| Background:MicroRNA(miRNA)controls the expression of target genes by degrading or inhibiting messenger RNA translation and interacts with DNA methylation modification.Studies have found that miRNA is involved in differentiation process of of bone marrow mesenchymal stem cells(hBMSCs)on the roughed titanium surface.In the process of stem cell differentiation,some miRNAs can act as tumor suppressor genes to regulate balance of gene function,and prevent cell malignancy.Tongue squamous cell carcinoma(TSCC)is one of the most common malignant tumors in the oral and maxillofacial region and the prognosis is poor.At present,the treatment of TSCC is still based on surgery.Patients often remove part or whole of their j aws due to the need for radical treatment,which seriously affects the quality of life of patients.Previous studies have shown that abnormal miRNA expression and DNA methylation can regulate the occurrence,development and metastasis of tumors.Some miRNAs are important in the osteogenic differentiation of stem cells and involved in tumors at the same time.Therefore,exploring the expression mechanism of miRNA in hBMSCs and TSCC is of great significance to the treatment of oral and maxillofacial surgery.Purpose:(1)To analyze the osteogenesis status and differentially expressed genes of hBMSCs on the roughened titanium surface and verify the expression of target gene and its osteogenic effects.(2)Explore the expression and mechanism of target gene in TSCC.Material and methods:(1)hBMSCs cell lines:Titanium specimens were divided into SLA group(test)and machined smooth group(control).The surface topography of the Ti surfaces were characterized using a scanning electron microscope.After osteogenic induction over a period of 7 days,the hBMSCs were immunostained and the fluorescence signal was observed and imaged using a confocal laser-scanning microscope.The degree of osteogenic differentiation was determined by alkaline phosphatase activity,Quantitative real time polymerase chain reaction(qRT-PCR)and Western blot of osteogenic marker genes.Expressions of mRNA and miRNA in hBMSCs seeded onto the SLA and control were identified by microarray and combined with DNA-methylation profiles at day 7.(2)TSCC cell lines:The relative expression of miR-29b in 60 paired primary TSCC samples and adjacent normal tissues was calculated by qRT-PCR.Analyse the luciferase activity of the the wild-type 3’UTR or mutant 3’UTR of DNMT3B with overexpression of miR-29b in CAL27 and SCC-15 cells.Analyse the mRNA and protein levels of DNMT3B in upon miR-29b overexpression.Analyse the miR-195 expression and methylation status of CpG islands upstream of miR-195 upon miR-29b overexpression and knocking down DNMT3B.Results:(1)hBMSCs cell lines:Relative expression of osteogenic marker genes in hBMSCs on the SLA treated Ti surface was significantly higher than that on the smooth Ti surface.2846 sites were significantly differentially methylated(covering 1841 genes).The osteogenesis-related genes IGF2/CPXM2 showing significant up-regulation/down-regulation in hBMSCs on SLA Ti surface(p<0.05).Different miRNA expression with 147 miRNAs downregulation and 13 upregulated.The differentially expressed miRNAs,with 15 of them associated to differentialy methylated sites,are associated with Wnt,cell junctions and pathways related to cancer and Proteoglycans in cancer.It was verified by qRT-PCR that miR29b was significantly highly expressed in hBMSCs on the roughened titanium surface at 7d(p<0.01),and the osteogenic factor Runx2 was significantly higher expressed after miR-29b was overexpressed in hBMSCs(p<0.05).(2)TSCC cell lines:Our data showed that miR-29b were significantly downregulated in TSCC compared with their matched nonmalignant tissues in 60 paired samples(p<0.05).miR-29b overexpression induced the demethylation of CpG islands upstream of miR-195 via targeting DNMT3B(p<0.01),leading to the upregulation of miR-195 in TSCC cell lines(p<0.05).Following DNMT3B silencing,the expression of miR195 was increased(p<0.01)and the methylation of CpG islands upstream of miR-195 was reduced.Conclusion:(1)Compared with the smooth titanium surface,hBMSCs on the roughened titanium surface had better osteogenic differentiation and there were significantly differently expressed methylation sites and miRNAs.The differentially expressed INS-IGF2 and CPMX2 genes were verified.High expression and possibility of osteogenesis of miR-29b was verified Differentially expressed miRNAs are enriched in osteogenic differentiation and tumor-related signal pathways;(2)In TSCC miR-29b is significantly down regulated,and miR-29b can regulate the expression of other genes by combining with DNMT3B in SCC-15 and CAL27. |
