CN109355252B

CN109355252B - Application of HOXD8 in preparation of product for promoting osteogenic differentiation of mesenchymal stem cells

Info

Publication number: CN109355252B
Application number: CN201811325070.9A
Authority: CN
Inventors: 庄乾宇; 仉建国; 惠尚懿; 范祖森; 邱贵兴; 吴志宏; 叶步青; 赵春华; 李静; 李娜; 王升儒; 林莞峰; 张延斌; 杨阳
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2018-11-08
Filing date: 2018-11-08
Publication date: 2020-11-06
Anticipated expiration: 2038-11-08
Also published as: CN109355252A

Abstract

The invention discloses application of a transcription factor HOXD8 in preparation of a product for promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC). We find that HOXD8 can promote cell proliferation in BM-MSC and increase expression levels of self-renewal related genes and osteogenic differentiation genes in BM-MSC, so that the HOXD8 can be used for preparing a product for promoting cell proliferation and osteogenic differentiation in BM-MSC, such as plasmids, recombinant expression vectors, transgenic cell lines and genetically engineered bacteria containing HOXD8 genes, and can be further used for treating Adolescent Idiopathic Scoliosis (AIS). The invention also discloses a pharmaceutical composition for preventing and/or treating AIS, which comprises an effective amount of HOXD8 and a pharmaceutically acceptable carrier, for treating AIS associated with BM-MSC osteogenic differentiation abnormality in a patient.

Description

Application of HOXD8 in preparation of product for promoting osteogenic differentiation of mesenchymal stem cells

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of a transcription factor HOXD8 related to osteogenic differentiation in preparation of a product for promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC).

Background

Adolescent Idiopathic Scoliosis (AIS) is a complex three-dimensional deformity of the spine, occurring primarily in girls 10 to 16 years of age during Adolescent growth. The scoliosis may have deformity in appearance, back pain and dysfunction, and severe patients will have physiological function problems such as limited cardiopulmonary function and the like, and the social activities are influenced to different degrees. Epidemiological survey results show a global incidence of AIS in adolescents of about 2-4%, of which about 10% of people diagnosed with AIS require treatment. Current treatments for AIS include brace therapy and surgical correction; among them, full-time bracing therapy may cause back pain and psychological disorders, while corrective surgery using pedicle screw instruments inevitably results in severe surgical trauma, and even permanent catastrophic nerve or blood vessel injury when the screws are poorly positioned. If the risk of the onset and progression of AIS can be detected as early as possible, appropriate treatment regimens may be timely taken, reducing the pain and inconvenience associated with treatment delays. New advances in the etiology and pathogenesis of AIS, such as the exploration of the molecular mechanisms of AIS pathogenesis, will likely provide more convenient methods for AIS detection and for progression prediction and treatment.

Studies have shown that AIS patients have abnormal bone growth and a sustained reduction in Bone Mineral Density (BMD) compared to gender and age matched controls. In 1982 Burner et al first reported a reduction in bone mass in patients with AIS. Approximately one third of AIS patients have poor BMD. Low BMD is reported to be a key prognostic factor for AIS girl curve progression. It is believed that reduced bone mass may be a major factor in AIS spinal deformity. Many studies have demonstrated a 27% to 38% reduction in BMD in AIS patients. In addition, longitudinal follow-up on skeletal maturation showed that there was a continuous decrease in bone mass in more than 80% of AIS girls, suggesting that the decrease in bone mass may be a lifelong systemic abnormality in bone metabolism in AIS patients.

Mesenchymal Stem Cells (MSCs) are present in the stroma of all mammalian organs and can differentiate into osteoblasts, adipocytes and chondrocytes. In addition, MSCs are essential in intramembranous and endochondral bone formation. We previously demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) of AIS patients exhibit reduced osteogenic differentiation capacity. The study of Park et al further confirmed the results of our study. Our and other studies have shown that AIS patients develop abnormal differentiation of MSCs during development of osteoblasts, chondrocytes and adipocytes, and in view of the functional characteristics of Mesenchymal Stem Cells (MSCs) in bone formation and resorption, we speculate that the abnormal osteogenic differentiation of MSCs is associated with the pathogenesis of AIS. However, how MSCs are abnormally regulated in AIS patients remains elusive.

Non-coding RNA (ncRNA) is called 'dark matter' in a living body, and the proportion of ncRNA in the whole genome is closely related to the complexity level among biological species. The complex and precise regulation function of the ncRNA in development and gene expression explains the complexity of the genome, and opens a new way for people to know the complexity of a living body from the dimension of a gene expression regulation network. There is increasing evidence that the development of a range of major diseases is associated with an imbalance in the regulation of non-coding RNAs.

Long non-coding rnas (lncrnas) have recently been considered to be transcripts of more than 200 nucleotides (nt) of a class of genes that have no protein coding ability. lncRNA is less conserved in various species, but is more tissue-specific than protein-encoding genes. Research has shown that lncRNA plays a broad role in gene regulation and other cellular processes. lncRNA is involved in a variety of biological processes including chromatin modification, transcriptional regulation, imprinting, and nuclear transport. LncRNA performs its function through a variety of mechanisms, including co-transcriptional regulation, regulation of gene expression, scaffolding of nuclear or cytoplasmic complexes, and pairing with other RNAs. We have recently reported that several lncrnas are involved in self-renewal maintenance of liver cancer stem cells. Recent studies have reported that several lncrnas regulate their respective adjacent protein-encoding genes, playing a key role in mesendoderm differentiation and cardiac development. However, the biological role of lncRNA in AIS pathogenesis is not clear.

