CN111166884A

CN111166884A - Application of Foxf1 gene in preparation of medicine for treating osteoporosis

Info

Publication number: CN111166884A
Application number: CN202010041695.3A
Authority: CN
Inventors: 江晓兵; 沈耿杨; 任辉; 尚奇; 梁德; 雷海青; 招文华; 张志达; 余翔; 黄锦菁; 何嘉辉; 汤凯; 梁梓扬; 唐晶晶
Original assignee: First Affiliated Hospital of Guangzhou University of Chinese Medicine
Current assignee: First Affiliated Hospital of Guangzhou University of Chinese Medicine
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2020-05-19
Anticipated expiration: 2040-01-15
Also published as: CN111166884B

Abstract

the invention relates to an application of a Foxf1 gene in preparation of a medicine for treating osteoporosis, which belongs to the technical field of biomedicine, wherein mouse and human mesenchymal stem cells (BMSC) and mouse bone marrow-derived macrophages (BMM) are utilized to research, research data show that Foxf1 silences and promotes BMSCs to form bones through Wnt/β -cantenin, and in vivo experiments prove that siFoxf1 (silences Foxf1) tail vein injection can reduce the bone mass loss of an ovariectomized mouse, so siFoxf1 can possibly play an important role in the treatment of PMOP and can be used for preparing a medicine for treating osteoporosis, particularly PMOP.

Description

Application of Foxf1 gene in preparation of medicine for treating osteoporosis

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of a Foxf1 gene in preparation of a medicine for treating osteoporosis.

Background

Postmenopausal osteoporosis (PMOP) is a metabolic disease in which the bone mass per unit volume is reduced, the bone microstructure is altered, the bone fragility is increased, and fractures are prone to occur after menopause. As the human beings enter an aging society, PMOP is increasingly developed year by year in countries around the world, causing a heavy economic burden to the society and families. Current drugs for treating PMOP are still limited. The recommended drugs for PMOP treatment, such as bisphosphonates and teriparatide, have serious side effects, such as nausea, epigastric pain, dyspepsia, gastritis, back pain, arthralgia, etc.; teriparatide needs to be injected daily and is only acceptable for one treatment over a 24-month period for life. Therefore, research on the pathogenesis of PMOP and search for the prevention and treatment target of PMOP become urgent clinical needs.

Disclosure of Invention

based on the above, there is a need to provide an application of Foxf1 gene in preparing a drug for treating osteoporosis, taking Foxf1 gene as a new target for treating osteoporosis, especially for PMOP, to silence Foxf1 to promote BMSCs osteogenesis and prevent ovariectomy-induced bone loss through Wnt/β -cantenin, and to provide a new therapeutic approach for treating osteoporosis, especially for PMOP.

Application of Foxf1 gene in preparation of drugs for treating osteoporosis.

the inventor utilizes mouse and human bone marrow mesenchymal stem cells (BMSC) and mouse bone marrow derived macrophages (BMM) to carry out research, and research data shows that Foxf1 silencing promotes the formation of BMSCs through Wnt/β -cantenin and prevents and treats ovariectomy-induced bone loss.

In one embodiment, the osteoporosis is post-menopausal osteoporosis.

The invention also discloses application of the reagent for silencing Foxf1 gene in preparing a medicine for treating osteoporosis.

In one embodiment, the agent that silences the Foxf1 gene comprises: foxf1 specific siRNA.

In one embodiment, the sense sequence of the Foxf 1-specific siRNA is selected from the group consisting of: the sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.

In one embodiment, the sense sequence of the Foxf 1-specific siRNA is selected from the group consisting of: and (b) an equivalent sequence which is subjected to substitution, deletion and addition modification of one or more bases with the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and can silence the Foxf1 gene.

In one embodiment, the Foxf1 specific siRNA has greater than 90% sequence similarity to the sequence shown in SEQ ID No.1 or SEQ ID No. 2.

The invention also discloses a medicine for treating osteoporosis, which comprises the following components: an agent that specifically silences the Foxf1 gene.

