目的:探究RNA干扰(RNAi)色氨酸5-单加氧酶激活蛋白(Ywhaz)基因对高糖诱导的肾小球系膜细胞增殖、凋亡的影响及可能作用机制。方法:在Lipofectamine 2000介导下,将siRNA阴性对照以及siRNA-Ywhaz分别转染高糖条件下培养的小鼠肾小球系膜细...目的:探究RNA干扰(RNAi)色氨酸5-单加氧酶激活蛋白(Ywhaz)基因对高糖诱导的肾小球系膜细胞增殖、凋亡的影响及可能作用机制。方法:在Lipofectamine 2000介导下,将siRNA阴性对照以及siRNA-Ywhaz分别转染高糖条件下培养的小鼠肾小球系膜细胞,以高糖未转染和正常培养的肾小球系膜细胞作为对照,实时定量PCR(RT-PCR)检测各组细胞中Ywhaz mRNA水平;噻唑蓝(MTT)实验检测细胞的增殖情况,流式细胞术检测细胞的凋亡率;RT-PCR检测各组细胞中炎症因子白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的表达量;蛋白质印迹法(Western blot)检测Ywhaz蛋白、丝裂原活化蛋白激酶(MAPK)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)和磷酸化丝裂原活化蛋白激酶-3(p-MKK3)的表达水平。结果: Ywhaz在高糖对照组细胞中的表达量显著高于正常对照组( P <0.05),RNA干扰后Ywhaz基因的表达量较高糖对照组显著降低( P <0.05);与高糖对照组相比,siRNA-Ywhaz组细胞的增殖能力明显降低( P <0.05),凋亡率显著增加( P <0.05),显著抑制IL-8和TNF-α以及p-p38 MAPK、p-MKK3的表达量( P <0.05)。结论:下调Ywhaz可降低p38 MAPK 信号通路关键蛋白 p-p38 MAPK、p-MKK3的表达以及炎症反应,从而抑制高糖诱导的肾小球系膜细胞增殖。展开更多
Objective Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated...Objective Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21（TRIM21） in osteosarcoma cell proliferation.Methods 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide（MTT） assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation（BIFC） were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.Results TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However,overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.Conclusion Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.展开更多
BACKGROUND Liver cancer is one of the most highly malignant cancers,characterized by easy metastasis and chemoradiotherapy resistance.Emerging evidence indicates that long noncoding RNAs(LncRNAs),including Lnc524369,a...BACKGROUND Liver cancer is one of the most highly malignant cancers,characterized by easy metastasis and chemoradiotherapy resistance.Emerging evidence indicates that long noncoding RNAs(LncRNAs),including Lnc524369,are highly involved in the initiation,progression,radioresistance,and chemoresistance of hepatocellular carcinoma(HCC).However,the function of Lnc524369 remains unclear.AIM To explore the function of Lnc524369 in HCC.METHODS To investigate the effect of Lnc524369,tissue from 41 HCC patients were analyzed using CCK8,migration,and invasion assays.Lnc524369 and YWHAZ(also named 14-3-3ζ)mRNA were detected by qPCR,and YWHAZ and RAF1 proteins were detected by western blot in liver cancer cell lines and human HCC tissues.The Cancer Cell Line Encyclopedia(CCLE)databases,STRING database,Human Protein Atlas database,and the TCGA database were used for bioinformatic analysis.RESULTS Lnc524369 was significantly upregulated in the nucleus of liver cancer cells and human HCC tissues.Overexpression of Lnc524369 was associated with the proliferation,migration,and invasion of liver cancer cells.YWHAZ and RAF1 proteins and YWHAZ mRNA were overexpressed in liver cancer,which could be attenuated by overexpression of Lnc524369.Lnc524369 and its downstream target YWHAZ and RAF1 proteins were negatively associated with overall survival time.CONCLUSION Lnc524369 might be a promising target of HCC as it can enhance liver cancer progression and decrease the overall survival time of HCC by activating the YWHAZ/RAF1 pathway.展开更多
