CN109568565A - Application of the NF90 in the biological agent of preparation regulation mesenchymal stem cell Osteoblast Differentiation - Google Patents

Abstract

The invention discloses application of the NF90 in the biological agent of preparation regulation mesenchymal stem cell (BM-MSC) Osteoblast Differentiation.We have found that rna binding protein NF90 is necessary to the Osteoblast Differentiation of BM-MSC, lncAIS is by enhancing the mRNA stability of transcription factor HOXD8 with NF90 protein binding to promote the Osteoblast Differentiation of BM-MSC in mesenchymal stem cell (BM-MSC)；Therefore, NF90 can be used to prepare to the biological agent of regulation BM-MSC Osteoblast Differentiation, such as comprising being overexpressed NF90 gene or plasmid, recombinant expression carrier, transgenic cell line, the genetic engineering bacterium of shNF90 can be expressed.The invention also discloses the pharmaceutical compositions for preventing and/or treating adolescent idiopathic scoliosis (AIS), it includes a effective amount of NF90 and pharmaceutically acceptable carriers, with the AIS extremely relevant to BM-MSC Osteoblast Differentiation for treating patient.

Description

NF90 is in the biological agent of preparation regulation mesenchymal stem cell Osteoblast Differentiation Using

Technical field

The present invention relates to biomedicine technical fields, specifically relate to rna binding protein NF90 in preparation and regulate and control medulla mesenchyma Application in the biological agent of stem cell (BM-MSC) Osteoblast Differentiation.

Background technique

Adolescent idiopathic scoliosis (Adolescent idiopathic scoliosis, AIS) is a kind of complicated Spinal three-dimensional deformity occurs mainly in during pubertal growth in 10 to 16 years old girls.Scoliosis progress is likely to occur outer See deformity, back pain, dysfunction, the physiological functions problems such as cardio-pulmonary function is limited, in various degree influence will occur in serious person Social activities.It is about 2-4% that epidemiology investigation result, which is shown in whole world AIS disease incidence in teenager, wherein about 10% The people for being diagnosed with AIS needs to treat.The treatment of AIS includes bracing and surgical correction at present；Wherein full-time branch Support therapy may cause back pain and mental handicape, and inevitably be led using the corrective procedure of pedicle screw instrument Serious operation wound is caused, permanent disaster nerve or vascular accident are even caused when screw position is bad.If can The risk of AIS morbidity and its progress is detected as early as possible, it is possible to be taken suitable therapeutic scheme in time, be reduced treatment delay and bring Pain and inconvenience.The molecular mechanism of AIS morbidity is such as explored in new development in the cause of disease and pathogenesis of AIS, it would be possible to More convenient method is provided for AIS detection and evolution prediction and treatment.

Studies have shown that the bone growth of AIS patient is abnormal, and bone mine compared with the control group of gender and age-matched Material density (BMD) continues to decline.The bone amount that nineteen eighty-two Burner et al. reports AIS patient for the first time is reduced.About three/ One AIS patient BMD is poor.It is reported that low BMD is the key that girl's AIS curve progress Prognostic Factors.It is believed that bone amount subtracts It less may be the principal element for leading to AIS deformity of spine.Many researchs confirm that the BMD of AIS patient reduces 27%~38%. In addition, longitudinal follow-up to skeletal maturation is shown, persistently there is bone amount reduction in girl AIS more than 80%, and bone amount is prompted to reduce It may be the lifelong system exception of AIS patient's bone metabolism.

Mescenchymal stem cell (MSC) is present in the matrix of all mammalian organs, can be divided into osteoblast, fat Cell and cartilage cell.In addition, MSC in film and cartilage formed in be all essential.We confirm AIS in the past The MSC of Bone Marrow of Patients (BM) shows the Osteoblast Differentiation ability of reduction.The research of Park et al. further demonstrates our grind Study carefully result.We and other people studies have shown that AIS patient MSC osteoblast, cartilage cell and fat cell development It is abnormal differentiation in the process, in view of functional character of the mescenchymal stem cell (MSC) in bon e formation and re-absorption, it is presumed that The abnormal Osteoblast Differentiation of MSC is related with the pathogenesis of AIS.However, being how to carry out abnormal adjusting to MSC in AIS patient Be still unintelligible.

Non-coding RNA (ncRNA) is referred to as in life entity " dark matter ", the ratio and biology of full-length genome shared by ncRNA The sophistication levels of inter-species have close correlation.The accurate regulation function of the complexity that ncRNA is played in development and gene expression The complexity of genome can be explained, while also recognizing the complexity of life entity from the dimension of gene expression regulation network for people Property opens the new world.More and more evidences show that a series of occurrence and development of major diseases and genetic regulation by non-coding RNAs are unbalance It is related.

Long-chain non-coding RNA (lncRNA) is considered as the transcription that a genoid is more than 200 nucleotide (nt) recently Object does not have protein coding capacity.LncRNA conservative is lower in various species, but compared with protein coding gene More tissue specificity.Studies have shown that lncRNA plays extensive effect in gene regulation and other cell processes. LncRNA participates in various bioprocess, including chromatin modification, transcriptional control, the marking and nuclear translocation.LncRNA passes through a variety of machines Its function of system performance, the adjusting including total transcriptional regulatory, gene expression, the bracket of core or cytoplasmic complex, and and other The pairing of RNA.We are recently reported the self-renewing maintenance that several lncRNA participate in liver-cancer stem cell.Nearest research report Road, several lncRNA adjust their own neighbouring protein coding gene, play in mesendoderm differentiation and heart development Key effect.However, biological action of the lncRNA in AIS pathogenesis is unclear.

