CN112029864A - Breast cancer miRNA detection kit and application thereof - Google Patents
Breast cancer miRNA detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses application of combined detection of expression levels of miR-1246, miR-141 and miR-520a-3p in preparation of a product for diagnosing breast cancer and a related detection kit. In addition, the invention also provides application of miR-1246, miR-141 and miR-520a-3p in preparation of a pharmaceutical composition for treating breast cancer and a related pharmaceutical composition. The kit can be used as one of the means for diagnosing the breast cancer, has the advantages of quick and convenient detection, high detection sensitivity, good specificity and low cost, can meet the detection requirements of most tumor patients, and has wide application range. The pharmaceutical composition has a good effect of inhibiting breast cancer, and has important reference significance and practical significance in breast cancer treatment.
Description
Technical Field
The invention relates to a miRNA detection kit related to breast cancer and application thereof, belonging to the field of biomedicine, in particular to the technical field of molecular biology.
Background
Breast cancer (breast cancer) is a malignant tumor that occurs in mammary gland epithelial tissue. Breast cancer occurs in 99% of women and only 1% in men. At present, breast cancer becomes a common tumor threatening the physical and mental health of women. The incidence of breast cancer worldwide has been on the rise since the end of the 70 s of the 20 th century. In the United states, 1 woman will have breast cancer in their lifetime. In recent years, the growth rate of the incidence rate of breast cancer in China is 1 to 2 percent higher than that of high incidence countries. According to 2009 breast cancer onset data published by the national cancer center and health department disease prevention and control agency 2012, it is shown that: the incidence of breast cancer of women in the national tumor registration area is 1 st of malignant tumors of women, the incidence of breast cancer of women is 42.55/10 ten thousand in total nationally, the incidence of breast cancer of women is 51.91/10 ten thousand in cities, and the incidence of breast cancer of women in rural areas is 23.12/10 ten thousand. Breast cancer has become a major public health problem in the current society. The early stage of breast cancer is clinically asymptomatic, the latent stage is averagely 12 years, and the early stage of breast cancer is usually middle and advanced stage when obvious symptoms exist.
MicroRNA (miRNA) is an endogenous, small RNA of about 20-24 nucleotides in length that has a number of important regulatory roles within the cell. Each miRNA may have multiple target genes, and several mirnas may also regulate the same gene. The complex regulatory network can regulate the expression of multiple genes through one miRNA or can finely regulate the expression of a certain gene through the combination of several miRNAs. It is speculated that mirnas regulate one third of the genes in humans. Recent studies have shown that approximately 70% of mammalian mirnas are located in the TUs region (TUs), and most of them are located in intronic regions. The location of some intronic mirnas is highly conserved across different species. mirnas are not only conserved in gene position, but also exhibit high homology in sequence. The conservation of miRNA is closely related to the importance of the function. The miRNA is closely related to the evolution of a target gene of the miRNA, and the research on the evolution history of the miRNA is helpful for further understanding the action mechanism and the function of the miRNA.
More and more literatures report that MicroRNA plays an important role in the occurrence, development and metastasis of cancer, and the inhibition or overexpression of some MicroRNAs can resist cancer. Therefore, it is necessary to investigate whether MicroRNA can also play a role in resisting cancer or delaying the development of cancer in the case of breast cancer.
Disclosure of Invention
In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide miRNAs markers associated with the development of breast cancer, which can be used as specific diagnostic markers for breast cancer; the invention also aims to provide application of the miRNAs markers in screening candidate drugs for treating breast cancer.
Therefore, the invention provides application of miRNAs and a reagent for jointly detecting the expression level of the miRNAs in preparing a product for diagnosing breast cancer, wherein the miRNAs are miR-1246, miR-141 and miR-520a-3 p.
