Objective:To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene.Methods:The shRNA eukaryotic expression vectors tar...Objective:To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene.Methods:The shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed.Inhibitory effect was detected by semiquantitative RT-PCR.The impacts on K562 cells by RNAi were studied by MTT assay,benzidine staining,light microscope and electron microscopy observation,cell cycles analysis,colony formation assay and RT-PCR.The expressions of erythroid differentiation correlated genes Gfi-lB,GPA and 7-globin were studied after being exposed to 0.2μmol/L imatinib for two days.Results:The shRNA eukaryotic expression vectors were successfully constructed.The expression of IER3IP1 gene was significantly inhibited with an inhibition efficiency of 76%(P〈0.01).Compared with the control groups,bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group(P〈0.01).The proliferation ability was enhanced(P〈0.01)and the proportion of cells at G0/G1 phase decreased but S phase increased(P〈0.05)in K562/shRNA-IER3IP1 group.Under electron microscopy,the amount of euchromatin increased but heterochromatin decreased.There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular.The percentage of benzidine staining positive cells and mRNA expression levels of Gfi-1B,GPA andγ-globin were all decreased after being exposed to 0.2μmol/L STI571 for two days in K562/shRNA-IER3IP1 group(P〈0.01).Conclusion:IER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells.IER3IP1 may play a role in erythroid differentiation and proliferation of K562 cells.展开更多
Background and aims:Liver fibrosis(LF)is the common pathological process of almost all liver diseases,but the pathogenesis is extremely complicated and has not been fully clarified.Therefore,we want to explore LF’s c...Background and aims:Liver fibrosis(LF)is the common pathological process of almost all liver diseases,but the pathogenesis is extremely complicated and has not been fully clarified.Therefore,we want to explore LF’s complex pathogenesis and identify key genetic markers that can predict LF prognosis.Methods:From Gene Expression Omnibus(GEO,https://www.ncbi.nlm.nih.gov/geo/),we contained datasets GSE84044 and GSE130970.And involved GSE15654 whose survival data were for the validation of our model’s prognostic value.Then,Bulk RNA sequencing was used to establish a LF-related prognostic model and to determine key genes.The key genes were later analyzed using single-cell RNA sequencing(scRNA-seq)and the role of macrophages in LF was investigated.Results:We established a comprehensive LF-related prognostic model(FMSig,i.e.,fibrosis macrophage-related prognostic signature)containing four genes(IER3,AKR1B10,ADCY1,and PGM1).FMSig can distinguish between survival outcomes in dataset GSE15654.IER3 showed a higher hazard ratio than the other three FMSig genes.Moreover,further scRNA-seq analysis showed that IER3 is highly expressed in myeloid cells while the other three genes were rarely expressed in six immune cell types and other cell clusters.After reclustering the myeloid cells into seven clusters,we found that IER3 was highly expressed in myeloid cell cluster 3,shown to be related to anti-inflammation and lipid metabolism functions.Based on pseudotime analysis,we suggest that myeloid cell type 3 is transformed from myeloid cell type 0 in the liver cirrhosis microenvironment.On immunohistochemical staining,LF showed significantly higher IER3 expression than healthy controls.Conclusion:This study established a predictive model,FMSig,to evaluate LF prognosis,and showed that IER3 and macrophages are related to LF.展开更多
基金supported by the National Natural Science Foundation of China(No.30171150)
文摘Objective:To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene.Methods:The shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed.Inhibitory effect was detected by semiquantitative RT-PCR.The impacts on K562 cells by RNAi were studied by MTT assay,benzidine staining,light microscope and electron microscopy observation,cell cycles analysis,colony formation assay and RT-PCR.The expressions of erythroid differentiation correlated genes Gfi-lB,GPA and 7-globin were studied after being exposed to 0.2μmol/L imatinib for two days.Results:The shRNA eukaryotic expression vectors were successfully constructed.The expression of IER3IP1 gene was significantly inhibited with an inhibition efficiency of 76%(P〈0.01).Compared with the control groups,bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group(P〈0.01).The proliferation ability was enhanced(P〈0.01)and the proportion of cells at G0/G1 phase decreased but S phase increased(P〈0.05)in K562/shRNA-IER3IP1 group.Under electron microscopy,the amount of euchromatin increased but heterochromatin decreased.There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular.The percentage of benzidine staining positive cells and mRNA expression levels of Gfi-1B,GPA andγ-globin were all decreased after being exposed to 0.2μmol/L STI571 for two days in K562/shRNA-IER3IP1 group(P〈0.01).Conclusion:IER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells.IER3IP1 may play a role in erythroid differentiation and proliferation of K562 cells.
基金funded by the National Natural Science Foundation of China(82270795)the Wisdom Ac-cumulation and Talent Cultivation Project of the 3rd University Xiangya Hospital of Central South(YX202009).
文摘Background and aims:Liver fibrosis(LF)is the common pathological process of almost all liver diseases,but the pathogenesis is extremely complicated and has not been fully clarified.Therefore,we want to explore LF’s complex pathogenesis and identify key genetic markers that can predict LF prognosis.Methods:From Gene Expression Omnibus(GEO,https://www.ncbi.nlm.nih.gov/geo/),we contained datasets GSE84044 and GSE130970.And involved GSE15654 whose survival data were for the validation of our model’s prognostic value.Then,Bulk RNA sequencing was used to establish a LF-related prognostic model and to determine key genes.The key genes were later analyzed using single-cell RNA sequencing(scRNA-seq)and the role of macrophages in LF was investigated.Results:We established a comprehensive LF-related prognostic model(FMSig,i.e.,fibrosis macrophage-related prognostic signature)containing four genes(IER3,AKR1B10,ADCY1,and PGM1).FMSig can distinguish between survival outcomes in dataset GSE15654.IER3 showed a higher hazard ratio than the other three FMSig genes.Moreover,further scRNA-seq analysis showed that IER3 is highly expressed in myeloid cells while the other three genes were rarely expressed in six immune cell types and other cell clusters.After reclustering the myeloid cells into seven clusters,we found that IER3 was highly expressed in myeloid cell cluster 3,shown to be related to anti-inflammation and lipid metabolism functions.Based on pseudotime analysis,we suggest that myeloid cell type 3 is transformed from myeloid cell type 0 in the liver cirrhosis microenvironment.On immunohistochemical staining,LF showed significantly higher IER3 expression than healthy controls.Conclusion:This study established a predictive model,FMSig,to evaluate LF prognosis,and showed that IER3 and macrophages are related to LF.
