CN117467765A

CN117467765A - Application of FGF6 in diagnosis and treatment of oral mucosa canceration

Info

Publication number: CN117467765A
Application number: CN202311429544.5A
Authority: CN
Inventors: 刘旭倩; 陈潇; 聂敏海
Original assignee: School/hospital Of Stomatology Southwest Medical University
Current assignee: School/hospital Of Stomatology Southwest Medical University
Priority date: 2023-10-31
Filing date: 2023-10-31
Publication date: 2024-01-30

Abstract

The invention discloses application of FGF6 in diagnosis and treatment of oral mucosa canceration, relates to oral mucosa precancerous lesions and oral mucosa cancers, provides products and application for distinguishing healthy people from oral mucosa precancerous lesions patients, also provides products and application for distinguishing oral mucosa precancerous lesions patients from oral mucosa cancers patients, also provides products and application for distinguishing healthy people from oral mucosa cancer patients, and also provides products and application for treating or treating oral mucosa precancerous lesions and oral mucosa cancers by a method, and provides beneficial help in assisting a subject in diagnosing oral mucosa precancerous lesions and oral mucosa cancers.

Description

Application of FGF6 in diagnosis and treatment of oral mucosa canceration

Technical Field

The invention belongs to the technical field of biology, and relates to application of FGF6 in diagnosis and treatment of oral mucosa canceration.

Background

OSCC can evolve from an oral mucosa underlying malignancy (oral potentially malignant disorders, OPMD). About 80% of OSCCs undergo various OPMD stages, such as oral leukoplakia, oral erythema, oral submucosal fibrosis. Clinical data suggests that the transition from OPMD to cancer may take years or more. Therefore, the key stage of diagnosis and treatment of OPMD is an effective way to control OSCC. OPMD was found early and highly susceptible populations were screened as a promising strategy for reducing OSCC incidence. Oral Leukoplakia (OLK) is one of the most common OPMD, with a incidence of 0.13-34.0% and a cancer incidence of 4-13%.

The gold standard for diagnosis of oral squamous cell carcinoma (oral squamous cell carcinoma, OSCC) is a biopsy, but is not suitable as a method for early diagnosis due to the characteristics of invasiveness, large postoperative influence, heavy psychological burden of patients and the like, and due to the conditions of tumor size, specificity of the material-taking part and the like. Therefore, how to find an early diagnosis method that is more efficient, low-cost and more acceptable to patients becomes a hotspot problem that is currently exploring the potential.

Because the oral cavity micro-ecological environment is complex, the metabolism of microorganisms, the oral cavity sanitation and the change of the health condition of mucous membrane can change saliva components and pH values to influence the detection of the markers; the urine components are relatively simple, are easily influenced by various factors such as living habits, drug administration conditions, water intake and the like, and have large fluctuation of urine protein properties and concentration at different stages of the same individual; cerebrospinal fluid is a blood ultrafiltrate, but has limitations that can only be used for diagnosis and monitoring of neurological diseases due to its anatomical location and physiological function specificity.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the following technical scheme:

the invention provides application of a reagent for detecting FGF6 markers in a sample in preparation of a product for diagnosing oral mucosa precancerous lesions.

Further, the detection reagent of the marker includes a reagent for specifically detecting the expression level of the FGF6 gene, or a reagent for specifically binding to a protein encoded by the FGF6 gene.

Further, the oral mucosa precancerous lesions include oral potential malignant diseases, oral epithelial dysplasia, and proliferative wart leukoplakia.

Further, the oral mucosa precancerous lesions include oral leukoplakia, oral lichen planus, oral mucosal erythema, oral submucosal fibrotic lesions, discoid lupus erythematosus, oral chronic candidiasis, and actinic cheilitis.

Further, the sample includes bone marrow, blood cells, serum, peripheral blood, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid, lavage, aspirate, scratch, bone marrow sample, tissue biopsy sample, surgical sample, secretions and/or excretions.

Further, the sample includes serum, peripheral blood.

As used herein, a marker refers to an entity or portion whose presence or level is characteristic of a particular state or event. In some embodiments, the presence or level of a particular marker may be characteristic of the presence or stage of a disease, disorder, or condition. By way of example only, in some embodiments, the term refers to a gene expression product that has characteristics of a particular tumor, tumor subclass, tumor stage, and the like. Alternatively or additionally, in some embodiments, the presence or level of a particular marker is associated with the activity (or activity level) of a particular signaling pathway, e.g., may be characteristic of a particular tumor type. The statistical significance of the presence or absence of a marker may vary from one specific marker to another. In some embodiments, detection of the marker is highly specific in that it reflects the high probability that the tumor belongs to a particular subclass. This specificity may occur at the expense of sensitivity (i.e., negative results may occur even if the tumor is one that is expected to express the marker). In contrast, a marker with high sensitivity may be less specific than a marker with lower sensitivity. According to the present invention, useful markers do not need to distinguish between specific subclasses of tumors with 100% accuracy.