In another aspect, the invention provides a breast cancer miRNA detection kit, which comprises a reagent for jointly detecting the expression levels of miR-1246, miR-141 and miR-520a-3p, wherein the reagent for detecting the expression levels of miR-1246, miR-141 and miR-520a-3p comprises a PCR reaction solution, the PCR reaction solution comprises a primer pair and a Taqman probe, and the sequences of the primer pair and the Taqman probe are as follows:
miR-1246-F：5’-TGTATCCTTGAATGGATTTT-3’；
miR-1246-R：5’-TCAATCCATAGGCTAGCAAT-3’；
miR-1246-Taqman-MGB：5’-FAM-AGTGGACACCTGACCCAAAG-MGB-3’；
miR-141-F：5’-CGGCCGGCCCTGGGTCCATC-3’；
miR-141-R：5’-GATGGCTCCCGGGTGGGTTC-3’；
miR-141-Taqman-MGB：5’-HEX-TTGTGAAGCTCCTAACACTG-MGB-3’；
miR-520a-3p-F：5’-CTCAGGCTGTGACCCTCCAG-3’；
miR-520a-3p-R：5’-TTGGACTGTTTCGGTTTGAG-3’；
miR-520a-3p-Taqman-MGB：5’-ROX-CTGTTGTCTGAGAGAA-MGB-3’。
preferentially, the 5 'end of the nucleotide sequence of the miR-1246Taqman probe is marked with an FAM fluorescent reporter group, the 3' end of the nucleotide sequence of the miR-1246Taqman probe is marked with an MGB quenching group, the 5 'end of the nucleotide sequence of the miR-141Taqman probe is marked with an HEX fluorescent reporter group, and the 3' end of the nucleotide sequence of the miR-141Taqman probe is marked with an MGB quenching group; the 5 'end of the nucleotide sequence of the miR-520a-3p Taqman probe is marked with an ROX fluorescent reporter group, and the 3' end is marked with an MGB quenching group.
Preferably, the PCR reaction solution further comprises a primer pair of an internal reference gene U6 and a Taqman probe, wherein the sequences of the primer pair and the Taqman probe are as follows:
U6-F：5’-TGCGGGTGCTCGCTTCGGCAGC-3’；
U6-R：5’-CCAGTGCAGGGTCCGAGGT-3’；
U6-Taqman-MGB：5’-CY5-CCTGCGCAA GGATGACACG-MGB-3’。
preferably, the 5 'end of the nucleotide sequence of the U6 Taqman probe is marked with a CY5 fluorescent reporter group, and the 3' end is marked with an MGB quenching group.
Preferably, the PCR reaction solution of the present invention further comprises 2 × One Step RT-PCR Buffer.
Preferably, the kit of the invention further comprises an enzyme mixture: PrimeScript RT Enzyme Mix.
Preferably, the reaction conditions of the kit of the present invention are: 5min at 42 ℃ and 10s at 95 ℃ as a first circulation step; 95 ℃ 5s, 58 ℃ 30s for 40 cycles of the second step, which was performed at the end of the extension for each cycle with detection of the fluorescence signal.
Preferably, the determination formula of the kit of the present invention is:
Y＝0.0261*AmiR-1246+0.0941*BmiR-141-0.0352*CmiR-520a-3p；
wherein A ismiR-1246Is the expression level of miR-1246, BmiR-141Is the expression level of miR-141, CmiR-520a-3pIs the expression level of miR-520a-3 p;
when Y is more than or equal to 0.05, judging the sample to be positive; when Y is less than 0.0499, the test result is negative.
In another aspect, the invention also provides application of miR-1246, miR-141 and miR-520a-3p genes, which is characterized in that the application is used for preparing a pharmaceutical composition for treating breast cancer.
Preferably, the pharmaceutical composition comprises miR-1246, an inhibitor of miR-141 gene and miR-520a-3 p.
Preferably, the inhibitor of the miR-1246 gene is si-miR-1246, and the sequence of the si-miR-1246 is shown in SEQ ID No. 1.
Preferably, the inhibitor of the miR-141 gene is si-miR-141, and the sequence of si-miR-1246 is shown in SEQ ID NO. 2.
