BACKGROUND Activated hepatic stellate cells(aHSCs)are the major source of cancer-associated fibroblasts in the liver.Although the crosstalk between aHSCs and colorectal cancer(CRC)cells supports liver metastasis(LM),t...BACKGROUND Activated hepatic stellate cells(aHSCs)are the major source of cancer-associated fibroblasts in the liver.Although the crosstalk between aHSCs and colorectal cancer(CRC)cells supports liver metastasis(LM),the mechanisms are largely unknown.AIM To explore the role of BMI-1,a polycomb group protein family member,which is highly expressed in LM,and the interaction between aHSCs and CRC cells in promoting CRC liver metastasis(CRLM).METHODS Immunohistochemistry was carried out to examine BMI-1 expression in LM and matched liver specimens of CRC.The expression levels of BMI-1 in mouse liver during CRLM(0,7,14,21,and 28 d)were detected by Western blotting(WB)and the quantitative polymerase chain reaction(qPCR)assay.We overexpressed BMI-1 in HSCs(LX2)by lentivirus infection and tested the molecular markers of aHSCs by WB,qPCR,and the immunofluorescence assay.CRC cells(HCT116 and DLD1)were cultured in HSC-conditioned medium(LX2 NC CM or LX2 BMI-1 CM).CM-induced CRC cell proliferation,migration,epithelial-mesenchymal transition(EMT)phenotype,and transforming growth factor beta(TGF-β)/SMAD pathway changes were investigated in vitro.A mouse subcutaneous xenotransplantation tumor model was established by co-implantation of HSCs(LX2 NC or LX2 BMI-1)and CRC cells to investigate the effects of HSCs on tumor growth and the EMT phenotype in vivo.RESULTS Positive of BMI-1 expression in the liver of CRLM patients was 77.8%.The expression level of BMI-1 continued to increase during CRLM in mouse liver cells.LX2 overexpressed BMI-1 was activated,accompanied by increased expression level of alpha smooth muscle actin,fibronectin,TGF-β1,matrix metalloproteinases,and interleukin 6.CRC cells cultured in BMI-1 CM exhibited enhanced proliferation and migration ability,EMT phenotype and activation of the TGF-β/SMAD pathway.In addition,the TGF-βR inhibitor SB-505124 diminished the effect of BMI-1 CM on SMAD2/3 phosphorylation in CRC cells.Furthermore,BMI-1 overexpressed LX2 HSCs promoted tumor growth and the EMT phenotype in vivo.CONCLUSION High expression of BMI-1 in liver cells is associated with CRLM progression.BMI-1 activates HSCs to secrete factors to form a prometastatic environment in the liver,and aHSCs promote proliferation,migration,and the EMT in CRC cells partially through the TGF-β/SMAD pathway.展开更多
目的:研究微小RNA-218(microRNA-218,miR-218)靶向B细胞特性莫洛尼鼠白血病病毒插入位点1(B-cell specific moloney leukemia virus insertion site 1,Bmi-1)抑制肺腺癌细胞增殖的作用及机制。方法:培养肺腺癌细胞株A549,分别转染miR-...目的:研究微小RNA-218(microRNA-218,miR-218)靶向B细胞特性莫洛尼鼠白血病病毒插入位点1(B-cell specific moloney leukemia virus insertion site 1,Bmi-1)抑制肺腺癌细胞增殖的作用及机制。方法:培养肺腺癌细胞株A549,分别转染miR-阴性对照(NC)核苷酸序列、miR-218核苷酸序列、NC质粒、Bmi-1质粒,采用平板克隆形成实验、CCK8实验检测细胞增殖水平,采用荧光定量PCR实验检测miR-218的表达,采用Western blot实验检测Bmi-1的表达,采用双荧光素酶报告基因实验检测miR-218与Bmi-1的靶向关系。建立移植瘤裸鼠,慢病毒转染miR-NC或miR-218核苷酸序列,检测移植瘤体积、质量及Bmi-1的表达。结果:与转染miR-NC核苷酸序列的miR-NC组比较,转染miR-218核苷酸序列的miR-218组A549细胞miR-218表达水平、增殖抑制率增加,克隆形成率、Bmi-1表达水平及野生型Bmi-1质粒的荧光素酶活性降低(P<0.05);与共转染miR-218核苷酸序列及NC质粒的miR-218+NC质粒组比较,共转染miR-218核苷酸序列及Bmi-1质粒的miR-218+Bmi-1质粒组A549细胞增殖抑制率降低,克隆形成率、Bmi-1表达水平增加(P<0.05);与慢病毒转染miR-NC核苷酸序列的miR-NC组比较,慢病毒转染miR-218核苷酸序列的miR-218组裸鼠移植瘤体积、质量及Bmi-1表达水平均降低(P<0.05)。结论:miR-218靶向下调Bmi-1表达并抑制肺腺癌细胞增殖。展开更多
基金Supported by National Natural Science Foundation of China,No.81472213the Health Commission of Zhejiang Province,No.2019ZD010 and No.2019ZD029+1 种基金the Science Technology Department of Zhejiang Province,No.LGF20H220001the Zhejiang Provincial Administration of Traditional Chinese Medicine,No.2021ZA088.
