CN114958856A - Application of long-chain non-coding RNA CYP1B1-AS1 AS breast cancer biomarker and treatment target - Google Patents
Application of long-chain non-coding RNA CYP1B1-AS1 AS breast cancer biomarker and treatment target Download PDFInfo
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- CN114958856A CN114958856A CN202210733639.5A CN202210733639A CN114958856A CN 114958856 A CN114958856 A CN 114958856A CN 202210733639 A CN202210733639 A CN 202210733639A CN 114958856 A CN114958856 A CN 114958856A
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Abstract
The invention discloses a long-chain non-coding RNA CYP1B1-AS1, the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention also discloses a CYP1B1-AS1 lentivirus overexpression vector, a lentivirus stable expression cell strain and application thereof in preparing a breast cancer diagnosis kit, a medicament for preventing or treating breast cancer or a prognosis product. CYP1B1-AS1 can be used for remarkably reducing expression in breast cancer and can be used AS a diagnostic marker of the breast cancer. The expression level of CYP1B1-AS1 is related to the prognosis of patients and can be used AS a prognostic marker of breast cancer. The up-regulation CYP1B1-AS1 can inhibit the malignant progression of breast cancer cells and has application value AS a gene therapy target. The up-regulation of the expression of CYP1B1-AS1 can improve the sensitivity of breast cancer cells to chemotherapeutic drugs. The invention can be used for the preparation of products for diagnosing and treating breast cancer, improving the sensitivity of targeted drugs and the like, and has certain clinical transformation prospect.
Description
Technical Field
The invention belongs to the technical field of tumor molecular biology, and relates to application of long-chain non-coding RNA CYP1B1-AS1 AS a breast cancer diagnosis and prognosis biomarker and a treatment target.
Background
Breast cancer, the first malignancy in worldwide incidence, is the leading cause of cancer death in women. The incidence of breast cancer in China is continuously increased, and the final treatment failure of patients is often caused due to the lack of timely and effective diagnosis and treatment. Therefore, the development mechanism of breast cancer is deeply explored, a new diagnosis and treatment target is searched, the application value of the breast cancer is excavated, and the clinical transformation potential of the breast cancer is developed.
Recent research results show that long non-coding RNA (incRNA) with the length of more than 200nt can interact with DNA, RNA or protein, participate in important regulation and control such as chromosome modification, transcription, intranuclear transportation, translation and posttranslational modification, act on various cell signal transduction pathways, and participate in the whole process of tumor development. Dysregulation of LncRNA expression is one of the characteristic changes in the human cancer transcriptome, with potential to become a tumor biomarker and therapeutic target molecule.
The invention relies on the fact that the unit is Jiangsu province tumor hospital, and the applicant undertakes a plurality of national and provincial natural science fund projects related to tumors and accumulates the research experience of abundant molecular markers related to the tumors. In recent years, genetic engineering techniques have been rapidly developed in tumor research, and gene diagnosis and molecular targeted therapy have shown great advantages in clinical treatment of various malignant tumors. Therefore, the pathogenesis of the breast cancer is deeply understood, the function of the related molecular marker in the disease development is determined, and the foundation can be laid for the individual accurate diagnosis and treatment of the breast cancer.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a long-chain non-coding RNA CYP1B1-AS1 beneficial to breast cancer diagnosis and treatment and disclosing the application potential of lncRNA CYP1B1-AS1 in solving the clinical current situation of breast cancer.
The invention also aims to solve the technical problem of providing the application of the CYP1B1-AS1 lentivirus overexpression vector and the lentivirus stable expression cell strain in preparing a breast cancer diagnosis kit, a medicine for preventing or treating breast cancer or a prognosis product.
The invention also aims to solve the technical problem of providing a fluorescence quantitative PCR detection kit.
The technical scheme is as follows: in order to solve the technical problems, the invention provides long-chain non-coding RNA CYP1B1-AS1, the nucleotide sequence of which is shown AS SEQ ID NO. 1.
The CYP1B1-AS1 gene is all called AS: cytochrome P450 family 1 subfamily B member 1-antisense RNA 1, located on chromosome 2 sense strand, with length 1776nt and polyA tail.
Seq ID NO.1
＞NR_027252.1Homo sapiens CYP1B1 antisense RNA 1(CYP1B1-AS1)，long non-coding RNA
The invention also comprises the biological function of CYP1B1-AS1 in breast cancer cells, and discloses the application value of the CYP1B1-AS1 AS a molecular therapeutic target.