In certain specific embodiments, the diagnosis varies from sample to sample. In a specific embodiment, serum or peripheral blood is used as the diagnostic sample, and the expression level of the marker FGF6 is increased continuously along with the canceration progress, and the expression level of the marker FGF6 shows a significant difference, particularly the expression level of the marker FGF6 in serum or peripheral blood of normal people is significantly lower than that of the marker FGF6 in serum or peripheral blood of patients with oral mucosa precancerous lesions. In a specific embodiment, the diagnostic sample uses tissue, where the expression level of the marker FGF6 is decreasing with the progression of the cancer and exhibits a significant difference, in particular FGF6 expression in normal human tissue is significantly lower than FGF6 expression in tissue of a patient with precancerous lesions of the oral mucosa.

The invention provides application of a reagent for detecting FGF6 markers in a sample in preparation of products for diagnosing precancerous lesions of oral mucosa and cancers of the oral mucosa.

Further, the oral mucosa precancerous lesion is an oral leukoplakia.

Further, the sample includes serum, tissue, peripheral blood.

The invention provides application of a reagent for detecting FGF6 markers in a sample in preparation of a product for diagnosing oral mucosa cancer.

Further, the sample includes serum, tissue, peripheral blood.

In certain specific embodiments, the diagnosis varies from sample to sample. In a specific embodiment, serum or peripheral blood is used as the diagnostic sample, and the expression level of the marker FGF6 is continuously increased along with the cancerous progression, and the marker FGF6 shows a significant difference, specifically that the expression level of FGF6 in the serum or peripheral blood of a patient suffering from precancerous lesions of the oral mucosa is significantly lower than that of FGF6 in the serum or peripheral blood of a patient suffering from cancers of the oral mucosa. In a specific embodiment, the diagnostic sample uses tissue, where the expression level of the marker FGF6 is decreasing with the progression of the cancer and exhibits a significant difference, in particular FGF6 expression in tissue of patients with precancerous lesions of the oral mucosa is significantly lower than FGF6 expression in tissue of patients with cancer of the oral mucosa.

The invention provides a product for diagnosing whether a subject is a precancerous lesion of the oral mucosa or is a cancer of the oral mucosa, the product comprising a reagent for detecting the gene or protein expression level of an FGF6 marker in a sample.

Further, the product comprises a kit, a primer, test paper, a nucleic acid membrane strip, a probe and a chip.

Further, the product comprises reagents for detecting the presence/absence or amount of the marker FGF6 gene, functional fragment, protein in a sample.

Further, the reagents include reagents for detecting the presence, absence and/or amount of FGF6 gene, functional fragment, protein in a sample by PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, taqMan probe, molecular beacon, two-hybrid probe, multiplex probe, ISH, microarray, southern blot, northern blot, multi-analyte profiling, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescent assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescent assay, immunohistochemical assay, dot blot assay, or slot blot assay.

In certain specific embodiments, the chip comprises a gene chip for detecting an oligonucleotide probe against an FGF6 gene at the level of transcription of the FGF6 gene and a protein chip comprising an antibody or ligand specific for an FGF6 protein.

In certain specific embodiments, the kit comprises a gene detection kit comprising reagents or chips for detecting the level of transcription of the FGF6 gene, a protein detection kit comprising reagents or chips for detecting the level of expression of the FGF6 protein.

In certain specific embodiments, the test strip comprises a gene test strip, a protein test strip.

The invention provides the use of an inhibitor of FGF6 for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of a precancerous lesion of the oral mucosa of a subject, or for the treatment and/or prophylaxis of a cancer of the oral mucosa of a subject.

Further, the oral mucosa precancerous lesion is an oral leukoplakia.

Further, the individual includes a human or non-human mammal.

Further, the subject is a human.

As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., tissue or organism or cell culture) as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or comprises biological tissue or fluid. In some embodiments, the biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; a cell-containing body fluid; a free floating nucleic acid; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological liquid; a skin swab; a vaginal swab; an oral swab; a nasal swab; irrigation or lavage fluid, such as catheter lavage fluid or bronchoalveolar lavage fluid; aspirate; scraping objects; a bone marrow sample; a tissue biopsy sample; a surgical sample; feces, other body fluids, secretions and/or excretions; and/or cells derived therefrom, etc. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the cells obtained are or include cells from an individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of: biopsies (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, stool, etc.), and the like. In some embodiments, as will be clear from the context, the term "sample" refers to a formulation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and/or by adding one or more agents to the primary sample).

The present invention provides a pharmaceutical composition for treating and/or preventing oral mucosa precancerous lesions, or for treating and/or preventing oral mucosa cancer, the pharmaceutical composition comprising a therapeutically effective amount and/or a prophylactically effective amount of an inhibitor of FGF 6.

As used herein, the term "inhibitor" refers to an entity, condition, or event whose presence, level, or extent correlates to a decrease in the level or activity of a target. In some embodiments, the inhibitor may act directly (in which case it directly exerts an effect on its target, e.g., by binding to the target); in some embodiments, the inhibitor may act indirectly (in which case it exerts its effect by interacting with and/or otherwise altering the modulator of the target such that the level and/or activity of the target is reduced). In some embodiments, an inhibitor is one whose presence or level is correlated to a reduced target level or activity relative to a particular reference level or activity (e.g., observed under appropriate reference conditions, such as the presence or absence of a known inhibitor, or the like).

