CN111304205A - circSPECC1 for treating brain glioma and application thereof - Google Patents
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Abstract
The invention relates to the field of gene medicine, in particular to circSPECC1 for treating brain glioma and application thereof. Use of circSPECC1 for the treatment of brain glioma in the manufacture of a medicament for the treatment of brain glioma. Has the advantages that: according to the project, based on the YB-1 gene of a glioma U251 cell, the circSPECC1 is found to interact with miR-615, the expression of HIP1 and Caspase9 genes is controlled in a targeted mode, the important effect is played in glioma invasion and proliferation, and the proliferation and invasion capacity of the glioma U251 cell can be effectively inhibited by inhibiting the circSPECC 1. The implementation of the project has great significance for filling up the gap of glioma biotherapy and perfecting the prior glioma comprehensive treatment measures, and also has profound significance for preventing and treating tumors of other tissues and organs.
Description
Technical Field
The invention relates to the field of gene medicine, in particular to circSPECC1 for treating brain glioma and application thereof.
Background
The current research situation and existing problems of glioma
Gliomas account for 30% of all nervous system tumors, and 80% of them are malignant brain tumors. Except for fibroastrocytomas, other glioma patients have very poor prognosis with survival rates of less than 10% 5 years after diagnosis, and scores on age diagnosis and pre-operative Karnofsky performance scales are predictors of conclusive survival. Over the past 30 years, conventional treatments for gliomas have included surgical resection, local radiation therapy, and temozolomide chemotherapy to the greatest extent. Although the median survival time for glioma patients increases from 6 months to 14.6 months, tumors still cause death in the vast majority of patients. Although much research is currently devoted to the molecular biological mechanisms underlying development of glioma, the conversion thereof into an effective therapeutic approach faces this enormous hurdle. Almost all patients are at risk of tumor recurrence. To date, no effective treatment regimen for preventing glioma development and recurrence has been established. Therefore, it is important to study the pathogenesis of glioma at the RNA level and develop a new diagnostic and therapeutic approach.
Role of circRANs in human tumors
Covalently closed single-stranded circular RNAs (circRNAs) are composed of introns or exons and are widely present in eukaryotic cells. Compared with messenger rna (mrna), circRNAs are usually expressed at a lower level and have a relatively stable structure, and most of the circRNAs are located in the cytoplasm and often function in a cell-type and tissue-specific manner, suggesting that circRNAs may be a new biomarker. In recent years, circRNAs have gradually become a focus in the field of RNA and cancer research, but the function of most of the circRNAs has not been discovered. Known circular RNAs can affect cancer biogenesis in a variety of ways, such as function as microrna (mirna) sponges, binding to RNA Binding Proteins (RBPs), as transcription factors and translation proteins. To date, various groups have screened and identified a variety of functional cancer-associated cyclic rnas (table 2). These cyclic rnas function as oncogenes or oncogenes in a variety of cancers and affect cancer phenotypes in a variety of ways.
Disclosure of Invention
The purpose of the invention is as follows: in order to provide circSPECC1 for treating brain glioma with better effect and the use thereof, the specific purpose is to see a plurality of substantial technical effects of the specific implementation part.