The invention screens miRNAs markers for breast cancer diagnosis and treatment by bioinformatics and the existing technical means of molecular biology, and selects miR-1246, miR-141 and miR-520a-3p from the miRNAs markers as combined molecular markers (in breast cancer, miR-1246 and miR-141 are up-regulated, and miR-520a-3p is down-regulated).
On the basis, the invention provides an application of jointly detecting the expression levels of miR-1246, miR-141 and miR-520a-3p in preparing a product for diagnosing breast cancer and a related detection kit. The kit can be used as one of the means for diagnosing the breast cancer, has the advantages of quick and convenient detection, high detection sensitivity, good specificity, low cost, wide application range and the like, and can meet the detection requirements of most tumor patients. The method comprises the following specific steps: (1) the invention designs and synthesizes a specific primer and a Taqman-MGB probe according to a conservative specific sequence, adopts a fluorescent quantitative PCR method to rapidly and sensitively detect 3 miRNAs, has high accuracy, specificity and sensitivity and good stability, and can realize rapid and effective detection on a sample to be detected. (2) As the quantitative detection technology-Taqman-MGB fluorescent quantitative PCR (Real-time PCR) is adopted, the method (Real-time PCR) has the advantages of single-tube closed operation, pollution prevention, high automation degree, strong specificity, Real-time monitoring and the like, and effectively solves the limitation that the traditional method can only carry out end-point detection. (3) Because the quenching group at the 3' end of the probe is a non-luminous fluorescent group and is closer to the position of the reporter group in space, the experimental sensitivity is higher and the specificity is stronger. (4) The Taqman-MGB probe fluorescent quantitative PCR method is simple, convenient and quick, the whole process (including sample adding, 3 miRNAs can be detected only by adding a sample once) can be completed within one hour, a computer automatically reports the result, electrophoresis and other subsequent work are not needed, the operation is convenient, and the pollution is reduced.
In addition, the invention also provides application of miR-1246, miR-141 and miR-520a-3p in preparation of a pharmaceutical composition for treating breast cancer and a related pharmaceutical composition. The pharmaceutical composition has a good effect of inhibiting breast cancer, and has important reference significance and practical significance in breast cancer treatment.
Drawings
FIG. 1 is a graph showing the results of the analysis of the expression levels of miRNAs in breast cancer tissues and paracarcinoma tissues.
Detailed Description
The invention is further illustrated below with reference to specific examples. The various starting materials mentioned in the following examples are all commercially available unless otherwise specified.
The invention, through extensive and intensive research, detects the expression level of miRNA in breast cancer by bioinformatics technology and molecular biology technology, finds miRNA fragments with obvious expression difference, and discusses the relationship between the miRNA fragments and the occurrence of breast cancer, thereby finding better ways and methods for the detection and the targeted therapy of breast cancer. Through screening, the method selects miR-1246, miR-141 and miR-520a-3p as combined molecular markers (in breast cancer, miR-1246 and miR-141 are up-regulated, but miR-520a-3p is down-regulated). Further experiments prove that the change of the expression levels of miR-1246, miR-141 and miR-520a-3p can influence the growth of breast cancer, and the miR-1246, miR-141 and miR-520a-3p can be used as drug targets for accurate treatment of breast cancer.
"biomarker" and "marker" are used interchangeably to refer to a molecular indicator of a specific biological property, biochemical characteristic or aspect, which can be used to determine the presence or absence and/or severity of a particular disease or condition. In the present invention, "marker" refers to a parameter associated with one or more biomolecules (i.e., "biomarker"), such as naturally or synthetically produced nucleic acids (i.e., individual genes, as well as coding and non-coding DNA and RNA). "marker" in the context of the present invention also includes reference to a single parameter which may be calculated or otherwise obtained by taking into account expression data from two or more different markers. In the present invention, the term "biomarker" refers to a gene, a fragment or a variant of a gene associated with breast cancer.
The full-length nucleotide sequences or fragments of miR-1246, miR-141 and miR-520a-3p can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method.