The invention also comprises a CYP1B1-AS1 lentivirus overexpression vector, wherein the CYP1B1-AS1 lentivirus overexpression vector contains the long-chain non-coding RNA CYP1B1-AS 1.
The invention also comprises a lentivirus stable expression cell strain, wherein the lentivirus stable expression cell strain is obtained by packaging the CYP1B1-AS1 lentivirus overexpression vector into virus particles to infect host cells.
Wherein the host cell is MCF7 or MDA-MB-231 cell.
The expression of CYP1B1-AS1 in breast cancer cell strains MCF7 and MDA-MB-231 is up-regulated through a lentivirus overexpression vector, and the up-regulation of CYP1B1-AS1 to inhibit the malignant progress of breast cancer cells is verified through a cell proliferation experiment, a clone formation experiment, flow cell cycle detection and the like.
The invention also comprises the application of the long-chain non-coding RNA CYP1B1-AS1 AS a breast cancer diagnosis marker.
The invention also comprises the application of the long-chain non-coding RNA CYP1B1-AS1, the CYP1B1-AS1 lentivirus overexpression vector and the lentivirus stable expression cell strain in the preparation of a breast cancer diagnosis kit, a medicament for preventing or treating breast cancer or a prognosis product.
The invention also comprises a fluorescent quantitative PCR detection kit, wherein the fluorescent quantitative PCR detection kit comprises a specific primer pair, and the sequences of the primer pair are shown as SEQ ID NO.2 and SEQ ID NO. 3.
The detection kit further comprises a reverse transcription reagent and a real-time fluorescence quantitative PCR reagent, and the real-time fluorescence quantitative PCR reagent is a preparation for detecting the CYP1B1-AS1 expression level in the breast cancer tissue.
The detection kit further comprises an internal reference GAPDH primer pair, wherein the sequences of the internal reference GAPDH primer pair are shown as SEQ ID NO.4 and SEQ ID NO. 5.
CYP1B1-AS1 detection primer for real-time fluorescent quantitative PCR is AS follows:
CYP1B1-AS1：
SEQ ID NO.2：Forward Primer(5’to3’)
SEQ ID NO.3：Forward Primer(5’to3’)
internal reference GAPDH:
SEQ ID NO.4：Forward Primer(5’to3’)
SEQ ID NO.5：Forward Primer(5’to3’)
the invention also comprises the long-chain non-coding RNA CYP1B1-AS1, the CYP1B1-AS1 lentivirus overexpression vector and the application of the lentivirus stable expression cell strain in preparing products for improving the sensitivity of breast cancer cells to chemotherapy.
The CYP1B1-AS1 lentivirus overexpression vector is LV6-Homo CYP1B1-AS1 recombinant plasmid, and is constructed by Shanghai Jima pharmaceutical technology Co.
The sequences of the primers plus the restriction enzyme sites NotI and NsiI used for constructing the CYP1B1-AS1 lentiviral over-expression vector are AS follows:
SEQ ID NO.6：Forward Primer(5’to3’)
SEQ ID NO.7：Forward Primer(5’to3’)
the invention also comprises a cell strain which is stably expressed and obtained by infecting cells after packaging the lentivirus recombinant vector LV6-Homo CYP1B1-AS 1.
The invention utilizes the lentivirus overexpression vector to up-regulate CYP1B1-AS1, which is beneficial to improving the sensitivity of breast cancer cells to the common chemotherapy medicament cisplatin.
Wherein, the application comprises the steps of adding cisplatin with different concentrations to treat breast cancer cells, and then adding CCK-8 to determine the influence of CYP1B1-AS1 on the sensitivity of cancer cell chemotherapy drugs.
Wherein the concentration of the cisplatin is 25 mug/mL, 12.5 mug/mL, 6.25 mug/mL, 3.13 mug/mL, 1.56 mug/mL and 0.78 mug/mL.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the invention discovers that CYP1B1-AS1 can be obviously expressed in the breast cancer in a down-regulation way for the first time and can be used AS a diagnostic marker of the breast cancer.
2. The invention discloses that the expression level of CYP1B1-AS1 is related to the prognosis of a patient for the first time and can be used AS a prognostic marker of breast cancer.
