BACKGROUND:Existing studies have made significant progress in PIWI-interacting RNAs(piRNAs)against osteoporosis,but the specific targets and related mechanisms by which piRNAs exert their functions remain to be explored.OBJECTIVE:To investigate the effects and downstream mechanisms of piR-7472 on the differentiation of mouse osteoblasts(MC3T3-E1 cells).METHODS:(1)Twelve C57/BL6J mice were randomly divided into a sham-operated and an ovariectomized group,with six mice in each group.Changes in bone mass and the expression of piR-7472 were detected using Micro-CT and RT-qPCR,respectively,at 8 weeks after surgery.(2)MC3T3-E1 cells were divided into NC mimics group,piR-7472 mimics group,NC inhibitor group,and piR-7472 inhibitor group.The mRNA expression of piR-7472,osteopontin,type I collagen,Runt-related transcription factor 2,and potassium voltage-gated channel modifier subfamily F member 1 were detected by RT-qPCR after 7 days of osteogenic induction.The protein expression of osteopontin,Runt-related transcription factor 2,bone morphogenetic protein 2,and potassium voltage-gated channel modifier subfamily F member 1(KCNF1)was detected using western blot assay.The expression of alkaline phosphatase was detected by alkaline phosphatase staining after 14 days of osteogenic induction,and the number of mineralized nodules was detected by alizarin red staining after 21 days of induction.Whether piR-7472 could bind to KCNF1 was observed by the dual luciferase reporter gene assay.RESULTS AND CONCLUSION:(1)Bone mineral density,bone volume fraction,bone trabecular thickness,bone trabecular number were significantly decreased and bone trabecular separation was significantly increased in ovariectomized mice,and piR-7472 in bone tissue was significantly down-regulated in osteoporotic mice.(2)Compared with the NC group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 were significantly increased,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly elevated,and the levels of mineralized deposition and alkaline phosphatase were increased in the piR-7472 mimics group.Compared with the NC inhibitor group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 was significantly downregulated,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly decreased,and the levels of mineralized deposition and alkaline phosphatase were reduced in the piR-7472 inhibitor group.(3)piR-7472 was found to interact with the potassium voltage-gated channel modifier subfamily F member 1 as predicted by the miRanda database.The dual luciferase reporter gene assay revealed that piR-7472 mimics could bind to and promote the expression of KCNF1.To conclude,piR-7472 can promote osteogenic differentiation of osteogenic precursor cells MC3T3-E1,and its mechanism of action may be achieved by promoting the expression of KCNF1.

BACKGROUND:Existing studies have made significant progress in PIWI-interacting RNAs(piRNAs)against osteoporosis,but the specific targets and related mechanisms by which piRNAs exert their functions remain to be explored.OBJECTIVE:To investigate the effects and downstream mechanisms of piR-7472 on the differentiation of mouse osteoblasts(MC3T3-E1 cells).METHODS:(1)Twelve C57/BL6J mice were randomly divided into a sham-operated and an ovariectomized group,with six mice in each group.Changes in bone mass and the expression of piR-7472 were detected using Micro-CT and RT-qPCR,respectively,at 8 weeks after surgery.(2)MC3T3-E1 cells were divided into NC mimics group,piR-7472 mimics group,NC inhibitor group,and piR-7472 inhibitor group.The mRNA expression of piR-7472,osteopontin,type I collagen,Runt-related transcription factor 2,and potassium voltage-gated channel modifier subfamily F member 1 were detected by RT-qPCR after 7 days of osteogenic induction.The protein expression of osteopontin,Runt-related transcription factor 2,bone morphogenetic protein 2,and potassium voltage-gated channel modifier subfamily F member 1(KCNF1)was detected using western blot assay.The expression of alkaline phosphatase was detected by alkaline phosphatase staining after 14 days of osteogenic induction,and the number of mineralized nodules was detected by alizarin red staining after 21 days of induction.Whether piR-7472 could bind to KCNF1 was observed by the dual luciferase reporter gene assay.RESULTS AND CONCLUSION:(1)Bone mineral density,bone volume fraction,bone trabecular thickness,bone trabecular number were significantly decreased and bone trabecular separation was significantly increased in ovariectomized mice,and piR-7472 in bone tissue was significantly down-regulated in osteoporotic mice.(2)Compared with the NC group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 were significantly increased,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly elevated,and the levels of mineralized deposition and alkaline phosphatase were increased in the piR-7472 mimics group.Compared with the NC inhibitor group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 was significantly downregulated,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly decreased,and the levels of mineralized deposition and alkaline phosphatase were reduced in the piR-7472 inhibitor group.(3)piR-7472 was found to interact with the potassium voltage-gated channel modifier subfamily F member 1 as predicted by the miRanda database.The dual luciferase reporter gene assay revealed that piR-7472 mimics could bind to and promote the expression of KCNF1.To conclude,piR-7472 can promote osteogenic differentiation of osteogenic precursor cells MC3T3-E1,and its mechanism of action may be achieved by promoting the expression of KCNF1.

