CN109136227B - Application of long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair - Google Patents

Abstract

The invention provides the application of long-chain non-coding RNA-HOXA-AS2 in the repair of bone tissue damage. Experiments show that overexpression of HOXA‑AS2 can significantly promote calcium ion deposition in mesenchymal stem cells, significantly enhance alkaline phosphatase activity in mesenchymal stem cells, and significantly increase osteogenic differentiation markers RUNX2 and SP7 in mesenchymal stem cells. , SPP1 gene expression, and can significantly inhibit the activity of NF-κB signaling.

Description

Application of long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair

Technical Field

The invention belongs to the technical field of stem cell bioengineering, and particularly relates to application of long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair.

Background

Repair and regeneration of bone damage has been a major problem in the medical community. For sports bone injuries, the medical field usually adopts traditional methods such as autologous bone transplantation, allogeneic bone transplantation, free bone transplantation with blood supply, biological material replacement and the like for treatment, but the methods have the problems of few sources, more complications, immunological rejection, iatrogenic infection and the like, and have ideal treatment effects. With the continuous development of bone tissue engineering technology, people gradually begin to apply tissue engineering methods to solve the problem of bone tissue injury repair. The mesenchymal stem cells have good proliferation capacity and multidirectional differentiation potential, and enter the visual field of people. Research shows that the mesenchymal stem cells can be separated and obtained from various tissues such as bone marrow, fat and the like; under specific conditions, mesenchymal stem cells can also be differentiated into various cells such as chondrocytes, osteoblasts and adipocytes; in addition, mesenchymal stem cells also have low immunogenicity. These unique characteristics make the mesenchymal stem cells become seed cells for repairing and regenerating damaged bone tissues clinically. A large body of research evidence suggests that: osteogenic differentiation of mesenchymal stem cells is affected by various regulatory factors at a molecular level, including important transcriptional regulatory factors, such as RUNX2 and SP7, which can regulate osteogenic differentiation of stem cells by regulating expression of osteogenic differentiation related genes, and in addition, many non-coding RNAs are involved in regulation of osteogenic differentiation of stem cells.

Long-chain non-coding RNAs are a class of small-molecule RNAs that do not code for proteins and are generally greater than 200 nucleotides in length, and their expression levels tend to be low and tissue-specific. Due to the low expression of long non-coding RNAs, the importance of such RNA molecules in biological function is often overlooked. In recent years, due to the development and application of high-throughput sequencing technology, long-chain non-coding RNA has been found to have very important regulation and control functions in various biological processes such as apoptosis, proliferation, differentiation and the like. For example, recently, a long non-coding RNA called pu.1-AS has been discovered, which can promote adipogenic differentiation of mesenchymal stem cells by blocking the expression of pu.1 gene. As another long non-coding RNA, called ANCR, it can regulate osteogenic differentiation of mesenchymal stem cells by modulating the expression of RUNX2 gene. These studies indicate that long non-coding RNAs play an important role in the regulation of stem cell differentiation into specific cell directions. Therefore, the aim of inducing stem cells to differentiate towards a specific direction can be achieved by changing the expression quantity of some important long-chain non-coding RNAs in the cells, so that a new solution is found for medical problems such as tissue injury repair and the like.

Disclosure of Invention

Based on this, the invention aims to provide the application of the long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair.

Another objective of the invention is to provide application of long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair detection.

The purpose of the invention is realized by the following technical scheme:

the application of the long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair is characterized in that the nucleotide sequence of the long-chain non-coding RNA-HOXA-AS2 is shown AS SEQ ID NO. 1 in a sequence table.

An overexpression vector pLV-HOXA-AS2 for repairing bone tissue injury is constructed by a HOXA-AS2 gene segment and a lentivirus empty vector pLV; the nucleotide sequence of the HOXA-AS2 gene fragment is shown AS SEQ ID NO. 2 in the sequence table, and the nucleotide sequence of the lentivirus empty vector pLV is shown AS SEQ ID NO. 3 in the sequence table.

The construction method of the over-expression vector pLV-HOXA-AS2 comprises the following steps: respectively carrying out double enzyme digestion on lentiviral vector pLV and HOXA-AS2 gene fragments by adopting restriction endonucleases BamH I and Xba I, carrying out ligation reaction on the enzyme-digested HOXA-AS2 gene fragment and linear lentiviral vector pLV by adopting a T4 DNA ligase system, then transforming competent cells, screening positive colonies, extracting plasmids of the positive colonies, and obtaining the over-expression vector pLV-HOXA-AS 2.

Further, the preparation method of the HOXA-AS2 gene fragment comprises the following steps: according to the nucleotide sequence of the long non-coding RNA-HOXA-AS2, performing double-strand synthesis on the HOXA-AS2 gene fragment by adopting a DNA synthesizer according to the principle of a solid phase phosphoramidite triester method, and simultaneously adding BamH I enzyme cutting sites and Xba I enzyme cutting sites on the 5 'end and the 3' end of the gene fragment respectively to obtain the HOXA-AS2 gene fragment containing specific enzyme cutting sites.

An inducible cell for repairing bone tissue injury, which is constructed by transfecting a cell vector with the over-expression vector pLV-HOXA-AS 2.

