CN111334509A - circSPECC1 for treating human kidney cancer and application thereof - Google Patents
circSPECC1 for treating human kidney cancer and application thereof Download PDFInfo
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Abstract
The invention relates to the field of gene medicine, in particular to circSPECC1 for treating human kidney cancer and application thereof. Use of circSPECC1 for treating human kidney cancer in the preparation of a medicament for treating human kidney cancer. Has the advantages that: the project discovers that circSPECC1 can perform targeted regulation on the expression of HIP1 (Huntingtin Interacting Protein 1) and Caspase9 genes through interaction with miR-615 based on human kidney cancer OS-RC-2 cell YB-1 gene, plays an important role in invasion and proliferation of human kidney cancer, and can effectively inhibit the proliferation and invasion capacity of human kidney cancer OS-RC-2 cells by inhibiting the circSPECC 1. The implementation of the project has great significance for filling up the gap of biological treatment of the human kidney cancer and perfecting the current comprehensive treatment measures of the human kidney cancer, and has far-reaching 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 human kidney cancer and application thereof.
Background
Present and existing problems of human kidney cancer research
The incidence of human kidney cancer, also known as human Renal Cell Carcinoma (RCC), has been increasing in recent years, accounting for 2% -3% of adult malignant tumors, 80-90% of Renal primary malignant tumors, and up to 40% of mortality. At present, the treatment methods of human kidney cancer mainly include surgery, chemotherapy, radiotherapy, biological treatment, thermotherapy and targeted therapy. However, even after surgical treatment of the patient in the early stage, metastasis will still occur in some patients. The prognosis of the metastatic renal cancer is poor, the metastatic renal cancer is not sensitive to treatment such as radiotherapy, chemotherapy and the like, and the 3-year average survival rate is lower than 5 percent. Although much research is currently being conducted to reveal the molecular biological mechanisms underlying the development of human renal cancer, the conversion to an effective therapeutic approach is faced with a significant hurdle. Almost all patients are at risk of tumor recurrence. To date, no effective treatment regimen has been established to prevent the occurrence and recurrence of renal cancer in humans. Therefore, it is important to study the pathogenesis of human kidney cancer at the RNA level and develop a new diagnostic and therapeutic method.
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 circular RNAs. These circular 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 the circSPECC1 for treating the human kidney cancer with better effect and the application thereof, the specific purpose is seen in a plurality of essential technical effects of the concrete implementation part.
In order to achieve the purpose, the invention adopts the following technical scheme:
a circSPECC1 for the treatment of human kidney cancer, 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 gcttctttgtcgccagatgc 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 human kidney cancer as described above in the manufacture of a medicament for the treatment of human kidney cancer.
The use of circSPECC1 for treating human kidney cancer as described above in the preparation of a medicament for promoting apoptosis of human kidney cancer cells OS-RC-2 and inhibiting the ability to reduce invasion of human kidney cancer cells OS-RC-2.
The circSPECC1 for treating human kidney cancer has the application of targeted regulation and control of the expression of HIP1 and Caspase9 genes through interaction with miR-615.
The application of YB-1, namely Y-box binding protein-1 in coordination with circSPECC1 in the preparation of medicaments for treating human kidney cancer.
Compared with the prior art, the invention adopting the technical scheme has the following beneficial effects: the project discovers that circSPECC1 performs targeted regulation on the expression of HIP1 and Caspase9 genes through interaction with miR-615 based on a YB-1 gene of a human kidney cancer OS-RC-2 cell, plays an important role in invasion and proliferation of human kidney cancer, and can effectively inhibit the proliferation and invasion capacity of the human kidney cancer OS-RC-2 cell by inhibiting the circSPECC 1. The implementation of the project has great significance for filling up the gap of biological treatment of the human kidney cancer and perfecting the current comprehensive treatment measures of the human kidney cancer, and has far-reaching 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 shows that the effect of circSPECC1 on the proliferation of OS-RC-2 in human renal cancer cells is suppressed;
FIG. 2 shows that the effect of circSPECC1 on the ability of human renal carcinoma cells to invade OS-RC-2 is suppressed;
FIG. 3 inhibition of circSPECC1 induces apoptosis in human renal carcinoma cells OS-RC-2.
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 effect of the Circ RAN in proliferation and metastasis of human renal cancer provides a new idea for preventing and treating human renal cancer 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 shows that YB-1 is over-expressed in human kidney cancer tissues and plays a key role in the aspects of the occurrence and drug resistance of human kidney cancer, but the regulation processes of transcription, translation and the like of a target gene are not clear. The research adopts RIP (RNA Binding Protein Immunoprecipitation Assay) technology to clarify the 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 occurrence and development of human renal carcinoma.
1. Further elucidating the YB-1 mediated biological function of key circRNA
2. The regulation mechanism of YB-1 and circRNA mediated by the same in the occurrence and development of human renal cancer is clarified.
