目的通过对食管鳞状细胞癌(ESCC)组织中含凝血酶敏感素基序的去整合素金属蛋白酶18(ADAMTS18)m RNA及蛋白表达情况的检测,探讨ADAMTS18在ESCC发生、发展中所起的作用及临床意义,为ESCC的临床早期诊断及预后评估提供新的客观参考指标。...目的通过对食管鳞状细胞癌(ESCC)组织中含凝血酶敏感素基序的去整合素金属蛋白酶18(ADAMTS18)m RNA及蛋白表达情况的检测,探讨ADAMTS18在ESCC发生、发展中所起的作用及临床意义,为ESCC的临床早期诊断及预后评估提供新的客观参考指标。方法采用半定量-逆转录聚合酶链反应(RT-PCR)的方法及免疫组化S-P的方法(生物素-链霉卵白素过氧化酶系统)对72例ESCC组织及癌旁正常组织中ADAMTS18 m RNA及蛋白的表达进行检测。结果 1ADAMTS18 m RNA在ESCC组织中的相对表达量为(0.361±0.115),在癌旁正常组织中表达量为(0.879±0.265),ESCC组织中ADAMTS18 m RNA表达量显著低于癌旁正常组织(P<0.01);ADAMTS18 m RNA在高/中分化鳞癌组中的相对表达量为(0.496±0.153),在低分化鳞癌组中为(0.232±0.088),低分化鳞癌组ADAMTS18 m RNA表达量显著低于高/中分化鳞癌组(P<0.05)。2ADAMTS18蛋白在ESCC组织中的阳性表达率为34.7%(25/72),在癌旁组织中的阳性表达率为93.1%(67/72),ADAMTS18蛋白在ESCC组织中阳性表达率显著低于癌旁组织(P<0.01);ADAMTS18蛋白在高/中分化鳞癌组中的阳性表达率为45.4%(20/44),在低分化鳞癌组中为17.8%(5/28),低分化鳞癌组ADAMTS18蛋白阳性表达率显著低于高/中分化鳞癌组(P<0.05)。结论 ADAMTS18基因在ESCC中的异常表达与ESCC的发生、发展及组织分化程度关系密切,检测ESCC组织中ADAMTS18基因的表达情况,有助于ESCC早期诊断及预后评估。展开更多
目的研究含血小板反应蛋白18型基序的解聚素样金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motif 18,ADAMTS18)在子宫内膜癌(endometrial cancer,EC)中的临床意义及生物学功能。方法采用免疫组织化学染色检测5...目的研究含血小板反应蛋白18型基序的解聚素样金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motif 18,ADAMTS18)在子宫内膜癌(endometrial cancer,EC)中的临床意义及生物学功能。方法采用免疫组织化学染色检测50对EC及癌旁组织中ADAMTS18的表达,采用单因素及多因素分析法评价ADAMTS18表达水平与患者临床特征及预后的相关性。通过转染ADAMTS18过表达质粒提高Ishikawa细胞内ADAMTS18的表达水平,通过MTT实验及流式细胞技术分别检测细胞增殖及细胞凋亡。结果ADAMTS18主要定位于细胞质中,与正常子宫内膜组织相比较,EC组织中ADAMTS18的表达水平明显降低(t=6.235,P=0.004)。ADAMTS18低表达与肿瘤直径>3 cm(χ^(2)=5.414,P=0.036)及较晚的FIGO分期(Ⅲ+Ⅳ期,χ^(2)=4.608,P=0.042)密切相关,并且ADAMTS18低表达的EC患者术后总生存期及无病生存期均较短。在体外,过表达ADAMTS18可以抑制Ishikawa细胞增殖(t=2.528,P=0.037)并促进其凋亡(t=2.684,P=0.027)。结论ADAMTS18低表达与EC恶性临床特征及不良预后密切相关,ADAMTS18在体外具有抗肿瘤增生能力。展开更多
Objective:To investigate the effect of curcumin on viability of clear cell renal cell carcinoma(cc RCC)and analyze its possible mechanism.Methods:In cell lines of A498 and 786-O,the effects of curcumin(1.25,2.5,5 and...Objective:To investigate the effect of curcumin on viability of clear cell renal cell carcinoma(cc RCC)and analyze its possible mechanism.Methods:In cell lines of A498 and 786-O,the effects of curcumin(1.25,2.5,5 and 10μmol/L)on the viability of cc RCC were analyzed at 24,48 and 72 h by MTT assay.The protein expression levels of ADAMTS18 gene,p65,phosphorylation p65(pp65),AKT,phosphorylation AKT(p AKT)and matrix metallopeptidase 2(MMP-2)before and after curcumin(10μmol/L)treatment were examined by Western blotting.Real-time PCR and methylation specific PCR(MSP)were applied to analyze the expression and methylation level of ADAMTS18 gene before and after curcumin treatment(10μmol/L).Results:Curcumin significantly inhibited the viability of A498 and 786-O cell lines in a dose-and time-dependent manner(P<0.01).Up-regulation of ADAMTS18 gene expression with