CN114045341B

CN114045341B - Application and product of oral submucosa fibrosis malignant progress or oral cancer early diagnosis and/or prognosis biomarker caused by oral submucosa fibrosis malignant progress

Info

Publication number: CN114045341B
Application number: CN202111445687.6A
Authority: CN
Inventors: 陈珺; 李文杰; 刘斌杰; 谢晓莉
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2024-03-01
Anticipated expiration: 2041-11-30
Also published as: CN114045341A

Abstract

The invention discloses application of a biomarker for early diagnosis and/or prognosis of oral cancer caused by malignant progression of oral submucosa fibrosis and a kit, wherein the biomarker comprises LINC02147 gene, and the kit comprises a primer for specifically amplifying LINC 02147. The LINC02147 RNA expression level in the sample can be accurately detected, and the method has early diagnosis and prognosis values for oral cancer and oral submucosal fibrosis cancer, and is simple to operate, high in accuracy and low in cost. The application of the reagent for detecting the biomarker in the sample in preparing an early diagnosis and/or prognosis product for oral cancer caused by the malignant progress of the oral submucosa fibrosis and the application of the biomarker in constructing a calculation model for predicting the malignant progress of the oral submucosa fibrosis and the oral cancer caused by the malignant progress of the oral submucosa fibrosis or a system embedded with the calculation model can guide doctors to timely take treatment strategies, measures and measures.

Description

Application and product of oral submucosa fibrosis malignant progress or oral cancer early diagnosis and/or prognosis biomarker caused by oral submucosa fibrosis malignant progress

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to application and a product of an early diagnosis and/or prognosis biomarker for oral cancer caused by malignant progression of oral submucosa fibrosis.

Background

Oral Submucosal Fibrosis (OSF) is a precancerous lesion of the oral cavity, and current research considers that its etiology is related to arecoline in betel nuts. OSF has the potential to progress to oral cancers with a rate of approximately 7% to 13%. OSF is generally atypical in early stage of canceration, once canceration is carried out, surgical excision is the optimal treatment means, but the optimal surgical time is missed when diagnosis is generally confirmed, and even if surgery is carried out, the beauty and functional damage of the face of the oral cavity can be caused, so that the canceration is monitored from the molecular level, the early diagnosis and the early treatment are realized, and the health cost and the economic cost of a patient can be greatly reduced.

The oncogene is generally inactivated in tumor cells, and thus restoring the function of the oncogene is considered as an effective method of inhibiting the development and progression of tumorigenesis. The cancer suppressor gene itself is expressed in normal cells and plays a role in suppressing cancer, so that the increase of the function of the cancer suppressor gene has no obvious side effect on the normal cells, and is a relatively safe tumor specific treatment method. Currently, there are tumor treatments aimed at restoring the function of oncogenes, such as p53 adenovirus. And with the continuous progress of molecular biology technology such as PCR, new cancer suppressor genes are continuously discovered and identified, thereby providing more opportunities for tumor diagnosis and treatment.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings in the background art, and provide an application and a product of an early diagnosis and/or prognosis biomarker for oral cancer caused by malignant progression of oral submucosa fibrosis.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

use of a reagent for detecting a biomarker in a sample, the biomarker comprising the LINC02147 gene, in the preparation of a product for early diagnosis and/or prognosis of malignant progression of oral submucosal fibrosis or oral cancer caused thereby. The research shows that LINC02147 participates in canceration of oral submucosal fibrosis and is a novel oral cancer suppressor gene.

For the above-described applications, preferably, the reagent comprises a reagent for detecting the expression level of LINC02147 RNA in a sample by a sequencing technique, a nucleic acid hybridization technique or a nucleic acid amplification technique.

Preferably, the sample comprises any one or more of tissue, saliva and blood.

Preferably, the reagent contains a primer for specifically amplifying LINC02147 or a probe for specifically recognizing LINC 02147.

Preferably, the primer for specifically amplifying LINC02147 comprises an LINC02147 upstream primer and an LINC02147 downstream primer, and the nucleotide sequences are as follows:

LINC02147 upstream primer: 5'-TGCTATCAGGGTTGGTTTCG-3';

LINC02147 downstream primer: 5'-AGTAGATTGCTGGAGCCAGTTC-3'.

Based on a general inventive concept, the invention also provides a primer for specifically amplifying LINC02147 gene, which is characterized by comprising an LINC02147 upstream primer and an LINC02147 downstream primer, wherein the nucleotide sequences are as follows:

LINC02147 upstream primer: 5'-TGCTATCAGGGTTGGTTTCG-3';

LINC02147 downstream primer: 5'-AGTAGATTGCTGGAGCCAGTTC-3'.

Based on one general inventive concept, the invention also provides a kit for detecting malignant progression of oral submucosa fibrosis or oral cancer caused by the malignant progression, which comprises a primer for specifically amplifying LINC02147, and specifically comprises an LINC02147 upstream primer and an LINC02147 downstream primer, wherein the nucleotide sequences of the primers are as follows:

LINC02147 upstream primer: 5'-TGCTATCAGGGTTGGTTTCG-3';

LINC02147 downstream primer: 5'-AGTAGATTGCTGGAGCCAGTTC-3'.

Preferably, the method further comprises: the nucleotide sequences of the upstream primer of GAPDH and the downstream primer of GAPDH are as follows:

GAPDH upstream primer: 5'-CCCACTCCTCCACCTTTGAC-3';

GAPDH downstream primer: 5'-TCTTCCTCTTGTGCTCTTGCTG-3'.