Expression of 1 circSPECC1 in human renal carcinoma
(1) And (3) carrying out high-throughput sequencing on YB-1 protein-RNA composite precipitates of OS-RC-2 cells 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) inhibit the effect of circSPECC1 on the proliferation of human renal carcinoma cells OS-RC-2.
On the molecular level, the action mechanism of inhibiting the influence of circSPECC1 on the proliferation of human renal carcinoma cells OS-RC-2 is researched.
(1) Inhibition of circSPECC1 promotes apoptosis of human renal cancer cells OS-RC-2.
(2) Inhibition of circSPECC1 decreases the invasive potential of human renal carcinoma cells OS-RC-2.
Beneficial effects (compared with the prior art)
At present, the conventional treatment methods for human kidney cancer mainly comprise surgical treatment, chemotherapy, radiotherapy, biological treatment, thermotherapy and targeted treatment. However, the prognosis and recurrence of human kidney cancer are still not effectively improved. Although much research is currently being conducted to reveal the molecular biological mechanisms underlying the development of human renal cancer, the conversion to an effective therapeutic approach is faced with a significant hurdle. Therefore, it is important to study the pathogenesis of human kidney cancer at the RNA level and develop a new diagnostic and therapeutic method.
Expression of circSPECC1 in human renal carcinoma (RNA binding protein immunoprecipitation assay, RIP)
Receive 2 × 107Each OS-RC-2 cell was treated according to the Magna RIPTM RNA-binding protein immunization Kit step of Millipore. 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)
Experimental group 3 blank control, negative control, si circSPECC1 (inhibiting circSPECC1 action), group 3 OS-RC-2 cells were diluted to 1 × 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 μ l DMSO into each well, shaking on shaking table at low speed for 10 min to dissolve residueThe formazan crystal is detected by an enzyme-labeling instrument, the absorption value of the formazan crystal at the wavelength of 450 nm is detected, and a cell proliferation curve is drawn. The experiment was repeated 3 times.
3 circSPECC1 and tumor cell metastasis (scratch test)
Experiments are divided into 3 groups, namely blank control, negative control and si circspec 1 (inhibiting the action of circspec 1), 5 parallel straight lines are drawn on each hole at the bottom of a 6-hole plate, 3 groups of cells are resuspended, 2.5 × 105/hole is respectively inoculated in the 6-hole plate and placed in an incubator to be cultured until the confluence degree reaches 90% -95%, the cells are washed for 3 times by PBS, 1 perpendicular line is drawn by 10 microliter of a gun head, a serum-free culture medium is added and placed in a 5% CO2 and is incubated at 37 ℃, finally, phases are collected under an inverted microscope (100 ×) at 0 h, 12 h and 24 h respectively, Image J software is adopted to calculate the scratch distance on the images, and the calculation formula is as follows, namely, the mobility (%) = (distance measured by 0 h-distance measured at preset time)/distance measured by 0 h × 100% and the experiments are repeated for 3 times.
4 circSPECC1 with tumor cell invasion (Transwell cell migration assay)
Collecting 3 groups of cells in logarithmic growth phase, and adjusting cell density to 2.5 × 105Selecting a Transwell chamber with the pore diameter of 0.8 mu m, placing the Transwell chamber in a 24-hole plate, adding 1 × 105 cells and 200 mu l of serum-free culture medium into the chamber, adding 600 mu l of complete culture medium into a lower chamber (the 24-hole plate), placing the lower chamber in an incubator, culturing for 36 h, taking out the Transwell chamber, wiping off the cells in the chamber by using a cotton swab, fixing the cells for 15 min by 4% paraformaldehyde, staining by using 0.1% crystal violet, rinsing 1-3 times by using PBS (phosphate buffer solution) until the background is white, observing the number of transmembrane cells under a microscope, randomly selecting 5 fixed visual fields (× 100) to photograph, evaluating the migration capacity of each group of cells, and repeating the experiment for 3 times.
5. The concentration of the circSPECC1 and the tumor cell apoptosis (annexin V-FITC/PI staining) is 1 × 10, the method of annexin V-FITC/PI is adopted to separate early apoptosis cells and late apoptosis cells, and the negative control and the sicircSPECC 1 cells in the logarithmic phase are prepared6Cell suspension in ml. Annexin V-FITC/PI kit was used to stain cells, protected from light for 35 min. Then, using BD Cell QuestPro software (BD Bio) by flow cytometrysciences) for data collection and analysis.
