The Preliminary Study And Functional Verification Of Long Non-coding RNA Involved In The Early Phase Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

Human bone marrow mesenchymal stem cells is derived from human bone marrow multipotent adult stem cells, it has the multipotent differentiation potential and no transplant rejection determine that it has renewable value on the clinical applications in tissue engineering field. In recent years, hBMSCs has large amount of advantages in building its maxillofacial bone defect repairing,so it has become a hot research. There are many reasons can cause facial deformities like Trauma, tumor, congenital malformations and other defects. not only causing the facial deformities, but also influenting dysfunction such as language, chewing, breathing. And it also distress the patient’s physical and psychological. Therefore, when we try to repair bone defects,we should take into account two aspects:functionality and aesthetics. Traditional bone defect repair method is generally divided into three categories: synthetic bone graft, autogenous bone graft and allograft. Due to the above-described repair method have their own drawbacks, people is not satisfactory with the results. But the bone tissue engineering involved in bone defect repairing provides a new method to avoid drawbacks that mentioned above.About the bone tissue engineering research,it always focus on seed cells, extracellular matrix material, growth and differentiation factors. The choice of seed cells is the main source of tissue engineering of the active ingredientand it also the most basic factor. Currently, the most widely used seed cells is human mesenchymal stem cells.Recent studies have found that LncRNA and human bone marrow mesenchymal stem cells (hBMSCs) may have some connection while osteogeny differentiation. In the process of the development and differentiation of osteoblasts, specific genes put on and off with a specific order to activate and control the different stages of osteogenic differentiation. Early differentiation determines the future development of cells. Thus,the impact of a regulatory mechanism for osteogenic differentiation LncRNA hBMSCs osteogenesis, it will lead a new research direction for maxillofacial bone defect repair. This study will investigate to determine the relationship between hMSCs and LncRNA.Recent studies found that mesenchymal stem cells into osteoblasts is regulated by a variety of molecular mechanisms, such as growth factors (Wnt family of bone morphogenetic protein family), transcription factor (β-catenin, Runx2) and miRNA and so on.The research about mechanism that mentioned above has made significant progress. Some scholars recently found that LncRNA have some connection in osteogenic differentiation hBMSCs, particularly closely related to human osteogenic differentiation.LncRNA is a transcription of non-coding RNA and the length above 200bp. They do not participate in protein coding but formed complex spatial structure.On the level of post-transcriptional or transcription, participating in a specific gene regulation and modification. With advances in detection techniques such as gene chips, RNA sequencing, a large number of LncRNA been identified and confirmed to participate in the regulation of cell differentiation and ontogeny and other important life processes in the post-transcriptional and transcriptional levels. Previous studies show LncRNA involved in regulating neuronal differentiation, muscle differentiation, epidermal differentiation, adipogenic differentiation.LncRNA can affecting the pluripotency markers transcription factors, the results is that it regulate and control the differentiation of embryonic stem cells. Similarly, the occurrence and development of bone differentiation is related to the regulation of transcription or LncRNA bone gene transcription after modification.Moreover, LncRNA in osteogenic differentiation expression,in different periods it have significant changes, we suggest that LncRNA osteoblasts and osteoclasts play a dynamic balance regulation in osteogenic differentiation.Currently, this area has great research space due to the short study period of LncRNA,and the less amount of research reports Concentrated on aspects which differentiate into bone in hBMSCs.Most of the literatures focused on the study of a single LncRNA.In this study, we use human LncRNA microarray technology to detect expression of osteogenic differentiation of hMSCs before and after having twice the amount of the difference and above LncRNA screening, select one large differences LncRNA expression by lentiviral vector interference and overexpression functional verification carried into the bone area. This experiment will be divided into the following three parts:Part I:Identification of osteogenic differentiation capacity of human bone marrow mesenchymal stem cells identification capabilities:ObjectiveHuman bone marrow mesenchymal stem cells (hBMSCs) is derived from human bone marrow multipotent adult stem cells, multipotent differentiation potential and no transplant rejection determine that it has important clinical value in the field of regenerative tissue engineering. In recent years, hBMSCs maxillofacial bone defects gradually become a hot topic, from the early osteogenic differentiation determine the future development of the cells. LncRNA mesenchymal stem cells (MSCs) have connection in the presence of osteogenic differentiation. Therefore, LncRNA for hBMSCs early osteogenic differentiation may have some regulatory function. To determine the relationship between them, the study will use stem cells osteogenic differentiation medium, to make it to the