In one embodiment, the sense sequence of the Foxf 1-specific siRNA is selected from the group consisting of: the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 are subjected to substitution, deletion and addition modification of one or more bases to obtain an equivalent sequence, and the equivalent sequence can silence the Foxf1 gene.

In one embodiment, the route of administration of the drug is intravenous.

Compared with the prior art, the invention has the following beneficial effects:

the inventor conducts research by using mouse and human bone marrow mesenchymal stem cells (BMSC) and mouse bone marrow derived macrophages (BMM), and research data shows that Foxf1 silences Wnt/β -cantenin to promote BMSCs to form bones, and in vivo experiments prove that siFoxf1 tail vein injection can reduce the bone mass loss of ovariectomized mice, therefore, siFoxf1 (silences Foxf1) may play an important role in the treatment of PMOP and can be used for preparing medicines for treating osteoporosis, particularly PMOP.

Drawings

FIG. 1 is the bone tissue of OVX mice in example 1, the expression level of Foxf1 in BMSC and BMM;

FIG. 2 is a schematic diagram showing the change of Foxf1 in the process of osteogenic differentiation and osteoclastic differentiation in example 1;

FIG. 3 is a graphical representation of the results of Foxf1 gene silencing in example 1 to enhance osteogenesis of BMSCs;

FIG. 4 is a schematic diagram showing the result of Foxf1 silencing activation of Wnt/β -catenin signaling pathway in example 1;

fig. 5 is a schematic representation of the reversion of siFoxf1 mediated osteogenesis of BMSCs by DKK1 in example 1;

FIG. 6 is a schematic diagram of the experimental design and results for preventing and treating bone mass loss caused by OVX in example 2 with siFoxf 1;

FIG. 7 is a graph showing the results of the siFoxf1 activation of the β -cantenin pathway to increase bone formation in vivo in example 2;

fig. 8 is a graphical representation of the results of increased Foxf1 expression in bone extracts from PMOP patients in example 3, and siffoxf 1 promoting osteogenesis in human BMSCs.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The starting materials used in the following examples are all commercially available unless otherwise specified.

The following animal experiments were approved by the ethical committee of the first subsidiary hospital of the university of traditional Chinese medicine, Guangzhou (TCMF 1-2019030);

statistical analysis methods in the following examples data analysis was performed using SPSS Statistics version 19.0 (IBM, usa). After checking distribution of positive and uniform variances, two groups of independent samples are compared by using a t test, and more than or equal to three groups of independent samples are analyzed by using variance. A P value of 0.05 or less is statistically significant. Data are expressed as mean ± standard deviation (s.d.).

Example 1

siFoxf1 promotes mouse bone marrow mesenchymal stem cell proliferation and osteogenic differentiation.

Firstly, a method is provided.

1. Removing Ovary (OVX) and making osteoporosis model.

To evaluate osteoporosis caused by OVX, 8-week-old C57BL/6 female mice were subjected to anesthesia excision of bilateral Ovaries (OVX), and a sham-operated group (only fat around ovaries was removed without ovaries excision) was set up, and the osteoporosis model was successfully created by taking the ovaries 6 weeks later.

2. And (5) culturing the cells.

A. Mouse bone marrow mesenchymal stem cell (BMSC) isolation and culture

Tibia and femur of 8-week-old C57BL/6 mice were aseptically dissected, and bone marrow cells were collected, cultured and passaged using mouse BMSC medium (cygen Biosciences, guangzhou, china). Cells from passage 5 to 9 were harvested for subsequent experiments.

B. BMM culture of mouse bone marrow-derived macrophages

mouse bone marrow-derived macrophages (BMM) were isolated by conventional methods and grown in α -MEM medium (Gibco, USA) containing 5ng/ml macrophage colony stimulating factor (M-CSF) (R & D Systems, USA), 1% penicillin and streptomycin (Gibco, USA) and 10% fetal bovine serum (Gibco, USA) in 100mm dishes.