Transcription Factors (TF) are proteins that play a key role in regulating gene Transcription, and can promote or inhibit the transcriptional expression of target genes by binding them, playing a very key role in various biological processes and disease development. Therefore, identifying, classifying and annotating transcription factors, and analyzing the regulation and function of transcription factors, etc., have been the hot spots and the basis of research. Homeobox genes (Hox genes) encode homeodomain-containing transcription factors (Hox transcription factors) that determine positional identity along the anteroposterior axes of animal embryos, which are widely expressed in adult tissues. In humans, 39 known HOX transcription factors are present in four separate clusters HOXA-D, located on four different chromosomes. The HOX gene regulates cell differentiation during embryonic development in many different lineages and developmental pathways. Studies have shown that mouse MSCs from different organs are characterized by different topographic (topographic) HOX codes, while fibroblasts from different anatomical parts of the human body express different HOX patterns. This indicates that the typical HOX code of the cells reflects the specific expression of functionally active HOX genes in different tissues.

In summary, given that the role of HOX gene in regulating BM-MSC osteogenic differentiation has not been characterized so far, we sought to further explore the role of lncRNA and HOX genes in regulating BM-MSC osteogenic differentiation and their association with the pathogenesis of Adolescent Idiopathic Scoliosis (AIS) in order to find suitable AIS treatment.

Disclosure of Invention

We found that HOXD8, which is a marker gene for BM-MSC, is expressed most highly in BM-MSC, the present invention seeks to further explore the association between lncRNA and HOXD8, and the role of HOXD8 in regulating osteogenic differentiation of BM-MSC, to further explore the pathogenesis and appropriate AIS treatment of Adolescent Idiopathic Scoliosis (AIS).

An object of the present invention is to provide the use of the transcription factor HOXD8 in the preparation of a product for promoting cell proliferation of bone marrow mesenchymal stem cells (BM-MSCs), we found that the transcription factor HOXD8 is highly expressed in human BM-MSCs and promotes cell proliferation in BM-MSCs.

In some embodiments of the invention, up-regulation of HOXD8 produced by infection of mesenchymal stem cells with a lentivirus overexpressing HOXD8 promotes cell proliferation of the mesenchymal stem cells, preferably the expression vector for the lentivirus overexpressing HOXD8 is the pSIN-EF2 plasmid.

In other embodiments of the present invention, the product for promoting cell proliferation of bone marrow mesenchymal stem cells is a plasmid, a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium comprising HOXD8 gene.

One aspect of the present invention provides a biological preparation for promoting cell proliferation of mesenchymal stem cells, wherein the biological preparation comprises a plasmid having HOXD8 gene, a recombinant expression vector, a transgenic cell line, or a genetically engineered bacterium.

It is another object of the present invention to provide use of the transcription factor HOXD8 in preparation of a product for promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs), and we found that the transcription factor HOXD8 is highly expressed in human BM-MSCs and increases expression levels of self-renewal-associated genes and osteogenic differentiation genes in BM-MSCs.

In some embodiments of the present invention, HOXD8 produced by infecting mesenchymal stem cells with a lentivirus overexpressing HOXD8, preferably the expression vector of the HOXD 8-overexpressing lentivirus is a pSicoR plasmid, upregulates the expression levels of self-renewal-associated genes and osteogenic differentiation genes in the mesenchymal stem cells.

In other embodiments of the present invention, the product for promoting osteogenic differentiation of mesenchymal stem cells is a plasmid, a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium comprising HOXD8 gene.

Still another aspect of the present invention provides a biological agent for promoting osteogenic differentiation of mesenchymal stem cells, wherein the biological agent is a plasmid, a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium comprising a gene having HOXD 8.

The inventor of the invention finds that LncAIS (gene symbol: ENST00000453347) is lncRNA which is significantly and differentially expressed in mesenchymal stem cells (BM-MSC) of bone marrow of AIS patients and is significantly down-regulated in BM-MSC of the AIS patients in related research on Adolescent Idiopathic Scoliosis (AIS); in addition, experiments of the invention also prove that the transcription factor HOXD8 in lncAIS-silenced BM-MSC is one of 10 transcription factors with the maximum expression and down-regulation amplitude, and LncAIS activates the transcription factor HOXD8 to transcribe; we further found that the transcription factor HOXD8 promotes cell proliferation in BM-MSC and increases the expression level of self-renewal-associated genes and osteogenic differentiation genes in BM-MSC, which are essential for osteogenic differentiation of BM-MSC.

As used herein, the term "down-regulation" or "down-regulation" refers to, for example, for a specific nucleotide sequence, such as a specific lncRNA sequence or HOXD8 gene sequence, a measurement of the amount of the sequence indicating a reduced level of expression of this sequence, for example, in a biological sample isolated from an AIS patient or an individual at risk of AIS, such as BM-MSC, compared to a control, such as a normal individual. Conversely, "expression up-regulation" or "up-regulation" means, for example, that for a specific nucleotide sequence, such as a specific lncRNA sequence or HOXD8 gene sequence, the measurement of the amount of the sequence indicates an increased level of expression of this sequence, for example, in a biological sample isolated from an AIS patient or an individual at risk of AIS, such as BM-MSC, compared to a normal individual.

