【作者】 刘静；

【导师】 张廷国；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2023， 博士

【摘要】 研究背景子宫内膜癌是子宫内膜上皮来源的恶性肿瘤,是女性生殖系统三大恶性肿瘤之一,好发于绝经后及围绝经期女性。近年来子宫内膜癌发病率逐年上升并趋向年轻化。在我国,子宫内膜癌发病率位居第二位,但是近年来在发达城市其发病率已超过宫颈癌而位居妇科恶性肿瘤首位。子宫内膜样癌占子宫内膜癌的大多数,早期子宫内膜样癌5年存活率较高,晚期病例5年生存率较低。子宫内膜样癌手术及放化疗后,复发转移率仍较高。针对以上情况,深入系统地探讨子宫内膜样癌生物学行为的发生、发展及浸润转移机制,寻找新的治疗靶点及切实有效的诊断和防治方法,具有非常重要的临床意义。亲环素(cyclophilin,Cyp)又名 PPI(Peptidyl proline trans isomerase),是亲免蛋白家族成员,属于免疫因子,具有肽基脯氨基顺反异构酶(PPIase)活性,对体内钙离子调控、蛋白质的正确折叠、装配和转运以及信号转导起着重要调节作用。CypB广泛分布于多种细胞和器官系统中,参与了蛋白质折叠、内质网应激反应、氧化还原调节反应及核糖体合成等多种进程,同时还是环孢素A、FK506、雷帕霉素等免疫抑制药物在细胞内的主要结合蛋白。同时,CypB与多种肿瘤的发病机制亦具有相关性,证实了 CypB与乳腺癌、胃癌、肠癌、胰腺癌、肝癌、胶质母细胞瘤、肺癌等肿瘤的发生发展均密切相关。迄今为止,CypB与子宫内膜样癌之间的关联尚未见报道。故本研究对以下问题进行探讨:①CypB在子宫内膜样癌中的表达特征如何?②其表达与子宫内膜样癌的预后是否有关联?③CypB对子宫内膜样癌细胞的生物学特性是否有影响?④如有影响,其影响的机制是什么?研究方法1、本研究通过免疫组化检测子宫内膜样癌(endometrial endometrioid cancer,EEC)组织标本、子宫内膜不典型增生(Endometrial atypical hyperplasia,EAH)组织标本及正常子宫内膜(Normal endometrium,NE)组织标本中CypB的表达情况,分析CypB表达与EEC临床病理特点(年龄、浸润深度、分化程度、淋巴结转移及临床分期)之间的关系。随后利用RT-qPCR检测子宫内膜样癌及癌旁组织中CypB mRNA的表达水平,并利用Kaplan-Meier统计法绘制生存曲线,采用Log-Rank检验对CypB表达与子宫内膜样癌患者总体生存率进行统计分析,探讨CypB表达与子宫内膜样癌患者生存率之间的关系。2、利用RT-qPCR和Western Blot进一步验证不同子宫内膜癌细胞系CypB的表达水平。利用JASPAR数据库、文献检索及RT-qPCR检测初步探索子宫内膜癌中CypB表达升高之原因。3、为验证CypB对子宫内膜癌细胞生物学特性的影响,进行体外细胞感染慢病毒以敲减CypB。应用CCK-8、平板克隆和BrdU实验检测敲低CypB表达后子宫内膜癌细胞增殖能力的变化;划痕和Transwell小室实验检测转染CypB后子宫内膜癌细胞的迁移和侵袭能力的变化;Hoechst染色、Western Blot及Caspase-Glo 3/7Assay细胞凋亡试剂盒检测抑制CypB后子宫内膜癌细胞的凋亡情况;流式细胞仪检测敲低CypB后子宫内膜癌细胞周期进程变化。4、构建以慢病毒为载体的敲低CypB表达的稳转子宫内膜癌细胞系并扩增子宫内膜癌细胞。通过皮下注射的方法将感染慢病毒的子宫内膜癌细胞注射到裸鼠体内。观察裸鼠皮下成瘤情况并分析CypB对体内对子宫内膜癌细胞增殖、侵袭及凋亡的影响。5、对慢病毒感染CypB的子宫内膜癌细胞及阴性对照组进行mRNA基因表达谱检测,筛选出影响显著的差异表达基因,对差异基因进行GO和KEGG信号通路富集分析,得到可能受CypB调控的下游信号通路。应用RT-qPCR及Western blot检测敲低CypB后子宫内膜癌细胞中筛选出的信号通路相关基因的表达情况。免疫组化验证差异显著的基因在子宫内膜样癌组织样本中的表达及其与CypB的相关性。6、体外条件下验证CypB影响子宫内膜癌的分子机制。应用CCK-8、平板克隆和BrdU实验检测同时敲低CypB及P21后子宫内膜癌细胞增殖能力的变化;划痕和Transwell小室实验检测转染LV-CypB-KD2及si-P21后子宫内膜癌细胞的迁移和侵袭能力的变化;Hoechst染色、Western Blot及Caspase-Glo 3/7Assay细胞凋亡试剂盒检测转染LV-CypB-KD2及si-P21后子宫内膜癌细胞的凋亡情况;流式细胞仪检测同时抑制CypB和P21后子宫内膜癌细胞周期进程变化。RT-qPCR和Western Blot实验进一步验证CypB调控P21的分子机制。结果1、CypB在子宫内膜样癌中高表达,与肿瘤分化程度呈显著正相关免疫组化结果显示CypB在NE、EAH及EEC中的阳性表达率依次升高,并存在显著差异。RT-qPCR结果表明CypB在子宫内膜样癌中的表达显著高于癌旁组织,且CypB的表达强度与子宫内膜样癌的分化程度呈明显正相关,但与患者年龄、浸润深度、淋巴结转移、临床分期及总体生存率无显著相关性。统计结果还显示脉管内癌栓及临床分期与患者总体生存率相关,而浸润深度与总体生存率无关。Cox比例风险回归模型显示,单因素分析中,肿瘤分化程度及FIGO分期影响子宫内膜样癌患者总体生存期,可以作为预测子宫内膜样癌患者预后的因子;多因素分析中,肿瘤分化程度及FIGO分期仍具有较高的相对危险度,是影响患者预后的独立因素。2、转录因子ATF6调控子宫内膜癌细胞中CypB的表达RT-qPCR及Western Blot结果表明HEC-1-B、Ishikawa子宫内膜癌细胞均具有较高的CypB表达水平;JASPAR数据库预测转录因子ATF6可调控CypB的表达;子宫内膜癌细胞中抑制AFT6后CypB表达显著降低。3、CypB促进子宫内膜癌细胞增殖、迁移、侵袭,同时抑制肿瘤细胞凋亡HEC-1-B、Ishikawa 子宫内膜癌细胞转染 LV-CypB RNAi 后,RT-qPCR 及 Western blot显示CypB表达明显降低;CCK-8增殖实验、细胞克隆实验及BrdU染色显示肿瘤细胞增殖能力显著下降;细胞划痕实验及Tanswell实验表明肿瘤细胞迁移及侵袭能力降低,而Hoechst染色、Caspase3/7活性检测及Western Blot检测BCL2和BAX的表达显示肿瘤细胞的凋亡增加。4、在子宫内膜癌中,CypB促进肿瘤的生长和浸润而抑制其凋亡构建小鼠子宫内膜癌动物模型,抑制肿瘤CypB的表达,Ki67免疫组化显示肿瘤生长能力显著降低;HE染色观察到肿瘤细胞的侵袭能力明显降低;Western Blot显示抑制CypB后Caspase-3及Caspase-7表达增加,表明肿瘤凋亡增加。5、下调CypB对PI3K-AKT信号通路有抑制作用全基因表达微阵列芯片检测显示敲低CypB共鉴定出1536个差异表达显著的mRNAs(倍数变化>1);KEGG信号通路分析显示,差异基因在代谢相关通路、PI3K-AKT、细胞周期及FOXO等通路中富集;抑制CypB的表达后,RT-qPCR显示子宫内膜癌细胞中PI3K、AKT、CDK1、VEGFA基因表达下调,P21基因表达上调;Western Blot发现p-PI3K、PI3K、p-AKT、AKT、CDK1、VEGFA 表达下调,P21 表达上调,提示下调CypB对PI3K-AKT信号通路有抑制作用。