CN113862365A - Application of lncRNA LINC00113 as a marker in the preparation of renal cancer detection reagents or detection kits - Google Patents
Application of lncRNA LINC00113 as a marker in the preparation of renal cancer detection reagents or detection kits Download PDFInfo
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Abstract
本发明涉及生物医药领域，特别涉及lncRNA LINC00113作为标志物在制备肾癌检测试剂或检测试剂盒中的应用。本发明实验结果表明：LINC00113在肾癌患者中表达下调，通过检测LINC00113的表达，判断受试者是否有患肾癌的风险，通过比较LINC00113的表达量，可以评估提示肾癌患者的远期预后和生存率，因此LINC00113可作为肾癌临床诊断评估的重要指标。The invention relates to the field of biomedicine, in particular to the application of lncRNA LINC00113 as a marker in the preparation of renal cancer detection reagents or detection kits. The experimental results of the present invention show that the expression of LINC00113 is down-regulated in renal cancer patients. By detecting the expression of LINC00113, it can be judged whether the subject has the risk of suffering from renal cancer. By comparing the expression of LINC00113, the long-term prognosis of renal cancer patients can be evaluated and prompted and survival rate, so LINC00113 can be used as an important indicator for clinical diagnosis and evaluation of renal cancer.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of lncRNA LINC00113 as a marker in preparation of a kidney cancer detection reagent or a detection kit.
Background
The incidence of Renal Cell Carcinoma (RCC) has been on the rising year-by-year trend in asia, the united states and europe, reaching approximately 3% annually, and causing approximately 90,000 deaths worldwide each year. At the time of initial diagnosis, 30% of patients have metastases and more than 30% eventually develop metastatic renal cell carcinoma (mRCC). For renal cell carcinoma that has metastasized, available therapies are often ineffective at reducing tumor growth or achieving remission with remission rates of 15% to 25%, so overall survival of mRCC patients is less than 1 year. Thus, early detection of metastatic renal cell carcinoma may improve patient prognosis. However, unlike gastrointestinal tumors, there is currently no biomarker for early diagnosis and monitoring of renal cell carcinoma. In order to design a therapeutic strategy with higher efficacy, it is crucial to decipher the molecular mechanisms involved in the development and progression of renal cell carcinoma.
High-throughput sequencing technology, also known as deep sequencing technology, has been emerging for over ten years and is widely used in the fields of basic research and clinical research. The technology has high sequencing flux and high sensitivity, can detect the expression of genes with low expression quantity, and can identify new transcription products.
Non-coding RNA (ncRNA) is RNA without protein coding properties, but since its discovery ncRNA plays a key role in vital activities and pathological processes. Recent researches show that lncRNA, miRNAs, mRNAs and proteins form a large and complex regulation network and participate in the development of various cell life processes and human diseases. At present, a great amount of lncRNA is reported to participate in the occurrence and development process of tumors, and the biological characteristics of the lncRNA can be used as a biomarker for tumor diagnosis and prognosis and a target point for accurate treatment. Although lncRNA has been studied widely in the past, the significance of lncRNA in molecular diagnosis of renal cancer has not been clarified. Therefore, there is an urgent need to find new lncRNAs and investigate their biological effects on kidney cancer to obtain potential therapeutic targets for diagnosis and gene therapy of gastric cancer.
Disclosure of Invention
In view of the above, aiming at the defects in the prior art, the lncRNA biomarker related to renal cell carcinoma is screened through a gene chip, lncRNA00113 with obvious differential expression is selected, the expression difference is detected in different stages of renal cancer tissues, and the expression level of the lncRNA is detected to judge whether a subject has renal cancer stages and prognosis, so as to provide support for clinically judging whether renal cancer is transferred.
In order to achieve the above object, the present invention provides the following technical solutions:
in a first aspect, the invention provides an application of lncRNA LINC00113 as a marker in preparation of a renal cancer detection reagent or a detection kit; the nucleotide sequence of the lncRNA LINC00113 is shown as SEQ ID No. 1.
In some embodiments of the invention, the lncRNA LINC00113 expression is significantly down-regulated.
In some embodiments of the invention, when the lncRNA LINC00113 expression is significantly down-regulated, the renal cancer is likely to occur or the renal cancer is less advanced and later, and the survival rate is low.
In a second aspect, the invention also provides a kidney cancer detection primer, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.2 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 3.
In a third aspect, the invention also provides a kidney cancer detection reagent, which comprises the kidney cancer detection primer and an acceptable auxiliary agent.
In some embodiments of the invention, the primer for amplifying the GAPDH reference gene is further included, and the primer comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.4 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 5.
In a fourth aspect, the present invention also provides a kidney cancer detection kit characterized by comprising the kidney cancer detection reagent according to claim 5 or 6.