Further, the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.

"composition" or "pharmaceutical composition" according to the present invention refers to a combination of two or more agents as described herein for co-administration or administration as part of the same regimen. Combinations of agents that are not required in all embodiments to produce a physical mixture, i.e., it is possible for the components of the composition to be applied as separate adjuvants; however, many patients or practitioners in the art may find it advantageous to prepare a composition that is a mixture of two or more ingredients in a pharmaceutically acceptable carrier, adjuvant, diluent or excipient, such that simultaneous administration of the constituent ingredients of the combination is possible.

In certain specific embodiments, non-therapeutic agents may be added to the pharmaceutical composition, for example, to provide or aid in a desired consistency or stabilization effect. Suitable pharmaceutical composition excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The invention provides application of FGF6 markers in constructing a computer model for diagnosing oral mucosa precancerous lesions or oral mucosa cancers.

The term "computer model" as used in the present invention refers to a model that predicts that a patient has a precancerous lesion of the oral mucosa or an oral mucosa cancer based on the obtained expression detection data of FGF6 markers.

The invention provides a system for diagnosing precancerous lesions of oral mucosa or diagnosing cancers of the oral mucosa by using a marker FGF6, which comprises the following steps: and the result judging unit is used for comparing the critical value of the marker obtained by the data processing unit with a set diagnostic value.

Further, the system comprises a nucleic acid or protein sample separation unit for separating a nucleic acid or protein sample from a sample provided by the test object.

Further, the system comprises a sequencing unit for sequencing the nucleic acid or protein sample to obtain sequencing results.

Further, the system comprises a data processing unit for detecting the expression level of the marker according to the sequencing result, and analyzing the obtained expression level to obtain the critical value of the marker.

The invention provides application of a marker FGF6 in screening candidate medicines for treating oral mucosa precancerous lesions or oral mucosa cancers, wherein the screening of the candidate medicines for treating the oral mucosa precancerous lesions or the oral mucosa cancers is performed through tissues or cells of the oral mucosa precancerous lesions or the oral mucosa cancers in vitro.

As used herein, the terms "improve," "increase," "inhibit," "decrease," or grammatical equivalents thereof indicate measured values relative to baseline or other reference. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., a single individual) in the absence of (e.g., before and/or after) a particular agent or treatment, or in the presence of an appropriate comparable reference agent under otherwise comparable conditions. In some embodiments, an appropriate reference measurement may be or include a measurement in a comparable system that is known or expected to respond in a particular manner in the presence of a relevant agent or treatment.

Drawings

FIG. 1 is a graph showing the expression of FGF6 in various types of cancer tissues in a TCGA dataset;

FIG. 2 is a graph showing FGF6 expression in normal and HNSC tissues in TCGA dataset; and (3) injection: a: FGF6 expression in normal and HNSC tissues; b: FGF6 expression in normal and HNSC pathological grading tissues, grade 1 representing high differentiation, grade 2 representing medium differentiation, grade 3 representing low differentiation, grade 4 representing carcinoma in situ;

FIG. 3 is a view of the histomorphology of nude mice; and (3) injection: a is constructing a disease model 14d; b is a disease model construction 30d;

FIG. 4 is a diagram of HSC-4 cells and HaCaT cell cultures; and (3) injection: a is HSC-4 cells; b is HaCaT cells; FIG. 5 is a graph showing green fluorescence expression after over-expressing lentivirus transfected cells; and (3) injection: a is HSC-4 cells; b is HaCaT cells; a is transfection for 24h; b is transfected for 48 hours; c is transfection for 72h;

FIG. 6 is a graph showing the relative expression of FGF6 mRNA in HSC-4 cells after lentiviral transfection;

FIG. 7 is a general view of animal modeling; and (3) injection: the first row is a control normal group, a control precancerous lesion tissue group and a control cancer group respectively; the second row is the normal group transfected by FGF6 slow virus, the group transfected by FGF6 slow virus and the group transfected by FGF6 slow virus;

FIG. 8 is a graph showing the difference in subcutaneous tumor-bearing weights of nude mice in the cancer group;

FIG. 9 is a graph of HE staining results; and (3) injection: the first row is a control normal group, a control precancerous lesion tissue group and a control cancer group respectively; the second row is the normal group transfected by FGF6 slow virus, the group transfected by FGF6 slow virus and the group transfected by FGF6 slow virus;

fig. 10 is a graph of negative control, FGF6 immunohistochemical staining results, notes: the first row is a control normal group, a control precancerous lesion tissue group and a control cancer group respectively; the second row is the normal group transfected by FGF6 slow virus, the group transfected by FGF6 slow virus and the group transfected by FGF6 slow virus;

Fig. 11 is a histogram of FGF6 immunohistochemical mean optical density values, notes: a is a normal group, b is a precancerous lesion tissue group, c is a cancer group, d is a control group, e is FGF6 lentiviral transfection group (< 0.05, <0.01, <0.001, < 0.0001);

fig. 12 is a graph of MDA content measurement results, notes: a is a normal group, b is a precancerous lesion tissue group, c is a cancer group, d is a control group, e is FGF6 lentivirus transfected group (< 0.05, <0.01, <0.001, < P);