NF90 albumen be the most important isomer protein of interleukins enhancer binding factor 3 (ILF3) protein family it One, which interacts with cell interior coding and non-coding RNA and viral beginning, and participation is related to cell development, cell The cell multiplexs function such as period and virus infection.Wherein NF90 is primarily present in nucleus, be a kind of adjusting gene expression or The rna binding protein of stable mRNA.

The present invention attempts to explore lncRNA (i.e. lncAIS) relevant to AIS and NF90 egg in mescenchymal stem cell (MSC) It interaction relationship between white and its is associated with AIS pathogenesis, to provide more convenient method for AIS treatment.

Summary of the invention

The present invention one is designed to provide NF90 and regulates and controls mesenchymal stem cell (BM-MSC) Osteoblast Differentiation in preparation Biological agent in application.It was found that lncAIS is by enhancing transcription factor with NF90 protein binding in BM-MSC For the mRNA stability of HOXD8 to promote the Osteoblast Differentiation of BM-MSC, NF90 is necessary to the Osteoblast Differentiation of BM-MSC；Therefore, NF90 can be used to prepare to the biological agent of regulation mesenchymal stem cell (BM-MSC) Osteoblast Differentiation, such as comprising being overexpressed NF90 gene or plasmid, recombinant expression carrier, transgenic cell line, the genetic engineering bacterium that shNF90 can be expressed.

The present inventor has found in the correlative study to adolescent idiopathic scoliosis (AIS), LncAIS (base Because of symbol: ENST00000453347) it is that significant difference is expressed in AIS Bone Marrow of Patients mescenchymal stem cell (BM-MSC) LncRNA is significantly lowered in AIS patient BM-MSC.The experiment proves that NF90 combines lncAIS in BM-MSC, And the interaction of lncAIS and NF90；We have found that first 10 turns lowered in the BM-MSC of the lncAIS silencing of selection It records in the factor, NF90 specifically binds the mRNA of transcription factor HOXD8, and the mRNA and NF90 of HOXD8 is in BM-MSC Interaction；Present invention demonstrates that lncAIS and NF90 interacts to maintain the stabilization of HOXD8 mRNA in normal BM-MSC Property, to promote the Osteoblast Differentiation of BM-MSC.

In some embodiments of the above-mentioned application of the present invention, the biological agent packet of the Osteoblast Differentiation of the regulation BM-MSC The NF90 gene containing overexpression or plasmid, recombinant expression carrier, transgenic cell line, the genetic engineering bacterium that shNF90 can be expressed.Into One step, in some embodiments of the invention, the overexpression NF90 gene plasmid is pSIN-EF2 or can express shNF90 Plasmid be pSicoR plasmid.Further, in other embodiments of the invention, the sequence of the shNF90 such as SEQ Shown in ID NO:7-9.

Another object of the present invention is to provide the biological agent of the Osteoblast Differentiation of regulation mesenchymal stem cell, packet The NF90 gene containing overexpression or plasmid, recombinant expression carrier, transgenic cell line, the genetic engineering bacterium that shNF90 can be expressed.? In some embodiments of biological agent of the present invention, the overexpression NF90 gene plasmid is pSIN-EF2 or can express shNF90 Plasmid be pSicoR plasmid.Further, in other embodiments of the invention, the sequence of the shNF90 such as SEQ Shown in ID NO:7-9.

Another aspect of the invention provides NF90 in preparation for preventing and/or treating adolescent idiopathic scoliosis (AIS) the application in pharmaceutical composition, wherein described pharmaceutical composition includes a effective amount of NF90 and pharmaceutically acceptable Carrier.

Another aspect of the present invention provides the adolescent idiopathic scoliosis (AIS) for preventing and/or treating patient Pharmaceutical composition, which is characterized in that described pharmaceutical composition include a effective amount of NF90 and pharmaceutically acceptable carrier, Optionally, described pharmaceutical composition also includes prevention or other medicaments for treating AIS.

As used herein, term " expression is lowered " or " downward " refer to for example for example specific for specific nucleotide sequence LncRNA sequence or NF90 gene order, the measurement of sequence amount show for example compared with normal individual, from AIS patient or In the biological sample such as BM-MSC of individual separation with AIS risk, the expression of the sequence is reduced.Conversely, " in expression Tune " or " up-regulation " refer to lncRNA sequence for example for example specific for specific nucleotide sequence or NF90 gene order, sequence amount Measurement show for example compared with normal individual, AIS patient or with AIS risk individual separation biological sample such as BM- In MSC, the expression of the sequence increases.

As used in the present invention, term " individual ", " object " or " patient " include but is not limited to people and other primates (such as Chimpanzee and other apes and monkey class).In some embodiments, the object or patient are people.

In the present invention, term " lncAIS (Ensembl genome database gene symbol: ENST00000453347) " Refer to the lncRNA with original series shown in the gene in shared nucleic acid database GeneBank international at present comprising natural Or synthesis source lncRNA.LncAIS analog refers to that the lncRNA is substituted, lacks or adds one or several cores Thuja acid, or still biologically active derivative or variant form after biology chemical modification.

In the present invention, " (ncbi database gene I/D: 3234) " refer to has shared nucleic acid international at present to HOXD8 to term The HOXD8 of original series shown in HOXD8 gene in database GeneBank comprising natural or synthesis source HOXD8 and Its analog.HOXD8 analog refers to that it is substituted, lacks or adds one or several nucleotide, or repairs by biology chemistry Still biologically active derivative or variant form after decorations.

