CN111394463B - Thyroid cancer related molecule and application thereof - Google Patents
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Abstract
The invention discloses a thyroid cancer related molecule and application thereof, and particularly relates to a molecule RUBCNL. The invention discloses that RUBCNL is up-regulated in thyroid cancer, and whether a subject has thyroid cancer or not can be assisted to diagnose by detecting the expression level of the RUBCNL. The invention also discloses application of the RUBCNL in constructing a calculation model for predicting thyroid cancer and application in treating thyroid cancer.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to a molecular diagnosis and treatment marker for thyroid cancer, wherein the marker is RUBCNL.
Background
Thyroid cancer is a malignant tumor that occurs in thyroid tissue, and is currently the most common malignant tumor of the endocrine system. Thyroid cancer can be classified into 4 types, the first of which is Papillary Thyroid Carcinoma (PTC), the most common of which is the pathological type (Bhaijee F, Nikiforov YE. molecular analysis of thyroid tumors [ J)]Endo cr Pathol,2011, 22: 126-. In recent years, thyroid cancer is the most common malignant tumor of endocrine system, and the prevalence rate is increasing year by year in the world. The growth rate of Thyroid Cancer has risen to The first stage of Cancer in China, and among all pathological types, papillary Thyroid Cancer accounts for about 80% -90% of Thyroid Cancer, is The most common pathological type, and has The highest Incidence (Luc GT, Morris, Andrew G, et al]Thyroid,2013,23(7): 885-. At present, the main treatment means aiming at papillary thyroid cancer is surgical excision, surgical incision and the like,131I and post-operative TSH suppression therapy, the prognosis is good for most patients with papillary thyroid cancer but recurrence and distant metastasis still occur in papillary thyroid cancer (Hu B, Wang Q, Wang YA, et al]Ce11,2016, 167(5): 1281-1295.). Therefore, the mechanism of papillary thyroid carcinoma is further studied, and a better method for preventing and treating the papillary thyroid carcinoma and preventing the papillary thyroid carcinoma from relapse and metastasis is favorably found.
The development of thyroid cancer is accompanied by the development and accumulation of various molecular genetic changes. In recent years, the research on the molecular pathogenic mechanism of thyroid cancer has made great progress, and the research proves that 60% -70% of thyroid cancer cases have at least 1 molecular genetic change and are different in different histopathological types, and the research on the function of genes in thyroid cancer has important significance for the personalized diagnosis and accurate medical treatment of thyroid cancer.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a molecular diagnosis marker related to thyroid cancer, and the individual diagnosis and treatment of a patient can be realized by diagnosing through the marker.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of a reagent for detecting the level of a gene and an expression product thereof in preparing a product for diagnosing thyroid cancer, wherein the gene is selected from RUBCNL.
Further, the product comprises reagents for detecting the level of RUBCNL in a sample by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunization technology.
Further, the agent is selected from:
an oligonucleotide probe that specifically recognizes the RUBCNL gene; or
Primers for specifically amplifying the RUBCNL gene; or
An antibody or ligand that specifically binds to a protein encoded by the RUBCNL gene.
Furthermore, the sequence of the primer for specifically amplifying the RUBCNL is shown as SEQ ID NO. 1-2.
Further, the thyroid cancer is follicular papillary thyroid cancer.
The invention provides a detection product for diagnosing thyroid cancer, which comprises a reagent for detecting the levels of a RUBCNL gene and an expression product thereof in a sample.
Further, the product comprises a chip, a kit or a nucleic acid membrane strip.
Further, the chip comprises a gene chip comprising an oligonucleotide probe for the RUBCNL gene for detecting the transcription level of the RUBCNL gene, a protein chip comprising a specific binding agent for the RUBCNL protein; the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the RUBCNL gene transcription level, and the protein detection kit comprises a reagent or a chip for detecting the RUBCNL protein expression level.
The invention provides application of RUBCNL in constructing a calculation model for predicting thyroid cancer.
The invention provides an application of RUBCNL in preparing a pharmaceutical composition for treating thyroid cancer.
Further, the pharmaceutical composition comprises an inhibitor of RUBCNL.
Further, the inhibitor is siRNA.
Further, the sequence of the siRNA is shown in SEQ ID NO. 5-6.
