CN110747273B - Application of long-chain non-coding RNA LINC00263 in preparation of drug resistance detection preparation for breast cancer - Google Patents
Application of long-chain non-coding RNA LINC00263 in preparation of drug resistance detection preparation for breast cancer Download PDFInfo
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Abstract
The invention discloses application of long-chain non-coding RNA LINC00263 in preparation of a drug resistance detection preparation for breast cancer. The invention can detect the expression level of the long-chain non-coding RNA LINC00263 in a breast cancer patient, provides a powerful molecular biological basis for predicting resistance to tamoxifen medication and 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 LINC00263 in preparation of a drug resistance detection reagent for breast cancer.
Background
Tamoxifen (tamoxifen) is a non-steroidal antiestrogen and is widely used in clinical medicine. This is a Selective Estrogen Receptor Modulator (SERM) of the triphenyldiene series, with estrogen agonist and antagonist properties. Thus, it may act as an estrogen in some tissues while preventing the effects of estrogen in other tissues. In breast tissue, tamoxifen is a potent antiestrogen and is therefore commonly used to treat women's hormone sensitive breast cancer. Unfortunately, up to half of ER α positive tumors have intrinsic or acquired resistance to endocrine therapy.
At present, the drug resistance mechanism of the endocrine treatment of breast cancer is complex, other drug treatment schemes are often selected after the endocrine treatment drug resistance condition appears, and the treatment requirement and the psychology of breast cancer patients cannot be met. Therefore, early judgment on endocrine treatment of breast cancer so as to select the optimal treatment scheme and remarkably improve the drug treatment efficiency is an important subject to be solved urgently in the field of breast cancer.
Long non-coding RNA (long non-coding RNA) is a general term for non-coding RNA with a length of 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 gradually paid more attention by researchers as an important regulatory molecule and the application value thereof in clinical diagnosis, chemotherapy sensitivity, prognosis evaluation and other aspects.
At present, no reference standard and no specific index exist for drug resistance of endocrine treatment of breast cancer, and the requirement for judging the effect of endocrine medication of breast cancer cannot be met. Therefore, determining the endocrine treatment effect to select the optimal treatment scheme and significantly improve the survival rate of patients becomes an important issue to be solved in the field of breast cancer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides application of long-chain non-coding RNA LINC00263 in preparation of a drug resistance detection preparation for breast cancer.
The long non-coding RNA LINC00263 maps to chromosome 10q24.31. The inventor finds that the expression of the long-chain non-coding RNA LINC00263 is up-regulated in tamoxifen resistant MCF-7 cells, the expression difference with tamoxifen sensitive MCF-7 cells is obvious (P < 0.01) (figure 1), the long-chain non-coding RNA LINC00263 can be used as a judgment standard for judging whether tamoxifen is resistant or not, and can be used as a biomarker for preparing medication guidance of breast cancer patients, namely, the long-chain non-coding RNA LINC00263 is used as a biomarker of a breast cancer tamoxifen medication resistance detection preparation.
Furthermore, the kit (medication guide kit) for detecting the drug resistance of the breast cancer tamoxifen, which has high cost performance and is easy to popularize and apply, can be provided.
The kit comprises a long non-coding RNA LINC00263 real-time fluorescent quantitative PCR specific primer and an ACTB internal reference real-time fluorescent quantitative PCR specific primer; the long-chain non-coding RNA LINC00263 real-time fluorescent quantitative PCR specific primers are as follows:
a forward primer: 5'TCGGATAGGAGTGTCAGG-3' (SEQ ID NO. 1);
reverse primer: 5-;
the ACTB internal reference real-time fluorescent quantitative PCR specific primers are as follows:
a forward primer: 5 'CACCATTGGCAATGAGCGTTC-3' (SEQ ID NO. 3);
reverse primer: 5 'AGGTCTTTGCGGATGTCCACGT-3' (SEQ ID NO. 4).
Use of long non-coding RNA LINC00263 in intramammary cancerThe method for detecting the patient with the secretory therapy comprises the following steps: (1) Collecting breast cancer tissues excised after the operation of an individual to be detected, and extracting total RNA; (2) Carrying out reverse transcription on the long-chain non-coding RNA LINC00263 into cDNA by taking the total RNA as a template; (3) Carrying out real-time fluorescent quantitative PCR amplification by using a long-chain non-coding RNA LINC00263 specific primer to obtain a relative expression quantity of 2 ΔΔCT When 2 is present ΔΔCT >5.23, the long-chain non-coding RNA LINC00263 is high in expression.
In conclusion, the invention can be used for detecting the expression level of the long-chain non-coding RNA LINC00263 in a breast cancer patient, provides a powerful molecular biological basis for predicting resistance to tamoxifen medication and has profound clinical significance and popularization.