In the present invention, " (ncbi database gene I/D: 3609) " refer to has shared nucleic acid international at present to NF90 to term The NF90 of original series shown in ILF3 gene in database GeneBank comprising natural or synthesis source NF90 and its class Like object.NF90 analog refers to that it is substituted, lacks or adds one or several nucleotide, or after biology chemical modification still Biologically active derivative or variant form.

The effective dose of NF90 can be carried out with the mode of administration and the severity of disease to be treated etc. in the present invention Corresponding adjustment.It is preferred it is a effective amount of can be each because usually determining by those of ordinary skill in the art's synthesis.The factor packet It includes but is not limited to: the pharmacokinetic parameter of NF90, the health status of subject, weight, administration route etc..

In some embodiments of the present invention, the carrier in conjunction with NF90 can exist for the NF90 that is suitable for commonly used in the art The carrier expressed in host cell such as liposome, chitosan or Lentiviral, the pharmaceutically acceptable auxiliary material Including not causing in but various excipient, diluent and the adjuvant of apparent side effect in drug, including but not limited to: purifying Water, physiological saline, buffer, glucose, water, glycerol, mannitol, ethyl alcohol, surfactant and salt such as sodium chloride, EDETATE SODIUM Deng.

Pharmaceutical composition of the invention may include classical medicament preparation.Root can be applied by any conventional route According to pharmaceutical composition of the invention, as long as target tissue can be obtained by the approach.

In some embodiments of the present invention, the expression vector is Lentiviral, it is preferable that the slow disease Malicious expression vector can be that pSicoR, pSIN-EF2 or pWPXL plasmid strike wherein being overexpressed the preferred pSIN-EF2 plasmid of NF90 The low preferred pSicoR plasmid of NF90.

In some embodiments of the present invention, described pharmaceutical composition also optionally comprising it is one or more other to controlling The effective drug of AIS is treated, these drugs are well-known to those skilled in the art.Pharmaceutical composition of the invention can be with other Treatment means are administered in combination, prevention and/or treatment for AIS.

The utility model has the advantages that

The inventors found that rna binding protein NF90 is related to Osteoblast Differentiation, in mesenchymal stem cell (BM-MSC) lncAIS is by enhancing the mRNA stability of transcription factor HOXD8 with NF90 protein binding to promote BM-MSC in Osteoblast Differentiation, NF90 is necessary to the Osteoblast Differentiation of BM-MSC；Therefore, NF90 can be used to prepare regulation marrow between fill The biological agent of matter stem cell (BM-MSC) Osteoblast Differentiation, such as comprising be overexpressed NF90 gene or can express shNF90 plasmid, Recombinant expression carrier, transgenic cell line, genetic engineering bacterium.The invention also discloses for preventing and/or treating teenager spy The pharmaceutical composition of hair property scoliosis (AIS), it includes a effective amount of NF90 and pharmaceutically acceptable carriers, to be used for The AIS extremely relevant to BM-MSC Osteoblast Differentiation for treating patient.

Detailed description of the invention

Result shown in a to h demonstrates LncAIS and lowers in the BM-MSC of AIS patient in Fig. 1.Specifically,

Fig. 1 a, which is shown, has carried out microarray analysis by the BM-MSC to healthy donors and AIS patient, obtained difference The clustering figure of the lncRNA of expression, wherein lncAIS is the lower maximum lncRNA of modulation in the BM-MSC of AIS patient.

Fig. 1 b shows that LncAIS is located on No. 1 chromosome of people, is accredited as conservative gene seat.

Fig. 1 c is shown through real-time qPCR to LncAIS in normal healthy donors (Normal) and the BM-MSC of AIS patient Transcript is compared, the results showed that lncAIS is significantly lowered in the BM-MSC of AIS patient source.

Fig. 1 d is shown through Northern trace in normal healthy donors (Normal) and the BM-MSC of AIS patient LncAIS expression is compared, the results showed that lncAIS is lowered in the BM-MSC of AIS patient.

Fig. 1 e is shown to be analyzed by code capacity assessment tool (CPAT), and LncAIS is without coding potentiality, and wherein XIST turns It records object to compare as Noncoding gene, GAPDH and RUNX2 are compareed as encoding gene.

Fig. 1 f, which is shown, shows that lncAIS is not generated using the outer translation measurement result that pcDNA4-myc-his plasmid carries out Any detectable peptide, wherein KLF4 is compareed as coding protein.

BM-MSC cell grade separation determination shown in Fig. 1 g the result shows that, lncAIS is mainly distributed on the thin of people BM-MSC In karyon, the positive control that wherein HMBS RNA, ACTIN RNA and GAPDH RNA are expressed as cytogene, U1RNA fills When the positive control of karyogene expression.

RNA fluorescence in situ hybridization (RNA-FISH) measurement result shown in Fig. 1 h shows relative to normal healthy donors (Normal), lncAIS is lowered in the BM-MSC of AIS patient, wherein red: lncAIS probe (probe)；Green: actin (Actin)；Nucleus is redyed by DAPI.

Result shown in a to l demonstrates LncAIS and NF90 and interacts to enhance the mRNA stability of HOXD8 in Fig. 2. Specifically,

Fig. 2 a, which is shown, assesses the consumption (shL ncAIS) for showing lncAIS in normal BM-MSC not shadow by RT-qPCR The expression of its contiguous gene is rung, wherein NS is indicated not significant, this shows that lncAIS its may adjust work in trans- middle performance With.

The RNA that Fig. 2 b show biotin labeling strikes low measurement, to identify from the possible of BM-MSC lysate LncAIS GAP-associated protein GAP, the results showed that NF90 combines lncAIS in BM-MSC.

Fig. 2 c, which is shown, demonstrates the interaction of lncAIS and NF90 by RNA drop-down (striking low) measurement, then passes through Anti- NF90 antibody carries out immunoblotting assay.

