CN111235274A - Screening method of laryngeal squamous carcinoma serum exosome marker and application of exosome source miR-941 - Google Patents
Screening method of laryngeal squamous carcinoma serum exosome marker and application of exosome source miR-941 Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular diagnosis and molecular biology, and particularly relates to a screening method of laryngeal squamous carcinoma serum exosome markers and application of exosome source miR-941. The screening method comprises the steps of screening out serum exosome miRNAs which are differentially expressed between a laryngeal squamous cell carcinoma patient and a healthy contrast person through high-throughput sequencing, and selecting miRNAs which are up-regulated and expressed in high expression quantity in the laryngeal squamous cell carcinoma patient as candidate miRNAs; then qRT-PCR verification is carried out on the candidate miRNAs in another group of amplified samples, and internal reference genes for qRT-PCR detection are screened out before verification; and finally, analyzing the diagnosis efficiency of the target miRNAs by adopting an ROC curve. The invention also provides a larynx squamous carcinoma serum exosome miRNA marker miR-941, provides a new way for minimally invasive diagnosis of larynx squamous carcinoma, can be used as a potential drug treatment target spot, and has great clinical practical value.
Description
Technical Field
The invention belongs to the technical field of molecular diagnosis and molecular biology, and particularly relates to a screening method of laryngeal squamous carcinoma serum exosome markers and application of exosome source miR-941.
Background
Squamous cell carcinoma of the larynx (laryngeal squamous carcinoma) is one of the most common malignant tumors of the head and neck, and is highly prevalent in northern regions of china, including the provinces of shanxi. The incidence of laryngeal squamous cell carcinoma gradually decreases over the last 40 years, while the 5-year survival rate does not increase and inversely decreases from 66% to 63%. Early symptoms of laryngeal squamous carcinoma are not significant, and approximately 60% of patients are not diagnosed until late stage (stage III or IV). Early diagnosis can increase the chance of successful treatment, while the anatomical location of the larynx is hidden, and early detection of laryngeal cancer is very difficult. Currently, the diagnosis of laryngeal squamous cell carcinoma mainly depends on endoscopy and pathological examination, which is an invasive detection mode, often causes different degrees of pain to patients, and usually needs multiple biopsies to confirm the diagnosis. Therefore, the development of a rapid, minimally invasive, highly sensitive diagnostic method and an effective and reliable biological marker is urgently needed in clinic.
Exosomes are membrane vesicles with a diameter of 30-150nm actively secreted by living cells, contain proteins, lipids and nucleic acids derived from maternal cells, and can be released into various body fluids including blood in the human body. Blood is a clinically common, minimally invasive and easily-obtained sample, and is convenient for dynamically observing various indexes. Exosomes and contents in blood can potentially reflect the structure and function of parent cells from which they are derived, thus mapping different pathophysiological states of the body without directly sampling the source cells, and have the potential to serve as a biological marker of tumors.
MicroRNA (miRNAs) is an endogenous non-coding RNA molecule, has the length of 19-22 nucleotides, is abnormally expressed in tumors, generally acts as a tumor suppressor or an oncogene, is closely related to the occurrence and development of the tumors, and is a potential tumor biological marker. Exosome miRNAs in circulating blood are used as tumor markers, and have the following advantages: the coating with the membrane on the outside can avoid the digestion of nuclease in blood, is more stable, is not easily influenced by extracellular environment, and has more reliable research result; the composition and abundance of the miRNAs in the exosome are different from those in the whole blood, and the miRNAs depend on the maternal cells and can better reflect the functional state of the maternal cells; some miRNAs in the exosome are enriched, which is beneficial to detecting low-abundance miRNAs.
Disclosure of Invention
In order to overcome the defects that the diagnosis of the laryngeal squamous cell carcinoma still needs histological examination, causes pain to patients and cannot be diagnosed as early as possible in the prior art, the invention provides a method for screening a laryngeal squamous cell carcinoma serum exosome miRNA marker. Based on the method, the invention provides a laryngeal squamous carcinoma serum exosome miRNA marker which is Hsa-miR-941 (miR-941).
