Tumor detection marker and application thereof in drug preparation

Abstract

The invention discloses a tumor detection marker and application thereof in medicine preparation, and belongs to the technical field of biomedicine. Clinical sample qRT-PCR (quantitative real-time polymerase chain reaction) shows that ENST00000412203.1 is obviously up-regulated in breast cancer tissues, the area (AUC) under the ROC curve reaches 0.9822, and the diagnosis efficiency is excellent. In addition, the invention further finds that the expression of the polypeptide in lymph node metastasis tissues is obviously improved, and metastasis specificity is displayed. Meanwhile, the expression of ENST00000412203.1 is knocked down through siRNA, it is found that proliferation, migration and invasion of breast cancer cells are remarkably inhibited, and therefore the siRNA can be used for preparing targeted therapy drugs for breast cancer and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a tumor detection biomarker and its application in drug preparation. Background Technology

[0002] Breast cancer has become one of the most common malignant tumors among women worldwide, and it is also the most prevalent malignant tumor among women in China. Globally, the diagnosis and screening of breast cancer primarily rely on mammography. However, mammography has poor imaging capabilities for dense breasts, especially since the proportion of dense breasts is high in Chinese and other Asian women, easily leading to false negative diagnoses and missed diagnoses, thus delaying optimal treatment. Furthermore, traditional serum tumor markers (such as CA15-3 and CEA) have limited sensitivity and specificity, making early and accurate diagnosis difficult. Therefore, developing novel diagnostic methods with higher sensitivity, stronger specificity, and non-invasiveness has become an urgent clinical problem to be solved.

[0003] Long non-coding RNAs (lncRNAs) are a class of RNA molecules longer than 200 nt that do not encode proteins. They play important regulatory roles in various biological processes, influencing cell proliferation, apoptosis, invasion, and migration through mechanisms such as epigenetic modification, chromatin remodeling, transcriptional regulation, and intracellular signaling pathways. In recent years, numerous studies have shown that some lncRNAs can stably exist in tumor cell exosomes or plasma, possessing advantages such as high tissue specificity, convenient detection, and non-invasiveness. They are considered highly promising liquid biomarkers for early screening, treatment monitoring, and recurrence prediction in breast cancer. However, currently, there is a lack of highly efficient and specific tumor biomarkers based on lncRNAs in clinical practice. Existing diagnostic methods still rely mainly on imaging and traditional protein biomarkers, which are insufficient to meet the needs of early diagnosis and personalized treatment. While research on lncRNAs as potential therapeutic targets has made progress, targeted drugs developed against their aberrant expression are still in the exploratory stage. Therefore, identifying and validating new breast cancer-related lncRNAs is of great value for improving the early diagnosis rate of breast cancer and developing new targeted therapies. Summary of the Invention

[0004] The purpose of this invention is to provide a tumor detection biomarker and its application in drug preparation.

[0005] To achieve the above objectives, the present invention provides the following technical solution: First, the present invention provides a biomarker for breast cancer detection, the biomarker being a long non-coding RNA ENST00000412203.1, the transcript sequence of which is shown in SEQ ID NO.1.

[0006] Preferably, the expression level of the biomarker in breast cancer tissue is significantly higher than that in adjacent normal tissue; The expression level of the biomarker in lymph node metastasis tissue was significantly higher than that in primary breast cancer tissue.

[0007] Secondly, the present invention provides the application of primers for detecting long non-coding RNA in the preparation of a PCR kit for detecting breast cancer, wherein the long non-coding RNA is long non-coding RNA ENST00000412203.1, and the transcript sequence of the long non-coding RNA ENST00000412203.1 is shown in SEQ ID NO.1.

[0008] Preferably, the sequences of the primers are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0009] Then, the present invention provides the application of an inhibitor that inhibits the expression of long non-coding RNA ENST00000412203.1 in the preparation of a breast cancer therapeutic drug, the transcript sequence of long non-coding RNA ENST00000412203.1 is shown in SEQ ID NO.1; The inhibitors include: (a) siRNA or shRNA; (b) Antisense oligonucleotides; (c) CRISPR / Cas system-mediated gene editing tools; (d) Small molecule compounds that target long non-coding RNA ENST00000412203.1.

[0010] Preferably, the inhibitor is siRNA.

