Long non-coding RNA HULC promotes proliferation and osteogenic differentiation of bone mesenchymal stem cells via down-regulation of miR-195.
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LncRNAs HULC has been reported to be important regulators in the development of various human diseases. However, the role of HULC in bone mesenchymal stem cells (BMSCs) remains unclear. The present study aimed to explore the regulatory effect of HULC on proliferation and osteogenic differentiation of BMSCs and the underlying mechanism.The expression of HULC and miR-195 in BMSCs were altered by transfection and measured by qRT-PCR. Cell viability was measured by the CCK-8 assay. Osteogenic differentiation of BMSCs was determined by evaluation of osteogenic markers (Ocn, ALP, Runx2, and Col-1) expression levels using Western blot and qRT-PCR. Furthermore, Western blot was performed to assess the expression of proliferation-related factors, Wnt/β-catenin and p38MAPK pathway-related factors.HULC overexpression significantly increased cell viability, down-regulated p21 expression but up-regulated CyclinD1 expression, and promoted the levels of osteogenic markers. However, the complete opposite effect was observed in HULC knockdown. Notably, miR-195 expression was negatively regulated by HULC and miR-195 exerted a reversed effect of HULC on BMSCs. Moreover, miR-195 mediated the regulatory effect of HULC on BMSCs proliferation and osteogenic differentiation, as miR-195 mimic abolished the effect of HULC overexpression on BMSCs. We also found that HULC overexpression enhanced the activation of Wnt/β-catenin and p38MAPK pathway through down-regulating miR-195.We revealed that HULC promoted proliferation and osteogenic differentiation of BMSCs. The potential mechanism might be involved in its negative regulation on miR-195 and enhanced activation of Wnt/β-catenin and p38MAPK pathway.Keywords:
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Objective To study the expression of insulin like growth factor binding proteins 3( IGFBP-3) during inhibition of resveratrol( Res) on cell proliferation. Methods The inhibitory effect of Res on BGC-823 cells was determined by MTT method; Real-time qRT-PCR and western blot were applied to detect the expression of IGFBP-3 in Res-treated BGC-823 cells. In addition,cytometry was used to determine the proliferation and apoptosis of Res-treated BGC-823 after knockdown of IGFBP-3 by siRNA. Results Upon Res( 20,40,80 and 160 μmol · L- 1) treatment,the viability of BGC-823 cells was( 82. 35 ± 10. 65) %,( 74. 30 ± 12. 36) %,( 62. 80 ± 14. 66) % and( 50. 75 ± 11. 14) %,respectively. The mRNA and protein expression of IGFBP-3 elevated as high as 2. 96-fold compared to the control group( P 0. 05). The cell viability of BGC-823 cells with IGFBP-3 knockdown was significantly higher than that of the wild type( P 0. 05) only at high Res concentration( 160 μmol·L- 1). Meanwhile,IGFBP-3 knockdown led to a significant decrease on cell apoptotic rate by Res( 160 μmol·L- 1) [( 20. 13 ± 9. 12) % vs( 35. 48 ± 11. 12) %,P 0. 05) ]. Conclusion Res can inhibit BGC-823 cell proliferation and promote cell apoptosis,the underlying mechanism of which may be related to the overexpression of IGFBP-3 in BGC-823 cells.
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Glucose-regulated protein 75 (Grp75) binds to p53 and inhibits its nuclear translocation, and thus plays a role in cell protection. To investigate whether the binding of Grp75 and p53 would influence the viability of cells, we constructed the eukaryotic expression vector of Grp75 deletion mutant. The deletion mutant gene was obtained by SOE-PCR (gene splicing by overlap extension) and then linked to the pcDNA3.0 vector. The constructed specific expression vector, pcDNA3.0/Grp75(Δ253-282), was identified by restriction enzymes and sequencing. Then we used liposome to transfect the specific vector into PC12 cells. The stable cell strain PC12/Grp75(Δ253-282)(+) was selected by G418 (1 mg/mL). Semi-quantitative RT-PCR and Western blot showed that Grp75 mRNA and protein expressions in PC12/Grp75(Δ253-282)(+) cells were higher than those in PC12 cells. The viability of cells undergoing 0 h, 3 h, 9 h, 18 h and 36 h of glucose deprivation respectively was measured by MTT assay. The results showed that the cell viability of PC12/Grp75(+) group was significantly higher than that of the other two groups, and the cell viability of PC12/Grp75(Δ253-282)(+) group was significantly higher than that of the PC12 group (P<0.05). Hoechst33324 staining was employed to detect cell apoptosis and the results were consistent with the MTT assay results. Western blot results indicated that the expression of p53 in PC12/Grp75(+) cells was lower than those in the other two groups, which might be due to the overexpression of Grp75. These results suggest that the protective role of Grp75 is partly associated with its binding to p53. The above results suggest that Grp75 deletion mutation could to some extent reduce the viability of cells.
