CN113322320A - Long non-coding RNA and application thereof in diagnosis and treatment of glioma - Google Patents
Long non-coding RNA and application thereof in diagnosis and treatment of glioma Download PDFInfo
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- CN113322320A CN113322320A CN202110586864.6A CN202110586864A CN113322320A CN 113322320 A CN113322320 A CN 113322320A CN 202110586864 A CN202110586864 A CN 202110586864A CN 113322320 A CN113322320 A CN 113322320A
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a long non-coding RNA and application thereof in diagnosis and treatment of glioma, wherein the long non-coding RNA is lncRNA LINC00689, and the nucleotide sequence of the long non-coding RNA is shown in Seq ID No. 1. The invention provides a molecular marker lncRNA LINC00689 for diagnosing glioma, wherein the high expression of lncRNA LINC00689 in the tumor of a patient with glioma is found through detection, so that the early diagnosis of glioma can be realized by detecting the expression of lncRNA LINC00689 in the patient with glioma, and the survival rate of the patient with glioma is improved.
Description
The technical field is as follows:
the invention relates to the field of genetic engineering, in particular to long non-coding RNA and application thereof in diagnosing and treating glioma.
Background art:
gliomas derived from neuroepithelia are the most common primary brain tumors and remain the leading cause of cancer-related deaths worldwide. Gliomas are classified into low-grade (WHO I-II) and high-grade (WHO III-IV) according to the severity of the tumor cells. Despite recent decades of progress in therapeutic approaches, the clinical efficacy of gliomas, and particularly glioblastoma, remains poor. Therefore, there is an urgent need to investigate molecular mechanisms involved in glioma pathogenesis, which may help to improve patient prognosis.
Only a small number of genes in the human genome encode proteins, and 98% of the genes are transcribed to form RNA, which cannot further eventually form proteins, and the RNA transcribed from these genes is called non-coding RNA. Previously, one considered these non-coding RNAs as transcription noises, and had no actual specific function. However, recent studies have shown that these non-coding RNAs have gene regulatory functions. The long non-coding RNA refers to non-coding RNA with the length of more than 200 basic groups, and is widely present in various tissues, and can participate in various life function regulation and control ways such as cell development and metabolism, and the like, including gene recombination, gene imprinting, cell cycle regulation, chromatin modification, transcription, translation, mRNA degradation and the like. Existing studies have shown that differences in expression of long non-coding RNAs are often closely linked to the development and progression of gliomas and post-operative recovery in patients.
Some abnormally expressed long-chain non-coding RNA can be used as a gene diagnosis target of glioma, and can be used as an early diagnosis of glioma patients and provide a new basis for judging cancer types. Therefore, the search for new glioma-related LncRNA gene markers is of great significance for realizing early diagnosis and treatment of glioma patients. With advances in technology, there is increasing evidence to support the involvement of long non-coding rnas (lncrnas) in the development and progression of gliomas. Recent studies have found that the long noncoding gene lncRNA00689(LINC00689) is associated with obesity and is involved in eukaryotic gene expression. However, it is not known whether LINC00689 plays a critical role in the progression of gliomas.
The invention content is as follows:
the technical problem to be solved by the invention is to provide a gene marker related to glioma occurrence and development, realize early diagnosis of a glioma patient by detecting the expression level of lncRNA LINC00689, and realize targeted treatment of the glioma patient by utilizing shRNA to target and interfere lncRNA LINC 00689.
The technical scheme of the invention is to provide a long non-coding RNA for diagnosing the prognosis condition of glioma or serving as a target of a drug for treating glioma, wherein the long non-coding RNA is lncRNA LINC00689, and the nucleotide sequence of the long non-coding RNA is shown in Seq ID No. 1. Early diagnosis of a patient with glioma is realized by detecting the expression level of IncRNA LINC00689, and meanwhile, targeted treatment of the patient with glioma is realized by utilizing shRNA to target and interfere with IncRNA LINC 00689.
Preferably, the application of the long non-coding RNA in preparing a kit or a chip for diagnosing glioma is also provided.