Background： MicroRNAs （miRNAs） are key regulators during tumor initiation and progression. MicroRNA-375 （MiR-375） has been proven to play a tumor-suppressive role in various types of human malignancies; however, ...Background： MicroRNAs （miRNAs） are key regulators during tumor initiation and progression. MicroRNA-375 （MiR-375） has been proven to play a tumor-suppressive role in various types of human malignancies; however, its biological role in clear cell renal cell carcinoma （ccRCC） remains unclear. The purpose of this study was to explore the biologic role as well as the underlying mechanism of miR-375 in ccRCC progression. Methods： Quantitative polymerase chain reaction （qPCR） was applied to test the expression of miR-375 in tissues and cell lines by t-test. Functional experiments were used to investigate the biological role of miR-375 utilizing a gain-of-function strategy. The target of miR-375 was investigated by bioinformatic analysis and further verified by luciferase reporter assay, qPCR, Western blotting, and functional experiments in vitro. Results： Our study demonstrated that miR-375 was significantly downregulated in ccRCC tissues （cancer vs. normal, 0.804 ±0.079 vs. 1.784 ± 0.200, t = 5.531 P 〈 0.0001 ） and cell lines, and loss ofmiR-375 expression significantly associated with advanced Fuhrman nuclear grades （Grade Ⅲ and Ⅳ vs. Grade Ⅰ and Ⅱ, 1.000 ± 0.099 vs. 1.731 ± 0.189, t = 3.262 P = 0.003）. Functional studies demonstrated that miR-375 suppressed ccRCC cell proliferation, migration, and invasion （all P 〈 0.05 in both 786-0 and A498 cell lines）. Multiple miRNA target prediction algorithms indicated the well-studied oncogene YWHAZ as a direct target ofmiR-375, which was further confirmed by the luciferase reporter assay, qPCR, and Western blotting. Moreover, restoration of YWHAZ could rescue the antiproliferation effect ofmi R-375. Conclusions： The data provide the solid evidence that miR-375 plays a tumor-suppressive role in ccRCC progression, partially through regulating YWHAZ. This study expands the antitumor profile ofmiR-375, and supports its role as a potential therapeutic target in ccRCC treatment.展开更多
文摘目的:探究RNA干扰(RNAi)色氨酸5-单加氧酶激活蛋白(Ywhaz)基因对高糖诱导的肾小球系膜细胞增殖、凋亡的影响及可能作用机制。方法:在Lipofectamine 2000介导下,将siRNA阴性对照以及siRNA-Ywhaz分别转染高糖条件下培养的小鼠肾小球系膜细胞,以高糖未转染和正常培养的肾小球系膜细胞作为对照,实时定量PCR(RT-PCR)检测各组细胞中Ywhaz mRNA水平;噻唑蓝(MTT)实验检测细胞的增殖情况,流式细胞术检测细胞的凋亡率;RT-PCR检测各组细胞中炎症因子白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的表达量;蛋白质印迹法(Western blot)检测Ywhaz蛋白、丝裂原活化蛋白激酶(MAPK)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)和磷酸化丝裂原活化蛋白激酶-3(p-MKK3)的表达水平。结果: Ywhaz在高糖对照组细胞中的表达量显著高于正常对照组( P <0.05),RNA干扰后Ywhaz基因的表达量较高糖对照组显著降低( P <0.05);与高糖对照组相比,siRNA-Ywhaz组细胞的增殖能力明显降低( P <0.05),凋亡率显著增加( P <0.05),显著抑制IL-8和TNF-α以及p-p38 MAPK、p-MKK3的表达量( P <0.05)。结论:下调Ywhaz可降低p38 MAPK 信号通路关键蛋白 p-p38 MAPK、p-MKK3的表达以及炎症反应,从而抑制高糖诱导的肾小球系膜细胞增殖。
基金partially supported by Guangzhou Science and Technology Project[20160701175201707010263]+5 种基金Natural Science Foundation of Guangdong Province[2016A0303130832016A030313420]Team Project of Natural Science Foundation of Guangdong Province[S2013030013315]Fundamental Research Funds for the Central Universities[Grant No.21609317ZX20170413]Guangdong Province Science and Technology Project YUEKEGUICAI[(2015)110-0024]
文摘Objective Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21（TRIM21） in osteosarcoma cell proliferation.Methods 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide（MTT） assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation（BIFC） were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.Results TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However,overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.Conclusion Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.
