CN111187840A

CN111187840A - Biomarker for early breast cancer diagnosis

Info

Publication number: CN111187840A
Application number: CN202010100992.0A
Authority: CN
Inventors: 罗忠兵
Original assignee: First Affiliated Hospital of Gannan Medical University
Current assignee: First Affiliated Hospital of Gannan Medical University
Priority date: 2019-02-20
Filing date: 2020-02-19
Publication date: 2020-05-22
Anticipated expiration: 2040-02-19
Also published as: CN109628600A; CN111187840B

Abstract

The invention discloses a biomarker for early breast cancer diagnosis, wherein the biomarker is LINC01929, and the expression level of LINC01929 in a cancer tissue is found to be remarkably up-regulated by detecting a tissue sample and a tissue beside the cancer of breast cancer in stages I-II, so that the LINC01929 can be applied to diagnosis and treatment of early breast cancer.

Description

Biomarker for early breast cancer diagnosis

Technical Field

The invention belongs to the field of biomedicine, and relates to a biomarker for early breast cancer diagnosis, wherein the biomarker is LINC 01929.

Background

Breast cancer (breast cancer) is one of the most common malignancies in women, usually occurring in mammary epithelial tissue. According to data statistics, the incidence of breast Cancer accounts for 7-10% of various malignant tumors in the whole body (Darabi, H et al. Breast Cancer prognosis and induced diagnosis basal on common genetic variation and Breast Cancer Res., 2012.14(1): p.R25.). There are 135 million new increases of breast Cancer worldwide, of which 42 million deaths occur in 2% increments each year (Wen, w., et., Prediction of Breast Cancer based on common genetic variants in recent animal research Res,2016.18(1): p.124.). More than 4 million women die of the disease every year in China, and although the Chinese is not a country with high incidence of breast cancer, the growth rate of the Chinese is far higher than that of other countries. Its pathogenesis has many factors, and genetic susceptibility and gene-environment interaction are closely related to its occurrence, progression and metastasis (Mavaddat, Net al., Prediction of Breast Cancer basal on profiling with common genetic variants. J Natl Cancer Inst,2015.107 (5)). Although a variety of oncogenes and cancer suppressor genes have been found through previous studies to improve the diagnosis rate and treatment effect of breast cancer, the problems cannot be completely solved. There is therefore a need for a thorough understanding of the molecular mechanisms of tumorigenesis, which provides new and effective therapeutic approaches for the treatment of breast cancer patients. Early diagnosis and early treatment of breast cancer are key to reducing breast cancer mortality before the cause of breast cancer is not completely found.

Long non-coding RNAs (lncRNAs) are a class of nucleotide transcripts lacking protein coding functions, comprising 200 to 100000 nucleotides, with highly conserved sequence elements and specific spatial secondary structures, present in the nucleus or cytoplasm (Hajjari, M., A. Khoshun, and YK. shin, Molecular function and regulation of long non-coding RNAs: fragments with Molecular roles in nucleic acids. Tumour Biol,2014.35(11): p.10645-63.). LncRNAs play an important role in epigenetic regulation, alternative splicing, RNA decay, cell differentiation, cell cycle control, and cell regulation processes such as cancer cell metastasis and drug resistance (Richtig, G., et al, Function and Clinical indications of Long Non-codingRNAs in Melanoma. int J Mol Sci,2017.18 (4)). With the intensive research on lncRNAs, more and more evidences indicate that lncRNAs participate in various biological processes and disease pathogenesis, especially tumorigenesis, and the lncRNAs are generally differentially expressed in various human tumors. To date, lncRNAs have made modest advances in tumors, such as HOTAIR and GWS5, which have been identified as oncogenes and cancer suppressor factors, respectively. However, the number of lncRNAs is enormous, only a few lncRNAs related to cancer have been reported, and research on lncRNAs in breast cancer is still in the initial stage. Therefore, the novel lncRNAs critical to the occurrence and development of breast cancer are still to be discovered and explored by researchers.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide the lncRNA marker related to the breast cancer, and the lncRNA marker is applied to clinic, so that the early diagnosis and treatment of the breast cancer are realized, the effective intervention is carried out, and the survival rate and the survival quality of a patient are improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of LINC01929 in preparing a product for diagnosing early breast cancer.