In order to achieve the purpose, the invention adopts the following technical scheme:
a circSPECC1 for the treatment of brain glioma, having the sequence:
gggcctttac aacaactaaa cggacaggca ttccagcccc acgggaattt tcagtaactg 60
tctcaagaga gaggtctgtg ccacgtggtc cctccaaccc caggaaatca gtgtccagtc 120
caacttcttc caacactccc actcctacga aacacctgag gaccccttcc acaaagccca 180
agcaagagaa tgaaggtgga gaaaaggctg cgcttgagtc ccaagttcgg gaacttttgg 240
cagaagccaa agcaaaagat agtgaaatta acaggcttcg aagtgaacta aagaaataca 300
aagagaaaag gactctgaac gctgagggga ctgatgcttt gggcccaaat gtcgatggaa 360
catcagtctc cccaggtgac acggaaccta tgataagagc tcttgaggag aagaacaaga 420
actttcagaa agagctttcc gatctagagg aagaaaaccg ggtcctgaag gagaaactga 480
tctatcttga gcactcccca aattcagaag gggcagcaag tcacactggc gacagcagct 540
gcccaacatc cataactcaa gagtcaagct tcggaagccc aactggaaat cagatgtcca 600
gtgacattga tgagtataaa aaaaacatac atggaaatgc attacggaca tcaggctcct 660
caagtagcga tgttaccaaa gcttctttgt cgccagatgc ttccgacttt gagcacatta 720
cagcagagac accctcaagg cccctgtcct ccaccagtaa cccctttaag agttcaaagt 780
gttctactgc tgggagttcc ccaaacagcg taagtgaatt gtccctggct tccctcacag 840
agaagataca aaagatggaa gaaaaccacc atagcactgc agaagaacta caggctactc 900
tacaagaatt atcagaccag caacaaatgg tacaggaatt gacagctgaa aatgagaagc 960
tggtggatga aaagacgatt ttagagacat cctttcatca gcatcgagag agggcagagc 1020
agctaagtca agaaaatgag aagctgatga atcttttaca agagcgagta aagaatgaag 1080
agcccaccac tcaggaagga aaaattattg aactggagca gaagtgcaca ggtattcttg 1140
aacagggccg ctttgaaaga gagaagctac tcaacattca gcagcagttg acctgtagct 1200
tgcggaaggt tgaggaagaa aaccaaggag ctttagaaat gattaaacgt ctgaaggaag 1260
aaaatgaaaa actgaatgag tttctagaac tggaacggca taataataac atgatggcca 1320
aaactttgga agagtgtaga gttaccttgg aagggctaaa aatggagaat ggatctttga 1380
agtctcattt gcagggtgag aagcagaaag ccacagaggc cagtgctgtg gagcagacgg 1440
cagagagctg cgaagttcaa gaaatgttga aagtagcccg agcagagaaa gatctactgg 1500
aactgtcttg caatgagctc agacaagaat tactaaaggc aaacggtgaa attaaacatg 1560
tttccagtct gctggccaag 1580。
use of circSPECC1 for the treatment of brain glioma as described above in the manufacture of a medicament for the treatment of brain glioma.
The use of circSPECC1 for treating glioma as described above in the preparation of a medicament for promoting apoptosis of glioma cells U251 and inhibiting the ability to reduce invasion of glioma cells U251.
The circSPECC1 for treating brain glioma has the application of targeted regulation and control of the expression of HIP1 and Caspase9 genes through interaction with miR-615.
The use of YB-1, namely Y-box binding protein-1, in combination with circSPECC1 in the preparation of a medicament for the treatment of brain glioma.
Compared with the prior art, the invention adopting the technical scheme has the following beneficial effects: according to the project, based on the YB-1 gene of a glioma U251 cell, the circSPECC1 is found to interact with miR-615, the expression of HIP1 and Caspase9 genes is controlled in a targeted mode, the important effect is played in glioma invasion and proliferation, and the proliferation and invasion capacity of the glioma U251 cell can be effectively inhibited by inhibiting the circSPECC 1. The implementation of the project has great significance for filling up the gap of glioma biotherapy and perfecting the prior glioma comprehensive treatment measures, and also has profound significance for preventing and treating tumors of other tissues and organs.
Drawings
To further illustrate the present invention, further description is provided below with reference to the accompanying drawings:
FIG. 1 inhibits the effect of circSPECC1 on brain glioma cell U251 proliferation;
FIG. 2 is a graph showing the effect of circSPECC1 on the U251 invasiveness of glioma cells;
FIG. 3 inhibition of circSPECC1 induced apoptosis in glioma cell U251.
Detailed Description
The patent provides a plurality of parallel schemes, and different expressions belong to an improved scheme based on a basic scheme or a parallel scheme. Each solution has its own unique features.