Preferably, the method further comprises: total RNA extraction reagent, RNA reverse transcription reaction solution and real-time fluorescence quantitative PCR reaction solution;

the RNA reverse transcription reaction solution comprises the following components: 1. Mu.L of Oligo dT at 0.5. Mu.g/. Mu.L, 4. Mu.L of 5 Xreaction Buffer, 1. Mu. L RNase Inhibitor, 2. Mu.L of 10mM dNTPs mix, 1. Mu. L Reverse Transcriptase, RNase-free water;

the real-time fluorescence quantitative PCR reaction liquid comprises the following components: 10. Mu.L 2X SYBR Green qPCR Mix, 1. Mu.L 10uM Forward primer, 1. Mu.L 10uM Reverse primer, 1. Mu.L cDNAtemplate, 7. Mu.L ddH ₂ O。

The invention also provides a method for evaluating the expression level of LINC02147, which comprises the following steps:

(1) Extracting total RNA of a sample;

(2) Carrying out RNA reverse transcription and real-time fluorescence quantitative PCR reaction on the total RNA of the sample obtained in the step (1) by adopting a primer for specifically amplifying LINC02147 gene;

(3) Pressing the buttonThe method calculates the analysis result.

In the above evaluation method, preferably, in the step (1), the sample is a tissue specimen of oral cancer, oral submucosa fibrosis or normal oral mucosa.

Based on one general inventive concept, the invention also provides an application of a biomarker in constructing a calculation model for predicting the malignant progress of oral submucosa fibrosis or oral cancer caused by the malignant progress of oral submucosa fibrosis or a system embedded with the calculation model, wherein the biomarker comprises LINC02147 gene.

For the above application, preferably, the calculation model uses the expression level of the biomarker as an input variable, and performs calculation by a bioinformatics method.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, LINC02147 is selected as a biomarker, can be used as an early diagnosis and prognosis marker of oral cancer caused by oral submucosal fibrosis, and is designed into relevant detection reagents (specific amplification primers), kits and calculation models aiming at the biomarker, so that the oral submucosal fibrosis malignant progress or an early diagnosis and/or prognosis product of oral cancer caused by the oral submucosal fibrosis malignant progress is further prepared, and the oral submucosal fibrosis canceration detection kit has the value of early diagnosis and prognosis.

2. The detection reagent and the kit provided by the invention can accurately detect the LINC02147 RNA expression level contained in a sample, and the LINC02147 RNA expression level is used as an input variable, and the operation is performed by a bioinformatics method, so that the kit can further have early diagnosis and prediction prognosis values for oral cancer and oral submucosal fibrosis cancer, and the kit is simple to operate, high in accuracy and low in cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the relative expression levels of LINC02147 in normal oral mucosa, submucosal fibrosis and oral cancer tissue specimens;

FIG. 2 is a graph showing the results of LINC02147 expression verification of oral cancer tissue and normal paracancerous tissue;

FIG. 3 is a K-M survival curve of LINC 02147;

FIG. 4 is the results of LINC02147 expression in oral cancers of different TNM stages;

FIG. 5 is a graph of LINC02147 diagnostic value ROC;

FIG. 6 is the results of a Cox regression analysis of LINC02147 (A is a single-factor Cox regression analysis, B is a multi-factor Cox regression analysis);

FIG. 7 is a nomogram (nomogram) constructed based on the expression level of LINC02147 to predict survival in patients with oral cancer;

FIG. 8 is a calibration curve for predicting 3-year survival and actual survival of patients with oral cancer based on a nomogram constructed;

FIG. 9 is a calibration curve for predicting 5 year survival and actual survival of patients with oral cancer based on a nomogram constructed;

FIG. 10 is a graph showing the results of LINC02147 expression verification of normal paracancerous and oral cancer tissues in dataset GSE 160042;

FIG. 11 is a ROC curve of the external database GSE160042 validating the diagnostic value of LINC 02147;

FIG. 12 is the relative expression of α -SMA after 40 μM arecoline treatment;

FIG. 13 shows the relative expression of COL1α1 after 40. Mu.M arecoline treatment;

FIG. 14 is a graph showing the relative expression of OSF and oral cancer cell level LICN02147 after 40. Mu.M arecoline treatment;

FIG. 15 is the relative expression of siRNA-LINC02147 24h after cell transfection of LINC02147 siRNA;

FIG. 16 is the relative expression of siRNA- α -SMA 24h after cell transfection with LINC02147 siRNA;

FIG. 17 is the relative expression of siRNA-COL 1. Alpha.1 after 24h of cell transfection of LINC02147 siRNA;

FIG. 18 shows the expression of proteins 24h after cell transfection of LINC02147 siRNA;

FIG. 19 is a graph showing the relative expression of siRNA-MCM2 24h after cell transfection of LINC02147 siRNA;

FIG. 20 is a graph showing the relative expression of siRNA-MCM3 24h after cell transfection of LINC02147 siRNA;

FIG. 21 is a graph showing the relative expression of siRNA-MCM5 after 24h of cell transfection of LINC02147 siRNA;

FIG. 22 is a schematic diagram showing the proliferation of sc-9 cells of LINC02147 knocked-out oral cancer cell line.

Detailed Description

The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.

Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.

Examples:

1. application of reagent for detecting LINC02147 gene in sample in preparation of oral submucosal fibrosis malignant progress or early diagnosis and/or prognosis products of oral cancer caused by oral submucosal fibrosis malignant progress

The invention discovers LINC02147 (Ensembl ID: ENSG 00000249797) is a novel cancer suppressor gene through bioinformatics and laboratory verification, has early diagnosis and prognosis values for oral submucosal fibrosis cancer, and can be used as an early diagnosis and prognosis marker for oral submucosal fibrosis malignant progress. The invention designs a primer and a kit for specifically amplifying LINC02147 by taking LINC02147 as a target point and a core.