Further, the product comprises an agent for detecting the expression level of LINC01929 in the sample.

Further, the reagent comprises a reagent for detecting the expression level of LINC01929 by reverse transcription PCR, real-time quantitative PCR, in-situ hybridization and a chip technology.

The invention provides a product for diagnosing breast cancer, which comprises a chip or a kit, wherein the chip or the kit comprises a reagent for detecting the expression level of LINC 01929.

Furthermore, the reagent for detecting the expression level of the LINC01929 in the chip comprises a probe for specifically recognizing the LINC01929 gene; the reagent for detecting the expression level of the LINC01929 in the kit comprises a primer for specifically amplifying the LINC01929 gene or a probe for specifically identifying the LINC01929 gene.

Further, the primer sequence of the specific amplification LINC01929 gene is shown in SEQ ID NO. 1-2.

In the present invention, the kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an auxiliary agent or a solvent, and instructions for use with the kit, wherein the instructions describe how to use the kit for detection, and how to use the detection results to determine tumor development and select a treatment regimen.

The invention provides application of LINC01929 in constructing a calculation model for predicting early breast cancer.

As the skilled person will be aware, the step of associating a marker level with a certain likelihood or risk may be carried out and carried out in different ways. Preferably, the measured concentrations of the marker and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.

Preferably, the mathematical algorithm applied in the marker combination is a logarithmic function. Preferably, the result of applying such a mathematical algorithm or such a logarithmic function is a single value. Such values can be readily correlated with, for example, an individual's risk for breast cancer or with other diagnostic purposes of interest that aid in the assessment of early breast cancer patients, based on underlying diagnostic questions. In a preferred manner, such a logarithmic function is obtained as follows: a) classifying individuals into groups, e.g., normal humans, individuals at risk for breast cancer, patients with breast cancer, etc., b) identifying markers that differ significantly between these groups by univariate analysis, c) logistic regression analysis to assess independent difference values of the markers that can be used to assess these different groups, and d) constructing a logistic function to combine the independent difference values. In this type of analysis, the markers are no longer independent, but represent a combination of markers.

The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in assessing breast cancer is selected from DA (i.e., linear, quadratic, regular discriminant analysis), Kernel method (i.e., SVM), non-parametric method (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based method (i.e., logistic regression, CART, random forest method, boosting method), or generalized linear model (i.e., logarithmic regression).

The invention provides application of LINC01929 in screening of candidate drugs for treating breast cancer.

Further, the screening steps were as follows:

treating a system expressing or containing the LINC01929 gene by using a substance to be screened; and

detecting the expression of the LINC01929 gene in the system;

wherein, if the substance to be screened can inhibit the expression level of the LINC01929 gene, the substance to be screened is a candidate drug for treating breast cancer.

The system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

The candidate substances include (but are not limited to): an interfering molecule, a nucleic acid inhibitor, a small molecule compound and the like designed aiming at the LINC01929 gene or the upstream or downstream gene thereof.

The invention provides application of LINC01929 in preparing a pharmaceutical composition for treating breast cancer.

Further, the pharmaceutical composition comprises an inhibitor of LINC 01929. The inhibitor is selected from: an interfering molecule which uses LINC01929 or a transcript thereof as a target sequence and can inhibit LINC01929 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

Further, the inhibitor is siRNA. In the specific embodiment of the invention, the sequence of the siRNA is shown in SEQ ID NO. 5-6.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier comprises (but is not limited to) diluents, binders, surfactants, humectants, adsorption carriers, lubricants, fillers, and disintegrating agents.

The pharmaceutical compositions of the invention may also be used in combination with other agents for the treatment of breast cancer, and other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

In the present invention, "marker", "biomarker", "genetic marker" may be used generically to refer to a molecular indicator having a specific biological property, biochemical characteristic or aspect, which may be used to determine the presence or absence of a particular disease or condition and/or the severity of a particular disease or condition.

In the present invention, the gene for transcribing LINC01929 is located in the 1 band of region 2 of human No. 18 chromosome, and ID is 101927229, and LINC01929 in the present invention includes wild type, mutant type or a fragment thereof. One representative LINC01929 is shown as NR _110743.1 disclosed in genebank. As will be appreciated by those skilled in the art, when performing bioinformatic analysis of the sequencing results, the sequencing results are typically aligned with a known genome, and the expression of the gene can be considered as long as the sequenced fragments can be aligned with the gene of interest.