The function of the Circ RAN in glioma proliferation and metastasis provides a new idea for preventing and treating glioma metastasis
YB-1 (Y-box binding protein-1) is a member of the cold shock protein superfamily, contains highly conserved nucleic acid sequences, and is a multifunctional protein. YB-1 is involved in a series of DNA/RNA dependent events, such as DNA repair, alternative splicing of mRNA, regulation of mRNA stability and translation, and plays an important biological role in cell proliferation, differentiation, stress response, and transformation of malignant cells. Early-stage research of project groups finds that YB-1 is over-expressed in brain glioma tissues and plays a key role in the aspects of brain glioma occurrence and drug resistance, but the regulation processes of transcription, translation and the like of target genes are not clear. The research adopts RIP (RNA Binding Protein Immunoprecipitation Assay) technology to clarify circRNAs in which YB-1 participates in regulation and further to clarify the biological functions of key circRNAs mediated by YB-1, thereby clarifying the regulation and control mechanism of YB-1 and the circRNAs mediated by YB-1 in the development of brain glioma.
1. Further elucidating the YB-1 mediated biological function of key circRNA
2. The regulation and control mechanism of YB-1 and circRNA mediated by the same in the development of brain glioma is clarified.
Expression of 1 circSPECC1 in brain gliomas
(1) And (3) carrying out high-throughput sequencing on the YB-1 protein-RNA composite precipitate of the U251 cell by adopting an RIP method.
The circSPECC1 gene sequence is:
gggcctttac aacaactaaa cggacaggca ttccagcccc acgggaattt tcagtaactg 60
tctcaagaga gaggtctgtg ccacgtggtc cctccaaccc caggaaatca gtgtccagtc 120
caacttcttc caacactccc actcctacga aacacctgag gaccccttcc acaaagccca 180
agcaagagaa tgaaggtgga gaaaaggctg cgcttgagtc ccaagttcgg gaacttttgg 240
cagaagccaa agcaaaagat agtgaaatta acaggcttcg aagtgaacta aagaaataca 300
aagagaaaag gactctgaac gctgagggga ctgatgcttt gggcccaaat gtcgatggaa 360
catcagtctc cccaggtgac acggaaccta tgataagagc tcttgaggag aagaacaaga 420
actttcagaa agagctttcc gatctagagg aagaaaaccg ggtcctgaag gagaaactga 480
tctatcttga gcactcccca aattcagaag gggcagcaag tcacactggc gacagcagct 540
gcccaacatc cataactcaa gagtcaagct tcggaagccc aactggaaat cagatgtcca 600
gtgacattga tgagtataaa aaaaacatac atggaaatgc attacggaca tcaggctcct 660
caagtagcga tgttaccaaa gcttctttgt cgccagatgc ttccgacttt gagcacatta 720
cagcagagac accctcaagg cccctgtcct ccaccagtaa cccctttaag agttcaaagt 780
gttctactgc tgggagttcc ccaaacagcg taagtgaatt gtccctggct tccctcacag 840
agaagataca aaagatggaa gaaaaccacc atagcactgc agaagaacta caggctactc 900
tacaagaatt atcagaccag caacaaatgg tacaggaatt gacagctgaa aatgagaagc 960
tggtggatga aaagacgatt ttagagacat cctttcatca gcatcgagag agggcagagc 1020
agctaagtca agaaaatgag aagctgatga atcttttaca agagcgagta aagaatgaag 1080
agcccaccac tcaggaagga aaaattattg aactggagca gaagtgcaca ggtattcttg 1140
aacagggccg ctttgaaaga gagaagctac tcaacattca gcagcagttg acctgtagct 1200
tgcggaaggt tgaggaagaa aaccaaggag ctttagaaat gattaaacgt ctgaaggaag 1260
aaaatgaaaa actgaatgag tttctagaac tggaacggca taataataac atgatggcca 1320
aaactttgga agagtgtaga gttaccttgg aagggctaaa aatggagaat ggatctttga 1380
agtctcattt gcagggtgag aagcagaaag ccacagaggc cagtgctgtg gagcagacgg 1440
cagagagctg cgaagttcaa gaaatgttga aagtagcccg agcagagaaa gatctactgg 1500
aactgtcttg caatgagctc agacaagaat tactaaaggc aaacggtgaa attaaacatg 1560
tttccagtct gctggccaag 1580。
Length: 1580 nt
(2) inhibition of the effect of circSPECC1 on the proliferation of brain glioma cells U251.
