Background:Reduced expression of tripartite motif-containing 3(TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly red...Background:Reduced expression of tripartite motif-containing 3(TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma(HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines(SK-Hep1,Hep3 B,Huh7,HepG2,Bel-7402) and one normal liver cell line(L02) were detected with Western blotting.HepG2 and Bel-7402 cells with low TRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3(LV-TRIM3),whereas Huh7 and Hep3 B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA(siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect ofTRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed that TRIM3 overexpression induced G_0/G_1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3 B cells by siTRIM3 led to significantly decreased percentages of both cells in the G_0/G_1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed that TRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G_0/G_1 phase.展开更多
黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞...黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞内。利用Western印迹、免疫细胞荧光法、免疫共沉淀、细胞核蛋白质与细胞质蛋白质分离和线粒体提取等技术,检测其在细胞内的表达与定位以及与其他蛋白质的相互作用情况。免疫细胞化学和蛋白质印迹结果显示,过表达的MAGEA1主要定位于细胞质,并部分与线粒体共定位。通过免疫共沉淀验证了MAGEA1与TRIM31、SNW1、HDAC1之间存在相互作用,并且发现MAGEA1可能主要与位于细胞质中的HDAC1相互作用。以上研究表明,MAGEA1可能参与不同的细胞内调节途径,部分与线粒体共定位;与TRIM31、SNW1、HDAC1相互作用,且可能主要与位于细胞质中的HDAC1蛋白相互作用,推测其可能参与到蛋白质泛素化和Notch信号通路。本研究的结果为后续深入研究MAGEA1的作用机制奠定了实验基础。展开更多
Histone methylation is a context-dependent modification that regulates gene expression,and the trimethylation of histone H3 lysine 27(H3K27me3)usually induces gene silencing.Overcoming colorectal cancer(CRC)chemoresis...Histone methylation is a context-dependent modification that regulates gene expression,and the trimethylation of histone H3 lysine 27(H3K27me3)usually induces gene silencing.Overcoming colorectal cancer(CRC)chemoresistance is currently a huge challenge,but the relationship between H3K27me3 modification and chemoresistance remains largely unclear.Here,we found that H3K27me3 levels positively correlated with the metastasis-free survival of CRC patients and a low H3K27me3 level predicted a poor outcome upon chemotherapeutic drug treatment.Oxaliplatin stimulation significantly induced the expression of H3K27 lysine demethylase 6A/6B(KDM6A/6B),thus decreasing the level of H3K27me3 in CRC cells.Elevation of H3K27me3 level through KDM6A/6B depletion or GSK-J4(a KDM6A/6B inhibitor)treatment significantly enhanced oxaliplatin-induced apoptosis.Conversely,when inhibiting the expression of H3K27me3 by EPZ-6438,an inhibitor of the histone methyltransferase EZH2,the proportion of apoptotic cells remarkably decreased.In addition,the combination of GSK-J4 and oxaliplatin significantly inhibited tumor growth in an oxaliplatin-resistant patient-derived xenograft model.Importantly,we revealed that oxaliplatin treatment dramatically induced NOTCH2 expression,which was caused by downregulation of H3K27me3 level on the NOTCH2 transcription initiation site.Thus,the activated NOTCH signaling promoted the expression of stemness-related genes,which resulted in oxaliplatin resistance.Furthermore,oxaliplatin-induced NOTCH signaling could be interrupted by GSK-J4 treatment.Collectively,our findings suggest that elevating H3K27me3 level can improve drug sensitivity in CRC patients.展开更多
基金primarily supported by grants from the National Natural Science Foundation of China(Nos.81472387 and 81402560)Guangdong Province Science and Technology Plan Project(No.2012A030400059)
文摘Background:Reduced expression of tripartite motif-containing 3(TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma(HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines(SK-Hep1,Hep3 B,Huh7,HepG2,Bel-7402) and one normal liver cell line(L02) were detected with Western blotting.HepG2 and Bel-7402 cells with low TRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3(LV-TRIM3),whereas Huh7 and Hep3 B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA(siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect ofTRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed that TRIM3 overexpression induced G_0/G_1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3 B cells by siTRIM3 led to significantly decreased percentages of both cells in the G_0/G_1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed that TRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G_0/G_1 phase.
文摘黑色素瘤相关抗原家族A1蛋白(melanoma associated antigen family A1,MAGEA1)在生殖细胞和多种组织学来源的肿瘤中有表达,但其作用机制尚不清楚。本研究通过构建带Flag和GFP标签的MAGEA1真核表达重组质粒,将其转染至HeLa、HEK293T细胞内。利用Western印迹、免疫细胞荧光法、免疫共沉淀、细胞核蛋白质与细胞质蛋白质分离和线粒体提取等技术,检测其在细胞内的表达与定位以及与其他蛋白质的相互作用情况。免疫细胞化学和蛋白质印迹结果显示,过表达的MAGEA1主要定位于细胞质,并部分与线粒体共定位。通过免疫共沉淀验证了MAGEA1与TRIM31、SNW1、HDAC1之间存在相互作用,并且发现MAGEA1可能主要与位于细胞质中的HDAC1相互作用。以上研究表明,MAGEA1可能参与不同的细胞内调节途径,部分与线粒体共定位;与TRIM31、SNW1、HDAC1相互作用,且可能主要与位于细胞质中的HDAC1蛋白相互作用,推测其可能参与到蛋白质泛素化和Notch信号通路。本研究的结果为后续深入研究MAGEA1的作用机制奠定了实验基础。
基金This work was supported by the National Program on Key Research(2018YFA0107500 and 2016YFC1302400)the National Natural Science Foundation of China(91742113 and 31570902)Natural Science Foundation of Shanghai(14ZR14-26300,18ZR1424400,18ZR1446400,and 18431902700).
文摘Histone methylation is a context-dependent modification that regulates gene expression,and the trimethylation of histone H3 lysine 27(H3K27me3)usually induces gene silencing.Overcoming colorectal cancer(CRC)chemoresistance is currently a huge challenge,but the relationship between H3K27me3 modification and chemoresistance remains largely unclear.Here,we found that H3K27me3 levels positively correlated with the metastasis-free survival of CRC patients and a low H3K27me3 level predicted a poor outcome upon chemotherapeutic drug treatment.Oxaliplatin stimulation significantly induced the expression of H3K27 lysine demethylase 6A/6B(KDM6A/6B),thus decreasing the level of H3K27me3 in CRC cells.Elevation of H3K27me3 level through KDM6A/6B depletion or GSK-J4(a KDM6A/6B inhibitor)treatment significantly enhanced oxaliplatin-induced apoptosis.Conversely,when inhibiting the expression of H3K27me3 by EPZ-6438,an inhibitor of the histone methyltransferase EZH2,the proportion of apoptotic cells remarkably decreased.In addition,the combination of GSK-J4 and oxaliplatin significantly inhibited tumor growth in an oxaliplatin-resistant patient-derived xenograft model.Importantly,we revealed that oxaliplatin treatment dramatically induced NOTCH2 expression,which was caused by downregulation of H3K27me3 level on the NOTCH2 transcription initiation site.Thus,the activated NOTCH signaling promoted the expression of stemness-related genes,which resulted in oxaliplatin resistance.Furthermore,oxaliplatin-induced NOTCH signaling could be interrupted by GSK-J4 treatment.Collectively,our findings suggest that elevating H3K27me3 level can improve drug sensitivity in CRC patients.