3. The invention first defines that the CYP1B1-AS1 can inhibit the malignant progress of the breast cancer cells and has application value AS a gene therapy target.
4. The invention firstly proves that the sensitivity of breast cancer cells to chemotherapeutic drugs can be improved by up-regulating the expression of CYP1B1-AS 1.
In conclusion, the invention can be used for the preparation of products for diagnosing and treating breast cancer, improving the sensitivity of targeted drugs and the like, and has certain clinical transformation prospect.
Drawings
FIG. 1 is a heat map analysis of the detection results of lncRNA chip of breast cancer tissues. Note: t, tumor tissue, N, paracancerous normal tissue.
FIG. 2TCGA data analysis of CYP1B1-AS1 expression in breast cancer.
The NCBI database of FIG. 3 shows the structural features of CYP1B1-AS 1.
FIG. 4LNCipedia database analyses CYP1B1-AS1 coding capacity and conservation.
FIG. 5 is a graph showing the application value of CYP1B1-AS1 detection in combination with clinical breast cancer tissue samples.
FIG. 6 is a graph showing the relationship between CYP1B1-AS1 expression level and prognosis of breast cancer patients.
FIG. 7 biological function study of CYP1B1-AS 1. A. qPCR measures the effect of up-regulating expression of CYP1B1-AS1 in breast cancer cells. B. The CCK-8 method detects the influence of the up-regulation of CYP1B1-AS1 on the proliferation capacity of the breast cancer cells. C. The clone formation experiment detects the influence of the up-regulated CYP1B1-AS1 on the clonality of single breast cancer cells. D. Flow cytometry analysis the effect of up-regulating CYP1B1-AS1 on the cell cycle of breast cancer. Note: the breast cancer cells up-regulated in CYP1B1-AS1 expression and their negative controls were labeled MCF7-exp, MCF7-NC and MDA-231-exp, MDA-231-NC, respectively.
FIG. 8 is a graph of the effect of over-expressing CYP1B1-AS1 on drug sensitivity in breast cancer cells.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 IncRNA chip detection of IncRNA-CYP 1B1-AS1 differentially expressed in breast cancer tissue
1. Experimental materials and methods
Approved by the ethical committee, 6 breast cancer tissues and paracancerous normal tissues were selected from the biological sample bank, and the expression profiles of lncRNA and mRNA in the tissues were examined using the array star human lncRNA chip, which was performed by shanghai Kangcheng biology. The chip can detect 40, 173 lncRNA and 20, 730 protein coding transcripts. Through the analysis of GeneSpring GX v12.1 software and the Quantille standardization, the differentially expressed lncRNAs or differentially expressed mRNAs with statistical significance between two groups of samples pass through the Fold change which is more than or equal to 2.0, and the P is less than 0.05 for screening.
2. Results of the experiment
In this example, the paracancer normal tissue is used AS a control, the Fold change is greater than or equal to 2, and the P is less than 0.05 AS a screening index, and it is found that CYP1B1-AS1 is significantly reduced in the breast Cancer tissue, and the GEPIA2 analysis of the Cancer Genome map (TCGA, the Cancer Genome Atlas) data indicates: CYP1B1-AS1 down-regulated expression in breast cancer (fig. 1, 2). CYP1B1-AS1 is an antisense lncRNA, has a polyA tail, is positioned on the sense strand of chromosome 2, has the length of 1776nt and is provided with the polyA tail. CYP1B1-AS1 did not have the ability to encode proteins (FIGS. 3, 4).
Example 2 application of CYP1B1-AS1 detection in preparation of breast cancer diagnosis and prognosis products
1. Experimental materials and methods
1.1 Breast cancer tissue sample Collection
30 primary breast cancer and paracarcinoma normal tissue samples were collected and immediately stored in liquid nitrogen until RNA extraction, approved by the ethical committee.
