Effects Of Long Noncoding RNA H19 On Cementoblast Differentiation,Mineralization And Proliferation

Tooth cementum is a thin layer of bone-like mineralized tissue,covering the root surface and synthesized by cementoblasts.The two ends of periodontal ligament fibers insert into the alveolar bone and the cementum,which helps to anchor the tooth to the surrounding alveolar bone.Cementum can be absorbed due to some pathological conditions such as periodontitis,inappropriate orthodontic treatments.Defects in the cementum weaken the attachment function and can even lead to tooth loss.Thus,the formation of cementum is considered to be the most critical part of periodontal regeneration.It's also important to study the regulatory mechanisms of differentiation and proliferation of cementoblasts.Long noncoding RNAs(lncRNAs)are defined as transcripts longer than 200 nucleotides without protein coding potential.More and more evidence suggests that lncRNAs play important roles in multiple cellular processes.lncRNA H19(H19)is highly conserved during evolution,suggesting an essential role in biological regulations.Previous studies demonstrated that H19 promoted the differentiation of osteoblasts.Considering the similarity between osteoblasts and cementoblasts,we wondered whether H19 could regulate the cementoblast differentiation.Interestingly,a recent study using RNA sequencing technology indicated that the H19 expression level of periodontitis tissues was much lower than the normal tissue from healthy people.It seems to support our conjecture.Therefore,this study aims to find out the relationship between H19 and cementoblasts and whether H19 could regulate the differentiation,mineralization and proliferation of cementoblasts.Part 1 The expression pattern of lncRNA H19 during cementoblast differentiationObjective: To find out the expression level of H19 during cementoblast differentiation.Methods: An immortalized murine cementoblast cell line OCCM-30 was used and cementoblast differentiation was induced using a mineral induction media containing 5%FBS,50 ?g/mL ascorbic acid and 10 mM Na?-glycerophosphate.RNA was collected at the indicated time(0,2,4,5,7d)using TRIzol reagent.After transcribing into cDNA,quantitative real-time polymerase chain reaction(qRT-PCR)was used to evaluate the expression levels of differentiation-related targets and H19.Results: 1.The ALP,RUNX2 and OSX mRNA levels were up-regulated under mineral induction conditions,which indicated that cementoblast differentiation was successfully induced.2.H19 expression was also increased during cementoblast differentiation of OCCM-30 cells,especially on day 7.Conclusions: H19 was up-regulated during the differentiation of cementoblasts.Part 2 The effects of lncRNA H19 on cementoblast differentiationObjective: To find out whether H19 could regulate the differentiation ability of cementoblasts by knocking down or up-regulating the H19 expression.Methods: OCCM-30 cells were infected with recombinant lentiviruses overexpressing H19.Then puromycin was used to select the stable H19 overexpression cells.Total RNA was collected with TRIzol reagent and H19 expression was detected by qRT-PCR.2.Smart Silencer targeting H19 and its negative control was transfected into OCCM-30 cells with Lipofectamine 3000 reagent.The knockdown efficiency was evaluated with qRT-PCR at indicated time.3.After knocking down or up-regulating H19,the expression levels of RUNX2,ALP,OCN,OSX and BSP were detected with Western Blot and qRT-PCR.Results: 1.Viral infection significantly increased the H19 expression while the transfection with Smart Silencer decreased the H19 expression.2.Overexpression of H19 increased the expression of RUNX2,OSX,ALP,BSP and OCN and knockdown of H19 decreased the expression of cementoblastic markers.Conclusions: H19 promoted the differentiation of cementoblasts.Part 3 The effects of lncRNA H19 on cementoblast mineralization and proliferationObjective: To find out whether H19 could regulate the mineralization and proliferation abilities of cementoblasts.Methods: OCCM-30 cells were cultured in vitro and the following operations were performed respectively after up-regulating or knocking down of H19.1.After 14 d of mineral induction,cells were fixed in 4% formaldehyde and incubated with 0.1% alizarin red staining.Photographs were captured,then 10% cetylpyridinium chloride was used to quantitatively analyze the mineralization degree.2.After 4 d of mineral induction,cells were fixed in 4% formaldehyde and ALP staining was performed.3.After 4 d of mineral induction,total protein was extracted and quantified.ALP activity was measured according to the instructions of an ALP activity kit.4.OCCM-30 cells were seeded into 96-well plates at a density of 5,000 cells per well.MTS assay was performed at 24,48,72,96 h and the OD values were read at 490 nm.Results: 1.The number of mineralized nodules significantly increased in the H19 overexpression group and decreased in the H19 knockdown group.2.Strong ALP staining was observed after the H19 up-regulation.3.The results of ALP activity assay suggested that H19 promoted the ALP activity.4.The results of MTS assay showed that there was no obvious difference of OD values among different groups.Conclusions: H19 increased the mineralization ability of OCCM-30 cells and had no effect on the proliferation ability of cementoblasts.