Fig. 2 d is shown incubates BM-MSC lysate together with anti-NF90 (Anti-ILF3) antibody, passes through immunoprecipitation (RIP) measurement demonstrates the interaction of lncAIS and NF90.

Fig. 2 e is shown lncAIS and NF90 common location in the nucleus of BM-MSC.Pass through RNA-FISH lncAIS BM-MSC is detected, immunofluorescence dyeing then is carried out to NF90.It is red: lncAIS probe；Green: NF90；Nucleus is by DAPI It redyes.

Fig. 2 f and Fig. 2 g are shown, with expression shNF90 the normal BM-MSC of slow-virus infection, respectively in MSC culture medium Culture in (Fig. 2 f) and OriCell MSC Osteoblast Differentiation culture medium (Fig. 2 g), real-time qPCR assessment result show that NF90 is consumed (shNF90) self-renewing related gene (NANOG, POU5F1 and SOX2) (Fig. 2 f) and Osteoblast Differentiation base in BM-MSC are inhibited Because of the expression of (ALPL, BSP, RUNX2, LPL and PPAR) (Fig. 2 g).

Fig. 2 h and Fig. 2 i are shown, with expression shNF90 the normal BM-MSC of slow-virus infection after OriCell MSC at Cultivated in bone differential medium, ALP dye (Fig. 2 h) and Von Kossa dyeing (Fig. 2 i) the result shows that, NF90 consume (shNF90) Osteoblast Differentiation of BM-MSC is inhibited.

Fig. 2 j show the interaction that the mRNA and NF90 that specify in analysis BM-MSC are measured by RIP；By BM-MSC Lysate incubates together with anti-NF90 antibody, then carries out RNA immunoprecipitation and real-time qPCR, the results showed that in selection In the BM-MSC of lncAIS silencing in the transcription factor of preceding 10 downwards, NF90 specifically binds the mRNA of HOXD8.

Fig. 2 k, which is shown, measures the interaction of the mRNA and NF90 of verifying HOXD8 in BM-MSC by RIP；By BM- MSC lysate incubates together with anti-NF90 antibody, then carries out RIP measurement.In addition as illustrated in figure 21, in Act D, treated Specified time point measures the HOXD8 mRNA stability from specified BM-MSC by real-time qPCR.The result shows that in BM-MSC LncAIS consumption (shLncAIS) eliminate NF90 and HOXD8 mRNA the region 3'-UTR interaction (Fig. 2 k), and because This causes HOXD8 mRNA to decay (Figure 21).Consistently, in the BM-MSC of AIS patient NF90 and HOXD8 mRNA the area 3'-UTR The interaction in domain is undetectable (Fig. 2 k), and also eliminates the stability (Fig. 2 l) of HOXD8 mRNA.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means.

The following are material therefor of the embodiment of the present invention and general approach

Antibody and reagent

Abcam (Cambridge, USA) is purchased from for the antibody of people HOXD8 (ab228450) and NF90 (ab89100).It is anti- The antibody of beta-actin (clone AC-74) comes from Sigma-Aldrich (St.Louis, USA).For Myc (clone 9E10) Antibody come from Santa Cruz Biotechnology (Santa Cruz, USA).For COL1A1 (BA0325), IBSP (BA2329) and the antibody of OPN (PA1432) comes from Boster (Wuhan, China).It is purchased from the secondary antibody of Alexa-594 conjugation Molecular probes Inc (Eugene, USA).Streptavidin pearl comes from Sigma-Aldrich (St.Louis, USA). A-protein/G pearl comes from Santa Cruz Biotechnology (Santa Cruz, USA).Alkaline phosphatase detecting reagent box Purchased from Millipore (Millerica, USA).SuperReal premixing plus qPCR buffer come from TIANGEN Biotech (BeiJing, China).OriCell BM-MSC Osteoblast Differentiation kit is from Cyagen (HUXMA-90021, China).Von Kossa Staining kit comes from Genmed Scientifics (Chinese Shanghai).Cell counting Kit -8 (CCK-8) comes from Dojindo (Kumamoto, Japan).Fast Green staining kit and Alcian Blue staining kit come from Xinhualvyuan Biotechnology (BeiJing, China).

Probe of the invention, primer, shLncAIS and shNF90 gene order

Table 1: lncAIS (11-228nt) probe sequence for RNA FISH

LncAIS probe	Probe sequence	Sequence number
			LncAIS probe #1	5’-CTTTCCCTGAGAAAAACCTCC-3’	SEQ ID NO:1
LncAIS probe #2	5’-AGGATTAGGAAGCCTCCTGC-3’	SEQ ID NO:2
			LncAIS probe #3	5’-CGCTTCTCTCTTCTACTGTCC-3’	SEQ ID NO:3

Table 2: low shLncAIS gene order is struck for lncAIS

Gene	Gene order	Sequence number
			shLncAIS#1	5’-TTCCTAATCCTGCTCCAG-3’	SEQ ID NO:4
shLncAIS#2	5’-ATAGACATCTGGTTTCTGG-3’	SEQ ID NO:5
			shLncAIS#3	5’-TTAGGACATAGACATCTG-3’	SEQ ID NO:6

Table 3: low shNF90 gene order is struck for NF90

Gene	Gene order	Sequence number
			shNF90#1	5’-AAACCCAGTGAAGCACAGG-3’	SEQ ID NO:7
shNF90#2	5’-AAGCCTGTCTGTTTCTTGC-3’	SEQ ID NO:8
			shNF90#3	5’-AATCCCATGCATCTGCAGC-3’	SEQ ID NO:9

Primer sequence in table 4:qRT-PCR analysis for cDNA amplification

Patient and sample

From 42 AIS patients (average age 14.5 years old, range 12-17 years old) and 25 healthy donors (average ages 14.9 Year, range 12-17 years old) obtain marrow (BM) aspirate.In AIS group, all patients receive comprehensive clinical and radiation It learns and checks, to exclude the other reasons of scoliosis and determine the diagnosis of AIS.In control group, 25 ages and gender matching Each of subject there is straight backbone and normal crooked test forward through physical examination.Into when research, their quilts Confirm no any relevant medical conditions or deformity of spine.The research is assisted through Ethics Committee, the Chinese Academy of Medical Sciences and Beijing Ratify with hospital.Before entering research, Written informed consent is obtained from all subjects and its parent.