3. RNA extraction and qRT-PCR

total RNA was extracted from BMSC and BMM with TRIzol reagent (Invitrogen), total RNA was reverse transcribed to cdna using PrimeScript RTMaster Mix (Perfect read Time; TaKaRa, japan), transcript levels were quantified using β -actin as an internal reference, SYBR Green PCR Master Mix (TaKaRa), gene expression levels were quantified using the 2- Δ Δ Ct method.

TABLE 1 primer sequences

4. Western blot

cells were lysed in RIPA lysate (Beyotime, china), proteins were isolated by 15% SDS-page (Bio-Rad, usa) and transferred onto PVDF (Millipore, shanghai, china), membranes were blocked in skim milk (5%) for 2h at room temperature, followed by incubation of primary antibodies, β -actin (1:500, Cell Signaling Technology, usa), Foxf1 (1:500, Biorbyt, usa) and β -cantenin (1: 500; Cell Signaling Technology, usa), washed 3 times with PBST, after 10 minutes each, membranes were incubated with the corresponding secondary antibodies (1: 3000; Cell Signaling Technology, usa) for 2 hours at room temperature, protein levels were determined by chemiluminescence (Bio-Rad, usa) Image J software, intensity was quantified using Image J software.

5. SiRNA mediated silencing and cell transfection

Foxf1 specific siRNA (target sequence GATCCGGCTAGCGAGTTTA, SEQ ID NO.4) with a sense sequence of GAUCCGGCUAGCGAGUUUA, SEQ ID NO.2, was used to silence Foxf1 and was also used as a negative control siRNA (siCtrl, from RiboBio, Guangzhou, China). siRNA transfection was performed using Lipofectamine RNAimax (Invitrogen, USA).

Foxf1 expression was subsequently verified by quantitative reverse transcription PCR (qRT-PCR), Western Blot (WB) and Immunofluorescence (IF).

6. Osteoblast differentiation assay

The BMSCs were cultured in osteogenic induction medium (Cyagen Biosciences, Guangzhou, China). Osteogenic induction medium was changed every three days. After 7 days of osteogenic induction, cells were subjected to alkaline phosphatase (ALP) staining (cloudy days, shanghai, china) and ALP activity (cloudy days, shanghai, china) assays. Mineral deposition was assessed by Von Kossa staining (solibao, china) 14 to 28 days after osteogenic induction.

7. ALP staining and Activity assay

Cells were fixed in 4% paraformaldehyde for 20 minutes and washed 3 times with distilled water. Next, cells were stained with BCIP/NBT alkaline phosphatase color kit (Beyotime, Shanghai, China). To determine ALP activity, ALP activity assay kit (bi yun day, shanghai, china) was used on 96-well plates. ALP activity was quantified by determining the absorbance at 405/650 nm.

8. Von Kossa staining

Calcium deposits were determined by Von Kossa staining. Cells were fixed in 4% paraformaldehyde for 20 minutes and washed 3 times with distilled water. Cells were incubated in 5% silver nitrate, exposed to uv light for 30 minutes, washed in 5% sodium sulfate and photographed.

9. Osteoclast differentiation

For osteoclast differentiation, BMMs were treated with M-CSF (5ng/ml) and receptor activator of NF-kB ligand (RANKL, 10ng/ml) (R & D Systems, USA) for 4-6 days. Then, the cells were fixed in 4% paraformaldehyde for anti-tartrate acid phosphatase (TRAP) (Sigma, usa) staining. TRAP + cells with at least three nuclei are considered osteoclasts.

10. Immunofluorescence

cells were cultured in confocal dishes to assess the expression levels of Foxf1, Runx2, Col1a1 and β -catenin proteins cells were fixed with 4% paraformaldehyde for 30 minutes, blocked with 0.3% Triton X-100 permeabilization and 1% bovine serum albumin for 30 minutes, then cells were incubated with primary antibodies Foxf1 (1: 500; Biorbyt), Runx2 (1: 1600; Cell signaling technology), Col1a1 (1: 500; Abcam, Cambridge, UK), cells were incubated with fluorescently coupled secondary antibodies (Beyotime, shanghai, china) for 60 minutes and photographed with 4', 6-diamidino-2-phenylindole (DAPI) (Beyotime, shanghai, china) for 5 minutes to stain by confocal laser scanning microscopy (Leica, germany).