The present invention provides a pharmaceutical composition for treating thyroid cancer comprising an inhibitor of RUBCNL.
Further, the inhibitor is siRNA.
Further, the sequence of the siRNA is shown in SEQ ID NO. 5-6.
The invention provides application of RUBCNL in screening candidate drugs for treating thyroid cancer.
Further, treating the culture system expressing or containing the RUBCNL gene or the protein encoded by the RUBCNL gene with a substance to be screened; and detecting the expression or activity of the RUBCNL gene or protein encoded thereby in said system; wherein, when the substance to be screened inhibits the level or expression activity of the RUBCNL gene, the substance to be screened is a candidate drug for treating thyroid cancer.
The invention has the advantages and beneficial effects that:
the invention discovers the molecular diagnosis marker which shows differential expression in the thyroid cancer for the first time, can judge whether a subject suffers from the thyroid cancer by detecting the level of the marker, diagnoses diseases by using the marker, has higher specificity and better sensitivity, and can guide a clinician to provide an accurate treatment scheme for the subject.
Drawings
FIG. 1 shows a graph for detecting the expression of RUBCNL gene in thyroid cancer tissue by QPCR.
FIG. 2 is a graph showing the cell proliferation in MTT assay.
FIG. 3 is a graph of cell invasion in a Transwell chamber.
Detailed Description
The invention utilizes a high-throughput sequencing method in combination with bioinformatics analysis to search for a marker which can be used for characterizing thyroid cancer by detecting differentially expressed genes in patients with thyroid cancer and normal persons. According to the invention, through analysis, the RUBCNL is found to be differentially expressed in thyroid cancer patients for the first time, and the RUBCNL is suggested to be applied to clinical diagnosis of thyroid cancer as a detection index.
RUBCNL
In the present invention, (gene ID: 80183) includes the human gene and the protein encoded by it, which is located in the region 4 of the long arm 1 of human chromosome 13. Currently, there are 8 transcripts in genebank, and the nucleic acid sequences are shown as NM-001286761.2, NM-001286762.3, NM-001286763.3, NM-001286764.3, NM-001286765.2, NM-001286766.2, NM-001349772.2, and NM-025113.4, respectively. A representative nucleic acid sequence is shown as NM-001286761.2, and the corresponding amino acid sequence is shown as NP-001273690.1.
The utility of the present invention is not limited to quantifying gene expression of any particular variant of the target gene of the present invention. It is known to those skilled in the art that when performing bioinformatic analysis, the sequenced sequence is usually aligned with a known gene, and the expression of the gene can be considered as long as the sequence can be aligned with the gene concerned, and therefore, when referring to differentially expressed genes, different transcripts, mutants or fragments thereof of the gene are also encompassed by the present invention.
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 present invention may utilize any method known in the art to determine the expression level of a gene.
The RUBCNL of the present invention are detected using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and protein immunization technology.
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.
Illustrative, non-limiting examples of nucleic acid hybridization techniques 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.
Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.
Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: 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). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).
Protein immunization techniques include sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.
Thus, the immunization method according to the present invention can be carried out using well-known methods. Any direct (e.g., using a sensor chip) or indirect method may be used in the detection of the biomarkers of the invention.
Chip, kit and nucleic acid membrane strip
The chip of the invention comprises a gene chip and a protein chip; the gene chip comprises a solid phase carrier; and oligonucleotide probes immobilized on the solid phase carrier in an ordered manner, wherein the oligonucleotide probes specifically correspond to a part or all of the sequence shown in RUBCNL. The protein chip comprises a solid phase carrier and an antibody or ligand of the RUBCNL-encoded protein fixed on the solid phase carrier. The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.
"antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.
"diabodies" (diabodies) refer to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) joined in the same polypeptide chain (VH-VL). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Diabodies may be bivalent or bispecific.
The present invention provides a kit useful for detecting the expression level of a RUBCNL gene or protein, comprising primers, oligonucleotide probes, ligands, and/or chips for RUBCNL detection and/or quantification. One or more selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.
The kit of the present invention may also contain instructions for use of the kit, which describe how to use the kit for detection, how to use the detection results to determine the progression of a disease, and how to select a treatment regimen.
The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.
In the present invention, a nucleic acid membrane strip comprises a substrate and a probe specifically recognizing RUBCNL immobilized on the substrate; the substrate may be any substrate suitable for immobilizing probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.