Table 1: primer sequences

LINC02147 was found to be expressed in normal oral mucosa, oral submucosal fibrosis, and oral cancer as follows:

1.1 discovery of LINC02147 to participate in oral submucosal fibrotic malignant progression based on GEO database

Two data sets, namely GSE125866 and GSE64216, are downloaded through a GEO database, wherein the GSE125866 data set comprises the following samples of normal oral mucosa: oral submucosal fibrosis: oral cancer = 2:8:8; GSE64216 contains the number of samples of normal oral mucosa: oral submucosal fibrosis: oral cancer = 2:2:4, differential expression of genes was calculated with R-pack Limma (chip data) and edge (sequencing data). Differential gene inclusion criteria: (1) log2 FC|is not less than 1 (i.e., the difference multiple of gene expression amounts is not less than 2 times or not more than 0.5 times); (2) the adjust-value is <0.05. Analysis shows that LINC02147 expression is reduced in the process of converting oral submucosal fibrosis into oral cancer.

1.2 clinical tissue sample validation:

the real-time fluorescence quantitative PCR detection LINC02147 expression in normal oral mucosa, oral submucosa fibrosis and oral cancer tissue is a method for carrying out marker tracking on PCR products by using fluorescent dyes or fluorescent marked specific probes, carrying out real-time online monitoring on the reaction process, analyzing the results by combining corresponding software, and calculating the initial template quantity of a sample to be detected. In the PCR reaction system, SYBR fluorescent dye is added, and after being specifically doped into DNA double chains, the SYBR fluorescent dye emits fluorescent signals, and SYBR dye molecules which are not doped into the chains do not emit any fluorescent signals, so that the increase of the fluorescent signals and the increase of PCR products are completely synchronous.

(1) Test reagent: highfidelity PFU DNA polymerase, trizol reagent, phosphate Buffer (PBS) and 0.25% (W/V) Trypsin/1 mM EDTA (Trypsin-EDTA) were purchased at Invitrogen (USA); fetal Bovine Serum (FBS) was purchased from Gibco (USA); DMEM medium was purchased from HyClone (USA); gotag polymerase is available from Promega (USA). cDNA reverse transcription kit used for reverse transcription Reaction (RT) was purchased from Takara (Tokyo, japan).

(2) The operation process is as follows:

(1) total RNA extraction

Trizol reagent from Invitrogen was used for isolation and extraction according to the method provided in the product specification. All vessels and water used for extracting RNA were subjected to enzyme-free treatment to ensure an enzyme-free environment in the experiment.

Taking oral cancer, oral submucosa fibrosis and normal oral mucosa tissue specimens refrigerated at the temperature of minus 80 ℃, adding lmLTrilzol into a 1.5mLEP tube, fully grinding in an ultrasonic refiner, and standing at room temperature for 5min. 200. Mu.L of chloroform was added thereto, the mixture was shaken for 10 seconds and allowed to stand for 2 minutes. Centrifuging at 4deg.C, 12000g×15min, and collecting supernatant. Add 500. Mu.L of isopropanol and gently mixMixing the liquid in the tube, and standing at room temperature for 30min. Centrifuge at 4℃at 12,000 g for 10min, discard supernatant. lmL 75% ethanol was added and the precipitate was gently washed. 4 ℃, 7500g,5min, discard supernatant. Air drying, adding proper amount of DEPC H ₂ O is dissolved.

(2) RNA concentration and purity detection

RNA concentration and purity were measured by measuring the absorbance of RNA solution at 260 and 280nm using a spectrophotometer by sucking L. Mu.L of each sample using RNase-free Water of constant volume RNA as a blank. And observing whether RNA is degraded or not in the MOPS formaldehyde denatured gel.

(3) RNA reverse transcription

The Takara RR037APrimeScript RTreagent Kit kit was used. The specific operation steps are as follows:

1) mu.L Oligo dT (0.5. Mu.g/. Mu.L) and 2.0. Mu.g Total RNA were added to the PCR vials, and RNase-free water was supplemented to 12. Mu.L; after mixing evenly, centrifugating, warm-bathing for 5min at 65 ℃, and immediately placing on ice.

2) The reaction system (carried out on ice) was prepared in the following proportions.

20 mu L of the mixture is blown and sucked uniformly and centrifuged for 10s.

Note that: if a plurality of samples exist, a Reaction mix can be prepared in advance, 5 times of Reaction Buffer, RNase Inhibitor, 10mM dNTPs mix and the like are added into a 0.5mL centrifuge tube together, and are blown and sucked by a 200 mu L pipettor, mixed evenly, split-packed into each 0.2mL PCR tube, and then corresponding templates are added.

3) After all samples are added, the tube cover is covered, centrifuged for 10s, and placed in a PCR instrument die, and the protective cover is covered.

4) The PCR instrument was turned on and the PCR procedure was set to:

42℃ 60min

70℃ 5min

this step may be performed using a water bath.

Note that: the enzyme can be deactivated by the reaction at 42 ℃ and 70 ℃ for 5min.

5) After the reaction was completed, the reaction mixture was stored at-20℃until use.

(4) Real-time fluorescent quantitative PCR reaction

The specific operation steps are as follows:

note that: the newly synthesized primer needs to be dissolved by adding water in advance: after centrifuging the centrifuge tube containing the primers shown in Table 1 at 1000rpm at room temperature for 3min, the tube lid was gently opened, a proper amount of autoclaved deionized water was added according to the primer synthesis instructions, and the mixture was blown and sucked with a gun to dissolve the primers sufficiently, followed by brief centrifugation for 15s. Typically, the primers are stored at a concentration of 100. Mu.M and used at a concentration of 10. Mu.M.

The ice box is prepared, and the required reagent is taken out from the refrigerator and then placed into the ice box to be melted. The primers and template may be thawed at room temperature and then placed on ice.

1) Pre-experiment: using SYBR Green qPCR Mix, it was determined whether the primers were specific primers and their optimal annealing temperatures. Using a 10. Mu.L pipette, add in a0.2 mL eight-way tube:

20 mu L of the total, the mixture is blown and sucked uniformly and centrifuged for 15s. If there are multiple samples, reaction mix may be prepared in advance, 2X SYBR Green qPCRMix, ddH2O, cDNAtemplate, etc. (if the same template is used) are added together into a 1.5mL centrifuge tube, and the mixture is pipetted and mixed with a 200. Mu.L pipette and dispensed into each 0.2mL octant. After all samples are added, the tube cover is covered, centrifuged for 10s, and the tube cover is placed in a mini fluorescent quantitative PCR instrument die to cover the protective cover.