11. β -catenin/TCF transcription reporter gene detection (TOP/FOPflash detection)

to determine the activation status of Wnt signaling, β -catenin/TCF transcriptional activity was determined by transfection of the TOPflash/FOPflash luciferase reporter plasmid (Addgene, USA.) cells were seeded (2X 10 per well)⁴) And cultured in 24-well plates for 12 h. Plasmids of either TOPflash (repeats with 3 TCF binding sites) or FOPflash (repeats with 3 TCF mutation sites) were transfected into cells. Luciferase activity was measured 48 hours after transfection using a luciferase assay system.

And II, obtaining a result.

1. Bone tissue from OVX mice, expression levels of Foxf1 in BMSC and BMM.

The results are shown in FIG. 1, where:

FIG. a shows q-RT PCR analysis of the mRNA expression level of Foxf1 in bone tissues of control and OVX mice, and the results of the analysis showed that the expression level of Foxf1 mRNA was significantly increased in vertebral tissues derived from OVX mice as compared with the control.

FIGS. b and c are a WB (Western blot) gel diagram of the expression level of Foxf1 protein in bone tissues of mice in the control group and OVX group, respectively, and an analysis of the expression level, and it was revealed that vertebral tissues derived from OVX mice showed a significant increase in the expression level of Foxf1 protein.

FIG. d shows the mRNA expression level of Foxf1 in BMSCs from control and OVX mice analyzed by q-RT PCR, and the results of the analysis showed that the BMSCs from OVX mice showed a significant increase in Foxf1 mRNA expression level.

FIGS. e and f are a WB (Western blot) gel image and an expression analysis of the expression level of Foxf1 protein in BMSCs of a control group and OVX group, respectively, and it was revealed that OVX mouse-derived BMSCs exhibited a significant increase in the expression level of Foxf1 protein.

FIG. g shows the Q-RT PCR analysis of the mRNA expression level of Foxf1 in the BMMS of the control group and the OVX group mice, and FIGS. h-i show the WB (Western blot) glue pattern and the WB (Western blot) expression level analysis of the Foxf1 protein expression level in the BMMS of the control group and the OVX group mice, respectively, and the results show that the expression levels of Foxf1 mRNA and protein in the BMMS of the OVX source are not significantly different from those of the control group.

2. Foxf1 decreased during osteogenic differentiation, with no significant change in osteoclastic differentiation.

The results are shown in FIG. 2, where:

panel a shows the q-RT PCR analysis of the mRNA expression level of Foxf1 in BMSCs over time during osteogenic differentiation, showing a significant decrease in Foxf1 mRNA expression following osteogenic differentiation.

Panels b and c show the expression level WB (western blot) of Foxf1 protein in BMSCs during osteogenic differentiation and analysis of the expression level, and the results show that Foxf1 protein expression is significantly reduced after osteogenic differentiation.

FIG. d shows the q-RT PCR analysis of the mRNA expression level of Foxf1 in BMMs as a function of time during osteoclastic differentiation, and FIGS. e-f show the WB (Western blot) gel pattern and the WB (Western blot) expression level analysis of Foxf1 protein expression level in BMMs as a function of time during osteoclastic differentiation, showing that there was no significant difference in the expression levels of Foxf1 mRNA and protein in OVX-derived BMMS.

3. Foxf1 gene silencing enhances osteogenesis of BMSCs.

The results are shown in FIG. 3, where:

panels a and b are representative images of ALP staining and Von Kossa staining, respectively, of siffoxf 1-transfected BMSCs. As can be seen, after siFoxf1 treatment, ALP staining was darker and the number of ALP positive cells was greater; more calcific deposits are deposited.

Panel c is a quantitative analysis of ALP activity of siFoxf1 transfected bone marrow mesenchymal stem cells. As can be seen from the graph, ALP activity was higher after siFoxf1 treatment, with the difference being statistically significant (p < 0.05).

