Variation Of OCT4 Promoter Methylation During Thy-1+Lin-Rat Bone Marrow Mesenchymal Stem Cells Differentiation Into Hepatocytes In Vivo

Our country is a hepatitis B superpower, which now hepatitis B carriers is up to 120 million, and 30 million patients with chronic hepatitis B. A significant number of patients with chronic hepatitis B break out repeatedly and chronic unhealed. Finally can be evolve for end-stage liver disease such as liver cirrhosis, portal hypertension, liver cancer and so on, at the same time affect the quality of life seriously. The most effective treatment for End-stage liver disease is liver transplantation, but need to face the shortage of donor liver source, treatment cost huge, ethical approval, and other issues. Currently emerging stem cell transplantation, means that via the autograft or allograft sources of stem cells after in vitro operation implant into the human body, for using disease treatment process. Such actions include stem cells in vitro separation, purification, amplification, modification, the establishment of the stem cells department, differentiation, frozen storage and recovery process. Stem cell transplantation that can effectively reverse liver function for the treatment of end-stage liver disease, and has obtained certain result in a small scale in small scale clinical trials, thereby serving as a bridge for the majority of patients waiting for transplantation, even reach the purpose of radical cure. At the same time, it can avoid the problem such as allograft rejection, ethics, and provides a new way of thinking for the treatment of end-stage liver disease.At present, there are a variety of protocols of bone marrow mesenchymal stem cells differentiation into all kinds of adult cells. Our experimental group had been screened out the bone marrow mesenchymal stem cells which has the phenotype to differentiation into hapetocytes. In vertical differentiation of stem cells, and its regulatory mechanism is very complex and regulated by the various factors of control network. Pluripotent OCT4 gene is in the vertex of stem cells regulation network which determined the fate of the stem cells proliferation and differentiation, and involved in the LIF, Wnt and Tgf-β, and other important regulating pathways with a variety of target genes together. Now a large number of experiments show that the epigenetic modification is closely related to the development of stem cell proliferation and differentiation, tumor evolution, its modification including the methylation of DNA and histones, acetylation, phosphorylation, ubiquitin, etc. While stem cell transplantation has achieved remarkable resμlts in a large number of animal experiments and a small scale clinical trials, however, there are also studies have shown that stem cells also exist tumorigenicity during differentiation. Therefore, the regulatory mechanism of bone marrow mesenchymal stem cells differentiation still need more in-depth researches.DNA methylation as the most common way of epigenetic modification, this article, from the aspects, explored the variation of OCT4 promoter in the process of rat bone marrow mesenchymal stem cells differentiation. Pyrosequencing was performed to dectect methylation frequency of all CpG sites in OCT4 promoter, in order to further reveal epigenetic regulation mechanism of bone marrow mesenchymal stem cells, providing more theory supports for application of stem cell transplantation in clinical more safer in the future.1. Materials1.1 Experimental cellsThe bone marrowmesenchymal stem cells extracted from vistar male rats, and phenotype Thy-1+Lin- was separated out by density gradient centrifugation and magnetic activated cell sorting. That is Thy-1+Lin- subgroup rat bone marrow mesenchymal stem cells(Thy-1+Lin-RBMMSCs), and were persevered in liquid nitrogen.1.2 Reagent and instrumentHigh sugar DMEM F12 medium(America, Hyclone),10% Australian premium fetal bovine serum(Isreal, Biolnd), Penicillin-Streptomycin(America, Gibco),0.25% trypsin -EDTA(America, Gibco), Recombinant human hepatocyte growth cytokines(America, Peprotech), dexamethasone(America, Gibco), DNA extraction kit(America, Promega), RNA extraction kit(America, Omega), Epitect bisμlfite kit (Germany, Qiagen), dNTP(China, Shenzhen genomics), Taq polymerase(America, KAPA), Pyrosequencing kit(Germany, Qiagen), cDNA first strand synthesis kit (Switzerland, Roche), qRT-PCR kit(Switzerland, Roche), PLATINUM Gel imaging system(Britain, UVItec), ND-1000 spectrophotometer(America, Nanodrop), ABI 9700 PCR instrument(America, Applied