down-regulation of ADAMTS18 gene methylation was reflected after curcumin treatment,accompanied by down-regulation of nuclear factorκB(NF-κk B)related protein(p65 and pp65),AKT related protein(AKT and p AKT),and NF-κB/AKT common related protein MMP-2.With ADAMTS18 gene overexpressed,the expression levels of p65,AKT and MMP2 were downregulated,of which were conversely up-regulated in silenced ADAMTS18(sh-ADAMTS18).The expression of pp65,p AKT and MMP2 in sh-ADAMTS18 was down-regulated after being treated with PDTC(NF-κB inhibitor)and LY294002(AKT inhibitor).Conclusion:Curcumin could inhibit the viability of cc RCC by down-regulating ADAMTS18 gene methylation though NF-κB and AKT signaling pathway.展开更多
Objective: To explore the effect of curcumin on the proliferation of renal cell carcinoma and analyze its regulation mechanism. Methods: In RCC cell lines of A498 and 786-O, the effects of curcumin(2.5, 5, 10 μmo/L) ...Objective: To explore the effect of curcumin on the proliferation of renal cell carcinoma and analyze its regulation mechanism. Methods: In RCC cell lines of A498 and 786-O, the effects of curcumin(2.5, 5, 10 μmo/L) on the proliferation were analyzed by Annexin V+PI staining. Besides, A498 was inoculated into nude mice to establish tumorigenic models, and the model mice were treated with different concentrations of curcumin(100, 200, and 400 mg/kg), once daily for 30 days. Then the tumor diameter was measured, the tumor cells were observed by hematoxylin-eosin staining, and the protein expressions of miR-148 and ADAMTS18were detected by immunohistochemistry. In vitro, after transfection of miR-148 mimics, miR-148 inhibitor or si-ADAMTS18 in cell lines, the expression of ADAMTS18 was examined by Western blotting and the cell survival rate was analyzed using MTT. Subsequently, Western blot analysis was again used to examine the autophagy phenomenon by measuring the relative expression level of LC3-Ⅱ/LC3-Ⅰ;autophagy-associated genes,including those of Beclin-1 and ATG5, were also examined when miR-148 was silenced in both cell lines with curcumin treatment. Results: Curcumin could inhibit the proliferation of RCC in cell lines and nude mice. The expressions of miR-148 and ADAMTS18 were upregulated after curcumin treatment both in vitro and in vivo(P<0.05). The cell survival rate was dramatically declined upon miR-148 or ADAMTS18 upregulated. However,si-ADAMTS18 treatment or miR-148 inhibitor reversed these results, that is, both of them promoted the cell survival rate. Conclusion: Curcumin can inhibit the proliferation of renal cell carcinoma by regulating the miR-148/ADAMTS18 axis through the suppression of autophagy in vitro and in vivo. There may exist a positive feedback loop between mi R-148 and ADAMTS18 gene in RCC.展开更多