[0011] Preferably, the siRNA includes siRNA-I and siRNA-II; The sense strand sequence of the siRNA-I is shown in SEQ ID NO.10, and the antisense strand sequence of the siRNA-I is shown in SEQ ID NO.11; The sense strand sequence of the siRNA-II is shown in SEQ ID NO.12, and the antisense strand sequence of the siRNA-II is shown in SEQ ID NO.13.

[0012] Preferably, the siRNA inhibits the proliferation, migration, and / or invasion of breast cancer cells by suppressing the expression of ENST00000412203.1.

[0013] Finally, this invention provides the application of siRNA in the preparation of cell inhibitors that inhibit the proliferation, migration and / or invasion of breast cancer cells, wherein the siRNA is the siRNA of long non-coding RNA ENST00000412203.1, and the transcript sequence of the long non-coding RNA ENST00000412203.1 is shown in SEQ ID NO.1.

[0014] Preferably, the siRNA includes siRNA-I and siRNA-II; The sense strand sequence of the siRNA-I is shown in SEQ ID NO.10, and the antisense strand sequence of the siRNA-I is shown in SEQ ID NO.11; The sense strand sequence of the siRNA-II is shown in SEQ ID NO.12, and the antisense strand sequence of the siRNA-II is shown in SEQ ID NO.13.

[0015] The beneficial effects of this invention are as follows: This invention provides a novel breast cancer tumor biomarker—the long non-coding RNA ENST00000412203.1, which is significantly highly expressed in breast cancer tissues (fold change approximately 2.96) and further upregulated in lymph node metastases, exhibiting significant tumor and metastasis specificity. Compared with other screened lncRNAs, ENST00000412203.1 demonstrates the best diagnostic efficacy, with an area under the ROC curve (AUC) of 0.9822, significantly higher than AC006449.1 (0.934) and ENST00000606834.1 (0.916). This significantly improves the sensitivity and specificity of early breast cancer diagnosis, compensating for the shortcomings of traditional imaging and serum biomarkers.

[0016] Meanwhile, this invention demonstrates through siRNA knockdown of ENST00000412203.1 that this RNA significantly inhibits the proliferation, migration, and invasion of breast cancer cells, indicating its important value as a potential therapeutic target and its potential to be used to develop targeted inhibitors (such as siRNA, ASO, or small molecule drugs), providing a new strategy for precision treatment of breast cancer. Attached Figure Description

[0017] Figure 1 This is a bar chart showing the relative expression levels of the three long non-coding RNAs of the present invention in 30 breast cancer tissues and 30 adjacent normal tissues. Figure 2This is a diagram showing the ROC diagnostic curves of the three long non-coding RNAs of this invention. Figure 3 This is a bar chart comparing the relative expression levels of the three long non-coding RNAs of the present invention in 30 primary breast cancer tumor tissues and 20 lymph node metastasis tissues. Figure 4 The bar chart shows the knockdown effect of the siRNA knockdown of ENST00000412203.1 in MCF-7 cells. Figure 5 Bar chart showing the 48-hour survival rate of MCF-7 and MDA-MB-231 cells after transfection with siRNA-II; Figure 6 Photograph (a) and cell count bar chart (b) of the Transwell assay results for the migration ability of MDA-MB-231 cells after transfection with siRNA-II. Figure 7 Photograph (a) and cell count bar graph (b) of the Transwell assay results for the invasive ability of MDA-MB-231 cells after transfection with siRNA-II. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0020] Example 1 (1) In this invention, the long non-coding genes ENST00000412203.1 (transcript sequence as shown in SEQ ID NO.1), AC006449.1 (transcript sequence as shown in SEQ ID NO.2), and ENST00000606834.1 (transcript sequence as shown in SEQ ID NO.3) that are highly expressed in tumors were screened from the GSE125677 dataset in the GEO database.

[0021] (2) Take 30 breast cancer tissues (aged 40-60 years, stage II-III, samples confirmed by pathology) and 30 adjacent normal breast tissues (normal breast tissue >2cm from the tumor edge), extract RNA using TRIzol method, determine concentration and purity using NanoDrop, check integrity by agarose electrophoresis and use it for subsequent operations; (3) Take 1 μg of RNA from each sample and synthesize cDNA using the PrimeScript RT Reagent Kit (TaKaRa); (4) Perform qPCR according to the following reaction system and conditions: qPCR reaction system: 10 μL SYBR Mix, 0.2 μL upstream primer (10 μM), 0.2 μL downstream primer (10 μM), 2 μL cDNA, ddH2O added to 20 μL.