6、CypB通过P21调节子宫内膜癌细胞生物学功能免疫组化结果显示,P21在NE、EAH及EEC中的阳性表达率依次降低,在组织样本中P21的表达与CypB的表达趋势相反。在子宫内膜癌细胞中抑制CypB的基础上抑制P21表达,较单纯抑制CypB相比,子宫内膜癌细胞的增殖、迁移、侵袭能力增加,而细胞的凋亡减低,提示CypB通过P21调节子宫内膜癌细胞生物学功能。7、CypB通过STAT5调控P21的表达Western Blot结果显示,子宫内膜癌细胞中,敲低CypB后p-STAT5的蛋白水平显著降低;抑制HEC-1-B中的STAT5后,P21 mRNA及蛋白水平均显著增加;当过表达CypB并抑制STAT5时,较单纯过表达CypB相比,P21表达显著增加。上述实验表明,CypB可通过STAT5调控P21的表达。结论1.在子宫内膜样癌中,CypB表达增加,并与肿瘤分化程度呈正相关;2.转录因子ATF6参与调控CypB表达;3.体内动物和体外细胞学实验均表明CypB的表达与子宫内膜癌细胞的增殖、迁移能力、侵袭能力呈正相关,而与肿瘤细胞的凋亡呈负相关;4.CypB通过STAT5的磷酸化抑制P21的表达进而调节子宫内膜癌的生物学功能。更多还原

【Abstract】 BackgroundEndometrial cancer is a malignant tumor of endometrial epithelial origin,which is one of the three major malignant tumors of the female reproductive system and occurs in postmenopausal and perimenopausal women.In recent years,the incidence of endometrial cancer has been increasing year by year and tends to be younger.In China,the incidence of endometrial cancer ranks second,but in recent years,its incidence has surpassed that of cervical cancer in developed cities and ranks first in gynecological malignancies.Endometrioid carcinoma accounts for the majority of endometrial cancers,with a high 5-year survival rate in early endometrioid carcinomas and a low 5-year survival rate in advanced cases.After surgery and chemoradiotherapy for endometrioid carcinoma,the recurrence and metastasis rate remains high.In view of the above conditions,it is of great clinical significance to deeply and systematically investigate the occurrence,development,invasion and metastasis mechanism of the biological behavior of endometrioid carcinoma,find new therapeutic targets and practical and effective diagnosis and prevention methods.Cyclophilin(Cyp),also known as PPI(Peptidyl proline trans isomerase),is a member of the immunophilin family and belongs to immune factors with peptidyl prolyl cis-trans isomerase(PPIase)activity,which plays an important role in calcium regulation,correct folding,assembly and transport of proteins,and signal transduction in vivo.CypB is widely distributed in a variety of cell and organ systems and is involved in various processes such as protein folding,ER stress response,redox regulation response and ribosome synthesis,while it is also the main binding protein of immunosuppressive drugs such as cyclosporine A,FK506,and rapamycin in cells.At the same time,CypB is also associated with the pathogenesis of a variety of tumors,confirming that CypB is closely related to the occurrence and development of breast cancer,gastric cancer,intestinal cancer,pancreatic cancer,liver cancer,glioblastoma,lung cancer and other tumors.So far,the association between CypB and endometrioid carcinoma has not been reported.Therefore,this study investigated the following questions:①What are the expression characteristics of CypB in endometrioid carcinoma?②Is there an association between its expression and the prognosis of endometrioid carcinoma?③Does CypB have an effect on the biological characteristics of endometrioid carcinoma cells?