CN107354210B - Application of CMYA5 gene and expression product thereof in laryngeal squamous cell carcinoma - Google Patents
Application of CMYA5 gene and expression product thereof in laryngeal squamous cell carcinoma Download PDFInfo
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Abstract
The invention discloses CMYA5 genes and application of expression products thereof in laryngeal squamous cell carcinoma, wherein CMYA5 has low expression in laryngeal squamous cell carcinoma tissues, and the increase of CMYA5 expression can reduce cell proliferation and invasion of the laryngeal squamous cell carcinoma.
Description
Technical Field
The invention belongs to the field of biomedicine, relates to CMYA5 genes and application of expression products thereof in laryngeal squamous cell carcinoma, and particularly relates to application in diagnosis and treatment of laryngeal squamous cell carcinoma.
Background
Laryngeal carcinoma is one of the most common head and neck tumors in the world, accounting for approximately 6% of all tumors. The average annual incidence of laryngeal cancer is more than 500,000, and the annual incidence of more than 200,000 new deaths is about 50-70% in 5 years worldwide. Despite the considerable advances in surgery, radiation therapy and chemotherapy in recent years, the long-term prognosis of patients with laryngeal cancer is still not ideal. Local recurrence of tumors, lymph node metastasis and distant metastasis are major causes affecting the prognosis of patients with laryngeal cancer. Malignant biological behavior of tumors requires activation and inactivation of specific proteins, and modulation of expression and function of these specific proteins may prevent malignant biological behavior of tumor cells.
With the intensive research in the field of canceration and the continuous development of molecular biology, it has been found that various genes are abnormally expressed in laryngeal cancer tissues, but the specific pathogenesis is still unclear. Therefore, the method explores the generation and development mechanisms of tumors from the direction of gene change, searches genes related to the generation and development of tumors, and has important significance in the aspects of early diagnosis, treatment, prognosis judgment, research and development of related medicaments and the like of tumors.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a gene related to the occurrence and development of laryngeal squamous cell carcinoma, and the diagnosis of laryngeal squamous cell carcinoma is realized by detecting the expression level of the gene.
The second object of the present invention is to provide a therapeutic target for laryngeal squamous cell carcinoma, which can achieve treatment of laryngeal squamous cell carcinoma by targeting the target.
The third object of the present invention is to provide a method for screening a potential substance for treating laryngeal squamous cell carcinoma, wherein whether the screened substance is a potential substance for treating laryngeal squamous cell carcinoma is judged by whether the substance can regulate the expression level of a biological target.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides CMYA5 genes and application of expression products thereof in preparing products for early diagnosis of laryngeal squamous cell carcinoma.
Further, the product comprises a reagent for detecting the expression level of CMYA5 genes and expression products thereof.
Further, the reagent comprises a reagent for detecting the expression levels of CMYA5 genes and expression products thereof by using RT-PCR, real-time quantitative PCR, in-situ hybridization, a chip or immunoassay technology.
Wherein, the reagent for detecting the expression levels of the CMYA5 genes and the expression products thereof by RT-PCR at least comprises a pair of primers for specifically amplifying the CMYA5 genes; the reagent for detecting the expression levels of the CMYA5 genes and the expression products thereof by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the CMYA5 genes; the reagent for detecting the expression levels of the CMYA5 genes and expression products thereof by using in situ hybridization comprises a probe hybridized with a nucleic acid sequence of the CMYA5 gene; the reagent for detecting the expression levels of the CMYA5 genes and the expression products thereof by using the chip comprises a probe hybridized with a nucleic acid sequence of CMYA5 or an antibody or a ligand specifically bound with CMYA5 protein; the reagent for detecting the expression level of CMYA5 genes and expression products thereof by immunoassay technology comprises an antibody and/or a ligand which are specifically bound with CMYA5 protein.
The invention provides a product for diagnosing laryngeal squamous carcinoma, which comprises a reagent for detecting the expression level of CMYA5 genes or proteins coded by the genes in a sample. The product includes (but is not limited to) a chip, a preparation, a kit.
Further, the reagents include probes that specifically recognize CMYA5 or primers that specifically amplify CMYA 5; or an antibody or ligand that specifically binds to a protein encoded by CMYA 5.
In the present invention, the product for diagnosing laryngeal squamous carcinoma can be used for detecting the expression levels of a plurality of genes related to laryngeal squamous carcinoma including CMYA5 and/or expression products thereof. The combined diagnosis of a plurality of genes can increase the accuracy of the diagnosis of the laryngeal squamous cell carcinoma.
