【作者】 王丽萍；

【导师】 李艳茹；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2022， 硕士

【摘要】 紧密连接（Tight Junctions,TJs）位于细胞的侧面顶端,发挥“门”和“栅栏”功能。当其结构和功能的完整性遭到破坏时,会引起肿瘤发生转移。紧密连接蛋白（claudins,CLDNs）作为组成TJs的重要成分,其表达异常会破坏TJs的完整性,导致细胞极性消失,肿瘤细胞的迁移和侵袭能力增强。CLDN6作为CLDNs家族成员之一,其结构也是由一个羧基末端、一个氨基末端、四个跨膜区域和两个细胞外环共同组成的。其羧基末端富含多个酪氨酸磷酸化位点,可被酪氨酸激酶磷酸化。研究发现,CLDN6与Src家族酪氨酸激酶（Src family kinases,SFKs）相互作用,招募SFKs到Y196和Y200位点上,并使Y196/Y200位点发生磷酸化,SFKs-SH2与CLDN6上磷酸化的Y196/Y200的结合加强,从而完全激活SFKs,进而激活其下游信号通路。Src是SFKs家族的核心成员,可作为膜受体与细胞质信号机制之间建立联系,从而调节各种基本的细胞过程。Src包含多个结构域,其中催化结构域SH1包含酪氨酸磷酸化位点Y419,使Src发生自磷酸化,活化的Src可以激活信号传导与转录激活因子3（Signal transducer and activator of transcription 3,STAT3）信号通路。STAT3参与多种肿瘤的增殖分化、免疫逃避、血管新生及侵袭等生物调控过程。STAT3一方面为活化的受体和非受体酪氨酸激酶传递信号,另一方面发挥转录因子的作用。活化的STAT3形成同源二聚体或异源二聚体,从细胞质直接进入到细胞核,与基质金属蛋白酶（Matrix Metalloproteinases,MMPs）基因的启动子区结合促进其表达,进而促进肿瘤细胞的迁移和侵袭。目前,关于CLDN6与Src相互作用,激活Src/STAT3信号通路的研究还未见报道。目的:探讨CLDN6通过激活Src/STAT3信号通路影响肝癌细胞迁移和侵袭的作用机制,为CLDN6作为肝癌潜在治疗靶点的研究提供实验依据。方法:1.CLDN6和Src在人肝癌组织中的表达及其与临床病理参数的关系1.1 CLDN6和Src在人肝癌组织中的表达及其相关性TCGA数据库分析人肝癌组织中CLDN6和Src的表达并分析两者之间的相关性。1.2人肝癌组织中CLDN6和Src的表达与临床病理学参数的关系免疫组织化学染色分析CLDN6和Src在人肝癌组织中的表达,并分析其与肝癌患者临床病理参数之间的关系。Spearman分析CLDN6与Src两者的相关性。1.3 CLDN6和Src在人肝癌细胞中的定位免疫荧光染色实验检测Hep G2细胞中CLDN6和Src的定位。2.CLDN6对人肝癌细胞迁移和侵袭能力的影响建立稳定沉默CLDN6的人肝癌细胞系（Hep G2-sh CLDN6）,RT-PCR和Western blot实验鉴定CLDN6的沉默效率。通过划痕实验,Transwell迁移实验和Transwell侵袭实验检测沉默CLDN6对Hep G2细胞迁移和侵袭能力的影响。3.CLDN6影响人肝癌细胞迁移和侵袭的作用机制Western blot实验检测Hep G2-sh CLDN6细胞中p-Src、Src、p-STAT3、STAT3、MMP2和MMP9的蛋白表达。使用Src激动剂处理Hep G2-sh CLDN6细胞,确定Src激动剂处理Hep G2-sh CLDN6细胞的最佳作用浓度和时间。通过划痕实验,Transwell迁移实验和Transwell侵袭实验检测Src激动剂处理后对Hep G2-sh CLDN6细胞迁移和侵袭能力的影响;Western blot实验检测Src激动剂处理后,Hep G2-sh CLDN6细胞中p-STAT3、STAT3、MMP2和MMP9的蛋白表达水平。结果:1.CLDN6和Src在人肝癌组织中的表达及其与临床病理参数的关系1.1 CLDN6和Src在人肝癌组织中的表达及其相关性利用TCGA数据库中肝癌患者数据进行分析,选取369例人肝癌组织和160例癌旁组织进行分析。结果显示,与癌旁组织相比,CLDN6和Src在人肝癌组织中均高表达（P<0.05）,两者之间呈正相关（P=0.0048<0.01,R=0.12>0）。1.2人肝癌组织中CLDN6和Src的表达与临床病理学参数的关系免疫组织化学染色实验结果显示,CLDN6和Src在人肝癌组织中高表达,其中CLDN6的高表达率为53.33%,Src的高表达率为86.67%。并且二者主要在细胞质中表达。CLDN6的高表达与肿瘤分化程度有关（P=0.016<0.05）,与患者的年龄、性别、淋巴结转移以及临床分期无关。而Src的高表达与肿瘤的淋巴结转移有关（P=0.045<0.05）,与患者的年龄、性别、肿瘤分化程度和临床分期无关。Spearman分析结果显示,在人肝癌组织中,CLDN6与Src表达呈正相关（P=0.0467<0.05,r=0.346>0）。1.3 CLDN6和Src在人肝癌细胞中的定位免疫荧光染色结果显示,CLDN6与Src在Hep G2细胞中共定位于核膜附近的细胞质区域。2.CLDN6对人肝癌细胞迁移和侵袭能力的影响成功构建稳定沉默CLDN6的人肝癌细胞株（Hep G2-sh CLDN6）。划痕实验结果显示,与空载组相比,Hep G2-sh CLDN6细胞的迁移能力明显下降,48h最为明显,差异具有显著性（P=0.0215<0.05）。Transwell迁移实验结果显示,与空载组相比,Hep G2-sh CLDN6细胞的迁移率明显下降,差异具有显著性（P<0.0001）。Transwell侵袭实验结果显示,与空载组相比,Hep G2-sh CLDN6细胞的侵袭能力明显减弱,差异具有显著性（P=0.0002<0.001）。3.CLDN6影响人肝癌细胞迁移和侵袭的作用机制Western blot实验结果观察到,与空载组相比,Hep G2-sh CLDN6细胞中Src的磷酸化水平明显下降(P_p-Src/Src=0.0272<0.05)。此外,Src下游的STAT3的磷酸化水平也明显下降(P_{p-STAT3/STAT3}=0.0402<0.05)、MMP2和MMP9的表达水平同样明显下降,差异具有显著性(P_MMP2=0.0348<0.05,P_MMP9=0.0465<0.05)。Src激动剂作用于人肝癌细胞的最佳作用浓度为4 n M（P=0.0316<0.05）,最佳作用时间为8 h（P=0.0046<0.01）。Src激动剂作用于Hep G2-sh CLDN6细胞后,划痕实验结果显示,与未加药组相比,Src激动剂组Hep G2-sh CLDN6细胞的迁移能力明显增强,48h最为明显,差异具有显著性（P=0.013<0.05）。Transwell迁移实验结果显示,与未加药组相比,Src激动剂组Hep G2-sh CLDN6细胞的迁移率明显增加,差异具有显著性（P=0.0164<0.05）。Transwell侵袭实验结果显示,与未加药组相比,Src激动剂组Hep G2-sh CLDN6细胞的侵袭能力明显增强,差异具有显著性（P=0.0387<0.05）。Src激动剂作用于Hep G2-sh CLDN6细胞后,Western blot结果显示,与未加药组相比,Src激动剂作用于Hep G2-sh CLDN6细胞后,p-STAT3与STAT3的比值显著增加(P_{p-STAT3/STAT3}=0.0356<0.05),MMP2和MMP9的表达亦显著上调(P_MMP2=0.0231<0.05,P_MMP9=0.000964<0.001)。结论:1.CLDN6和Src在人肝癌组织中高表达,CLDN6表达与肿瘤分化程度有关,Src表达与肿瘤淋巴结转移有关,两者呈正相关。2.CLDN6和Src共定位于人肝癌细胞核膜附近的胞质区域。3.CLDN6通过激活Src/STAT3信号通路促进人肝癌细胞的迁移和侵袭。更多还原