Note that: in the case where the template concentration is sufficient, the template may be diluted and then detected.

Opening the instrument and computer program, and setting a gradient Real-Time program to be:

a dissolution profile procedure was added.

After the end of the procedure, the dissolution curve was checked, the effect of the primers was detected, and the primers (i.e., the primers described in Table 1) and annealing temperatures were selected which gave the best product specificity (single dissolution curve peak, high peak).

2) After the applicable primer and annealing temperature are determined, the fluorescent quantitative detection of the required sample is carried out.

Using a 10. Mu.L pipette, add in a0.2 mL eight-way tube:

20 mu L of the total, the mixture is blown and sucked uniformly and centrifuged for 15s. If there are multiple samples, the reaction mix can be pre-configured to give 2X SYBR Green qPCR Mix, ddH ₂ O, forward primer, reverse primer (if the same primer is used) were added together into a 1.5mL centrifuge tube, and the mixture was pipetted with a 200. Mu.L pipette and aliquoted into each 0.2mL octant tube (three replicates for each sample and internal controls were provided). After all samples are added, the tube cover is covered, centrifuged for 10s, and the tube cover is placed in a fluorescent quantitative PCR instrument die to cover the protective cover.

a dissolution profile procedure was added.

After the reaction is finished, checking curve data obtained by the program, adjusting the curve to be a linear curve, moving a reading line to a lower part of the curve, reading a Ct value and copying.

(5) And (3) judging a fluorescent quantitative PCR result: the internal reference gene is GAPDH gene, and the RNA content of each sample is detected. The sample holes and the internal reference holes are respectively provided with 2And (5) repeating the holes, and taking average Ct values. Δct=ct _{Target gene} -Ct _GAPDH Then 2 ^-△Ct Is the expression level of LINC02147 relative to the reference gene GAPDH in the tissue; ΔΔct= Δct _{Tissue to be tested} -△Ct _{Control tissue} Multiple variation of 2 ^-△△Ct I.e., the amount of expression in the test tissue relative to the control tissue.

(2) Analysis of results: 10 cases of normal oral mucosa, oral Submucosal Fibrosis (OSF) and oral cancer (OSCC) tissue samples were collected clinically, and qPCR verification revealed that LINC02147 expression gradually decreased from normal mucosa, oral submucosal fibrosis to oral cancer, and the difference was statistically significant (P < 0.05) (FIG. 1). The detection reagent and the kit provided by the invention can accurately detect the expression level of LINC02147 RNA contained in a sample.

2. Application of LINC02147 gene in construction of computational model for predicting oral submucosal fibrosis malignant progress or oral cancer caused by oral submucosal fibrosis malignant progress or system embedded with computational model

The expression level of the biomarker is used as an input variable, and the operation is performed by a bioinformatics method. The TCGA verification and prognosis and diagnostic value evaluation process is as follows:

2.1Kaplan-Meier method survival analysis:

(1) The operation steps are as follows:

(1) RNA-seq data and clinical data for head and neck squamous cell carcinoma were downloaded from The Cancer GenomeAtlas (TCGA) (http:// tcgadata. Nci. Nih. Gov /), with no history of malignancy or history of neoadjuvant treatment for 327 oral cancer patients who were included in the analysis.

(2) The expression level of LINC02147 in oral and normal paracancerous tissues was analyzed using limma package, t-test based on TCGA.

(3) The correlation of LINC02147 with the total survival (OS) of patients with oral cancer was calculated using the Kaplan-Meier (K-M) method. According to the gene expression condition of each sample, selecting an optimal cut-off value as a threshold value of a high expression group and a low expression group through X-tile software. Patients are classified according to a threshold. The X-tile graph provides an intuitive way to evaluate the relationship between variables and survival. The X-tile map was drawn using version 3.6.1 of X-tile software (university of Kyoto medical college, new York, CT, USA). Survival was calculated using "survivinal" in "R" (https:// cran. R-project. Org/web/packages/survivinal/index. HtmL) and K-M curves were plotted using "ggplot 2".

(4) The diagnostic value of LINC02147 was assessed using ROC analysis, with the pROC software package used to draw a test subject operating characteristic (ROC) curve, the area under the ROC curve (AUC) representing the diagnostic value. When AUC is greater than 0.7, we consider LINC02147 as a diagnostic marker with better specificity and sensitivity.

(5) To further confirm the prognostic effect of LINC02147, expression levels of LINC02147 in different TNM stages of oral cancer patients were analyzed based on TCGA using Limma package, t-test.

(3) Analysis of results: the expression of the TCGA database is verified that the LINC02147 expression in the oral cancer is obviously lower than that of the tissue beside the normal cancer (figure 2), and is consistent with the qPCR result of a clinical tissue sample; survival analysis found that patients with low expression of LINC02147 had a worse prognosis than patients with higher expression of LINC02147 (fig. 3); analysis of the relationship between LINC02147 expression level and clinical stage of oral cancer found that: LINC02147 expression was significantly higher in patients with stage I oral cancer than in II, III, IV (fig. 4), and these results suggest that LINC02147 is proportional to prognosis of oral cancer, with lower LINC02147 expression leading to poorer prognosis of oral cancer. ROC curve analysis found that the area under the curve auc=0.893, indicating that LINC02147 has good diagnostic value (fig. 5).

2.2Cox regression analysis to determine if LINC02147 is an independent prognostic factor

(1) The operation process is as follows:

(1) 327 oral cancer patient data are encoded and then input into a computer to establish a database. 9 indexes such as sex, age, pathological Borders grade, lymphatic invasion condition, peri-nerve invasion condition, T stage, N stage, TNM stage and LINC02147 expression condition are selected.