On a molecular level, the action mechanism of inhibiting the influence of circSPECC1 on the proliferation condition of the brain glioma cells U251 is researched.
(1) Inhibition of circSPECC1 promotes apoptosis of brain glioma cell U251.
(2) Inhibition of circSPECC1 decreased the invasive potential of brain glioma cells U251.
Beneficial effects (compared with the prior art)
Currently, the conventional treatment for gliomas includes the largest extent of surgical resection, local radiation therapy, and temozolomide chemotherapy. However, the prognosis and recurrence of glioma are still not effectively improved. Although much research is currently devoted to the molecular biological mechanisms underlying development of glioma, the conversion thereof into an effective therapeutic approach faces this enormous hurdle. Therefore, it is important to study the pathogenesis of glioma at the RNA level and develop a new diagnostic and therapeutic approach.
Expression of circSPECC1 in brain gliomas (RNA binding protein immunoprecipitation assay, RIP)
Receive 2X 107U251 cells were treated according to the Millipore Magna RIPTM RNA-Binding protein immunopropractination Kit procedure. The rabbit anti-human HuR antibody and normal rabbit IgG antibody are used in 5 microgram amount, and the cell lysate and the antibody are incubated overnight at 4 deg.c. When a specific protein in a cell is captured by a specific antibody, a protein-RNA complex is obtained. Then, the protein is removed by digestion with proteinase K and the RNA molecule is extracted. In the experimental process, the magnetic beads are repeatedly washed by RIP washing buffer solution to remove some non-specific adsorption as much as possible, and finally the obtained RNA molecules are subjected to high-throughput sequencing according to the experimental requirements. And in the analysis of results, the RNA molecules combined with the YB-1 protein are found by comparing the FPKM values of the RNAs of the YB-1-RIP group and the RNA of the Input group. The screening conditions of the analysis are that the log2(Fold change) of the FPKM value IP/Input is more than or equal to 1, the P value is less than or equal to 0.01, and the q value is less than or equal to 0.01. 2 circSPECC1 and tumor cell proliferation (MTT cell proliferation assay)
Experiment was divided into 3 groups: blank control, negative control, si circspec 1 (inhibiting circspec 1 effect). 3 groups of U251 cells were diluted to 1X 106mL, 2000 cells/well were added to 96-well plates, respectively. Each set was provided with 3 multiple wells. After the cells were attached and cultured for 24 h, 48h, 72h, 96 h, 50. mu.l XTT solution was added to each well, the plates were wrapped with aluminum foil and incubated at 37 ℃ for 4 h. Adding 150 mu l of DMSO into each hole, placing the hole on a shaking table, oscillating at low speed for 10 min, dissolving residual formazan crystals, detecting the absorption value of 450nm by using a microplate reader, and drawing a cell proliferation curve. The experiment was repeated 3 times.
3 circSPECC1 and tumor cell metastasis (scratch test)
Experiment was divided into 3 groups: blank control, negative control, si circspec 1 (inhibiting circspec 1 effect). 5 parallel lines were drawn on each well at the bottom of the 6-well plate. Resuspending 3 groups of cells, respectively inoculating 2.5 multiplied by 105/hole in a 6-hole plate, and culturing in an incubator until the confluency reaches 90-95%. The cells were washed 3 times with PBS, and then 1 vertical line was drawn with a 10. mu.l pipette tip, and then incubated in a 5% CO2 incubator at 37 ℃ in the presence of serum-free medium. Finally, the phase is collected under an inverted microscope (100 x) at 0 h, 12 h and 24 h respectively, and the scratch distance on the picture is calculated by adopting Image J software, wherein the calculation formula is as follows: mobility (%) = (distance measured at 0 h-distance measured at predetermined time)/distance measured at 0 h × 100%. The experiment was repeated 3 times.