文摘BACKGROUND Activated hepatic stellate cells(aHSCs)are the major source of cancer-associated fibroblasts in the liver.Although the crosstalk between aHSCs and colorectal cancer(CRC)cells supports liver metastasis(LM),the mechanisms are largely unknown.AIM To explore the role of BMI-1,a polycomb group protein family member,which is highly expressed in LM,and the interaction between aHSCs and CRC cells in promoting CRC liver metastasis(CRLM).METHODS Immunohistochemistry was carried out to examine BMI-1 expression in LM and matched liver specimens of CRC.The expression levels of BMI-1 in mouse liver during CRLM(0,7,14,21,and 28 d)were detected by Western blotting(WB)and the quantitative polymerase chain reaction(qPCR)assay.We overexpressed BMI-1 in HSCs(LX2)by lentivirus infection and tested the molecular markers of aHSCs by WB,qPCR,and the immunofluorescence assay.CRC cells(HCT116 and DLD1)were cultured in HSC-conditioned medium(LX2 NC CM or LX2 BMI-1 CM).CM-induced CRC cell proliferation,migration,epithelial-mesenchymal transition(EMT)phenotype,and transforming growth factor beta(TGF-β)/SMAD pathway changes were investigated in vitro.A mouse subcutaneous xenotransplantation tumor model was established by co-implantation of HSCs(LX2 NC or LX2 BMI-1)and CRC cells to investigate the effects of HSCs on tumor growth and the EMT phenotype in vivo.RESULTS Positive of BMI-1 expression in the liver of CRLM patients was 77.8%.The expression level of BMI-1 continued to increase during CRLM in mouse liver cells.LX2 overexpressed BMI-1 was activated,accompanied by increased expression level of alpha smooth muscle actin,fibronectin,TGF-β1,matrix metalloproteinases,and interleukin 6.CRC cells cultured in BMI-1 CM exhibited enhanced proliferation and migration ability,EMT phenotype and activation of the TGF-β/SMAD pathway.In addition,the TGF-βR inhibitor SB-505124 diminished the effect of BMI-1 CM on SMAD2/3 phosphorylation in CRC cells.Furthermore,BMI-1 overexpressed LX2 HSCs promoted tumor growth and the EMT phenotype in vivo.CONCLUSION High expression of BMI-1 in liver cells is associated with CRLM progression.BMI-1 activates HSCs to secrete factors to form a prometastatic environment in the liver,and aHSCs promote proliferation,migration,and the EMT in CRC cells partially through the TGF-β/SMAD pathway.
文摘目的:研究微小RNA-218(microRNA-218,miR-218)靶向B细胞特性莫洛尼鼠白血病病毒插入位点1(B-cell specific moloney leukemia virus insertion site 1,Bmi-1)抑制肺腺癌细胞增殖的作用及机制。方法:培养肺腺癌细胞株A549,分别转染miR-阴性对照(NC)核苷酸序列、miR-218核苷酸序列、NC质粒、Bmi-1质粒,采用平板克隆形成实验、CCK8实验检测细胞增殖水平,采用荧光定量PCR实验检测miR-218的表达,采用Western blot实验检测Bmi-1的表达,采用双荧光素酶报告基因实验检测miR-218与Bmi-1的靶向关系。建立移植瘤裸鼠,慢病毒转染miR-NC或miR-218核苷酸序列,检测移植瘤体积、质量及Bmi-1的表达。结果:与转染miR-NC核苷酸序列的miR-NC组比较,转染miR-218核苷酸序列的miR-218组A549细胞miR-218表达水平、增殖抑制率增加,克隆形成率、Bmi-1表达水平及野生型Bmi-1质粒的荧光素酶活性降低(P<0.05);与共转染miR-218核苷酸序列及NC质粒的miR-218+NC质粒组比较,共转染miR-218核苷酸序列及Bmi-1质粒的miR-218+Bmi-1质粒组A549细胞增殖抑制率降低,克隆形成率、Bmi-1表达水平增加(P<0.05);与慢病毒转染miR-NC核苷酸序列的miR-NC组比较,慢病毒转染miR-218核苷酸序列的miR-218组裸鼠移植瘤体积、质量及Bmi-1表达水平均降低(P<0.05)。结论:miR-218靶向下调Bmi-1表达并抑制肺腺癌细胞增殖。