The laryngeal squamous carcinoma serum exosome marker is prepared by the following steps:
(1) collecting serum samples of a laryngeal squamous carcinoma patient and a healthy contrast person, extracting exosomes, identifying, and extracting exosome RNA;
(2) carrying out high-throughput sequencing in a small sample amount, screening out serum exosome miRNAs which are differentially expressed between a laryngeal squamous cell carcinoma patient group and a healthy control group, and selecting miRNAs which are up-regulated and expressed in high expression amount in the laryngeal squamous cell carcinoma patient as candidate miRNAs;
(3) verifying the candidate miRNAs in the enlarged sample by adopting a qRT-PCR method; before verification, firstly determining an internal reference gene of a qRT-PCR experiment, wherein the screening process comprises the following steps: screening RNAs with stable expression from the RNA sequencing data and literature reports in the step (2) as candidate internal reference genes, carrying out comprehensive analysis by 5 internal reference analysis algorithms after qRT-PCR detection, and screening the internal reference genes for qRT-PCR verification;
(4) the ROC curve evaluates the diagnostic efficacy of the miRNAs validated in step (3), i.e. the ability to identify patients with laryngeal squamous cell carcinoma and healthy individuals.
The screening method of the laryngeal squamous carcinoma serum exosome marker comprises the following steps:
(1) collecting serum samples of a laryngeal squamous carcinoma patient and a healthy contrast person, extracting exosomes, identifying, and extracting exosome RNA;
(2) carrying out high-throughput sequencing in a small sample amount, screening out serum exosome miRNAs which are differentially expressed between a laryngeal squamous cell carcinoma patient group and a healthy control group, and selecting miRNAs which are up-regulated and expressed in high expression amount in the laryngeal squamous cell carcinoma patient as candidate miRNAs;
(3) verifying the candidate miRNAs in the enlarged sample by adopting a qRT-PCR method; before verification, firstly determining an internal reference gene of a qRT-PCR experiment, wherein the screening process comprises the following steps: screening RNAs with stable expression from the RNA sequencing data and literature reports in the step (2) as candidate internal reference genes, carrying out comprehensive analysis by 5 internal reference analysis algorithms after qRT-PCR detection, and screening the internal reference genes for qRT-PCR verification;
(4) the ROC curve evaluates the diagnostic efficacy of the miRNAs validated in step (3), i.e. the ability to identify patients with laryngeal squamous cell carcinoma and healthy individuals.
The application of the laryngeal squamous cell carcinoma serum exosome marker is applied to the preparation of laryngeal squamous cell carcinoma diagnostic reagents.
The laryngeal squamous carcinoma diagnostic kit consists of a serum exosome, an RNA extraction reagent, miR-941 specific amplification primers and a universal PCR amplification reagent; forward primer for miR-941: 5'-GCACCCGGCTGTGT-3' are provided.
The application of the kit for diagnosing the laryngeal squamous cell carcinoma, and the application of the kit containing the miR-941 detection reagent in screening and diagnosing the laryngeal squamous cell carcinoma.
The laryngeal squamous carcinoma drug treatment target is miR-941, and the nucleotide sequence of the miR-941 is as follows: CACCCGGCUGUGUGCACAUGUGC are provided.
The current research considers that the serum exosome miRNAs of the tumor patients are mainly actively secreted by tumor cells, and different from free miRNAs passively released after the death of the tumor cells, the serum exosome miRNAs can truly reflect the functional state of the original maternal cells. The invention screens the laryngeal squamous carcinoma serum exosome miRNAs with potential diagnosis value by adopting high-throughput sequencing for the first time, and further verifies the expression difference and the diagnosis value in other expanded samples to achieve an optimal evaluation system, so that the selected serum exosome miRNAs have more clinical guidance, provide theoretical basis for developing and diagnosing non-invasive biomarkers of laryngeal squamous carcinoma in future and have great clinical practical value.