Biosystems), LightCycler 480IISystem PCR instrument(Switzerland, Roche), Inverted fluorescence microscope(Japan, Olympus), PyroMark Q96 ID pyrophosphate sequencing instrument(Germany, Qiagen).2. Method2.1 Cell culture and induction2.1.1 Cell cultureWe cultured the Thy-1+Lin- RBMMSCs persevered in liquid nitrogen after recovery ing in basal medium containing high sugar DMEM F12 medium supplemented with 10% Australian premium fetal bovine serum (Biolnd, Israel), Penicillin-Streptomycin (America, Gibco). The cells were incubated at 37℃ in the presence of 5% CO2. The medium was replaced once in 3 day, and the cells were subcultured using 0.25% trypsin-EDTA (America, Gibco) digestion after reaching 80% confluence.2.1.2 Cell inductionThe induction protocol published by our research group in pre-study was adopted, which using recombinant human hepatocyte growth cytokines and dexamethasone as inducer. The third generation of cells grew well were with induction medium containing basal medium supplemented 25ng/ml hepatocyte growth factor (HGF) (America, Peprotech) and 10-7mmol/L dexamethasone (America, Gibco). The medium was replaced once in 3d until to 14d. During this differentiation period, the morphology of the cells was observed dynamically and recorded by inverted microscope(Japan, Olympus) in the four stages: non-differentiation, Od,7d, and 14d. At the same time, samples (at least containing 1×107 cells) in each timing were reserved in liquid nitrogen container for future experiment. The non-differentiation cells were set as negative control group, other groups were set as Od group,7d group,14d group according to point-in-time of cells differentiation. Repeat cases were 3.2.2. The detection of relative gene mRNA during Thv-1+Lin- RBMMSCs differentiation2.2.1 RNA extractionRNA was extracted from each 25cm2 culture bottle (about 1×107) in every group using RNA extraction kit (America, Omega) according to manufacturer’s instruction. RNA concentration and quality were detected by ND-1000 spectrophotometry(America, Nanodrop). All the samples’OD was greater than 1.9, and persevered in -20℃ for standby application.2.2.2 First chain cDNA synthesisPrimers were designed and compounded by Shanghai Sangon Company. ALB primer:upstream was 5’-TACACCCAGAAAGCACCTCA-3’and downstream was 5’-TACACCCAGAAAGCACCTCA-3’. OCT4 primer:upstream was5’-GGACACCTGGCTTCAGACTT-3’ and downstream was5’-TCCCTCCACAGAACTCGTATG-3’.GADPH primer:upstream was5’-ACCACAGTCCATGCCATCAC-3’and downstream was5-TCCACCACCACCCTGTTGCTGTA-3’. All reagents in cDNA first strand synthesis kit (Switzerland, Roche) after melting on ice,20μl reaction system was confented according to manufacturer’ instruction and was performed by ABI 9700 PCR instrument(America, Applied Biosystems). Reaction parameter was set as 25℃ 10min,50 ℃ 60min,85 ℃ heating 5min to inactivated reverse transcriptase. Then taking ice-bath immediately. It could stored in -20℃ for a long time.2.2.3 Quantitative real time PCR(qRT-PCR) reactionAll reagents in qRT-PCR kit (Switzerland, Roche) after melting on ice, and 50μl reaction system was confented according to manufacturer. Then close 96 orifice by Seal plate membrane,1500×g centrifuge 2 min.96 orifice was put in Roche LightCycler 480 PCR instrument(Switzerland, Roche)to perprerate reaction. Reaction parameter was set as 95 ℃ denaturation 10s,60 ℃ annealing 20s,72 ℃ extension 20s,45 cycles amplification. Relative quantitive method was adopt to calculate PCR results.2.3 Analysis of OCT4 promoter methylation2.3.1 DNA extractionDNA was extracted from each 25cm2 culture bottle (about 1×107) in every group using DNA extraction kit (America, Promega) according to manufacturer’s instruction. DNA concentration and quality were detected by ND-1000 spectrophotometry(America, Nanodrop). All the samples’OD was greater than 1.6, and persevered in -20℃ for standby application.2.3.2 DNA bisulfite conversionAccording to the instruction of Epitect bisulfite kit (Germany, Qiagen), all reagents were added into 200μl PCR tube and placed in room temperature after thorough blending. DNA conversion reaction was performed by PCR instrument. Reaction parameter was set as 95 ℃ denaturation 5min,60 ℃ renaturation 25min,95℃ denaturation 5min,60 ℃ renaturation 85min,95℃ denaturation 5min,60 ℃ renaturation 175min, Spending at night in 20 ℃ cooling.2.3.3 Methylation PCR reactionThe OCT4 core promoter region was selected from upstreamof transcription start sites -364bp to+1bp, containing 4 CpG sites, and the 4th CpG site is GC box. Primers was designed by software PyroMark Assay Design 2.0. The distance