CN115141808A - Hsa _ circ _0001493 and hsa _ circ _0070562 inhibit osteogenic differentiation - Google Patents
Hsa _ circ _0001493 and hsa _ circ _0070562 inhibit osteogenic differentiation Download PDFInfo
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Abstract
The invention belongs to the technical field of regenerative medicine, and particularly relates to a method for inhibiting osteogenic differentiation by hsa _ circ _0001493 and hsa _ circ _0070562. According to research, the two genes hsa _ circ _0001493 and hsa _ circ _0070562 which are closely related to regulation of osteogenic differentiation can be knocked down to inhibit osteogenic differentiation of mesenchymal stem cells, so that hsa _ circ _0001493 and hsa _ circ _0070562 can be used for inhibiting osteogenic differentiation of mesenchymal stem cells, and ectopic osteogenesis is overcome; on one hand, a brand-new action target is provided for the research and development of small molecular chemical tools for regulating and controlling the osteogenic directed differentiation of the stem cells, and on the other hand, a solid foundation is laid for the clinical transformation application of the bone tissue engineering technology based on the stem cells.
Description
Technical Field
The invention belongs to the technical field of regenerative medicine, and particularly relates to hsa _ circ _0001493 and hsa _ circ _0070562 for inhibiting osteogenic differentiation, which are expected to be used for inhibiting ectopic bone regeneration.
Background
Ectopic ossification (HO) refers to abnormal bone tissue formed outside the normal skeletal system, which is often secondary to trauma such as limb fracture, joint replacement, spinal cord injury, brain injury and burn, and is easily developed around hip, elbow and knee joint, and currently, there is no effective means for clinically preventing and treating traumatic ectopic ossification.
Human mesenchymal stem cells have the potential to differentiate into tissues such as bone, cartilage, fat, muscle, tendon, etc., and a plurality of preclinical and clinical studies show that the human mesenchymal stem cells are important seed cells for generating new bone in stem cell tissue engineering. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells is a complex process, and various transcription factors, signal pathways and epigenetic regulatory factors play an important role in the process and participate in the process of accurately controlling cell fate. Circular RNA (circular RNA) is a large class of circularly closed single-stranded RNA generated by reverse splicing and was first identified in 1993, and the function of naturally expressed circular RNA was first studied in 2013. More and more studies have shown that circRNA plays an important biological role in cell senescence, proliferation and differentiation. Hsa _ circ _0001439 and Hsa _ circ _0070562 are two circRNA molecules expressed in human bone marrow-derived MSCs, but their effects on osteogenic differentiation and bone formation of human mesenchymal stem cells are not clear.
Disclosure of Invention
In order to overcome the defects of the prior art, the research of the invention discovers that the knock-down hsa _ circ _0001493 or the knock-down hsa _ circ _0070562 can inhibit osteogenic differentiation of mesenchymal stem cells, is expected to be applied to overcoming the problem of ectopic osteogenesis, and provides a new thought for clinical transformation application of a bone tissue engineering technology based on the mesenchymal stem cells.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention provides application of a reagent for inhibiting hsa _ circ _0001493 and/or hsa _ circ _0070562 in preparation of a medicament for inhibiting mesenchymal stem cell osteogenic differentiation.
The invention also provides the application of the agent for inhibiting hsa _ circ _0001493 and/or hsa _ circ _0070562 in preparing a medicament for inhibiting ectopic bone regeneration.
According to research, the invention discovers that the knock-down of Hsa _ circ _0001493 and Hsa _ circ _0070562 can inhibit osteogenic differentiation, on one hand, the invention provides an application basis for circRNA in the process of inhibiting mesenchymal stem cell osteogenic differentiation, and provides a brand-new theoretical and experimental basis for pertinently applying small molecular compounds to inhibit the activity of target proteins and overcoming the treatment of ectopic osteogenesis; on the other hand, a new idea is provided for the clinical transformation application of Hsa _ circ _0001493 and Hsa _ circ _0070562 in the bone tissue engineering technology based on the mesenchymal stem cells.
Preferably, the mesenchymal stem cell is a human bone marrow-derived mesenchymal stem cell.
Preferably, the inhibition is inhibition of expression of hsa _ circ _0001493 and/or hsa _ circ _0070562.
Preferably, the agent is an siRNA.
Further, the siRNA is a nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 and/or a nucleotide sequence shown in SEQ ID No.3 and SEQ ID No. 4.
The invention also provides a non-treatment-purpose mesenchymal stem cell osteogenic differentiation inhibition method, namely the mesenchymal stem cell osteogenic differentiation inhibition method is achieved by reducing the expression of Hsa _ circ _0001493 and/or Hsa _ circ _0070562.