(2) By using χ ² And (3) checking the prior single-factor Cox regression analysis, and performing multi-factor Cox regression analysis on indexes with obvious significance in the single-factor analysis to judge whether LINC02147 is an independent prognosis influence factor. In the Cox regression model of the model,and taking a power number with a certain independent variable coefficient as e, wherein the obtained value is the HR value. Considering the practical significance of HR values in clinical studies, when HR>1, the independent variable is a dangerous factor; when HR is<1, the argument is a protection factor. Data analysis was performed using SPSS20.0 statistical software. All statistical analyses were performed at P<0.05 is significant.

(2) Results: single factor Cox regression analysis showed that LINC02147 (hr=0.56 (0.38-0.83), p=0.004) is a protective factor for oral cancer (i.e. the lower the expression, the worse the prognosis of oral cancer), lymphatic vessel invasion (hr=1.66 (1.11-2.48), p=0.014), peri-nerve invasion (hr=1.91 (1.28-2.87), p=0.002), TNM staging (hr=2.22 (1.41-3.51), p=0.001) is a risk factor for oral cancer (fig. 6A). Further multifactorial Cox regression analysis showed that LINC02147 (hr=0.52 (0.30-0.90), p=0.020) is an independent protective factor for oral cancer; TNM staging (hr=2.17 (1.14-4.16), p=0.019) and peri-nerve invasion (hr=1.75 (1.11-2.77), p=0.016) are independent risk factors for oral cancer. This part of the experiment further illustrates the poor prognosis of oral cancer patients with LINC02147 low expression (fig. 6B).

2.3 construction of a model nomogram for predicting the total survival of patients with oral cancer for 3 years and 5 years

Nomograms (nomograms) are widely used in oncology and medicine as a predictive tool for the incidence or prognosis of a certain event. The nomogram can integrate the individual probability of clinical events generated by the risk factors related to prognosis, so that the pursuit of a novel medical mode of combining biology and clinic is realized, and the development of personalized medical treatment in a new era is promoted. The Nomogram predictive model is more advantageous than traditional TNM staging systems and has therefore been proposed as an alternative or a new standard, becoming an important component of modern medical decisions. According to the invention, through researching the expression of LINC02147 in oral cancer and the influence of LINC02147 on the prognosis of a patient, a nomogram prediction model of the overall survival rate of the oral cancer patient is constructed, so that more accurate prognosis information can be provided and personalized medical decision can be made.

(1) The operation process is as follows: 327 oral cancer patients based on TCGA construct a nomogram prediction model of the overall survival rate of the patients for 3 years and 5 years by using R language; evaluating the discrimination of the model using a consistency index (concordance index, C-index); internal verification is carried out on the model by adopting a Bootstrap method, and the prediction consistency of the model is evaluated

(2) Results: by single-factor and multi-factor Cox regression analysis of 327 oral cancer patients in the TCGA database, it is clear that LINC02147, TNM staging and peri-nerve invasion are independent influencing factors for prognosis of oral cancer patients. Based on the 3 variables, a nomogram prediction model of survival rate of patients with oral cancer for 3 years and 5 years was constructed, and by using the model, scores (Points) corresponding to LINC02147, TNM stage (TNM-stage) and peri-nerve invasion (perineural invasion) of the patients were obtained, and the sum of the scores (Total Points) of the variables was correlated with 3-year survival and 5-year survival, and predicted values of survival rate for 3 years and 5 years were obtained (FIG. 7). Discrimination evaluation: the overall survival of patients with oral cancer had a C-index value of 0.624 (95% ci=0.577-0.670, p=3e-04) for the nomogram predictive model, suggesting that the nomogram has a better differentiation. Consistency evaluation: as can be seen from the correction graph (Calibration curve), the overall survival rate predicted value and the actual observed result of the oral cancer patient in 3 years and 5 years are high in coincidence, and especially the overall survival rate predicted in 3 years is basically consistent with the actual observed value (fig. 8 and 9), which suggests that the consistency and accuracy of the nomogram model are high.

3. External database verification

GSE160042 was taken from GEO, which data set contained oral cancer tissue (10 samples) and paired adjacent non-cancerous tissue (10 samples). The data in this dataset was used to further verify the expression, prognosis and diagnostic value of LINC 02147. The method is the same as before.

Analysis of results: the GSE160042 dataset further verifies the expression of LINC 02147. As shown in fig. 10, LINC02147 was significantly less expressed in oral cancers than in paired paracancerous non-cancerous tissues. Further evaluation of the diagnostic value of LINC02147 resulted in auc=0.950 consistent with TCGA results, demonstrating that LINC02147 has the ability to distinguish oral cancers from normal controls (fig. 11).

4. In vitro cell level verification of expression of LINC02147

(1) Main reagents and instruments: high sugar DMEM (Hyclone, USA), fetal bovine serum (Gibco, USA), trypsin (Solarbor) containing EDTA0.25%, inverted microscope, 10mL disposable pipette, 1000. Mu.L (1 mL) pipette, 3mL disposable Papanicolaou pipette, glass plate (sterilized), disposable culture dish, 15mL centrifuge tube, 1000. Mu.L tip, ophthalmic scissors, scalpel handle, tip blade, periodontal probe, 25cm ² Cell culture flasks (1).