[0022] qPCR reaction conditions: 95°C for 10 min; 95°C for 15 s, 60°C for 1 min (40 cycles); 95°C for 15 s, 60°C for 1 min, then slowly increase the temperature from 60°C to 95°C at a rate of 0.5°C / 5 s, and continuously collect fluorescence signals.

[0023] The primer design for ENST00000412203.1 is as follows: Upstream primer: 5'-ACCTGCACGTACACATCCAG-3', SEQ ID NO.4; Downstream primer: 5'-GGGAGCTTTTGAGCCAGGAT-3', SEQ ID NO.5; The primer design for AC006449.1 is as follows: Upstream primer: 5'-GGTGGGGTGTCTTGGTTTCA-3', SEQ ID NO.6; Downstream primer: 5'-GGCCCACTTAGAGCGAAACT-3', SEQ ID NO.7; The primer design for ENST00000606834.1 is as follows: Upstream primer: 5'-CCTTTGCTAGAGCTGGCACA-3', SEQ ID NO.8; Downstream primer: 5'-AGTAGTGAGGGGCAGAGAGG-3', SEQ ID NO.9; (5) The internal reference is GAPDH, and the relative expression is calculated using the 2^(-ΔΔCt) method.

[0024] The results of differential expression of three long non-coding RNAs are as follows: Figure 1 As shown, the ROC curves of the three long non-coding RNAs are as follows: Figure 2 As shown.

[0025] from Figure 1 The results showed that in breast cancer tissue, the expression levels of long non-coding RNA ENST00000412203.1 were 2.955±0.971, long non-coding RNA AC006449.1 were 2.253±0.699, and long non-coding RNA ENST00000606834.1 were 1.940±0.657. from Figure 2 The results show that the AUC value of long non-coding RNA ENST00000412203.1 is 0.9822, the AUC value of long non-coding RNA AC006449.1 is 0.934, and the AUC value of long non-coding RNA ENST00000606834.1 is 0.916.

[0026] The results above show that, in terms of gene expression level and diagnostic value, the long non-coding RNA ENST00000412203.1 is significantly better than the long non-coding RNAs AC006449.1 and ENST00000606834.1.

[0027] Example 2 (1) Take 30 breast cancer tissues and 20 lymph node metastasis tissues, extract RNA using the TRIzol method, determine the concentration and purity using NanoDrop, check the integrity by agarose electrophoresis and use it for subsequent operations; (2) Take 1 μg of RNA from each sample and use a reverse transcription kit to synthesize cDNA; (3) Perform qPCR according to the following reaction system and conditions: qPCR reaction system: 10 μL SYBR Mix, 0.2 μL upstream primer (10 μM), 0.2 μL downstream primer (10 μM), 2 μL cDNA, ddH2O added to 20 μL.

[0028] qPCR reaction conditions: 95°C for 10 min; 95°C for 15 s, 60°C for 1 min (40 cycles); 95°C for 15 s, 60°C for 1 min, then slowly increase the temperature from 60°C to 95°C at a rate of 0.5°C / 5 s, and continuously collect fluorescence signals.

[0029] The primer design for ENST00000412203.1 is the same as in Example 1; (4) The internal reference is GAPDH, and the relative expression is calculated using the 2^(-ΔΔCt) method.

[0030] The expression differences of three non-coding RNAs in breast cancer metastatic tissues are as follows: Figure 3 As shown, from Figure 3 The results showed that only ENST00000412203.1 expression was significantly increased in lymph node metastasis tissues, while the expression levels of long non-coding RNAs AC006449.1 and ENST00000606834.1 showed no significant difference. This indicates that ENST00000412203.1 may be closely related to the metastasis of breast cancer cells. Therefore, it was selected for further research in this invention.

[0031] Example 3 (1) Based on the transcript sequence of ENST00000412203.1, the following two siRNAs were designed and named siRNA-I and siRNA-II, respectively. The specific sequences are shown below: siRNA-I Chain of Justice: 5'-AGUAGAAGGUGUUCCUUAGAU-3', SEQ ID NO.10; Antisense chain: 5'-CUAAGGAACACCUUCUACUUA-3', SEQ ID NO.11; siRNA-II Chain of Justice: 5'-UUCAAUUUUAAGUAGAAGGUG-3', SEQ ID NO.12; Antisense chain: 5'-CCUUCUACUUAAAAUUGAAGU-3', SEQ ID NO.13; The siRNA was synthesized by Gemma Genetics, and the si-NC was provided by the company.