The present invention may utilize any method known in the art to determine the expression level of a gene. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level.

Some methods of detection or quantification of lncRNA levels are known in the art and are all suitable for use in the methods provided herein to measure levels of biomarkers. Exemplary methods include, but are not limited to, northern blots (northern blots), ribonuclease protection assays, and PCR-based methods.

The assay method may vary depending on the type of lncRNA information desired. Exemplary methods include, but are not limited to, Northern blots (Northern blots) and PCR-based methods (e.g., qRT-PCR). The method of qRT-PCR and the like can also accurately quantify the amount of lncRNA in the sample.

The invention has the advantages and beneficial effects that:

the invention discovers that the differential expression of the LINC01929 is related to the occurrence and development of the breast cancer for the first time, and whether a subject suffers from early breast cancer can be judged by detecting the expression level of the LINC 01929.

The invention discloses a method for screening a candidate drug for treating breast cancer, which judges whether a substance to be screened is the candidate drug for treating the breast cancer by detecting whether the substance to be selected can reduce the expression level of LINC 01929.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC01929 gene in breast cancer tissue by QPCR.

FIG. 2 is a diagram showing CCK-8 detecting the effect of LINC01929 gene on breast cancer cell proliferation.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening of Gene markers associated with early Breast cancer

1. Sample collection

30 samples of stage I-II breast cancer tissue and their corresponding paracancerous tissue samples were collected, 5 samples were randomly selected for high throughput sequencing, all cases received no chemotherapy or radiotherapy prior to surgery, other neoplastic diseases, autoimmune diseases and severe chronic diseases were excluded, all patients had known informed consent, and were approved by the institutional ethics committee.

2. Preparation of RNA samples

Cutting tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1 min; standing at room temperature for 10min to completely decompose nucleoprotein; then adding 200 mul of chloroform (chloroform), covering a pipe cover tightly, violently shaking for 15s, standing for 10min at normal temperature, centrifuging for 15min at the temperature of 4 ℃ and the rpm of 11000; transferring the water sample layer into a new centrifuge tube, and adding 500 mul of isopropanol; after being reversed and mixed evenly, the mixture is stood for 10min at normal temperature, and then centrifuged for 15min at 11000rpm after being 4 ℃; carefully sucking away the liquid with a gun, leaving the precipitate at the bottom of the tube, adding 1ml of 75% ethanol, shaking on an oscillator for 5s, washing the precipitate once, and centrifuging at 4 ℃ and 8000rpm for 5 min; then carefully removing the supernatant, drying the precipitate for 10min, and adding a proper amount of water to dissolve the precipitate for 10 min.

3. Total RNA quantitation and purity analysis

The extracted RNA is subjected to agarose gel electrophoresis, the concentration and purity of the extracted RNA are detected by using Nanodrop2000, the integrity of the RNA is detected by the agarose gel electrophoresis, and the RIN value is determined by Agilent 2100. The total amount of RNA required for single library construction is 5 mug, the concentration is more than or equal to 200 ng/mug, and the OD260/280 is between 1.8 and 2.2.

4. Construction of cDNA library

Ribosomal RNA was removed from total RNA using the Ribo-Zero kit from Epicentre; randomly breaking the complete RNA sequence by using metal ions, and randomly breaking the RNA into small fragments of about 200 bp; the construction of cDNA library was carried out using IlluminaTruseq TM RNA sample Prep Kit.

5. Sequencing

2X 150bp sequencing was performed using the Illumina X-Ten sequencing platform.

6. High throughput transcriptome sequencing data analysis

Deletion of non-detectable lncRNA differential expression analysis of reads numbers using DESeq2 in the R-3.3.3 tool, differential expression lncRNA screening criteria: FDR <0.05, abs (log2FC) > 2.

7. Results

The results show that LINC01929 is significantly up-regulated in expression levels in early breast cancer tissues compared to paracancerous tissues.

Example 2 QPCR sequencing verification of differential expression of LINC01929 Gene

1. Large sample QPCR validation of LINC01929 was performed using 30 previously collected tissue samples of stage I-II breast cancer and paracancerous tissue samples.