Preferably, the application of the long non-coding RNA in preparing a real-time fluorescent quantitative PCR kit for diagnosing glioma is also provided.
And the application of the long non-coding RNA in the preparation of drugs or drug compositions for treating glioma.
The invention also provides an application of the marker for identifying the long non-coding RNA in preparing a diagnostic product for judging the treatment prognosis of glioma, wherein the marker comprises but is not limited to:
(1) a primer combined with long noncoding lncRNA LINC00689 or a fluorescence-labeled primer combined with long noncoding lncRNA LINC 00689;
(2) a small molecule compound or biomacromolecule which is combined with long noncoding lncRNA LINC 00689; wherein the biological macromolecules include, but are not limited to: an antibody or functional fragment of an antibody, a fluorescently labeled antibody or functional fragment of an antibody, an RNA-binding protein or functional fragment thereof, a fluorescently labeled RNA-binding protein or functional fragment thereof.
The forward sequence of the primer is shown in Seq ID No.2, and the reverse sequence of the primer is shown in Seq ID No. 3.
The invention also discloses a kit or a chip for judging the treatment prognosis condition of glioma, and the reagent or the kit comprises the marker.
The invention also provides application of the inhibitor for inhibiting the long non-coding RNA in preparation of a medicine for diagnosing or treating glioma, wherein the inhibitor comprises shRNA for inhibiting the expression of the long non-coding lncRNA LINC00689 gene. Alternatively, it may be a small molecule compound that inhibits the long non-coding RNA; or a biological macromolecule that inhibits the long non-coding RNA, wherein the biological macromolecule includes, but is not limited to: an antibody or functional fragment of an antibody, an enzyme with high substrate specificity or a functional fragment thereof.
In addition, the invention also discloses a medicine or a medicine composition containing the inhibitor and used for treating glioma.
And the application of the medicine or the medicine composition in treating glioma.
The pharmaceutical composition further comprises other pharmaceutically acceptable carriers and/or auxiliary materials compatible with the shRNA, wherein the pharmaceutically acceptable carriers and/or the auxiliary materials comprise but are not limited to adhesives, diluents, surfactants and fillers.
Preferably, the route of use of the pharmaceutical composition includes, but is not limited to, intravenous, oral, intramuscular, and subcutaneous.
Compared with the prior art, the invention has the following advantages after adopting the scheme:
the invention provides a molecular marker lncRNA LINC00689 for diagnosing glioma, wherein the high expression of lncRNA LINC00689 in the tumor of a patient with glioma is found through detection, so that the early diagnosis of glioma can be realized by detecting the expression of lncRNA LINC00689 in the patient with glioma, and the survival rate of the patient with glioma is improved;
secondly, the invention provides an application of the shRNA of lncRNA LINC00689 in preparing a glioma drug composition, and the drug composition can be used as a new therapeutic drug for treating glioma patients, and has important clinical value and application prospect.
Description of the drawings:
FIG. 1, LINC00689 expression profile in glioma tissues and paraneoplastic tissues.
FIG. 2, ROC curves for LINC00689 expression in glioma tissues and paraneoplastic tissues.
FIG. 3, effect of LINC00689 transfected shRNA on the expression of LINC00689 in glioma U251 and U87 cells.
Figure 4, interference with LINC00689 effect on glioma U251 and U87 cell proliferation.
FIG. 5, LINC00689 regulates the miR-338-3p/PKM2 pathway in glioma.
The specific implementation mode is as follows:
the invention will be further described with respect to specific embodiments in conjunction with the following drawings:
it should be noted that in the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the invention may be practiced otherwise than as specifically described herein. Accordingly, the present invention is not limited to the specific embodiments disclosed in the following description. Techniques not described in detail correspond to standard methods well known to the person skilled in the art.
