| BackgroundCaries are a group of diseases that occur in the hard tissue of teeth and are mainly caused by four linked factors(i.e.bacteria,oral environment,host and time).It is a common disease.WHO has listed the dental caries,tumor and cardiovascular diseases as three key diseases that need prevention and treatment.Delayed treatment of dental caries will affect pulp,causing pulpits or periapical periodontitis.The pulp tissue is located in the pulp cavity inside the tooth,which mainly contains nerve,blood vessel,lymph and connective tissue,etc.It has a vascular system but no collateral vessels.Besides,it lies in the hard dentin and is unable to withstand volume change.Therefore,when inflammation occurs,irreversible inflammation easily occurs.Root canal therapy is currently the most effective treatment for pulpal and periapical diseases.However,due to the complexity of root canal system,many complications may occur during and after treatment.Therefore,pulp tissue engineering is now a research hotspot.Diabetes mellitus is a highly prevalent metabolic disease characterized by hyperglycemia caused by insufficient insulin secretion and function.Studies have proved that hyperglycemia is associated with long-term damage,dysfunction or failure of various organs,especially eyes,kidneys,nerves,heart and blood vessels.In mouth diseases,diabetes mellitus is closely related to periodontal disease,but whether it is related to dental pulp disease has not been determined yet.Diabetic patients are susceptible to bacterial or opportunistic infections,which may cause widespread circulatory disorders.Thus,atherosclerosis deposits mainly in vascular lumen,especially in capillary tissue.The main manifestation was basement membrane thickening.This change reduces the microbicidal activity of polymorphonuclear leukocytes and prevents the transmission of humoral and cellular components of the immune system.The main blood vessels in dental pulp are microvessels.Whether this pathological phenomenon can also occur in dental pulp of diabetic patients remains to be further studied.Calcification is a common pathological phenomenon in dental pulp tissue.Several studies have found that pulp blood flow in diabetic patients decreases[1].The incidence of intramedullary calcification is higher[2].Its calcification is mostly sickle-shaped.Increased expression of osteopontin and alkaline phosphatase in rat dental pulp cells induced by high glucose has also been reported[2].These two indices can be used as important biological markers for calcification of dental pulp tissue.These studies have confirmed that high sugar microenvironment may lead to calcification of dental pulp tissue.Meanwhile,pulp calcification is one of the disadvantageous factors in root canal therapy,which may lead to failure of root canal therapy.Therefore,the connection between hyperglycemia and dental pulp tissue deserves further study.Long non-coding RNA(LncRNA)is a kind of RNA that does not participate in protein coding.Besides binding with DNA and protein,LncRNA can also form complex through base pairing with other RNA and participate in many physiological and pathological processes.In recent years,LncRNA has been a research hotspot,but its mechanism is still not fully clear.Salmena put forward the hypothesis of competitive endogenous RNA(ceRNA)in 2011[3].This hypothesis was first proposed that miRNAs as the Core Network was composed of LncRNA,circular RNA(circRNA)and RNA.LncRNA can play a role in regulating RNA by competing for one or more miRNAs.Therefore,in recent years,the research on LncRNA has focused on its role as ceRNA to regulate various physiological and pathological processes.Metastasis-associated lung adenocarcinoma transcript 1 MALAT1 is an important member of the LncRNA family.Some studies have found that MALAT1 has obvious expression changes in high glucose microenvironment.MALAT1 can participate in the occurrence of diabetic complications caused by high glucose,and regulate this process through miRNAs.Cell mineralization is a complex process.It has been found that MALAT1 can regulate this process.It can regulate the expression of many mineralization-related factors,such as ALP,RUNX2,OPN and OCN.Although there have been many studies on MALAT1 in recent years,and it has been found that MALAT1 participates in many biological processes,its expression in dental pulp cells has not been reported.The regulatory mechanism of MALAT1 is still unclear,and its role in mineralization process has not yet been determined.To sum up,we designed this research project to culture dental pulp cells in highglucose microenvironment,explore the possibility of mineralization and the role of LncRNA-MALAT1 in this process,and further explore the mechanism of MALAT1 as ceRNA to regulate the mineralization of dental pulp cells,and understand the effect of high-glucose on dental pulp tissue.It can also be used as a supplement to the mechanism of mineralization of dental