞中被证明是一个增殖抑制因子,同时,它在胰腺癌中是一个抑癌基因,其高表达与较好预后相关,但IRF1在胰腺癌中是否抑制细胞增殖尚不清楚,IRF1与FOXP1的关系也未见报道。本文研究了FOXP1在胰腺癌中表达及其与胰腺癌生物学行为的关系,并深入研究了机制,证明了FOXP1在胰腺癌中通过与IRF1启动子特定区域结合调控IRF1转录水平,进而影响细胞增殖相关生物学行为,FOXP1在胰腺癌中是一个抑癌基因。材料方法:(一)FOXP1在体内外与胰腺癌细胞增殖以及胰腺癌生长和预后的关系1.临床标本中分析FOXP1与胰腺癌大小和预后的关系:采用免疫组化染色检测了72对胰腺癌及其癌旁组织中FOXP1的表达,并分析二者之间的差异,及FOXP1表达与患者总生存期和肿瘤最大直径之间的关系。2.胰腺癌细胞系的选择:通过Western blotting检测人正常胰腺导管上皮细胞系HPDE及4种常用人胰腺癌细胞系CFPAC1,SW1990,PANC1和CAPAN1中FOXP1的蛋白表达水平;通过CCLE数据库检索比较SW1990,PANC1细胞与所有其他胰腺癌细胞系的FOXP1 m RNA表达水平。3.FOXP1对胰腺癌细胞系增殖相关生物学行为影响的检测:采用FOXP1过表达质粒瞬时转染PANC1和SW1990细胞,通过CCK8观察细胞增殖,通过PI染色+流式细胞术检测细胞周期,PI/Annexin V双染+流式细胞术检测细胞凋亡。4.胰腺癌裸鼠模型构建及FOXP1对肿瘤生长和增殖凋亡的影响:通过pc DNA3.1(+)-FOXP1转染+G418筛选构建稳定表达FOXP1的PANC1细胞系,通过腋下皮下注射稳转细胞系构建裸鼠成瘤模型,通过游标卡尺测量和瘤体称重评估肿瘤生长,通过HE染色和FOXP1免疫组化鉴定模型,通过Ki67免疫组化和TUNEL染色评估细胞增殖和凋亡。(二)FOXP1通过IRF1影响胰腺癌细胞增殖相关生物学行为1.预测和筛选FOXP1可能的下游效应分子:使用TCGA数据库、GTRD和GSEA数据库预测得到FOXP1可能的下游效应分子,通过q PCR筛选得到候选下游效应分子IRF1,通过Western blotting检测FOXP1过表达对PANC1和SW1990细胞IRF1蛋白表达水平的影响,通过裸鼠皮下瘤免疫组化染色检测FOXP1过表达在体内对胰腺癌细胞IRF1蛋白表达水平的影响;2.FOXP1通过IRF1影响胰腺癌细胞增殖相关生物学行为:通过FOXP1过表达+IRF1敲减的rescue实验处理PANC1和SW1990细胞,通过CCK8观察细胞增殖,通过PI染色+流式细胞术检测细胞周期,PI/Annexin V双染+流式细胞术检测细胞凋亡。3.IRF1对胰腺癌细胞CDK2表达以及细胞增殖相关生物学行为的影响:采用IRF1过表达质粒瞬时转染处理PANC1和SW1990细胞,通过Western blotting检测CDK2表达,通过CCK8观察细胞增殖,通过PI染色+流式细胞术检测细胞周期,PI/Annexin V双染+流式细胞术检测细胞凋亡。通过裸鼠皮下瘤免疫组化染色检测比较FOXP1过表达组和对照组的CDK2蛋白表达水平。(三)FOXP1在IRF1启动子区域结合位点的确定通过JASPAR网站预测FOXP1在IRF1启动子区域的结合位点;通过Ch IP-PCR和Ch IP-q PCR确认FOXP1在IRF1启动子区域的结合位点;通过双萤光素酶报告基因实验检测野生型和结合位点突变型启动子应答于FOXP1并激活下游基因转录的能力。结果:(一)FOXP1在体内外与胰腺癌细胞增殖以及胰腺癌生长和预后的关系1.FOXP1表达与胰腺癌患者肿瘤大小以及总生存期的关系:72对胰腺癌及其癌旁组织免疫组化染色结果显示FOXP1在癌组织中的表达显著低于癌旁组织(P<0.001);根据FOXP1免疫组化染色评分的中位数值将72例癌组织分为FOXP1高表达组和低表达组,K-M检验显示FOXP1高表达组的OS显著优于低表达组(P<0.001);对72例癌组织的FOXP1表达水平和肿瘤大小进行了相关性分析,结果显示两者呈负相关(P<0.0001)。根据以上结果,我们推测,在胰腺癌中FOXP1可能是一个抑癌基因。2.胰腺癌细胞系的选择:Western blotting检测人正常胰腺导管上皮细胞系HPDE和4种常用人胰腺癌细胞系CFPAC1,SW1990,PANC1以及CAPAN1中FOXP1的蛋白表达水平,结果显示,所有细胞系的FOXP1蛋白表达水平均存在统计学差异(P<0.01),各细胞系两两比较则显示,FOXP1在SW1990和PANC1细胞中的表达水平均显著低于其他胰腺癌细系和HPDE细胞(P<0.01)。CCLE数据库数据也显示,在所有胰腺癌细胞系中,SW1990和PANC1细胞的FOXP1表达处于较低水平,我们决定选择这两个细胞系进行后续实验。3.FOXP1影响了胰腺癌细胞系的增殖相关生物学行为:采用FOXP1过表达质粒瞬时转染PANC1和SW1990细胞,Western blotting验证了FOXP1过表达的效果(P<0.01),FOXP1过表达显著抑制细胞增殖(P<0.01),导致细胞周期G1期阻滞(P<0.01),并显著促进细胞凋亡(P<0.01)。4.FOXP1在裸鼠模型中影响了胰腺癌生长等生物学行为:胰腺癌裸鼠模型构建及FOXP1对肿瘤生成和增殖凋亡的影响:oe FOXP1/Vector-PANC1细胞皮下接种裸鼠后成功成瘤,HE染色表明动物模型构建成功;oe FOXP1-PANC1细胞接种裸鼠的肿瘤体积和重量显著低于Vector-PANC1细胞接种裸鼠(P<0.01),免疫组化显示FOXP1在oe FOXP1-PANC1细胞接种的肿瘤内表达显著高于Vector-PANC1细胞接种的肿瘤(P<0.01),细胞增殖的标志物Ki67免疫组化显示oe FOXP1-PANC1细胞接种组肿瘤内增殖细胞百分比显著低于Vector-PANC1细胞接种组肿瘤(P<0.01);TUNEL染色表明oe FOXP1-PANC1细胞接种的肿瘤内细胞凋亡显著高于Vector-PANC1细胞接种的肿瘤(P<0.01)。以上结果表明FOXP1可抑制胰腺癌的生长和细胞增殖,并促进细胞凋亡。(二)FOXP1通过调控IRF1表达进而影响胰腺癌细胞增殖相关生物学行为1.预测和筛选FOXP1可能的下游效应分子:使用TCGA数据库、GTRD和GSEA数据库预测得到FOXP1可能的下游效应分子IRF1和CDKN1A,FOXP1过表达显著促进了PANC1和SW1990细胞IRF1 m RNA的表达(P<0.01),但对CDKN1A m RNA的表达水平无显著影响(p>0.05);FOXP1过表达显著促进PANC1和SW1990细胞IRF1蛋白表达(P<0.01),裸鼠皮下瘤免疫组化染表明FOXP1过表达在体内显著促进了胰腺癌细胞IRF1蛋白表达(P<0.01)。以上结果表明IRF1可能是FOXP1下游效应分子。