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Objective To explore the role of long noncoding RNA-MGC (lnc-MGC) on the trans-differentiation of human peritoneal mesothelial cells (HPMCs) induced by high glucose. Methods The immortalized HPMCs were used to establish control group and high glucose group (60mmol/L) respectively. Cells in control group were cultured with ordinary cell medium, and in high glucose group were stimulated with high glucose medium for 72h. Changes of lnc-MGC expression in the both groups were measured by RT-PCR, and the changes of mRNA and protein expression of E-Cadherin, α smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), type Ⅰ collagen (COL-1) and type Ⅲ collagen (COL-3) in epithelial cells of both groups were measured by RT-PCR and Western blotting. The HPMCs were transfected with lentivirus, and then the changes of the above indexes were observed after up- and down-regulation of lnc-MGC. Results By high glucose stimulation of HPMCs for 72h, RT-PCR results showed that the expressions of lnc-MGC, α-SMA, CTGF, COL-1 and COL-3 mRNA increased obviously (P

Objective To explore the effect of long noncoding RNA-ATB (LncRNA-ATB) on phenotypic transition and proliferation of human peritoneal mesothelial cells (HPMCs) induced by high glucose. Methods HPMCs used in experiment were divided into three groups: control group, mannitol group and hypertonic glucose group. HPMCs in control group received no treatment, and in hypertonic glucose group and mannitol group were treated with 50mmol/L D-glucose and isotonic mannitol for 72 hours, respectively. Real-time PCR was employed to detect the mRNA expression of LncRNA-ATB, E-cadherin, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), Cyclin D1, cyclin dependent kinase inhibitor 4 (CDK4), protein 27 (p27) and proliferating cell nuclear antigen (PCNA). Western blotting was performed to detect the proteins expression of E-cadherin, α-SMA, CTGF, Cyclin D1, CDK4, p27 and PCNA, and flow cytometry was used to test the cell cycle. Lentivirus artifice was used to up- or down-regulate the expression of LncRNA-ATB in untreated HPMCs. Real-time PCR was employed to detect the mRNA expression of E-cadherin, α-SMA and CTGF, Western blotting was performed to detect the proteins expression of E-cadherin, α-SMA and CTGF, and flow cytometry was used to test the cell cycle. Results It is revealed by Real-time PCR, Western blotting and flow cytometry that the expressions increased of LncRNA-ATB, α-SMA, CTGF, Cyclin D1, CDK4 and PCNA induced by hypertonic glucose, and decreased of E-cadherin and p27 (P

Objective To explore the effects of p53 up-regulated modulator of apoptosis (PUMA)-α protein on the apoptosis and fibrosis of human peritoneal mesothelial cells (HPMCs) induced by high glucose. Methods HPMCs were induced by 50mmol/L D type glucose or mannitol for 72 hours respectively, flow cytometry was employed to detect the rate of apoptotic cells, and the expression levels of apoptosis- and fibrosis-related proteins were detected by Western blotting. The untreated HPMCs were transfected with Lenti-PUMA-α, and the treated cells were transfected with shRNA-PUMA-α, the number of apoptotic cells and the expression levels of apoptosis- and fibrosis-related proteins were detected with the methods mentioned above. Results Flow cytometry showed that the rate of apoptotic HPMCs increased after being induced by high glucose for 72 hours, and Western blotting revealed that the expression levels of pro-apoptotic and pro-fibrotic related proteins increased, but the arrestins of apoptosis-and fibrosis-related proteins decreased. Up-regulation of PUMA-α promoted apoptosis and fibrosis, while down-regulation of PUMA-α alleviated apoptosis and fibrosis of HPMCs. Conclusion High glucose may accelerate apoptosis and fibrosis of HPMCs by up-regulating the expression of PUMA-α. DOI: 10.11855/j.issn.0577-7402.2017.09.06

