CN108192975B - Application of long-chain non-coding RNA LINC00559 as biomarker in preparation of lung squamous carcinoma prognosis detection preparation - Google Patents
Application of long-chain non-coding RNA LINC00559 as biomarker in preparation of lung squamous carcinoma prognosis detection preparation Download PDFInfo
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Abstract
The invention discloses application of a long-chain non-coding RNA LINC00559 as a biomarker in preparation of a lung squamous carcinoma prognosis detection preparation. The expression level of the long-chain non-coding RNA LINC00559 in the squamous cell lung carcinoma patient can be detected, a powerful molecular biological basis is provided for prognosis prediction of the squamous cell lung carcinoma patient, and the method has profound clinical significance and popularization.
Description
Technical Field
The invention belongs to the field of tumor molecular biology, and particularly relates to application of a long-chain non-coding RNA LINC00559 as a biomarker in preparation of a lung squamous cell carcinoma prognosis detection reagent.
Background
Squamous cell carcinoma of the lung, which is one of the most prominent pathological types of lung cancer, is derived from malignant changes of bronchial epithelial cells, and the number of cases accounts for about 30 percent of non-small cell lung cancer. The conventional treatment methods for squamous cell lung carcinoma mainly comprise surgical treatment, chemotherapy and molecular targeted therapy, the 5-year survival rate of patients is lower than 15%, and about 40 million people are killed worldwide each year. At present, there is no reference standard and no specific index for prognosis determination of squamous cell lung carcinoma patients, and thus the method is far not suitable for the requirement of prognosis determination of squamous cell lung carcinoma patients. Therefore, the determination of prognosis of squamous cell lung carcinoma patients so as to select the optimal treatment scheme and significantly improve the survival rate of patients becomes an important issue to be solved urgently in the field of thoracic surgery.
Long non-coding RNA (long non-coding RNA) is a general term for non-coding RNA with length more than 200 nt. Initial studies thought it to be "noise" of the transcriptome, but life sciences over the last decade found that they are involved in multiple biological regulatory processes. Researches show that the long-chain non-coding RNA can be used as a very important epigenetic regulation factor in human genome, and can regulate and control DNA methylation, histone modification or chromatin remodeling to silence or activate genes through epigenetic regulation, transcription regulation, post-transcriptional regulation and other mechanisms. In recent years, long-chain non-coding RNA has been paid more attention by researchers as an important regulatory molecule and its application value in clinical diagnosis, chemotherapy sensitivity, prognosis evaluation and the like. There are studies showing that HOTAIR expression is increased in both primary breast tumors and metastases, and the level of HOTAIR expression in primary tumors can be used to effectively predict cancer metastasis and death. The long-chain non-coding RNA PCA3 is highly overexpressed in prostate cancer, and the long-chain non-coding RNA is found in urine and is quite convenient for clinical detection. The long-chain non-coding RNA LALR1 accelerates the cell cycle process and promotes the hepatocyte proliferation by activating Wnt/beta-catenin signals. The drug target intervention long-chain non-coding RNA LALR1 is adopted to induce liver regeneration, and the drug target intervention long-chain non-coding RNA LALR1 is possibly beneficial to liver failure and liver transplantation treatment. It follows that long non-coding RNAs have great potential as molecular markers for diagnosis, prognosis and therapy.
At present, there is no reference standard and no specific index for prognosis determination of squamous cell lung carcinoma patients, and thus the method is far not suitable for the requirement of prognosis determination of squamous cell lung carcinoma patients. Therefore, the determination of prognosis of squamous cell lung carcinoma patients so as to select the optimal treatment scheme and significantly improve the survival rate of patients becomes an important issue to be solved urgently in the field of thoracic surgery.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of a long-chain non-coding RNA LINC00559 as a biomarker in preparation of a lung squamous cell carcinoma prognosis detection preparation.
Long non-coding RNA LINC00559 maps to chromosome 13q31.3, GeneBank accession no: NR-047489.1 (the sequence of the long-chain non-coding RNA LINC00559 is shown in SEQ NO: 1). In the previous research work, the inventor extracts RNA from lung squamous carcinoma tissues, carries out real-time fluorescence quantitative PCR analysis on the expression of the long-chain non-coding RNA LINC00559 after reverse transcription, and finds that: the expression of the long-chain non-coding RNA LINC00559 is up-regulated in lung squamous carcinoma tissues, and is remarkably different from the expression in normal human lung tissues (P <0.0001) (figure 1). And prognosis of patients with squamous cell lung carcinoma with different expression levels of long-chain non-coding RNA LINC00559 is obviously different, Kaplan-Meier survival analysis shows that the survival rate of patients with squamous cell lung carcinoma with high expression of long-chain non-coding RNA LINC00559 is obviously lower than that of patients with low expression (P0.0041) (figure 2), ratio risk model prognosis analysis shows that the risk rate of long-chain non-coding RNA LINC00559 is less than 1(HR 0.2414), which indicates that patients with low expression prognosis of patients with high expression of long-chain non-coding RNA LINC00559 have poor prognosis, and the high expression of long-chain non-coding RNA LINC00559 is an independent risk factor for predicting the prognosis risk of patients with squamous cell lung carcinoma. Therefore, the long-chain non-coding RNA LINC00559 can be used as a judgment standard for prognosis of squamous cell lung carcinoma, can be used as a biomarker for preparing a prognostic preparation for patients with squamous cell lung carcinoma, and further can provide a prognostic prediction kit for squamous cell lung carcinoma, which has high cost performance and is easy to popularize and apply.