In some embodiments of the invention, enzymes and reagents commonly used in qRT-PCR and/or reverse transcription reactions are also included, preferably reverse transcriptase, buffers, dNTPs, DEPC water and Taq enzyme.
The experimental results of the invention show that: the expression of LINC00113 is down-regulated in a renal cancer patient, whether a subject is at risk of suffering from the renal cancer is judged by detecting the expression of LINC00113, and the long-term prognosis and survival rate of the renal cancer patient can be evaluated and prompted by comparing the expression amount of LINC00113, so that LINC00113 can be used as an important index for clinical diagnosis and evaluation of the renal cancer.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Figure 1 shows a heatmap analysis of lncRNA differentially expressed in normal and renal cancer groups by high throughput sequencing;
FIG. 2 shows positional information of LINC00113 in a chromosome;
FIG. 3 shows qRT-qPCR validation of the difference in expression of LINC00113 in the jaw normal group of renal cancer tissues;
FIG. 4 shows the overall survival analysis curve for renal cancer patients with high and low expression of LINC 00113;
FIG. 5 shows disease-free survival analysis curves for renal cancer patients with high and low expression of LINC 00113.
Detailed Description
The invention discloses application of lncRNA LINC00113 as a marker in preparation of a kidney cancer detection reagent or a detection kit, and a person skilled in the art can realize the detection by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a non-coding RNA marker, which is LINC00113, and the nucleotide sequence of the marker is shown in SEQ ID No. 1. When LINC00113 expression levels are significantly down-regulated compared to normal tissue, the patient is indicated for risk of renal cancer. Lower expression of LINC00113 suggests that the renal cancer of the patient has poor long-term prognosis and low survival rate.
As a preferred embodiment of the present invention, the marker is a marker for diagnosis of kidney cancer.
The invention also provides a reagent for verifying the marker, wherein the reagent comprises a primer for specifically amplifying LINC00113, and the nucleotide sequence is shown as SEQ ID No. 2.
The invention also provides a kit for detecting the marker, which comprises a primer for amplifying the GAPDH reference gene, a primer for reverse transcription PCR and real-time quantitative PCR, a nucleotide sequence shown as SEQ ID No.3, enzymes and reagents commonly used for qRT-PCR and reverse transcription reaction, such as reverse transcriptase, buffer solution, dNTPs, DEPC water and Taq enzyme.
The expression of LINC00113 is reduced in a renal cancer patient, whether a subject has the risk of suffering from the renal cancer is judged by detecting the expression of LINC00113, and the long-term prognosis and survival rate of the renal cancer patient can be evaluated and prompted by comparing the expression amount of LINC00113, so that LINC00113 can be used as an important index for clinical diagnosis and evaluation of the renal cancer.
In the application of lncRNA LINC00113 as a marker in preparing a kidney cancer detection reagent or a detection kit, raw materials and reagents used in the method can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 chip screening of LINC00113
1. 4 cases of clinically paired kidney cancer tissues and 4 cases of clinically paired normal tissues are selected
2. Using a chipAgilent Human lncRNA(4*180K，042818)To finish8Individual samples were tested and analyzed (fig. 1).
3. And (4) screening candidate lncRNA. The most pronounced downregulation of LINC00113 was selected to study its role in renal cancer.
Example 2 expression of LINC00113 in renal cancer group
1. The position of LINC00113 in the chromosome is shown in FIG. 2, and the sequence information is shown in SEQ ID No. 1.
2. LINC00113qRT-PCR primers are designed, and the upstream and downstream primers are SEQ ID No. 2.
3. 90 normal tissues and 90 cancer tissues collected in our hospital are stored in liquid nitrogen to avoid RNA degradation. All the collected samples were confirmed by pathological examination.
4. Extraction of tissue Total RNA
Grinding a mortar used for tissue, wrapping products such as a pestle and the like with tinfoil paper, baking for 4 hours at 200 ℃, and cooling for later use;
precooling the mortar and pestle by using liquid nitrogen before grinding, taking the collected tissues out of the liquid nitrogen, immediately putting the tissues into the mortar, adding the liquid nitrogen for repeatedly grinding until the tissues become powder, adding 1ml of RNAiso Plus to cover all the powder, and transferring the powder into a 1.5ml of EP tube after the RNAiso Plus is melted;
③ adding 200 mul of chloroform after cracking by Trizol method, oscillating and mixing evenly, standing for 2min at room temperature, 12000g, centrifuging for 15min at 4 ℃;
transferring the supernatant to a new 1.5ml EP tube, adding isopropanol with the same volume, reversing, uniformly mixing, standing at room temperature for 10min, 12000g, centrifuging at 4 ℃ for 10min, and removing the supernatant;
fifthly, adding 1ml of 75% ethanol into the EP tube of the fourth step, reversing and washing the RNA precipitate, centrifuging for 5min at 4 ℃ at 12000g, and removing the supernatant;
fifthly, performing instantaneous centrifugation, namely placing an EP tube, removing supernatant, drying at room temperature until precipitate is transparent, and adding 30 mu l of DEPC water;
seventhly, identifying the quality of the total RNA of the tissue through electrophoresis, wherein A260/A280 is 1.8-2.0, and 28S: when 18S is 1.8 to 2.0, RNA quality is good and it can be used for subsequent studies. The reagents and consumables used in the whole process are RNase-free.