Drawings
FIG. 1 shows the real-time fluorescent quantitative PCR analysis of the expression difference of the long non-coding RNA LINC00263 in tamoxifen sensitivity and drug resistance.
Detailed Description
EXAMPLE 1 preparation of Long non-coding RNA LINC00263 for tamoxifen guidance kit (50 reactions)
50ml of 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
8.10 mM 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 LINC00263 real-time fluorescent quantitative PCR specific primer:
a forward primer of 5'TCGGATAGGAGTGTCAGG-3' (SEQ ID NO. 1),
reverse primer 5'TTCAGTTGCTTCAGGTCAT-3' (SEQ ID NO. 2),
13.10 μ M internal reference ACTB real-time fluorescent quantitative PCR specific primers:
the forward primer is 5 'CACCATTGGCAATGAGCGTTC-3' (SEQ ID NO. 3),
the reverse primer is 5.
Example 2 detection of Long non-coding RNA LINC00263 in Breast cancer cells
1. Preservation of breast cancer tissue: collecting breast cancer tissues to be detected, storing the breast cancer tissues in a freezing storage tube filled with RNA stable solution, and placing the breast cancer tissues in a refrigerator at the temperature of minus 80 ℃ for later use.
2. Extraction of RNA from tissues: taking a proper amount of sample, adding liquid nitrogen into a mortar baked for 6-8h at 180 ℃, grinding the sample into powder, adding 1ml of Trizol mortar sample 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-layer water phase, adding 0.5ml of precooled isopropanol into a new centrifugal tube, uniformly mixing, standing for 20min at-20 ℃ in a refrigerator, and centrifuging for 10min at 12000g at 4 ℃; 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 the RNA are measured by a spectrophotometer, and the OD260/280 ratio is between 1.8 and 2.0 and stored at the temperature of minus 80 ℃.
3. Long non-coding RNA LINC00263 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
Reverse transcription first step conditions: 5min at 65 ℃ as shown in Table 2:
TABLE 2
| Composition (I) | Dosage/tube |
| 5 × reverse transcription buffer | 4μl |
| Base triphosphate deoxynucleotide (10 mM) | 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 LINC00263 specific primer: the specific primer sequence of the long non-coding RNA LINC00263 is synthesized by Shanghai biological engineering Co., ltd.
The transcription product is diluted 5 times and mixed evenly. The 20 μ L reaction system is shown in table 3:
TABLE 3
| Composition (I) | Dosage/tube |
| SYBR Premix Ex Taq | 10μl |
| LINC00263 specific primer (10. Mu.M) | 0.5μl |
| cDNA products | 1μl |
| Enzyme-free water | To 20μl |
Real-time fluorescent quantitative PCR reaction program: 95 ℃ for 3 minutes, 40 cycles, 95 ℃ for 10 seconds, 60 ℃ for 30 seconds.
The specific primer sequence of the long-chain non-coding RNA LINC00263 is as follows:
a forward primer of 5'TCGGATAGGAGTGTCAGG-3' (SEQ ID NO. 1),
reverse primer 5'TTCAGTTGCTTCAGGTCAT-3' (SEQ ID NO. 2),
5、-2 ΔΔCT measurement 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.
Sequence listing
<110> university of Zhongnan
Application of long-chain non-coding RNA LINC00263 in preparation of drug resistance detection preparation for breast cancer
<141> 2019-10-18
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> null(null)
<400> 1
tcggatagga gtgtcagg 18
<210> 2
<211> 19
<212> DNA
<213> null(null)
<400> 2
ttcagttgct tcaggtcat 19
<210> 3
<211> 22
<212> DNA
<213> null(null)
<400> 3
caccattggc aatgagcggt tc 22
<210> 4
<211> 22
<212> DNA
<213> null(null)
<400> 4
aggtctttgc ggatgtccac gt 22
Claims (2)
1. The application of the primer for detecting the expression level of the long-chain non-coding RNA LINC00263 in preparing a drug resistance detection preparation for breast cancer is characterized in that the primer is a real-time fluorescent quantitative PCR specific primer for detecting the long-chain non-coding RNA LINC00263, and the specific primer has the following sequence: a forward primer: 5 'TCGGATAGGAGTGTCAGG-3'; reverse primer: 5 'TTCAGTTGCTTCAGGTCAT-doped 3'.
2. The application of the primer for detecting the expression level of the long-chain non-coding RNA LINC00263 in the preparation of a drug resistance detection kit for breast cancer is characterized in that the primer is a real-time fluorescent quantitative PCR specific primer for detecting the long-chain non-coding RNA LINC00263, and the specific primer has the following sequence: a forward primer: 5 'TCGGATAGGAGTGTCAGG-3'; reverse primer: 5 'TTCAGTTGCTTCAGGTCAT-doped 3'.
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