FIG. 13 is Fe ²⁺ Content measurement result graph, note: a is the normal group, b is the group of precancerous lesions, c is the group of carcinomas, d is the control group, e is the FGF6 lentivirus transfected group (×p)<0.05，**P<0.01，***P<0.001，****P<0.0001)；

Fig. 14 is a graph of the analysis results of the differential expression of FGF6 by healthy and pre-oral mucosal lesion patients and oral mucosal cancer patients in test set 2; wherein, A is the differential expression of FGF6 in a squamous carcinoma group and normal group (serum), and B is the differential expression of FGF6 in a squamous carcinoma group and white spot group (serum);

FIG. 15 is a graph showing WB validating FGF6 expression in each group of sera of oral mucosal carcinoma;

FIG. 16 is a graph showing WB validating FGF6 expression in groups of tissues with oral mucosa canceration;

FIG. 17 is a graph showing the expression of FGF6 in groups of tissues with cancerous oral mucosa by an immune group assay;

FIG. 18 is a ROC graph of test set 1 for diagnosing healthy and oral mucosa cancer using TCGA database tissue samples to plot FGF 6;

Fig. 19 is a ROC graph of test set 2 for FGF6 for healthy humans with oral mucosa carcinoma, oral mucosa precancerous lesions and oral mucosa carcinoma using real world collected serum samples;

FIG. 20 is a ROC graph of a validation set using real world collected serum samples to draw FGF6 for diagnosis of precancerous lesions and cancers of the oral mucosa, healthy and oral mucosa in healthy humans;

fig. 21 is a ROC graph of a validation set using real world collection tissue samples to draw FGF6 for oral mucosal precancerous lesions and oral mucosal cancers, healthy subjects and oral mucosal cancers.

Detailed Description

Example 1

1. Experimental method

1) TCGA dataset analysis

From the TCGA (https:// TCGA-data. Nci. Nih. Gov/TCGA /) dataset, 520 HNSCs of raw data and corresponding clinical information were obtained altogether, and FGF6 expression in normal and HNSC tissues was analyzed.

2) Animal disease model establishment for oral mucosa canceration process

(1) Cell preparation: recovering and amplifying a large amount of HaCaT and HSC-4 cells, and constructing an animal model when the cell quantity is enough. After HaCaT and HSC-4 cells are digested and centrifuged, cell count is carried out, and a proper amount of serum-free DMEM high-sugar culture medium is added and mixed uniformly to make the cell density of the mixture be about 1.0X10 ⁷ And each mL.

(2) Animal preparation: nude mice are fed in SPF-class environment of animal experiment center of southwest medical university after being purchased back, and model construction is carried out after 1 week of feeding without abnormal conditions.

(3) Animal disease model construction: 5 groups of nude mice are injected with HaCaT cell suspension subcutaneously at the back of the right side of the nude mice, each of which is 0.2mL, and the injection sites are marked, and after stable epithelial-like structures are formed for two weeks, HSC-4 cell suspension is injected into the basal membrane area of the epithelial-like layers. Notice that: (1) two cell suspensions are prepared as much as possible, so that the situation that the total amount of the cell suspensions is insufficient due to residues in the inner wall of a container is avoided; (2) the injector should be gently blown repeatedly before absorbing the cell suspension each time to ensure the cell density to be uniform; (3) after the injection is finished, the needle head is taken out after being kept under the skin of the nude mice for a plurality of seconds, so that the leakage caused by the immediate taking out of the needle head is avoided; (4) the above procedures are strictly sterile, and the death of nude mice due to cell contamination or pathogen infection is avoided. The condition of the injection site of the nude mice is observed every day after injection, and the change condition of the package blocks is observed periodically when the macroscopic package blocks appear, and the condition of the package blocks is reduced, absorbed and the like.

(4) Drawing materials: the nude mice of each group are respectively obtained from the 7d, 14d, 21d, 30d and 37d parts after the injection of HSC-4 cell suspension is finished, the obtained tissues are divided into two parts, one part is fixed in 10% neutral formalin for standby, and the other part is stored in a refrigerator at the temperature of minus 80 ℃ for standby.

3) Tissue morphology observations

(1) Tissue embedding

Tissues were embedded after 24h fixation with 10% neutral formalin.

a) Tissue fixation and dehydration: placing the tissue into a disposable plastic embedding frame, sequentially placing into AF fixing solution (prepared by 95% ethanol and concentrated formaldehyde according to the proportion of 9:1), soaking for 15min, dehydrating with 80% ethanol for 1h, dehydrating with 95% ethanol for 1h, dehydrating with absolute ethanol I for 1.5h, and dehydrating with absolute ethanol II for 1.5h.

b) Tissue transparency: the plastic embedding frame with the tissue is soaked in xylene for 40min.

c) Tissue waxing: the tissue was taken out of the plastic embedding frame, dried in a fume hood for 10min, and then put into a paraffin box with constant temperature of 60 ℃ for 45min for waxing, and repeated twice.

d) Tissue embedding: after the wax dipping is completed, selecting a metal embedding frame with proper size, placing a tissue into the bottom of the metal embedding frame (taking care of the placement direction of a tissue block), firstly dripping a small part of paraffin to be primarily fixed, then continuously dripping paraffin to fill the whole metal embedding frame, finally covering the plastic embedding frame on the upper part of the metal embedding frame, placing the plastic embedding frame on a refrigerator to solidify wax blocks, taking out a wax block mark group after the wax liquid is completely solidified, and placing the plastic embedding frame into a refrigerator at the temperature of minus 20 ℃ for standby.