The invention provides a CMYA5 gene and application of an expression product thereof, which is used for screening potential substances for treating laryngeal squamous cell carcinoma.
Further, the steps for screening potential substances for treating laryngeal squamous cell carcinoma are as follows:
treating a system expressing or containing the CMYA5 genes or proteins encoded thereby with a candidate substance; and
detecting the expression or activity of CMYA5 genes or proteins encoded by the genes in the system;
wherein, if the candidate substance can promote the expression or activity of CMYA5 gene or its encoded protein (preferably significantly increased, such as more than 20%, preferably more than 50%, more preferably more than 2 times), it indicates that the candidate substance is a potential substance for preventing or treating laryngeal squamous cell carcinoma.
The invention provides a method for screening potential substances for preventing or treating laryngeal squamous cell carcinoma, which comprises the following screening steps:
treating a system expressing or containing the CMYA5 genes or proteins encoded thereby with a candidate substance; and
detecting the expression or activity of CMYA5 genes or proteins encoded by the genes in the system;
wherein, if the candidate substance can promote the expression or activity of CMYA5 gene or its encoded protein (preferably significantly increased, such as more than 20%, preferably more than 50%, more preferably more than 2 times), it indicates that the candidate substance is a potential substance for preventing or treating laryngeal squamous cell carcinoma.
In the test group, the candidate substance is added into a system expressing or containing CMYA5 genes or proteins coded by the genes; and/or detecting the expression or activity of the CMYA5 genes or the proteins encoded by the genes in the system of the test group and comparing the expression or activity with the control group, wherein the control group is the system without adding the expression of the candidate substance or containing the CMYA5 genes or the proteins encoded by the genes; if the expression or activity of CMYA5 gene or its coded protein in the test group is higher than that in the control group, the candidate substance is the potential substance for preventing or treating lung adenocarcinoma.
Further, the steps further include: further cell experiments and/or animal experiments are carried out on the obtained potential substances to further select and determine substances useful for preventing, alleviating or treating laryngeal squamous cell carcinoma from the potential substances.
In the present invention, the system includes (but is not limited to): a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system. The candidate compounds include (but are not limited to): nucleic acid promoters, protein binding molecules designed for the CMYA5 gene or genes upstream or downstream thereof.
The invention provides CMYA5 genes and application of expression products thereof in preparing a pharmaceutical composition for treating laryngeal squamous cell carcinoma.
Further, the pharmaceutical composition comprises CMYA5 genes, proteins or promoters thereof. The CMYA5 proteins include full length CMYA5 protein, mature CMYA5 protein, CMYA5 DNA binding domains, or CMYA5 active fragments containing said domains; the promoter comprises CMYA5 gene expression products, promoted miRNA, promoted transcription regulatory factor, or promoted targeted small molecule compounds.
Further, the pharmaceutical composition includes an enhancer for enhancing functional expression of CMYA 5.
The invention provides a pharmaceutical composition for treating laryngeal squamous cell carcinoma, which comprises the following components: promoters of functional expression of CMYA 5; and/or
A pharmaceutically acceptable carrier.
Further, the promoter includes substances that improve the stability of the CMYA5 genes or their expression products, up-regulate the expression level of the CMYA5 genes or their expression products, and increase the effective acting time of the CMYA5 genes or their expression products.
The pharmaceutically acceptable carrier includes (but is not limited to) diluents, binders, surfactants, humectants, adsorptive carriers, lubricants, fillers, disintegrants.
Drawings
FIG. 1 is a graph showing the detection of CMYA5 gene expression in laryngeal squamous carcinoma tissues using QPCR;
FIG. 2 is a graph showing the expression of CMYA5 proteins in laryngeal squamous carcinoma tissues detected by Western blot;
FIG. 3 is a graph of CMYA5 transfection in laryngeal squamous carcinoma cells; wherein panel A is a graph of the effect of transfection on CMYA5mRNA expression in laryngeal squamous carcinoma cells using QPCR; FIG. B is a graph showing the effect of Western blot on CMYA5 proteins in laryngeal squamous carcinoma cells after transfection;
FIG. 4 is a graph showing the effect of CMYA5 genes on laryngeal squamous carcinoma cell proliferation measured by MTT;
FIG. 5 is a graph of CMYA5 effect on laryngeal squamous carcinoma cell migration using a cell scratch test;
FIG. 6 is a graph showing the effect of CMYA5 on laryngeal squamous cell carcinoma cell invasion, as measured using a Transwell cell.