The lung squamous carcinoma prognosis detection kit comprises a long-chain non-coding RNA LINC00559 real-time fluorescent quantitative PCR specific primer: a forward primer: 5'-GGCTGTGATATGAAAGTTGGCTC-3' (SEQ ID NO. 2); reverse primer: 5'-CAACTACATATCAAGCCAGGC-3' (SEQ ID NO. 3).
Preferably, the kit further comprises ACTB internal reference real-time fluorescence quantitative PCR specific primers: a forward primer: 5'-GAACCCCAAGGCCAACCGCGAG-3' (SEQ ID NO. 4); reverse primer: 5'-CAGTGGTACGGCCAGAGGCGTAC-3' (SEQ ID NO. 5).
The detection method of using the long-chain non-coding RNA LINC00559 in the prognosis of the squamous cell lung carcinoma patient comprises the following steps: (1) collecting lung squamous carcinoma tissues excised after the operation of an individual to be detected, and extracting total RNA; (2) using total RNA as template to make long chain non-RNAThe coding RNA LINC00559 is reversely transcribed into cDNA; (3) carrying out real-time fluorescent quantitative PCR amplification by using a long-chain non-coding RNA LINC00559 specific primer to obtain a relative expression amount of 2ΔΔCTWhen 2 is presentΔΔCT>2.92, the long-chain non-coding RNA LINC00559 is high-expression.
In conclusion, the detection preparation can be used for detecting the expression level of the long-chain non-coding RNA LINC00559 in the squamous cell lung carcinoma patients, provides a powerful molecular biological basis for prognosis prediction of the squamous cell lung carcinoma patients, and has profound clinical significance and popularization.
Drawings
FIG. 1 shows that real-time fluorescence quantitative PCR analysis is carried out on the expression difference of a long-chain non-coding RNA LINC00559 in squamous cell lung carcinoma tissues and normal lung tissues;
FIG. 2 is a graph showing the relationship between LINC00559 long non-coding RNA and the prognosis of 35 patients with squamous cell lung carcinoma.
Detailed Description
Example 1 preparation of Long non-coding RNA LINC00559 for Lung squamous cell carcinoma prognosis test kit (50 reactions)
50ml RNA stabilizing solution
2. Isopropanol 100ml
3. Chloroform 100ml
4.Trizol 50ml
5. 10ml of enzyme-free water
6.1 uM random reverse transcription primer 50ul
7.5 Xreverse transcription buffer 200ml
8.10mM deoxynucleotide triphosphate base 100ul
9.40U/. mu.l RNase inhibitor 500ul
10.200U/. mu.l MMLV reverse transcriptase 50ul
11.Premix Ex Taq 50ul
12.10. mu.M long-chain non-coding RNA LINC00559 real-time fluorescent quantitative PCR specific primer:
forward primer 5'-GGCTGTGATATGAAAGTTGGCTC-3' (SEQ ID NO.2),
reverse primer 5'-CAACTACATATCAAGCCAGGC-3' (SEQ ID NO.3),
13.10 μ M internal reference ACTB real-time fluorescent quantitative PCR specific primers:
the forward primer was 5'-GAACCCCAAGGCCAACCGCGAG-3' (SEQ ID NO.4),
the reverse primer was 5'-CAGTGGTACGGCCAGAGGCGTAC-3' (SEQ ID NO. 5).
Example 2 detection of Long non-coding RNA LINC00559 in Lung squamous carcinoma tissue
1. Preservation of squamous cell lung carcinoma tissue: collecting lung squamous carcinoma tissues to be detected, storing the lung squamous carcinoma tissues in a freezing storage tube filled with RNA stable solution, and storing the lung squamous carcinoma tissues in a refrigerator at the temperature of minus 80 ℃ for later use.