Objective To explore the role of Kruppel-like factor 4 (KLF-4) in phenotypic transition of human peritoneal mesothelial cells (HPMCs) induced via high glucose. Methods HPMCs were induced by 50mmol/L glucose for 72 hours, the expressions of epithelium-cadherin (E-cadherin), KLF-4, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and miRNA-143 were detected by Real-time PCR and Western blotting, respectively. The treated cells were transfected with LV-KLF-4 and inhibitor, the untreated cells were transfected with shRNA-KLF-4 and mimic. The mRNA and protein expressions of KLF-4, E-cadherin, α-SMA, CTGF and miRNA-143 were detected by Real-time PCR and Western blotting, respectively. Results Real-time PCR showed that the expression of E-cadherin decreased and of α-SMA, CTGF and miRNA-143 increased, but of KLF-4 not changed in high glucose treated cells. Western blotting showed that the expression of KLF-4 and E-cadherin decreased. Up-regulating KLF-4 increased the expression of E-cadherin, but decreased the expression of α-SMA and CTGF. Down-regulating KLF-4 decreased the expression of E-cadherin, but augment the expression of α-SMA and CTGF. Conclusion High glucose may induce the down-regulation of KLF-4 protein, and SRF- miRNA-143-KLF-4 signal pathway axis may be involved in the process of HPMC phenotypic transition. DOI: 10.11855/j.issn.0577-7402.2016.12.06

Objective To explore the mechanism of transcription factor Twist on promoting peritoneal fibrosis of human peritoneal mesothelial cells (HPMCs) in patients undergoing peritoneal dialysis (PD). Methods The HPMCs in peritoneal fluid after dialysis from patients underwent peritoneal dialysis was collected and cultured. The expression and subcellular localization of E-cadherin, α-SMA, Twist and Bmi-1 were assayed by immunofluorescence and Western blotting. The expression of Twist, Bmi-1, E-cadherin and α-SMA were determined by immunofluorescence and Western blotting after the plasmids pcDNA3.1-twist and empty vector pcDNA3.1 were transfected into HPMCs with Lipofectamine 2000 in vitro. Twist, Bmi-1, E-cadherin and α-SMA were determined by Western blotting and immunofluorescence after silencing the expression of Twist by small interfering RNA (siRNA) and empty vector pSlience. Bmi-1, E-cadherin and α-SMA were assessed by Western blotting and immunofluorescence after silencing the expression of Bmi-1 by siRNA. Results The expression of E-cadherin decreased gradually and the expression of Twist, α-SMA and Bmi-1 increased along with the increase in PD time as determined with immunofluorescence and Western blotting. Compared with that expressed in the HPMCs trasfected with empty vector pcDNA3.1, the expression of E-cadherin significantly reduced and the expressions of α-SMA, Twist and Bmi-1 increased significantly after transfection of the plasmids pcDNA3.1-twist (P

Objective To explore the effects of twist over-expression on the transdifferentiation of human peritoneal mesothelial cells(HPMCs).Methods The HPMCs were treated separately with 50mmol/L(high glucose group) and 5.6mmol/L(control group) D-glucose for 48h.The protein expression of twist,E-cadherin,Fsp1 and α-SMA were detected by Western blotting.The mRNA and protein expression of twist,E-cadherin,Fsp1 and α-SMA were tested by qRT-PCR and Western blotting after transfecting the plasmids pcDNA3.1-twist and empty vector pcDNA3.1 into HPMCs with Lipofectamine 2000 in vitro.The protein expression of twist,E-cadherin,Fsp1 and α-SMA were tested by Western blotting after silencing the twist expression by siRNA,empty vector pSlience and the parental cells.Results Compared with control group,the expression of twist,Fsp1 and α-SMA increased,and of E-cadherin decreased in high glucose group(P < 0.05).Compared with the empty vector group,after the over-expression of twist in HPMCs,the expression of E-cadherin decreased,and of Fsp1 and α-SMA increased in high glucose group(P < 0.05).Silencing the twist expression by siRNA,the expression of E-cadherin increased and of α-SMA and Fsp1 decreased in siTwist transfacted cells compared with pSlicencer-transfacted cell and parental cells(P < 0.05),and finally reversed the phenotype of MMT.Conclusion The over-expression of twist may promote the mesothelial-to-mesenchymal transition of HPMCs.