2.FOXP1通过IRF1影响胰腺癌细胞增殖相关生物学行为:(1)通过FOXP1过表达+IRF1敲减的rescue实验处理PANC1和SW1990细胞,q PCR和Western blotting检测表明:在PANC1和SW1990两个细胞中,FOXP1过表达显著促进了IRF1蛋白和m RNA表达(si NC+oe FOXP1 vs.si NC+Vector,P<0.01);而IRF1敲减则显著逆转了FOXP1过表达引起的IRF1蛋白和m RNA表达水平上升(si IRF1+oe FOXP1 vs.si NC+oe FOXP1,P<0.01);提示我们的rescue实验模型构建是成功的。(2)进一步研究FOXP1对IRF1的调控对胰腺癌生物学行为的影响,结果表明在PANC1和SW1990两个细胞中,FOXP1过表达显著抑制细胞增殖(si NC+oe FOXP1 vs.si NC+Vector,P<0.01)、引起细胞周期G1期阻滞(si NC+oe FOXP1 vs.si NC+Vector,P<0.01)并促进细胞凋亡(si NC+oe FOXP1 vs.si NC+Vector,P<0.01);IRF1敲减则显著逆转了FOXP1过表达引起的细胞增殖抑制(si IRF1+oe FOXP1 vs.si NC+oe FOXP1,P<0.01)、细胞周期G1期阻滞(si IRF1+oe FOXP1 vs.si NC+oe FOXP1,P<0.01)和细胞凋亡(si IRF1+oe FOXP1 vs.si NC+oe FOXP1,P<0.01),证明FOXP1通过IRF1造成了以上生物学效应。3.IRF1对胰腺癌细胞CDK2表达以及细胞增殖相关生物学行为的影响:IRF1过表达显著促进了PANC1和SW1990细胞CDK2蛋白表达(P<0.01),同时显著抑制了细胞增殖、导致细胞周期G1期阻滞和并促进细胞凋亡(P<0.01)。裸鼠皮下瘤免疫组化染色表明FOXP1过表达组CDK2蛋白表达水平显著高于对照组(P<0.01)。(三)FOXP1通过与IRF1启动子区特定序列结合激活其转录1.通过JASPAR网站预测FOXP1在IRF1启动子区的结合位点,位点1位于-717~-702,位点2位于-552~-540;在PANC1细胞中进行Ch IP-PCR和Ch IP-q PCR实验,表明FOXP1与两个位点都有结合,但主要与位点1结合(P<0.01);双萤光素酶报告基因实验表明,结合位点1突变型启动子完全丧失了应答于FOXP1并激活下游基因转录的能力(p>0.05),结合位点2突变型启动子部分丧失了应答于FOXP1并激活下游基因转录的能力(P<0.01),进一步表明两个位点对于FOXP1转录激活IRF1都很重要,但位点1更为重要。结论:1.FOXP1在胰腺癌中起抑癌作用,并影响肿瘤的生长与预后。2.FOXP1通过调控IRF1,进而影响胰腺癌的增殖凋亡等生物学行为。3.FOXP1主要与IRF1启动子区域的-717~-702位点和-552~-540位点结合,进而激活IRF1的转录。更多还原

【Abstract】 Background and purpose :The continuously increasing incidence and high mortality rate of pancreatic cancer make it a major challenge in the field of public health.The mechanism of pancreatic cancer initiation and development is currently a research hotspot,and research in this field can help identify diagnostic markers and new therapeutic targets.As a transcription factor,FOXP1 can initiate transcription of different downstream molecules and play different roles in different tumors,both promoting and inhibiting cancer.Bioinformatics predictions show that FOXP1 has thousands of potential target genes,many of which play important roles in cell proliferation and apoptosis in the development of tumors.Although the role of FOXP1 in pancreatic cancer remains to be elucidated currently,abnormal cell proliferation and apoptosis plays a key role in the initiation and development of pancreatic cancer.Therefore,we hypothesize that FOXP1 plays a role in the initiation and development of pancreatic cancer,but it is unclear whether it has a tumor suppressive or tumor promoting role in pancreatic cancer,and its downstream effector molecules in pancreatic cancer are also unclear.To address these questions,we studied the expression of FOXP1 in pancreatic cancer and its association with the biological behavior of pancreatic cancer,and investigated the underlying mechanisms,in order to provide new targets for the prevention and treatment of pancreatic cancer.Materials and methods:(1)The association between FOXP1 expression and pancreatic cancer cell proliferation,growth,and prognosis in vivo and in vitro:Analysis of the association between FOXP1 and pancreatic cancer size and prognosis in clinical specimens: Immunohistochemical staining was used to detect FOXP1 expression in 72 pairs of pancreatic