This study sought to explore the relationship between the change in ventricular electrical remodeling caused by mechano-electrical feedback and the expression of L-type Ca2+ -channel and/or sarcoplasmic reticulum Ca2+ -ATPase in the rabbits with congestive heart failure (CHF). 138 rabbits were divided into two groups (CHF and control). We measured the ventricular monophasic action potential duration (MAPD) and ventricular effective refractory period (VERP) during ventricular pacing at the stimulus frequency of 220/240/260 bpm in these rabbits. Rapid atrial pacing (260/min) was given for 30 minutes. The MAPD and VERP were measured again. Then ventricular fibrillation was induced by S1S2S3 program stimulation. We extracted the total RNA from the myocardium respectively and detected L-type Ca2+ -channel mRNA and sarcoplasmic reticulum Ca2+ -ATPase mRNA by use of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In group CHF, with the increasing of preload/afterload, L-type Ca2+ -channel mRNA was up regulated after rapid atrial pacing when compared with that in control groups (P < 0.05). There was no significant change in sarcoplasmic reticulum Ca2+ -ATPase mRNA after rapid atrial pacing when compared with controls (P > or = 0.05). The changes in MAPD90 and VERP were related with the extent of L-type Ca2+ -channel mRNA up regulation. But the changes in MAPD90 and VERP were not significantly related with the extent of sarcoplasmic reticulum Ca2+ -ATPase mRNA up regulation. These findings suggest that Mechano-Electrical Feedback could increase the regional changes of ventricular electrical remodeling in rabbits with CHF and so to predispose them to ventricular arrhythmia. The changes may be related with the up regulation of L-type Ca2+ -channel mRNA, but not with sarcoplasmic reticulum Ca2+ -ATPase mRNA.
Action Potentials ; Animals ; Calcium Channels, L-Type ; genetics ; metabolism ; Cardiac Pacing, Artificial ; Electric Conductivity ; Electrophysiology ; Female ; Heart Failure ; metabolism ; physiopathology ; Male ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Random Allocation ; Refractory Period, Electrophysiological ; Sarcoplasmic Reticulum ; genetics ; metabolism ; Ventricular Remodeling

Objective To examine the gene expression levels of matrix metalloproteinase-1(MMP-1), tissue inhibitor of metalloproteinase-1 ( TIMP-1) in peripheral blood mononuclear cells (PBMC) and sera in the patients with chronic hepatitis B (CHB) and to investigate the value of message RNA(mRNA) expression of MMP-1 and TIMP-1 for diagnosing liver fibrosis. Methods PBMC and sera samples were collected from 37 CHB patients and 20 healthy controls. The total RNA isolated from PBMC was reversely transcribed into cDNA. The mRNA levels of MMP-1 and TIMP-1 in PBMC were examined by real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR). The serum levels of MMP-1 and TIMP-1 were determined by sandwich enzyme-linked immunosorbent assay (ELISA). Liver tissues were obtained from all these patients by biopsy and subsequently used for evaluating liver fibrosis stages (S). Intergroup comparison was performed by non parametric test. The correlation analysis was performed by Spearman. Results The MMP-1 and TIMP-1 mRNA levels in PBMC from healthy controls were low. The MMP-1 mRNA levels in PBMC from CHB patients were not significantly different from those in healthy controls,while the TIMP-1 mRNA levels were remarkably higher in CHB patients' PBMC compared to healthy controls. Both the MMP-1 mRNA levels in PBMC and the MMP-1 protein levels in sera were not significantly different among CHB patients at different disease stages and healthy controls (χ~2 =8. 960,P=0.111l ;χ~2 =7. 898, P = 0.211). However, the TIMP-1 mRNA levels in PBMC and the TIMP-1 protein levels in sera increased gradually along with the disease progressed from S1 to S4. The TIMP-1 mRNA levels in PBMC were (1.67±0. 84) lg copy/μL, (3. 48±2. 08) lg copy/μL,(5. 86±3. 47) lgcopy/μL and (8. 14 ± 6. 48) lg copy/μL from stage 1 to 4 respectively, while the protein levels of TIMP-1 in sera were (233. 73±64. 84) ,μg/L, (262. 10±71. 12) μg/L, (301. 15±62. 74)μg/L and(381. 15 ± 152. 75)μg/L, respectively. The differences between each stages were statistically significant (χ~2'= 14. 290, P=0.002,χ~2 = 12.209, P=0. 007). The TIMP-1 mRNA levels in PBMC and the TIMP-1 serum levels were positively correlated with liver fibrosis stage (r=0. 752, P<0. 01;r=0. 530, P=0. 008). Conclusions The TIMP-1 mRNA level in PBMC and TIMP-1 protein level in serum are closely related with liver fibrosis stages. These two parameters, especially the TIMP-1 mRNA level in PBMC, can be potentially new markers for diagnosing liver fibrosis.