Detailed Description
The invention discovers the significant down-regulation of CMYA5 in laryngeal squamous cell carcinoma for the first time through a high-throughput sequencing technology. Experiments prove that by increasing the expression level of CMYA5, the growth and invasion of laryngeal squamous cell carcinoma cells can be effectively inhibited, and the CMYA5 genes can be used for clinical diagnosis and treatment of laryngeal squamous cell carcinoma.
CMYA5 genes
CMYA5 is located on 3-band long arm 3 of human chromosome 5, and CMYA5 in the present invention includes wild type, mutant or fragment thereof. A representative CMYA5 gene sequence is shown in CMYA5 gene (NC-000005.10) in GeneBank, the current International public nucleic acid database.
The full-length nucleotide sequence of human CMYA5 or its fragment can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.
The present invention may utilize any method known in the art for determining gene expression. 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.
Diagnostic product
In the present invention, the product for diagnosing laryngeal squamous carcinoma may be in any form, including (but not limited to) a chip, a preparation, a kit, as long as it can detect the expression level of CMYA5 genes or expression products thereof.
The chip of the invention comprises: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown in CMYA 5.
Specifically, suitable probes can be designed according to the genes of the present invention, and immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays as desired.
The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.
The solid phase carrier of the present invention can be made of various materials commonly used in the field of gene chip, such as but not limited to plastic products, microparticles, membrane carriers, etc. The plastic products can be combined with antibodies or protein antigens through a non-covalent or physical adsorption mechanism, and the most common plastic products are small test tubes, small beads and micro reaction plates made of polystyrene; the micro-particles are microspheres or particles polymerized by high molecular monomers, the diameter of the micro-particles is more than micron, and the micro-particles are easy to form chemical coupling with antibodies (antigens) due to the functional groups capable of being combined with proteins, and the combination capacity is large; the membrane carrier comprises microporous filter membranes such as a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane and the like.
The CMYA5 chip can be prepared by conventional methods for manufacturing biochips known in the art. For example, if a modified glass slide or silicon wafer is used as the solid support, and the 5' end of the probe contains a poly-dT string modified with an amino group, the oligonucleotide probe can be prepared into a solution, and then spotted on the modified glass slide or silicon wafer using a spotting apparatus, arranged into a predetermined sequence or array, and then fixed by standing overnight, thereby obtaining the gene chip of the present invention.
The invention provides a kit which can be used for detecting expression of CMYA 5. Preferably, the preparation or the kit further comprises a marker for marking the RNA sample, and a substrate corresponding to the marker. In addition, the kit may further include various reagents required for RNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like. In addition, the kit also comprises an instruction manual and/or chip image analysis software. The kit can be also attached with an instruction book of the kit, wherein the instruction book describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.
Accelerator and pharmaceutical composition
Based on the findings of the inventors, the present invention provides the CMYA5 promoter, which includes substances that improve stability of CMYA5 genes or expression products thereof, up-regulate expression levels of CMYA5 genes or expression products thereof, and increase effective action time of CMYA5 genes or expression products thereof. The promoter may be a CMYA5 gene expression product, a promoting miRNA, a promoting transcriptional regulator, or a promoting targeted small molecule compound.
In the present invention, there are various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like.
The invention also provides a pharmaceutical composition, which contains an effective amount of the CMYA5 accelerant and a pharmaceutically acceptable carrier. The composition can be used for treating laryngeal squamous cell carcinoma. Any of the aforementioned CMYA5 accelerators may be used in the preparation of the composition. The pharmaceutical composition can be used for supplementing the deletion or deficiency of endogenous CMYA5 proteins and treating laryngeal squamous carcinoma caused by CMYA5 reduction by improving the expression of CMYA5 genes or proteins.
The pharmaceutically acceptable carrier includes (but is not limited to) diluents, binders, surfactants, humectants, adsorptive carriers, lubricants, fillers, disintegrants.
Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, 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 zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.
The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.