One aspect of the present invention provides the use of HOXD8 in the preparation of a pharmaceutical composition for the prevention and/or treatment of Adolescent Idiopathic Scoliosis (AIS), wherein said pharmaceutical composition comprises an effective amount of HOXD8 and a pharmaceutically acceptable carrier.

Yet another aspect of the present invention provides a pharmaceutical composition for the prevention and/or treatment of Adolescent Idiopathic Scoliosis (AIS) in a patient, wherein said pharmaceutical composition comprises an effective amount of HOXD8 and a pharmaceutically acceptable carrier, optionally said pharmaceutical composition further comprises a further agent for the prevention or treatment of AIS.

The invention also provides a kit for detecting the expression level of HOXD8 in mesenchymal stem cells, which is characterized by comprising a primer pair for specifically amplifying HOXD8 and an instruction book, wherein the primer pair comprises a forward primer shown as SEQ ID NO. 33 and a reverse primer shown as SEQ ID NO. 34.

As used herein, the term "individual", "subject" or "patient" includes, but is not limited to, humans and other primates (e.g., chimpanzees and other apes and monkey species). In some embodiments, the subject or patient is a human.

In the present invention, the term "lncAIS (Ensembl genome database gene symbol: ENST 00000453347)" refers to lncRNA having the original sequence of the gene shown in GeneBank, which is currently the international consensus nucleic acid database, and includes lncRNA of natural or synthetic origin and its derivatives or variant forms that have been substituted, deleted or added with one or several nucleotides or that have been biologically modified and still have biological activity.

In the present invention, the term "HOXD 8(NCBI database gene ID: 3234)" refers to HOXD8 having the original sequence shown by HOXD8 gene in GeneBank, the present international common nucleic acid database, which includes HOXD8 and its analogs, which are naturally or synthetically derived. HOXD8 analog refers to a derivative or variant form thereof, which is substituted, deleted or added with one or several nucleotides, or which is biologically modified and still biologically active.

The effective dose of HOXD8 according to the invention can be adjusted accordingly, depending on the mode of administration and the severity of the disease to be treated, etc. The preferred effective amount can be determined by one of ordinary skill in the art by combining various factors. Such factors include, but are not limited to: pharmacokinetic parameters of HOXD8 or its analogs, health status of the treated patient, body weight, route of administration, and the like.

In some embodiments of the invention, the vector to which HOXD8 is conjugated may be a vector of the type commonly used in the art for expression of HOXD8 in host cells, such as a liposome, chitosan, or lentiviral expression vector, and the pharmaceutically acceptable excipients include various excipients, diluents, and adjuvants that are used in medicine but do not cause significant side effects, including but not limited to: purified water, physiological saline, buffer, glucose, water, glycerol, mannitol, ethanol, surfactants and salts such as sodium chloride, sodium EDTA and the like.

The pharmaceutical compositions of the present invention may include classical pharmaceutical formulations. The pharmaceutical composition according to the present invention may be administered by any conventional route as long as the target tissue is available by the route. In some embodiments of the invention, the pharmaceutical composition is in a form suitable for: direct naked lncRNA injection, liposome-encapsulated RNA direct injection, plasmid DNA carried by reproduction-defective bacteria or target DNA carried by replication-defective adenovirus, etc.

In some embodiments of the invention, the expression vector is a lentiviral expression vector, preferably the lentiviral expression vector may be a pSicoR, pSIN-EF2 or pwxl plasmid, wherein over-expression of HOXD8, preferably the pSIN-EF2 plasmid, and knock-down of HOXD8, preferably the pSicoR plasmid.

In some embodiments of the invention, the pharmaceutical composition further optionally comprises one or more other agents effective in treating AIS, which agents are well known to those skilled in the art. The pharmaceutical composition of the present invention may be administered in combination with other therapeutic means for the prevention and/or treatment of AIS.

Has the advantages that:

the inventors of the present invention found that the transcription factor HOXD8 is associated with osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs), and HOXD8 can promote cell proliferation of BM-MSCs and increase expression levels of self-renewal-associated genes and osteogenic differentiation genes in BM-MSCs, so that it can be used to prepare products for promoting cell proliferation and osteogenic differentiation in BM-MSCs, such as plasmids containing the transcription factor HOXD8 gene, recombinant expression vectors, transgenic cell lines, genetically engineered bacteria, to further treat Adolescent Idiopathic Scoliosis (AIS). The invention also discloses a pharmaceutical composition for preventing and/or treating AIS, which comprises an effective amount of HOXD8 and a pharmaceutically acceptable carrier, for treating AIS associated with BM-MSC osteogenic differentiation abnormality in a patient.

Drawings

The results shown in fig. 1a to h demonstrate that LncAIS are down-regulated in the BM-MSCs of AIS patients. In particular, the amount of the solvent to be used,

FIG. 1a is a graph showing the cluster analysis of differentially expressed lncRNA obtained by microarray analysis of BM-MSC of healthy donors and AIS patients, where lncAIS is the lncRNA with the greatest downregulation in BM-MSC of AIS patients.

