【作者】 王娟；

【导师】 台桂香；

【作者基本信息】 吉林大学 ， 免疫学， 2017， 博士

【摘要】 Mucin 1(MUC1)作为一种癌基因,可以通过多种信号通路调节多种人类腺癌的发生发展。前期研究显示,MUC1在肝细胞癌(HCC)组织中过表达,可以促进HCC细胞的增殖,抑制细胞凋亡,并通过JNK-TGF-β信号调控HCC的发生发展。然而,MUC1是否可以促进HCC细胞的迁移和侵袭,仍不清楚。前期研究还显示,MUC1在HCC细胞中可以通过JNK-AP-1通路诱导自分泌TGF-β。TGF-β作为一种多功能的细胞因子,在癌细胞中TGF-β可以通过促进细胞的增殖、侵袭和转移等过程,促进癌症的发生发展。前期研究显示,MUC1可以促进TGF-β由抑癌作用转变为促癌作用。因此,我们猜想在HCC细胞中MUC1是否可以通过诱导自分泌TGF-β,从而促进HCC细胞的迁移和侵袭,是否可以以MUC1和JNK作为靶点有效治疗HCC。本研究,我们采用MUC1基因沉默和过表达HCC细胞系作为研究对象,在体外通过划痕实验、Transwell迁移和侵袭实验研究MUC1对HCC细胞迁移和侵袭能力的影响,通过Western blotting、RNAi、ELISA等方法对其作用分子机制进行研究。进一步,本研究在体外通过WST-1检测RNAi技术和JNK抑制剂靶向抑制MUC1或JNK对HCC细胞增殖能力的影响,在体内通过建立裸鼠皮下移植瘤模型分析靶向抑制MUC1和JNK对HCC生长的影响,并通过免疫组织化学技术进行MUC1-JNK-TGF-β信号途径与HCC生长抑制相关性分析。本研究结果表明,MUC1可以促进JNK激活,JNK的激活,一方面通过AP-1诱导TGF-β/TβRI促进Smad2C的磷酸化,另一方面可以直接促进Smad2L的磷酸化,磷酸化的Smad2L/C可以同时上调MMP-9介导的HCC细胞的迁移和侵袭。通过RNAi技术及JNK抑制剂靶向抑制MUC1或JNK,可以抑制HCC的生长,进一步证实MUC1通过JNK-TGF-β信号促进HCC的发生发展。本研究揭示JNK-TGF-β信号途径是MUC1发挥癌蛋白作用的新途径,提示MUC1作为癌基因,可能是TGF-β由抑癌转变为促癌作用的重要因素之一,提示MUC1和JNK是HCC治疗的理想靶点,为肝癌的靶向治疗提供新策略。更多还原

【Abstract】 Mucin 1(MUC1),as an oncogene,it can promote the progression and tumorigenesis of many human adenocarcinomas via various signaling pathways.Our previous studies have shown that MUC1 is overexpressed in hepatocellular carcinoma(HCC)tissues,and promotes the proliferation of HCC cells,while inhibits the apoptosis of HCC cells.In addition,MUC1 can enhance the progression and tumorigenesis of HCC through c-Jun N-terminal kinase(JNK)-transforming growth factor beta(TGF-β)signaling pathway.However,whether MUC1 could promote the migration and invasion of HCC cells remains unclear.In our recent study,we showed that MUC1 induced autocrine TGF-β in HCC cells via JNK-activator protein 1(AP-1)pathway.TGF-β,a multifunctional cytokine,accelerates the progression of established cancers by promoting cell proliferation,invasion,and metastasis.Moreover,MUC1 can convert TGF-β signaling from tumor-supression to oncogenesis.Thus,we hypothesis that MUC1 might activate TGF-β signaling,of which promote the migration and invasion of HCC cells.Furthermore,whether it can provide a new strategy for the treatment of HCC through MUC1-JNK signaling pathway targeted therapy,of which needs to be further studied.In this study,MUC1 gene silencing and overexpressing HCC cells were used to determine if MUC1 could promote the migration and invasion of HCC cells by wound-healing,transwell migration,and matrigel invasion assays,and clarify the molecular mechanisms by Western blotting,RNA interference(RNAi),ELISA and so on.In addition,the inhibition effects on the proliferation of HCC cells by targeting inhibition of MUC1 and JNK were investigated by WST-1 cell viability assay in vitro and by establishing subcutaneous xenograft model in nude mice in vivo,and the correlation between MUC1-JNK-TGF-β signaling pathway and HCC growth inhibition was analyzedby immunohistochemistry staining assays.The present study includes the following contents:The expression of MUC1 in MUC1 gene silencing and overexpressing HCC cells was detectedThe expression of MUC1 in MUC1 gene silencing and overexpressing HCC cells was detected by Western blotting,and the results showed that the expression of MUC1 in MUC1 gene silencing MR1-D4 and MR1-D9 cells was significant reduced compared to the control cells,while the expression of MUC1 in MUC1 overexpressing Bel-7402-MUC1 and Hep3B-MUC1 cells was significant increased compared to the control cells,suggesting that these MUC1 gene silencing and overexpressing HCC cells are qualified for the following studies.The effect of MUC1 on HCC cell migration and invasion1.To determine the effect of MUC1 on the migration of HCC cells,wound-healing and transwell migration assays were performed,and the results showed that the migration of HCC cells was reduced when MUC1 was gene silenced,while the