【Abstract】 Tight junctions（TJs）are located at the top of the side of the cell and function as"gates"and"fences".But when the integrity of its structure and function is destroyed,it will cause tumor metastasis and invasion.As an important component of TJs,abnormal expression of CLDNs will destroy the integrity of TJs,resulting in loss of cell polarity and enhanced migration and invasion ability of tumor cells.As a member of CLDNs family,CLDN6 structure is also composed of a carboxyl terminal,an amino terminal,four transmembrane regions and two extracellular loops.Its carboxyl terminal is rich in multiple tyrosine phosphorylation sites and can be phosphorylated by tyrosine kinases.CLDN6 interacts with Src family tyrosine kinases（SFKs）to recruit SFKs to Y196 and Y200,and phosphorylates Y196/Y200.SFKs-SH2 binds to Y196/Y200 phosphorylated on CLDN6,thereby fully activating SFKs.The phosphorylation of SFKs activates its downstream signaling pathways.Src is a core member of the SFKs family and serves as a membrane receptor to establish connections with cytoplasmic signaling mechanisms to regulate various basic cellular processes.Src contains multiple domains,among which the catalytic domain SH1 contains the tyrosine phosphorylation site Y419,which leads to the autophosphorylation of Src,and the activated Src can activate the STAT3 signaling pathway.STAT3 is involved in the biological regulation of proliferation and differentiation,immune avoidance,angiogenesis and invasion of various tumors.STAT3 signals both activated receptor and non-receptor tyrosine kinases and acts as a transcription factor.Activated STAT3 forms homo-dimer or hetero-dimer,which directly enters the nucleus from the cytoplasm and binds to the promoter of MMPs to promote its expression,thus promoting the migration and invasion of tumor cells.At present,there are no reports on the interaction between CLDN6 and Src to activate the Src/STAT3 signaling pathway.Objective:To explore the mechanism of CLDN6 affecting the migration and invasion ability of hepatocellular carcinoma cells by activating the Src/STAT3 signaling pathway and to provide experimental basis for the study of CLDN6 as a potential therapeutic target for hepatocellular carcinoma.Methods:1.Expression of CLDN6 and Src in human hepatocellular carcinoma tissue and its relationship with clinicopathological parameters1.1 Expression and correlation of CLDN6 and Src in human hepatocellular carcinoma tissueTCGA database was used to analyze the expression of CLDN6 and Src in human hepatocellular carcinoma tissues and analyze the correlation between them.1.2 Relationship between CLDN6 and Src