Figure 1b shows that LncAIS is located on human chromosome 1, which is identified as a conserved locus.

FIG. 1c shows comparison of LncAIS transcripts in Normal healthy donors (normals) and AIS patient BM-MSCs by real-time qPCR, indicating significant downregulation of lncAIS in AIS patient-derived BM-MSCs.

FIG. 1d shows comparison of LncAIS expression in BM-MSC of Normal healthy donors (Northern) and AIS patients by Northern blotting, indicating that lncAIS is down-regulated in BM-MSC of AIS patients.

FIG. 1e shows that LncAIS has no coding potential as analyzed by the coding Capacity assessment tool (CPAT), where XIST transcript was used as the non-coding gene control and GAPDH and RUNX2 were used as the coding gene control.

FIG. 1f shows the results of the outer translation assay using pcDNA4-myc-his plasmid, which KLF4 was used as a protein-encoding control, showing that lncAIS does not produce any detectable peptide.

The BM-MSC cell fractionation assay results shown in fig. 1g indicate that lncas is mainly distributed in the nuclei of human BM-MSCs, with HMBS RNA, ACTIN RNA and GAPDH RNA as positive controls for cytoplasmic gene expression, and U1 RNA as a positive control for nuclear gene expression.

The results of the RNA fluorescent in situ hybridization (RNA-FISH) assay shown in FIG. 1h indicate that lncAIS is down-regulated in the BM-MSC of AIS patients relative to Normal healthy donors (Normal), with red: lncAIS probe (probe); green: actin (Actin); nuclei were counterstained with DAPI.

The results shown in a to l in fig. 2 demonstrate that LncAIS activate HOXD8 transcription. In particular, the amount of the solvent to be used,

FIG. 2a is a heat map showing transcriptome microarray analysis of lncAIS silenced (shLnccAIS) and shCtrl-treated BM-MSCs, wherein the top 10 down-regulated Transcription Factors (TF) in the BM-MSCs of shlncAIS are listed.

FIG. 2b shows comparative real-time qPCR analysis of BM-MSCs of shCtrl and shLncAIS, which indicates that HOXD8, which is a marker gene for BM-MSC, is most expressed in BM-MSC among the first 10 down-regulated TFs in BM-MSC.

FIG. 2c shows the depletion of HOXD8 by infecting normal BM-MSCs with a lentivirus expressing shHOXD8, followed by detection of cell proliferation using the CCK-8 kit; the results showed that HOXD8 depletion (shHOXD8) inhibited BM-MSC proliferation compared to shCtrl-treated cells.

FIGS. 2d and 2e show that infection of normal BM-MSCs with lentivirus expressing shHOXD8 to deplete HOXD8, culturing the designated BM-MSCs in MSC maintenance medium (FIG. 2d) and OriCell MSC osteogenic differentiation medium (FIG. 2e), respectively, and real-time qPCR assessment results showed that HOXD8 depletion suppressed the expression levels of self-renewal-associated genes (NANOG, POU5F1 and SOX2) (FIG. 2d) and osteogenic differentiation genes (ALPL, BSP, RUNX2, LPL and PPAR) (FIG. 2e) in BM-MSCs.

FIGS. 2f and 2g show that infection of normal BM-MSCs with lentivirus expressing shHOXD8 to deplete HOXD8, BM-MSCs cultured in OriCell MSC osteogenic differentiation medium, ALP staining (FIG. 2f) and Von Kossa staining (FIG. 2g) results indicate that osteogenic differentiation was inhibited by HOXD8 depletion.

FIG. 2h shows that HOXD8 overexpression significantly increases the rate of cell proliferation in normal BM-MSCs when cultured in MSC medium after infection of normal BM-MSCs with a lentivirus overexpressing HOXD8(oeHOXD 8).

FIGS. 2i and 2j show that after infection of normal BM-MSC with lentivirus overexpressing HOXD8(oeHOXD8), cultured in MSC medium (FIG. 2i) and OriCell MSC osteogenic differentiation medium (FIG. 2j), respectively, real-time qPCR assessment results showed that HOXD8 overexpression increased the expression levels of self-renewal-associated genes (NANOG, POU5F1, and SOX2) (FIG. 2i) and osteogenic differentiation genes (ALPL, BSP, RUNX2, LPL, and PPAR) (FIG. 2j) in BM-MSC.

FIGS. 2k and 2l show that when normal BM-MSCs were cultured in OriCellMSC osteogenic differentiation medium after infection with a lentivirus overexpressing HOXD8(oeHOXD8), ALP staining (FIG. 2k) and Von Kossa staining (FIG. 2l) indicated that HOXD8 overexpression enhances osteogenic differentiation of BM-MSCs.