The stabilizer includes human serum protein, L-amino acid, sugar and cellulose derivatives L-amino acid may further include any one of glycine, cysteine and glutamic acid, the saccharide includes monosaccharide such as glucose, mannose, galactose, fructose, etc., sugar alcohol such as mannitol, cellulose, xylitol, etc., disaccharide such as sucrose, maltose, lactose, etc., polysaccharide such as dextran, hydroxypropyl starch, chondroitin sulfide, hyaluronic acid, etc., and their derivatives, the cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and carboxymethylcellulose sodium, the surfactant includes ionic or nonionic surfactant such as polyoxyethylene alkyl ester, sorbitan monoacyl ester, fatty acid glyceride, the additive buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and their corresponding salts (their alkali metal or basic rare earth metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt), the isotonic agent includes potassium chloride, sodium chloride, sugar and glycerol, and the chelating agent includes sodium ethylenediaminetetraacetate and citric acid.
The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical composition of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.
The mode of introducing the drug of the present invention into a tissue or cell can be classified into an in vitro mode or an in vivo mode. The in vitro method comprises introducing a drug containing CMYA5 genes or a drug containing CMYA5 proteins into cells, and transplanting or returning the cells into the body. The in vivo approach involves directly injecting drugs containing CMYA5 genes or drugs containing CMYA5 proteins into the tissues in vivo.
The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.
The pharmaceutical composition of the invention can also be used in combination with other drugs for the treatment of squamous cell carcinoma of the larynx, and the other therapeutic compounds can be administered simultaneously with the main active ingredient, 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.
In the present invention, the term "sample" is used in its broadest sense. It is intended to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass liquids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, and the like. However, such samples should not be construed as limiting the type of sample that is suitable for use in the present invention.
In the specific embodiment of the invention, the experiments are repeated at least 3 times, the result data are expressed in a mean value plus or minus standard deviation manner, statistical analysis is carried out by using SPSS18.0 statistical software, and pairing comparison of cancer tissues and normal paracancer mucosa tissues adopts t test, and the statistical significance is considered when P is less than 0.05.
The following examples are intended to illustrate the invention in further detail with reference to the accompanying drawings and examples, and are not intended to limit the scope of the invention the experimental procedures, for which specific conditions are not indicated in the examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L aboratoryPress,1989), or according to the manufacturer's recommendations.
Example 1 screening of Gene markers associated with laryngeal squamous cell carcinoma
1. Sample collection
Each of 6 laryngeal squamous carcinoma tissues and corresponding normal mucosal tissue samples were collected, and informed consent was obtained from the patients and given consent by the tissue ethics committee.
2. Preparation of RNA samples
1) Adding liquid nitrogen, grinding tissue to powder, adding 1ml TRIzol (Invitrogen) solution, blowing, mixing, fully cracking tissue, and standing for 5 min;
2) centrifuging at 12000rpm at 4 deg.C for 5min, and transferring the supernatant to 1.5ml RNase free EP tube;
3) adding 200 μ l chloroform, shaking vigorously and mixing well for 30s to make the water phase and organic phase contact sufficiently, standing at room temperature for 15 min;
4) separating at 12000g at 4 deg.C for 15min, centrifuging the solution into three layers, transferring RNA to the upper water phase, and transferring to another new RNase free EP tube;
5) adding 0.5ml isopropanol, gently mixing well, standing at room temperature for 10 min;
6) centrifuging at 12000g for 10min at 4 deg.C, precipitating RNA by adding 75% ethanol with the same volume as RNAioso Plus, centrifuging at 7500g at 4 deg.C for 5min, and removing supernatant;
7) washing twice with 75% ethanol, and air drying on a super clean bench; the precipitate was dissolved with 30. mu.l of DEPC water.
8) Mass analysis of RNA samples
The concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The concentration is more than or equal to 200 ng/mul, and the OD260/280 is between 1.8 and 2.2.
3. Removal of rRNA
Ribosomal RNA was removed from total RNA using Ribo-Zero kit.
4. Construction of cDNA library
The construction of cDNA library was carried out using the Truseq RNA sample Prep Kit from Illumina, the detailed procedures were as described in the specification.
5. Sequencing on machine
The cDNA library was sequenced using the Hiseq4000 sequencing platform, the specific procedures were as described in the specification.
6. High throughput transcriptome sequencing data analysis
Bioinformatics analysis is carried out on the sequencing result, RNA-seq reading positioning is carried out by using TopHat v1.3.1, the relative abundance of the transcript is calculated by normalizing the number of RNA-seq fragments by Cufflinks v1.0.3, differential expression is detected by using cuffdiff, and mRNA is considered to be significantly differentially expressed when the p value is less than 0.001, | log2(Fold _ change) normalized | > 2.