cancer and adjacent tissues,and the differences between them were analyzed,as well as the association between FOXP1 expression and overall survival and maximum tumor diameter of the patients.Selection of pancreatic cancer cell lines: FOXP1 protein expression levels in human normal pancreatic ductal epithelial cell line HPDE and four commonly used human pancreatic cancer cell lines CFPAC1,SW1990,PANC1,and CAPAN1 were detected by Western blotting;FOXP1 m RNA expression levels in SW1990 and PANC1 cells were compared to all other pancreatic cancer cell lines using the CCLE database.Detection of the effects of FOXP1 on pancreatic cancer cell proliferation and other biological behaviors: FOXP1 overexpression plasmids were transiently transfected into PANC1 and SW1990 cells.Cell proliferation was observed using CCK8 assay,cell cycle was detected by PI staining and flow cytometry,and cell apoptosis was detected by PI/Annexin V double staining and flow cytometry.Construction of a pancreatic cancer nude mouse model and detection of the effects of FOXP1 on tumor growth,proliferation,and apoptosis: Stable FOXP1-expressing PANC1 cell lines were constructed by transfection with pc DNA3.1(+)-FOXP1+G418 screening.A nude mouse tumor model was established by subcutaneously injecting stable transfected cell lines into the axilla.Tumor growth was evaluated by caliper measurement and tumor weight.The model was identified by HE staining and FOXP1 immunohistochemistry,and cell proliferation and apoptosis were evaluated by Ki67 immunohistochemistry and TUNEL staining.(1)FOXP1 regulates biological behaviors of pancreatic cancer cells through IRF11.Prediction and screening of potential downstream effectors of FOXP1: FOXP1 downstream effectors were predicted using TCGA,GTRD,and GSEA databases,and candidate downstream effector IRF1 was screened by q PCR.The effect of FOXP1 overexpression on IRF1 protein expression was detected by western blotting in PANC1 and SW1990 cells,and the effect of FOXP1 overexpression in vivo on IRF1 protein expression was detected by immunohistochemistry in subcutaneous tumors of nude mice.2.FOXP1 regulates biological behaviors of pancreatic cancer cells through IRF1:PANC1 and SW1990 cells were treated with FOXP1 overexpression and IRF1 knockdown rescue experiments,and cell proliferation was observed using CCK8 assay,cell cycle was detected using PI staining and flow cytometry,and apoptosis was detected using PI/Annexin V double staining and flow cytometry.3.Effect of IRF1 on CDK2 expression and biological behaviors of pancreatic cancer cells: PANC1 and SW1990 cells were transiently transfected with IRF1 overexpression plasmids,and CDK2 expression was detected by western blotting.Cell proliferation was observed using CCK8 assay,cell cycle was detected using PI staining and flow cytometry,and apoptosis was detected using PI/Annexin V double staining and flow cytometry.The protein expression level of CDK2 was compared between the FOXP1 overexpression group and the control group by immunohistochemistry in subcutaneous tumors of nude mice.