【作者】 徐伟；

【导师】 陆荣柱；

【作者基本信息】 江苏大学 ， 病理学与病理生理学， 2021， 硕士

【摘要】 背景与目的:钙敏感受体（calcium-sensing receptor,CaSR）在不同组织器官中表达水平的差异性与肿瘤关系密切,这种差异性使其在不同的肿瘤类型中发挥促进或抑制肿瘤发生发展的作用,但其在肝癌中的作用尚不明确。3,3’-二吲哚甲烷（3,3’-diindolylmethane,DIM）为十字花科植物的重要活性产物,具有显著的抗肝癌效应,但其具体机制尚不清楚;在本课题中,我们选取HepG2和SMMC-7721人肝癌细胞株为研究对象,探讨CaSR在肝癌细胞中的表达及其对细胞行为学的影响,同时阐明其在DIM抑制肝癌细胞增殖、迁移和侵袭效应中的作用及分子机制,为CaSR应用于肝癌的分子诊断及DIM应用于肝癌的临床治疗提供理论依据。方法:1.采用免疫荧光法和蛋白质免疫印迹法（Western Blot）检测两株肝癌细胞HepG2和SMMC-7721中CaSR蛋白表达水平。利用CaSR特异性抑制剂NPS-2143以及CaSR si RNA作用于两株肝癌细胞,从药理学及基因学层面观察抑制CaSR表达后,对细胞增殖、迁移及侵袭能力的影响,同时检测ERK及p-ERK蛋白,探索ERK信号通路在其中发挥的作用。2.MTT法检测DIM分别作用肝癌细胞株HepG2、SMMC-7721以及人正常肝细胞株HL-7702后,细胞增殖能力的变化,进而选择合适作用时间和作用浓度用于后续实验;Transwell实验检测DIM作用HepG2和SMMC-7721后细胞迁移和侵袭能力改变;Western Blot检测DIM对肝癌细胞增殖相关蛋白PCNA、迁移侵袭相关蛋白MMP-2和MMP-9表达水平的影响。3.采用Western Blot和q RT-PCR法观察DIM作用HepG2和SMMC-7721细胞后CaSR以及CaSR m RNA的变化,同时检测ERK及p-ERK蛋白表达水平的改变;4.采用CaSR特异性激动剂GdCl₃、ERK特异性抑制剂U0126预处理后与DIM联用,观察激活CaSR或抑制ERK磷酸化后对细胞增殖、迁移及侵袭能力的影响,探讨DIM在肝癌细胞中抑制增殖、迁移和侵袭的效应与ERK以及CaSR的关系。结果:1.Western Blot和免疫荧光发现HepG2和SMMC-7721细胞中存在CaSR表达且CaSR蛋白存在于细胞膜和细胞质中;CaSR抑制剂NPS-2143以及CaSR si RNA作用后,肝癌细胞增殖、迁移和侵袭能力受到显著抑制,p-ERK水平降低而总ERK水平保持不变。2.DIM呈时间-剂量依赖性显著抑制HepG2和SMMC-7721细胞增殖能力,同时以剂量-效应关系显著抑制HepG2和SMMC-7721细胞迁移和侵袭能力。Western Blot结果表明DIM作用后肝癌细胞增殖相关蛋白PCNA、迁移侵袭相关蛋白MMP-2和MMP-9蛋白水平逐渐降低。3.Western Blot和q RT-PCR结果表明DIM呈浓度依赖性抑制CaSR蛋白和CaSR m RNA的表达,同时也显著降低了p-ERK蛋白表达,而总ERK蛋白水平不变。4.采用GdCl₃激活CaSR后,DIM诱导的肝癌细胞增殖、迁移及侵袭能力下降被抑制,降低的p-ERK蛋白水平同样得到缓解;选取ERK抑制剂U0126与DIM联用,进一步抑制了肝癌细胞增殖、迁移和侵袭能力。结论:下调HepG2和SMMC-7721细胞中CaSR表达通过调节ERK信号通路显著抑制了细胞的增殖、迁移和侵袭能力;DIM呈浓度依赖性抑制肝癌HepG2和SMMC-7721细胞增殖、迁移及侵袭能力,且抑制效应主要通过CaSR/ERK信号通路实现,提示CaSR是DIM抗肝癌效应的潜在靶点,且主要通过抑制ERK信号通路来介导。更多还原