④If there is an effect,what is the mechanism of its effect?Methods1.In this study,we detected the expression of CypB in endometrial endometrioid carcinoma(EEC)tissue samples,endometrial dysplasia(EAH)tissue samples and normal endometrium(NE)tissue samples by immunohistochemistry,and analyzed the relationship between CypB expression and clinicopathological characteristics(age,depth of invasion,differentiation,lymph node metastasis and clinical stage)of EEC.Subsequently,RT-qPCR was used to detect the expression levels of CypB mRNA in endometrioid carcinoma and adjacent non-cancerous tissues,and Kaplan-Meier statistics was used to draw survival curves,and Log-Rank test was used to statistically analyze CypB expression and the overall survival rate of patients with endometrioid carcinoma to investigate the relationship between CypB expression and the survival rate of patients with endometrioid carcinoma.2.RT-qPCR and Western blot were used to further validate the expression levels of CypB in different endometrial cancer cell lines.JASPAR database,literature search and RT-qPCR were used to preliminarily explore the causes of elevated CypB expression in endometrial cancer.3.To verify the effect of CypB on the biological characteristics of endometrial cancer cells,in vitro cell infection with lentivirus was performed to knockdown CypB.CCK-8,plate cloning and BrdU assay were used to detect the changes of proliferation ability of endometrial carcinoma cells after knockdown of CypB expression;scratch and transwell chamber assay were used to detect the changes of migration and invasion ability of endometrial carcinoma cells after transfection of CypB;Hoechst staining,Western Blot and caspase-Glo 3/7 Assay apoptosis kit were used to detect the apoptosis of endometrial carcinoma cells after inhibition of CypB;flow cytometry was used to detect the changes of cycle progression of endometrial carcinoma cells after knockdown of CypB.4.Stable transfer endometrial carcinoma cell lines and amplicon endometrial carcinoma cells with lentiviral knockdown of CypB expression were constructed.Lentivirus-infected endometrial cancer cells were injected subcutaneously into nude mice.To observe the subcutaneous tumor formation in nude mice and analyze the effect of CypB on the proliferation,invasion and apoptosis of endometrial carcinoma cells in vivo.5.Endometrial cancer cells infected with lentiviral CypB and negative controls were examined for mRNA gene expression profiles to select differentially expressed genes with significant effects,and GO and KEGG signaling pathway enrichment analysis of differential genes was performed to obtain downstream signaling pathways that may be regulated by CypB.RT-qPCR and Western blot were used to detect the expression of selected signaling pathway-related genes in endometrial cancer cells after knockdown of CypB.Immunohistochemistry was performed to validate the expression of genes with significant differences in endometrioid carcinoma tissue samples and their association with CypB.6.To verify the molecular mechanism of CypB affecting endometrial cancer