(2) Primary culture of human oral mucosa fibroblasts (human oral mucosa fibroblasts, hOMFs) (tissue mass method)

Taking 5mm of relatively healthy periodontal gum (meeting ethical requirements) of 12-25 years old, immediately putting the gum into a PBS solution containing 10% of double antibody (1000 uI/mL) (the PBS solution containing the double antibody is prepared one day before operation), and taking the gum to an ultra-clean workbench of a laboratory within 2 hours;

placing the objects required by the experiment into an ultra-clean workbench ultraviolet lamp for disinfection for 30 minutes;

at 25cm ² Dripping 1mL of FBS (1 d-2 d of Babbit irrigation) into the cell culture flask, wetting the whole culture flask, and then placing the cell culture flask into a 37 ℃ incubator;

preparing 10% PBS solution containing double antibodies, DMEM medium containing 10% double antibodies and DMEM medium containing 15% FBS;

repeatedly (3-4 times) flushing and blowing the tissue blocks by using 10% of double-antibody PBS solution;

transferring the tissue block into a glass culture dish, repeatedly flushing and blowing the tissue block with 10% double-resistant DMEM medium (3-4 times), discarding supernatant, and dividing the tissue block into 5mm pieces with a tissue shear and a 15 th circular blade ² The tissue pieces were then placed into the bottom of a T-25 cell culture flask (connective tissue near the basement membrane was attached to the bottom) taken out of a 37℃incubator with ophthalmic forceps, each tissue piece being 5mm apart.

Dropping 1mL of DMEM medium containing 15% FBS, putting into an incubator with 37 ℃ and 5% CO2 and 95% saturated humidity, taking out the DMEM medium containing 2mL of 15% FBS after 24 hours, changing liquid once every 3-4 days, observing the growth condition of cells under a microscope, culturing for 5-7 days, and then observing that oral mucosa fibroblasts climb out from the periphery of a tissue block, wherein the cells are in a fusiform or spindle shape, have a plurality of longer cell protrusions (2-4), plump and uniform cytoplasm, and have round or oval nuclei and 1-3 nucleoli.

(3) Passage of human oral mucosa fibroblasts (human oral mucosa fibroblasts, hOMFs)

When the cells climb up to 70% -80% of the bottle bottom, the first passage is cultured according to 1-2 passages.

Sucking the liquid in the culture flask, and washing with PBS to remove residual serum and cell debris;

digestion: 2mL of 0.25% trypsin-0.02% EDTA digested for 2min (not more than 4 min), and cell gap increase and cell rounding under an inverted microscope;

terminating digestion: digestion was stopped with 0.5ml DMEM medium with 15% fbs;

then adding 3mL of sterile PBS liquid, blowing (50 times) into single-cell suspension by using a Pasteur dropper, and transferring to a 15mL centrifuge tube;

and (3) centrifuging: centrifuging at 1000r/min for 10min;

removing supernatant, adding 4mL of DMEM with 15% FBS, lightly blowing with a Pasteur dropper uniformly, evenly distributing the mixture into 2T-25 culture flasks, and then supplementing the culture solution to 3-4 mL;

and forming stable passage cells, and taking 3-5 passages of cells for subsequent experiments.

(4) Construction of oral submucosal fibrotic cell model

Experimental reagent: betulin (Sigma-Aldrich, USA), the remaining reagents were identical to the clinical tissue sample expression validation experiments (fig. 12 and 13), demonstrating that 40 μm betulin treatment of hOMFs could successfully construct an oral submucosal fibrocyte model.

(5) Culture of SCC9 cell as one of oral cancer cell lines

1) Resuscitates cells: the frozen tube containing 1mL of cell suspension was thawed by shaking in a 37℃water bath and 4mL of medium was added and mixed well. Centrifuging at 1000RPM for 4min, discarding supernatant, adding 1-2ml culture medium, and blowing uniformly. All cell suspensions were then added to the flask for overnight incubation (or the cell suspension was added to a 10cm dish, about 8ml of medium was added, and incubated overnight). The next day the fluid was changed and the cell density was checked.

2) Cell passage: the cell density reaches 80% -90%, and subculture can be performed. Discarding culture supernatant, and washing cells with PBS without calcium and magnesium ions for 1-2 times; adding 1ml of digestive juice (0.25% Trypsin-0.53mM EDTA) into a culture flask, placing into a culture box at 37 ℃ for digestion for 1-2 minutes, observing the digestion condition of cells under a microscope, if most of the cells become round and fall off, rapidly taking back to an operating table, tapping the culture flask for several times, and adding a small amount of culture medium to stop digestion; supplementing culture medium at a ratio of 6-8 mL/bottle, gently stirring, sucking out, centrifuging at 1000RPM for 4min, removing supernatant, supplementing 1-2mL culture solution, and blowing; the cell suspension was prepared at 1: the 2 ratio was divided into new dishes or flasks containing 8ml of medium.

(6) qPCR detects the expression level of LINC02147 in normal oral mucosa fibroblasts (hOMFs), oral submucosal fibrotic cell model (40. Mu.M arecoline stimulated hOMFs cells for 24 h) and oral cancer cell line SCC9 cells. The procedures of total RNA extraction, RNA concentration and purity detection, RNA reverse transcription and real-time fluorescent quantitative PCR reactions are referred to above.

(7) Analysis of results: compared with normal oral mucosa fibroblasts, oral submucosal fibrosis cell model (40 mu M arecoline treatment hOMFs for 24 hours) and LINC02147 expression in SCC9 cells of an oral cancer cell line are obviously down-regulated (figure 14), which is consistent with the analysis results of clinical tissue samples qPCR and bioinformatics.

The use of a biomarker (LINC 02147) in the construction of a computational model for predicting the progression of oral submucosal fibrosis malignancy or oral cancer resulting therefrom, or a system incorporating said computational model,

5. in vitro cell experiments prove that down-regulating LINC02147 promotes oral submucosa fibrosis, oral submucosa fibrosis and oral submucosa fibrosis canceration

5.1 cell transfection experiments:

(1) Main reagents and instruments: LINC02147 siRNA and negative control siRNA were synthesized by the company ebo, guangzhou, siRNA oligonucleotide sequences were as follows: 5'-GTCCTCACGTGGCCTCTTT-3', 5'-CAAGATCAAGGTGCTATCA-3',5'-CTGGCTTGTAGACAGCTAT-3',5'-CAGGGTTGGTTT CGGCTGTG-3',5'-CAAGATCAAGGTGCTATCAG-3',5' -TCCTCACGTGGCCTCTTTGT-3’；Ribo FECT ^TM CPTransfection Kit transfection kit.