基金the Medical and Health Science and Technology Project of Zhejiang Province(No.2021KY043)Chinese Foundation for Hepatitis Prevention and Control-TianQing Liver Disease Research Fund Subject(No.TQGB2020168).
文摘BACKGROUND Liver cancer is one of the most highly malignant cancers,characterized by easy metastasis and chemoradiotherapy resistance.Emerging evidence indicates that long noncoding RNAs(LncRNAs),including Lnc524369,are highly involved in the initiation,progression,radioresistance,and chemoresistance of hepatocellular carcinoma(HCC).However,the function of Lnc524369 remains unclear.AIM To explore the function of Lnc524369 in HCC.METHODS To investigate the effect of Lnc524369,tissue from 41 HCC patients were analyzed using CCK8,migration,and invasion assays.Lnc524369 and YWHAZ(also named 14-3-3ζ)mRNA were detected by qPCR,and YWHAZ and RAF1 proteins were detected by western blot in liver cancer cell lines and human HCC tissues.The Cancer Cell Line Encyclopedia(CCLE)databases,STRING database,Human Protein Atlas database,and the TCGA database were used for bioinformatic analysis.RESULTS Lnc524369 was significantly upregulated in the nucleus of liver cancer cells and human HCC tissues.Overexpression of Lnc524369 was associated with the proliferation,migration,and invasion of liver cancer cells.YWHAZ and RAF1 proteins and YWHAZ mRNA were overexpressed in liver cancer,which could be attenuated by overexpression of Lnc524369.Lnc524369 and its downstream target YWHAZ and RAF1 proteins were negatively associated with overall survival time.CONCLUSION Lnc524369 might be a promising target of HCC as it can enhance liver cancer progression and decrease the overall survival time of HCC by activating the YWHAZ/RAF1 pathway.
基金This work was supported by the grants from the National Natural Science Foundation of China （No. 81702521） and Provincial Natural Science Foundation of Shandong （No. ZR2017PH019 and No. ZR2018BH018）.
文摘Background： MicroRNAs （miRNAs） are key regulators during tumor initiation and progression. MicroRNA-375 （MiR-375） has been proven to play a tumor-suppressive role in various types of human malignancies; however, its biological role in clear cell renal cell carcinoma （ccRCC） remains unclear. The purpose of this study was to explore the biologic role as well as the underlying mechanism of miR-375 in ccRCC progression. Methods： Quantitative polymerase chain reaction （qPCR） was applied to test the expression of miR-375 in tissues and cell lines by t-test. Functional experiments were used to investigate the biological role of miR-375 utilizing a gain-of-function strategy. The target of miR-375 was investigated by bioinformatic analysis and further verified by luciferase reporter assay, qPCR, Western blotting, and functional experiments in vitro. Results： Our study demonstrated that miR-375 was significantly downregulated in ccRCC tissues （cancer vs. normal, 0.804 ±0.079 vs. 1.784 ± 0.200, t = 5.531 P 〈 0.0001 ） and cell lines, and loss ofmiR-375 expression significantly associated with advanced Fuhrman nuclear grades （Grade Ⅲ and Ⅳ vs. Grade Ⅰ and Ⅱ, 1.000 ± 0.099 vs. 1.731 ± 0.189, t = 3.262 P = 0.003）. Functional studies demonstrated that miR-375 suppressed ccRCC cell proliferation, migration, and invasion （all P 〈 0.05 in both 786-0 and A498 cell lines）. Multiple miRNA target prediction algorithms indicated the well-studied oncogene YWHAZ as a direct target ofmiR-375, which was further confirmed by the luciferase reporter assay, qPCR, and Western blotting. Moreover, restoration of YWHAZ could rescue the antiproliferation effect ofmi R-375. Conclusions： The data provide the solid evidence that miR-375 plays a tumor-suppressive role in ccRCC progression, partially through regulating YWHAZ. This study expands the antitumor profile ofmiR-375, and supports its role as a potential therapeutic target in ccRCC treatment.