(2) Tissue section

Trimming the wax block, making the wax block into uniform continuous and complete slices with the thickness of 5 mu m, then spreading, fishing out and baking the slices (the baking time is at least 2 h), marking the slices, and then placing the slices in a slicing box for standby.

(3) Tissue HE staining

a) Slice dewaxing and hydration: before dewaxing, firstly placing the slices on a slice baking machine for 20min at 60 ℃, cooling to room temperature, dewaxing with dimethylbenzene for 12min, and repeating for 2 times; absolute ethyl alcohol, 95% alcohol, 80% alcohol, 70% gradient alcohol for 2min each; the ultrapure water was rinsed for 2s.

b) Dyeing: (1) hematoxylin staining for 5min; (2) washing with ultrapure water for 2s; (3) acidifying with 1% hydrochloric acid alcohol for 1s;

(4) washing with ultrapure water for 2s; (5) returning 1% ammonia water to blue for 1min; (6) washing with ultrapure water for 2s; (7) eosin staining for 1min;

(8) washing with ultrapure water for 2s; (9) dehydrating 70% alcohol, 80% alcohol and 95% alcohol for 2s respectively; dehydrating absolute ethyl alcohol for 10min;the xylene is transparent for at least 5min; />And (5) sealing the neutral resin.

c) HE dyeing result judgment

All sections are subjected to pathological diagnosis by two high-annual-resource pathologists according to the WHO standard in 2005, and the success rate of constructing the identification model is improved.

4) HSC-4 cell transfection and construction of stable transgenic lines

(1) Digesting and centrifuging HSC-4 cells with good growth state, no pollution and cell density of more than 80%, preparing cell suspension, and counting cells to reach cell density of 3×10 ⁴ ～5×10 ⁴ Inoculating cells into 6-well plate at a ratio of one mL/one, inoculating 2mL of each well, slightly shaking 6-well plate in cross to uniformly distribute cells, labeling, grouping, cell types and time, and placing at 37deg.C with 5% CO ₂ Culturing in an incubator for 24 hours;

(2) Taking out the over-expressed FGF6 slow virus, empty vector slow virus and polybrene from a refrigerator at the temperature of minus 80 ℃ in advance, slowly dissolving the over-expressed FGF6 slow virus, preparing a cell culture solution containing polybrene (5 mug/mL) for standby, and simultaneously calculating the addition amount of each pore virus according to MOI=60 and the virus titer of each virus;

(3) Taking out the pore plate from the incubator after 24 hours, observing the cell state under an optical microscope, wherein the cell state is good, the distribution is uniform, and slow virus transfection can be carried out when more than 90% of cells are attached;

(4) Removing old culture solution in a 6-hole plate, washing cells for 3 times by using PBS buffer solution, adding 2mL of over-expression virus liquid and polybrene, DMEM basic culture medium in each hole of FGF6 lentivirus transfection group (overexpression group, namely OE group), sequentially adding the culture solution containing polybrene and the over-expression lentivirus into the 6-hole plate according to the calculated virus addition amount, and gently shaking and uniformly mixing in a cross manner;

(5) The empty vector lentivirus transfection group (control group), wherein each hole contains 2mL of empty vector virus liquid and polybrene, DMEM basic culture medium, and the culture liquid containing polybrene and the empty vector lentivirus are sequentially added into a 6-hole plate according to the calculated virus addition amount and are mixed uniformly by slight cross shaking;

(6) Blank group (blank group) was added to 2mL DMEM basal medium;

(7) After lentivirus is added for 10-12 hours, observing the cell state for liquid exchange, discarding the culture solution with virus liquid and polybrene, adding 2mL of complete cell culture solution into each hole, putting into an incubator for continuous culture, and if the cell state is bad, changing the liquid for 8 hours in advance;

(8) And adding puromycin 48h after transfection, preparing a screening culture medium according to the optimal puromycin concentration (2 mug/mL) of the HSC-4 cell stable transgenic strain screened by the early experiment, changing the screening culture medium every 2-3 days to ensure that the total puromycin concentration is 2 mug/mL, and screening for about one week to obtain the stable transgenic strain of the HSC-4 cell.

(9) Stable transgenic plants can be passaged and frozen according to proper proportion according to cell density, and can also be subjected to subsequent experiments according to cell amplification amount.