The method for constructing the inducible cell comprises the following steps: co-transfecting an overexpression vector pLV-HOXA-AS2, a lentivirus packaging plasmid pSPAX2 and a lentivirus packaging plasmid pMD2G into a packaging cell by adopting a liposome transfection reagent, and collecting lentivirus supernatant after culturing; and then adding the lentivirus supernatant into a cell vector, and culturing to obtain the inducible cell stably expressing the long-chain non-coding RNA-HOXA-AS 2.

The application of the long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair detection is disclosed, wherein the nucleotide sequence of the long-chain non-coding RNA-HOXA-AS2 is shown AS SEQ ID NO:1 in a sequence table.

A kit for bone tissue damage repair detection, which comprises specific primers for detecting the expression level of HOXA-AS 2; the specific primer comprises:

an upstream primer: 5'-GTTCAGCTCAAGTTGAACATA-3' the flow of the air in the air conditioner,

a downstream primer: 5'-AAACCTTGTAGATAGCTTGAG-3' are provided.

A bone tissue damage repair detection method comprises the following steps:

1) taking a sample to be detected, and extracting the total RNA of the sample by adopting an RNA extraction kit;

2) carrying out reverse transcription reaction on the extracted total RNA by adopting a reverse transcription kit to obtain cDNA;

3) and (3) carrying out fluorescent quantitative PCR reaction by using cDNA obtained by reverse transcription reaction AS a template and adopting the specific primer, and determining the expression level of HOXA-AS2 in the sample.

Further, use 2^-ΔΔCTAnd (4) calculating the expression amount of the HOXA-AS2 in the sample to be detected by a relative quantification method.

The invention provides a long-chain non-coding RNA-HOXA-AS2 for repairing bone tissue damage and application thereof in detection of bone tissue damage. Experiments prove that the over-expression HOXA-AS2 can remarkably promote the deposition of calcium ions in the mesenchymal stem cells, remarkably enhance the activity of alkaline phosphatase in the mesenchymal stem cells, remarkably improve the expression quantity of osteogenic differentiation markers RUNX2, SP7 and SPP1 genes in the mesenchymal stem cells, and remarkably inhibit the activity of NF-kappa B signals (NF-kappa B is a signal path for inhibiting osteogenic differentiation of the mesenchymal stem cells). Therefore, the long-chain non-coding RNA-HOXA-AS2 not only can be used AS a clinical inducer for bone tissue damage repair and regeneration, but also can be an important molecular marker for bone tissue damage repair and regeneration.

The overexpression vector pLV-HOXA-AS2 and the inducible cell containing the overexpression vector pLV-HOXA-AS2 provided by the invention can specifically express long-chain non-coding RNA-HOXA-AS2, and can be applied to repair and regeneration of bone tissue injury.

Drawings

FIG. 1 is an electrophoretogram of an enzyme digestion product of a lentivirus expression vector pLV-HOXA-AS2, wherein 1 is a lentivirus empty vector control group, 2 is a positive plasmid cloning experimental group, and Marker is a Marker;

FIG. 2 is a graph showing the results of detecting the expression level of lentiviral expression vector pLV-HOXA-AS2, wherein vec is a lentiviral empty vector control group, and HOXA-AS2-flag is a lentiviral expression vector experimental group;

FIG. 3 is a graph showing the results of detection of calcium ion deposition in MenSCs cells, wherein vec is a lentivirus empty vector control group, and HOXA-AS2-flag is a lentivirus expression vector experimental group;

FIG. 4 is a graph showing the results of detection of alkaline phosphatase activity in MenSCs cells, wherein vec is a lentivirus empty vector control group, and HOXA-AS2-flag is a lentivirus expression vector experimental group;

FIG. 5 is a graph showing the results of measurement of the expression level of an osteogenic differentiation marker in MenSCs cells, wherein vec is a lentiviral empty vector control group, and HOXA-AS2-flag is a lentiviral expression vector experimental group;

FIG. 6 is a graph showing the results of measurement of acetylation levels of the K310 site of RelA subunit in MenSCs cells, wherein vec is a lentiviral empty vector control group, and HOXA-AS2-flag is a lentiviral expression vector experimental group.

Detailed Description

The first embodiment is as follows: acquisition of HOXA-AS2 Gene fragment

According to the RNA nucleotide sequence (RefSeq sequence number: NR _122069.1, shown AS SEQ ID NO:1 in a sequence table) of long-chain non-coding RNA-HOXA-AS2 in NCBI database and the specific enzyme cutting sites of BamH I and Xba I on lentiviral vector pLV, a DNA chem192 synthesizer is adopted to carry out double-strand synthesis on a HOXA-AS2 gene fragment according to the solid phase phosphoramidite triester method principle, and two specific enzyme cutting sites of BamH I (GGATCC) and Xba I (TCTAGA) are respectively added on the 5 'end and the 3' end to obtain a HOXA-AS2 gene fragment containing the specific enzyme cutting sites, wherein the nucleotide sequence is shown AS SEQ ID NO:2 in the sequence table.

Example two: construction of Lentiviral expression vector pLV-HOXA-AS2

Respectively carrying out double digestion on a lentivirus empty vector pLV (the nucleotide sequence of which is shown AS SEQ ID NO:3 in a sequence table) and a HOXA-AS2 gene fragment obtained in the first embodiment by adopting restriction endonucleases BamH I and Xba I, carrying out a ligation reaction on the HOXA-AS2 gene fragment subjected to the digestion and a linear lentivirus empty vector pLV by adopting a T4 DNA ligase system, then transforming competent cells, screening positive colonies, and extracting a plasmid of the positive colonies to obtain a lentivirus expression vector pLV-HOXA-AS 2.