Cell separation, culture and Osteoblast Differentiation measurement

From AIS patient and healthy donors collector's myeloid tissue.All experiments are entrusted according to Chinese Academy of Medical Sciences's ethics The program that member can ratify with BJ Union Hospital carries out.As follows (Zhuang Q, Mao W, Xu P, Li H, Sun Z, Li S, et al.Identification of Differential Genes Expression Profiles and Pathways of Bone Marrow Mesenchymal Stem Cells of Adolescent Idiopathic Scoliosis Patients by Microarray and Integrated Gene Network Analysis.Spine 2016,41 (10): 840-855) separation and culture people BM-MSC.With being supplemented with 10%FBS and 100U/ml penicillin and 100mg/ml The DMEM of streptomysin cultivates people 293T cell.Slow virus is generated in 293T cell using standard scheme.Use lipofectin (Invitrogen) it is transfected.It strikes low for shRNA and is overexpressed experiment, target sequence is building up in pSicoR plasmid.Slowly Virus is generated by 293T cell.It screens most effective shRNA in 3 kinds of shRNA constructs and is used for following experiment.In each measurement It independent strike low cell line using three and carries out biology repetition.At least four independent experiments are carried out to repeat as biology.Example Such as, low shlncAIS gene order is struck as shown in SEQ ID NO:4-6 in the above table 2 for lncAIS；It is struck for NF90 low ShNF90 gene order as shown in SEQ ID NO:7-9 in the above table 3.For induced osteogenesis differentiation, by third generation BM-MSC It is seeded in six orifice plates, and is handled according to the scheme of manufacturer with Osteogenic Induction Medium.Replacement in culture medium every 3 days is primary.

Wound healing assay

Culture dish is coated 1 hour at 37 DEG C with 0.1% gelatin (v/v).By 1 × 10⁶A BM-MSC plating cells with Generate the single layer converged.Cell is cultivated to adhere to and spread completely.By scraping cell monolayer manually with p200 pipette tip To generate wound.The first image is obtained using reference point marker.Cell is cultivated 24 hours in incubator for tissue culture.Pass through matching The shooting area of first image obtains the second image.

Encode Potentials

According to the explanation of manufacturer, by coding Potential Evaluation tool (CPAT) in http: // The coding potentiality of lncAIS are analyzed on the website of lilab.research.bcm.edu/cpat/.XIST transcript is used as non-coding Genetic contrast.GAPDH and RUNX2 is compareed as encoding gene.

Cell grade separation analysis

According to the manufacturer's instructions, BM-MSC is cracked using NE-PER core and cytoplasmic extraction reagents box (Pierce), Then core and cytoplasm classification separation are carried out.RNA is extracted using TRIzol Reagent (Invitrogen), then uses RNeasy Kit (Qiagen, Valencia, CA, USA) purifying.By M-MLV reverse transcriptase (Promega) and qRT-PCR analyze into Row reverse transcription.The positive control that ACTIN RNA and GAPDH RNA are expressed as cytogene.U1RNA serves as karyogene expression Positive control.

ALP and Von Kossa dyeing

According to the scheme of manufacturer, dyed using ALP staining kit monitoring ALP.According to the scheme of manufacturer, use Von Kossa staining kit monitors mineral deposition.Image is obtained with Nikon EclipseTi microscope (Nikon, Japan). Quantify the color intensity of mineral deposition by ImageJ.

Northern trace

Total serum IgE is extracted from BM-MSC with TRIzol.The 10 μ g RNA from each sample are subjected to denaturing formaldehyde agarose Then electrophoresis is transferred to positively charged NC film with 20 × SSC buffer (3.0M NaCl and 0.3M sodium citrate, pH7.0). Film is subjected to UV crosslinking, and is visited with the rna probe such as IncAIS (11-228nt) by the way that the biotin labeling generated is transcribed in vitro Needle incubates together.According to the manufacturer's instructions, it is used for the Streptavidin detection biotin signal of HRP- conjugation Cheniluminescent detection of nucleic acids module.

RNA FISH

The lncAIS probe of fluorescence conjugation is generated according to the scheme of Biosearch Technologies.For RNA FISH LncAIS (11-228nt) probe set sequences as shown in SEQ ID NO:1-3 in the above table 1.By BM-MSC and DNA probe group Hybridization, then with specified antibody dyeing.With Olympus FV1200 laser scanning co-focusing microscope (Olympus, Japan) Obtain image.