expression and clinicopathological parameters in human hepatocellular carcinoma tissuesImmunohistochemical staining was used to analyze the expression and localization of CLDN6 and Src in the tissues of patients with hepatocellular carcinoma,and the relationship between CLDN6 and Src and clinicopathological parameters of hepatocellular carcinoma was analyzed.Spearman correlation co-efficient was used to analyze the correlation between CLDN6 and Src.1.3 Localization of CLDN6 and Src in human hepatoma cellsLocalization of CLDN6 and Src in Hep G2 cells were detected by immunofluorescence assay.2.Effects of CLDN6 on migration and invasion of human hepatoma cellsA stable CLDN6 silencing human hepatoma cell line（Hep G2-sh CLDN6）was established,and the silencing efficiency of CLDN6 was determined by RT-PCR and Western blot.The effects of CLDN6 silencing on migration and invasion of Hep G2cells were detected by wound heal assay,Transwell migration assay and Transwell invasion assay.3.The mechanism of CLDN6 affecting migration and invasion of human hepatoma cellsThe protein expressions of p-Src、Src、p-STAT3、STAT3、MMP2 and MMP9 in Hep G2-sh CLDN6 cells were detected by Western blot assay.Hep G2-sh CLDN6cells were treated with Src activator to determine the optimal concentration and time of Src activator treatment on Hep G2-sh CLDN6 cells.The effects of Src activator treatment on migration and invasion of Hep G2-sh CLDN6 cells were detected by wound heal test,Transwell migration test and Transwell invasion test.The protein expression levels of p-STAT3、STAT3、MMP2 and MMP9 in Hep G2-sh CLDN6 cells treated with Src activator were determined by Western blot assay.Results:1.Expression of CLDN6 and Src in human hepatocellular carcinoma tissue and its relationship with clinicopathological parameters1.1 Expression and correlation of CLDN6 and Src in human hepatocellular carcinoma tissueThe data of HCC patients in TCGA database were analyzed,including 369 HCC tissues and 160 paracancer tissues.The results showed that CLDN6 and Src were highly expressed in HCC tissues compared with paracancer tissues（P<0.05）,and there was a positive correlation between them（P=0.0048<0.01,R=0.12>0）.1.2 Relationship between CLDN6 and Src expression and clinicopathological parameters in human hepatocellular carcinoma tissueImmunohistochemical staining results showed that CLDN6 and Src were highly expressed in human hepatocellular carcinoma tissues,and they were mainly expressed in the cytoplasm.The high expression rate of CLDN6 and Src was 53.33%and86.67%respectively.The high expression of CLDN6 was correlated with the degree of tumor differentiation（P=0.016<0.05）,but not correlated with the age,gender,lymph node metastasis and clinical stage of patients.The high expression of Src was associated with lymph node metastasis（P=0.045<0.05）,but not correlated with age,gender,degree of tumor differentiation and clinical stage.Spearman correlation analysis showed that CLDN6 was positively correlated with Src expression in human hepatocellular