7. Results
The RNA-seq results show that there are 665 genes differentially expressed in laryngeal squamous carcinoma patients, 450 genes up-regulated and 215 genes down-regulated, wherein the expression level of the genes CMYA5 in laryngeal squamous carcinoma tissues is significantly lower than that in normal paracancerous mucosal tissues.
Example 2QPCR sequencing verification of differential expression of CMYA5 genes
1. Large sample QPCR validation was performed on CMYA5 gene differential expression. 50 cases of the normal paracancerous mucosal tissue and the laryngeal squamous cell carcinoma tissue of the laryngeal squamous cell carcinoma patient were selected in accordance with the sample collection method of example 1.
2. The specific procedure for RNA extraction was as described in example 1.
3. Reverse transcription
3. Reverse transcription: mRNA reverse transcription was performed using the FastQuant cDNA first strand synthesis kit (cat # KR 106). The method comprises the following specific steps:
(1) 5 × g of DNA Buffer 2.0. mu.l, total RNA 1. mu.g, RNase Free ddH was added2O, heating to 42 ℃ in a water bath for 3min until the total volume is 10 mu l;
(2) a20. mu.l reaction system was constructed, 10 × Fast RT Buffer 2.0. mu. L Enzyme Mix 1.0. mu.l,FQ-RT Primer Mix 2.0μl，RNase Free ddH2adding O5.0 mul into the mixed solution in the step (1) after mixing, and mixing uniformly;
(3) heating in water bath at 42 deg.C for 15min, heating at 95 deg.C for 3min, and storing at-20 deg.C for use.
4. QPCR amplification
(1) Primer design
QPCR amplification primers were designed based on the coding sequences of the CMYA5 gene and the housekeeping GAPDH gene in Genebank and synthesized by Bomaide.
Primer sequence for CMYA5 genes:
the forward primer sequence is 5'-CAATCGCTCCTCCATCTAA-3' (SEQ ID NO. 1);
the reverse primer sequence is 5'-GGCACCTTATCTACTACATCA-3' (SEQ ID NO. 2).
Primer sequence of GAPDH gene:
the forward primer sequence is 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO. 3);
the reverse primer sequence is 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO. 4).
(2) PCR reaction system including 0.6. mu.l each of forward and reverse primers, 10. mu.l of 2 × SuperReal Premix Plus, 2. mu.l of DNA template, ddH2O 7.4μl，50×ROX Reference Dye △2. mu.l of sterile distilled water, 4.8. mu.l.
(3) The PCR reaction conditions are that × 40 cycles are carried out at 95 ℃ for 15min, (95 ℃ for 10s, 55 ℃ for 30s and 72 ℃ for 32s), the PCR reaction is carried out at 95 ℃ for 15s, 60 ℃ for 60s and 95 ℃ for 15s on an ABI 7300 type fluorescence quantitative PCR instrument, the target band is determined by melting curve analysis and electrophoresis, and the relative quantification is carried out by a delta CT method.
5. Results
Results as shown in fig. 1, CMYA5 expression was down-regulated in laryngeal squamous carcinoma tissues compared to laryngeal squamous carcinoma normal paracancerous mucosal tissues, with the differences statistically significant (P <0.05), consistent with high-throughput sequencing results.
Example 3 detection of differential expression of CMYA5 proteins by Western immunoblotting assay
1. Extraction of total tissue protein
Shearing tissue with scissors, placing into a glass homogenizer in ice, mixing RIPA lysate and PMSF at a ratio of 100:1, adding RIPA lysate of corresponding amount into tissue specimen of 20mg per 100 μ l lysate, grinding tissue with glass homogenizer until it is fully lysed, sucking the lysed liquid into EP tube, centrifuging at 14000rpm at 4 deg.C for 5min, and collecting supernatant.
2. Total protein concentration determination
The protein concentration was determined according to the instructions of the BCA protein concentration determination kit.
3. SDS-PAGE electrophoresis
8% of separation gel and 5% of concentrated gel were prepared and electrophoresed according to the instruction of SDS-PAGE gel preparation kit.