5. Reverse transcription
The procedure was performed according to the instruction of 037a kit of TaKaRa. The reverse transcription system comprises the following specific steps of 20 mu l:
1. mu.g of RNA in (i) above was prepared in Microtube, and the volume was adjusted to 13. mu.l and mix 7. mu.l in DEPC water, and 20. mu.l in total.
② mixing and then centrifuging instantly;
placing the Microtube in the PCR instrument, and sequentially carrying out three temperature reactions: 15min at 37 ℃; keeping the temperature at 85 ℃ for 15 s; keeping the temperature at 4 ℃ for cooling, and directly using the obtained cDNA solution for PCR amplification, qRT-PCR and the like.
6、qRT-PCR
Real-time fluorescent quantitative PCR was performed using SYBR-Green dye method (Real-time qPCR Master Mix, Takara, Dalian) to detect the expression level of LINC00113 in normal and renal cancer tissues. The specific operation is as follows:
firstly, the cDNA is obtained in the steps, and the reaction system is as follows: SYBR 10. mu.l, Forward Primer (10. mu.M) 0.5. mu.l, Reverse Primer (10. mu.M), 0.5. mu.l, cDNA 2. mu.l, water was added to a total volume of 20. mu.l. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; 95 ℃ for 5 s; 60 ℃ for 20 s; 72 ℃ for 10 s; 40 reaction cycles.
The model of a real-time fluorescent quantitative PCR instrument is Roche RT-PCRLC480 II;
by using
Calculating the expression difference:
the specific calculation method of qPCR: and obtaining CT values of the lncRNA and the internal reference GAPDH through real-time fluorescence quantification QPCR Roche RT-PCR LC480 II, calculating an average value, subtracting the CT value of the GAPDH from the CT value of the lncRNA to obtain a delta Ct value, subtracting the delta Ct value of a paracancer tissue from the delta Ct value of the cancer tissue to obtain the delta Ct value, and calculating a value of 2-delta Ct.
7. The statistical method comprises the following steps: statistical analysis is carried out by adopting SPSS17.0 statistical software, all data are expressed in the form of mean plus or minus standard deviation, comparison among groups is tested by adopting LSD-t and Dunnettt-test method, P <0.05 is statistical significance, and difference is obvious.
Effect analysis
As shown in fig. 3, in 90 cases of kidney cancer and paracancerous normal tissue, the expression of LINC00113 was significantly reduced in kidney cancer tissue (P ═ 0.0004), indicating that LINC00113 is a potential target for diagnosing kidney cancer.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488726A (en) * | 2017-09-25 | 2017-12-19 | 中国人民解放军南京军区南京总医院 | A kind of kidney prognosis evaluation biomarker and its detection reagent and application |
| CN111701025A (en) * | 2020-06-16 | 2020-09-25 | 宜兴市人民医院 | Drugs for the treatment of renal cell carcinoma and their applications |
| CN112646885A (en) * | 2020-12-17 | 2021-04-13 | 核工业总医院 | Renal cell carcinoma miRNA molecular marker and application thereof |
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- 2021-11-01 CN CN202111282896.3A patent/CN113862365A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488726A (en) * | 2017-09-25 | 2017-12-19 | 中国人民解放军南京军区南京总医院 | A kind of kidney prognosis evaluation biomarker and its detection reagent and application |
| CN111701025A (en) * | 2020-06-16 | 2020-09-25 | 宜兴市人民医院 | Drugs for the treatment of renal cell carcinoma and their applications |
| CN112646885A (en) * | 2020-12-17 | 2021-04-13 | 核工业总医院 | Renal cell carcinoma miRNA molecular marker and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| TIAN,Z.H.等: "Systematic identification of key genes and pathways in clear cell renal cell carcinoma on bioinformatics analysis", ANN.TRANSL.MED. * |
| YAO,X.L.等: "LncRNA ENST00113 promotes proliferation, survival, and migration by activating PI3K/Akt/mTOR signaling pathway in atherosclerosis", MEDICINE * |
| 佚名: "Homo sapiens long intergenic non-protein coding RNA 113 (LINC00113), long non-coding RNA,登录号：NR_024357.2", GENBANK * |
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Application publication date: 20211231 |