The invention is based on a high-throughput sequencing method, and is used for sequencing the serum exosome RNA of 6 laryngeal squamous cell carcinoma patients and 6 healthy contrast persons to obtain miRNA expression profiles and miRNAs with differential expression, then selecting miRNAs with differential up-regulation expression and high expression quantity in the laryngeal squamous cell carcinoma patients, carrying out qRT-PCR verification in the other 50 laryngeal squamous cell carcinoma patients and 25 healthy contrast persons, and carrying out diagnosis efficiency evaluation by applying an ROC curve. The result shows that the serum exosome miR-941 provided by the invention is closely related to laryngeal squamous cell carcinoma, can be used for clinical diagnosis and treatment of laryngeal squamous cell carcinoma, and lays a foundation for development of clinically-related diagnosis and treatment reagents or chips and the like.
Compared with the prior art, the invention has the following advantages:
the invention is based on high-throughput RNA sequencing analysis, combined with qRT-PCR method to carry out verification in a large sample, and carries out diagnosis efficiency evaluation through ROC curve analysis, thereby screening out the marker suitable for diagnosing the laryngeal squamous cell carcinoma serum exosome, and the invention is a simple and effective screening method.
The miRNA marker for the laryngeal squamous cell carcinoma serum exosome provided by the invention is remarkably increased in the serum exosome of a laryngeal squamous cell carcinoma patient, is a very reliable marker for diagnosing laryngeal squamous cell carcinoma by using a serum exosome sample, provides a new way for clinical diagnosis, and is a potential drug therapy target spot.
The kit provided by the invention can be used for simply, conveniently and quickly diagnosing laryngeal squamous cell carcinoma and has reliable diagnosis result.
Drawings
FIG. 1 is a characterization of serum exosomes. A. Observing the morphology and size of exosome by a transmission electron microscope (Bar is 200 nm); NTA detecting the particle size distribution of exosome; C. the expression of the exosome marker protein was detected by immunoblotting.
FIG. 2 shows the results of RNA sequencing screening for serum exosome-differential miRNAs between 6 laryngeal squamous cell carcinoma patients and 6 healthy controls. A. Clustering heatmaps of the differential miRNAs; B. differential miRNAs volcano pattern showing up-regulation of 34 miRNAs and 41 down-regulation in laryngeal squamous cell carcinoma patients.
FIG. 3 is a qRT-PCR validation of candidate miRNAs in 50 laryngeal squamous cell carcinoma patients and 25 healthy controls showing only miRNAs with statistically significant differences between groups, and panels A and B show the qRT-PCR detection of miR-941 and miR-27a-5p, respectively.
FIG. 4 is a ROC curve for the diagnosis of laryngeal squamous carcinoma by serum exosome miRNAs. A. ROC analysis result of miR-941. The AUC value is 0.797, and the diagnostic efficacy is higher; ROC analysis result of miR-27a-5 p. The AUC value was 0.692, and the diagnostic efficacy was low.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, and it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Extracting and identifying serum exosomes and extracting exosome RNA:
step 1, collection of serum samples
Acquiring an object: a total of 87 sera samples were collected, 56 of the histopathologically confirmed laryngeal squamous carcinoma patients and 31 of the sera of healthy controls. The laryngeal squamous carcinoma patients are originated from otorhinolaryngology and head and neck surgery of the first hospital of Shanxi medical university without the history of radiotherapy and chemotherapy and the history of acute and chronic inflammatory diseases, and are used as an experimental group. The healthy control group subjects were from the hospital's physical examination center, had no history of acute and chronic inflammatory disease and malignancy, and were age and gender matched to the experimental group. The study was approved by the university of Shanxi medical research ethics Committee, and each subject signed an informed consent.
The method comprises the following specific steps: peripheral blood was collected in a serum procoagulant tube and centrifuged within 2 hours to extract serum. Blood samples are centrifuged at 1200 Xg for 10min at 4 ℃, supernatant is sucked and centrifuged at 3000 Xg for 15min at 4 ℃, and light yellow supernatant is serum and is frozen at-80 ℃ for later use.