4. Western detection
1) Electrotransfer
And (3) putting the PVDF membrane into a methanol solution for activating for 5min, and putting the PVDF membrane into a membrane transferring buffer solution for balancing for 20 min. Taking out the PAGE gel, putting the PAGE gel into a membrane transferring buffer solution, cutting off the corresponding PAGE gel, putting the PAGE gel, the filter paper, the PVDF membrane, the PAGE gel and the filter paper in sequence from bottom to top into a semi-dry membrane transferring instrument, and transferring the membrane for 1.5h at constant pressure of 25V;
2) immunological hybridization
Taking out the PVDF membrane, washing the PVDF membrane by PBS, placing the washed PVDF membrane in a 5% BSA solution, shaking and sealing the PVDF membrane for 2 hours at room temperature, placing the PVDF membrane in a hybridization bag, adding a primary antibody for overnight, washing the PVDF membrane by a TBST buffer solution, adding a corresponding secondary antibody, incubating the PVDF membrane for 2 hours at room temperature, and washing the PVDF membrane by the TBST buffer solution.
3) DAB color development
The method comprises the following steps of dropwise adding a freshly prepared DAB color development solution into a PVDF membrane after the PVDF membrane is slightly dried, scanning and recording after the PVDF membrane develops color, carrying out semi-quantitative gray scale analysis on a strip by using β -actin as an internal reference and adopting a Quantity One gel imaging analysis system, repeating the experiment for 3 times, and taking an average gray scale value as a result;
5. results
The results are shown in fig. 2, where CMYA5 proteins were expressed at significantly lower levels in laryngeal squamous carcinoma tissue than normal paracancerous mucosal tissue.
Example 4 overexpression of the CMYA5 genes
1. Cell culture
Human laryngeal squamous carcinoma cell line Hep2, cultured in RPMI1640 medium containing 10% fetal calf serum and 1% P/S at 37 deg.C and 5% CO2And culturing in an incubator with relative humidity of 90%. The liquid is changed for 1 time in 2-3 days, the cells grow well and grow in a monolayer adherent manner. Passage was routinely digested with 0.25% EDTA-containing trypsin.
2. Transfection
1) Treatment of cells prior to transfection
One day before transfection, 3-5 × 10 are planted on 6-hole culture plates5And (3) culturing each cell/hole in an antibiotic-free culture medium for one day, wherein the cell density is 30-50% during transfection, and the cell/hole is replaced by a serum-free culture medium before transfection.
2) Construction of Gene overexpression vectors
Specific PCR amplification primers were synthesized based on the sequence of CMYA5 in GeneBank, and the primer sequences were as follows:
a forward primer: 5'-CCGGGTACCGCCACCATGGCGAGCCGCGATAGCA-3' (SEQ ID NO.5)
Reverse primer: 5'-CGGCTCGAGCTTGTGCCTTACAGAATCCGGGGGCTC-3' (SEQ ID NO.6)
Two restriction sites KpnI and XhoI are added to the 5 'end primer and the 3' end primer respectively. cDNA obtained by extracting and reverse transcribing lung adenocarcinoma patient tissues is used as an amplification template, the cDNA sequence is inserted into a eukaryotic cell expression vector pcDNA3.1 subjected to double enzyme digestion of KpnI and XhoI by restriction endonucleases, and the obtained recombinant vector pcDNA3.1-1 is connected for subsequent experiments.
The experiment was divided into three groups: a control group (Hep2), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2), wherein the siRNA of the negative control group has no homology with the sequence of CMYA5 genes.
3) Transfection
The lung adenocarcinoma cells were divided into 3 groups, which were respectively a control group (A549), a blank control group (transfected pcDNA3.1-NC), and an experimental group (transfected pcDNA3.1-1). Transfection of the vector was performed using liposome 2000, and the specific transfection method was performed as indicated in the specification. The transfection concentrations of the pcDNA3.1 empty vector and pcDNA3.1-1 were 0.5. mu.g/ml.
3. QPCR detection of expression levels of CMYA5 genes
1) Extraction of Total RNA from cells
The RNA in the cells was extracted using Qiagen's cell RNA extraction kit, and the experimental procedures were performed according to the instructions.
2) The reverse transcription procedure was as in example 2.
3) The QPCR amplification procedure was as in example 2.
4. Western blot detection of expression level of CMYA5 protein
1) Extraction of Total cellular protein
Cells from different treatment groups at log phase were collected and washed with pre-chilled PBS. Mixing RIPA cell lysate and PMSF at a ratio of 100:1, adding 150 μ l of the lysate into cells, standing on ice for 30min, scraping the lysed cells with a cell scraper, sucking the lysed liquid into an EP tube with a pipette, and centrifuging at 14000rpm at 4 ℃ for 5 min. The centrifuged supernatant was carefully collected.