1. Enzyme digestion of lentivirus empty vector pLV

The cleavage reaction system was as follows (20. mu.L):

the enzyme digestion reaction conditions are as follows: the reaction was carried out at 37 ℃ for 4 hours.

2. Enzyme digestion of HOXA-AS2 gene fragment

The cleavage reaction system was as follows (20. mu.L):

the enzyme digestion reaction conditions are as follows: the reaction was carried out at 37 ℃ for 4 hours.

3. Ligation of HOXA-AS2 Gene fragment and lentivirus empty vector pLV

The ligation reaction was as follows (10. mu.L):

the connection reaction conditions are as follows: incubate overnight at 16 ℃.

4. Transformation of competent cells

Adding 5 μ L of the ligation reaction product into 50 μ L of Escherichia coli DH5 α competent cells, cooling on ice for 5 min, heating in 42 deg.C water bath for 90 s, and cooling on ice for 10 min; the transformant was then plated on LB plates containing ampicillin resistance (final concentration: 50. mu.g/. mu.L) and cultured overnight at 37 ℃; single colonies were picked, added to LB medium containing ampicillin resistance (final concentration: 50. mu.g/. mu.L), and cultured overnight on a shaker at 37 ℃ and 200 rpm.

5. Extracting and identifying positive plasmid

Plasmid extraction kit (purchased from Beijing Tiangen Biochemical technology company, model DP116) is adopted, the operation is carried out according to the instruction of the kit, the plasmid of the positive colony is extracted, and restriction enzymes BamH I and Xba I are adopted for enzyme digestion identification.

The cleavage reaction system was as follows (20. mu.L):

the enzyme digestion reaction conditions are as follows: the reaction was carried out at 37 ℃ for 4 hours.

Taking the enzyme digestion reaction product, carrying out electrophoresis by using 1% agarose gel, and identifying positive clones. The electrophoresis result is shown in FIG. 1, and the target band appears at 1kb after electrophoresis, namely the HOXA-AS2 gene fragment. The identification result shows that the construction of the lentivirus expression vector pLV-HOXA-AS2 is successful.

Example three: lentiviral packaging

HEK293T cells were inoculated in a six-well culture plate for culture, DMEM complete medium containing 10% Fetal Bovine Serum (FBS) was used for culture in a cell culture box at 37 ℃, and when the cell density reached about 70%, lentivirus expression vector pLV-HOXA-AS2 and lentivirus empty vector pLV were transfected into HEK293T cells, respectively, using Lipofectamine 3000 liposome transfection reagent (purchased from Thermo Fisher, Inc., model L3000015) for lentivirus packaging. The method comprises the following specific steps:

1) two 1.5mL EP tubes are respectively taken and respectively added with 150 mu L serum-free DMEM medium;

2) adding 1 μ g of lentiviral expression vector pLV-HOXA-AS2, 0.5 μ g of lentiviral packaging plasmid pSPAX2, and 0.5 μ g of lentiviral packaging plasmid pMD2G into one of the EP tubes, and mixing well;

3) adding the Lipofectamine 3000 liposome transfection reagent into another EP tube, and uniformly mixing;

4) quickly adding the plasmid solution obtained in the step 2) into the transfection reagent solution in the step 3), carrying out vortex oscillation for 5 seconds to fully mix the plasmid solution and the transfection reagent solution uniformly, and then standing for 10 minutes at room temperature;

5) dropwise adding the mixed solution obtained in the step 4) into a HEK293T cell culture plate, slightly shaking and uniformly mixing, placing the mixed solution into a 37 ℃ incubator for culture, replacing a fresh DMEM complete culture medium containing 10% FBS after 6 hours, and continuing to culture;

6) after 48 hours of culture, the supernatant of HEK293T cells (lentivirus supernatant) was collected, centrifuged, and cell debris removed.

Similarly, lentiviral empty vector pLV was transfected into HEK293T cells as experimental control following the procedure described above.

Example four: determination of expression amount of lentivirus expression vector pLV-HOXA-AS2

The lentivirus supernatant obtained in example three was infected with blood-derived mesenchymal stem cells (MenSCs), 48 hours after infection, total RNA in MenSCs cells was extracted using Trizol kit (purchased from Takara corporation, model 9109), and reverse transcription was performed on the total RNA, and then the expression amount of the lentivirus expression vector pLV-HOXA-AS2 was determined by fluorescent quantitative PCR (qRT-PCR).

1. Extraction of Total RNA

And (2) inoculating MenSCs cells into a six-well culture plate, culturing in a cell culture box at 37 ℃ by using a DMEM complete culture medium containing 10% FBS, discarding the original culture medium when the cell density reaches about 50%, taking 1mL of each of the lentivirus empty vector supernatant and the lentivirus expression vector supernatant obtained in the third embodiment, respectively adding the lentivirus empty vector supernatant and the lentivirus expression vector supernatant into the MenSCs cells, respectively adding 1mL of fresh DMEM complete culture medium, and respectively adding 2 mu L of polybrene with the concentration of 4 mu g/mu L for infection. After 8 hours of infection, fresh DMEM complete medium was replaced and incubation in a cell incubator at 37 ℃ was continued for 48 hours. Finally, the Trizol kit is adopted, the operation is carried out according to the instruction of the kit, the MenSCs cells are cracked, and the total RNA of the MenSCs cells is extracted.