(2) The method comprises the following specific steps:

inoculating cells: hOMFs (1X 10) ⁴ ) Inoculating the cells into a 24-pore plate culture hole, and transfecting when the cells grow normally and the confluence degree reaches 30-50%;

transfection: for each transfected sample, please prepare as follows: preparation of transfection complex: at 30 μl1 XriboFECT ^TM 1.5. Mu.L of 20. Mu.M siRNA stock solution (v 3) was added to CP Buffer (v 2) and gently mixed; then 3. Mu.l riboFECT was added ^TM CP Reagent (v 4), gently beating and mixing, and incubating for 0-15 min at room temperature to prepare the transfection complex. The transfection complex was added drop-wise to cells already containing the appropriate amount of the double antibody-free complete medium (v 1) and gently mixed. The plates were placed in a CO2 incubator at 37℃for 24h.

And (3) effect detection: detection of RNA level (RNAi silencing assay): the target molecule acted by the siRNA is RNA of a target gene, and the target RNA is sheared under the action of a RISC system, so that the detection of the RNA level of the target gene can directly reflect whether the siRNA plays a corresponding role or not, and the specific inhibition efficiency can be calculated through qRT-PCR. The RNA expression of the target gene can be detected to be obviously reduced within 24-72 hours after siRNA transfection.

5.2 Western Blot (Western Blot) experiments

(1) Reagent and consumable: protein extraction reagent (modified RIPA formulation), BCA protein quantification kit, 5×reduction sample buffer, 10×tris-Glycine-SDS running buffer, coomassie blue staining solution, 10×tbstph8.0 wet transfer buffer, PVDF membrane (0.22 μm pore size), PMSF, protease inhibitor, blocking solution, primary antibody: anti-alpha-SMA (Abcam, ab 124964), anti-COL1 alpha 1 (Abcam, ab 260043), anti-beta-actin (Immunoway, YM 3028); and (2) secondary antibody: a secondary antibody labeled with horseradish peroxidase; fresco cryo centrifuge, multiSman 3 microplate reader, electrophoresis tank, wet transfer electrophoresis tank, electrophoresis apparatus, chemi Doc XRS imaging system

(2) The steps are as follows:

extraction of total protein: (1) preparing cell lysate, adding according to a ratio of 100:1Adding RIPA and PMSF, mixing, and placing on ice for use. In the cell number of 1×10 ⁷ 1ml of lysate was added. (2) Cells were collected, medium was aspirated, and cells were washed 2-3 times with pre-chilled PBS (containing PMSF). The PBS was aspirated off, a suitable volume of pancreatin was added to digest the cells, a suitable amount of complete medium was added to each well to terminate the pancreatin digestion and blow, and the cells were collected in a 15ml centrifuge tube, centrifuged at 800rpm for 5min. (3) Adding appropriate amount of cell lysate into each tube, blowing with gun head to fully suspend cells, transferring into 1.5mLEP tube, and standing on ice for 5-10min. d.12000rpm, centrifuging at 4 ℃ for 5min, sucking the supernatant, split charging and storing at-80 ℃ for testing or standby.

And (1) quantitative determination of protein by BCA method, namely preparing working solution by adding 1 volume of BCA reagent B (50:1) into 50 volumes of BCA reagent A according to the number of standard substances and samples, and fully and uniformly mixing. (2) Dilution of standard: the freeze-dried standard is diluted with 0.9% NaCl or PBS to 2000. Mu.l/ml stock solution, and then the stock solution is diluted to 25-2000. Mu.g/ml. (3) 25 μl of standard and samples of the appropriate concentration range were added to the microwells of the 96-well plate, respectively. (4) 200 μl BCA working fluid was added to each well and mixed well (preferably with a gun to increase the speed, otherwise the time difference of addition could affect the final result). (5) The wells were covered and incubated at 37℃for 30 minutes. (6) Cooled to room temperature and measured for a562 with a microplate reader. (7) In Excel, the absorption values of the standard samples (used BSA) are all selected, the graph in point insert is selected, the xy scatter graph (with protein concentration as abscissa and OD value as ordinate, Y (OD) =ax (concentration) +b) is selected to generate a linear equation formula, and when the protein concentration is measured, the Y value is substituted to calculate the X value, and the concentration of the sample protein is measured.

Protein expression levels were determined by Western Blot: (1) protein sample preparation, namely, after the extracted cell total protein is detected by BCA concentration, the concentration of each sample to be detected is regulated by double distilled water and 6xSDS loading buffer to keep the same, the sample to be detected with the regulated concentration is heated at 100 ℃ for 5min to fully denature the protein, and the protein is preserved at-80 ℃ for standby. (2) The formulations of the 12% split gum and the 4% concentrated gum are shown in Table 2.