(2)Identification of FOXP1 binding sites in the IRF1 promoter regionThe binding sites of FOXP1 in the IRF1 promoter region were predicted using the JASPAR website.The binding sites of FOXP1 in the IRF1 promoter region were confirmed by Ch IP-PCR and Ch IP-q PCR.The ability of wild-type and binding site mutant promoters to respond to FOXP1 and activate downstream gene transcription was detected by dual luciferase reporter gene experiments.Results:(1)The association between FOXP1 expression,pancreatic cancer cell proliferation,growth and prognosis in vitro and in vivo:1.FOXP1 is down-regulated in pancreatic cancer and affects tumor growth and prognosis: Immunohistochemical staining of 72 pairs of pancreatic cancer and adjacent tissues showed that FOXP1 expression in cancer tissues was significantly lower than that in adjacent tissues(p < 0.001).Based on the median value of the FOXP1 immunohistochemical staining score,the 72 cancer tissues were divided into a high expression group and a low expression group.The Kaplan-Meier test showed that the OS of the FOXP1 high expression group was significantly better than that of the low expression group(p < 0.001).Correlation analysis between FOXP1 expression levels and tumor size in 72 cancer tissues showed a negative correlation between the two(p < 0.001).Based on these results,we speculate that FOXP1 may be a tumor suppressor gene in pancreatic cancer.2.Selection of pancreatic cancer cell lines: Western blotting was used to detect FOXP1 protein expression levels in the human normal pancreatic duct epithelial cell line HPDE and four commonly used human pancreatic cancer cell lines CFPAC1,SW1990,PANC1 and CAPAN1.The results showed that there was a statistically significant difference in FOXP1 protein expression levels among all cell lines(p < 0.05).Comparisons between each pair of cell lines showed that FOXP1 expression levels were significantly lower in SW1990 and PANC1 cells than in other pancreatic cancer cell lines and HPDE cells(p < 0.05).CCLE database data also showed that FOXP1 expression levels were relatively low in SW1990 and PANC1 cells among all pancreatic cancer cell lines.Therefore,we decided to use these two cell lines for further experiments.3.FOXP1 affects the biological behavior of pancreatic cancer cell lines such as proliferation: FOXP1 overexpression plasmids were transiently transfected into PANC1 and SW1990 cells,and Western blotting was used to verify the overexpression effect of FOXP1(p < 0.05).FOXP1 overexpression significantly inhibited cell proliferation(p < 0.05),caused G1 phase arrest of the cell cycle(p < 0.05),and significantly promoted cell apoptosis(p < 0.05).4.FOXP1 affects the biological behavior of pancreatic cancer cells in nude mouse models: Construction of nude mouse models of pancreatic cancer and the effect of FOXP1 on tumor generation and proliferation and apoptosis: oe FOXP1/Vector-PANC1 cells were subcutaneously implanted