2) Determination of Total protein concentration
The protein concentration was determined according to the instructions of the BCA protein concentration determination kit.
3) SDS-PAGE electrophoresis
8% of separation gel and 5% of concentrated gel were prepared and electrophoresed according to the instruction of SDS-PAGE gel preparation kit.
4) Western detection
See example 3 for details of the procedure.
5. Results
The results are shown in fig. 3, where CMYA5 in the transfected CMYA5 group was over-expressed compared to the non-transfected group versus the transfected empty plasmid group, with the difference being statistically significant (P < 0.05).
Example 5 Effect of CMYA5 genes on laryngeal squamous carcinoma cell proliferation
MTT experiment is adopted to detect the effect of CMYA5 gene on the proliferation capacity of laryngeal squamous carcinoma cells.
1. Taking cells with good growth conditions, conventionally digesting the cells into a single cell suspension, counting the cells, and diluting the cells into a cell suspension with proper concentration;
2. diluting in 96-well culture plateThe cells of the different treatment groups of (2) were seeded with 2000 cells per well, at least 3 parallel wells were set, 37 ℃ and 5% CO2Culturing for 24 h;
3. taking out 3- well cells 1, 2, 3, 4 and 5 days after inoculation every day, detecting the OD value of 490nm by an MTT method, counting and calculating the average value;
4. removing supernatant before detection, washing with culture solution for 3 times, adding 100 μ l MTT serum-free culture medium solution (0.2mg/ml) into each well, and continuously culturing at 37 deg.C for 4 hr;
5. terminating the culture, carefully removing the supernatant, adding 150. mu.l DMSO into each well, shaking for 10min to dissolve the crystals sufficiently, measuring the Optical Density (OD) value on a microplate reader at 490nm, and plotting the cell growth curve with time as the horizontal axis and optical density as the vertical axis.
6. Results
The results are shown in fig. 4, the proliferation of the cells in the experimental group is obviously inhibited compared with the control, and the difference has statistical significance (P <0.05), which indicates that CMYA5 overexpression has the effect of inhibiting the proliferation of cancer cells.
Example 7 Effect of the CMYA5 genes on apoptosis of laryngeal squamous carcinoma cells
The effect of CMYA5 genes on apoptosis was examined using flow cytometry.
1. The cell culture procedure was as in example 3.
2. The cell transfection procedure was as in example 3.
3. Step (ii) of
1) Cells from different treatment groups in the logarithmic growth phase were trypsinized and blown into cell suspensions and counted. Get 106Centrifuging the cell suspension at 1000rpm for 5 min;
2) discarding the supernatant, adding 195. mu.l Annexin V-FITC binding solution to gently resuspend the cells;
3) adding 5 μ l Annexin V-FITC, mixing, and incubating at room temperature in dark for 10 min;
4) centrifuging at 1000rpm for 5min, discarding the supernatant, and adding 190 μ l Annexin V-FITC binding solution to gently resuspend the cells;
5) and adding 10 mu l of Propidium Iodide (PI) staining solution, mixing gently, placing in ice bath and in dark, detecting the apoptosis condition by using a flow cytometer, repeating all experiments for 3 times, and taking an average value of results.
4. As a result:
the results show that the apoptosis rate of the cells in the experimental group has no obvious change compared with the control group, which indicates that CMYA5 has little influence on the apoptosis of laryngeal squamous carcinoma cells.
Example 8 cell scratch test
1. Add 1ml of fibronectin 50. mu.g/ml per well to 6 well plates and put in a refrigerator at 4 ℃ overnight;
2. discarding the rest fibronectin solution, washing with serum-free medium, subjecting the cells of different groups in logarithmic growth phase to trypsinization and resuspension, inoculating into 6-well plate paved with fibronectin, wherein each group of cells has 2 multiple wells, each well has 5 × 105(ii) individual cells;
3. placing the mixture at 37 ℃ in 5% CO2Culturing in an incubator overnight;
4. when the cells grow to be about 90 percent fused, drawing a fine trace without the cells by using a Tip head of 10 mul, washing off the fallen cells by using PBS solution, and adding a serum-free culture medium for continuous culture;
5. the healing condition of the cell scratch is observed at 0h and 48h after scratching respectively and photographed. The experiment was repeated 3 times and the results averaged.