4 circSPECC1 with tumor cell invasion (Transwell cell migration assay)
Collecting 3 groups of cells in logarithmic growth phase, adjusting cell density to 2.5 × 105And/ml. Transwell chambers with a pore size of 0.8 μm were selected and placed in 24-well plates, and 1X 105 cells and 200. mu.l of serum-free medium were added to the chambers, respectively. Each of the lower chambers (24-well plates) was filled with 600. mu.l of complete medium and placed in an incubator for 36 hours. Taking out the Transwell chamber, wiping off cells in the chamber by using a cotton swab, fixing for 15 min by using 4% paraformaldehyde, dyeing by using 0.1% crystal violet, and rinsing for 1-3 times by using PBS until the background is white. Finally, the number of the transmembrane cells is observed under a microscope, 5 fixed fields (multiplied by 100) are randomly selected for photographing, and the migration capacity of each group of cells is evaluated. The experiment was repeated 3 times.
5. CircSPECC1 and tumor cell apoptosis (Annexin V-FITC/PI staining) Annexin V-FITC/PI method was used to separate early apoptotic cells from late apoptotic cells. Negative control and si circSPECC1 cells were prepared at 1 × 10 concentration in log phase6Cell suspension in ml. Annexin V-FITC/PI kit was used to stain cells, protected from light for 35 min. Data acquisition and analysis were then performed by flow cytometry using BD Cell QuestPro software (BD Biosciences). Creatively, the above effects exist independently, and the combination of the above results can be completed by a set of structure.
FIG. 1 shows the effect of inhibiting circSPECC1 on the proliferation of brain glioma cells U251. FIG. 1 shows the effect of circSPECC1 on the proliferation of glioma cells U251. 3 groups of U251 cells were diluted to 1X 106mL, 2000 cells/well were added to 96-well plates, respectively. Each set was provided with 3 multiple wells. After the cells were attached and cultured for 24 h, 48h, 72h, 96 h, 50. mu.l XTT solution was added to each well, the plates were wrapped with aluminum foil and incubated at 37 ℃ for 4 h. Adding 150 mu l of DMSO into each hole, placing the hole on a shaking table, oscillating at low speed for 10 min, dissolving residual formazan crystals, detecting the absorption value of 450nm by using a microplate reader, and drawing a cell proliferation curve. The experiment was repeated 3 times. It was observed that cell proliferation was significantly lower in the siSPECC1 groups at 48h and 72h than in the control and siR-NC groups. It is shown that the circSPECC1 can inhibit the proliferation of tumor cells after being inhibited.
Note: crol is blank control; SiR-NC is negative siRNA control; SiSPECC1 is siRNA of circSPECC1
FIG. 2 shows the effect of inhibiting circSPECC1 on the U251 invasiveness of brain glioma cells. Collecting 2 groups of cells in logarithmic growth phase, adjusting cell density to 2.5 × 105And/ml. Selecting Transwell chamber with pore diameter of 0.8 μm, placing in 24-well plate, adding 1 × 105Individual cells and 200. mu.l serum-free medium. Each of the lower chambers (24-well plates) was filled with 600. mu.l of complete medium and placed in an incubator for 36 hours. Taking out the Transwell chamber, wiping off cells in the chamber by using a cotton swab, fixing for 15 min by using 4% paraformaldehyde, dyeing by using 0.1% crystal violet, and rinsing for 1-3 times by using PBS until the background is white. Finally, the number of the transmembrane cells is observed under a microscope, 5 fixed fields (multiplied by 100) are randomly selected for photographing, and the migration capacity of each group of cells is evaluated. The experiment was repeated 3 times. It was observed that the number of cell migrations was significantly lower in the siSPECC1 group at 48h than in the siR-NC group. It is shown that circSPECC1 can inhibit the invasiveness of tumor cells after being inhibited.
Note: SiR-NC is negative siRNA control; SiSPECC1 is siRNA of circSPECC1
FIG. 3 shows that inhibition of circSPECC1 induces apoptosis in brain glioma cell U251. CircSPECC1 and tumor cell apoptosis (Annexin V-FITC/PI staining) Annexin V-FITC/PI method was used to separate early apoptotic cells from late apoptotic cells. Negative control and si circSPECC1 cells were prepared at 1 × 10 concentration in log phase6Cell suspension in ml. Annexin V-FITC/PI kit was used to stain cells, protected from light for 35 min. Data acquisition and analysis were then performed by flow cytometry using BDCell QuestPro software (BD Biosciences). It was observed that the apoptosis number was significantly higher in the siSPECC1 group than in the siR-NC group. It is shown that circSPECC1 can promote apoptosis of tumor cells after being inhibited.