carcinoma tissue（P=0.0467<0.05,r=0.346>0）.1.3 Expression and localization of CLDN6 and Src in human hepatoma cellsThe results of immunofluorescence assay showed that CLDN6 and Src were highly expressed in Hep G2 cells and co-located in the cytoplasmic region near the nuclear membrane.2.Effects of CLDN6 on migration and invasion of human hepatoma cellsA stable CLDN6 silenced human hepatoma cell line Hep G2-sh CLDN6 was successfully constructed.Wound heal test results showed that compared with the vector group,migration ability of Hep G2-sh CLDN6 cells was significantly decreased,the most obvious at 48 h（P=0.0215<0.05）.Transwell migration assay showed that the mobility of Hep G2-sh CLDN6 cells decreased significantly compared with the vector group,and the difference was significant（P<0.0001）.The results of transwell invasion assay showed that the invasion ability of Hep G2-sh CLDN6 cells was significantly reduced compared with that of the vector group（P=0.0002<0.001）.3.The mechanism of CLDN6 affecting migration and invasion of human hepatoma cellsWestern blot results showed that phosphorylation of Src decreased significantly in Hep G2-sh CLDN6 cells compared with the vector group(P_p-Src/Src=0.0272<0.05).In addition,phosphorylation of STAT3（downstream of Src）also decreased significantly(P_{p-STAT3/STAT3}=0.0402<0.05),MMP2 and MMP9 expression levels were also significantly decreased(P_MMP2=0.0348<0.05,P_MMP9=0.0465<0.05).The optimal concentration of Src activator on human hepatoma cells was 4 n M（P=0.0316<0.05）and the optimal reaction time was 8 h（P=0.0046<0.01）.After the treatment of Hep G2-sh CLDN6 cells with Src activator,the results of wound heal test showed that the migration ability of Hep G2-sh CLDN6 cells in Src activator group was significantly enhanced compared with that in the untreated group,the most obvious at 48h,and the difference was significant（P=0.013<0.05）.Transwell migration assay showed that the cell mobility of Hep G2-sh CLDN6 in Src activator group was significantly increased compared with that in untreated group（P=0.0164<0.05）.Transwell invasion assay showed that the invasion ability of Hep G2-sh CLDN6 cells in Src activator group was significantly enhanced compared with that in untreated group（P=0.0387<0.05）.Western blot results showed that the ratio of p-STAT3 to STAT3 in Hep G2-sh CLDN6 cells treated with Src activator was significantly increased compared with that in Hep G2-sh CLDN6 cells(P_{p-STAT3/STAT3}=0.0356<0.05),MMP2 and MMP9 were significantly up-regulated(P_MMP2=0.0231<0.05,P_MMP9=0.000964<0.001).Conclusions:1.CLDN6 and Src are highly expressed in human liver cancer tissues,CLDN6expression is related to the degree of tumor differentiation,and Src expression is related to lymph node metastasis of tumor.They are positively correlated.2.CLDN6 and Src are co-locate in the cytoplasmic region near the nuclear membrane of human hepatoma cell.3.CLDN6 promotes migration and invasion of human hepatoma cells by activating the Src/STAT3 signaling pathway.更多还原