1.2RNA extraction, reverse transcription and qPCR reaction
Extracting total RNA from cells by using Trizol reagent (Invitrogen), wherein the total number of cells extracting RNA in one time is not less than 10 6 Using OD 260/280 Detecting the purity of RNA when OD 260/280 Between 1.9 and 2.1, can be used for subsequent detection.
cDNA was synthesized using the PrimeScript RT kit (TaKaRa) with a total of 1. mu.g of RNA and stored at-20 ℃ until use. The primers for CYP1B1-AS1 are AS follows: an upstream primer: 5'-ACTGGTATGACCACCCGAGA-3', respectively; a downstream primer: 5'-ACTGGCATACTGGTCACTGC-3', Tm 60 ℃. Primers for internal control GAPDH are as follows, upstream primers: 5'-ATGGGTGTGAACCATGAGAA-3', respectively; a downstream primer: 5'-GTGCTAAGCAGTTGGTGGTG-3', Tm 60 ℃. Primers were synthesized by Shanghai Biotech.
Real-time fluorescent quantitative PCR detection and melting curve analysis were carried out using LightCycler PCR instrument of Roche, USA, using TB from Takara
Premix Ex Taq TM II, carrying out PCR reaction by using the kit, amplifying 40 cycles by using a two-step method, collecting fluorescence signals, analyzing an amplification curve and a melting curve by using software carried by an instrument, calculating a mean cycle threshold (Ct), and using 2 -ΔΔCt The expression quantity of the relative gene is obtained by calculation.
The specific operation and reaction system are as follows:
1.2.1RNA extraction
Adding Trizol into the tissue sample according to the concentration of 50-100 mg/mL. The electric homogenizer is used for homogenizing fully for about 1-2min, and the cells are transferred to a centrifuge tube after being completely lysed.
② 0.2mL of chloroform (0.2 mL of chloroform is added to 1mL of Trizol) is added into the centrifuge tube containing the lysate, and the mixture is fully shaken and mixed for 20s on a shaker, and is placed for 5min at room temperature.
③ 12000rpm centrifugation at 4 ℃ for 10min, then absorbing the upper water phase containing the total RNA into a new centrifuge tube, and absorbing about 0.5mL of the upper water phase per mL of Trizol. The uptake of DNA and proteins in the organic phase and the intermediate layer should be avoided.
Adding isopropanol with the same volume as the upper aqueous phase, reversing for several times, uniformly mixing, and precipitating for 5min at room temperature. Centrifuge at 12000rpm for 15min at 4 ℃.
Fifthly, abandoning the supernatant, adding 1mL of 75% ethanol into each milliliter of Trizol, and slightly reversing and uniformly mixing to clean the RNA precipitate twice. Centrifuging at 12000rpm at 4 deg.C for 2min, discarding liquid, and air drying at room temperature.
Sixthly, adding proper DEPC treatment water to dissolve the RNA precipitate. The mixture was stored at-80 ℃.
1.2.2 reverse transcription:
(ii) removal of genomic DNA
Reaction conditions are as follows: preserving at 42 deg.C, 2min, 4 deg.C.
Reverse transcription reaction:
the above reaction product was added to the following reagents:
the reaction conditions are 37 ℃ and 15 min; 5s at 85 ℃; and preserving at 4 ℃.
1.2.3PCR amplification:
the reverse transcribed cDNA was pipetted 2. mu.L into the following reaction system:
1.3Kaplan-Meier Plotter analyses the relationship of CYP1B1-AS1 expression to the prognosis of breast cancer patients.
1.4 data processing and analysis
The Wilcoxon signed rank test is used to compare breast cancer tissue to paracancerous normal tissue samples. Statistical analysis was performed using GraphPad Prism 8.0 software. Values of P less than 0.05 were considered statistically significant (, P < 0.05,. P < 0.01,. P < 0.001).
2. Results of the experiment
In this example, the RT-PCR was used to detect CYP1B1-AS1 in 30 cases of breast cancer tissues and paracancer normal tissues, and it was found that CYP1B1-AS1 in cancer tissues was significantly lower than paracancer normal tissues, and a receiver operating characteristic curve (ROC) showed that the area under the curve (AUC) was 0.909, [ 95% confidence interval: 0.839-0.979, P < 0.001], suggesting that CYP1B1-AS1 has higher diagnostic value (figure 5).
In addition, Kaplan-Meier Plotter analysis showed that: the breast cancer patients with high CYP1B1-AS1 have higher relapse-free survival Rate (RFS) and overall survival rate (OS), and show the application value AS the clinical prognostic factor of the breast cancer (figure 6).