Microarray analysis

According to the manufacturer's instructions, RNA is extracted from BM MSC using TRIzol Reagent (Invitrogen), then It is purified with RNeasy kit (Qiagen, Valencia, CA, USA).Use One-Cycle Target Labeling and Control Reagents (Affymetrix, SantaClara, CA, USA) generates cDNA, and is marked and tried with GeneChip WT Agent box (Affymetrix, Santa Clara, CA, USA) generates cRNA.By the fragmentation (≤200nt) of biotin labeling CRNA hybridizes at 45 DEG C with Affymetrix GeneChip Human transcript array 2.0 (Affymetrix) 16 hours.It is washed in Affymetrix Fluidics Station 450 and dyes GeneChip.Using being mounted onIn Scanner3000 7GGeneChipCommand Console (AGCC) scanning GeneChips.Use Affymetrix default analysis setting and global scaling as method for normalizing, uses Robust Multichip Analysis (RMA) algorithm analyzes data.The value provided is log2 RMA signal strength.Microarray data is to step on Record number (GSE110359) is stored in GEO.

RNA strikes low test

With biotin RNA label mixture (Roche) in vitro obtain biotin labeling lncAIS overall length (ariyoshi) and Then antisense RNA incubates together with the nuclear extract separated from BM-MSC.(pull down) is pulled down by Streptavidin pearl Rna binding protein.Drop-down component is separated with SDS-PAGE, then carries out Silver stain.Pass through LTQ Orbitrap XL mass spectrography point Analysis carries out immunoblotting by the lncAIS differential band being enriched with or with specified antibody.

RNA immunoprecipitation (RIP) measurement

1% formaldehyde treated of BM-MSC, then with the RIPA buffer (50mM Tris-HCl [pH for being free of RNase 7.4], 150mM NaCl, 0.5% NaTDC, 0.1%SDS, 5mM EDTA, 2mM PMSF, 20mg/ml Aprotinin, 20mg/ml leupeptin, 10mg/ml pepsin inhibitor A, 150mM benzenecarboximidamide, 1%Nonidet P-40 and RNase inhibit Agent) dissolution.Sample is ultrasonically treated and is centrifuged on ice.Supernatant is removed in advance and is incubated together with specified antibody, so Albumin A/G pearl immunoprecipitation is carried out afterwards.Total serum IgE is extracted from eluent.LncAIS enrichment is analyzed by qPCR.Expand for cDNA Increase primer sequence to be listed in table 4.

Chromatin imrnunoprecipitation (ChIP) measurement

Quantitative ChIP is carried out according to standard scheme (Upstate).It will be from the BM-MSC (2 × 10 being fixed in 1% formaldehyde⁶) (ultrasonic treatment is incubated overnight together with 4 μ g antibody at 4 DEG C the chromatin of shearing to 200-500bp), then uses salmon sperm DNA/ protein sepharose 4B carries out immunoprecipitation.After washing, elution and crosslinking reversion, purifying from each ChIP sample and The DNA of corresponding input sample, and analyzed using qPCR.It is listed in table 4 for cDNA amplimer sequence.

Internal heterotopic osteogenesis

It will in total 2 × 10⁶HA/TCP ceramic powders (the National that personal BM-MSC and about 100mg is moistened Engineering Research Center for Biomaterials, Chengdu, China) it is incubated overnight at 37 DEG C. Cell is subcutaneously implanted to the back surfaces of 8 week old NOD/SCID mouse.Implantation material is harvested after 8 weeks, it is solid in 4% paraformaldehyde Fixed, the decalcification in 10%EDTA is embedded in paraffin, is then sliced and dyes.Bone tissue is contaminated by quickly green dyeing At green.Cartilaginous tissue is dyed into blue to indicate osseous maturation by alcian blue dyeing.

Statistical analysis

Unpaired student t is used in the present invention to examine as statistical analysis.Use Microsoft Excel or SPSS 13 carry out statistical calculations.As P < 0.05, P value is significant.

Embodiment

Embodiment 1: identification LncAIS is the significant difference lowered in AIS Bone Marrow of Patients mescenchymal stem cell (BM-MSC) The lncRNA of expression

In order to identify crucial lncRNA involved in adolescent idiopathic scoliosis (AIS), we are to from 5 health Donor and the BM-MSC of 12 AIS patients have carried out microarray analysis.Have in the BM-MSC of normal BM-MSC and AIS patient 1483 lncRNA show differential expression, wherein 718 up-regulations, 765 downwards, as shown in Figure 1a.In the BM-MSC of AIS patient In in the lower maximum lncRNA of modulation, we are absorbed in us and are known as lncAIS's (gene symbol: ENST00000453347) The lncRNA not being characterized.LncAIS is located on No. 1 chromosome of people, includes 4 exons, is the transcript of overall length 476nt. LncAIS is accredited as conservative gene seat, as shown in Figure 1 b.

Real-time qPCR is carried out according to routine operation, is analyzed in the BM-MSC of BM-MSC and AIS patient of normal healthy donors LncAIS transcript, expand the cDNA primer of lncAIS as shown in SEQ ID NO:10-11 in above-mentioned table 4.BM-MSC comes from 20 healthy donors and 30 AIS patients.Relative gene expression multiple is standardized as endogenous beta-actin, and is counted as Average value ± S.D.*, P < 0.01.As a result it confirms compared with the BM-MSC from healthy donors, in AIS patient source BM-MSC Middle lncAIS is significantly lowered, as illustrated in figure 1 c.

The lncAIS table in the BM-MSC of the BM-MSC and AIS patient of normal healthy donors is detected by Northern trace It reaches.The probe (its sequence is as shown in SEQ ID NO:1-3 in the above table 1) of the lncAIS (11-228nt) of 217nt is marked, is used In northern engram analysis.RNA is extracted from the BM-MSC of specified normal healthy donors BM-MSC and AIS patient respectively, 18S rRNA (1482-1725nt) is used as loading control.BM-MSC comes from 3 healthy donors and 3 AIS patients.Such as Fig. 1 d Shown, Northern trace further demonstrates, and lncAIS is lowered in the BM-MSC of AIS patient, and display only has lncAIS in figure One transcript.