under in vitro conditions.CCK-8,plate cloning and BrdU assay were used to detect the changes of proliferation ability of endometrial carcinoma cells after simultaneous knockdown of CypB and P21;scratch and transwell chamber assay were used to detect the changes of migration and invasion ability of endometrial carcinoma cells after transfection with LV-CypB-KD2 and si-P21;Hoechst staining,Western Blot and Caspase-Glo 3/7 Assay apoptosis kit were used to detect the apoptosis of endometrial carcinoma cells after transfection with LV-CypB-KD2 and si-P21;flow cytometry was used to detect the changes of cycle progression of endometrial carcinoma cells after simultaneous inhibition of CypB and P21.RT-qPCR and Western Blot experiments were performed to further validate the molecular mechanism of P21 regulation by CypB.Results1.CypB is highly expressed in endometrial endometrioid cancerand is significantly positively correlated with tumor differentiationThe immunohistochemical results showed that the positive expression rates of CypB in NE,EAH and EEC increased in turn,and there were significant differences.RT-qPCR results showed that the expression of CypB in endometrioid carcinoma was significantly higher than that in adjacent non-cancerous tissues,and the expression intensity of CypB was significantly positively correlated with the differentiation of endometrioid carcinoma,but not significantly correlated with patient age,depth of invasion,lymph node metastasis,clinical stage and overall survival rate.The statistical results also showed that intravascular tumor thrombus and clinical stage were associated with overall survival,while depth of invasion was not associated with overall survival.Cox proportional hazards regression model showed that in univariate analysis,tumor differentiation and FIGO stage affected the overall survival of patients with endometrioid carcinoma and could be used as factors to predict the prognosis of patients with endometrioid carcinoma;in multivariate analysis,tumor differentiation and FIGO stage still had a high relative risk and were independent factors affecting the prognosis of patients.2.Transcription factor ATF6 regulates CypB expression in endometrial carcinoma cellsRT-qPCR and Western blot results showed that HEC-1-B and Ishikawa endometrial carcinoma cells had high CypB expression levels;the JASPAR database predicted that the transcription factor ATF6 could regulate the expression of CypB;CypB expression was significantly decreased after inhibition of AFT6 in endometrial carcinoma cells.3.CypB promotes endometrial cancer cell proliferation,migration,and invasion,while inhibiting tumor cell apoptosisAfter HEC-1-B and Ishikawa endometrial carcinoma cells were transfected with LV-CypB RNAi,RT-qPCR and Western Blot showed that CypB expression was significantly decreased;CCK-8 proliferation assay,cell cloning assay and BrdU staining showed that tumor cell proliferation was significantly decreased;cell scratch assay and Tanswell assay showed that tumor cell migration and invasion were decreased,while Hoechst staining,Caspase3/7 activity