direction of bone cells after induction of differentiation were identified as osteogenic differentiation for LncRNA screening LncRNA functional verification preparation.Methods1. The purchase of the primary hBMSCs in vitro amplification culture After 7 days of osteogenic induction and differentiation, the P5 generation of hBMSCs was observed in the cells.2. HBMSCs was induced into osteogenic differentiation, and the hBMSCs was recorded as the experimental group (D7).After 7 days of differentiation,DO was compared with the hBMSCs before the differentiation of bone, and the control group (the control group) was recorded. The alkaline phosphatase (ALP) staining and alizarin red staining method, identification of the results of induction and differentiation of bone.3. Detection of osteoblast-specific transcription factor (Osterix, ALP, OPN) expression by real-time PCR to identify osteoblasts results.ResultsThe original hBMSCs were cultured in vitro to P5 after generation of cell growth in good condition, the shuttle showed long lines. After osteogenic induction medium osteogenic differentiation, the cells become short cords polygonal cells to accelerate cell proliferation after gathering the form of pellets. Then proliferation rate decreases, showing a small amount of brown nodules.2.ALP staining showed that after induction of cell surface adhesion 7d hBMSCs dark blue dye; without induce hardly adhere dye; D7 group compared with the group D0, D7 group has significant ALP staining situation. ALP staining showed that, hBMSCs 7d after osteogenic differentiation induction can show certain osteogenic differentiation. Alizarin red staining showed that after induction of cell surface 7d hBMSCs visible nodules of calcium deposits; without induced no calcium nodules; D7 group compared with the group D0, D7 group has obvious alizarin red staining Happening.3.5th generation hBMSCs osteoblast differentiation induction 7d extract total RNA, 2-ΔΔCT analysis result, the data show that after hBMSCs 7d induced osteoblast-specific transcription factor ALP, OCN and Runx2 expression of an uptrend, the difference there was statistically significant (P<0.05). Among them, the most obvious is the increase ALP, ALP is hBMSCs osteogenic genes play a role in the early days, the results illustrate hBMSCs osteogenic differentiation of bone induction ability 7d for ALP plays a major role.Part II:Screening and validation of differential expression of LncRNA in human bone marrow mesenchymal stem cells before and after osteogenic differentiationObjectiveThe early hBMSCs osteogenic differentiation for 7 days, LncRNA microarray screening test to LncRNA before and after osteogenic differentiation differentially expressed.At the same time,selected more than four LncRNA conduct further explore the role of LncRNA in hMSCs osteogenic differentiation process. In order to further the role of LncRNA in the osteogenic differentiation process, we will construct the slow virus vector of LncRNA, and prepare for the change of the osteogenic efficacy after hMSCs transfection.Methods1. In this study, DO and D7 group cell samples were extracted with Trizol reagent for total RNA, Total RNA quality inspection qualified for in vitro amplification and fluorescence labeling, labeling process with Jingxin biochip general Labeling Kit (CapitalBio). The product is labeled in 45 ℃ with Agilent’s 4 x 180 K people lncRNA V3.0 chip (containing 37,581 people lncRNA specific probe and 34,235 people mRNA probe) hybridized overnight. And using the matching software Feature Extraction Agilent (v10.7) for the analysis of the picture, and then extract data. Then use CeneSpring Agilent software to carry out the normalization and difference analysis of the data (not less than 2 times the difference multiple).the image is analyzed and extracted data. Then use the Agilent CeneSpring software for data normalization and variance analysis (difference of not less than 2 times the multiples).2. From the large difference in the expression of LncRNA, select 4 LncRNA to verify, two of which raised the other two down, by quantitative PCR identification accuracy of the results. At the same time, select these four LncRNA for further functional verification.Results1.5th generation hBMSCs osteoblast differentiation induced 7d were LncRNA microarray. It was found that after osteogenic differentiation induction 7d, where more than 2-fold upregulated LncRNA have 923, down more than 2 times there 993; It shows that raised more than four times the LncRNA have 225, down more than 4 times there 120; raised more than eight times the LncRNA have 60, down more than 8 times have 17 bars. Positive clones were sequenced delivery life technologies company reaction, sequencing results consistent with the expected results.2.RT-PCR validation results showed that compared with the DO group, LncRNA ENST00000523786.1 and LncRNA ENST00000436715.1 (H19) expression was significantly up-regulated (P<0.05) in group D7; LncRNA HIT000218960, LncRNA ENST00000502125.2 group in D7 expression was significantly lower (P<0.05). LncRNA microarray results are consistent with the results of real-time PCR.Part III:Study on function of LncRNA ENST00000502125.2ObjectiveThis section will provide access to information LncRNA sequence according ENSEMBL database, by extracting total RNA hMSCs cells, RT-PCR to obtain the desired DNA fragment was constructed LncRNA lentiviral vector transfected clones cells give lentivirus particles transfected hMSCs, explore LncRNA ENST00000502125.2 in human bone marrow mesenchymal stem cell osteogenic differentiation of important functions, observe the changes in its osteogenic efficacy. Predict the mesenchymal