2. Reverse transcription of cDNA

The extracted total RNA is digested with DNase and RNase inhibitor to remove residual DNA components from the extracted total RNA, and the concentration of the extracted total RNA is measured with a spectrophotometer. Then, the total RNA extracted was subjected to reverse transcription using a reverse transcription kit (purchased from Takara corporation, model 2641A) in accordance with the instructions of the kit to obtain cDNA.

The reverse transcription reaction system is as follows:

the total RNA content was 1.0. mu.g,

random primers (25. mu.M) 2.0. mu.L,

DEPC water was added to a total volume of 12. mu.L;

adding the reactants into a DNA synthesizer, reacting for 10 minutes at 70 ℃ to open an RNA structure, and then immediately placing on ice for reacting for 3-5 minutes to enable an RNA template to be combined with a Random Primer (Random Primer).

Subsequently, adding the following components into the reaction system:

the reaction system is mixed evenly and added into a DNA synthesizer to react for 10 minutes at 30 ℃, then react for 1 hour at 42 ℃ and react for 15 minutes at 70 ℃.

3. Determination of expression level of Long non-coding RNA-HOXA-AS2

According to the RNA nucleotide sequence (RefSeq sequence number: NR _122069.1, shown AS SEQ ID NO:1 in a sequence table) of long-chain non-coding RNA-HOXA-AS2 in NCBI database, a specific primer capable of detecting the expression level of HOXA-AS2 is designed, and the primer sequence is AS follows:

HOXA-AS2 upstream primer: 5'-GTTCAGCTCAAGTTGAACATA-3' the flow of the air in the air conditioner,

HOXA-AS2 downstream primer: 5'-AAACCTTGTAGATAGCTTGAG-3' are provided.

The cDNA obtained by reverse transcription reaction is used AS a template, a qPCR kit (purchased from Thermo Fisher company, and the model is K0251) is adopted, operation is carried out according to the instruction of the kit, fluorescent quantitative PCR (qRT-PCR) reaction is carried out, and the expression quantity of the long-chain non-coding RNA-HOXA-AS2 is measured.

The qRT-PCR reaction system was as follows (20. mu.L):

the two-step method reaction conditions of qRT-PCR are as follows: pre-denaturation at 95 ℃ for 7 min; denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ and extension for 30 seconds, and 40 cycles.

By using 2^-ΔΔCTThe results of comparative analysis of the difference in the expression levels of HOXA-AS2 in the lentiviral expression vector pLV-HOXA-AS2 and the lentiviral empty vector pLV with respect to the quantification method are shown in FIG. 2. The determination result shows that compared with the lentiviral empty vector pLV, the expression amount of HOXA-AS2 of the lentiviral expression vector pLV-HOXA-AS2 is remarkably increased (the value of P is less than 0.001, and the statistical significance is achieved), and the lentiviral expression vector pLV-HOXA-AS2 can effectively over-express HOXA-AS 2.

2^-ΔΔCTRelative quantification method: performing fluorescent quantitative PCR reaction by respectively adopting a qPCR kit by taking GAPDH as an internal reference gene and a lentivirus empty vector pLV as a control group; and CT values (fluorescence threshold cycle number) of the GAPDH reference gene and HOXA-AS2 in the lentiviral expression vector pLV and HOXA-AS2 in the lentiviral empty vector pLV were determined, respectively. Subtracting the CT value of the GAPDH reference gene from the CT value of the lentivirus expression vector pLV-HOXA-AS2 to obtain delta CT 1; subtracting the CT value of the GAPDH reference gene from the CT value of the lentiviral empty vector pLV to obtain delta CT 2; the difference between the HOXA-AS2 expression levels of the lentiviral expression vector pLV-HOXA-AS2 and the lentiviral empty vector pLV was 2^-ΔΔCTWherein Δ CT is Δ CT 1- Δ CT 2.

Specific primers for the fluorescent quantitative PCR reaction of the GAPDH reference gene are as follows:

GAPDH upstream primer: 5'-CATGAGAAGTATGACAACAGCCT-3' the flow of the air in the air conditioner,

GAPDH downstream primer: 5'-AGTCCTTCCACGATACCAAAGT-3' are provided.

Example five: effect of Long-chain non-coding RNA-HOXA-AS2 on osteogenic differentiation of mesenchymal Stem cells

1. Effect of Long-chain non-coding RNA-HOXA-AS2 on calcium ion deposition in mesenchymal Stem cells

And (2) inoculating MenSCs cells into a six-well culture plate, culturing in a cell culture box at 37 ℃ by using a DMEM complete culture medium containing 10% FBS, discarding the original culture medium when the cell density reaches about 50%, taking 1mL of each of the lentivirus empty vector supernatant and the lentivirus expression vector supernatant obtained in the third embodiment, respectively adding the lentivirus empty vector supernatant and the lentivirus expression vector supernatant into the MenSCs cells, respectively adding 1mL of fresh DMEM complete culture medium, and respectively adding 2 mu L of polybrene with the concentration of 4 mu g/mu L for infection. After 8 hours of infection, fresh DMEM complete medium was replaced, and then culture was continued in a cell culture box at 37 ℃, and fresh DMEM complete medium was replaced every two days for three weeks. After the incubation, the cells were washed with PBS buffer, then fixed with 1mL of 70% ethanol for 30 minutes, and then stained with 1% alizarin red for 1 minute. The effect of over-expression of HOXA-AS2 on calcium ion deposition in MenSCs cells was observed by taking pictures under a microscope, and the results are shown in FIG. 3.