5) Animal disease model construction of FGF6 lentivirus interfering normal mucous membrane canceration process

The experiment simulates three stages, namely a normal stage, an oral mucosa potential malignant disease (oral potential malignant disorders, OPMD) stage and a mucosa canceration stage, wherein each stage comprises two groups of FGF6 lentivirus transfection groups and a control group, and each group comprises 6 nude mice, and the total number of the nude mice is 36. In the normal stage, the FGF6 slow virus transfection group is subcutaneously injected into the back of the right side of a nude mouse with the suspension of the HaCaT cells transfected with the FGF6 slow virus, and the control group is injected with the suspension of the HaCaT cells transfected with the empty vector slow virus; in the OPMD stage, firstly, injecting a HaCaT cell suspension into the back of the right side of a nude mouse in a subcutaneous mode, and after a stable epithelial-like structure is formed for two weeks, injecting a HSC-4 cell suspension transfected with FGF6 lentivirus into an epithelial-like basement membrane area, and injecting a HSC-4 cell suspension transfected with empty vector lentivirus into the epithelial-like basement membrane area in a control group; in the mucosa canceration stage, the FGF6 slow virus transfection group is subcutaneously injected into the right back of a nude mouse with HSC-4 cell suspension of the FGF6 slow virus, and the control group is injected with HSC-4 cell suspension of the empty vector slow virus transfection, and each nude mouse is subcutaneously injected with 0.2mL.

6) Tissue Malondialdehyde (MDA) content detection

(1) Sample processing: taking out each group of tissue samples from a refrigerator at the temperature of minus 80 ℃, weighing about 0.1g of tissue, and adding 1mL of extracting solution for ice bath homogenization; at the temperature of 8000g, centrifuging for 10min at the temperature of 4 ℃, taking supernatant, and placing the supernatant on ice for testing;

(2) Sample adding: the measuring tube is respectively added with 300 mu L of MDA detection working solution, 100 mu L of sample and 100 mu L of reagent III; the blank tube is respectively added with 300 mu L of MDA detection working solution, 100 mu L of distilled water and 100 mu L of reagent III, wherein the blank tube is processed for 2 times;

(3) Keeping the mixed solution in 100deg.C water bath for 60min (covering tightly to prevent water loss), cooling in ice bath, cooling 10000g, centrifuging at room temperature for 10min;

(4) Absorbing 200 mu L of supernatant of each tube into a 96-well plate, and respectively measuring the absorbance of each sample at 532nm and 600nm (preheating by an enzyme-labeled instrument for more than 30 min);

(5) MDA content calculation: the MDA content was calculated according to the instructions.

7) Tissue immunohistochemical staining

(1) Slice dewaxing and hydration

Roasting the slices for 20min at 60 ℃ before dewaxing, cooling to room temperature, dewaxing with dimethylbenzene for 12min, and repeating for 2 times; absolute ethyl alcohol, 95% alcohol, 80% alcohol, 70% alcohol for 2min; washing with ultrapure water for 2s;

(2) Antigen thermal repair

Placing the slices into a beaker filled with 0.01M citrate buffer solution (PH=6.0) at normal temperature, heating in a constant-temperature water bath kettle, setting the temperature to 96 ℃, stopping water bath when the water bath temperature reaches 96 ℃ for 20min, naturally cooling to room temperature, taking out the slices, flushing with PBS buffer solution for 3min, repeatedly flushing for 3 times, and sucking liquid around the slice tissues;

(3) Dropwise adding a reagent 1 (endogenous peroxidase blocking agent) to the tissue on the slice, incubating for 10min at room temperature, then flushing with PBS buffer solution for 3min, repeatedly flushing for 3 times, and sucking the liquid around the tissue of the slice;

(4) Dripping a reagent 2 (goat sealing serum) into the tissue on the slice, incubating for 10-15 min at room temperature, pouring out, not flushing, and sucking the liquid around the tissue of the slice;

(5) Incubation resistance: dripping primary antibodies (the dilution ratio of the primary antibodies is FGF6:1:200, BCL2:1:200, cyclin D1:1:100, pERK 1:500, ERK 1:200, caspase9:1:200, pAKT 1:100, AKT 1:200, FGFR4:1:200, FGFR 1:200, PI3K 1:200, ACSL4:1:200 and GPX4:1:200 respectively) into tissues on the sections, placing the sections in a wet box, incubating for 2 hours at 37 ℃ (midway notice to observe the dry plates) or incubating overnight at 4 ℃, and taking PBS buffer solution as a negative control instead of the primary antibodies; washing with PBS buffer solution for 3min, repeatedly washing for 3 times, and sucking the liquid around the tissue of the slice;

(6) Dropwise adding a reagent 3 (biomarker goat anti-mouse/rabbit IgG) into tissues on the sections, incubating for 10-15 min at room temperature, then flushing for 3min by using PBS buffer, repeatedly flushing for 3 times, and sucking up liquid around the tissues of the sections;

(7) Dropwise adding a reagent 4 (horseradish enzyme labeled streptavidin working solution) into the tissues on the sections, incubating for 10-15 min at room temperature, then flushing for 3min by using PBS buffer solution, repeatedly flushing for 3 times, and sucking the liquid around the tissues of the sections;

(8) DAB color development: dripping DAB working solution (the preparation ratio of the solution A to the solution B is 20:1, and the DAB working solution is prepared by uniformly mixing the solution A and the solution B in a dark place) and incubating for 5-10 min at room temperature, observing color change under a microscope, and immediately flushing with ultrapure water to terminate the reaction after the expected color change is reached;

(9) Hematoxylin counterstain: hematoxylin staining for 4min, ultrapure water washing for 2s,1% hydrochloric acid-alcohol acidification for 1s, ultrapure water washing for 2s,1% ammonia returning to blue for 1min, and ultrapure water washing for 2s;

(10) Slice dehydration: sequentially dehydrating 70% alcohol, 80% alcohol, 95% alcohol and absolute ethanol for 2s;

(11) Xylene was transparent for 10min, gel-sealed, and imaged under a positive microscope and analyzed.