migration of HCC cells was increased when MUC1 was overexpressed,suggesting that MUC1 could promote the migration of HCC cells.2.To determine the effect of MUC1 on the invasion of HCC cells,transwell invasion assays was performed,and the results showed that the invasion of HCC cells was reduced when MUC1 was gene silenced,while the invasion of HCC cells was increased when MUC1 was overexpressed,suggesting that MUC1 could promote the invasion of HCC cells.MUC1 promotes the migration and invasion of HCC cells through JNK-AP-1-TGF-β signaling pathway1.The effect of MUC1-induced autocrine TGF-β on the migration and invasion of HCC cells(1)The autocrine TGF-β1 levels in both MUC1 gene silencing and overexpressing HCC cells were detected by ELISA,and the results showed that MUC1 enhanced the autocrine TGF-β in HCC cells.(2)Different doses of exogenous TGF-β1 were added to the culture media of HCC cells,and the results of transwell migration and invasion assays showed MUC1-induced TGF-β could promote the migration and invasion of HCC cells.2.The effect of blocking TGF-β signaling pathway on HCC cell migration and invasion(1)To verify the effect of blocking TGF-β signaling pathway on HCC cell migration and invasion,SB431542,an inhibitor of TβRI,was added to the culture media of HCC cells,and the results showed that SB431542 could significant inhibit MUC1-induced HCC cell migration and invasion.(2)The expression of TGF-β1 in HCC cells was silenced by RNAi,and the transfection and silencing efficiency was detected by fluorescence microscopy and RT-q PCR,and the results showed that the transfection efficiency of si RNAs reached 95%and the silencing efficiency of the TGF-β1 gene induced by TGF-β1 si RNA1 and TGF-β1si RNA2 reached approximately 80.35% and 65.83%,respectively.The effect of silencing TGF-β1 on HCC cell migration and invasion were detected by transwell migration and invasion assays,and the results showed that inhibiting the TGF-β signaling by RNAi inhibited MUC1-induced HCC cell migration and invasion.These results further suggest that MUC1-induced TGF-β upregulates HCC cell migration and invasion.3.MUC1-induced the activation of JNK promotes the migration and invasion of HCC cells(1)To investigate the effect of MUC1 on JNK activation,Western blotting was performed,and the results showed that MUC1 enhanced the activation of JNK.(2)The HCC cells were treated with different doses of exogenous TGF-β1 or TGF-β1 si RNAs,and the effect of different doses of exogenous TGF-β1 or TGF-β1si RNAs on the activation of JNK was detected by Western blotting,and the results showed that in the presence of 500 ng/l exogenous TGF-β1,which was similar to the concentration of the autocrine TGF-β1 or TGF-β1 si RNAs,there were no significant differences in the activation of JNK in the HCC cells.Taken together,these results indicate that JNK activation is enhanced by MUC1 but not MUC1-induced TGF-β in HCC cells.(3)The HCC cells were treated with JNK inhibitor SP600125,the results of Western blotting showed that SP600125 could obvious inhibit the activation of JNK;the results of ELISA showed that SP600125 could significantly downregulate the autocrine TGF-β1;the results of transwell migration and invasion assays showed that MUC1-induced HCC cell migration and invasion were almost abolished by SP600125,and the inhibition effect of SP600125 was much greater than blocking TGF-β signaling on HCC cell migration and invasion.These results reveal that MUC1-enhanced activation of JNK promotes the migration and invasion of HCC cells partly through TGF-β signaling.4.MUC1 promotes the migration and invasion of HCC cells through the JNK-AP-1-TGF-β signaling pathwayThe HCC cells were treated with AP-1 inhibitor Curcumin,and the result of ELISA showed that Curcumin could inhibit the autocrine