FIG. d shows the mRNA expression levels of the osteogenic markers Runx2, Alp, Osx, Ocn and Col1a1 in BMSCs transfected with siFoxf1, and the results of the analysis showed that the mRNA expression of the osteogenic markers Runx2, Osx, Alp, Ocn and Col1a1 was significantly increased after treatment with siFoxf 1.

FIG. e is immunofluorescence analysis of BMSCs transfected by siFoxf1, and shows that Runx2 fluorescence expression is enhanced after treatment of siFoxf1 (the scale bar in the figure is 40 μm), and FIG. f is fluorescence quantitative analysis of bone formation marker Runx2 protein expression in BMSCs transfected by siFoxf1, and the analysis result shows that the treatment of siFoxf1 promotes the protein expression level of Runx 2.

FIG. g is immunofluorescence analysis of BMSCs transfected by siFoxf1, which shows that Col1a1 fluorescence expression is enhanced after treatment of siFoxf1 (the scale bar in the figure is 40 μm), and FIG. h is fluorescence quantitative analysis of bone formation marker Col1a1 protein expression in BMSCs transfected by siFoxf1, and the analysis result shows that the protein expression level of Col1a1 is promoted by treatment of siFoxf 1.

4. Foxf1 silencing activates the Wnt/β -catenin signaling pathway.

The results are shown in FIG. 4, where:

panel a is a luciferase report, and experiments show that siFoxf1 treatment significantly increased the activity of Topflash, indicating that Wnt/β -catenin pathway was activated.

panels b and c show the expression level WB (Western blotting) gel map of β -catenin protein in BMSCs transfected by siFoxf1 and the analysis of the expression level, and the results show that the treatment of siFoxf1 significantly promotes the expression level of β -catenin protein.

FIG. d is immunofluorescence analysis of BMSCs transfected with siFoxf1, showing that the fluorescence expression of β -catenin is enhanced after treatment with siFoxf1 (the scale bar in the figure is 40 μm), and FIG. e is fluorescence quantitative analysis of the expression of β -catenin protein in BMSCs transfected with siFoxf1, and the analysis result shows that the treatment with siFoxf1 induces more β -catenin protein to accumulate in the cell nucleus.

FIG. f shows the mRNA expression levels of β -catenin, Oct4, Cyclin D1, C-myc and CD44mRNA in BMSCs transfected with siFoxf1, and the analysis results show that the siFoxf1 treatment significantly increases the expression levels of β -catenin, Oct4, Cyclin D1, C-myc and CD44 mRNA.

5. siFoxf1 mediated osteogenesis of BMSCs was reversed by the Wnt pathway inhibitor DKK1(Dickkopf-1 protein).

The results are shown in FIG. 5, where:

panel a is a luciferase report and experiments show that DKK1 treatment (0.5 μ g/ml) reversed the increase in Topflash activity induced by siFoxf1 treatment.

panels b and c show the expression level WB (Western blotting) gel map of β -catenin protein in BMSCs transfected by siFoxf1 and the analysis of the expression level, and the results show that the DKK1 treatment reverses the increase of the expression level of β -catenin protein induced by siFoxf1 treatment.

FIG. d shows the mRNA expression levels of Runx2 and Col1a1 in BMSCs transfected with siFoxf1, and the analysis results show that DKK1 treatment reverses the increase of Runx2 and Col1a1 mRNA expression levels induced by siFoxf1 treatment.

Panel e shows ALP activity results for BMSCs treated with DKK1 and transfected with siFoxf1, showing darker ALP staining and higher ALP positive cell numbers after siFoxf1 treatment.

Panel f is a representative image of Von Kossa staining of BMSCs treated with DKK1 and transfected with siFoxf1, showing greater calcific deposits after siFoxf1 treatment.

Example 2

siFoxf1 inhibited bone mass loss by ovariectomy in mice.

Materials and methods

1. Laboratory animal

Animal experiments were approved by the ethical committee of the first subsidiary hospital of the university of medicine, guangzhou (TCMF 1-2019030).