Detailed Description

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

The following are materials and general methods used in the examples of the present invention

Antibodies and reagents

Antibodies against human HOXD8(ab228450) were purchased from Abcam (Cambridge, USA). Antibodies against human RNA polymerase II (clone 4H8) were purchased from Active Motif (Carlsbad, USA). Anti- β -actin (clone AC-74) antibody was from Sigma-Aldrich (st. louis, USA). Antibodies against Myc (clone 9E10) were from Santa Cruz biotechnology (Santa Cruz, USA). Antibodies against COL1A1(BA0325), IBSP (BA2329) and OPN (PA1432) were from Boster (Wuhan, China). Secondary antibodies conjugated to Alexa-594 were purchased from Molecular probes Inc (Eugene, USA). Streptavidin beads were from Sigma-Aldrich (st. louis, USA). Protein A/G beads were from Santa Cruz Biotechnology (Santa Cruz, USA). Alkaline phosphatase detection kits were purchased from Millipore (Millerica, USA). SuperReal premix plus qPCR buffer was from TIANGEN Biotech (Beijing, China). The oriCell BM-MSC osteogenic differentiation kit is from Cyagen (HUXMA-90021, China). The Von Kossa staining kit is from Genmed sciences (shanghai, china). Cell counting kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan). The Fast Green staining kit and the Alcian Blue staining kit were from Xinhuaalvuan Biotechnology (Beijing, China).

The probe, the primer, the shLncAIS and shHOXD8 gene sequences of the invention

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

lncAIS probe	Probe sequence	Sequence numbering
			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: shLncAI gene sequence for lncAIS knock-down

Gene	Gene sequences	Sequence numbering
			shLncAIS#1	5’-TTCCTAATCCTGCTCCAG-3’	SEQ ID NO:4
shLncAIS#2	5’-ATAGACATCTGGTTTCTGG-3’	SEQ ID NO:5

Table 3: shHOXD8 gene sequence for HOXD8 knock-down

Gene	Gene sequences	Sequence numbering
			shHOXD8#1	5’-GCTCGTCTCCTTCTCAAAT-3’	SEQ ID NO:6
shHOXD8#2	5’-GGCCGAGCTGGTACAATAT-3’	SEQ ID NO:7
			shHOXD8#3	5’-GACAAACCTACAGTCGCTT-3’	SEQ ID NO:8

Table 4: primer sequences for cDNA amplification in qRT-PCR analysis

Patient and sample

Bone Marrow (BM) aspirates were obtained from 42 AIS patients (mean age 14.5 years, range 12-17 years) and 25 healthy donors (mean age 14.9 years, range 12-17 years). In the AIS group, all patients received a comprehensive clinical and radiological examination to exclude other causes of scoliosis and to determine a diagnosis of AIS. In the control group, each of 25 age and gender matched subjects had a straight spine and normal forward curvature test by physical examination. Upon entry into the study, they were confirmed to be free of any associated medical illness or spinal deformity. The study was approved by the ethical committee of the Chinese academy of medical sciences and the Beijing-coordinated hospital. Written informed consent was obtained from all subjects and their parents prior to study entry.

Cell isolation, culture and osteogenic differentiation assay

Human bone marrow tissue was collected from AIS patients and healthy donors. All experiments were performed according to the procedure approved by the ethical committee of the chinese medical academy of sciences and the cooperation of beijing with hospitals. Human BM-MSCs are isolated and cultured as described below (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 genetic Steel Cells of additive Idiopathic Scolosis Patents by micro and Integrated Gene Network analysis. spine 2016,41(10):840 855). Human 293T cells were cultured in DMEM supplemented with 10% FBS and 100U/ml penicillin and 100mg/ml streptomycin. Lentiviruses were produced in 293T cells using standard protocols. Transfection was performed using lipofectin (Invitrogen). For shRNA knockdown and overexpression experiments, the target sequence was constructed into the pSicoR plasmid. Lentiviruses are produced by 293T cells. The most potent shRNA among the 3 shRNA constructs was selected for the following experiment. Biological replicates were performed using three independent knockdown cell lines in each assay. At least four independent experiments were performed as biological replicates. For example, the sequence of the shlncAIS gene used for the knockdown of lncAIS is shown as SEQ ID NOS: 4-5 in Table 2 above; the shHOXD8 gene sequence for HOXD8 knock-down is shown as SEQ ID NO 6-8 in Table 3 above. To induce osteogenic differentiation, third generation BM-MSCs were seeded in six-well plates and treated with osteogenic induction medium according to the manufacturer's protocol. The medium was changed every 3 days.

Wound healing test

The dishes were coated with 0.1% gelatin (v/v) for 1 hour at 37 ℃. Will be 1 × 10⁶Individual BM-MSC cells were plated to generate confluent monolayers. Cells were cultured to complete adhesion and spreading. Wounds were created by manually scraping cell monolayers with a p200 pipette tip. A first image is acquired using the reference point markers. Cells were cultured in a tissue culture incubator for 24 hours. The second image is acquired by matching the shooting area of the first image.

Coding potential analysis

The coding potential of lncAIS was analyzed on the website of http:// lilab. research.bcm.edu/CPAT/by the Coding Potential Assessment Tool (CPAT) according to the manufacturer's instructions. XIST transcript was used as a non-coding gene control. GAPDH and RUNX2 were used as coding gene controls.

Cell fractionation analysis

BM-MSCs were lysed using NE-PER nuclear and cytoplasmic extraction kit (Pierce) according to the manufacturer's instructions, followed by nuclear and cytoplasmic fractionation. RNA was extracted using TRIzol Reagent (Invitrogen) and then purified using RNeasy kit (Qiagen, Valencia, Calif., USA). Reverse transcription was performed by M-MLV reverse transcriptase (Promega) and qRT-PCR analysis. ACTIN RNA and GAPDH RNA were used as positive controls for cytoplasmic gene expression. The U1 RNA served as a positive control for nuclear gene expression.