pulp cells,and lay a foundation for exploring more effective treatment methods in the future.Materials and MethodsChapterⅠ Effects of high glucose microenvironment on proliferation,cloning and mineralization in DPCsHealthy and complete teeth extracted due to orthodontics or impacted extraction in clinical patients who aged between 19 and 25 years old.We cultured the DPCs by modified enzyme digestion tissue block method.In the primary culture from 4d to 10d,DPCs could be observed beside the tissue block and growing adherently to the culture flask.When the cell density reached 80%,cell passage cultivation was carried out.The positive rates of CD44,CD29 and CD90 in DPCs were detected by flow cytometry to identify the DPCs.The P3 DPCs were used for MTT proliferation experiment.The cells were divided into high-glucose group(DMEM containing 10%fetal bovine serum and 50mmol/L glucose)and control group(DMEM containing 10%fetal bovine serum).The time points were 1d,3d,5d and 7d.We set up six parallel holes at each time point in each group,and OD values at 490 wavelengths of each hole were detected by micrometer.We used P3 DPCs in clone formation experiment,after pancreatic enzyme digestion and inoculated the DPCs in the density of 103 cells in 60 mm dish.And DPCs divided into high-glucose group(DMEM containing 10%fetal bovine serum and 50 mmol/L glucose)and control group(DMEM containing 10%fetal bovine serum).In the 1d and 7d after culture,we observed the colony formation under the inverted microscope.In the 14d after culture,the DPCs were fixed by 4%paraformaldehyde fixed at room temperature and stained by crystal violet,then we observed colony formation under the inverted microscope and calculate the number of colony formation.The DPCs were divided into high-glucose mineralization induction group.After 7d and 14d induction,alizarin red staining,qRT-PCR and Western blot experiments were performed.1.Alizarin red staining:After 7d and 14d of mineralization induction,4%paraformaldehyde was fixed at room temperature and stained with 2%alizarin red.The formation of mineralized nodules was observed under the inverted microscope and photographed.Alizarin red staining was eluted with cetylpyridine chloride and its OD value at the wavelength of 562 was detected for semi-quantitative analysis.2.qRT-PCR:After 7 and 14 days of mineralization induction,RNA was extracted and reverse transcribed,the expressions of mineralization related factors,like TGF-β1,TGF-β2,BMP2,BMP4,BMPR1,SMAD2,RUNX2,MSX2,SP7,ALP,DMP1 and DSPP were detected by qRT-PCR.3.Western blot:After mineralization induction,the protein was extracted at 7 and 14 days,the expressions of mineralization related factors,like TGF-β,BMP2,BMP4,RUNX2,MSX2,ALP,DMP1 and DSPP were detected by Western blot.Chapter Ⅱ Effect of LncRNA-MALAT1 on mineralization of DPCs in high glucose microenvironmentThe P3 DPCs were used for experiments,after induction of high-glucose mineralization,the expression level of LncRNA-MALAT1 was detected.Then siRNA-MALAT1(divided into the first,second and third strands)and siRNANcontrol were used to carry out instantaneous gene knockout of DPCs using liposome as carrier.At 48h after transfection,the expression level of MALAT1 after gene knockout was detected by qRT-PCR.The P2 DPCs was selected for experiments,and lentiviruses expressing MALAT1 were inhibited according to the third strand of siRNA.To determine the MOI of lentivirus infected cells and the optimal infection conditions.sh-MALAT1 blank vector and sh-MALAT1 was transfected into DPCs after 72h,then use 10%FBS DMEM containing 2μg/mL of purinomycin to remove the untransfected cells.RNA was extracted and transcribed by qRT-PCR on 7 and 14days after mineralization induction to detect the expression of TGF-β1,BMP2,MSX2,SP7,ALP and DSPP.Then DPCs were stained with 2%alizarin red and the formation of mineralized nodules was observed under the inverted microscope and photographed.Alizarin red staining was eluted with cetylpyridine chloride and its OD value at the wavelength of 562 was detected for semi-quantitative analysis.Chapter Ⅲ LncRNA-MALAT1 regulates the mineralization mechanism of DPCsThe P3 DPCs were used for experiments to prepare cell slides.When the cell density was 30%,the medium was changed to high sugar mineralization induction medium.After 14 days,the in situ hybridization experiment was conducted to detect the expression site of MALAT1 in DPCs.The P3 DPCs were selected for experiments,and the cells were divided into control group and high-glucose group.After 14 days of culture,the sequencing experiment was conducted to detect the differential expression of miRNA in DPCs after high-glucose culture and explore the possible pathway.Three up-regulated miRNAs and three down-regulated miRNAs were randomly selected,primers were designed,and the sequencing results were verified by qRT-PCR.The software was used to predict miRNAs that might bind to MALAT1.The P3 DPCs were used in the