Objective To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis(RIF)progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18(ADAMTS18)methylation.Methods N...Objective To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis(RIF)progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18(ADAMTS18)methylation.Methods NRK-49F cells RIF model were induced with transforming growth factorβ1(TGF-β1).Effects of different concentrations of curcumin(0,10,20,and 30μmol/L)on cell proliferation,cell cycle,cell apoptosis as well as cyclin D1 expression were analyzed by cell counting kit-8,flow cytometry and Western blot,respectively.ADAMTS18 methylation levels were determined by methylation-specific polymerase chain reaction.ADAMTS18,fibronectin(FN),type I collagen(Col-I)and alpha-smooth muscle actin(α-SMA)mRNA and protein expressions were analyzed by real-time PCR(RT-PCR)and Western blot,respectively.Meanwhile,cells were treated with 50 mmol/L 5-aza-2′-deoxycytidine(5-aza-dC,demethylation agent)for 72 h.Effect of curcumin on extracellular matrix(ECM)deposition was evaluated by immunochemical staining and Western blot.NRK-49F cells were transfected with ADAMTS18 small interfering RNA and grouped into a normal control,ADAMTS18-knock-out(KO),and ADAMTS18-KO+30μmol/L curcumin groups,and whether curcumin can reverse the effect of ADAMTS18 knockdown on RIF was evaluated.Results Compared with the control group,TGF-β1 significantly inhibited the proliferation of NRK-49F cells,blocked the G1/G0 phase,promoted cell apoptosis and inhibited cyclin D1 expression(P<0.01).Among the different concentrations of curcumin,30μmol/L curcumin significantly reversed these processes(P<0.01).Immunochemical staining and Western blot results showed that curcumin significantly inhibited the deposition of FN,Col-I andα-SMA(P<0.01).Curcumin and 5-zaz-dC had synergistic effects,promoting ADAMTS18 expression,removing ADAMTS18 methylation,and reducing ECM deposition.ADAMTS18 knockdown promoted ECM accumulation,and curcumin reversed this process(P<0.01).Conclusion TGF-β1-induced fibrosis in NRK-49F cells.Curcumin promoted ADAMTS18 expression,reduced ECM accumulation,and alleviated RIF progression by inhibiting ADAMTS18 methylation.展开更多
文摘目的通过对食管鳞状细胞癌(ESCC)组织中含凝血酶敏感素基序的去整合素金属蛋白酶18(ADAMTS18)m RNA及蛋白表达情况的检测,探讨ADAMTS18在ESCC发生、发展中所起的作用及临床意义,为ESCC的临床早期诊断及预后评估提供新的客观参考指标。方法采用半定量-逆转录聚合酶链反应(RT-PCR)的方法及免疫组化S-P的方法(生物素-链霉卵白素过氧化酶系统)对72例ESCC组织及癌旁正常组织中ADAMTS18 m RNA及蛋白的表达进行检测。结果 1ADAMTS18 m RNA在ESCC组织中的相对表达量为(0.361±0.115),在癌旁正常组织中表达量为(0.879±0.265),ESCC组织中ADAMTS18 m RNA表达量显著低于癌旁正常组织(P<0.01);ADAMTS18 m RNA在高/中分化鳞癌组中的相对表达量为(0.496±0.153),在低分化鳞癌组中为(0.232±0.088),低分化鳞癌组ADAMTS18 m RNA表达量显著低于高/中分化鳞癌组(P<0.05)。2ADAMTS18蛋白在ESCC组织中的阳性表达率为34.7%(25/72),在癌旁组织中的阳性表达率为93.1%(67/72),ADAMTS18蛋白在ESCC组织中阳性表达率显著低于癌旁组织(P<0.01);ADAMTS18蛋白在高/中分化鳞癌组中的阳性表达率为45.4%(20/44),在低分化鳞癌组中为17.8%(5/28),低分化鳞癌组ADAMTS18蛋白阳性表达率显著低于高/中分化鳞癌组(P<0.05)。结论 ADAMTS18基因在ESCC中的异常表达与ESCC的发生、发展及组织分化程度关系密切,检测ESCC组织中ADAMTS18基因的表达情况,有助于ESCC早期诊断及预后评估。