Serum exosomes were isolated using ExoQuick reagent (System Biosciences, Mountain View, CA, USA) as described. Mu.l of serum was mixed with 125. mu.l of ExoQuick reagent, incubated at 4 ℃ for 1 hour, the ExoQuick/serum mixture was centrifuged at 1500 Xg for 30 minutes at 4 ℃ and the supernatant discarded, and then centrifuged again at 1500 Xg for 5 minutes at 4 ℃ and the supernatant discarded sufficiently to avoid touching the bottom pale yellow exosome pellet, and the exosome pellet was dissolved in 50. mu.l of PBS.
Detecting the form and size of the exosome by a transmission electron microscope: diluting the exosome sample in the step 2 by using a proper amount of PBS, dripping 10 mu l of the diluted exosome sample on a copper mesh, standing for 1 minute, absorbing liquid on the surface of the copper mesh by using filter paper, dripping 10 mu l of 2% uranyl acetate on the copper mesh, standing for 1 minute, absorbing liquid on the surface of the copper mesh by using the filter paper, and standing for 15 minutes. The observation was carried out with a transmission electron microscope FEI TecnaG 2 spirit (Thermo-Fischer, Waltham, MA, USA) at a voltage of 120kV and photographed. As shown in fig. 1A, serum exosomes with typical saucer-like structures were isolated from this experiment.
Nta (nanoparticle Tracking analysis) assay for exosome particle size: and (3) diluting the exosome sample in the step 1 with a proper amount of PBS, and observing and detecting the exosome sample in a nanoparticle tracking analyzer NanoSight LM 10. As shown in FIG. 1B, the vesicle size isolated in this experiment was mainly around 110nm, which is consistent with the exosome size distribution.
Detection of exosome marker proteins by immunoblotting: detection was performed using an Exo-Check Exosome antibody array (System Biosciences, Mountain View, Calif., USA) as described. Adding 600 mul of lysis buffer solution into 300 mul of exosome protein sample in the step 2, swirling for 15 seconds, sucking 600 mul of sample-lysis solution mixed solution, mixing the mixed solution with 9.4mL of binding buffer solution uniformly, adding the mixed solution to a pre-wetted antibody array membrane, and incubating overnight at 4 ℃ on a shaking table; the next day, membranes were washed, 10mL of detection buffer was added, incubated on a shaker for 2 hours, then washed and imaged. As shown in fig. 1C, the serum exosomes extracted in this experiment expressed exosome-associated proteins such as CD81, CD63, Alix, flo 1, ICAM1, EpCAM, ANXA5 and TSG101, but did not express the cell contamination marker protein GM130 (cis golgi matrix protein).
Step 4, extracting serum exosome RNA
Exosome RNA was extracted with TRIzol reagent. Adding 1mL of TRIzol into the exosome sample in the step 2, sucking, pumping, uniformly mixing, standing at room temperature for 10 minutes, and fully releasing exosome RNA; adding 0.2mL of chloroform, manually and violently oscillating the tube body for 15 seconds, and standing for 10 minutes at room temperature; centrifuging at 12000 Xg for 15min at 4 deg.C, separating the liquid layers, wherein the upper water phase is enriched with RNA, and carefully absorbing the upper water phase into 1.5mL RNase-free centrifuge tube; adding isopropanol with the same volume, reversing up and down for 5-10 times, mixing, adding 10ng glycogen for sedimentation assistance, and standing at-20 deg.C overnight; centrifuging at 12000 Xg for 15min at 4 deg.C, and discarding the supernatant; washing the precipitate with 1mL of precooled 75% ethanol for 2 times, drying at room temperature for 3-5 minutes, adding a proper amount of RNase-free water to dissolve the precipitate, and storing at-80 ℃ for later use.