into nude mice,and tumors were successfully formed,as confirmed by HE staining.The tumor volume and weight of the oe FOXP1-PANC1 cell group were significantly lower than those of the Vector-PANC1 cell group(p < 0.05).Immunohistochemical staining showed that FOXP1 expression in tumors formed by oe FOXP1-PANC1 cells was significantly higher than that in tumors formed by Vector-PANC1 cells(p < 0.05),while the expression of the proliferation marker Ki67 was the opposite(p < 0.05).TUNEL staining showed that apoptosis was significantly higher in tumors formed by oe FOXP1-PANC1 cells than in tumors formed by Vector-PANC1 cells(p < 0.05).These results indicate that FOXP1 can inhibit the growth and proliferation of pancreatic cancer cells and promote cell apoptosis.(2)FOXP1 affects pancreatic cancer cell proliferation and other biological behaviors by regulating the expression of IRF11.Prediction and screening of possible downstream effector molecules of FOXP1: Using TCGA,GTRD and GSEA databases,the possible downstream effector molecules of FOXP1,IRF1 and CDKN1 A,were predicted and FOXP1 overexpression significantly promoted the expression of IRF1 m RNA in PANC1 and SW1990 cells(P<0.01),but had no significant effect on the expression level of CDKN1 A m RNA(p>0.05).(p>0.05);FOXP1 overexpression significantly promoted IRF1 protein expression in PANC1 and SW1990 cells(P<0.01),and immunohistochemical staining of nude mice subcutaneous tumors showed that FOXP1 overexpression significantly promoted IRF1 protein expression in pancreatic cancer cells in vivo(P<0.01).The above results suggested that IRF1 may be a downstream effector molecule of FOXP1.2.FOXP1 affects biological behaviors such as proliferation of pancreatic cancer cells through IRF1:(1)PANC1 and SW1990 cells were treated by FOXP1 overexpression + IRF1 knockdown rescue assay,and q PCR and Western blotting assays showed that in both PANC1 and SW1990 cells,the expression of FOXP1 and IRF1 protein and m RNA was significantly higher in the oe FOXP1 group than in the corresponding Vector group(P<0.01),while the expression of FOXP1 protein and m RNA in the IRF1 knockdown group was not significantly different from its corresponding NC group regardless of whether FOXP1 was overexpressed or not(P>0.05)and the expression of IRF1 protein and m RNA in the IRF1 knockdown group was significantly lower than its corresponding NC group regardless of whether FOXP1 was overexpressed or not(P<0.01).The above results indicated that our rescue experimental model construction was successful.(2)Further study of the effect of FOXP1 regulation on IRF1 on biological behavior of pancreatic cancer showed that in both PANC1 and SW1990 cells with or without knockdown of IRF1,cell proliferation in the oe FOXP1 group was significantly lower than its corresponding Vector group(P<0.01)and apoptosis was significantly higher in the oe FOXP1 group than in the corresponding Vector group(P<0.05).The cell cycle assay also revealed that the proportion of G1-phase cells