Preferably, reducing the expression of hsa _ circ _0001493 and/or hsa _ circ _0070562 is achieved by gene silencing or gene knockout. That is, mesenchymal stem cells are inhibited from osteogenic differentiation by transfecting mesenchymal stem cells with siRNA double-stranded nucleic acid that silences or knockouts hsa _ circ _0001493 and/or hsa _ circ _0070562.
The invention also provides a medicament for inhibiting osteogenic differentiation of mesenchymal stem cells, the medicament comprising an agent that inhibits hsa _ circ _0001493 and/or hsa _ circ _0070562.
The invention also provides a medicament for inhibiting ectopic bone regeneration, the medicament comprising an agent that inhibits hsa _ circ _0001493 and/or hsa _ circ _0070562.
Preferably, both said medicaments further comprise a pharmaceutically acceptable carrier.
Further, the carrier is a functional pharmaceutical adjuvant acceptable in the pharmaceutical field, and comprises a surfactant, a suspending agent, an emulsifier and some novel pharmaceutical high polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid and the like.
Preferably, the dosage forms of the two medicaments are clinically acceptable conventional dosage forms.
Furthermore, the dosage form of the medicament is not particularly limited, and the medicament can be prepared into injections, tablets, capsules, suppositories, powder injections and the like which are well known to those skilled in the art. The formulations prepared may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) and, if certain drugs are unstable under gastric conditions, may be formulated as enteric coated tablets.
Compared with the prior art, the invention has the beneficial effects that:
the invention discovers two genes closely related to the regulation of osteogenic differentiation, namely hsa _ circ _0001493 and hsa _ circ _0070562 through research. And the knock-down hsa _ circ _0001493 and/or hsa _ circ _0070562 can inhibit osteogenic differentiation of mesenchymal stem cells, so that the regulation and control effect of hsa _ circ _0001493 and hsa _ circ _0070562 on osteogenic differentiation of mesenchymal stem cells is disclosed, and hsa _ circ _0001493 and hsa _ circ _0070562 can be used for inhibiting osteogenic differentiation of mesenchymal stem cells so as to overcome ectopic osteogenesis; on one hand, a brand-new action target is provided for the research and development of small molecular chemical tools to regulate the osteogenic directed differentiation of the stem cells, and on the other hand, a solid foundation is laid for the clinical transformation application of the bone tissue engineering technology based on the stem cells.
Drawings
FIG. 1 shows the cyclization sites of hsa _ circ _0070562 and hsa _ circ _0001493 as determined by shotgun sequencing of the qPCR products;
FIG. 2 shows the expression levels of Hsa _ circ _0001493 Hsa _ circ _0070562 in human mesenchymal stem cells cultured in osteogenic medium for 0, 3, and 7 days;
FIG. 3 shows the effect of knocking down Hsa _ circ _0001493 and Hsa _ circ _0070562 on osteogenic differentiation of human mesenchymal stem cells (knocking down efficiency of A shows successful knocking down Hsa _ circ _0001493 and Hsa _ circ _0070562 but does not affect their respective parental gene expression), the left side in the figure shows the last nucleotide sequence (non-underlined part) of Exon Exon14 of Hsa _ circ _0001493, the start nucleotide sequence (underlined part) of Exon Exon8, the dotted line shows the corresponding sequence of designed si, the right side in the figure shows the last nucleotide sequence (non-underlined part) of Exon Exon3 of Exon _ circ _0070562, the start nucleotide sequence (000underlined part) of Exon Exon3, the dotted line shows the corresponding sequence of designed si, and B shows the knocking down of Hsa _ circ _ 000323 and 14945, bone marrow stem cells were stained down after induction of bone marrow induction by ALP _ cisa _ cich 32 and ALP _ cisc _ C _ 32.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example identification and sequencing of 1hsa _circ _0001493, hsa _ circ _0070562
1. Cell culture
Human bone marrow mesenchymal stem cells are from healthy volunteersApproved by the ethics committee of the eighth hospital affiliated to zhongshan university. The proliferation medium used was low-sugar DMEM (purchased from Gibco) supplemented with 10% fetal bovine serum (purchased from sequoyiani). MSCs cultured to the P3 generation (see the literature: peng W, li Y, huang L. Effects and safety of the antigenic sensory stem cell infiltration in active and inhibitory specific properties of cells in vitro failed NSAIDs: a 20-welk clinical trial [ J20-welk clinical trial ]]Cell Transplantation,2014,23 (10): 1293-303.) in a petri dish, using a proliferation medium at 37 ℃ and 5% CO 2 Then, culture was performed, half of the culture was changed every 3 days for 1 time, until the cells were observed to grow to 80% confluency under an inverted microscope, and then, 0.25% trypsin was used for digestion and passage, and the P3-P5-generation MSCs after passage were used in the subsequent related experiments.
2. RNA extraction, reverse transcription into cDNA, design qPCR primers spanning the cyclization sites of Hsa _ circ _0001493, hsa _ circ _0070562 (Hsa _ circ _ 0001493.