Example 1
Detecting the expression condition of LINC00689 in glioma tissues and normal brain tissues
Study object
56 cases of glioma tissues and adjacent non-tumor tissues are selected for experiments, the tissues are from people hospitals in Zhejiang province, all tissue specimens are examined and approved by ethical committees of hospitals, and patients from all tissue specimens sign informed consent. The glioma tissue is proved to be glioma through pathological detection (when the glioma tissue is removed, the patient has not been treated by chemotherapy, radiotherapy and the like).
Tissue RNA extraction
(1) Taking out the tissue specimen from a refrigerator at minus 80 ℃, taking about 50mg of the tissue specimen to put into a mortar, adding a small amount of liquid nitrogen, rapidly grinding, adding a small amount of liquid nitrogen again to grind after the tissue becomes soft, and repeating for three times;
(2) adding 1ml Trizol, homogenizing for 2 minutes by using an automatic homogenizer, transferring the tissue specimen into a centrifuge tube after homogenizing, and standing for 5 minutes at room temperature;
(3) centrifuging at 12000rpm for 5min, collecting supernatant, and removing precipitate;
(4) adding 0.2mL of chloroform, shaking and uniformly mixing, and standing at room temperature for 15 minutes;
(5) centrifuging at 12000rpm at 4 deg.C for 15 min;
(6) transferring the supernatant into another centrifuge tube, adding equal volume of precooled isopropanol, mixing uniformly, and standing for 8 minutes on ice;
(7) centrifuging at 12000rpm at 4 deg.C for 10 min, removing supernatant, and retaining precipitate;
(8) adding 1ml of 75% ethanol into the precipitate, gently shaking the centrifugal tube, and suspending the precipitate;
(9) centrifuging at 8000rpm at 4 deg.C for 5min, and carefully removing supernatant with a pipette;
(10) the RNA was dried by standing at room temperature for 10 minutes, and then the precipitate was dissolved in 50. mu.l of DEPC water;
(11) the purity and concentration of total RNA from the tissue was determined using a micro uv spectrophotometer.
Reverse transcription to obtain cDNA
The procedure was performed according to the instructions of the reverse transcription kit of TaKaRa.
Reaction conditions are as follows: 15 minutes at 37 ℃, 5 seconds at 85 ℃ and 4 ℃.
Fluorescent quantitative PCR detection
Amount of reagent used
TB Green Premix Ex Taq II(Tli RNaseH Plus)(2×)12.5μl
Upstream primer (10. mu.M) 1. mu.l
Downstream primer (10. mu.M) 1. mu.l
Sterilized Water 8.5. mu.l
Total volume 25. mu.l
qPCR was performed in an Applied Biosystems 7500 sequence detection system, and GAPDH was used as an internal control.
Reaction conditions are as follows: at 95 ℃ for 5min, at 95 ℃ for 5s, at 60 ℃ for 30s, and circulating for 40 cycles; 60s at 75 ℃. And (3) processing real-time quantitative PCR data by adopting a 2-delta-delta Ct method, and calculating the expression change of LINC 00689.
Primer sequences
LINC00689 primer is Forward 5'-AAGAAGGTGAACGCTGGAGA-3' (Seq ID No.2)
Reverse 5’-CTGGGAAGTGTAACGGCAAT-3’(Seq ID NO.3)
The GAPDH primer sequence is Forward 5'-AGCCACATCGCTCAGACAC-3' (SEeq NO:4)
Reverse 5’-GCCCAATACGACCAAATCC-3’(Seq NO:5)
Results
The results of fluorescent quantitative PCR (FIG. 1) show that the expression of LINC00689 in glioma tissues is significantly higher than that in adjacent non-tumour tissues compared to paraneoplastic tissues, the difference is statistically significant (P <0.05), and ROC curves are drawn for the expression level of LINC00689, and the results are shown in FIG. 2. We found that overall survival of glioma patients expressing high levels of LINC00689 was significantly reduced (P ═ 0.0019), suggesting that LINC00689 may be a potential indicator of poor prognosis.
Example 2
Knock-down of LINC00689 Gene
shRNA design
Slow vector mediated LINC00689 shRNA (shLINC00689) and non-targeting (NT) shRNA were obtained from RiboBio (Guangzhou, China).