[0032] (2) MCF-7 cells were seeded in 6-well plates at a rate of 1×10^5 cells / well and cultured until the cells reached 70%-80% confluence. (3) Take 50 pmol siRNA and add it to 250 μL of serum-free Opti-MEM medium. Add 5 μL of Lipofectamine 3000 to 250 μL of Opti-MEM and incubate at room temperature for 5 minutes. (4) Mix the two tubes and incubate at room temperature for 20 minutes to form a complex. Replace the cell culture medium with serum-free Opti-MEM, add the transfection complex, and gently shake well. Set up 3 replicates for each group. (5) After incubating at 37°C for 6 hours, replace with DMEM medium containing 10% FBS and continue culturing for 48 hours; (6) 48 hours after transfection, RNA was extracted using the TRIzol method, and cDNA was synthesized by reverse transcription. The relative expression level of ENST00000412203.1 was determined according to Example 1.

[0033] from Figure 4 The results show that the inhibition rate of siRNA-I was 73.67%, while that of siRNA-II was 83.44%. The interference effect of siRNA-II was significantly better than that of siRNA-I. Therefore, siRNA-II was selected for the experiment in this invention.

[0034] Example 4 (1) After 24 hours of transfection culture, MCF-7 cells and MDA-MB-231 cells transfected with si-NC and si-RNA-II were digested with 0.25% trypsin-EDTA to prepare single-cell suspensions; (2) Seed 2×10^3 cells / well in 96-well plates and continue to culture for 48 hours. Each group has 5 replicate wells. (3) After the culture is completed, add 10 μL of CCK-8 solution to each well, incubate at 37°C for 2 h, and measure the OD value at 450 nm using a microplate reader; (4) Calculate the cell survival rate according to the formula: cell survival rate = (experimental group OD - blank OD) / (si-NC group OD - blank OD) ×100%.

[0035] Test results as follows Figure 5 As shown, from Figure 5 The results showed that for MCF-7 cells, the cell survival rate decreased to 69.23±2.46% after transfection with si-RNA-II, and for MDA-MB-231 cells, the cell survival rate decreased to 61.74±3.84% after transfection with si-RNA-II.

[0036] The results above show that inhibiting ENST00000412203.1 with si-RNA-II can effectively suppress cell proliferation.

[0037] Example 6 (1) MDA-MB-231 cells transfected with si-NC and si-RNA-II were digested with 0.25% trypsin-EDTA to prepare a single-cell suspension, which was adjusted to 1×10^6 cells / mL. (2) Add 600 μL of DMEM medium containing 10% FBS as a chemokine to the lower chamber of the Transwell chamber (8 μm pore size, Corning); (3) Add 100 μL of MDA-MB-231 cell suspension transfected with si-NC and si-RNA-II to the upper chamber of the Transwell chamber, respectively; (4) Place the Transwell chamber in a 37℃, 5% CO2 incubator for 24 h; (5) After the culture is completed, take out the chamber and gently wipe the surface of the upper chamber with a cotton swab dipped in PBS to completely remove the unmigrated cells.

[0038] (6) Immerse the chamber in pre-cooled methanol for 20 min to fix, then rinse with PBS 2-3 times; (7) After staining with 0.1% crystal violet solution for 20 min, rinse repeatedly with PBS until the background is clear and there is no excess dye; (8) Dry the chamber, observe and photograph the migrating cells on the chamber membrane using an inverted microscope, randomly select 5 fields of view to count the number of migrating cells, and calculate the average value.

[0039] The results obtained are as follows Figure 6 As shown, from Figure 6 It can be seen that the number of MDA-MB-231 cells transfected with si-RNA-II decreased significantly, indicating that inhibiting ENST00000412203.1 can effectively inhibit the migration ability of breast cancer cells.