Table 2:12% separator gum and 4% concentrated gum configuration

(3) Preparing gel: and (3) after the lower edges of two glass plates with different sizes are aligned, placing the two glass plates into a clamping groove to be clamped tightly so as to avoid glue leakage, vertically clamping the two glass plates on a glue preparation clamp to perform glue filling, slowly adding lower glue along one side edge of the glass plates, immediately sealing with methanol after the glue filling is finished, sucking the methanol with filter paper after the lower glue is solidified, filling the mixed upper glue, inserting an electrophoresis comb, and solidifying after the glue is gelled. (4) And (3) electrophoresis, namely loading a protein sample to be tested according to the uniform adjusted concentration and equal volume, carrying out electrophoresis for 30min at an initial voltage of 70V, adjusting the voltage to 90V when the protein sample is electrophoresed to the junction of the upper layer glue and the lower layer glue, and stopping electrophoresis when the pre-dyed protein is separated to a proper degree. (5) Transfer film (wet transfer method): placing the film transfer device in a 4 ℃ environment, and carrying out 300mA constant current; PVDF membrane with 0.22um aperture, and membrane transferring time of 75min. Cutting off a gel block corresponding to the target protein according to the indication of the molecular weight of the pre-dyed protein, cutting a PVDF film with the size corresponding to the gel block, preparing filter paper special for film transfer, and placing the filter paper in a wet transfer printing instrument from a negative electrode to a positive electrode according to the following sequence: filter paper, glue, PVDF film, filter paper. (6) Closing: the PVDF membrane after membrane transfer was placed in TBST solution containing 5% skimmed milk powder and placed on a shaker for 2h at room temperature to bind to non-specific binding sites on the PVDF membrane. (7) Incubating primary antibodies: specifically labeled α -SMA, COL1 α1 antibody and GAPDH reference antibody (all of which were purchased from Abcam, USA) were diluted 1:1000 by volume ratio of TBST solution containing 5% nonfat dry milk, and the blocked PVDF membranes were placed in the corresponding detection antibody solutions, placed on a shaker, incubated overnight at 4 ℃, primary antibody solution recovered, and washed 6 times with TBST. (8) The conjugated horseradish peroxidase-labeled secondary antibody was diluted with a TBST solution containing 5% nonfat milk powder at a volume ratio of 1:5000, incubated for 1h at room temperature, and the membrane was washed 8 times with TBST solution. (9) Proteins were detected using the Chemi Doc XRS imaging system.

5.3CCK-8 detection of cell proliferation

With 96-well plates, 1X 10 cells were grown per well ⁵ The individual oral cancer cell lines SCC-9 cells were cultured for 24 hours and repeated three times. Then respectively using LINC02147 siRNA and negative control siRNA transfected SCC-9 cells, absorbance at 450nm was checked with a microplate reader at 0h,12h,24h,48h after transfection. The final cell count results are the average of 3 samples, expressed as mean ± standard deviation. The method comprises the following specific steps:

(1) the oral cancer cell line, SCC9 cells, transfected with LINC02147 siRNA and negative control siRNA at equal concentration were inoculated into 96-well plates at 100. Mu.l/well and incubated in an incubator for 1 hour.

(2) The CCK-8 solution was thawed in a water bath at 37℃for 5 minutes, and then 10. Mu.l of CCK-8 reagent was added to each well (this step was carefully done to avoid air bubbles from affecting the OD).

(3) D0 is the absorbance value of 450nm measured by an enzyme-labeled instrument after 4 hours of initial culture, then the culture solution is sucked out, 100 mu l of 10% FBS RPM 1640 culture solution containing 10% CCK8 is added into each hole, the culture is continued, the absorbance at 450nm is checked by the enzyme-labeled instrument at the 0 th hour, 12h,24h and 48h after transfection, and proliferation curves are drawn and analyzed after completion.

5.4 data analysis SPSS20.0 software was used. All data are expressed as mean ± standard deviation (±sd). The comparison between the two groups adopts t test, and the analysis of more than three groups of data adopts One WayANOVA. P <0.05 is statistically significant.

Analysis of results: the present experiment performed qPCR assays 24h after LINC02147 siRNA transfection (qPCR methods, steps referenced above), and the results showed significant down-regulation of LINC02147 expression relative to the control group (fig. 15), demonstrating that cell transfection was effective; and after being transfected by LINC02147 siRNA for 24 hours, the expression of an oral submucosa fibrosis marker alpha-SMA and COL1 alpha 1 are obviously increased in the levels of RNA (figure 16 and figure 17) and protein (figure 18), which indicates that the down regulation of LINC02147 expression in hOMFs cells can promote the occurrence and development of oral submucosa fibrosis; after being transfected with LINC02147 siRNA for 24 hours, the expression of the early diagnosis markers MCM2, MCM3 and MCM5 of the oral cancer is obviously increased (FIG. 19, FIG. 20 and FIG. 21), which shows that the down-regulation of the expression of LINC02147 can promote the canceration of hOMFs cells.

Down-regulating LINC02147 expression in the SCC-9 cells of the oral cancer cell line promoted cell proliferation of SCC-9 (FIG. 22), suggesting that down-regulating LINC02147 expression may promote oral cancer progression.

Conclusion: the reduced expression of LINC02147 indicates that patients with malignant progression of oral submucosa are ill-predicted, and LINC02147 has early diagnosis and prediction of oral cancer and oral submucosa fibrosis cancer. The detection reagent and the kit provided by the invention can accurately detect the LINC02147 RNA level in a sample, and have early diagnosis and prediction values for oral cancer and oral submucosal fibrosis cancer, and are simple to operate, high in accuracy and low in cost.

Sequence listing

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Claims

1. Use of a reagent for detecting a biomarker in a sample, wherein the biomarker comprises the LINC02147 gene, in the preparation of an early diagnosis and/or prognosis product of oral submucosal fibrosis malignant progression or oral squamous cell carcinoma caused thereby.

2. The use of claim 1, wherein the reagent comprises a reagent for detecting the expression level of LINC02147 RNA in a sample by a sequencing technique, a nucleic acid hybridization technique or a nucleic acid amplification technique.

3. The use according to claim 2, wherein the sample comprises any one or more of tissue, saliva, blood.

4. The use according to claim 2, wherein the reagent comprises a primer for specifically amplifying LINC02147 or a probe for specifically recognizing LINC 02147; the primers for specifically amplifying LINC02147 comprise an LINC02147 upstream primer and an LINC02147 downstream primer, and the nucleotide sequences are as follows:

LINC02147 upstream primer: 5'-TGCTATCAGGGTTGGTTTCG-3';

LINC02147 downstream primer: 5'-AGTAGATTGCTGGAGCCAGTTC-3'.

5. Use of a biomarker in constructing a computational model for predicting the progression of oral submucosal fibrosis malignancy or oral squamous cell carcinoma resulting therefrom, or a system in which said computational model is embedded, characterized in that said biomarker comprises the LINC02147 gene.

6. The use according to claim 5, wherein the computational model is operated by bioinformatics methods using the expression level of the biomarker as an input variable.