The test result shows that compared with a lentivirus empty vector control group, the overexpression of the long-chain non-coding RNA-HOXA-AS2 can obviously promote the deposition of calcium ions in MenSCs cells, namely, can promote the osteogenic differentiation of the MenSCs cells.

2. Effect of Long non-coding RNA-HOXA-AS2 on alkaline phosphatase Activity in mesenchymal Stem cells

And (2) inoculating MenSCs cells into a six-well culture plate, culturing in a cell culture box at 37 ℃ by using a DMEM complete culture medium containing 10% FBS, discarding the original culture medium when the cell density reaches about 50%, taking 1mL of each of the lentivirus empty vector supernatant and the lentivirus expression vector supernatant obtained in the third embodiment, respectively adding the lentivirus empty vector supernatant and the lentivirus expression vector supernatant into the MenSCs cells, respectively adding 1mL of fresh DMEM complete culture medium, and respectively adding 2 mu L of polybrene with the concentration of 4 mu g/mu L for infection. After 8 hours of infection, fresh DMEM complete medium was replaced, and then culture was continued in a cell culture box at 37 ℃, and fresh DMEM complete medium was replaced every two days for three weeks. After the culture, the cells were washed with PBS buffer, 1mL of 70% ethanol was added to fix the cells for 30 minutes, and then the alkaline phosphatase activity in MenSCs cells was measured using BCIP/NBT substrate color development kit (purchased from Sigma, type B1911) according to the instructions of the kit, and the results are shown in FIG. 4.

The test result shows that compared with a lentivirus empty vector control group, the overexpression of the long-chain non-coding RNA-HOXA-AS2 can obviously enhance the activity of alkaline phosphatase in MenSCs cells, namely can promote the osteogenic differentiation of the MenSCs cells.

3. Influence of long-chain non-coding RNA-HOXA-AS2 on mesenchymal stem cell osteogenic differentiation marker gene expression level

Specific primers capable of detecting the expression levels of RUNX2, SP7 and SPP1 genes are designed according to the mRNA nucleotide sequences (the RefSeq sequence numbers of the mRNA nucleotide sequences are respectively NM-001024630.3, NM-001300837.1 and NM-001251830.1) of osteogenic differentiation markers RUNX2, SP7 and SPP1 in NCBI databases, and the primer sequences are as follows:

RUNX2 upstream primer: 5'-GGACGAGGCAAGAGTTTCAC-3' the flow of the air in the air conditioner,

primer downstream of RUNX 2: 5'-GAGGCGGTCAGAGAACAAAC-3' are provided.

SP7 upstream primer: 5'-CACAGCTCTTCTGACTGTCTG-3' the flow of the air in the air conditioner,

SP7 downstream primer: 5'-CTGGTGAAATGCCTGCATGGAT-3' are provided.

SPP1 upstream primer: 5'-AGCCAATGATGAGAGCAATG-3' the flow of the air in the air conditioner,

SPP1 downstream primer: 5'-TCCTTACTTTTGGGGTCTAC-3' are provided.

And (2) inoculating MenSCs cells into a six-well culture plate, culturing in a cell culture box at 37 ℃ by using a DMEM complete culture medium containing 10% FBS, discarding the original culture medium when the cell density reaches about 50%, taking 1mL of each of the lentivirus empty vector supernatant and the lentivirus expression vector supernatant obtained in the third embodiment, respectively adding the lentivirus empty vector supernatant and the lentivirus expression vector supernatant into the MenSCs cells, respectively adding 1mL of fresh DMEM complete culture medium, and respectively adding 2 mu L of polybrene with the concentration of 4 mu g/mu L for infection. After 8 hours of infection, fresh DMEM complete medium was replaced, and then culture was continued in a cell culture box at 37 ℃, and fresh DMEM complete medium was replaced every two days for three weeks. After the culture is finished, the MenSCs cells are lysed by using a Trizol kit according to the instructions of the kit, and the total RNA of the MenSCs cells is extracted.

The reverse transcription reaction was performed on the extracted total RNA using the reverse transcription kit according to the reverse transcription method of example four to obtain cDNA. Using the cDNA obtained by the reverse transcription reaction as a template, a fluorescent quantitative PCR (qRT-PCR) reaction was performed to determine the expression levels of the osteogenic differentiation markers RUNX2, SP7, SPP1 genes.

The qRT-PCR reaction system for the RUNX2 gene was as follows (20. mu.L):

the qRT-PCR reaction system for the SP7 gene was as follows (20. mu.L):

the qRT-PCR reaction system of the SPP1 gene is as follows (20. mu.L):

the qRT-PCR reaction conditions described above were as follows: pre-denaturation at 95 ℃ for 7 min; denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ and extension for 30 seconds, and 40 cycles.