Cell culture
Human glioma cell lines (U87 and U251) were cultured in high-glucose DMEM medium (10% fetal bovine serum) under the following conditions: the cells were incubated at 37 ℃ in a 5% CO2 incubator.
shRNA transfection
(1) The day before transfection, 2X 105 HepG2 cells were inoculated onto 6-well cell culture plates, cultured at 37 ℃ for 24 hours in a 5% CO2 incubator, and then transfected according to the instructions;
(2) the experiment groups are divided into a control group, an sh-NC group and an sh-RNA group.
Fluorescent quantitative PCR detection of knocked-down LINC00689 gene expression
(1) Total RNA extraction from cells
Adding 500 mu L Trizol into each hole of a 6-hole cell culture plate, standing at room temperature for 5 minutes, fully cracking, and transferring to a centrifuge tube;
the remaining steps were the same as those of the tissue RNA extraction step of example 1 (the respective amounts added were scaled down).
(2) The reverse transcription reaction procedure and conditions were the same as in example 1.
(3) The fluorescent quantitative PCR detection procedure and conditions were the same as in example 1.
Results
As can be seen from FIG. 3, the LINC00689 expression level of the sh-RNA is remarkably reduced (P <0.05) compared with that of the control group, which indicates that the synthetic sh-RNA designed by the invention can inhibit the expression of LINC 00689.
Example 3
Cell proliferation assay
Transfected cells were digested, centrifuged and resuspended, and after counting 100. mu.l, 3X 103 cells were added to each well of a 96-well plate. And (3) continuously culturing for 0h, 24h, 48h, 72h and 96h in a complete culture medium of 10% in an incubator containing 5% CO2 at 37 ℃, adding a CCK-8 solution with the volume of 10% into the holes, and incubating for 2h in a dark place. The absorbance was measured at a wavelength of 450nm under a microplate reader and subjected to statistical analysis. And drawing a growth curve.
Results
As can be seen from FIG. 4, after the experiment group is transfected with sh-RNA, compared with the sh-NC group, the cell proliferation is inhibited, the proliferation rate is obviously reduced, and at 72h, the difference has statistical significance, which indicates that the proliferation of glioma cells can be effectively inhibited by knocking down LINC00689 through sh-RNA.
Example 3
Western Blot to detect the influence of interference LINC00689 on PKM2
(1) Inoculating two 2X 105 cells on a 6-hole culture plate one day before transfection, culturing for 24h at 37 ℃ in a 5% CO2 cell culture box, and then transfecting the cells according to an instruction, wherein the experiment is divided into an sh-NC group and an sh-RNA group;
(2) after transfection for 48h, after washing cells with PBS, 100. mu.l of RIPA cell lysate was added to each well;
(3) after full cracking, transferring the mixture into a centrifuge tube, centrifuging the mixture at 10000g/min and 4 ℃ for 10 minutes, and taking the supernatant to a new centrifuge tube;
(4) sucking 2 μ l, quantifying protein by BCA method, adding 5 Xloading buffer solution, and boiling for 10 min;
(5) centrifuging at 12000r/min for 5 minutes to obtain a prepared protein sample;
(6) assembling an electrophoresis tank, and preparing 5% of upper layer glue and 12% of lower layer glue;
(7) adding 20 mu g of protein sample and a protein Marker indicator into each hole;
(8) adding a newly configured electrophoresis buffer solution into the electrophoresis tank, starting electrophoresis under the conditions that the voltage of the upper layer gel is 80v and the voltage of the lower layer gel is 120v, and finishing electrophoresis when the bromophenol blue indicator moves to the bottom of the gel;
(9) assembling a film transferring clamp, loading the film transferring clamp into a transferring groove filled with transferring liquid, and transferring the film for 1.5 hours at 200 mA;
(10) transferring the PVDF membrane after membrane conversion into 5% skimmed milk powder, sealing for 1h at room temperature;
(11) washing the membrane for 3 times by TBST, incubating beta-actin, C-myc and Cyclin-D1 primary antibody diluent for 5min each time, and incubating overnight by shaking at 4 ℃;
(12) washing the membrane for 3 times by TBST, incubating a secondary antibody for 5min each time, incubating at room temperature, and incubating for 1h by a shaking table;
(13) in a dark room, the luminescent solution is quickly dripped on the PVDF membrane for developing.