In the present invention, gene expression can be determined using any method known in the art. 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. The mirnas of the present invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques. Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.
Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.
The nucleic acid amplification technique of the invention is selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.
The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA autocatalytically synthesizes multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength, and pH, wherein the multiple RNA copies of the target sequence autocatalytically generate additional copies; the ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of a target nucleic acid; other amplification methods include, for example: nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using an RNA replicase (commonly referred to as Q β replicase); a transcription-based amplification method; and self-sustained sequence amplification.
Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.
The invention provides a kit which can be used for detecting the expression of miR-1246, miR-141 and miR-520a-3 p.
In certain embodiments, the kit comprises one or more probes that specifically bind to mRNA of one or more biomarkers. In certain embodiments, the kit further comprises a wash solution. In certain embodiments, the kit further comprises reagents for performing hybridization assays, mRNA isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kit may be customized for home use, clinical use, or research use. For example, the kit provided by the invention is based on qRT-PCR experimental sources, the invention not only provides a primer for detecting miR-1246, miR-141 and miR-520a-3p, but also provides a specific detection method, and on the basis, the invention provides a qRT-PCR detection kit for detecting miR-1246, miR-141 and miR-520a-3p expression levels and application of the kit in breast cancer diagnosis.
Such a kit may employ, for example, a test strip, membrane, chip, tray, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The solid support of the kit can be, for example, a plastic, a silicon wafer, a metal, a resin, a glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide. The biological sample may be, for example, a cell culture, cell line, tissue, oral tissue, gastrointestinal tissue, organ, organelle, biological fluid, blood sample, urine sample, or skin inhibitor and drug (composition).
Based on the discovery of the inventor, the invention also provides an inhibitor of miR-1246 and miR-141, the property of the inhibitor is not important for the invention, as long as the inhibitor inhibits the functional expression of miR-1246 and miR-141 genes, for example, the inhibitor of the invention can be an interfering molecule which takes miR-1246 and miR-141 genes as target sequences and can inhibit miR-1246 and miR-141 genes, and comprises: 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. The inhibitors are useful as substances for down-regulating miR-1246 and miR-141, and can be used for treating breast cancer.
As a preferable mode of the invention, the inhibitor of miR-1246 and miR-141 is siRNA specific to miR-1246 and miR-141. As used herein, the inhibitor used in combination with miR-520a-3p can achieve the purpose of inhibiting the occurrence and development of breast cancer.
The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.
The pharmaceutical composition comprises inhibitors of miR-1246 and miR-141, miR-520a-3p and a pharmaceutically-acceptable carrier. The pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, disintegrants. In embodiments of the invention, the pharmaceutical compositions of the invention target the inhibition of the occurrence and development of breast cancer. In addition, in another embodiment of the present invention, the pharmaceutical composition of the present invention has a good effect of inhibiting the occurrence and development of breast cancer in a nude mouse tumor model.
Of course, the pharmaceutical compositions of the invention may also be combined 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.
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1: expression level detection of miR-1246, miR-141 and miR-520a-3p in breast cancer
The specific operation processes (such as primer, miRNA extraction and fluorescent quantitative RT-PCR reaction) of the embodiment 1 are participated in the expression quantity (2) of miR-1246, miR-141 and miR-520a-3p in the tissues and the paracarcinoma tissues of 30 breast cancer patients-△△CTValue) is detected and analyzed. The specific results are shown in FIG. 1. As can be seen from the figure, the expression levels of miR-1246 and miR-141 in breast cancer are obviously up-regulated, and the expression level of miR-520a-3p is obviously down-regulated.
Expression levels (2) of miR-1246, miR-141 and miR-520a-3p in human breast cancer cells (MCF7) by using the above method-△△CTValue) is detected andthe results of the analysis are shown in the following table:
| miRNA | delta Ct value (target miRNA Ct value-U6 Ct value) |
| miR-1246 | 5.82 |
| miR-141 | 6.23 |
| miR-520a-3p | -0.38 |
As can be seen from the table, the expression level of miR-1246 and miR-141 is far higher than that of U6, and the expression level of miR-520a-3p is lower than that of U6.