was significantly higher in the oe FOXP1 group than in the corresponding Vector group,regardless of whether IRF1 was knocked down(P<0.05).The proportion of cells in S phase was significantly higher in the oe FOXP1 group than in the corresponding Vector group(P<0.05).We also found that in both PANC1 and SW1990 cells,regardless of FOXP1 overexpression,cell proliferation was significantly higher in the IRF1 knockdown group than in the corresponding NC group(P<0.01),while apoptosis was significantly lower than in the corresponding NC group(P<0.05).and apoptosis was significantly lower than that in the corresponding NC group(P<0.05).The cell cycle assay also revealed that the proportion of G1 phase cells in the IRF1 knockdown group was significantly lower than that in the corresponding NC group,regardless of whether FOXP1 was overexpressed or not(P<0.05).The proportion of cells in S phase was significantly higher than that in the corresponding NC group(P<0.05).These phenomena were particularly evident in the oe FOXP1 group.The above results suggested that IRF1 knockdown significantly reversed the decrease in cell proliferation,cell cycle G1 phase arrest and increase in apoptosis caused by FOXP1 overexpression(P<0.05),demonstrating that FOXP1 caused the above biological effects through IRF1.3.Effect of IRF1 on CDK2 expression and cell proliferation in pancreatic cancer cells: IRF1 overexpression significantly promoted CDK2 protein expression in PANC1 and SW1990 cells(P<0.01),while significantly inhibiting cell proliferation,causing cell cycle G1 phase arrest and promoting apoptosis(P<0.01).Immunohistochemical staining of nude mice subcutaneous tumors showed that the expression level of CDK2 protein was significantly higher in the FOXP1 overexpression group than in the control group(P<0.01).(3)FOXP1 activates its transcription by binding to specific sequences in the promoter region of IRF11.The binding sites of FOXP1 in IRF1 promoter region were predicted by JASPAR website,site 1 was located at-717~-702 and site 2 was located at-552~-540;Ch IP-PCR and Ch IP-q PCR experiments in PANC1 cells showed that FOXP1 bound to both sites,but mainly to site 1(P<0.01);the double fluorophore reporter gene assay showed that the binding site 1 mutant promoter completely lost the ability to respond to FOXP1 and activate downstream gene transcription(p>0.05),and the binding site 2 mutant promoter partially lost the ability to respond to FOXP1 and activate downstream gene transcription(P<0.01),further indicating that both sites are important for FOXP1 transcriptional activation of IRF1 are important,but locus 1 is more important.Conclusions:1.FOXP1 plays an oncogenic role in pancreatic cancer and affects tumor growth and prognosis.2.FOXP1 affects the biological behaviors of pancreatic cancer,such as proliferation and apoptosis,through the regulation of IRF1.3.FOXP1 mainly binds to the-717~-702 sites and-552~-540 sites in the promoter region of IRF1,and then activates the transcription of IRF1.更多还原