The results of the experiment are shown in fig. 5, LINC00689 silencing significantly reduced the level of PKM2 protein in U251 and U87 cells. While miR-338-3p knock-down reversed LINC00689 silencing to induce PKM2 inhibition in glioma cells, and the two were summed up, our results showed that LINC00689 acts as a cerRNA to interact with miR-338-3p and enhances PKM2 expression in glioma.
In conclusion, the sh-RNA of LINC00689 provided by the invention can be used for preparing a pharmaceutical composition for treating glioma.
The foregoing is illustrative of the preferred embodiments of the present invention only and is not to be construed as limiting the claims. All the equivalent structures or equivalent process changes made by the description of the invention are included in the scope of the patent protection of the invention.
Sequence listing
<110> Hangzhou college of medicine
Zhejiang province people hospital
First subsidiary Hospital of medical college of Sian traffic university
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<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcccaatacg accaaatcc 19
Claims (10)
1. A long non-coding RNA for diagnosing glioma prognosis or serving as a drug target for treating glioma is characterized in that the long non-coding RNA is lncRNA LINC00689, and the nucleotide sequence of the long non-coding RNA is shown in Seq ID No. 1.
2. Use of the long non-coding RNA of claim 1 in the preparation of a kit or chip for the diagnosis of glioma.
3. Use of the long non-coding RNA of claim 1 in the preparation of a real-time fluorescent quantitative PCR kit for the diagnosis of glioma.
4. Use of a marker for identifying the long non-coding RNA of claim 1 in the preparation of a diagnostic product for prognosis of glioma treatment, wherein said marker includes but is not limited to:
(1) a primer combined with long noncoding lncRNA LINC00689 or a fluorescence-labeled primer combined with long noncoding lncRNA LINC 00689;
(2) a small molecule compound or biomacromolecule that binds long noncoding lncRNA LINC 00689.
5. The use according to claim 4, wherein the forward sequence of the primer is shown as Seq ID No.2 and the reverse sequence of the primer is shown as Seq ID No. 3.
6. A kit or chip for determining the prognosis of a glioma treatment comprising the marker of claim 4 or 5.
7. Use of an inhibitor of the long non-coding RNA of claim 1 in the manufacture of a medicament for diagnosing or treating glioma, wherein the inhibitor comprises shRNA that inhibits the expression of the long non-coding incrna LINC00689 gene.
8. A medicament or pharmaceutical composition for the treatment of glioma comprising the inhibitor of claim 7.
9. Use of the medicament or pharmaceutical composition of claim 8 for the treatment of glioma.
10. Use of the long non-coding RNA of claim 1 for the preparation of a medicament or pharmaceutical composition for the treatment of glioma.
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| CN108504737A (en) * | 2018-04-11 | 2018-09-07 | 中南大学湘雅医院 | A kind of application of long-chain non-coding RNA TRALR |
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| CN111424091A (en) * | 2020-04-20 | 2020-07-17 | 中国医学科学院北京协和医院 | Marker for differential diagnosis of benign and malignant thyroid follicular tumor and application thereof |
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| CN108504737A (en) * | 2018-04-11 | 2018-09-07 | 中南大学湘雅医院 | A kind of application of long-chain non-coding RNA TRALR |
| CN110872630A (en) * | 2019-12-12 | 2020-03-10 | 湖南中医药大学第一附属医院((中医临床研究所)) | Long-chain non-coding RNA for glioma diagnosis and application thereof |
| CN111424091A (en) * | 2020-04-20 | 2020-07-17 | 中国医学科学院北京协和医院 | Marker for differential diagnosis of benign and malignant thyroid follicular tumor and application thereof |
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