Example 2
RNA sequencing screening of serum exosome differential miRNAs of laryngeal squamous carcinoma patients and healthy control groups:
serum exosome RNAs were used for this experiment in 6 laryngeal squamous cell carcinoma patients and 6 healthy controls.
Step 1, sample detection, library construction and sequencing
(1) Total RNA sample detection: the total amount of RNA and the fragment distribution were accurately determined using a highly sensitive Agilent 2100pic 600.
(2) Library construction: after the Sample is detected to be qualified, a library is constructed by using Small RNA Sample Pre Kit, the 3 'end and the 5' end of the Small RNA are utilized to have special structures (the 5 'end has a complete phosphate group, and the 3' end has a hydroxyl group), total RNA is used as an initial Sample, the joints are directly added to the two ends of the Small RNA, and then the cDNA is synthesized by reverse transcription. And then, carrying out PCR amplification, carrying out PAGE gel electrophoresis to separate a target DNA fragment, and cutting and recycling the gel to obtain the cDNA library.
(3) And (4) library inspection: after the library is constructed, firstly using Qubit2.0 to carry out preliminary quantification, diluting the library to 1 ng/mu l, then using Agilent 2100 to detect the insert size of the library, and after the insert size meets the expectation, using a Q-PCR method to accurately quantify the effective concentration of the library (the effective concentration of the library is more than 2nM) so as to ensure the quality of the library.
(4) And (3) machine sequencing: and after the library is qualified, carrying out HiSeq/MiSeq sequencing according to the effective concentration and the requirement of the target offline data volume.
And (3) sequencing to obtain raw reads, wherein the raw reads contain low-quality reads with connectors, and in order to ensure the quality of information analysis, the raw reads are processed to obtain clean reads. And screening small RNA in a certain length range for clean reads of each sample for subsequent analysis. The length-screened small RNAs were mapped to the reference sequence using Bowtie and then aligned in miRBase to identify known miRNAs, and new miRNAs were predicted using miRDeep2 and mirewo software. Then, the expression level of miRNAs was counted, and expression level normalization was performed using TPM (bridges per million reads). Thereafter, the differential expression analysis of miRNAs was performed between the experimental group and the control group by using DESeqR software, and the screening was evaluated from the two aspects of fold difference and significance level, and the threshold value of the screening was set as: log2(fold change) ≧ 0.5 and the P-value < 0.05. The results are shown in fig. 2A, and cluster analysis shows that the expression pattern of serum exosome miRNAs of laryngeal squamous carcinoma patients is obviously different from that of healthy controls; as shown in fig. 2B, 34 miRNAs were significantly highly expressed and 41 miRNAs were significantly less expressed in laryngeal squamous carcinoma patients relative to healthy controls.
Example 3
Real-time fluorescent quantitation (qRT-PCR) verification of serum exosome differential miRNAs of laryngeal squamous carcinoma patients and healthy control groups:
no known stable reference gene exists in the serum exosome, and the experiment firstly carries out screening and identification on the reference gene and then carries out qRT-PCR verification on miRNAs differentially expressed in the experiment II. In this experiment, serum exosome RNAs of 50 laryngeal squamous carcinoma patients and 25 healthy controls were used for qRT-PCR validation of differentially expressed miRNAs, wherein serum exosome RNAs of 7 laryngeal squamous carcinoma patients and 7 healthy controls were simultaneously used for qRT-PCR detection in internal reference screening. The specific implementation steps are as follows:
step 1, reference Gene screening
In the invention, serum exosome RNAs stably expressed in laryngeal squamous cell carcinoma patients (experimental group) and healthy controls (control group) are screened from RNA sequencing data and documents in the second experiment as candidate internal reference genes, and then five internal reference analysis algorithms are used for comprehensive analysis and selection.