2. Removing Ovary (OVX) and making osteoporosis model

To evaluate OVX-induced osteoporosis, 8-week-old C57BL/6 female mice were anesthetized to resect bilateral Ovaries (OVX). siRNA tail vein injection was started on day 2 after OVX surgery. We injected siFoxf1(7mg kg-1), siCtrl (7mg kg-1) or PBS (0.2ml) into the tail vein of OVX or sham operated mice for 6 weeks (n ═ 8 per group). The siFoxf1 target sequence in the in vivo mouse experiment was GATCCGGCTAGCGAGTTTA (SEQ ID NO.4), and its siRNA sense sequence was SEQ ID NO. 2.

3、micro-CT

Scanning was performed using a Skyscan 1172Micro-CT imaging system (Skyscan, Belgium) at a spatial resolution of 12 mm (X-ray source 80kV/100 μ A). To assess the L4 vertebral microstructure, trabecular bone volume fraction (BV/TV,%), trabecular number (tb.n,/mm), trabecular thickness (tb.th, mm) and trabecular spacing (tb.sp, mm) were calculated within the defined region of interest.

4. Bone tissue morphometry

For histological analysis, 25mg/kg calcein was injected intraperitoneally on day 8 and day 2 before drawing the material. Bone histomorphology was assessed using hard tissue sections of undecalcified bone samples. The bone histomorphometric parameters evaluated were bone formation rate (BFR/BS, d. mu. m 3. mu. m-2. d-1), bone mineral deposition rate (MAR, μm per day), mineralized surface (MS/BS,%), osteoblast number (N.ob/B.Pm,/mm), osteoclast number (N.oc/B.Pm,/mm) and osteoclast surface (oc.S/BS,%).

5. Enzyme-linked immunosorbent assay (ELISA)

ELISA kit (Fountain Hills, USA) measures serum concentrations of ALP and TRAP5 b.

And II, obtaining a result.

1. SiFoxf1 can be used for preventing and treating bone loss caused by OVX.

The results are shown in FIG. 6, where:

FIG. a is a schematic diagram of the experimental design in mice, specifically 8-week old C57BL/6 female mice were anesthetized to remove bilateral Ovaries (OVX). siRNA tail vein injection was started on day 2 after OVX surgery. OVX or sham operated mice were injected intravenously with siFoxf1(7mg kg-1), siCtrl (7mg kg-1) or PBS (0.2ml) for 6 weeks (n ═ 8 per group). Lumbar vertebra and serum were obtained 6 weeks later, and lumbar micro-CT, bone histomorphology, qpcr, WB, ELISA were analyzed. The target sequence for siFoxf1 in the in vivo mouse experiment was GATCCGGCTAGCGAGTTTA (SEQ ID NO. 4).

Fig. b is a representative image of a micro CT scan of mouse lumbar 4 vertebrae (scale bar 1.0 mm) showing that trabecular bone thickening, spacing reduction, and bone microstructure improvement after siFoxf1 treatment.

And the results of analysis on the bone microstructure and bone tissue morphology of the vertebral body trabecular bone micro-CT comprise BV/TV, Tb.N, Tb.Th, Tb.Sp, bone formation parameters (N.ob/B.Pm, MS/BS, MAR, BFR/BS) and bone absorption parameters (N.ob/B.Pm and Oc.S/BS), and show that the results show that after the siFoxf1 treatment, BV/TV, Tb.N and Tb.Th are increased, Tb.Sp is decreased, N.ob/B.Pm, MS/BS, MAR and BFR/BS are increased, and N.oc/B.Pm and Oc.S/BS are not obviously changed.

The results show that siFoxf1 can prevent and treat bone loss caused by OVX.

2. siFoxf1 activates the β -cantenin pathway to increase bone formation in vivo.

The results are shown in FIG. 7, where:

panels a-c show mRNA expression levels of Runx2, Col1a1 and TRAP, respectively, in bone tissue after siFoxf1 treatment, and the analysis results show that siFoxf1 treatment increased the expression levels of Runx2 and Col1a1 mrnas in bone tissue, but had no effect on the expression level of TRAP mRNA.