between promoter CpG sites was considered longer, and therefore, divided into three sections to ensure veracity of sequencing. Primer of 1st section:forward was5’-GAAGGTTTATTTGGTTGT-3’, reverse was5’-CCAATCCCACCCTCTAACCTTAA-3’and sequencing was5’-GAAGGTTTATTTGGTTGT-3’. Primer of 2nd section:forward was5’-GAGGAATGTGGAGAAGTGAAAGAGATAG-3’,reverse was5’-GAGGAATGTGGAGAAGTGAAAGAGATAG-3’and sequencing was 5’-AGTTTTTTAGATTTTAGGTAAATT-3’. Primer of 3rd section:forward was 5’-TGGAGAAGTGAAAGAGATAGAGT-3’, reverse was 5’-CCAATCCCACCCTCTAACCTTAA-3’and sequencing was 5’-CTTAACCTCTAACCCC-3’.50μl reaction system was confented according to manufacturer’ instruction and was performed by ABI 9700 PCR instrument. Reaction parameter was set as 95℃ pre-heating 3min,94℃ denaturation 3min,50℃ annealing 30s,72 ℃ extenstion 1min,40 cycles amplification,72 ℃ cooling 7min. PCR product quality was detected by Agarose gel electrophoresis.2.3.4 Pyrosequencing2p.1 reaction combinded beads was added into 96 holes PCR reaction plate according to instruction. Then combinded beads on the probe and product of methylation PCR were put into annealing buffer containing 1.5μl sequencing primers, 85 ℃ denaturation 2min, cooling to room temperature, to mark primers annealing hybrid with template. The substrate mixture, enzyme mixture and 4 kinds of dNTP(China, Shenzhen genomics) were added into reagent compartment successively. Reagent compartment and 96 holes PCR reaction plate were put in PyroMark Q96 ID pyrophosphate sequencing instrument(Germany, Qiagen) to perform sequencing according to the manufacturer’s instructions. The methylation frequency of all CpG sites was analyzed automatically by software Pyro Q-CpG.2.4 Data analysisStatistical analysis software SPSS version 19.0 was used to calculate experimental results. Gene expression and methylation frequency were calculated by one-way analysis of variance(one-way ANOVA), when homogeneity of variance using LSD testing and teterogeneity of variane using Dunnett T3 testing. P<0.05 means difference was statistical significantly.3. Results3.1 The morphology of Thy-1+Lin RBMMSCs differentiationThy-1+Lin-RBMMSCs after recovery were spread equally, transparent, uniform and oval in basal medium. About 24h later, most of the spindle-shaped RBMMSCs were clustered, and colonies showed circinate pattern. After altering induction medium, RBMMSCs did not presented remarkable change in Od. In 7d, the cells differentiation into a spindle or polygon shape without any swirling. The cells presented more circular and organelles increased with prolonged culture to 14d.3.2 OCT4 and ALB mRNA expression during Thy-1+Lin- RBMMSCs differentiationAfter altering induction medium, ALB and OCT4 mRNA expression in Od were no apparent change compared with non-differentiation group. ALB mRNA expression in 7d and 14d were 5.22 times(P=0.025) and 14.7 times(P=0.003) higher compared with the non-differentiation group. However, OCT4 mRNA expression was significantly decreased to 0.23 times(P=0.000) and 0.055 times(P=0.000) accordingly.3.3 Analysis of OCT4 promoter methylation frequencyOCT4 promoter was subjected to methylation bisulfite sequencing during Thy-1+Lin-RBMMSCs induced into mature hepatocytes. In these bisμlfite sequencing maps, sequencing peaks were distinct, with limited background interference and sequence resμlts were identical with that of GeneBank data. Former 3 CpG sits of upstream presented different methylation frequency in the predicted 4 CpG sites. We set a limit value of 20% with that CpG methylation frequency above this limit considered as hypermethylation, and below 20% as hypomethylation. The methylation frequency at CpG sites 1-2-3 was increased constantly during cells differentiation.7d group was significant higher(P=0.000) than Od and non-differentiation groups; 14d group was significant higher(P=0.000) than 7d, Od and non-differentiation groups. But CpG 4 was no statistical significance(P>0.05) in all groups, means that GC box were no apparent change.4. Conclusion1. During Thy-1+Lin-RBMMSCs induced into mature hepatocytes, OCT4 mRNA expression sustain decrease, on the contrary, the methylation frequency of OCT4 promoter increase. It means that OCT4 mRNA expression may mediated by its promoter methylation, its promoter hypermethylation may inhibit mRNA expression, which may involve into regulation segment of stem cell differentiation.2. CpG 4, that is GC box had no obvious variation during Thy-1+Lin-RBMMSCs differentiation. It means that GC box may not mediate the regulation of Thy-1+Lin-RBMMSCs differentiation.