The product for diagnosing thyroid cancer can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to thyroid cancer) including a RUBCNL gene, and can be used for simultaneously detecting a plurality of markers of thyroid cancer, so that the accuracy of thyroid cancer diagnosis can be greatly improved.
The present invention provides the use of RUBCNL in the construction of a computational model for predicting thyroid cancer, the steps of correlating marker levels with a certain likelihood or risk can be implemented and realized 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.
The invention provides the use of a RUBCNL for the preparation of a pharmaceutical composition for the treatment of thyroid cancer, said pharmaceutical composition being an inhibitor of RUBCNL. Such inhibitors of RUBCNL include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules. Wherein the nucleic acid inhibitor is selected from: an interfering molecule targeting a RUBCNL or a transcript thereof and capable of inhibiting RUBCNL 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. The protein binding molecule is selected from: an agent that specifically binds to a RUBCNL protein, such as an antibody or ligand capable of inhibiting the activity of a RUBCNL protein.
The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier includes (but is not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as simple syrup, glucose solution, starch solution, cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as dry starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate; absorption accelerators quaternary ammonium compounds, sodium lauryl sulfate, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, and the like.
The agents of the invention may also be used in combination with other agents for treating thyroid cancer, and the other therapeutic compounds may be administered simultaneously with the primary active ingredient (e.g., an inhibitor of RUBCNL), even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient. A partial dose of the main ingredient (e.g., a rub bnl inhibitor) may be administered concurrently with other therapeutic compounds, while other doses may be administered separately.
Detailed Description
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. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.
Example 1 screening of Gene markers associated with thyroid cancer
1. Sample collection
4 samples of follicular papillary thyroid carcinoma and paracancerous tissue were collected separately, and patients with other malignancies were excluded.
2. Preparation of RNA samples
Extraction of tissue total RNA Using TRIZOL method
1) Cutting tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1 min; standing at room temperature for 10min to completely decompose nucleoprotein.
2) Adding 200 μ l chloroform (chloroform), covering the tube, shaking vigorously for 15s, and standing at room temperature for 10 min.
3) Centrifuge at 11000rpm for 15min at 4 ℃.
4) Transferring the water sample layer into a new centrifuge tube, and adding 500 mul of isopropanol; after the mixture was inverted and mixed, the mixture was left standing at room temperature for 10 min.
5) Centrifuge at 11000rpm for 15min at 4 ℃.
6) The liquid was carefully aspirated off with a gun, the precipitate was left at the bottom of the tube, 1ml of 75% ethanol was added, the mixture was shaken on a shaker for 5s, and the precipitate was washed once.
7) Centrifuge at 8000rpm for 5min at 4 ℃.
8) Carefully removing the supernatant, drying the precipitate for 10min, and adding appropriate amount of water to dissolve the precipitate for 10 min.
9) And detecting the concentration of the RNA, and identifying the yield and purity of the RNA.
3. Construction of cDNA library
Ribosomal RNA was removed from total RNA using the Ribo-Zero kit from Epicentre; the complete RNA is randomly broken into small fragments of about 200bp by using metal ions.
The construction of cDNA library was carried out by using the Truseq RNA sample Prep Kit from Illumina, the detailed procedures are described in the specification.
4. Sequencing
And detecting a qualified library, adding NaOH to denature the qualified library into a single chain, adding the denatured and diluted library into the FlowCell, hybridizing the denatured and diluted library with a linker on the FlowCell, completing bridge PCR amplification on the cBot, and sequencing by using an Illumina X-Ten sequencing platform.
5. High throughput transcriptome sequencing data analysis
Bioinformatics analysis was performed on the sequencing results, and the number of reads for the genes was analyzed using the DESeq package in the R software, setting the screening criteria to P < 0.05.
6. Results
Analysis of high-throughput sequencing results shows that the expression level of the RUBCNL gene in follicular papillary thyroid carcinoma tissues is remarkably higher than that of paracarcinoma tissues.
Example 2 QPCR sequencing validation of differential expression of RUBCNL Gene
1. Large sample QPCR validation was performed on the differential expression of the RUBCNL gene. 30 cancer tissue samples and a tissue sample adjacent to the cancer were collected according to the sample collection method in example 1.