ALP and Von Kossa staining

ALP staining was monitored using the ALP staining kit according to the manufacturer's protocol. Mineral deposition was monitored using Von Kossa staining kit according to the manufacturer's protocol. Images were obtained with a Nikon EclipseTi microscope (Nikon, Japan). The colour intensity of mineral deposits was quantified by ImageJ.

Northern blotting

Total RNA was extracted from BM-MSC using TRIzol. Mu.g of RNA from each sample was subjected to formaldehyde denaturing agarose electrophoresis and then transferred to positively charged NC membranes using 20 XSSC buffer (3.0M NaCl and 0.3M sodium citrate, pH 7.0). The membrane is UV cross-linked and incubated with a biotin-labeled RNA probe, such as the IncAIS (11-228nt) probe, produced by in vitro transcription. Detection of biotin signal with HRP-conjugated streptavidin was used in the cheniuuminescent nucleic acid detection module according to the manufacturer's instructions.

RNA FISH

Fluorescently conjugated lncas probes were generated according to the protocol of Biosearch Technologies. The sequences of the lncAIS (11-228nt) probe set for RNA FISH are shown in SEQ ID NOS: 1-3 in Table 1 above. BM-MSCs were hybridized to DNA probe sets and then stained with the indicated antibodies. Images were obtained with an Olympus FV1200 laser scanning confocal microscope (Olympus, Japan).

Microarray analysis

RNA was extracted from BM MSCs using TRIzol Reagent (Invitrogen) and then purified using RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. cDNA was generated using One-Cycle Target Labeling and control Reagents (Affymetrix, Santa Clara, Calif., USA) and cRNA was generated using the GeneChip WT Labeling kit (Affymetrix, Santa Clara, Calif., USA). Biotin-labeled fragmented (. ltoreq.200 nt) cRNA was hybridized with Affymetrix GeneChip Human transcript array 2.0(Affymetrix) at 45 ℃ for 16 h. GeneChip was washed and stained in Affymetrix Fluidics Station 450. Use and install in

Scanner 30007G

GeneChipCommand Console (AGCC) scans GeneChips. Data were analyzed using the RobustMultichip Analysis (RMA) algorithm using Affymetrix default Analysis settings and global scaling as normalization methods. The values given are log2 RMA signal strength. Microarray data was stored in GEO under accession number (GSE 110359).

RNA knock-down assay

Biotin-labeled lncas full length (sense) and antisense RNA were obtained in vitro with biotin RNA labeling cocktail (Roche) and then incubated with extracts isolated from BM-MSCs. The RNA binding protein was pulled down (pull down) by streptavidin beads. The pull-down fractions were separated by SDS-PAGE and then silver stained. Differential bands enriched by lncas were analyzed by LTQ Orbitrap XL mass spectrometry or immunoblotted with the indicated antibodies.

RNA Immunoprecipitation (RIP) assay

BM-MSCs were treated with 1% formaldehyde and then solubilized with RNase-free RIPA buffer (50mM Tris-HCl [ pH7.4], 150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 5mM EDTA, 2mM PMSF, 20mg/ml aprotinin, 20mg/ml leupeptin, 10mg/ml pepstatin A, 150mM benzamidine, 1% Nonidet P-40, and RNase inhibitor). Samples were sonicated on ice and centrifuged. The supernatant was previously cleared and incubated with the indicated antibodies, followed by protein a/G bead immunoprecipitation. Extracting total RNA from the eluate. LncAIS enrichment was analyzed by qPCR. The primer sequences for cDNA amplification are listed in Table 4 above.

Chromatin immunoprecipitation (ChIP) assay

ChIP was quantified according to standard protocol (Upstate). Will be selected from BM-MSC (2X 10) fixed in 1% formaldehyde⁶) Sheared chromatin (sonicated to 200-500bp) was incubated with 4 μ g of antibody overnight at 4 ℃ and then immunoprecipitated with salmon sperm DNA/protein agarose beads. After washing, elution, and cross-linking reversal, DNA from each ChIP sample and the corresponding input sample was purified and analyzed using qPCR. The primer sequences for cDNA amplification are listed in Table 4 above.

Ectopic bone formation in vivo

Will be 2X 10 in total⁶The individual BM-MSC were incubated with approximately 100mg of wet HA/TCP ceramic powder (national engineering Research Center for Biomaterials, Chengdu, China) overnight at 37 ℃. Cells were implanted subcutaneously on the dorsal surface of 8-week-old NOD/SCID mice. The implants were harvested after 8 weeks, fixed in 4% paraformaldehyde, decalcified in 10% EDTA, embedded in paraffin, then sectioned and stained. The bone tissue was stained green by rapid green staining. Cartilage tissue was stained blue by alcian blue staining to indicate bone maturation.

Statistical analysis

Unpaired student's t-test was used as a statistical analysis in the present invention. Statistical calculations were performed using Microsoft Excel or SPSS 13. When P <0.05, the P value was significant.