3. Sequencing:
the qPCR product and the primers are sent to Ai Ji Biotech limited for shotgun sequencing. The sequencing results showed (figure 1) that unique circularization sites were detected in MSC for Hsa _ circ _0001493 and Hsa _ circ _00705623, indicating that two circRNAs were indeed present in MSC.
Example 2 variation of expression levels of Hsa _ circ _0001493 and Hsa _ circ _0070562 in osteogenic differentiation of human mesenchymal stem cells
The P3 generation MSC human bone marrow mesenchymal stem cells are inoculated into a six-hole plate, the temperature is 37 ℃, and the content is 5%CO 2 Culturing under the condition, adding osteogenic differentiation medium (proliferation medium containing 0.2mM vitamin C, 10mM beta-glycerophosphate sodium and 100nM dexamethasone), collecting RNA on days 0, 3 and 7, respectively, and detecting the expression change (Relative RNA expression) of hsa _ circ _0001493 and hsa _ circ _0070562 by qPCR.
The results show (FIG. 2) that the expression level of human mesenchymal stem cells was increased in the bone-forming cultures for 0 days, 3 days, and 7 days, hsa _ circ _0001493 and hsa _ circ _0070562.
Example 3 knockdown of hsa _ circ _0001493 and hsa _ circ _0070562 inhibits osteogenic differentiation of human mesenchymal stem cells
1. siRNA sequence design:
inhibition of the osteogenic differentiation of human mesenchymal stem cells was observed by designing the expression of siRNA (small interfering RNA) silencing genes specifically matching the circularization site of circRNA to inhibit the expression of hsa _ circ _0001493 and/or hsa _ circ _0070562, si _ circ _0001493 and/or si _ circ _0070562 and control sirnas purchased from Ai Ji, guangzhou, where the sequences of the sirnas are as follows:
si_circ_0001493：
si_circ_0070562：
negative Control (Control siRNA):
2. application of siRNA to MSC
24h before transfection, P3-P4 generation MSC was inoculated into a dodecawell plate, when human bone marrow mesenchymal stem cells grew to 70% -80% confluence, the medium (proliferation medium of example 1) was discarded, serum-free medium was added, siRNA was transfected using Lipofectamine RNAiMAX (purchased from Ensifier Elite), after overnight culture, osteogenic differentiation medium was changed, and half-change of liquid was performed every 2 days. Transfection can be added once in the middle according to requirements; alkaline phosphatase ALP and alizarin red staining were performed on days 7 and 14, respectively, and ALP activity on day7, i.e., ALP activity on day7 (U/g Pro/15 min), and ARS staining quantification on day 14, i.e., ARS staining quantification on day 14 (OD at 562 nm) (si transfection method, ALP staining and alizarin red staining method, published in the publications: li, J., P.Wang, Z.Xie, S.Wang, S.Cen, M.Li, and W.Liu, et al.2019."TRAF4 positional Regulation the Osteogenetic Differentiation of sensory Stem Cells activation as an E3 Ubicin lipid ligand to gradient Smurf2." Cell Death & Cell Differentiation 26 (12): 52-2666).
3. The results showed (fig. 3) that the si _ circ _0001493 and si _ circ _0070562 groups showed significantly reduced ALP staining and ARS staining, and significantly reduced osteogenic capacity, compared to the control group.
As can be seen from the comprehensive examples 1-3, the expression levels of Hsa _ circ _0001493 and Hsa _ circ _0070562 are gradually increased in the osteogenic differentiation process of the mesenchymal stem cells; knockdown of Hsa _ circ _0001493 and Hsa _ circ _0070562 can inhibit osteogenic differentiation of mesenchymal stem cells. Therefore, the silencing of Hsa _ circ _0001493 and Hsa _ circ _0070562 can inhibit the osteogenic differentiation capacity of the mesenchymal stem cells, and provides a new idea for promoting the application of the mesenchymal stem cell small molecule target and the clinical transformation application of the bone tissue engineering technology based on the mesenchymal stem cells.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> Zhongshan university subsidiary eighth Hospital (Shenzhen Futian)
<120> hsa _ circ _0001493 and hsa _ circ _0070562 inhibit osteogenic differentiation
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA/RNA
<213> hsa _ circ _0001493-si-1 sense strand (Artificial Sequence)
<400> 1
uugccuaaaa uguuggcuca adtdt 25
<210> 2
<211> 25
<212> DNA/RNA