2. RNA extraction procedure as in example 1

3、QPCR

1) Reverse transcription reaction

Reverse transcription of lncRNA was performed using the FastQ μ ant cDNA first strand synthesis kit (cat # KR106) from Tiangen.

First, remove the genomic DNA reaction, add 5 XgDNA B. mu.ffer 2.0. mu.l, total RNA 1. mu.g, RNase Free ddH to the tube₂O to make the total volume 10. mu.l, heating in a water bath at 42 ℃ for 3min, and adding 10 Xfast RT B. mu.ffer 2.0. mu.l, RT Enzyme Mix 1.0. mu.l, FQ-RT Primer Mix 2.0. mu.l, RNase Free ddH₂O5.0 μ l, mixing, adding into the above test tube, mixing to give 20 μ l, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3 min.

2) Primer design

A QPCR amplification primer is designed according to the coding sequences of a LINC01929 gene and a GAPDH gene in Genebank, and when the primer design of the LINC01929 is carried out, common sequences of different transcription product sequences are selected for design, and specific primer sequences are as follows:

LINC01929 gene:

the forward primer is 5'-TCCTCTCATACCACTAACATC-3' (SEQ ID NO. 1);

the reverse primer was 5'-GCACCAACTTCAAGACAAT-3' (SEQ ID NO. 2).

GAPDH gene:

the forward primer is 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO. 3);

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO. 4).

3) QPCR amplification assay

Amplification was performed using SuperReal PreMix Plus (SYBR Green) (cat # FP 205).

A20. mu.l reaction was used: 2 XSuperReal PreMix Plus 10. mu.l, forward and reverse primers (10. mu.M) 0.6. mu.l each, 5 XROX Reference Dye^△2. mu.l, DNA template 2. mu.l, sterilized distilled water 4.8. mu.l. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.

The amplification procedure was: 95 degrees 15min, (95 degrees 10s, 55 degrees 30s, 72 degrees 32s) x 40 cycle.

4) Sample RealTime PCR detection

After 10-fold dilution of cDNA of each sample, 2 μ l of cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification. Simultaneously performing dissolution curve analysis at 60-95 deg.C, and determining target band by dissolution curve analysis and electrophoresis, 2^-ΔΔCTThe method is used for relative quantification.

4. Statistical analysis

The experiment was repeated 3 times, and all data were expressed as mean ± standard deviation (mean ± SD). Comparisons between two groups were performed using a two-sided Student's t test, and three and more groups were analyzed using one-way anova. All results were plotted using graphpad software, with P <0.05 defined as statistically significant differences. ROC curve analysis was performed on variable LINC01929 using SPSS to determine the diagnostic potency, sensitivity and specificity of the gene.

5. Results

The QPCR result is shown in figure 1, compared with the tissue beside the cancer, the expression of LINC01929 is up-regulated in the early breast cancer tissue, and the difference has statistical significance (P < 0.05); 29 cancer tissue samples with up-regulated expression exist in the samples, and 1 cancer tissue sample without significant difference exists in the samples, which suggests that LINC01929 can be used as a molecular marker for diagnosis and treatment of breast cancer.

ROC curve analysis shows that LINC01929 can be used as a biomarker for diagnosing breast cancer, the area under the curve is 0.967, and specific values are shown in Table 1, which indicates that LINC01929 expression has high sensitivity and specificity on diagnosis of breast cancer.

TABLE 1 area under the curve

Variable of inspection result LINC01929

a. Under the nonparametric assumption

b. Zero hypothesis: real area is 0.5

Example 3 Effect of LINC01929 on Breast cancer cells

1. Cell culture

The BT474 cell line of Luminal type B breast cancer was cultured in DMEM medium containing 10% fetal bovine serum (Gibco) in 5% CO₂And culturing at 37 deg.C in a constant temperature incubator. The growth of the cells was observed every day, and the cells were subcultured by changing the medium every other day.

2. Transfection

The siRNNA aiming at LINC01929 is designed and synthesized by Shanghai Ji code pharmaceutical technology Limited, and the contrast is universal siRNA-NC.

The siRNA-LINC01929 sequence of the silent LINC01929 is shown as follows.