Examples

Example 1: identification of LncAS as a significantly differentially expressed lncRNA that is down-regulated in bone marrow mesenchymal stem cells (BM-MSC) of AIS patients

To identify key lncrnas involved in Adolescent Idiopathic Scoliosis (AIS), we performed microarray analysis of BM-MSCs from 5 healthy donors and 12 AIS patients. 1483 lncRNA showed differential expression in normal BM-MSC and AIS patients' BM-MSC, with 718 up-regulated and 765 down-regulated, as shown in FIG. 1 a. Among the lncRNAs with the greatest downregulation amplitude in BM-MSCs of AIS patients, we focused on the uncharacterized lncRNA which we called lncAIS (gene symbol: ENST 00000453347). lncas is located on human chromosome 1, contains 4 exons, and is a full-length 476nt transcript. lncas were identified as conserved loci as shown in figure 1 b.

Real-time qPCR was performed according to the routine procedure to analyze the incais transcripts in BM-MSC of normal healthy donors and BM-MSC of AIS patients, and cDNA primers for amplification of incais were as shown in SEQ ID NOs 9-10 in table 4 above. BM-MSC were from 20 healthy donors and 30 AIS patients. Relative gene expression fold was normalized to endogenous β -actin and counted as mean ± s.d. P < 0.01. The results demonstrate a significant downregulation of lnciss in AIS patient derived BM-MSCs compared to BM-MSCs from healthy donors, as shown in figure 1 c.

IncAIS expression in BM-MSC of normal healthy donors and in BM-MSC of AIS patients was examined by Northern blot. The probes for lncAIS (11-228nt) of 217nt, whose sequences are shown in SEQ ID NOS: 1-3 in Table 1 above, were labeled and used for northern blot analysis. RNA was extracted from designated normal healthy donor BM-MSC and AIS patient BM-MSC, respectively, and 18S rRNA (1482-1725nt) was used as loading control. BM-MSC were from 3 healthy donors and 3 AIS patients. As shown in fig. 1d, Northern blot further confirmed lncas down-regulation in BM-MSC of AIS patients, showing only one transcript of lncas.

Analysis by a coding ability assessment tool (CPAT) showed that lncas has no coding potential; where XIST transcripts were used as non-coding gene controls and GAPDH and RUNX2 were used as coding gene controls, as shown in FIG. 1 e. The lncAIS transcript was cloned into pcDNA4-myc-his plasmid and transfected into 293T cells for 48 hours. Expression of Myc-fusion protein was analyzed by immunoblotting with anti-Myc antibody. KLF4 was used as a control for coding proteins. In vitro translation assays showed that lncas did not produce any detectable peptides, as shown in figure 1 f.

BM-MSC is firstly cracked, then nuclear and cytoplasmic fractionation and RNA extraction are carried out, and then qRT-PCR analysis is carried out. HMBS RNA, ACTIN RNA and GAPDH RNA were used as positive controls for cytoplasmic gene expression. The U1 RNA served as a positive control for nuclear gene expression. N: the nuclear fraction. C: cytoplasmic fraction. The primer pairs used for cDNA amplification in the qRT-PCR analysis are listed in table 4 above. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. Cell fractionation assay results showed that lncAIS is mainly distributed in the nuclei of human BM-MSC, as shown in FIG. 1 g.

lncAIS was visualized in BM-MSC by RNA fluorescent in situ hybridization (RNA-FISH) assay followed by immunofluorescent staining. Red: a lncAIS probe; green: actin; nuclei were counterstained with DAPI. Scale bar, 20 μm. The lncAIS probe sequence is shown in SEQ ID NO 1-3 in Table 1 above. Over 100 representative cells were observed. RNA fluorescence in situ hybridization (RNA-FISH) further demonstrated the downregulation of lncAIS in BM-MSC of AIS patients and the distribution of lncAIS in nuclei, as shown in FIG. 1 h.

In conclusion, we revealed that lncAIS is highly expressed in normal human BM-MSC, but is significantly down-regulated in AIS patients BM-MSC.

Example 2: determination that LncAIS activates HOXD8 transcription

Transcriptome microarray analysis comparisons of lnciss-silenced (shLncAIS) and shCtrl-treated BM-MSCs (the sequence of the shlnciss gene for lnciss knockdown is shown in SEQ ID NOs: 4-5 in table 2 above) and results are shown in the heat map of fig. 2a, in which 10 Transcription Factors (TFs) with the largest downregulation amplitude were selected from lnciss-silenced BM-MSCs; and their expression level in normal BM-MSC was examined. Data were from two independent experiments using BM-MSCs derived from 2 healthy donors. Each matched shCtrl and shLncAIS is from the same healthy BM-MSC donor.

As shown in FIG. 2b, the real-time qPCR comparative analysis of shCtrl and shLncAIS BM-MSC showed that HOXD8, which is a BM-MSC marker gene, was most expressed in normal BM-MSC among the 10 TFs with the largest down-regulation range among shLncAIS-treated BM-MSCs. Data were from two independent experiments using BM-MSCs derived from 2 healthy donors. Each matched shCtrl and shLncAIS is from the same healthy BM-MSC donor. However, the role of HOXD8 in regulating osteogenic differentiation of BM-MSCs has not been characterized currently.