(1) Screening candidate reference genes. And selecting miRNAs with small variation coefficient and moderate expression level in sequencing data as candidate internal reference miRNAs. First, miRNAs with significant difference in expression level (P <0.05) between the experimental group and the control group, or miRNAs with TPM value <1, were excluded; secondly, miRNAs with a ratio of average TPM (experimental group) to average TPM (control group) of <0.75 or >1.3 were excluded; finally, the total variation coefficient of each miRNA is calculated (standard deviation/average), the variation coefficients are sorted from small to large, and the first 8 miRNAs with the average TPM value of more than 50 are selected as candidate internal reference genes, namely miR-30a-5p, -532-5p, -181a-5p, -425-5p, -363-3p, -424-3p, -181b-5p and-181 a-2-3 p. In addition, U6 and miR-16-5p reported in the literature are also incorporated into candidate reference genes to be analyzed.
(2) And (3) detecting candidate reference genes qRT-PCR and analyzing reference software.
a. The above 10 candidate internal reference genes were subjected to qRT-PCR assay in serum exosomes of 7 additional laryngeal squamous cell carcinoma patients and 7 healthy controls.
Reverse transcription: reverse transcription was performed using the All-in-one TMmiRNA First-Strand cDNA Synthesis Kit (Genecopoeia, Rockville, Md., USA) in a reaction system comprising: 1 μ L of 2.5UMu.l Poly (alpha-glucosidase), 1. mu.L RTase Mix, 5. mu.L 5 × Reaction Buffer, 50ngRNA and corresponding volume of ddH2O (RNase/DNase-free), 25. mu.l of total reaction system. The reaction conditions of the method are as follows: the reaction was carried out at 37 ℃ for 60 minutes and at 85 ℃ for 5 minutes.
Real-time fluorescent quantitative PCR (qRT-PCR): the method adopts a ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) reagent to carry out qRT-PCR detection, and the reaction system comprises the following steps: 10 μ L of 2 XMASTER Mix, 0.4 μ L of 10 μ M forward primer, 0.4 μ L of 10 μ M reverse primer, 0.4 μ L of reference dye 1, 5 μ L of diluted cDNA, and 3.8 μ L of LH2O, 20 μ l total, 3 replicate wells per miRNA were detected. The PCR was carried out using a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, Massachusetts, USA) fluorescent quantitative PCR instrument under the following reaction conditions: the reaction was carried out at 95 ℃ for 3 minutes, at 95 ℃ for 10 seconds, at 60 ℃ for 30 seconds, and for 40 cycles.
b. For Ct values detected by qRT-PCR in a, a comprehensive analysis was performed using 5 algorithms BestKeeper, NormFinder, geNorm, Δ Ctmethod, and RefFinder. miR-30a-5p, miR-532-5p and U6 were finally selected as the optimal reference gene combination.
For clinical application, the miRNAs with up-regulated expression and high expression (TPM is more than or equal to 50) in laryngeal squamous cell carcinoma patients are selected from 75 miRNAs with differential expression in the second experiment to carry out a large sample qRT-PCR verification. A total of 9 miRNAs were selected, which were: miR-941, -27a-5p, -1246, -452-5p, -1-3p, -7-5p, -3529-3p, -24-3p and-223-5 p. The reverse transcription and qRT-PCR experimental method is the same as the experimental implementation step 1(1), and miR-30a-5p, miR-532-5p and U6 in the step 1 are used as internal reference genes to carry out data calibration. Quantitative Delta Ct ═ Ct of target miRNAs relative to internal referencePurpose(s) to-CtGeometric mean of three reference genesBy log10(2-ΔCt) The relative expression level is determined by the method. The Mann-Whitney test was used to assess the statistical differences in the relative expression levels of miRNAs between the two groups, as P<0.05 was considered statistically significant. The results are shown in FIGS. 2A and 2B, and the expression levels of miR-941 and miR-27a-5P in laryngeal squamous carcinoma serum exosome are significantly higher than those of healthy controls (P)<0.05). It is composed ofHis 7 miRNAs were not statistically different between the two groups.