6. Results
The results are shown in fig. 5, the migration distance of the cells after in vitro scratching is obviously reduced compared with the control group, and no significant difference exists between the control groups, which indicates that CMYA5 overexpression can inhibit the migration of laryngeal cancer cells.
Example 9 cell invasion assay
1. Transwell cell preparation
50 mg/L of Matrigel gel was diluted with 4 ℃ precooled serum-free medium at a ratio of 1:8, mixed well, coated on the upper surface of the bottom membrane of the Transwell cell, air-dried at 4 ℃, 60. mu.l to 80. mu.l of the diluted Matrigel gel (3.9. mu.g/. mu.l) was placed on a polycarbonate membrane of the Transwell upper chamber with a pore size of 8 μm so that all the micropores on the membrane were covered with Matrigel, and placed at 37 ℃ for 30min to polymerize Matrigel into a gel.
2. Preparing a cell suspension
The cells of different treatment groups in logarithmic growth phase were trypsinized and resuspended in serum-free medium, and the cell concentration was adjusted to 5 × 104One per ml.
3. Cell seeding
2ml of cell suspension was added to the upper chamber of the Transwell, 1ml of complete medium containing 10% fetal bovine serum was added to the lower chamber, and the mixture was placed in a matched 6-well plate and incubated at 37 ℃ with 5% CO2Culturing for 20-24h under the condition; the Transwell chamber was removed and the cotton swab wiped to remove Matrigel and non-membrane-penetrating cells from the upper chamber.
4. Dyeing process
After the cell culture is finished, taking out the Transwell chamber, wiping off Matrigel glue on the upper chamber surface and cells which do not penetrate through the membrane with a cotton swab, fixing the lower chamber surface with 95% alcohol for 15min, staining with hematoxylin for 2min, and randomly taking 5 high-power lenses under an inverted microscope for visual field observation, counting and photographing. Counting the number of cells on the lower surface of the chamber, namely the number of cells penetrating the Matrigel gel, taking the average number as an experimental result, representing the invasiveness of the tumor cells by the number of the cells, repeating the experiment for 3 times, and arranging 3 compound holes in each group of the cells.
5. Results
The results are shown in fig. 6, the number of cells penetrating the polycarbonate membrane of the Transwell chamber is obviously reduced in the experimental group compared with the control group, and the control group has no obvious difference, which indicates that CMYA5 overexpression can reduce the invasion of laryngeal squamous cell carcinoma.
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
<110> Beijing, the deep biometric information technology GmbH
<120> CMYA5 gene and application of expression product thereof in laryngeal squamous cell carcinoma
<160>6
<170>PatentIn version 3.5
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Claims (7)
1. The application of the reagent for detecting the expression level of CMYA5 genes is characterized in that the reagent is used for preparing a product for early diagnosing laryngeal squamous carcinoma.
2. The use as claimed in claim 1 wherein the reagents include reagents for detecting the expression levels of the CMYA5 genes by RT-PCR, real time quantitative PCR, in situ hybridization, chip or immunoassay techniques.
3. The use according to claim 2, characterized in that the reagents comprise probes specifically recognizing the CMYA5 genes or primers specifically amplifying the CMYA5 genes; or an antibody or ligand that specifically binds to a protein encoded by the CMYA5 genes.
4. Use of the CMYA5 genes or expression products thereof for screening potential substances for treating laryngeal squamous cell carcinoma.
5. A method for screening potential substances for preventing or treating laryngeal squamous carcinoma is characterized in that the screening steps are as follows:
treating a system containing the CMYA5 genes or proteins encoded thereby with a candidate substance; and
detecting the expression of CMYA5 genes or the activity of the protein coded by the genes in the system;
wherein, if the candidate substance can increase the expression of CMYA5 gene or the activity of protein coded by CMYA5 gene, the candidate substance is a potential substance for preventing or treating lung adenocarcinoma.
6. Use of the CMYA5 genes or their expression products for the preparation of a pharmaceutical composition for the treatment of laryngeal squamous cell carcinoma.
7. The use according to claim 6, wherein the pharmaceutical composition comprises CMYA5 genes, their encoded proteins or CMYA5 gene functional expression promoters.
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| Identification of novel mutations in endometrial cancer patients by whole-exome sequencing;Ya-Sian Chang et al.;《INTERNATIONAL JOURNAL OF ONCOLOGY》;20170320;第1783页左栏最后一段 * |
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