The results of examining the expression levels of the osteogenic differentiation markers RUNX2, SP7, and SPP1 genes by a 2- Δ Δ CT relative quantification method are shown in fig. 5. The measurement results show that overexpression of long-chain non-coding RNA-HOXA-AS2 significantly increased the expression amounts of osteogenic differentiation markers RUNX2, SP7, and SPP1 genes in MenSCs cells compared to the lentiviral empty vector control group (the value of P was less than 0.05, the value of P was less than 0.01, and had statistical significance), i.e., facilitated osteogenic differentiation of MenSCs cells.

4. Effect of Long-chain non-coding RNA-HOXA-AS2 on osteoblast differentiation regulatory signal pathways in mesenchymal stem cells

NF-kB is one of the most important inflammatory signal pathways, and has important regulation and control functions in biological processes such as cell proliferation, differentiation, apoptosis and the like, and researches show that the activation of NF-kB signals can inhibit the osteogenic differentiation of mesenchymal stem cells. The influence of over-expression of HOXA-AS2 on the osteogenic differentiation of mesenchymal stem cells can be reflected by detecting the influence of over-expression of HOXA-AS2 on NF-kB signal activity.

And (2) inoculating MenSCs cells into a six-well culture plate, culturing in a cell culture box at 37 ℃ by using a DMEM complete culture medium containing 10% FBS, discarding the original culture medium when the cell density reaches about 50%, taking 1mL of each of the lentivirus empty vector supernatant and the lentivirus expression vector supernatant obtained in the third embodiment, respectively adding the lentivirus empty vector supernatant and the lentivirus expression vector supernatant into the MenSCs cells, respectively adding 1mL of fresh DMEM complete culture medium, and respectively adding 2 mu L of polybrene with the concentration of 4 mu g/mu L for infection. After 8 hours of infection, fresh DMEM complete medium was replaced, and then culture was continued in a cell culture box at 37 ℃, and fresh DMEM complete medium was replaced every two days for three weeks. After the culture was completed, the cells were lysed, and then the effect of over-expression of HOXA-AS2 on the acetylation level of K310 site in RelA subunit of NF- κ B was examined by Western blotting (Western Blot) assay (it was found that the higher the acetylation level of K310 site, the stronger the NF- κ B signal activity), and the results are shown in FIG. 6.

The specific steps of the Western Blot (Western Blot) experiment are as follows: adding the 1 xSDS sample-carrying buffer solution into a cell culture plate for cell lysis, centrifuging the lysate, and performing electrophoresis by using 8% polyacrylamide gel; transferring the electrophoresis product to a PVDF membrane through a half-glycoside membrane converter, and carrying out sealing treatment; then, RelA antibody (purchased from Santa Cruz Corp., model: sc-109), K310 acetylated RelA antibody (purchased from Cell Signaling, model: 3045s) and GAPDH antibody (purchased from Cell Signaling, model: 2118) were added, incubated overnight at 4 ℃, washed with TBST buffer, and then, horseradish peroxidase-coupled secondary antibody was added, incubated at room temperature for 1 hour, washed with TBST buffer, and then, ECL was added to carry out development reaction, and changes in protein expression level were observed.

Test results show that compared with a lentivirus empty vector control group, the overexpression long-chain non-coding RNA-HOXA-AS2 can obviously reduce the acetylation level of K310 sites in RelA subunits, so that the activity of NF-kB signals is inhibited, and osteogenic differentiation of MenSCs cells can be promoted.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> New countryside medical college

<120> application of long-chain non-coding RNA-HOXA-AS2 in bone tissue injury repair

