| Purpose:To investigate the effects of long non-coding RNA(lncRNA)urothelial carcinoma associated 1(UCA1)on the osteogenic differentiation and adipogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs),and to further reveal the molecular mechanism of osteoporosis,in order to find potential targets for diagnosis and treatment,and to break through the limitations of existing clinical treatment.methods:1.Culture human bone marrow mesenchymal stem cells.2.Two kinds of lentivirus vectors(UCA1a and UCA1b)and empty vectors were constructed to infect hBMSCs,respectively.The experiment was divided into four groups: Control1 for blank Control group;Control2 for infection with empty lentivirus vector;UCA1a for high expression of UCA1a;UCA1b for high expression of UCA 1b.3.Conduct Real-Time quantitative reverse transcription polymerase chain reaction(Real-Time RT-PCR)to detect the expression of UCA1 in the above four groups of cells.4.After successfully overexpressing UCA1,the four groups of BMSCs were induced to differentiate into osteogenic and adipogenic cells,respectively.5.Samples were taken at 7,14 and 21 days,and the operation was technically repeated three times.6.Oil red O staining was performed at 7d,14 d and 21 d to observe the results of adipogenic differentiation.Alizarin red staining was conducted at 7d,14 d and 21 d to observe the results of osteogenic differentiation.7.Conduct Real-Time RT-PCR synchronously to detect the expression level of osteogenic genes,like Col1,ALP,OPN and SP7,and the expression levels of adipogenic genes PPAR? and LPL.8.The data are dealed by 2^(-??Ct)method,and the data between groups are compared by one-way ANOVA Tukey's multiple comparisons test.* P < 0.05,** P <0.0001,* means the difference is statistically significant.Results:1.Construction of high-expressing UCA1 modelsThe two designed UCA1 sequences,UCA1 a and UCA1 b,could significantly increase the expression of UCA1 in hBMSCs,compared with the control group,p<0.0001,the difference was statistically significant.2.The effect of up-regulation of UCA1 on the adipogenic differentiation of hBMSCsThe results of Oil red O staining showed that there was no significant difference after 7 days of adipogenic induction.And after 14 and 21 days of adipogenic induction,the proportion of adipocytes in experimental groups was higher than that in control groups.Real-Time RT-PCR showed that after overexpression of UCA1,PPAR? and LPL,the key Gene of lipid synthase in hBMSCs,had no significant difference at 7 days compared with Control 2.The expression levels were significantly up-regulated at 14 and 21 days,Pmax = 0.0216,p < 0.05,with statistical significance.There was no significant difference between the two Control groups.3.The effect of up-regulation of UCA1 on the osteogenic differentiation of hBMSCsAlizarin red staining results showed that there was no significant difference in each group after 7 days of osteogenic induction,and the results were strongly positive after 14 and 21 days of osteogenic induction.Real-Time RT-PCR results showed that after overexpression of UCA1,the expression level of Col1 in hBMSCs was higher than that in the control2 group after 7days of osteogenic induction.The expression of ALP was up-regulated at 14 and 21 days after hBMSCs osteogenesis,Pmax = 0.0197,p < 0.05.The expression level of Col1,OPN and SP7 of hBMSCs in the UCA1 overexpression groups at 14 days were all higher than those in the control groups,p < 0.05.There was no significant difference between the two control groups.Conclusions:1.Two hBMSCs cell lines with stable and high UCA1 expression were successfully established by lentiviral vector.2.UCA1 overexpression can promote the adipogenic differentiation and osteogenic differentiation of hBMSCs,which provides a certain reference value for subsequent in-depth research.3.UCA1 may serve as a potential target for the diagnosis and treatment of osteoporosis in the future.However,the mechanism needs to be further studied. |
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