Example 3 biological function of CYP1B1-AS1 in Breast cancer cells
1. Experimental materials and methods
1.1 cell culture
Breast cancer cell lines MCF7 and MDA-MB-231 were purchased from shanghai cell bank of chinese academy of sciences and cultured according to ATCC protocol: MCF7 cells were cultured in RPMI 1640 containing 10% fetal bovine serum and incubated at 37 ℃ with 5% CO 2 An incubator. MDA-MB-231 cells were cultured using L-15 containing 10% FBS and placed in an air incubator at 37 ℃. 293T cells were purchased from Shanghai cell Bank of Chinese academy of sciences and cultured in DMEM containing 10% FBS.
1.2 establishment of cell line stably expressing lentivirus
The stable expression cell strain of CYP1B1-AS1 is established by using a lentivirus overexpression vector (LV6, Shanghai Jima pharmaceutical technology Co., Ltd.): the sequences of primers for amplifying the target gene HomoCYP1B1-AS1 and enzyme cutting sites NotI and NsiI are AS follows: an upstream primer: 5'-AGGGTTCCAAGCTTAAGCGGCCGCCAACTATGGTTGCTAACACAAGAATCGGCA-3', downstream primer: 5'-GATCCATCCCTAGGTAGATGCATTTTTTTTTTTTTTTTTTTTTTTTTTATTAAACTG-3', Tm is 55 ℃; cloning a target gene Homo CYP1B1-AS1 to a lentivirus overexpression vector (LV6, Shanghai Jima pharmaceutical technology Co., Ltd.) to obtain LV6-Homo CYP1B1-AS1 recombinant plasmid (H2470-2), transfecting 293T cells with the lentivirus overexpression vector LV6-Homo CYP1B1-AS1 recombinant plasmid and the lentivirus overexpression vector LV6 (negative control) by using liposome Lipofectamine 3000(Invitrogen) respectively for virus packaging, collecting virus liquid in cell culture supernatant after 72H, MCF7 and MDA-MB-231 cells are respectively infected, puromycin 10 mu g/mL is added after 48 hours for screening, and MCF7 and MDA-MB-231 cells which stably express CYP1B1-AS1 and negative controls (marked AS MCF7-exp, MCF7-NC and MDA-231-exp and MDA-231-NC respectively) thereof are obtained. Construction and packaging of lentiviral expression vectors was performed by Shanghai Jima pharmaceutical technology, Inc. The effect of over-expressing CYP1B1-AS1 was examined using the RT-PCR method in example 2, and the results are shown in FIG. 7A.
1.3 cell proliferation assay
CCK-8 was used to test the proliferative capacity of MCF7-exp, MCF7-NC and MDA-231-exp, MDA-231-NC cells. The treated test cells were plated in 96-well plates (3X 10 cells per well) 3 One), the culture solution containing 10% of CCK-8 was replaced and incubated for 2 hours at 0h, 24h, 48h, 72h and 96h, respectively, and the absorbance was measured at a wavelength of 450 nm. CCK-8 was purchased from the institute of Homon chemistry, Japan.
1.4 clone formation experiments
MCF7-exp, MCF7-NC and MDA-231-exp, MDA-231-NC cells were seeded in six-well plates containing 2mL RPMI 1640(MCF7) or L-15(MDA-MB-231) culture, respectively, at gradient densities of 300, 500, 800 cells, and gently swirled to disperse the cells evenly. Culturing in cell culture box for 1-2 weeks. When macroscopic colonies appeared in the dish, the supernatant was discarded and washed 2 times with PBS. Cells were fixed for 15min by adding 5mL of 4% paraformaldehyde. Removing the fixing solution, adding a proper amount of crystal violet staining solution for staining for 20min, then slowly washing off the staining solution by running water, and observing the cell clone formation under a microscope after drying.
1.5 cell cycle assays
Cell cycle analysis was performed using propidium iodide staining: MCF7-exp, MCF7-NC and MDA-231-exp, MDA-231-NC cells were trypsinized, washed with PBS, and fixed with 70% ethanol at-20 ℃. After centrifugation, the cells were incubated in hypotonic solution containing 50. mu.g/mL propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100 and 20. mu.g/mL for 30min at 37 ℃. The cells were then analyzed in linear mode using a flow cytometer.
1.6 data processing and analysis
t-test and analysis of variance (ANOVA) were used for comparison between groups. Statistical analysis was performed using GraphPad Prism 8.0 software. Values of P less than 0.05 were considered statistically significant (, P < 0.05,. P < 0.01).