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To investigate the expression level of microRNA-194 in myocardial injury induced by lipopolysaccharide (LPS) and its underlying mechanism.LPS-induced H9c2 cardiomyocytes injury model was established. The expression level of microRNA-194 at different treatment time points was detected. Survival and apoptosis of cardiomyocytes were detected after overexpression or knockdown of microRNA-194. The target genes of microRNA-194 were predicted by bioinformatics analysis. The relationship between microRNA-194 and target genes was verified by the dual luciferase reporter analysis and Western blot. The effects of microRNA-194 mimics and overexpression plasmid pcDNA3/Slc7a5 on the cardiomyocyte apoptosis were investigated by MTT assay. Expressions of relative genes involved in Wnt/β-catenin pathway during the process of LPS-induced cardiomyocytes injury were detected by qRT-PCR and Western blot.The expression level of microRNA-194 was increased in LPS-induced H9c2 cardiomyocytes injury model in a time-dependent manner. Overexpressed microRNA-194 directly bound to the target gene Slc7a5 and inhibited its expression. Transfection of microRNA-194 mimics increased apoptosis of H9c2 cells, which was rescued by overexpression of pcDNA3/Slc7a5. MicroRNA-194 was capable of promoting cardiomyocyte apoptosis by activating Wnt/β-catenin pathway.MicroRNA-194 promotes cardiomyocyte apoptosis and participates in myocardial injury induced by endotoxemia via activating Wnt/β-catenin pathway.
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The colorectal neoplasia differentially expressed (CRNDE) gene is a long noncoding RNA (lncRNAs) that is upregulated in colorectal cancer and glioma. Here, we investigated the regulatory function of CRNDE in gastric cancer (GC).CRNDE and miR-145 expression were assayed by qRT-PCR, and E2F3 protein expression was measured by western blotting. A luciferase reporter assay was used to detect the direct regulation of miR-145 by CRNDE. Cell viability and colony formation of human GC cells were detected using MTT and colony formation assay, respectively.CRNDE was highly expressed in GC cell lines and tissues; overexpression of CRNDE increased GC cell viability and promoted colony formation. Knockdown of CRNDE did not result in loss of expression-related effects on cell proliferation and colony formation. Further investigation revealed that the miR-145 target gene E2F3 was strongly expressed following CRNDE competitive molecular sponging of miR-145.CRNDE acted as a growth-promoting lncRNA in GC and maybe a potential target of GC treatment.
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To investigate the effect and mechanism of CPNE7 siRNA on proliferation and osteogenic differentiation in human periodontal ligament cells (hPDLs).hPDLs were isolated by enzyme digestion, transfected with pSUPER-CPNE7 in order to knock down CPNE7. The expression of CPNE7 mRNA and protein was detected by Western blot and RT-PCR; ELISA was carried out for the activity of NF-κB; hPDLs were pretreated with PDTC (10 μmol/L) for 30 min, CCK8 was used to evaluate the proliferation; ALP activity was assayed with ELISA; The expression of RUNX2, OSX and OCN was measured with RT-PCR and Western blot. The data were analyzed with SPSS11.0 software package.After transfection with pSUPER-CPNE7, CPNE7 expression was significantly decreased (P<0.05). ELISA assay indicated that CPNE7 siRNA enhanced NF-κB activity. CCK8 and RT-PCR assay showed that CPNE7 siRNA inhibited cell proliferation, ALP activity and decreased the expression of RUNX2, OSX and OCN in hPDLs; however, these effects were abolished by PDTC.CPNE7 siRNA inhibits the proliferation and osteogenic differentiation of hPDLs through NF-κB pathway.
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Small nucleolar non-coding RNA(snoRA)23 is upregulated in human pancreatic ductal adenocarcinoma. However, to the best of our knowledge, the role of snoRA23 in hepatocellular carcinoma progression has not been determined. MTT and colony formation assays were used to assess the cell viability and proliferation of HCC cells with snoRA23 knocked down, respectively, and a lymphatic vessel formation assay was used to determine tube formation ability of Human dermal lymphatic endothelial cells treated with conditioned media from HCC cell cultures. The results showed that snoRA23 knockdown attenuated cell viability, colony formation,and lymphatic vessel formation in HCC cells. snoRA23 was correlated with the prolonged overall survival of patients with HCC. Additionally, snoRA23 knockdown downregulated the Wnt/?-catenin signaling pathway by decreasing Wnt3a expression and ?-catenin levels.?-methylacyl-CoA racemase (AMACR) levels were notably decreased by snoRA23 depletion. Finally, it was confirmed that AMACR overexpression partially rescued snoRA23-modulated HCC tumorigenesis. The results of the present study provide further insight into the role of non-coding RNAs in the development and progression of HCC.