By code capacity assessment tool (CPAT), analysis shows that, lncAIS is without coding potentiality；Wherein XIST transcript is used Make Noncoding gene control, GAPDH and RUNX2 to compare as encoding gene, as shown in fig. le.LncAIS transcript is cloned into PcDNA4-myc-his plasmid, and be transfected into 293T cell 48 hours.With anti-Myc antibody by immunoblotting, Myc- is analyzed The expression of fusion protein.KLF4 is compareed as coding protein.It is any detectable that In Vitro Translation measurement shows that lncAIS is not generated Peptide, as shown in Figure 1 f.

First BM-MSC is cracked, then carries out core and cytoplasm classification separation and RNA extraction, then carries out qRT-PCR analysis. The positive control that HMBS RNA, ACTIN RNA and GAPDH RNA are expressed as cytogene.U1 RNA serves as karyogene table The positive control reached.N: nuclear leve point.C: cytoplasm fraction.Each primer pair for cDNA amplification in qRT-PCR analysis is recited in In the above table 4.Data are from three independent experiments for using the BM-MSC from 3 healthy donors.Cell grade separation determination The result shows that lncAIS is mainly distributed in the nucleus of people BM-MSC, as shown in Figure 1 g.

LncAIS is observed in BM-MSC by RNA fluorescence in situ hybridization (RNA-FISH) measurement, then carries out being immunized glimmering Light dyeing.It is red: lncAIS probe；Green: actin；Nucleus is redyed by DAPI.Scale bar, 20 μm.LncAIS probe Sequence is as shown in SEQ ID NO:1-3 in the above table 1.Observe more than 100 typical cells.RNA fluorescence in situ hybridization (RNA- FISH it) further demonstrates, the distribution of lncAIS downward and lncAIS in nucleus in the BM-MSC of AIS patient, such as Shown in Fig. 1 h.

It is highly expressed in normal person BM-MSC in short, we disclose lncAIS, and it is significant in AIS patient BM-MSC It lowers.

Embodiment 2: determine that LncAIS and NF90 interacts to enhance the mRNA stability of HOXD8

Regulation of the LncRNA usually with its neighbouring protein coding gene is positively correlated.However, as shown in Figure 2 a, passing through QRT-PCR assessment is it was found that the expression that the consumption of lncAIS does not influence its contiguous gene in BM-MSC (is used for lncAIS Low shlncAIS gene order is struck as shown in SEQ ID NO:4-6 in the above table 2), wherein Relative gene expression multiple variation It is calculated as average value ± S.D.NS, not significantly.Data are from three independence realities for using the BM-MSC from 3 healthy donors It tests.This shows that lncAIS may be in trans- middle its adjustment effect of performance.

Then the RNA for carrying out biotin labeling strikes low measurement, to identify the possible lncAIS from BM-MSC lysate GAP-associated protein GAP.It is compareed, is then analyzed by mass spectrometry, with BM-MSC's using overall length lncAIS transcript (justice) and antisense sequences Lysate carries out biotin-RNA drop-down, as shown in Figure 2 b, determines that NF90 combines lncAIS in BM-MSC.NF90 is leucocyte The protein of interleukin enhancer binding factor 3 (ILF3) family is a kind of RNA combination egg for adjusting gene expression or stable mRNA It is white.

As shown in Figure 2 c, the interaction of lncAIS and NF90 are demonstrated by RNA drop-down measurement, then by anti- NF90 antibody carries out immunoblotting assay.SEQ ID NO:7-9 in low shNF90 gene order such as the above table 3 is struck for NF90 It is shown；Data are from three independent experiments for using the BM-MSC from 3 healthy donors.

BM-MSC lysate is incubated together with anti-NF90 antibody, then carries out RIP measurement.It extracts RNA and reversely turns Record analyzes LncAIS transcript by real-time qPCR, is recited in the above table 4 for each primer pair expanded of cDNA in qRT-PCR In.Data are from three independent experiments for using the BM-MSC from 3 healthy donors.As shown in Figure 2 d, pass through immunoprecipitation (RIP) measurement demonstrates the interaction of lncAIS and NF90.

RNA strikes low (Fig. 2 c) and the result of RNA- immunoprecipitation (RIP) measurement (Fig. 2 d) confirms lncAIS's and NF90 Interaction.

As shown in Figure 2 e, by lncAIS and NF90 common location in the nucleus of BM-MSC.It is used by RNA-FISH LncAIS detects BM-MSC, then carries out immunofluorescence dyeing to NF90.It is red: lncAIS probe；Green: NF90；Nucleus It is redyed by DAPI.Scale bar, 50 μm.LncAIS probe sequence is as shown in SEQ ID NO:1-3 in the above table 1.It observes and is more than 100 typical cells.Data are from three independent experiments for using the BM-MSC from 3 healthy donors.

Above-mentioned experimental data shows lncAIS and NF90 protein binding in BM-MSC.

Osteoblast Differentiation and the pathogenesis of AIS how are adjusted in order to further determine NF90, we are situated between by slow virus The shRNA led exhausts NF90 in normal BM-MSC.