Example 2: establishment of expression level kit for jointly detecting miR-1246, miR-141 and miR-520a-3p
1. Design of primers and Taqman-MGB probes
According to the sequences of miR-1246, miR-141 and miR-520a-3p, Primer 5.0 software is used for designing primers and probes. Obtaining a plurality of pairs of specific primers and probes, and finally determining a group of optimal primers and MGB probes respectively through comparison and screening. In particular, see the following table:
as the TaqMan-MGB probe has the following advantages compared with the traditional TaqMan-TAMRA probe: (1) increase of TM value: on average 15bases can increase the temperature by 18 ℃, which can shorten the length of the probe, especially greatly help the design of sequences with high AT content, and increase the difference of TM value between the paired and unpaired templates. (2) Improving the signal-to-noise ratio: since the quencher group at the 3' end of the probe is a non-luminescent fluorophore group and is spatially closer to the reporter group. Therefore, the fluorescence quantitative experiment result is more accurate and the resolution ratio is higher.
Extraction of miRNA
Extraction of miRNA was performed using an extraction Kit (TaqMan ABC miRNA Purification Kit) purchased from Thermo Fisher, USA. The specific operation steps are detailed in the specification.
And (3) determining the concentration of the extracted miRNA sample and the ratio of OD260/OD280 to control the sample quality, and selecting the sample with the ratio of OD260/OD280 between 1.8-2.0 for subsequent experiments.
3. Fluorescent quantitative RT-PCR reaction
The fluorescent quantitative PCR reaction solution was prepared using One-Step PrimeScript RT-PCR Kit (Bao bioengineering Daizian Co., Ltd., product No. RR064A) as follows:
the fluorescent quantitative PCR reaction conditions are as follows: 5min at 42 ℃ and 10s at 95 ℃ as a first circulation step; 95 ℃ 5s, 58 ℃ 30s for 40 cycles of the second step, which was performed at the end of the extension for each cycle with detection of the fluorescence signal.
4. Kit judgment standard
The two indexes are fitted through a Logistic regression equation, and the sensitivity and specificity after fitting are higher than those of the single indexes. Through data analysis, a judgment formula (regression equation) is obtained as follows:
Y＝0.0261*A miR-1246+0.0941*BmiR-141-0.0352*CmiR-520a-3p；
wherein A ismiR-1246Is the expression level of miR-1246 (2)-△△CTValue), BmiR-141Is the expression level of miR-141 (2)-△△CTValue), CmiR-520a-3pIs the expression level of miR-520a-3p (2)-△△CTA value);
when Y is more than or equal to 0.05, judging the sample to be positive; when Y is less than 0.0499, the test result is negative.
Example 3: detection of clinical samples by kit
The detection kit in example 2 is adopted to express miR-1246, miR-141 and miR-520a-3p in serum of 30 breast cancer patients and 30 healthy people (2)-△△CTValue) is detected. And carrying out regression analysis on the detection results by using origin software, wherein the single detection index results of miR-1246, miR-141 and miR-520a-3p and the combined results of miR-1246, miR-141 and miR-520a-3p are shown in the following table.
As shown in the table above, the P values of each index and the combined index are all <0.05, which indicates that each detection index is significantly related to the prediction of breast cancer. The combined prediction accuracy of miR-1246, miR-141 and miR-520a-3p reaches 96.8% (area under ROC curve), and is higher than the prediction rate of a single detection index and the prediction rate of a pairwise combination (both are less than 90%). The regression analysis proves that the sensitivity and specificity of the kit for three-index combined detection are 95 percent and 99 percent respectively higher than those of a single index and those of a pair of combined indexes (the sensitivity is less than 85 percent and the specificity is less than 97 percent), and all three-index combined detection has obvious advantages in breast cancer.