The sense strand is 5'-AAUAUGCACCAACUUCAAGAC-3' (SEQ ID NO.5)

The antisense strand is 5'-CUUGAAGUUGGUGCAUAUUUC-3' (SEQ ID NO.6)

Lipofectamin from Invitrogen was used^TM2000, transfecting siRNA of LINC01929 to breast cancer BT474 cells in a logarithmic growth phase, preparing cells planted in a 6-well plate in an incubator in advance before cell transfection, and changing the liquid of the cells in the 6-well plate and continuously culturing 24 hours after transfection. The experiment was divided into 3 groups, a blank control group (BT474), a negative control group (siRNA-NC) and an experimental group (transfected siRNA).

3. QPCR detection of the expression level of LINC01929 in cells

Total cellular RNA was extracted using Trizol followed by reverse transcription and real-time quantitative PCR detection as in example 1.

4. CCK-8 detection of the Effect of LINC01929 on Breast cancer cell proliferation

The breast cancer cells transfected with siRNA-LINC01929 are used as an experimental group, the cells transfected with siRNA-NC are used as a control group, the cells are added into a 96-well plate, the number of the cells added into each well is 5000, each group is provided with 5 multiple wells, 10 mu l of CCK-8 detection solution is added into the cell wells when the cells are cultured for 72h, the 96-well plate is continuously placed into a cell incubator to be incubated for about 4h, an enzyme-labeling instrument is used for detecting the absorbance value of each well at the wavelength of 450nm and recording data, and the proliferation condition of the cells is detected according to the detected OD value.

5. Statistical analysis

All data are expressed as mean ± standard deviation (mean ± SD). Comparisons between two groups were performed using a two-sided Student's t test, and three and more groups were analyzed using one-way anova. All results were analyzed using GraphPad Software, with P <0.05 defined as statistically significant differences.

6. Results

The siRNA transfection results showed that, with the expression level of LINC01929 in the blank control group set as 1 as a reference, the expression level of LINC01929 in the transfected blank control group (relative expression level of 1) was significantly decreased compared to the expression level of LINC01929 in the transfected siRNA-NC group (relative expression level of 0.920 ± 0.036), and the expression level of LINC01929 in the transfected siRNA-LINC01929 experimental group (relative expression level of 0.117 ± 0.035) was significantly decreased, with statistical significance for the difference (experimental group vs blank control group, P ═ 0.0005; experimental group vs siRNA-NC group, P ═ 0.0005), whereas there was no significant difference between the siRNA-NC group and the blank control group (P ═ 0.0615)

CCK-8 cell proliferation activity results As shown in FIG. 2, the OD450(1.420 +/-0.087) of the experimental group transfected with siRNA is obviously lower than that of the control group (0.856 +/-0.0456) transfected with siRNA-NC, which indicates that LINC01929 can influence the proliferation activity of breast cancer cells, and the expression level of LINC01929 can change the proliferation capacity of the breast cancer cells.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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Claims

The application of LINC01929 in preparing a product for diagnosing early breast cancer.
2. The use according to claim 1, wherein the product comprises an agent for measuring the level of LINC01929 expression in a sample.
3. The use according to claim 2, wherein the reagents comprise reagents for detecting the expression level of LINC01929 by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, chip technology.
4. A product for diagnosing early breast cancer, which is characterized by comprising a chip or a kit, wherein the chip or the kit comprises a reagent for detecting the expression level of LINC 01929.
5. The product according to claim 4, characterized in that the reagents for detecting the expression level of LINC01929 in the chip comprise a probe specifically recognizing the LINC01929 gene; the reagent for detecting the expression level of the LINC01929 in the kit comprises a primer for specifically amplifying the LINC01929 gene or a probe for specifically identifying the LINC01929 gene.
6. The product according to claim 5, wherein the primer sequence for specifically amplifying the LINC01929 gene is shown in SEQ ID No. 1-2.
Application of LINC01929 in constructing a calculation model for predicting early breast cancer.
The application of LINC01929 in screening candidate drugs for treating breast cancer.
Application of LINC01929 in preparing a pharmaceutical composition for treating breast cancer.
10. The use according to claim 9, wherein the pharmaceutical composition comprises an inhibitor of LINC01929, preferably the inhibitor is siRNA.