Note: SiR-NC is negative siRNA control; SiSPECC1 is siRNA of circSPECC1
List of abbreviations
| Serial number | Abbreviations | Full scale |
| 1 | circSPECC1 | Covalently closed single stranded circular RNA SPECC1 |
| 2 | miR-615 | Micro RNA-615 |
| 3 | HIP1 | Huntington protein binding protein 1 |
| 4 | Caspase 9 | Apoptotic protease 9 |
| 5 | siR-NC | Small interference RNA-negative control |
| 6 | siSPECC1 | Small interfering RNA- |
| 7 | OS-RC-2 | Human kidney cancer cell strain |
| 8 | YB-1 | Y-box binding protein 1 |
| 9 | RIP | RNA binding |
| 10 | Transwell | Transfer cell |
| 11 | AnnexinV | Modal catenin V |
| 12 | RBPs | RNA binding proteins |
| 13 | HuR | Human antigen R (RNA binding protein) |
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to illustrate the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and the invention is to be limited to the embodiments described above.
Sequence listing
<110> first subsidiary hospital of the university of west-safety traffic
<120> circSPECC1 for treating brain glioma and use thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>3
<211>1580
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
gggcctttac aacaactaaa cggacaggca ttccagcccc acgggaattt tcagtaactg 60
tctcaagaga gaggtctgtg ccacgtggtc cctccaaccc caggaaatca gtgtccagtc 120
caacttcttc caacactccc actcctacga aacacctgag gaccccttcc acaaagccca 180
agcaagagaa tgaaggtgga gaaaaggctg cgcttgagtc ccaagttcgg gaacttttgg 240
cagaagccaa agcaaaagat agtgaaatta acaggcttcg aagtgaacta aagaaataca 300
aagagaaaag gactctgaac gctgagggga ctgatgcttt gggcccaaat gtcgatggaa 360
catcagtctc cccaggtgac acggaaccta tgataagagc tcttgaggag aagaacaaga 420
actttcagaa agagctttcc gatctagagg aagaaaaccg ggtcctgaag gagaaactga 480
tctatcttga gcactcccca aattcagaag gggcagcaag tcacactggc gacagcagct 540
gcccaacatc cataactcaa gagtcaagct tcggaagccc aactggaaat cagatgtcca 600
gtgacattga tgagtataaa aaaaacatac atggaaatgc attacggaca tcaggctcct 660
caagtagcga tgttaccaaa gcttctttgt cgccagatgc ttccgacttt gagcacatta 720
cagcagagac accctcaagg cccctgtcct ccaccagtaa cccctttaag agttcaaagt 780
gttctactgc tgggagttcc ccaaacagcg taagtgaatt gtccctggct tccctcacag 840
agaagataca aaagatggaa gaaaaccacc atagcactgc agaagaacta caggctactc 900
tacaagaatt atcagaccag caacaaatgg tacaggaatt gacagctgaa aatgagaagc 960
tggtggatga aaagacgatt ttagagacat cctttcatca gcatcgagag agggcagagc 1020
agctaagtca agaaaatgag aagctgatga atcttttaca agagcgagta aagaatgaag 1080
agcccaccac tcaggaagga aaaattattg aactggagca gaagtgcaca ggtattcttg 1140
aacagggccg ctttgaaaga gagaagctac tcaacattca gcagcagttg acctgtagct 1200
tgcggaaggt tgaggaagaa aaccaaggag ctttagaaat gattaaacgt ctgaaggaag 1260
aaaatgaaaa actgaatgag tttctagaac tggaacggca taataataac atgatggcca 1320
aaactttgga agagtgtaga gttaccttgg aagggctaaa aatggagaat ggatctttga 1380
agtctcattt gcagggtgag aagcagaaag ccacagaggc cagtgctgtg gagcagacgg 1440