As shown in figure 2f, it with the slow-virus infection BM-MSC of expression shNF90, and cultivates 3 days, passes through in MSC culture medium The expression of real-time qPCR assessment self-renewing related gene.As shown in Figure 2 g, by BM-MSC in OriCell MSC skeletonization point Change and cultivated 6 days in culture medium, assesses the expression of Osteoblast Differentiation gene in specified BM-MSC by real-time qPCR.For Each primer pair that cDNA is expanded in qRT-PCR is recited in the above table 4.Relative gene expression multiple variation is standardized as endogenous Property beta-actin, and it is counted as average value ± S.D.*, P < 0.01.Data are originated from the BM- of 3 healthy donors from using Three independent experiments of MSC.The result shows that, NF90 consumption inhibits self-renewing dependency basis in BM-MSC shown in Fig. 2 f and 5g Because of the expression of (NANOG, POU5F1 and SOX2) and Osteoblast Differentiation gene (ALPL, BSP, RUNX2, LPL and PPAR).

BM-MSC is cultivated to induced osteogenesis differentiation in 6 days in OriCell MSC Osteoblast Differentiation culture medium, in Osteoblast Differentiation Progress ALP dyeing in 6th day, as shown in fig. 2h.BM-MSC is cultivated 6 days in OriCell MSC Osteoblast Differentiation culture medium and is induced Osteoblast Differentiation carries out Von Kossa dyeing to indicate the 12nd day mineral deposit, as shown in fig. 2i.Data come from the source of using From three independent experiments of the BM-MSC of 3 healthy donors.Scale bar, 10 μm.Quantify the color of mineral deposition by ImageJ Strength Changes are calculated as average value ± S.D by intensity.*, P < 0.01.ALP dyes (Fig. 2 h) and Von Kossa dyeing (Fig. 2 i) The suppressed Osteoblast Differentiation of result verification.

The above experimental data shows that NF90 is necessary to the Osteoblast Differentiation of BM-MSC.

It is reported that the area 3'-UTR of NF90 and PARP1mRNA interacts to maintain its mRNA stability.We pass through The interaction of the mRNA and NF90 that are specified in RIP measurement analysis BM-MSC.By BM-MSC lysate together with anti-NF90 antibody It incubates, then carries out RNA immunoprecipitation and real-time qPCR.Data are from three of BM-MSC used from 3 healthy donors Independent experiment.As shown in figure 2j, in the BM-MSC for the lncAIS silencing that we select in the transcription factor of preceding 10 downwards, The mRNA of NF90 specific binding HOXD8.

The interaction of the mRNA and NF90 of HOXD8 in BM-MSC is demonstrated by RIP measurement.By BM-MSC lysate It is incubated together with anti-NF90 antibody, then carries out RIP measurement.Data from use from 3 healthy donors BM-MSC three A independent experiment, as shown in Fig. 2 k.As illustrated in figure 21, in Act D treated specified time point, by real-time qPCR measure come From the HOXD8 mRNA stability of specified BM-MSC.Data are shown as average value ± S.D.**, P < 0.01.Data come from the source of using From three independent experiments of the BM-MSC of 3 healthy donors.

The result shows that, the lncAIS in BM-MSC eliminates the area 3'-UTR of NF90 Yu HOXD8 mRNA shown in Fig. 2 k The interaction in domain, and HOXD8 mRNA is therefore caused to decay (Figure 21).Consistently, as shown in Fig. 2 k, the BM-MSC of AIS patient The interaction in the region 3'-UTR of middle NF90 and HOXD8 mRNA is undetectable, and also eliminates HOXD8 mRNA's Stability (Fig. 2 l).

The experimental results showed that, to maintain, HOXD8 mRNA's in normal BM-MSC is steady for lncAIS and NF90 interaction above It is qualitative.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Chinese Academy of Medical Sciences Beijing Union Medical College Hospital

<120>application of the NF90 in the biological agent of preparation regulation mesenchymal stem cell Osteoblast Differentiation

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Claims (10)

1. Application of the 1.NF90 in the biological agent of preparation regulation mesenchymal stem cell Osteoblast Differentiation.
2. 2. application as described in claim 1, which is characterized in that in mesenchymal stem cell lncAIS by with NF90 egg White combination enhances the mRNA stability of transcription factor HOXD8 to promote the Osteoblast Differentiation of mesenchymal stem cell.
3. 3. application as claimed in claim 1 or 2, which is characterized in that the regulation mesenchymal stem cell Osteoblast Differentiation Biological agent includes plasmid, recombinant expression carrier, transgenic cell line, the gene for being overexpressed NF90 gene or capable of expressing shNF90 Engineering bacteria.
4. 4. application as claimed in claim 3, which is characterized in that the plasmid for being overexpressed NF90 gene is pSIN-EF2 or energy The plasmid for expressing shNF90 is pSicoR plasmid.
5. 5. application as claimed in claim 3, which is characterized in that the sequence of the shNF90 is as shown in SEQ ID NO:7-9.
6. 6. regulating and controlling the biological agent of the Osteoblast Differentiation of mesenchymal stem cell, which is characterized in that it includes be overexpressed NF90 base Cause or plasmid, recombinant expression carrier, transgenic cell line, the genetic engineering bacterium that shNF90 can be expressed.
7. 7. biological agent as claimed in claim 6, which is characterized in that the plasmid for being overexpressed NF90 gene is pSIN-EF2 Or can express the plasmid of shNF90 is pSicoR plasmid.
8. 8. biological agent as claimed in claim 6, which is characterized in that the sequence of the shNF90 such as SEQ ID NO:7-9 institute Show.
9. 9.NF90 is preparing answering in the pharmaceutical composition for preventing and/or treating adolescent idiopathic scoliosis (AIS) With, which is characterized in that described pharmaceutical composition includes a effective amount of NF90 and pharmaceutically acceptable carrier.
10. 10. the pharmaceutical composition for preventing and/or treating adolescent idiopathic scoliosis (AIS), which is characterized in that described Pharmaceutical composition includes a effective amount of NF90 and pharmaceutically acceptable carrier.