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Objective To investigate cell proliferation and invasiveness of cervical cancer HeLa229 cell after knockdown of NF-κB signaling pathway by P65 siRNA.Methods RNA interference was employed for specific inhibition of the expression of P65.HeLa229 cell was divided into transfected group and untransfected group.Cell viability was detected by MTT after the HeLa229 cells were transfected with or without P65 siRNA for 24,48,72 h.The sensitivity to 5-Fu of the HeLa229 cell,transfected with or without P65 siRNA,was evaluated also by MTT.Boyden chamber experiment in vitro was used to detect the invasion of HeLa229 cell.Results P65 siRNA inhibited the cell proliferation as compared with the untransfected cells.Proliferations of both cells transfected with and without P65 siRNA were inhibited in a concentration-dependent manner,while at the same concentration of 5-Fu the viability of transfected HeLa229 cells was significantly suppressed(P0.05).Compared with the untransfected cells,the number of cells which traversed through Matrigel was decreased obviously.ConclusionRNAi targeting of P65 has anti-proliferative effects,inhibits the invasiveness and increases the 5-Fu sensitivity ofthe ESCC cells,suggesting that NF-κB might be a target for cancer treatment.
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Objective To create a MIN6 cell line overexpressing murine Reg3α cDNA and to investigate its cell viability character under low glucose concentrations.Methods The full-length murine Reg3α cDNA was inserted into the plasmid pcDNA3.1(-).Then MIN6 cells were transfected with the Reg3α-pcDNA3.1 and pcDNA3.1 vector alone to establish Reg3α-overexpression and empty vector MIN6 cell line.Reg3α protein in the low glucose concentration cell medium was detected by Western blot.Cell viability was evaluated by an MTT reduction conversion assay.Results Three Reg3α-overexpression MIN6 cells were confirmed by real-time PCR and Western blot.Reg3α expression was barely detectable in the cells transfected with the empty vector alone.In contrast,the levels of Reg3α mRNA and protein in three pcDNA-Reg3α-transfected clones were increased by an average 10-and 6-fold,respectively.Western blot also revealed Reg3α protein release into the culture medium.In MTT cell proliferation assay,compared to vector-transfection alone,three clones of Reg3α-overexpression MIN6 cells exhibited 2-fold increases in cell number over 7 days when cultured in the presence of 10 mL/L fetal bovine serum and 5.5 mmol/L glucose.Conclusion The Reg3α-overexpression MIN6 cells have been established.Reg3α protein was detectable in culture medium,supporting an endocrine action,and Reg3α overexpression promoted islet cell growth.
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Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into adipocytes, osteoblasts, or chondrocytes. A mutually inhibitory relationship exists between osteogenic and adipogenic lineage commitment and differentiation. Such cell fate decision is regulated by several signaling pathways, including Wnt and bone morphogenetic protein (BMP). Accumulating evidence indicates that microRNAs (miRNAs) act as switches for MSCs to differentiate into either osteogenic or adipogenic lineage. Different miRNAs have been reported to regulate a master transcription factor for osteogenesis, such as Runx2, as well as molecules in the Wnt or BMP signaling pathway, and control the balance between osteoblast and adipocyte differentiation. Here, we discuss recent advancement of the cell fate decision of MSCs by miRNAs and their targets. [BMB Reports 2015; 48(6): 319-323]
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MicroRNAs comprise a group of non-coding small RNAs (17-25 nt) involved in post-transcriptional regulation that have been identified in various plants and animals. Studies have demonstrated that miRNAs are associated with stem cell self-renewal and differentiation and play a key role in controlling stem cell activities. However, the identification of specific miRNAs and their regulatory roles in the differentiation of multipotent mesenchymal stromal cells (MSCs) have so far been poorly defined. We isolated and cultured human MSCs and osteo-differentiated MSCs from four individual donors. miRNA expression in MSCs and osteo-differentiated MSCs was investigated using miRNA microarrays. miRNAs that were commonly expressed in all three MSC preparations and miRNAs that were differentially expressed between MSCs and osteo-differentiated MSCs were identified. Four underexpressed (hsa-miR-31, hsa-miR-106a, hsa-miR-148a, and hsa-miR-424) and three novel overexpressed miRNAs (hsa-miR-30c, hsa-miR-15b, and hsa-miR-130b) in osteo-differentiated MSCs were selected and their expression were verified in samples from the fourth individual donors. The putative targets of the miRNAs were predicted using bioinformatic analysis. The four miRNAs that were underexpressed in osteo-differentiated MSCs were predicted to target RUNX2, CBFB, and BMPs, which are involved in bone formation; while putative targets for miRNAs overexpressed in osteo-differentiated MSCs were MSC maker (e.g., CD44, ITGB1, and FLT1), stemness-maintaining factor (e.g., FGF2 and CXCL12), and genes related to cell differentiation (e.g., BMPER, CAMTA1, and GDF6). Finally, hsa-miR-31 was selected for target verification and function analysis. The results of this study provide an experimental basis for further research on miRNA functions during osteogenic differentiation of human MSCs.
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