<213> hsa _ circ _0001493-si-1 antisense strand (Artificial Sequence)
<400> 2
uugagccaac auuuuaggca adtdt 25
<210> 3
<211> 25
<212> DNA/RNA
<213> hsa _ circ _0070562-si-3 sense strand (Artificial Sequence)
<400> 3
uugcagaugu guagaauuca adtdt 25
<210> 4
<211> 25
<212> DNA/RNA
<213> hsa _ circ _0070562-si-3 antisense strand (Artificial Sequence)
<400> 4
uugaauucua cacaucugca adtdt 25
<210> 5
<211> 23
<212> DNA/RNA
<213> NC sense chain (Artificial Sequence)
<400> 5
uucuccgaac gugucacgud tdt 23
<210> 6
<211> 23
<212> DNA/RNA
<213> NC antisense strand (Artificial Sequence)
<400> 6
acgugacacg uucggagaad tdt 23
<210> 7
<211> 20
<212> DNA/RNA
<213> Hsa_circ_0001493 Forward Primer(Artificial Sequence)
<400> 7
tgccagcaaa ttttggcaga 20
<210> 8
<211> 20
<212> DNA/RNA
<213> Hsa_circ_0001493 Reverse Primer(Artificial Sequence)
<400> 8
acatggtacc aaccgcacaa 20
<210> 9
<211> 25
<212> DNA/RNA
<213> Hsa_circ_0070562 Forward Primer(Artificial Sequence)
<400> 9
ttattggata cacctgtcaa gactc 25
<210> 10
<211> 20
<212> DNA/RNA
<213> Hsa_circ_0070562 Reverse Primer (Artificial Sequence)
<400> 10
ctgttccatc aggcttgctt 20
Claims (10)
1. Application of a reagent for inhibiting hsa _ circ _0001493 and/or hsa _ circ _0070562 in preparation of a medicament for inhibiting osteogenic differentiation of mesenchymal stem cells.
2. Use of an agent that inhibits hsa _ circ _0001493 and/or hsa _ circ _0070562 in the manufacture of a medicament for inhibiting ectopic bone regeneration.
3. The use of claim 1, wherein the mesenchymal stem cells are human bone marrow-derived mesenchymal stem cells.
4. Use according to claim 1 or 2, wherein the inhibition is inhibition of expression of hsa _ circ _0001493 and/or hsa _ circ _0070562.
5. The use of claim 1 or 2, wherein the agent is an siRNA.
6. The use according to claim 1 or 2, wherein the siRNA is the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2 and/or the nucleotide sequence shown in SEQ ID No.3, SEQ ID No. 4.
7. A non-treatment-purpose mesenchymal stem cell osteogenic differentiation inhibition method is characterized in that the purpose of inhibiting mesenchymal stem cell osteogenic differentiation is achieved by reducing the expression of Hsa _ circ _0001493 and/or Hsa _ circ _0070562.
8. The method of claim 7, wherein the reduction of expression of Hsa _ circ _0001493 and/or Hsa _ circ _0070562 is achieved by gene silencing or gene knockout.
9. A medicament for inhibiting osteogenic differentiation of mesenchymal stem cells, the medicament comprising an agent that inhibits hsa _ circ _0001493 and/or hsa _ circ _0070562.
10. A medicament for use in inhibiting ectopic bone regeneration, which medicament comprises an agent that inhibits hsa _ circ _0001493 and/or hsa _ circ _0070562.
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| US20180021340A1 (en) * | 2015-02-13 | 2018-01-25 | The Brigham And Women's Hospital, Inc. | Methods and compositions for the treatment or prevention of abnormal bone formation in a soft tissue |
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| CN111733184A (en) * | 2020-07-29 | 2020-10-02 | 和元生物技术（上海）股份有限公司 | Adenovirus packaging method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180021340A1 (en) * | 2015-02-13 | 2018-01-25 | The Brigham And Women's Hospital, Inc. | Methods and compositions for the treatment or prevention of abnormal bone formation in a soft tissue |
| CN111500578A (en) * | 2020-04-22 | 2020-08-07 | 辽宁省肿瘤医院 | Circ RNA-FTO for regulating and controlling osteogenic differentiation and tissue regeneration of ADSCs and application thereof |
| CN111733184A (en) * | 2020-07-29 | 2020-10-02 | 和元生物技术（上海）股份有限公司 | Adenovirus packaging method |
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| Title |
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| JIANHUA HAN等: "Circular RNA expression pattern in cisplatin-resistant osteosarcoma cells", 《TRANSL CANCER RES》, vol. 9, no. 1, pages 264 * |
| MEI-LI ZHENG等: "Expression profile of circular RNAs in epicardial adipose tissue in heart failure", 《CHINESE MEDICAL JOURNAL》, vol. 133, no. 21, pages 2569 - 2570 * |
| SHAN WANG等: "RNA Sequencing Reveals the Expression Profiles of circRNAs and Indicates Hsa_circ_0070562 as a Pro-osteogenic Factor in Bone Marrow-Derived Mesenchymal Stem Cells of Patients With Ankylosing Spondylitis", 《FRONT. GENET.》, vol. 13, pages 1 - 16 * |
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