2. Results of the experiment
The lentivirus overexpression vector in the embodiment can stably up-regulate the expression of CYP1B1-AS1 in breast cancer cells MCF7 and MDA-MB-231. Cell proliferation experiments, colony formation experiments and flow cell cycle detection results show that the up-regulation of the expression of CYP1B1-AS1 can effectively inhibit the proliferation of cells (figure 7).
Example 5 application of CYP1B1-AS1 in preparation of a product for improving sensitivity of breast cancer cells to chemotherapy
1. Experimental materials and methods
1.1 drug susceptibility test
MCF7-exp, MCF7-NC and MDA-231-exp, MDA-231-NC cells at 5X 10 per well 3 Each was seeded in a 96-well plate and after 24h incubation cisplatin (dissolved in physiological saline) was added to the cells at various concentrations (25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.13. mu.g/mL, 1.56. mu.g/mL, 0.78. mu.g/mL). An equal volume of solvent was added as a control with a drug concentration of 0. After 48h of treatment, the culture was replaced with 100. mu.L of a reaction solution containing 10% CCK-8 (10% CCK-8 was prepared in RPMI 1640 or L-15 culture solution, respectively, and prepared on site), incubated for 3h, and the absorbance was measured at a wavelength of 450nm to prepare a dose-response curve.
1.2 data processing and analysis
ANOVA was used for group comparisons and statistical analysis was performed using GraphPad Prism8 software. Values of P less than 0.05 were considered statistically significant (, P < 0.05,. P < 0.01).
2. Results of the experiment
In this example, we treated breast cancer cells with cisplatin at various concentrations and measured the cell activity by adding CCK-8, and the results showed that up-regulation of CYP1B1-AS1 expression is beneficial to increase the sensitivity of breast cancer cells to chemotherapeutic drugs (FIG. 8).
Sequence listing
<110> tumor hospital in Jiangsu province
<120> application of long-chain non-coding RNA CYP1B1-AS1 AS breast cancer biomarker and treatment target
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Claims (10)
1. The long-chain non-coding RNA CYP1B1-AS1 is characterized in that the nucleotide sequence is shown AS SEQ ID NO. 1.
The CYP1B1-AS1 lentiviral overexpression vector, wherein the CYP1B1-AS1 lentiviral overexpression vector comprises the long-chain non-coding RNA CYP1B1-AS1 of claim 1.
3. A stable lentivirus-expressing cell line obtained by infecting host cells with the CYP1B1-AS1 lentiviral over-expression vector of claim 2 packaged AS viral particles.
4. The use of the long non-coding RNA CYP1B1-AS1 of claim 1 AS a diagnostic marker for breast cancer.
5. The long-chain non-coding RNA CYP1B1-AS1 of claim 1, CYP1B1-AS1 lentiviral over-expression vector of claim 2, and the application of the lentiviral stable expression cell strain of claim 3 in the preparation of a breast cancer diagnosis kit, a drug for preventing or treating breast cancer or a prognostic product.
6. A fluorescent quantitative PCR detection kit is characterized by comprising a specific primer pair, wherein the sequences of the primer pair are shown as SEQ ID NO.2 and SEQ ID NO. 3.
7. The fluorescent quantitative PCR detection kit of claim 6, wherein the detection kit further comprises a reverse transcription reagent and a real-time fluorescent quantitative PCR reagent.
8. The fluorescent quantitative PCR detection kit of claim 6, further comprising an internal reference GAPDH primer pair, wherein the sequences of the internal reference GAPDH primer pair are shown as SEQ ID No.4 and SEQ ID No. 5.
9. The use of the long non-coding RNA CYP1B1-AS1 of claim 1, the CYP1B1-AS1 lentiviral over-expression vector of claim 2, and the lentivirus stable expression cell strain of claim 3 in the preparation of a product for increasing the sensitivity of breast cancer cells to chemotherapy.
10. The use of claim 9, wherein the use comprises treating breast cancer cells with cisplatin at various concentrations and then measuring the effect of CYP1B1-AS1 on cancer cell chemotherapeutic drug sensitivity by adding CCK-8.
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| GENBANK ACCESSION：NR_027252.1: "Homo sapiens CYP1B1 antisense RNA 1 (CYP1B1-AS1), long non-coding RNA", 《GENBANK》 * |
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