8) Tissue ferrous ion (Fe) ²⁺ ) Content determination

(1) Reagent preparation: before detection, balancing the reagent in the kit to room temperature, preparing standard protection liquid and 100 mu mol/L iron standard according to the specification, and diluting the standard to different concentrations;

(2) Sample processing: taking 0.1g of fresh tissue, adding buffer solution for homogenating, centrifuging 10000g for 10min, and taking supernatant for later use;

(3) Sample addition

(1) Standard tube: 300. Mu.L of standard substances with different concentrations are respectively added into corresponding 1.5mL EP tube to be measured: 300. Mu.L of sample was taken and added to the corresponding 1.5mL EP tube; (2) 150. Mu.L of a color-developing solution was added to each tube of step (1); (3) uniformly mixing, and incubating for 10min at 37 ℃; (4) centrifuge each tube at 12000g for 10min; (5) taking 300 mu L of each tube supernatant in the step (4) and adding the supernatant into a 96-well plate; (6) the OD value of each hole at 593nm is measured by an enzyme label instrument, and Fe is calculated according to the specification ²⁺ The content is as follows.

9) Statistical method

Statistical analysis and mapping were performed using Image J software and GraphPad Prism 9.0, with independent sample t-test for comparison between two groups, single factor analysis of variance for comparison between more than two groups, and differences were statistically significant when P < 0.05.

2. Experimental results

1) TCGA dataset results

As shown in fig. 1, FGF6 is expressed in HNSC tissues in various types of cancer tissues; and FGF6 was expressed in normal tissue more than HNSC tissue (fig. 2A); as shown in fig. 2B, FGF6 was expressed differently in each pathological grading tissue of HNSC, and the expression difference of FGF6 between normal tissue and each group of tissues of HNSC hyperdifferentiation, medium differentiation, low differentiation was statistically significant, while the expression difference between HNSC hyperdifferentiation, medium differentiation, low differentiation, and carcinoma in situ was not statistically significant.

2) Results of oral mucosa cancerous Process animal disease model criteria

HE results show that 14d is the construction period of the OPMD stage standard model, and the cell polymorphism is visible under the lens, so that the nuclei are densely stained, the nucleolus is increased, the nuclear pulp proportion is increased and the like; 30d is the model construction period of the mucosa canceration stage, and cancer cells and necrotic tissues are visible under the mirror, and the result is shown in fig. 3.

3) HSC-4 cells and HaCaT cell culture results

As shown in FIG. 4, the results of both cell cultures were shown for HSC-4 cells in an epithelial-like morphology, nuclei in a polymorphic form, haCaT cells in a paving stone-like shape, cells in an oblate shape, no protrusions, plump cytoplasm, and nuclei in a round or oval shape.

4) Over-expression FGF6 lentiviral transfection

HSC-4 cells and HaCaT cells are transfected by the over-expression FGF6 lentivirus, after 24h of transfection, the HSC-4 cells and the HaCaT cells transfected by the over-expression FGF6 lentivirus have green fluorescence expression under the mirror, and after 72h of transfection, the green fluorescence expression is strongest, and the result is shown in figure 5.

5) RT-qPCR (reverse transcription-quantitative polymerase chain reaction) verification of FGF6 over-expression effect

FGF6 lentiviral transfected group (OE group) HSC-4 cells had higher relative expression of FGF6 mRNA than the blank group (blank group) and the differences were statistically significant (P < 0.0001); whereas the relative amount of FGF6 mRNA expressed in empty vector transfected (control group) HSC-4 cells was not statistically different from that in the blank group (FIG. 6).

6) Lentivirus interference oral mucosa canceration process disease model result

The model of the disease of the chronic virus interfering with the canceration process of the oral mucosa is generally shown in figure 7, the model of a nude mouse of a control precancerous lesion tissue group is successful, the FGF6 chronic virus transfected precancerous lesion tissue group is in a tumor trend, the histopathological characterization of squamous cell carcinoma is prone to happen, and the squamous cell carcinoma is visible under the lens; the tumor-bearing tissue weight of the nude mice in the FGF6 lentiviral transfected cancer group is different from that of the nude mice in the control cancer group, the difference has statistical significance (P < 0.001) (figure 8), and the differentiation degree of the tumor-bearing squamous carcinoma tissue of the nude mice in the FGF6 lentiviral transfected cancer group is lower than that of the nude mice in the control cancer group, so that large-area necrotic tissue can be seen under the mirror (figure 9).