TGF-β;the results of transwell migration and invasion assays showed that Curcumin could inhibit MUC1-induced HCC cell migration and invasion.The results indicate that MUC1 promotes the migration and invasion of HCC cells through the JNK-AP-1-TGF-β signaling pathway.MUC1 promotes the migration and invasion of HCC cells through JNK-mediated p Smad2L/C-MMP-9 signaling pathway1.MUC1 enhances the phosphorylation of Smad2L/C in HCC cellsThe effect of MUC1 on the phosphorylation of Smad2 L and Smad2 C in HCC cells was detected by Western blotting,and the results showed that MUC1 enhanced the phosphorylation of Smad2L/C in HCC cells.2.The autocrine TGF-β enhances the phosphorylation of Smad2 C in HCC cells(1)The HCC cells were stimulated with different doses of exogenous TGF-β1,the results of Western blotting showed that a low concentration of exogenous TGF-β1,which was similar to the concentration of the autocrine TGF-β1,could promote the phosphorylation of Smad2 C,whereas had no effect on the phosphorylation of Smad2 L.(2)The TGF-β1 was silenced by TGF-β1 si RNAs in HCC cells,the results of Western blotting showed that the phosphorylation of Smad2 C was decreased,while the phosphorylation of Smad2 L was not affected.3.JNK promotes the phosphorylation of Smad2L/C,while autocrine TGF-β only promotes the phosphorylation of Smad2 C in HCC cellsTo further determine the mechanism of how Smad2L/C was phosphorylated,JNK inhibitor SP600125 and TβRI inhibitor SB431542 were applied individually or in combination in HCC cells as well as AP-1 inhibitor Curcumin.Western blotting showed that SP600125 inhibited the phosphorylation of Smad2L/C,while SB431542 inhibited the phosphorylation of Smad2 C but not the phosphorylation of Smad2 L.In addition,the results showed that the phosphorylation of Smad2 C was significant inhibited by Curcumin,while Curcumin had no inhibitory effect on the phosphorylation of Smad2 L in HCC cells.Taken together,these results demonstrate that MUC1-induced JNK activation not only enhances the phosphorylation of Smad2 C through TGF-β/TβRI,but also directly enhances the phosphorylation of Smad2 L.4.MUC1 enhances the expression of MMP-9 in HCC cellsThe m RNA expression levels of PAI-1,MMP-2,MMP-9,Rho A,Rho B,and RAC-1 in HCC cells were detected by RT-q PCR,and the protein expression of MMP-9 was detected by Western blotting,and the results showed that only the expression of MMP-9was positive correlated with the migration and invasion of HCC cells.5.JNK and autocrine TGF-β promote the expression of MMP-9 in HCC cellsThe HCC cells were stimulated with different doses of exogenous TGF-β1,TGF-β1si RNA,JNK inhibitor SP600125 and/or TβRI inhibitor SB431542,or AP-1 inhibitor Curcumin,the results of Western blotting showed that TGF-β1 had a dose-dependent effect on the expression of MMP-9;TGF-β1 gene silencing inhibited the expression of MMP-9;SP600125 and SB431542 applied individually or in combination inhibited the expression of MMP-9;Curcumin inhibited the expression of MMP-9.In summary,these results demonstrate that MUC1-induced JNK activation not only enhances the phosphorylation of Smad2 C by TGF-β/TβRI through AP-1,but also directly enhances the phosphorylation of Smad2 L,and then both of them collaborate to upregulate the MMP-9-mediated cell migration and invasion of HCC cells.The effect of targeted inhibition of MUC1 and JNK on HCC cell proliferation in vitro1.MUC1 gene silencing inhibits the proliferation of HCC cell in vitro(1)The effect of MUC1 stable gene silencing on HCC cell proliferation was analyzed by WST-1 cell viability assay,and the results showed that the viability of MR1-D4 cells was reduced in a time-dependent manner compared to the control cells.