2. RNA extraction procedure as in example 1
3. Real-time quantitative PCR detection
1) Reverse transcription: the operation was carried out using a reverse transcription kit (Takara code: DRR047A) of TAKARA.
a. Removal of genomic DNA
Add 5 XgDNA Eraser B. mu.ffer 2.0. mu.l, gDNA Eraser 1.0. mu.l, total RNA 1. mu.g, and RNase Free ddH into the tube2O to make the total volume to 10 μ l, heating in water bath at 42 deg.C for 2 min.
b. Reverse transcription reaction
Will be provided with
2 4.0μl，
Enzyme Mix I 1.0μl，RT Primer Mix 1.0μl，RNase Free ddH2O4.0. mu.l was added to the above test tube and mixed together to give 20. mu.l, which was then heated in a water bath at 37 ℃ for 15min and 85 ℃ for 5 s.
2) QPCR amplification
a. Primer design
Primers were designed based on the gene sequences of RUBCNL and GADPH, and the specific primer sequences were as follows:
the RUBCNL gene (5 'to 3'):
TATATCAGCAATGGAGAA(SEQ ID NO.1)；
TGTATGAAGAAGTAGAAGA(SEQ ID NO.2)。
GAPDH gene:
AATCCCATCACCATCTTCCAG(SEQ ID NO.3)；
GAGCCCCAGCCTTCTCCAT(SEQ ID NO.4)。
QPCR amplification assay
By using
Ex TaqTMII (Takara Code: DRR081) kit is configured with a PCR reaction system in a Thermal Cycler
PCR amplification and reverse reaction are carried out on a Time System amplification instrumentAfter completion of the reaction, the amplification curve and the dissolution curve of Real Time PCR were confirmed, and relative quantification was performed by the Δ Δ CT method.
Prepare 25. mu.l reaction:
Reaction conditions are as follows: 30s at 95 ℃ (5 s at 95 ℃, 30s at 60 ℃) multiplied by 40
4. Results
The QPCR results are shown in fig. 1, and the expression of rubbcnl in thyroid cancer tissues is significantly up-regulated, and the difference is statistically significant (P <0.05), which is consistent with the high throughput sequencing results, wherein 31 samples of rubbcnl up-regulated, 26 samples of thyroid cancer tissues, and 5 samples of para-carcinoma tissues suggest that rubbcnl has high application value in the diagnosis of thyroid cancer.
Example 3 and functional study of the RUBCNL Gene
1. Design and Synthesis of RUBCNL interfering RNA
The interfering siRNA-RUBCNL aiming at the RUBCNL is designed and synthesized by Shanghai Ji code pharmaceutical technology Limited company, the control is general siRNA-NC, and the sequence of the siRNA-RUBCNL is as follows:
sequence 5 'to 3':
AUUUGCAAAUCUCUUUCACAU(SEQ ID NO.5)；
GUGAAAGAGAUUUGCAAAUGC(SEQ ID NO.6)。
2. cell culture
5% CO at 37 ℃2In the incubator, K1 cells were cultured in DMEM medium containing 10% FBS, and K1 cells in logarithmic growth phase were used for the experiment. The experiment was divided into K1 group (blank control i.e. cells not transfected), negative control group: siRNA-NC group; experimental groups: siRNA-RUBCNL group.
3. Transfection
The day before the experiment, 6-well plates were plated with serum-free medium without double antibody, and the cell density was 6X 105A hole. Transfection was initiated when the degree of cell fusion reached 70%. Taking 1.5ml of EP tube, each tubeOPTI-MEM was added to each of the diluted siRNAs, and the mixture was allowed to stand at room temperature for 5 min. Adding OPTI-MEM into another 1.5ml EP tube, diluting Lipofectamine2000 with each tube, and standing at room temperature for 5 min; the diluted siRNA was gently mixed with Lipofectamine2000 and allowed to stand at room temperature for 20 min. Adding the mixed solution into each 6-pore plate containing the OPTI-MEM, and slightly and uniformly mixing the mixture front, back, left and right; after incubation in the incubator for 6h, the transfection solution was changed to a serum-free medium without double antibody.
4. Real-time PCR assay
After 48h of transfection and culture of each group of cells, total RNA of the cells was extracted by Trizol method, reverse transcription and real-time quantitative PCR detection were performed according to the method of example 2.