HOXD8 was then depleted in normal BM-MSCs by lentivirus-mediated shRNAs. BM-MSCs were infected with lentiviruses expressing shHOXD8 (shHOXD8 gene sequence for HOXD8 knock-down is shown as SEQ ID NOS: 6-8 in Table 3 above), cultured in MSC medium for 3 days, and then cell proliferation was detected using a CCK-8 kit. The absorbance change was calculated as mean ± s.d. P < 0.01. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. As shown in fig. 2c, HOXD8 depletion inhibited BM-MSC proliferation compared to shCtrl-treated cells.

The indicated BM-MSCs were cultured in MSC maintenance medium and the expression level of self-renewal associated genes was assessed by real-time qPCR. BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days, and the expression level of osteogenic differentiation genes was evaluated by real-time qPCR. The primer pairs used for cDNA amplification in qPCR are listed in table 4 above. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. As shown in fig. 2d and 2e, HOXD8 depletion suppressed the expression levels of self-renewal-associated genes (NANOG, POU5F1, and SOX2) and osteogenic differentiation genes (ALPL, BSP, RUNX2, LPL, and PPAR) in BM-MSCs.

BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days to induce osteogenic differentiation, and ALP staining was performed on day 6 of osteogenic differentiation, as shown in fig. 2 f. BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days to induce osteogenic differentiation, and Von Kossa staining was performed to indicate mineral deposition on day 12, as shown in fig. 2 g. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. Scale bar, 10 μm. The colour intensity of mineral deposits was quantified by ImageJ and the intensity variation was calculated as mean ± s.d. P < 0.01. ALP staining (fig. 2f) and Von Kossa staining (fig. 2g) results indicate that osteogenic differentiation was inhibited by HOXD8 depletion.

BM-MSCs were infected with HOXD 8-overexpressing lentivirus and cultured in MSC medium for 3 days, followed by detection of cell proliferation using the CCK-8 kit. Inoculation of 1X 10 per well³Personal BM-MSC. 24 hours after inoculation, CCK-8 reagent was loaded. OD450 was measured every 2 days. The absorbance change was calculated as mean ± s.d. P<0.01. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. As shown in fig. 2h, the results indicate that HOXD8 overexpression (oeHOXD8) significantly increased the rate of cell proliferation in normal BM-MSCs.

The indicated BM-MSCs were cultured in MSC maintenance medium and the expression levels of self-renewal associated genes (NANOG, POU5F1 and SOX2) were assessed by real-time qPCR, as shown in figure 2 i. BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days as shown in FIG. 2 j. Expression levels of osteogenic differentiation genes were assessed in designated BM-MSCs by real-time qPCR. The primer pairs used for cDNA amplification in qPCR are listed in table 4 above. Relative gene expression fold changes were normalized to endogenous β -actin and counted as mean ± s.d. P < 0.01. Data represent three independent experiments using BM-MSCs derived from 3 healthy donors. As shown in fig. 2i and 2j, the results indicate that overexpression of HOXD8(oeHOXD8) increases the expression levels of self-renewal-related genes (NANOG, POU5F1, and SOX2) and osteogenic differentiation genes in BM-MSCs.

BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days to induce osteogenic differentiation, and ALP staining was performed on day 6 of osteogenic differentiation, as shown in fig. 2 k. BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days to induce osteogenic differentiation, and Von Kossa staining was performed to indicate mineral deposition on day 12, as shown in fig. 2 l. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. Scale bar, 10 μm. The colour intensity of mineral deposits was quantified by ImageJ and the intensity variation was calculated as mean ± s.d. P < 0.01. As shown in FIGS. 2k and 2l, the results indicate that overexpression of HOXD8 enhances osteogenic differentiation of BM-MSCs.

In conclusion, the above experimental results indicate that HOXD8 is essential for the osteogenic differentiation of BM-MSCs.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing coordination hospital of Chinese academy of medical sciences

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Claims

1. Application of over-expressed HOXD8 gene in preparation of products for promoting cell proliferation of mesenchymal stem cells, wherein the over-expressed HOXD8 gene can promote cell proliferation of the mesenchymal stem cells.

2. The use of claim 1, wherein HOXD8 produced by infection of mesenchymal stem cells with a lentivirus overexpressing HOXD8 upregulates cell proliferation of mesenchymal stem cells.

3. Use according to claim 2, wherein the expression vector of the lentivirus overexpressing HOXD8 is the pSIN-EF2 plasmid.

4. The use according to claim 1 or 2, wherein the product promoting cell proliferation of mesenchymal stem cells is a lentivirus-mediated cell overexpressing HOXD8 gene.

5. The application of the over-expressed HOXD8 gene in preparing a product for promoting osteogenic differentiation of bone marrow mesenchymal stem cells is characterized in that the over-expressed HOXD8 gene can increase the expression level of self-renewal related genes and osteogenic differentiation genes in the bone marrow mesenchymal stem cells.

6. The use of claim 5, wherein infection of the mesenchymal stem cells with a lentivirus overexpressing HOXD8 produces HOXD8 that upregulates expression levels of self-renewal-associated genes and osteogenic differentiation genes in the mesenchymal stem cells.

7. The use of claim 6, wherein the expression vector of the lentivirus overexpressing HOXD8 is the pSIN-EF2 plasmid.

8. The use according to claim 5, wherein the product promoting osteogenic differentiation of mesenchymal stem cells is a cell overexpressing the HOXD8 gene effected in a lentivirus-mediated manner.