assay and Western Blot detection of BCL2 and BAX expression showed that tumor cell apoptosis was increased.4.In endometrial cancer,CypB promotes tumor growth and invasion while inhibiting their apoptosisA mouse model of endometrial carcinoma was established to inhibit the expression of CypB,Ki67 immunohistochemistry showed that the tumor growth ability was significantly reduced;HE staining observed that the invasion ability of tumor cells was significantly reduced;Western Blot showed that the expression of Caspase-3 and Caspase-7 was increased after inhibiting CypB,indicating that tumor apoptosis was increased.5.Downregulation of CypB has an inhibitory effect on PI3K-AKT signaling pathwayWhole gene expression microarray microarray detection showed that a total of 1536 mRNAs with significant differential expression(fold change>1)were identified by knockdown of CypB;KEGG signaling pathway analysis showed that differential genes were enriched in metabolism-related pathways,PI3K-AKT,cell cycle and FOXO pathways;after inhibiting the CypB,RT-qPCR showed that PI3K,AKT,CDK1,and VEGFA gene expression was down-regulated and P21 gene expression was up-regulated in endometrial cancer cells;Western Blot revealed that p-PI3K,PI3K,p-AKT,AKT,CDK1,and VEGFA expression was down-regulated and p21 expression was up-regulated,suggesting that down-regulation of CypB inhibited PI3K-AKT signaling pathway.6.CypB Regulates Biological Functions of Endometrial Cancer Cells via P21The immunohistochemical results showed that the positive expression rates of p21 in NE,EAH and EEC decreased in turn,and the expression trend of p21 in tissue samples was opposite to that of CypB.Inhibition of CypB on the basis of inhibition of P21 expression in endometrial cancer cells,compared with inhibition of CypB alone,endometrial cancer cells proliferation,migration,invasion increased,while apoptosis decreased,suggesting that CypB regulates biological functions of endometrial cancer cells via P21.7.CypB regulates expression of p21 through STAT5Western Blot results showed that in endometrial carcinoma cells,the protein level of p-STAT5 was significantly decreased after knockdown of CypB;after inhibition of STAT5 in HEC-1-B,both P21 mRNA and protein levels were significantly increased;when CypB was overexpressed and STAT5 was inhibited,P21 expression was significantly increased compared with overexpression of CypB alone.The above experiments showed that CypB could regulate the expression of p21 through STAT5.Conclusion1.In endometrial endometrioid cancer,CypB expression is increased and positively correlated with tumor differentiation;2.Transcription factor ATF6 is involved in the regulation of CypB expression;3.In vivo animal and in vitro cytological experiments showed that the expression of CypB was positively correlated with the proliferation,migration and invasion of endometrial carcinoma cells,but negatively correlated with the apoptosis of tumor cells;4.CypB inhibits the expression of P21 through the phosphorylation of STAT5 and then regulates the biological function of endometrial cancer.更多还原