experiments,after transfected with sh-MALAT1 and induced to mineralization for 7d and 14d,then detected the expression of miR92b-3p.Chapter Ⅳ Effect of LncRNA-MALAT1 on mineralization direction induction in DPCs through miR-92b-3pThe P3 of DPCs was used for experiments to design miR-92b-3p mimics and miR-92b-3p inhibitor.Lipofectamine 2000 was used as the vector for instantaneous transfection of DPCs,including Mimics negative control group,miR-92b-3p mimics,Inhibitor negative control group and miR-92b-3p inhibitor,then the DPCs were inducted to mineralization for 7d and 14d.The expression of miR-92b-3p was detected by qRT-PCR.In addition,the expression of minerality-related factors was detected by qRT-PCR and Western Blot.And these factors include TGF-β1,BMP2,MSX2,ALP and DSPP.Statistical AnalysisStatistical analysis was performed by analysis of T-test by using SPSS 19.0 software.Statistical significance was set at P<0.05.Results1.DPCs were successfully separated and culturedThis experiment used the modified enzyme digestion tissue block method to separate and culture DPCs.And then detected the surface markers by flow cytometry.We tested the molecular markers CD44,CD29,CD90 which the positive expression rate of 99.98%,100%and 99.98%respectively.The results indicated that the cells isolated and cultured were mesenchymal and derived from pulp tissue,which could be identified as DPCs and applied in all subsequent experiments.2.DPCs cultured in high-glucose microenvironment can maintain the proliferation and cloning ability of cellsWe used MTT experiment and clone formation experiment to detect the proliferation and clone formation ability of DPCs.DPCs entered the logarithmic growth stage at 3d,and the high-glucose group entered the plateau stage after 5d,while the control group did not see a significant plateau stage during the experiment.The OD value of control group and high-glucose group at 7d showed significant statistical difference(P<0.05).The cloning formation experiment found that DPCs had the ability of cloning formation under control group and high glucose group.On the second day of inoculation,DPCs were found to be single-cell adherents.At 7d,it was found that both groups of cells were cloned.After 14d staining with crystal violet,the formed clonal structure can appear more apparent,and there is no significant difference between the number of clones formed between the two groups(P>0.05).3.High-glucose microenvironment can promote the mineralization ability of DPCsDPCs mineralized induced 7d and 14d,alizarin red staining found mineralized nodules which is formed by the high-glucose group were more than the other group.When calcium ions are precipitated by cetylpyridine chloride,we detected the wavelength in the OD value of 562,found that the OD value in high-glucose group at 14d was significantly higher than that in control group,and the results are statistically significant(P<0.05).The results of qRT-PCR showed that,on the 7d of mineralization induction,the expressions of mineralization related factors including TGF-β1,TGF-β2,BMP2,BMP4,BMPR1,SMAD2,RUNX2,MSX2,SP7,ALP,DMP1 and DSPP were significantly higher in the high-glucose group than in the control group.All the other factors were statistically significant except for DMP1 and DSPP.At 14d after mineralization induction,the expressions of mineralization related factors except BMPR1 were significantly increased and the results were statistically significant(P<0.05,P<0.01).The expression of minerality-related factors,like TGFβ,BMP2,BMP4,RUNX2,MSX2,ALP,DMP1 and DSPP was detected by Western blot,and the expression was higher in the group with high glucose,the grey value was detected and the results were statistically significant(P<0.01).4.Effect of LncRNA-MALAT1 on mineralization of DPCs in high glucose microenvironmentAt 7d and 14d after mineralization induction,the expression of LncRNAMALAT1 in the high-glucose group was significantly higher by qRT-PCR,and the results were statistically significant(P<0.01).We designed three siRNA-MALAT1 based on this result,and detected the expression of MALAT1 48h after transfection.It was found that all three designed siRNA-MALAT1 could down-regulate the expression of MALAT1 in DPCs,and the results were statistically significant(P<0.01),among which the third strand was the most down-regulated with the most obvious results.According to the results of siRNA transfection,sh-MALAT1 was designed with the third strand,and DPCs was transfected with the best infection condition(MOI was 30 and P infected fluid).DPCs transfected with sh-MALAT1 and blank vector were used to induce mineralization for 7d and 14d.Then we dected the expressions of mineralization-related factors by qRT-PCR.The results showed that the expressions of TGF-β1,BMP2,MSX2,SP7,ALP and DSPP in sh-MALAT1 group were decreased,and the results were statistically significant(P<0.05,P<0.01).The expression of related factors on the protein level was