Panels d and e show ELISA measurements of serum ALP after siFoxf1 treatment showing that siFoxf1 treatment increased the expression level of serum ALP, while no significant difference was observed in the expression level of serum TRAP5 b.

Panel f shows a qRT-PCR analysis of Foxf1 expression levels in Sham and OVX bone tissue after siFoxf1 treatment, demonstrating a significant reduction in Foxf1 expression levels in Sham and OVX bone tissue using siFoxf 1.

Panel g shows qRT-PCR analysis of the expression levels of β -catenin mRNA in vivo by Sham and OVX after siFoxf1 treatment, showing that siFoxf1 increases the expression levels of β -catenin mRNA in vivo.

panel h shows the WB (western blot) glue pattern and expression analysis of Foxf1 protein and β -catenin protein expression levels in Sham and OVX after siFoxf1 treatment, and shows that siFoxf1 delivery decreased the expression level of Foxf1 protein and increased the expression level of β -catenin protein in Sham and ovariectomized bone tissues.

Example 3

siFoxf1 promotes osteogenic differentiation of human mesenchymal stem cells.

A material and a method.

1. Preparation of PMOP patient-derived vertebral bone samples

All protocols were approved by the ethical committee of the first hospital affiliated with the university of traditional Chinese medicine, Guangzhou (ethical No.: ZYYECK [2016] 028). The study included 26 spine-related surgical patients from spinal surgery, the first subsidiary hospital of the university of traditional Chinese medicine, Guangzhou, and vertebral bone samples were collected. We obtained written informed consent from all patients. Inclusion criteria were as follows:

PMOP group: 1)55-80 years old female (average lumbar vertebra BMD T value, T is less than or equal to-2.5); 2) fracture of lumbar vertebra in 2 weeks of lumbar vertebroplasty or internal fixation; 3) the laboratory index is normal; 4) no ingestion of drugs that affect bone metabolism (e.g., corticosteroids, aluminum-containing antacids, and heparin); 5) systemic diseases without affecting bone metabolism (such as secondary osteoporosis, osteogenesis imperfecta, diabetes); 6) no liver or renal insufficiency.

Control group: 1) non-postmenopausal women who undergo spinal surgery for degenerative diseases of the lumbar spine (e.g., spondylolisthesis and lumbar stenosis); 2) there is no osteoporosis or other metabolic disease.

2. Isolation and culture of human mesenchymal stem cells (BMSCs)

Human BMSCs (Cyagen Biosciences, Guangzhou, China) were cultured in an incubator containing 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin at 37 ℃ for subsequent experiments.

3. SiRNA mediated silencing and cell transfection

Foxf1 specific siRNA (target sequence GTGTGACCGAAAGGAGTTT, SEQ ID NO.3) with a sense sequence of GUGUGACCGAAAGGAGUUU, SEQ ID NO.1, and negative control siRNA (siCtrl) (RiboBio, Guangzhou, China) were used to silence Foxf 1. siRNA transfection was performed using Lipofectamine RNAimax (Invitrogen, USA).

4. Osteoblast differentiation assay

BMSCs were cultured in osteogenic induction medium (cygen Biosciences, guangzhou, china). Osteogenic induction medium was changed every three days. Mineral deposition was assessed by Von Kossa staining (solibao, china) 14 to 28 days after osteogenic induction.

5. ALP staining and Activity assay

6. RNA extraction and qRT-PCR

Total RNA was extracted from BMSC and BMM using TRIzol reagent (Invitrogen), total RNA was reverse-transcribed into cDNA using PrimeScript RTMaster Mix (Perfect read Time; TaKaRa, Japan), transcript levels were quantified using β -actin as an internal reference, SYBR Green PCR Master Mix (TaKaRa), gene expression levels were quantified using the 2- Δ Δ Ct method.

7. Western blot

And II, obtaining a result.