(2)The effect of MUC1 gene silencing by transient transfected with MUC1-targeted si RNA on HCC cell proliferation was analyzed by WST-1 cell viability assay,fluorescence microscopy,and Western blotting,and the results showed that the transfection efficiency was above 95% and MUC1-si RNA could significantly inhibit the expression of MUC1.Moreover,MUC1-si RNA inhibited the proliferation of HCC cells,but the inhibitory effect of MUC1-si RNA decreased as time progressed.These results indicate that MUC1 gene silencing by MUC1-stable-knockdown is much more effective than MUC1-transient-knockdown in inhibiting the proliferation of HCC cells in vitro.2.Blocking the activity of JNK inhibits the proliferation of HCC cells in vitro(1)The effect of JNK inhibitor SP600125 on HCC cell proliferation was analyzed by WST-1 cell viability assay and Western blotting,and the results showed that SP600125 significant inhibited the activation of JNK and inhibited the proliferation of HCC cells in dose-and time-dependent manner.(2)The effect of blocking JNK activity by transient transfected with JNK-targeted si RNA on HCC cell proliferation was analyzed by WST-1 cell viability assay,fluorescence microscopy,and Western blotting,and the results showed that the transfection efficiency was above 95% and JNK-si RNA could significantly inhibit the expression of JNK.Moreover,JNK-si RNA inhibited the proliferation of HCC cells,but the inhibitory effect of JNK-si RNA decreased as time progressed.These results indicate that blocking the activity of JNK by SP600125 is much more effective than JNK-si RNA in inhibiting the proliferation of HCC cells in vitro.The effect of silencing the expression of MUC1 and blocking the activity of JNK on the growth of tumors in vivoSubcutaneous transplant tumor models were established in BALB/c nude mice using SMMC-7721 HCC cells,and these mice were intratumorally injected with JNK inhibitor SP600125,JNK-si RNA,or MUC1-si RNA,and the results showed that the tumors in the mice treated with SP600125 were much smaller than the control group,while there was no significant difference in the tumors when treated with JNK-si RNA or MUC1-si RNA.The inhibition effects of silencing the expression of MUC1 and blocking the activity of JNK on HCC growth are through the JNK-TGF-β signaling pathway in vivoTo verify the molecular mechanism by which inhibiting the expression of MUC1 and the activity of JNK reduced the growth of HCC cells in vivo,immunohistochemical staining was preformed to detect the expression of MUC1,p-JNK,TGF-β,p Smad3 L,p Smad3 C,c-Myc,p21,p Smad2 L,p Smad2 C,and MMP-9,and the results showed that inhibiting the expression of MUC1 and the activity of JNK inhibited the expression of p-JNK,TGF-β,p Smad3 L,c-Myc,p Smad2 L,p Smad2 C,and MMP-9,while enhanced the expression of p Smad3 C and p21.These results demonstrate that inhibiting the expression of MUC1 and the activity of JNK reduces the growth of HCC cells through the JNK-TGF-β signaling pathway in vivo.ConclusionIn summary,our results demonstrate that MUC1-induced JNK activation not only enhances the phosphorylation of Smad2 C by TGF-β/TβRI through AP-1,but also directly enhances the phosphorylation of Smad2 L,and then both of them collaborate to upregulate the MMP-9-mediated cell migration and invasion of HCC cells.Silencing the expression of MUC1 and blocking the activity of JNK by RNAi or JNK inhibitor suppress the growth of tumors in mice,demonstrating that MUC1 promotes the progression and tumorigenesis of HCC through JNK-TGF-β signaling pathway.This study clarifies that JNK-TGF-β signaling pathway is a novel pathway by which MUC1 acts as an oncogene,reveals that MUC1,as an oncogene,may convert TGF-β signaling from tumor-suppression to oncogenesis,indicates that MUC1 and JNK are attractive targets for HCC therapy,providing new strategies for the treatment of liver cancer.更多还原