Example 4
ROC curve evaluates the diagnostic efficacy of serum exosome miRNAs:
to further test the discriminatory power of miR-941 and miR-27a-5p, we performed diagnostic efficacy assessment using ROC curve analysis and calculated the area under the curve (AUC value). The results are shown in fig. 4A, the AUC value of miR-941 is 0.797, and the 95% Confidence Interval (CI) is 0.676-0.918; fig. 4B shows that the AUC value of miR-27a-5p is 0.672, and 95% CI is 0.54-0.804. As can be seen, the miR-941 has high diagnostic efficiency (> cutoff value is 0.7), and can be used as an ideal laryngeal squamous cell carcinoma diagnostic marker.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention.
Claims (6)
1. Laryngeal squamous carcinoma serum exosome marker, characterized in that: the preparation method comprises the following steps:
(1) collecting serum samples of a laryngeal squamous carcinoma patient and a healthy contrast person, extracting exosomes, identifying, and extracting exosome RNA;
(2) carrying out high-throughput sequencing in a small sample amount, screening out serum exosome miRNAs which are differentially expressed between a laryngeal squamous cell carcinoma patient group and a healthy control group, and selecting miRNAs which are up-regulated and expressed in high expression amount in the laryngeal squamous cell carcinoma patient as candidate miRNAs;
(3) verifying the candidate miRNAs in the enlarged sample by adopting a qRT-PCR method; before verification, firstly determining an internal reference gene of a qRT-PCR experiment, wherein the screening process comprises the following steps: screening RNAs with stable expression from the RNA sequencing data and literature reports in the step (2) as candidate internal reference genes, carrying out comprehensive analysis by 5 internal reference analysis algorithms after qRT-PCR detection, and screening the internal reference genes for qRT-PCR verification;
(4) the ROC curve evaluates the diagnostic efficacy of the miRNAs validated in step (3), i.e. the ability to identify patients with laryngeal squamous cell carcinoma and healthy individuals.
2. The screening method of the laryngeal squamous carcinoma serum exosome marker is characterized in that: the method comprises the following steps:
(1) collecting serum samples of a laryngeal squamous carcinoma patient and a healthy contrast person, extracting exosomes, identifying, and extracting exosome RNA;
(2) carrying out high-throughput sequencing in a small sample amount, screening out serum exosome miRNAs which are differentially expressed between a laryngeal squamous cell carcinoma patient group and a healthy control group, and selecting miRNAs which are up-regulated and expressed in high expression amount in the laryngeal squamous cell carcinoma patient as candidate miRNAs;
(3) verifying the candidate miRNAs in the enlarged sample by adopting a qRT-PCR method; before verification, firstly determining an internal reference gene of a qRT-PCR experiment, wherein the screening process comprises the following steps: screening RNAs with stable expression from the RNA sequencing data and literature reports in the step (2) as candidate internal reference genes, carrying out comprehensive analysis by 5 internal reference analysis algorithms after qRT-PCR detection, and screening the internal reference genes for qRT-PCR verification;
(4) the ROC curve evaluates the diagnostic efficacy of the miRNAs validated in step (3), i.e. the ability to identify patients with laryngeal squamous cell carcinoma and healthy individuals.
3. The application of the laryngeal squamous carcinoma serum exosome marker is characterized in that: is applied to the preparation of the laryngeal squamous cell carcinoma diagnostic reagent.
4. Larynx squamous carcinoma diagnostic kit, characterized by: the kit consists of a serum exosome, an RNA extraction reagent, a miR-941 specific amplification primer and a universal PCR amplification reagent; forward primer for miR-941: 5'-GCACCCGGCTGTGT-3' are provided.
5. The application of the laryngeal squamous carcinoma diagnostic kit is characterized in that: the application of the kit containing the miR-941 detection reagent in screening and diagnosing laryngeal squamous cell carcinoma.
6. The laryngeal squamous carcinoma drug treatment target is characterized in that: the laryngeal squamous carcinoma drug treatment target is miR-941, and the nucleotide sequence of miR-941 is as follows: CACCCGGCUGUGUGCACAUGUGC are provided.
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