文摘目的研究含血小板反应蛋白18型基序的解聚素样金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motif 18,ADAMTS18)在子宫内膜癌(endometrial cancer,EC)中的临床意义及生物学功能。方法采用免疫组织化学染色检测50对EC及癌旁组织中ADAMTS18的表达,采用单因素及多因素分析法评价ADAMTS18表达水平与患者临床特征及预后的相关性。通过转染ADAMTS18过表达质粒提高Ishikawa细胞内ADAMTS18的表达水平,通过MTT实验及流式细胞技术分别检测细胞增殖及细胞凋亡。结果ADAMTS18主要定位于细胞质中,与正常子宫内膜组织相比较,EC组织中ADAMTS18的表达水平明显降低(t=6.235,P=0.004)。ADAMTS18低表达与肿瘤直径>3 cm(χ^(2)=5.414,P=0.036)及较晚的FIGO分期(Ⅲ+Ⅳ期,χ^(2)=4.608,P=0.042)密切相关,并且ADAMTS18低表达的EC患者术后总生存期及无病生存期均较短。在体外,过表达ADAMTS18可以抑制Ishikawa细胞增殖(t=2.528,P=0.037)并促进其凋亡(t=2.684,P=0.027)。结论ADAMTS18低表达与EC恶性临床特征及不良预后密切相关,ADAMTS18在体外具有抗肿瘤增生能力。
基金Supported by the Beijing Natural Science Foundation(No.7204316)Beijing Traditional Chinese Medicine Development Fundation(No.QN-2020-03)Peking University Medicine Fund of Fostering Young Scholars’Scientific&Technological Innovation(No.BMU2020PYB018)。
文摘Objective:To investigate the effect of curcumin on viability of clear cell renal cell carcinoma(cc RCC)and analyze its possible mechanism.Methods:In cell lines of A498 and 786-O,the effects of curcumin(1.25,2.5,5 and 10μmol/L)on the viability of cc RCC were analyzed at 24,48 and 72 h by MTT assay.The protein expression levels of ADAMTS18 gene,p65,phosphorylation p65(pp65),AKT,phosphorylation AKT(p AKT)and matrix metallopeptidase 2(MMP-2)before and after curcumin(10μmol/L)treatment were examined by Western blotting.Real-time PCR and methylation specific PCR(MSP)were applied to analyze the expression and methylation level of ADAMTS18 gene before and after curcumin treatment(10μmol/L).Results:Curcumin significantly inhibited the viability of A498 and 786-O cell lines in a dose-and time-dependent manner(P<0.01).Up-regulation of ADAMTS18 gene expression with down-regulation of ADAMTS18 gene methylation was reflected after curcumin treatment,accompanied by down-regulation of nuclear factorκB(NF-κk B)related protein(p65 and pp65),AKT related protein(AKT and p AKT),and NF-κB/AKT common related protein MMP-2.With ADAMTS18 gene overexpressed,the expression levels of p65,AKT and MMP2 were downregulated,of which were conversely up-regulated in silenced ADAMTS18(sh-ADAMTS18).The expression of pp65,p AKT and MMP2 in sh-ADAMTS18 was down-regulated after being treated with PDTC(NF-κB inhibitor)and LY294002(AKT inhibitor).Conclusion:Curcumin could inhibit the viability of cc RCC by down-regulating ADAMTS18 gene methylation though NF-κB and AKT signaling pathway.