cagagagctg cgaagttcaa gaaatgttga aagtagcccg agcagagaaa gatctactgg 1500
aactgtcttg caatgagctc agacaagaat tactaaaggc aaacggtgaa attaaacatg 1560
tttccagtct gctggccaag 1580
Claims (6)
1. A circSPECC1 for the treatment of brain glioma, having the sequence:
gggcctttac aacaactaaa cggacaggca ttccagcccc acgggaattt tcagtaactg 60
tctcaagaga gaggtctgtg ccacgtggtc cctccaaccc caggaaatca gtgtccagtc 120
caacttcttc caacactccc actcctacga aacacctgag gaccccttcc acaaagccca 180
agcaagagaa tgaaggtgga gaaaaggctg cgcttgagtc ccaagttcgg gaacttttgg 240
cagaagccaa agcaaaagat agtgaaatta acaggcttcg aagtgaacta aagaaataca 300
aagagaaaag gactctgaac gctgagggga ctgatgcttt gggcccaaat gtcgatggaa 360
catcagtctc cccaggtgac acggaaccta tgataagagc tcttgaggag aagaacaaga 420
actttcagaa agagctttcc gatctagagg aagaaaaccg ggtcctgaag gagaaactga 480
tctatcttga gcactcccca aattcagaag gggcagcaag tcacactggc gacagcagct 540
gcccaacatc cataactcaa gagtcaagct tcggaagccc aactggaaat cagatgtcca 600
gtgacattga tgagtataaa aaaaacatac atggaaatgc attacggaca tcaggctcct 660
caagtagcga tgttaccaaa gcttctttgt cgccagatgc ttccgacttt gagcacatta 720
cagcagagac accctcaagg cccctgtcct ccaccagtaa cccctttaag agttcaaagt 780
gttctactgc tgggagttcc ccaaacagcg taagtgaatt gtccctggct tccctcacag 840
agaagataca aaagatggaa gaaaaccacc atagcactgc agaagaacta caggctactc 900
tacaagaatt atcagaccag caacaaatgg tacaggaatt gacagctgaa aatgagaagc 960
tggtggatga aaagacgatt ttagagacat cctttcatca gcatcgagag agggcagagc 1020
agctaagtca agaaaatgag aagctgatga atcttttaca agagcgagta aagaatgaag 1080
agcccaccac tcaggaagga aaaattattg aactggagca gaagtgcaca ggtattcttg 1140
aacagggccg ctttgaaaga gagaagctac tcaacattca gcagcagttg acctgtagct 1200
tgcggaaggt tgaggaagaa aaccaaggag ctttagaaat gattaaacgt ctgaaggaag 1260
aaaatgaaaa actgaatgag tttctagaac tggaacggca taataataac atgatggcca 1320
aaactttgga agagtgtaga gttaccttgg aagggctaaa aatggagaat ggatctttga 1380
agtctcattt gcagggtgag aagcagaaag ccacagaggc cagtgctgtg gagcagacgg 1440
cagagagctg cgaagttcaa gaaatgttga aagtagcccg agcagagaaa gatctactgg 1500
aactgtcttg caatgagctc agacaagaat tactaaaggc aaacggtgaa attaaacatg 1560
tttccagtct gctggccaag 1580。
2. use of circSPECC1 for the treatment of brain glioma according to claim 1 in the preparation of a medicament for the treatment of brain glioma.
3. Use of circSPECC1 for the treatment of glioma according to claim 1 in the preparation of a medicament for promoting apoptosis of brain glioma cell U251 and inhibiting the ability to reduce the invasiveness of brain glioma cell U251.
4. The use of circSPECC1 for the treatment of brain glioma according to claim 1 for the targeted modulation of the expression of HIP1 and Caspase9 genes by interaction with miR-615.
Use of YB-1, i.e. Y-box binding protein-1 in combination with circSPECC1 in the preparation of a medicament for the treatment of glioma.