The results are shown in FIG. 8, where:

panel a is BMD analysis of control and PMOP groups; the analysis result shows that the bone density of the PMOP group is obviously reduced compared with that of the control group

FIGS. b and c are graphs showing the expression levels of mRNA of Runx2 and Col1a1 in PMOP patient-derived bone tissue, and the results of qRT-PCR analysis show that the expression levels of mRNA of Runx2 and Col1a1 are significantly reduced in PMOP patient-derived bone tissue.

FIGS. d and e show the expression levels of Foxf1 mRNA and protein in PMOP patient-derived bone tissue, respectively, and the results of qRT-PCR and WB analyses indicate that Foxf1 mRNA and protein expression levels in PMOP patient-derived bone tissue are significantly increased.

Graphs f and g show that the expression levels of Runx2 and Col1a1 mrnas in PMOP patients are changed with BMD levels, respectively, and the results of correlation analysis show that the expression levels of Runx2 and Col1a1 mrnas in PMOP patients are reduced with the reduction of BMD levels.

Panel h shows the expression level of Foxf1 mRNA in PMOP patients as a function of BMD levels, and the results of the correlation analysis indicate that Foxf1 mRNA expression levels in PMOP patients increase with decreasing BMD levels.

FIGS. i and j show the relationship between the expression level of Foxf1 and the mRNA levels of Runx2 and Col1a1 in the bone tissue of PMOP patients, respectively, and the correlation analysis results show that the expression level of Foxf1 is negatively correlated with the decrease of the mRNA levels of Runx2 and Col1a1 in the bone tissue of PMOP patients.

Panel k is a representative image of Von Kossa staining of human BMSCs treated with siFoxf1, showing increased calcific deposits following siFoxf1 treatment.

FIG. l is a quantitative analysis of ALP activity of siFoxf1 transfected human mesenchymal stem cells, showing that ALP staining was darker and ALP positive cell number was increased after siFoxf1 treatment.

Panel m shows Foxf1, Runx2 and Col1a1 mRNA expression levels in siFoxf1 treated human BMSCs, and qRT-PCR analysis results show that siFoxf1 treatment can significantly reduce Foxf1 mRNA expression levels and increase Runx2 and Col1a1 mRNA expression levels.

The above results indicate that siFoxf1 can promote osteogenic differentiation of mouse BMSC, and Foxf1 expression in vertebral bone of PMOP patients is increased, and Foxf1 is negatively correlated with BMD, Runx2 and cola1A 1.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

Application of <120> Foxf1 gene in preparation of medicine for treating osteoporosis

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gugugaccga aaggaguuu 19

<210>2

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gauccggcua gcgaguuua 19

<210>3

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gtgtgaccga aaggagttt 19

<210>4

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gatccggcta gcgagttta 19

Claims

Use of the Foxf1 gene in the preparation of a medicament for the treatment of osteoporosis.
2. The use according to claim 1, wherein the osteoporosis is post-menopausal osteoporosis.
3. Use of an agent that silences the Foxf1 gene in the manufacture of a medicament for treating osteoporosis.
4. The use of claim 3, wherein the agent that silences the Foxf1 gene comprises: foxf1 specific siRNA.
5. The use of claim 4, wherein the sense sequence of the Foxf1 specific siRNA is selected from the group consisting of: the sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.
6. The use of claim 5, wherein the sense sequence of the Foxf1 specific siRNA is selected from the group consisting of: and (b) an equivalent sequence which is subjected to substitution, deletion and addition modification of one or more bases with the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and can silence the Foxf1 gene.
7. The use according to claim 6, wherein the Foxf1 specific siRNA has greater than 90% similarity to the sequence shown in SEQ ID No.1 or SEQ ID No. 2.
8. A medicament for the treatment of osteoporosis, comprising: an agent that specifically silences the Foxf1 gene.
9. The drug for treating osteoporosis of claim 8, wherein the target sequence of the Foxf1 specific siRNA is selected from the group consisting of: the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 are subjected to substitution, deletion and addition modification of one or more bases to obtain an equivalent sequence, and the equivalent sequence can silence the Foxf1 gene.
10. The drug for the treatment of osteoporosis of claim 8, wherein the route of administration of the drug is intravenous injection.