[0040] Example 7 (1) Take the BD Matrigel out of the -80°C freezer and place it in the 4°C freezer overnight to thaw into a liquid state. Then dilute Matrigel with pre-cooled serum-free DMEM medium at a ratio of 1:8 to about 50 μg / mL. (2) After thorough mixing, take 60 μL of diluted gel and spread it evenly on the upper chamber membrane of Transwell. Place the Transwell chamber after gel spreading in a 37℃, 5% CO2 incubator for 30-60 min to allow Matrigel to polymerize into a gel barrier. (3) MDA-MB-231 cells transfected with si-NC and si-RNA-II were digested with 0.25% trypsin-EDTA to prepare a single-cell suspension, which was adjusted to 1×10^6 cells / mL. (4) Add 600 μL of DMEM medium containing 10% FBS as a chemokine to the lower chamber of the Transwell chamber; (5) Add 100 μL of MDA-MB-231 cell suspension transfected with si-NC and si-RNA-II to the upper chamber of the Transwell chamber, respectively; (6) Place the Transwell chamber in a 37℃, 5% CO2 incubator and incubate for 24 h; (7) After the culture is completed, take out the chamber and gently wipe the surface of the upper chamber with a cotton swab dipped in PBS to completely remove the unmigrated cells.

[0041] (8) Immerse the chamber in pre-cooled methanol for 20 min to fix, then rinse with PBS 2-3 times; (9) After staining with 0.1% crystal violet solution for 20 min, rinse repeatedly with PBS until the background is clear and there is no excess dye; (10) Dry the chamber, observe and photograph the invading cells on the chamber membrane using an inverted microscope, randomly select 5 fields of view to count the number of migrating cells, and calculate the average value.

[0042] The resulting graph is shown below. Figure 7 As shown, from Figure 7 The results showed that the number of invasive cells in MDA-MB-231 cells transfected with si-RNA-II was also significantly reduced, indicating that inhibiting ENST00000412203.1 can effectively inhibit the invasive ability of breast cancer cells.

Claims

1. A biomarker for breast cancer detection, characterized in that, The biomarker is the long non-coding RNA ENST00000412203.1, and the transcript sequence of the long non-coding RNA ENST00000412203.1 is shown in SEQ ID NO.

1.

2. The marker according to claim 1, characterized in that, The expression levels of the biomarkers in breast cancer tissues were significantly higher than those in adjacent normal tissues; The expression level of the biomarker in lymph node metastasis tissue was significantly higher than that in primary breast cancer tissue.

3. The application of a primer for detecting long non-coding RNA in the preparation of a PCR kit for detecting breast cancer, characterized in that, The long non-coding RNA is long non-coding RNA ENST00000412203.1, and the transcript sequence of the long non-coding RNA ENST00000412203.1 is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The sequences of the primers are shown in SEQ ID NO.4 and SEQ ID NO.

5.

5. The application of an inhibitor of long non-coding RNA ENST00000412203.1 expression in the preparation of a breast cancer therapeutic drug, characterized in that, The transcript sequence of the long non-coding RNA ENST00000412203.1 is shown in SEQ ID NO.1; The inhibitors include: (a) siRNA or shRNA; (b) Antisense oligonucleotides; (c) CRISPR / Cas system-mediated gene editing tools; (d) Small molecule compounds that target long non-coding RNA ENST00000412203.

1.

6. The application according to claim 5, characterized in that, The inhibitor is siRNA.

7. The application according to claim 6, characterized in that, The siRNA includes siRNA-I and siRNA-II; The sense strand sequence of the siRNA-I is shown in SEQ ID NO.10, and the antisense strand sequence of the siRNA-I is shown in SEQ ID NO.11; The sense strand sequence of the siRNA-II is shown in SEQ ID NO.12, and the antisense strand sequence of the siRNA-II is shown in SEQ ID NO.

13.

8. The application according to claim 7, characterized in that, The siRNA inhibits the proliferation, migration, and / or invasion of breast cancer cells by suppressing the expression of ENST00000412203.

1.

9. The application of a siRNA in the preparation of a cell inhibitor that inhibits the proliferation, migration, and / or invasion of breast cancer cells, characterized in that, The siRNA is the siRNA of the long non-coding RNA ENST00000412203.1, and the transcript sequence of the long non-coding RNA ENST00000412203.1 is shown in SEQ ID NO.

1.

10. The application according to claim 9, characterized in that, The siRNA includes siRNA-I and siRNA-II; The sense strand sequence of the siRNA-I is shown in SEQ ID NO.10, and the antisense strand sequence of the siRNA-I is shown in SEQ ID NO.11; The sense strand sequence of the siRNA-II is shown in SEQ ID NO.12, and the antisense strand sequence of the siRNA-II is shown in SEQ ID NO.13.

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