2. Extraction of RNA from tissues: taking a proper amount of specimen, adding liquid nitrogen into a mortar baked for 6-8h at 180 ℃, grinding the specimen into powder, adding 1ml of Trizol mortar specimen into the mortar, grinding the powder into liquid, transferring the liquid into a centrifuge tube, adding 200 mu l/ml of chloroform into the centrifuge tube, shaking the liquid for 15-30s by hand, standing the liquid on ice for 5min, and centrifuging the liquid for 15min at 12000g at 4 ℃; carefully taking the upper water phase into a new centrifugal tube, adding 0.5ml of precooled isopropanol, uniformly mixing, standing for 20min in a refrigerator at the temperature of-20 ℃, and centrifuging for 10min at the temperature of 4 ℃ at 12000 g; discarding supernatant, adding 1-2ml ethanol diluted with 75% ribozyme-free water, mixing, centrifuging at 4 deg.C for 5min at 7500g, discarding supernatant, drying at room temperature for 5-10min, and adding 10-20 μ l ribozyme-free water to dissolve RNA. The concentration and the quality of RNA are measured by spectrophotometry, the OD260/280 ratio is between 1.8 and 2.0, and the RNA is stored at the temperature of minus 80 ℃.
3. Long non-coding RNA LINC00559 transcription: a reverse transcription kit from Thermo was used. The system for 20. mu.L reverse transcription reaction is shown in Table 1:
TABLE 1
| Composition (I) | Dosage/tube |
| Random reverse transcription primer (1uM) | 1ul |
| RNA samples | 2ug |
| Ribozyme-free water | To 12ul |
Reverse transcription first step conditions: 5min at 65 ℃ as in table 2:
TABLE 2
| Composition (I) | Dosage/ |
| 5 × reverse transcription buffer | 4μl |
| Base triphosphate deoxynucleotide (10mM) | 2μl |
| RNase inhibitor (40U/. mu.l) | 1μl |
| MMLV reverse transcriptase (200U/. mu.l) | 1μl |
| Products of the first PCR | 12μg |
Reverse transcription second step procedure: 5 minutes at 25 ℃, 60 minutes at 42 ℃ and 5 minutes at 70 ℃.
4. Carrying out real-time quantitative PCR by using a long-chain non-coding RNA LINC00559 specific primer: the specific primer sequence of the long-chain non-coding RNA LINC00559 is synthesized by Shanghai biological engineering Co.
The transcription product is diluted 5 times and mixed evenly. 20 μ L reaction system as in Table 3:
TABLE 3
Real-time fluorescent quantitative PCR reaction program: 95 ℃ for 3 minutes, 40 cycles, 95 ℃ for 10 seconds, 60 ℃ for 30 seconds.
The sequence of the long-chain non-coding RNA LINC00559 specific primer is as follows:
forward primer 5'-GGCTGTGATATGAAAGTTGGCTC-3' (SEQ ID NO.2),
reverse primer 5'-CAACTACATATCAAGCCAGGC-3' (SEQ ID NO.3),
5、-2ΔΔCTmeasurement of the index: the experimental data were analyzed by a relatively quantitative analysis method with ACTB as an internal reference gene and using GraphPad Prism software. Analysis shows that the expression of the long-chain non-coding RNA LINC00559 in 35 lung squamous carcinoma patients is obviously up-regulated and has significant difference (P) compared with the expression of the long-chain non-coding RNA LINC00559 in normal lung tissues<0.0001)。
6. Prognosis determination
Through the follow-up of 35 patients or family members of squamous cell lung carcinoma, we asked the first onset time, treatment condition, recurrence status and death time of the patients in detail, and the follow-up time is 1-60 months. And selecting an expression value of fluorescent quantitative PCR analysis as a reference standard from selected patients with squamous cell lung carcinoma, wherein the expression value of the long-chain non-coding RNA LINC00559 is high in expression when the obtained results are subjected to descending order arrangement and is higher than the median, and the expression values of the long-chain non-coding RNA LINC00559 are 18 cases in total, and the expression values of the other long-chain non-coding RNA LINC00559 are low in expression and are 17 cases in total. Through Kaplan-Meier survival analysis, the survival time of the patient with high expression of the long-chain non-coding RNA LINC00559 is shorter than that of the patient with low expression of the long-chain non-coding RNA LINC00559, and the prognosis is poor. The difference is statistically significant (P ═ 0.0041). Ratio risk model prognostic analysis shows that the risk rate of the long-chain non-coding RNA LINC00559 is less than 1(HR is 0.2414), which indicates that the prognosis of patients with low expression prognosis of patients with high expression of the long-chain non-coding RNA LINC00559 is poor, and the high expression of the long-chain non-coding RNA LINC00559 is an independent risk factor for predicting the prognosis risk of squamous cell lung carcinoma patients.
The research also shows that the long-chain non-coding RNA LINC00559 can be used as a specific molecular marker for prognosis of patients with squamous cell lung carcinoma.
SEQUENCE LISTING
<110> Hunan ya Hospital MaoChao of the university of China
<120> Long non-coding RNA
Application of LINC00559 as biomarker in preparation of lung squamous carcinoma prognosis detection preparation
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<170> PatentIn version 3.5
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Claims (2)
1. The application of the long-chain non-coding RNA LINC00559 as a biomarker in preparing lung squamous cell carcinoma prognosis detection preparations.
2. The application of the long-chain non-coding RNA LINC00559 as a biomarker in preparing a lung squamous carcinoma prognosis detection kit.
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