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