【Abstract】 Objective: The difference of CaSR expressions in various tissues and organs is closely to tumors,which makes it plays a role in promoting or inhibiting the development of various types of tumors.However,its role in hepatocellular carcinoma(HCC)remains unclear.3,3-diindolylmethane(DIM)is a vital active product derived from cruciferous vegetables.DIM has prominent anti-hepatoma effects,but its specific mechanism remains unclear.In this study,we revealed the expression of CaSR and its influence on cell behavior of HCC cell lines.Meanwhile,we clarified the role and molecular mechanism of CaSR in the effects of proliferation,migration and invasion inhibited by DIM on human HepG2 and SMMC-7721 HCC cell lines,so as to provide theoretical basis for application of CaSR in molecular diagnosis and DIM in the clinical treatment of HCC.Methods 1.The expression of CaSR protein in two HCC cells HepG2 and SMMC-7721 were detected by Western blot and immunofluorescence.NPS-2143,a CaSR specific inhibitor and CaSR si RNA were used to observe the effects of inhibition of CaSR on the proliferation,migration and invasion ability on two HCC cell lines from the pharmacological and genetic levels.Meanwhile,ERK and p-ERK protein expression levels were detected to explore the role of ERK signaling pathway on the abovementioned effects.2.After treatment with DIM on HepG2 and SMMC-7721 cells,the cell proliferation level was detected by MTT,so as to choose an appropriate treatment time and concentration.Transwell assay was applied to detect cell migration and invasion ability after treatment with DIM.The expression of proliferating cell nuclear antigen(PCNA)and proteins closely related to migration and invasion,such as matrix metalloproteinases(MMP-2,MMP-9)was assayed by western blot.3.Western blot and q RT-PCR were applied to observe the changes of CaSR and CaSR m RNA after treatment with DIM in HepG2 and SMMC-7721 cells,also the ERK and p-ERK protein expressions were assayed.4.Gd Cl3,a CaSR specific agonist and ERK specific inhibitor U0126 were used in combination with DIM to explore the influences of activation of CaSR or inhibition of ERK phosphorylation on cell proliferation,migration and invasion ability,and thus to investigate the relationship between CaSR and ERK on inhibitory effects induced by DIM in HCC cells.Results 1.Western blot and immunofluorescence indicated the CaSR protein expression level in HepG2 and SMMC-7721 cells and existed in the cell membrane and cell nucleus.After treatment with CaSR inhibitor NPS-2143 and CaSR si RNA,the proliferation,migration and invasion ability of HCC cells were significantly suppressed and the p-ERK level was decreased while the total ERK protein level remained unchanged.2.DIM significantly suppressed the proliferation in a time-dosedependent manner,and also DIM significantly inhibited the migration and invasion ability in a concentration dependent manner in HepG2 and SMMC-7721 cells.Western blot showed that the PCNA,MMP-2 and MMP-9 protein expressions decreased gradually.3.Western blot and q RT-PCT showed that DIM decreased the expression of CaSR protein and m RNA in a dose-dependent manner.Meanwhile,DIM remarkedly decreased p-ERK protein expression while the total ERK protein level remained unchanged.4.After activation of CaSR by Gd Cl3,the decreased proliferation,migration and invasion ability of HCC cells induced by DIM was restored,and also the reduced p-ERK level was alleviated.The combination of ERK inhibitor U0126 with DIM further decreased the cell proliferation,migration and invasion ability of HCC cells.Conclusion Downregulation of CaSR expressions significantly inhibited cell proliferation,migration and invasion by regulating ERK signaling pathway in HepG2 and SMMC-7721 cell lines.DIM inhibited proliferation,migration and invasion ability of HepG2 and SMMC-7721 cells in a concentration-dependent manner.The inhibitory effects of DIM were mainly carried out through the CaSR/ERK signaling pathway,suggesting that CaSR was a potential target of DIM’s anti-HCC effects,which was mainly achieved by inhibiting ERK signaling pathway.更多还原