5. MTT assay
Collecting cells in logarithmic growth phase, adjusting cell concentration to 1 × 103Adding 100 μ L of each cell suspension into a 96-well plate, setting a zero-setting hole, placing at 37 deg.C and 5% CO2Culturing in an incubator, taking out cells in 48h, adding 20 mu L MTT solution into each hole, incubating in the incubator for 2h, adding 150 mu L DMSO, and shaking for 10min to fully dissolve crystals. The 490nm wavelength is selected, the light absorption value of each pore is measured on an enzyme linked immunosorbent instrument, and the result is recorded.
6. Transwell experiment
The cells were plated on Transwell chambers using 30. mu.L of 1:8 diluted Matrigel gel, hydrated after 2h with 100. mu.L of DMEM medium, and plated with transfected K1 cells 2X 105One, the lower chamber was filled with 500. mu.L of medium containing 10% FBS, and 3 wells per set. Placing in an incubator for 48 h. Then the chamber was taken out, washed with PBS 3 times, the upper chamber cells were gently wiped off with a cotton swab, fixed with absolute methanol for 30min, stained with 0.5% crystal violet for 30min, washed with PBS 3 times, air dried naturally, and observed under a microscope.
7. Statistical method
The experiments were performed in 3 replicates, and the results were expressed as mean ± sd, and the difference between the two was determined by paired t-test, which was considered statistically significant when P < 0.05.
8. Results
1) The expression level of RUBCNL (0.28 + -0.085) after siRNA-RUBCNL transfection in the experimental group was significantly lower than that in the control group (K1 vs siRNA-RUBCNL, P value 0.0047 ═ 0.917 + -0.0451) compared to the control group (K1 vs siRNA-NC, P value 0.0853, ns) in the control group transfected with RUBCNL (K1) as a reference, and the expression level of RUBCNL in the negative control transfected with siRNA-NC was not significantly changed (K1 vs siRNA-NC, P value 0.0853, ns).
2) The MTT assay results are shown in fig. 2, and compared with the negative control group siRNA-NC OD, the experimental group siRNA-rubbnl had significantly reduced OD value, and the difference was statistically significant (siRNA-NC vs siRNA-rubbnl, P ═ 0.0031 ═ x), indicating that the proliferation of K1 was significantly inhibited after knockdown of rubbnl.
3) The results of the cell invasion experiments are shown in fig. 3, and the number of cells passing through the basement membrane is obviously reduced after knocking down the RUBCNL compared with the control group, and the difference is statistically significant (P ═ 0.0169 ═ C); the above results indicate that knocking down RUBCNL can inhibit the invasive ability of cancer cells in vitro.
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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<120> thyroid cancer-associated molecule and use thereof
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<213> Artificial Sequence (Artificial Sequence)
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Claims (9)
1. Use of a reagent for detecting gene levels in the manufacture of a product for diagnosing follicular papillary thyroid carcinoma wherein the gene is RUBCNL.
2. The use of claim 1, wherein the product comprises reagents for detecting the level of RUBCNL in a sample by sequencing, nucleic acid hybridization, or nucleic acid amplification techniques.
3. Use according to claim 2, wherein said agent is selected from:
an oligonucleotide probe that specifically recognizes the RUBCNL gene; or
Primers for specifically amplifying the RUBCNL gene.
4. The use of claim 3, wherein the primer sequence for specifically amplifying the RUBCNL gene is shown in SEQ ID No. 1-2.
5. The use of claim 1, wherein the product comprises a chip, a kit, or a nucleic acid membrane strip.
6. The use of claim 5, wherein the chip comprises a gene chip comprising oligonucleotide probes for the RUBCNL gene for detecting the level of transcription of the RUBCNL gene; the kit comprises a gene detection kit, and the gene detection kit comprises a reagent or a chip for detecting the transcription level of the RUBCNL gene.
7. Application of RUBCNL in construction of a computational model for predicting thyroid cancer.
Use of an inhibitor of RUBCNL for the preparation of a pharmaceutical composition for the treatment of thyroid cancer, wherein said inhibitor is siRNA.
9. The use of claim 8, wherein the siRNA has a sequence as shown in SEQ ID No. 5-6.
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