detected by Western Blot,and it was found that the expression of sh-MALAT1 was decreased after transfection.The gray value indicated that the result was statistically significant(P<0.05,P<0.01).Alizarin red staining showed that there were few mineralized nodules formed after transfection with sh-MALAT1.OD value of the calcium ions deposited by shMALAT1 was significantly lower than that of the control group(P<0.01).5.LncRNA-MALAT1 regulates the mineralization mechanism of DPCsSince LncRNA-MALAT1 can promote the mineralization of DPCs,in order to explore its mechanism,the main expression sites of LncRNA-MALAT1 were explored by in situ hybridization experiment.It was found that LncRNA-MALAT1 was mainly expressed in the cytoplasm of DPCs.Next,DPCs were cultured in high glucose,and two groups of the cells were sent to sequencing test to detect the differential expression of miRNAs.The results showed that 134 miRNAs were significantly up-regulated expression,and 115 miRNAs down-regulated expression.Results of Pathway analysis showed that there were 281 signaling pathways involved.We selected three up-regulated miRNAs and three down-regulated miRNAs randomly for verification,and the results of qRT-PCR were consistent with sequencing.miRNAs likely to bind to MALAT1 were predicted by software,both of which contained miR-92b-3p.The expression of miR-92b-3p in DPCs after transfected by sh-MALAT1 was detected by qRT-PCR,and the results showed that the expression of miR-92b-3p was significantly increased after sh-MALAT1 transfected,and the results were statistically significant(P<0.01).6.Effect of miR-92b-3p on mineralization direction induction in DPCsThe DPCs were divided into Mimics negative control group,miR-92b-3p mimics group,Inhibitor negative control group,and miR-92b-3p inhibitor group.After 7d and 14d of mineralization induction,the expression of miR-92b-3p was detected by qRT-PCR,and the expression of miR-92b-3p was significantly upregulated after mimics transfer,while the expression of miR-92b-3p was significantly down-regulated after inhibitor transfer,and the results were statistically significant(P<0.01).Mineralization related factors were detected by qRT-PCR on 7d and 14d after mineralization induction,and the results showed that TGF-β1,MSX2 and ALP were significantly down-regulated in miR-92b-3p mimics on 7d after mineralization induction,but significantly up-regulated in miR-92b-3p inhibitor.On 14d after mineralization induction,TGF-β1,BMP2,MSX2,ALP and DSPP were also significantly down-regulated in miR-92b-3p mimics,but significantly up-regulated in miR-92b-3p inhibitor(P<0.05,P<0.01).Western Blot was used to detect the protein expression at the protein level,and the trend was consistent with the mRNA level.After mineralization,alizizine red staining was performed on the cells in the four groups,and it was found that mineralized nodules in the miR-92b-3p mimics group were less formed,while mineralized nodules in the miR-92b-3p inhibitor group were significantly increased.The semi-quantitative test results showed that their OD values were statistically significant(P<0.05,P<0.01).Conclusion1.The cells were isolated and cultured by modified enzyme digestion tissue block method in this experiment,and subpassage was conducted according to the cell culture method.The surface markers of the cells isolated and cultured were detected,and the markers of the highly expressed mesenchymal stem cells were found,confirming that the cells isolated and cultured were dental pulp cells.2.Using 50mmol/L glucose to simulate the high-glucose microenvironment,it was found that high-glucose could maintain the proliferation and cloning ability of DPCs.Moreover,after the induction of mineralization,DPCs in the high-glucose mineralization group showed high expression of mineralization related factors,which significantly promoted mineralization.3.LncRNA-MALAT1 can be highly expressed by DPCs in a high-glucose microenvironment,and after DPCs were transformed into sh-MALAT1 and induced to mineralization,the expression of mineralization related factors was downregulated,suggesting that LncRNA-MALAT1 plays an important role in regulating mineralization.4.LncRNA-MALAT1 is mainly expressed in cytoplasm under the induction of high sugar mineralization,and it may regulate mineralization through miRNA.5.According to the sequencing results,it can be found that there are multiple miRNAs with differential expressions in the high-glucose induced DPCs,among which miR-92b-3p is down-regulated.LncRNA-MALAT1 can regulate miR-92b-3p through software prediction,indicating that MALAT1 can regulate mineralization through miR-92b-3p as ceRNA,and the expression of miR-92b-3p is significantly up-regulated after inhibiting the expression of MALAT1.6.miR-92b-3p not only participates in the mineralization process of DPCs,but also participates in it through targeted regulation of TGF-β1. |
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