<160> 13

<170> PatentIn version 3.1

<210> 1

<211> 1048

<212> RNA

<213> Artificial sequence

<223> Long non-coding RNA-HOXA-AS2

<400> 1

gaaaaggaaa cgccaagaca tagaaaacca cgcttttccc gtaggaagaa 50

ccgatgatga gccctgatga aagaaggaag aagacccgct gtctgcgaag 100

gcctaaaggc cgcggttgcc aggaaccgtg gagggccaac tcctcccaac 150

cgccctggtg caaagtccca cgcggcgaag agttttggag cagcgcttac 200

ctagaaagat gtttaaattc tgaaccagga attgtctcca actccaggcg 250

ctcagggaat cgccttttcc ggtgtccagg cgctctgcag acaaataaac 300

agcagaagca aatggtcacc gagccggcag tcagctttct gggagtggga 350

gatgatgggg aaagaggaaa gaatcgtccg ctcgccggac cctggcttgg 400

agaagttctg cgctccgctg ggactctgcg ggccctttgc gtctacagac 450

ctatccctgc cccgactacc ccttcactca gacccaggga aggacacgtt 500

tctatgcctt acagagactt gaagcctgaa agcctggcca agtttgggaa 550

gaagaggagc cctctcagag ctcaagagcc ttcctactct ttggaacttt 600

tccacagtag gccaagcttg acaagagttc agctcaagtt gaacatacat 650

acacacactc tcacacacaa attatgtgag ccgtcagaat ccaagtgaat 700

ccagctcaag ctatctacaa ggtttttaca tgcaaggtca agtatctcaa 750

tccagaggac ttttgttttc ttaatgaaaa gcttagaaaa cacatgaatc 800

ttagattttt aatgtttttt aaatggagtt tattcttagc acatggcttt 850

ctatgtagcc acatcacaat ttgtacagtt ccacataagt ctaaatgcac 900

tcccctctcc ccaaagaccg tgccccagaa ggggacaaca gtatctctgt 950

aacagtgtct taaataaatg caagtaagaa aaactaacat gtcacaccta 1000

ccatcaaggt ctacacatct taagaattaa ataatcttgt gaggtcca 1048

<210> 2

<211> 1066

<212> DNA

<213> Artificial sequence

<223> HOXA-AS2 Gene fragment containing specific cleavage site

<400> 2

cgcggatccg aaaaggaaac gccaagacat agaaaaccac gcttttcccg 50

taggaagaac cgatgatgag ccctgatgaa agaaggaaga agacccgctg 100

tctgcgaagg cctaaaggcc gcggttgcca ggaaccgtgg agggccaact 150

cctcccaacc gccctggtgc aaagtcccac gcggcgaaga gttttggagc 200

agcgcttacc tagaaagatg tttaaattct gaaccaggaa ttgtctccaa 250

ctccaggcgc tcagggaatc gccttttccg gtgtccaggc gctctgcaga 300

caaataaaca gcagaagcaa atggtcaccg agccggcagt cagctttctg 350

ggagtgggag atgatgggga aagaggaaag aatcgtccgc tcgccggacc 400

ctggcttgga gaagttctgc gctccgctgg gactctgcgg gccctttgcg 450

tctacagacc tatccctgcc ccgactaccc cttcactcag acccagggaa 500

ggacacgttt ctatgcctta cagagacttg aagcctgaaa gcctggccaa 550

gtttgggaag aagaggagcc ctctcagagc tcaagagcct tcctactctt 600

tggaactttt ccacagtagg ccaagcttga caagagttca gctcaagttg 650

aacatacata cacacactct cacacacaaa ttatgtgagc cgtcagaatc 700

caagtgaatc cagctcaagc tatctacaag gtttttacat gcaaggtcaa 750

gtatctcaat ccagaggact tttgttttct taatgaaaag cttagaaaac 800

acatgaatct tagattttta atgtttttta aatggagttt attcttagca 850

catggctttc tatgtagcca catcacaatt tgtacagttc cacataagtc 900

taaatgcact cccctctccc caaagaccgt gccccagaag gggacaacag 950

tatctctgta acagtgtctt aaataaatgc aagtaagaaa aactaacatg 1000

tcacacctac catcaaggtc tacacatctt aagaattaaa taatcttgtg 1050

aggtccatct agagca 1066

<210> 3

<211> 8865

<212> DNA

<213> Artificial sequence

<223> empty lentiviral vector pLV

<400> 3

aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa 50

cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc 100

cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca 150

gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag 200

atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta 250

gaccagatct gagcctggga gctctctggc taactaggga acccactgct 300

taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc 350

tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 400

tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg 450

aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 500

ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta 550

gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg 600

gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa 650

gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac 700

gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa 750

atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 800

atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag 850

agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac 900

aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag 950

gaggagatat gagggacaat tggagaagtg aattatataa atataaagta 1000

gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt 1050

ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1100

tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg 1150

gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 1200

gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg 1250

gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag 1300

gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac 1350

cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1400

tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac 1450

acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa 1500

gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt 1550

ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 1600

gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt 1650

gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1700

caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga 1750

gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta 1800

tcggttaact tttaaaagaa aaggggggat tggggggtac agtgcagggg 1850

aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa 1900

aaacaaatta caaaattcaa aattttatcg atgcctcccc gtcaccaccc 1950

cccccaaccc gccccgaccg gagctgagag taattcatac aaaaggactc 2000

gcccctgcct tggggaatcc cagggaccgt cgttaaactc ccactaacgt 2050

agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc 2100

atcactgagg tggagaagag catgcgtgag gctccggtgc ccgtcagtgg 2150

gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 2200

caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 2250

atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 2300

ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc 2350

cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta 2400

cgggttatgg cccttgcgtg ccttgaatta cttccacgcc cctggctgca 2450

gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt 2500

cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 2550

tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc 2600

ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgat 2650

atcctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa 2700

gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc 2750

ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc 2800

caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 2850

cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc 2900

ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg 2950

ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt 3000

gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc 3050

atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct 3100

cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 3150

atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg 3200

gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt 3250

ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt 3300

caggtgtcgt gaaaactacc cctgagctcc ttaaggttaa cgccaccatg 3350

gactacaaag acgatgacga caagtctaga gaattcggat ccaatattcc 3400

cgggctcgag ccatggaagc ttgatatcta actgactgaa ccggtggtac 3450

cgatccacgc gtctccggcc tagggataac agggtaatcc gctagcccct 3500

ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc 3550

cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg 3600

caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta 3650

ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 3700

aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc 3750

gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg 3800

gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag 3850

tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc 3900

aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta 3950

tgggatctga tctggggcct cggtacacat gctttacatg tgtttagtcg 4000

aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct 4050

ttgaaaaaca cgatgataat atggccacac tagagatcca ccggtcgcca 4100

ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 4150

cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac 4200

gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc 4250

aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 4300

gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga 4350

agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg 4400

gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 4450

cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga 4500

ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg 4550

cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 4600

aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga 4650

ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct 4700

gagcggccgc gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat 4750

tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 4800

gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 4850

ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 4900

ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 4950

cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 5000

cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg 5050

cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 5100

ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac 5150

ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc 5200

cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 5250

cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 5300

cccgcctgga attcgagctc ggtaccttta agaccaatga cttacaaggc 5350

agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 5400

taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 5450

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 5500

cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt 5550

gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt 5600

tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 5650

tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact 5700

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 5750

ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 5800

actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac 5850

tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 5900

atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc 5950

tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac 6000

gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt 6050

cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 6100

gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 6150

cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga 6200

cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 6250

gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 6300

ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 6350

tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6400

ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 6450

tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 6500

actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 6550

ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 6600

ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac 6650

aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 6700

atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 6750

gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6800

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 6850

tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6900

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 6950

gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 7000

tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 7050

tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 7100

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 7150

tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 7200

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 7250

catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 7300

aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 7350

gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7400

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 7450

ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 7500

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 7550

atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 7600

tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 7650

cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7700

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 7750

aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7800

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 7850

ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 7900

agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 7950

taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag 8000

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 8050

ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 8100

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 8150

acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 8200

gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 8250

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8300

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 8350

ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 8400

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 8450

ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 8500

ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 8550

ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 8600

aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga 8650

caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 8700

gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct 8750

cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 8800

ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac 8850

aaaagctgga gctgc 8865

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<223> HOXA-AS2 upstream primer

<400> 4

gttcagctca agttgaacat a 21

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<223> HOXA-AS2 downstream primer

<400> 5

aaaccttgta gatagcttga g 21

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<223> RUNX2 upstream primer

<400> 6

ggacgaggca agagtttcac 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<223> RUNX2 downstream primer

<400> 7

gaggcggtca gagaacaaac 20

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<223> SP7 upstream primer

<400> 8

cacagctctt ctgactgtct g 21

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<223> SP7 downstream primer

<400> 9

ctggtgaaat gcctgcatgg at 22

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<223> SPP1 upstream primer

<400> 10

agccaatgat gagagcaatg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<223> SPP1 downstream primer

<400> 11

tccttacttt tggggtctac 20

<210> 12

<211> 23

<212> DNA

<213> Artificial sequence

<223> GAPDH upstream primer

<400> 12

catgagaagt atgacaacag cct 23

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence

<223> GAPDH downstream primer

<400> 13

agtccttcca cgataccaaa gt 22

Claims (6)

1. Use of an overexpression vector pLV-HOXA-AS2 for the preparation of an agent for the repair of damaged bone tissue, characterized in that: is constructed by a HOXA-AS2 gene fragment and a lentivirus empty vector pLV; the nucleotide sequence of the HOXA-AS2 gene fragment is shown AS SEQ ID NO. 2 in the sequence table, and the nucleotide sequence of the lentivirus empty vector pLV is shown AS SEQ ID NO. 3 in the sequence table.

2. The use according to claim 1, wherein the overexpression vector pLV-HOXA-AS2 is constructed by a method comprising the steps of: respectively carrying out double enzyme digestion on lentiviral vector pLV and HOXA-AS2 gene fragments by adopting restriction endonucleases BamH I and Xba I, carrying out ligation reaction on the enzyme-digested HOXA-AS2 gene fragment and linear lentiviral vector pLV by adopting a T4 DNA ligase system, then transforming competent cells, screening positive colonies, extracting plasmids of the positive colonies, and obtaining the over-expression vector pLV-HOXA-AS 2.

3. The method of claim 2, wherein: the preparation method of the HOXA-AS2 gene fragment comprises the following steps: according to the nucleotide sequence of the long non-coding RNA-HOXA-AS2, performing double-strand synthesis on the HOXA-AS2 gene fragment by adopting a DNA synthesizer according to the principle of a solid phase phosphoramidite triester method, and simultaneously adding BamH I enzyme cutting sites and Xba I enzyme cutting sites on the 5 'end and the 3' end of the gene fragment respectively to obtain the HOXA-AS2 gene fragment containing specific enzyme cutting sites.

4. Use of an inducible cell in an agent for bone tissue injury repair, characterized in that: constructed from HEK293T cells transfected with the over-expression vector pLV-HOXA-AS2 of claim 1.

5. The use of claim 4, wherein said inducible cell is constructed by a method comprising the steps of: co-transfecting an overexpression vector pLV-HOXA-AS2, a lentivirus packaging plasmid pSPAX2 and a lentivirus packaging plasmid pMD2G into a packaging cell by adopting a liposome transfection reagent, and collecting lentivirus supernatant after culturing; then, the lentivirus supernatant is added into a cell vector, and the cell vector is cultured to obtain the inducible cell stably expressing the long-chain non-coding RNA-HOXA-AS 2.

6. The application of the kit in a reagent for bone tissue injury repair detection is characterized by comprising a specific primer for detecting the expression level of a HOXA-AS2 gene; the specific primer comprises:

an upstream primer: 5'-GTTCAGCTCAAGTTGAACATA-3' the flow of the air in the air conditioner,

a downstream primer: 5'-AAACCTTGTAGATAGCTTGAG-3' are provided.

Images