Example 4: preparation of pharmaceutical composition for treating breast cancer
From the 3 previous examples, the expression levels of miR-1246, miR-141 and miR-520a-3p can be used as diagnostic markers of breast cancer, and the expression levels of miR-1246 and miR-141 are remarkably up-regulated and the expression level of miR-520a-3p is remarkably down-regulated in breast cancer tissues. We speculate that the miR-1246, the interference of miR-141 and miR-520a-3p can be used for treating the breast cancer to a certain extent, so that a new possibility and direction are provided for the targeted treatment of the breast cancer.
In view of the above, we designed and synthesized si-miR-1246 (the specific sequence is shown in SEQ ID NO. 1), si-miR-141 (the specific sequence is shown in SEQ ID NO. 2) and miR-520a-3p (the specific sequence is shown in SEQ ID NO. 3).
Example 5: validation of pharmaceutical compositions
The nude mouse tumorigenesis model is used for carrying out test treatment verification on related medicaments, and the specific scheme is as follows:
NOD/SCID/IL2r γ (null) mice (NSG) (8week-old) (Hunan Slek Sedrida). All mice were injected subcutaneously at a corresponding concentration of 0.6X 105MCF7 cells. Relevant pharmaceutical compositions were injected subcutaneously into mice at fixed points, and experimental groups were as follows: 1) blank control group, no drug combination was injected; 2) in the si-miR-1246 group, si-miR-1246 is injected; 3) in the si-miR-141 group, si-miR-141 is injected; 4) miR-520a-3p group, miR-520a-3p is injected; 5) in the si-miR-1246+ si-miR-141 group, si-miR-1246 and si-miR-141 are injected at the same time; 6) in the group of si-miR-1246+ miR-520a-3p, si-miR-1246 and miR-520a-3p are injected at the same time; 7) the group of si-miR-141+ miR-520a-3p is injected with si-miR-141 and miR-520a-3p at the same time; 8) and the si-miR-1246+ si-miR-141+ miR-520a-3p group is simultaneously injected with si-miR-1246, si-miR-141 and miR-520a-3 p. Measuring and recording the change of the nude mouse transplanted tumor volume in real time, and using V ═ A × B2/2(mm3) The equation calculates the tumor volume, where a is the maximum diameter and B is the perpendicular diameter. When the tumor volume reaches 1,000mm3At time, mice were sacrificed, tumors excised and weighed. The results are shown in the following table:
as can be seen from the table, the si-miR-1246+ si-miR-141+ miR-520a-3p group has the best treatment effect, two treatment groups are used together, the single treatment group is used at the first time, and all treatment groups are better than the blank control group.
The above description is not intended to limit the invention, nor is the invention limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the spirit of the invention.
Sequence listing
<110> Chen Wei nation
<120> breast cancer miRNA detection kit and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> si-miR-1246 Sequence [ Artificial Sequence ]
<400> 1
aaacctcgtc ctcacctgta 20
<210> 2
<211> 21
<212> DNA
<213> si-miR-141 Sequence [ Artificial Sequence ]
<400> 2
ggtccatctt ccagtacagt g 21
<210> 3
<211> 85
<212> DNA
<213> miR-520a-3p Sequence [ Artificial Sequence ]
<400> 3
ctcaggctgt gaccctccag agggaagtac tttctgttgt ctgagagaaa agaaagtgct 60
tccctttgga ctgtttcggt ttgag 85
Claims (10)
1. The breast cancer miRNA detection kit is characterized by comprising a reagent for jointly detecting the expression levels of miR-1246, miR-141 and miR-520a-3p, wherein the reagent for detecting the expression levels of miR-1246, miR-141 and miR-520a-3p comprises a PCR reaction liquid, the PCR reaction liquid comprises a primer pair and a Taqman probe, and the sequences of the primer pair and the Taqman probe are as follows:
miR-1246-F：5’-TGTATCCTTGAATGGATTTT-3’；
miR-1246-R：5’-TCAATCCATAGGCTAGCAAT-3’；
miR-1246-Taqman-MGB：5’-FAM-AGTGGACACCTGACCCAAAG-MGB-3’；
miR-141-F：5’-CGGCCGGCCCTGGGTCCATC-3’；
miR-141-R：5’-GATGGCTCCCGGGTGGGTTC-3’；
miR-141-Taqman-MGB：5’-HEX-TTGTGAAGCTCCTAACACTG-MGB-3’；
miR-520a-3p-F：5’-CTCAGGCTGTGACCCTCCAG-3’；
miR-520a-3p-R：5’-TTGGACTGTTTCGGTTTGAG-3’；
miR-520a-3p-Taqman-MGB：5’-ROX-CTGTTGTCTGAGAGAA-MGB-3’。
2. the kit according to claim 1, wherein the 5 'end of the nucleotide sequence of the miR-1246Taqman probe is labeled with a FAM fluorescent reporter group, the 3' end of the nucleotide sequence of the miR-1246Taqman probe is labeled with an MGB quencher group, the 5 'end of the nucleotide sequence of the miR-141Taqman probe is labeled with a HEX fluorescent reporter group, and the 3' end of the nucleotide sequence of the miR-141Taqman probe is labeled with an MGB quencher group; the 5 'end of the nucleotide sequence of the miR-520a-3p Taqman probe is marked with an ROX fluorescent reporter group, and the 3' end is marked with an MGB quenching group.
3. The kit according to claim 1, wherein the PCR reaction solution further comprises a primer pair of an internal reference gene U6 and a Taqman probe, and the sequences of the primer pair and the Taqman probe are as follows:
U6-F：5’-TGCGGGTGCTCGCTTCGGCAGC-3’；
U6-R：5’-CCAGTGCAGGGTCCGAGGT-3’；
U6-Taqman-MGB：5’-CY5-CCTGCGCAA GGATGACACG-MGB-3’；
the 5 'end of the nucleotide sequence of the U6 Taqman probe is marked with a CY5 fluorescent reporter group, and the 3' end is marked with an MGB quenching group.
4. The kit according to claim 1, wherein the PCR reaction solution further comprises 2 x One Step RT-PCR Buffer.
5. The kit of claim 1, further comprising an enzyme cocktail: PrimeScript RT Enzyme Mix.
6. The kit according to claim 1, wherein the reaction conditions of the kit are: 5min at 42 ℃ and 10s at 95 ℃ as a first circulation step; 95 ℃ 5s, 58 ℃ 30s for 40 cycles of the second step, which was performed at the end of the extension for each cycle with detection of the fluorescence signal.
7. The kit according to claim 1, wherein the determination formula of the kit is: y is 0.0261AmiR-1246+0.0941*BmiR-141-0.0352*CmiR-520a-3p；
Wherein A ismiR-1246Is the expression level of miR-1246, BmiR-141Is the expression level of miR-141, CmiR-520a-3pIs the expression level of miR-520a-3 p;
when Y is more than or equal to 0.05, judging the sample to be positive; when Y is less than 0.0499, the test result is negative.
8. The use of the miR-1246, miR-141 and miR-520a-3p genes of claim 1 for preparing a pharmaceutical composition for treating breast cancer.
9. The use according to claim 8, wherein the pharmaceutical composition comprises miR-1246, an inhibitor of the miR-141 gene and miR-520a-3 p.
10. The pharmaceutical composition according to claim 9, wherein the inhibitor of the miR-1246 gene is si-miR-1246, and the sequence of si-miR-1246 is shown in SEQ ID No. 1; the inhibitor of the miR-141 gene is si-miR-141, and the sequence of si-miR-1246 is shown in SEQ ID No. 2.
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| US20200157631A1 (en) * | 2012-06-21 | 2020-05-21 | Ruprecht-Karls-UIniversität Heidelberg | CIRCULATING miRNAs AS MARKERS FOR BREAST CANCER |
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| CN111154878A (en) * | 2020-02-14 | 2020-05-15 | 北京粒基生物科技有限公司 | Application of group of miRNAs in preparation of markers for screening early breast cancer |
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