6. The experimental method of circSPECC1 for the treatment of brain glioma of claim 1, comprising the steps of;
expression of circSPECC1 in brain gliomas (RNA binding protein immunoprecipitation assay, RIP)
Receive 2X 107U251 cells, according to Millipore company Magna RIPTM RNA-Binding protein immunopropraction Kit steps processing cells; the usage amount of rabbit anti-human HuR antibody and normal rabbit IgG antibody is 5 mug, thinCarrying out rotary incubation on the cell lysate and the antibody at 4 ℃ overnight; when a specific protein in a cell is captured by using a specific antibody, a protein-RNA complex is obtained; then, digesting with proteinase K to remove proteins and extract RNA molecules; in the experimental process, the RIP washing buffer solution is used for repeatedly washing the magnetic beads so as to remove some non-specific adsorption as much as possible, and finally the obtained RNA molecules are subjected to high-throughput sequencing according to the experimental requirements; during result analysis, the RNA molecules combined with the YB-1 protein are found by comparing the FPKM values of the RNAs of the YB-1-RIP group and the RNA of the Input group; the screening conditions of the analysis are that log2(Fold change) of IP/Input of FPKM value is more than or equal to 1, P value is less than or equal to 0.01, q value is less than or equal to 0.01; 2 circSPECC1 and tumor cell proliferation (MTT cell proliferation assay)
Experiment was divided into 3 groups: blank control, negative control, si circspec 1 (inhibiting circspec 1 effect); 3 groups of U251 cells were diluted to 1X 106mL, 2000 cells/well added to 96 well plates, respectively; each group is provided with 3 multiple holes; after the cells adhere to the wall, culturing for 24 h, 48h, 72h and 96 h, respectively adding 50 mul XTT solution into each well, wrapping the culture plate with aluminum foil, and incubating for 4 h at 37 ℃; adding 150 mu l of DMSO into each hole, placing the mixture on a shaking table to oscillate at a low speed for 10 min, dissolving residual formazan crystals, detecting an absorption value of 450nm by using an enzyme labeling instrument, and drawing a cell proliferation curve; the experiment was repeated 3 times;
circSPECC1 and tumor cell metastasis (scratch test)
Experiment was divided into 3 groups: blank control, negative control, si circspec 1 (inhibiting circspec 1 effect); drawing 5 parallel straight lines on each hole at the bottom of the 6-hole plate; resuspending 3 groups of cells, respectively inoculating 2.5 multiplied by 105/hole in a 6-hole plate, and placing in an incubator for culture until the confluence reaches 90-95%; washing cells for 3 times by PBS, marking 1 vertical line by a 10-microliter gun head, adding a serum-free culture medium, placing in a 5% CO2 incubator at 37 ℃ for incubation; finally, the phase is collected under an inverted microscope (100 x) at 0 h, 12 h and 24 h respectively, and the scratch distance on the picture is calculated by adopting Image J software, wherein the calculation formula is as follows: mobility (%) = (distance measured at 0 h-distance measured at predetermined time)/distance measured at 0 h × 100%; the experiment was repeated 3 times;
CircSPECC1 and tumor cell invasion (Transwell cell migration assay)
Collecting 3 groups of cells in logarithmic growth phase, adjusting cell density to 2.5 × 105Per ml; selecting a Transwell chamber with the pore diameter of 0.8 mu m, placing the Transwell chamber in a 24-pore plate, and respectively adding 1 multiplied by 105 cells and 200 mu l of serum-free culture medium into the chamber; adding 600 mul of complete culture medium into each lower chamber (24-hole plate), and culturing in an incubator for 36 h; taking out the Transwell chamber, wiping off cells in the chamber by using a cotton swab, fixing for 15 min by using 4% paraformaldehyde, dyeing by using 0.1% crystal violet, and rinsing for 1-3 times by using PBS (phosphate buffer solution) until the background is white; finally, observing the number of the cell penetrating the membrane under a microscope, randomly selecting 5 fixed visual fields (multiplied by 100) for photographing, and evaluating the migration capacity of each group of cells; the experiment was repeated 3 times;
the method for separating early apoptosis cells and late apoptosis cells from tumor cell apoptosis (annexin V-FITC/PI staining) by using the annexin V-FITC/PI method is adopted in the circSPECC 1; negative control and si circSPECC1 cells were prepared at 1 × 10 concentration in log phase6A cell suspension in ml; annexin V-FITC/PI kit was used to stain cells, protected from light for 35 min; data acquisition and analysis were then performed by flow cytometry using BD Cell QuestPro software (BD Biosciences).
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