基金Beijing Traditional Chinese Medicine Development Fundation(No.QN-2020-03)。
文摘Objective: To explore the effect of curcumin on the proliferation of renal cell carcinoma and analyze its regulation mechanism. Methods: In RCC cell lines of A498 and 786-O, the effects of curcumin(2.5, 5, 10 μmo/L) on the proliferation were analyzed by Annexin V+PI staining. Besides, A498 was inoculated into nude mice to establish tumorigenic models, and the model mice were treated with different concentrations of curcumin(100, 200, and 400 mg/kg), once daily for 30 days. Then the tumor diameter was measured, the tumor cells were observed by hematoxylin-eosin staining, and the protein expressions of miR-148 and ADAMTS18were detected by immunohistochemistry. In vitro, after transfection of miR-148 mimics, miR-148 inhibitor or si-ADAMTS18 in cell lines, the expression of ADAMTS18 was examined by Western blotting and the cell survival rate was analyzed using MTT. Subsequently, Western blot analysis was again used to examine the autophagy phenomenon by measuring the relative expression level of LC3-Ⅱ/LC3-Ⅰ;autophagy-associated genes,including those of Beclin-1 and ATG5, were also examined when miR-148 was silenced in both cell lines with curcumin treatment. Results: Curcumin could inhibit the proliferation of RCC in cell lines and nude mice. The expressions of miR-148 and ADAMTS18 were upregulated after curcumin treatment both in vitro and in vivo(P<0.05). The cell survival rate was dramatically declined upon miR-148 or ADAMTS18 upregulated. However,si-ADAMTS18 treatment or miR-148 inhibitor reversed these results, that is, both of them promoted the cell survival rate. Conclusion: Curcumin can inhibit the proliferation of renal cell carcinoma by regulating the miR-148/ADAMTS18 axis through the suppression of autophagy in vitro and in vivo. There may exist a positive feedback loop between mi R-148 and ADAMTS18 gene in RCC.
基金Supported by National Natural Science Foundation of China(No.82200740)。
文摘Objective To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis(RIF)progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18(ADAMTS18)methylation.Methods NRK-49F cells RIF model were induced with transforming growth factorβ1(TGF-β1).Effects of different concentrations of curcumin(0,10,20,and 30μmol/L)on cell proliferation,cell cycle,cell apoptosis as well as cyclin D1 expression were analyzed by cell counting kit-8,flow cytometry and Western blot,respectively.ADAMTS18 methylation levels were determined by methylation-specific polymerase chain reaction.ADAMTS18,fibronectin(FN),type I collagen(Col-I)and alpha-smooth muscle actin(α-SMA)mRNA and protein expressions were analyzed by real-time PCR(RT-PCR)and Western blot,respectively.Meanwhile,cells were treated with 50 mmol/L 5-aza-2′-deoxycytidine(5-aza-dC,demethylation agent)for 72 h.Effect of curcumin on extracellular matrix(ECM)deposition was evaluated by immunochemical staining and Western blot.NRK-49F cells were transfected with ADAMTS18 small interfering RNA and grouped into a normal control,ADAMTS18-knock-out(KO),and ADAMTS18-KO+30μmol/L curcumin groups,and whether curcumin can reverse the effect of ADAMTS18 knockdown on RIF was evaluated.Results Compared with the control group,TGF-β1 significantly inhibited the proliferation of NRK-49F cells,blocked the G1/G0 phase,promoted cell apoptosis and inhibited cyclin D1 expression(P<0.01).Among the different concentrations of curcumin,30μmol/L curcumin significantly reversed these processes(P<0.01).Immunochemical staining and Western blot results showed that curcumin significantly inhibited the deposition of FN,Col-I andα-SMA(P<0.01).Curcumin and 5-zaz-dC had synergistic effects,promoting ADAMTS18 expression,removing ADAMTS18 methylation,and reducing ECM deposition.ADAMTS18 knockdown promoted ECM accumulation,and curcumin reversed this process(P<0.01).Conclusion TGF-β1-induced fibrosis in NRK-49F cells.Curcumin promoted ADAMTS18 expression,reduced ECM accumulation,and alleviated RIF progression by inhibiting ADAMTS18 methylation.