stem cells in human bone marrow into the molecular mechanism of bone differentiation, LncRNA provide new targets for bone tissue engineering research will reveal LncRNA molecular regulation of specific sites osteogenic differentiation of hMSCs.Methods1.According to the ENSEMBL database to provide information LncRNA ENST00000436715.1 (H19) length of 861bp, the length of LncRNA HIT000218960 is 229bp, LncRNA ENST00000502125.2 length of 1930bp.2 Extraction hMSCs (P5-generation) cell total RNA by Trizol method, RT-PCR to obtain the desired DNA fragments.3. Design a suitable primer to obtain the desired DNA sequenced to confirm the correct late lentiviral vectors.4. Select which LncRNA amplification can be effective, according to preliminary microarray experiments LncRNA sequence information obtained primers were designed, constructed LncRNA overexpression and shRNA lentivirus vector vector after sequencing to confirm the correct insert fragment, a large number of plasmid, transfection 293TN cells for packaging recombinant virus was observed by GFP lentiviral vector transfection efficiency, more than 80% of the cells with green fluorescent qualified were constructed LncRNA negative control lentivirus particles.5. The gain of function studies:experimental groups:Select LncRNA ENST00000502125.2 overexpression lentivirus vector was transfected hMSCs quantitative detection of overexpression lentivirus vector transfection efficiency after osteogenic medium for 7 days, alkali phosphatase staining and alizarin red staining and detection by Real-time PCR to check LncRNA ENST00000502125.2 expression.control group:the empty vector transfected hMSCs cultured in osteogenic medium for 7 days, alkaline phosphatase staining, alizarin red staining and by Real-time PCR detection of LncRNA ENST00000502125.2 expression.6. loss of function studies:experimental groups:LncRNA ENST00000502125.2-lentivirus RNAi vector transfected hMSCs quantitative detection of lentivirus RNAi vector transfection efficiency after osteogenic medium for 7 days, be alkaline phosphatase staining, alizarin red staining and detection by Real-time PCR to check LncRNA ENST00000502125.2 expression, control group:the empty vector transfected hMSCs cultured in osteogenic medium for 7 days, alkaline phosphatase staining, alizarin red staining and by Real-time PCR to check LncRNA ENST00000502125.2expression.Results1.LncRNA ENST00000436715.11 and HIT000218960 use TAKARA onestep kitto do RT-PCR, after completion of the reaction, take 2ul electrophoresis, fragment size and ENSEMBL database information is inconsistent, the experiment was repeated three times, the result is consistent.2. Real-time PCR primers LncRNA detection, LncRNA ENST00000436715.1 and LncRNA HIT000218960 failed to amplify the expected product does not match with the theoretical amplification product was considered low LncRNA abundance or hMSCs subcultured to 5 generations after the above two LncRNA is not expressed.3. LncRNA ENST00000502125.2 synthetic primers as a template hMSCs RT-PCR reactions, LncRNA ENST00000502125.2 can be effectively amplified.4 LncRNA ENST00000502125.2 RT-PCR after effective amplification and recover LncRNA products. NheI+NotI enzyme cut ENST00000502125.2 LncRNA product, and the same treatment of pCDH-CMV-MCS-EF1-coGFP carrier connection, the transformation of E. coli DH5a, After that,colony PCR with primers obtained 9 positive clones of LncRNA ENST00000502125.2.5. The positive clones were sequenced delivery life technologies company reaction, sequencing results are consistent with the expected results.6. The control vector and an interference vector virus packaging,293NT transfected cells, using puromycin, the cells were collected, expressing cells in each group LncRNA ENST00000502125.2 detected by quantitative PCR methods. Interference sequence did not affect virus packaging, viruses can interfere with a good transfected cells 293NT, quantitative results show sequence showed some interference effects.7. The results of overexpression lentivirus vector transfection efficiency display: transfected cells expressing viral LncRNA ENST00000502125.2 transcript levels increased significantly, which successfully passed the lentivirus transfected hMSCs obtained LncRNA ENST00000502125.2 cells in the outer endogenous expression.8. After ALP, alizarin red staining shows that in hMSCs transfected into overexpressed LncRNA ENST00000502125.2 and after induction of differentiation into bone, hMSCs osteogenic differentiation capacity was significantly weaker than in the control group, which describes LncRNAENST00000502125.2 in the hMSC osteoblastic differentiation plays a negative correlation, ie inhibition of osteogenic differentiation.Conclusions1.hMSCs under appropriate in vitro environment can be induced osteogenic differentiation into osteoblasts.2. LncRNA high-throughput microarray technology to screen out hMSCs osteogenic differentiation of bone differentiation for 7 days before a significant difference between the expression of LncRNA, and draw LncRNA expression profile characteristics, LncRNA expression by real-time PCR validation part of the difference, Exclude chip false positive results.3.LncRNA ENST00000502125.2 in hBMSCs osteogenic differentiation induced expression decreased, indicating that it is in hBMSCs osteogenic differentiation plays a negative role.4. The results showed that hBMSCs was enhanced after transfection with ENST00000502125.2 LncRNA slow virus vector. The results showed that the expression of hBMSCs was enhanced, and the transfection of the vector was reversed. It shows that ENST00000502125.2 LncRNA has the ability to inhibit the osteogenic differentiation of hBMSCs.