FIG. 1 shows the effect of circSPECC1 on the proliferation of human renal carcinoma cells OS-RC-2.group 3 OS-RC-2 cells were diluted to 1 × 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 450 nm 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 that the influence of circSPECC1 on the invasion ability of human renal carcinoma cells OS-RC-2. OS-RC-2 cells of group 2 in logarithmic growth phase were collected and the cell density was adjusted to 2.5 × 105Selecting Transwell cell with pore diameter of 0.8 μm, placing in 24-well plate, adding 1 × 10 to each cell5Adding 600 mu l of complete culture medium into each lower chamber (a 24-pore plate), placing the lower chamber in an incubator for 36 h, taking out a Transwell chamber, wiping off cells in the chamber by using a cotton swab, fixing the cells in the chamber by 4% paraformaldehyde for 15 min, then staining by using 0.1% crystal violet, rinsing by using PBS for 1-3 times until the background is white, finally observing the number of transmembrane cells under a microscope, randomly selecting 5 fixed visual fields (× 100) for photographing, and evaluating the migration capacity of each group of cells, repeating the experiment for 3 times, observing that the migration number of the cells in the 48h and siSPECC1 groups is obviously lower than that of the siR-NC group, and indicating that the invasion capacity of tumor cells can be inhibited after the circSPECC1 is inhibited.
Note: SiR-NC is negative siRNA control; SiSPECC1 is siRNA of circSPECC1
FIG. 3 inhibition of circSPECC1 induces apoptosis in human renal carcinoma cells OS-RC-2. As for circSPECC1 and tumor cell apoptosis (Annexin V-FITC/PI staining), Annexin V is adoptedSeparation of early apoptotic cells and late apoptotic cells by FITC/PI method A negative control and si circSPECC1 cells were prepared at a concentration of 1 × 106Cell 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 |
| 2 | miR-615 | Micro RNA-615 |
| 3 | HIP1 | Huntington protein binding protein 1 |
| 4 | Caspase 9 | Apoptotic protease 9 |
| 5 | siR-NC | Small interfering 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) |
| 14 | DNA | Deoxyribonucleic acid |
| 15 | RNA | Ribonucleic acid |
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 human kidney cancer 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 human kidney cancer, 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 human kidney cancer according to claim 1 in the manufacture of a medicament for the treatment of human kidney cancer.
3. Use of circSPECC1 for the treatment of human renal cancer according to claim 1, in the preparation of a medicament for promoting apoptosis of human renal cancer cell OS-RC-2 and inhibiting the ability to decrease invasion of human renal cancer cell OS-RC-2.
4. The use of circSPECC1 for the treatment of human renal carcinoma according to claim 1, for the targeted regulation of HIP1 and Caspase9 gene expression through 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 human renal cancer.
6. The experimental method of circSPECC1 for the treatment of human kidney cancer according to claim 1, comprising the steps of;
expression of circSPECC1 in human Kidney cancer (RNA binding protein immunoprecipitation assay, RIP)
Receive 2 × 107OS-RC-2 cells, treated according to the protocol of Magna RIPTM RNA-Binding protein immunopropractination Kit from Millipore; the usage amount of the rabbit anti-human HuR antibody and the normal rabbit IgG antibody is 5 mu g, and the cell lysate and the antibody are incubated overnight at 4 ℃ in a rotating way; 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)
Experimental groups 3, blank control (Ctrl), negative control (siR-NC), si circSPECC1 (inhibiting circSPECC1 action), sequence GCCAAGGGGCCTTTACAACAA, and 3 groups of OS-RC-2 cells diluted to 1 × 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 for 10 min at a low speed, dissolving residual formazan crystals, detecting an absorption value of 450 nm by using an enzyme labeling instrument, and drawing a cell proliferation curve; the experiment was repeated 3 times;
CircSPECC1 and tumor cell invasion (Transwell cell migration assay)
Collecting 3 groups of cells in logarithmic growth phase, and adjusting cell density to 2.5 × 105The method comprises the steps of selecting a Transwell chamber with the aperture of 0.8 mu m, placing the Transwell chamber into a 24-hole plate, adding 1 × 105 cells and 200 mu l of serum-free culture medium into the chamber respectively, adding 600 mu l of complete culture medium into a lower chamber (the 24-hole plate) respectively, placing the lower chamber in an incubator for culturing for 36 hours, taking out the Transwell chamber, wiping off the cells in the chamber by using a cotton swab, fixing the cells for 15 minutes by using 4% paraformaldehyde, dyeing the cells by using 0.1% crystal violet, rinsing the cells for 1-3 times by using PBS until the background is white, observing the number of transmembrane cells under a microscope, randomly selecting 5 fixed visual fields (× 100) for photographing, evaluating the migration capacity of each group of cells, and repeating the experiment for 3 times;
the method for separating early apoptosis cell and late apoptosis cell of circSPECC1 and tumor cell apoptosis (Annexin V-FITC/PI staining) adopts Annexin V-FITC/PI method, and negative control and si circSPECC1 cell in logarithmic phase are prepared to have concentration of 1 × 106A 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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022111637A1 (en) * | 2020-11-27 | 2022-06-02 | 中国科学院分子细胞科学卓越创新中心 | Nucleic acid molecule binding to yb-1 protein |
| CN112980953A (en) * | 2021-02-08 | 2021-06-18 | 浙江大学 | Clear cell carcinoma of kidney circRNA biomarker, screening method and diagnostic kit |
| CN112980953B (en) * | 2021-02-08 | 2022-07-08 | 浙江大学 | Clear cell carcinoma of kidney circRNA biomarker, screening method and diagnostic kit |
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