7) Tissue immunohistochemical results

As shown in fig. 10 and 11, immunohistochemistry analyzed FGF6 expression differences in each group of tissues, and the results showed that: FGF6 showed a down-regulation trend in expression levels in the control normal group, the control precancerous lesion tissue group, and the control cancer group; the expression level of FGF6 in the FGF6 lentivirus transfected normal group and FGF6 lentivirus transfected precancerous lesion tissue group and FGF6 lentivirus transfected cancer group also shows a downregulation trend; the above expression differences were all statistically significant (P < 0.05).

8) MDA content (lipid oxidation level) measurement results

As shown in fig. 12, a. The MDA content of FGF6 lentivirus transfected normal group was down-regulated compared to control normal group; FGF6 lentivirus transfected with MDA content down-regulated in the pre-cancerous lesions compared to the control pre-cancerous lesions; the MDA content of the FGF6 lentivirus transfected carcinoma group was also down-regulated compared to the control carcinoma group. MDA content in a control normal group, a control precancerous lesion tissue group and a control cancer group shows a down-regulation trend; the content in the FGF6 lentivirus transfected normal group, the FGF6 lentivirus transfected precancerous lesion tissue group and the FGF6 lentivirus transfected cancer group also shows a down-regulation trend. The difference in MDA content in each group was statistically significant (P < 0.05).

9) Ferrous ion content measurement results

As shown in FIG. 13, a. FGF6 lentivirus transfected Fe in the normal group compared to the control normal group ²⁺ Content is regulated down; FGF6 lentivirus transfected Fe of the pre-cancerous lesions compared to the control pre-cancerous lesions ²⁺ Content is regulated down; fe of FGF6 lentiviral transfected carcinoma group compared to control carcinoma group ²⁺ The content is also down-regulated. Fe (b) ²⁺ The content in the control normal group, the control precancerous lesion tissue group and the control cancer group shows a down-regulation trend, and the content in the FGF6 slow virus transfected normal group, the FGF6 slow virus transfected precancerous lesion tissue group and the FGF6 slow virus transfected cancer group also shows a down-regulation trend. Fe in each group ²⁺ The differences in content are all statistically significant (P)<0.05)。

Example 2

1. Experimental method

1) Sample source

Test set 1: the study object source is a TCGA database, and the original tissue data and corresponding clinical information of 520 HNSCs are obtained in total and used for subsequent analysis.

Test set 2: the results of the sequencing analysis of 3 samples of normal human serum, 3 samples of OLK serum and 3 samples of OSCC serum collected clinically are shown in tables 1 and 2, wherein,

table 1 shows the normal mucosa group of squamous carcinoma group vs

Table 2 shows squamous carcinoma group vs oral leukoplakia group

TABLE 1

TABLE 2

Verification set: from real world clinical samples, 13 healthy human samples are obtained in total, 7 patients with precancerous lesions of oral mucosa are obtained, 30 patients with cancerous lesions of oral mucosa are obtained, and the samples are serum and tissue samples, so that the correctness of diagnosing precancerous lesions of oral mucosa or diagnosing cancers of oral mucosa by using the marker FGF6 is verified. The clinical patient profile statistics in test set 2 and in validation set are shown in table 3.

TABLE 3 Table 3

Inclusion and exclusion criteria for the oral mucosa precancerous lesions patients were as follows:

inclusion criteria:

1) The age is 18 years old and less than 80 years old.

2) All conform to those WHO promulgated by WHO regarding precancerous lesions of the oral mucosa and have been diagnosed by pathological detection.

3) And (5) performing primary diagnosis.

4) Neither local nor systemic glucocorticoids nor severe cardiovascular diseases or any liver, kidney or other organ dysfunction are used.

5) Patients were included to learn about the study and signed informed consent.

Exclusion criteria:

1) Other oral diseases are accompanied by precancerous lesions of the oral mucosa.

2) Periodontitis.

3) Salivary gland diseases.

4) Systemic diseases.

5) Drugs affecting immune function, antibacterial drugs and hormone drugs are used in about 1 month.

6) Combining malignant tumors/combining other malignant tumors (non OSCC).

7) Patients undergoing chemotherapy and radiation therapy.

8) Patients who received organ transplants.

9) Patients with a positive history of chronic viral disease.

10 Pregnant or lactating women.

11 A) diagnostic ambiguity.

Inclusion exclusion criteria for the oral mucosa cancer patients were as follows:

inclusion criteria:

1) The specimen needs to be independently confirmed as OSCC by two pathologists.

2) OSCC radical cure was performed for the first time.

3) No treatment of any form has been accepted in the past. The patients do not receive anti-tumor treatment such as chemotherapy, radiotherapy and the like.

4) The age is 18 years old and less than 80 years old.

5) Sex is not divided.

6) The score of the Carlsberg functional status is > 70.

7) The clinical stage is II-III.

8) Patient clinical data and follow-up data are complete.

Exclusion criteria:

1) With malignant tumors in other areas.

2) With diseases of the blood system, serious dysfunction of vital organs, high risk of thrombosis and active bleeding.

3) With immunodeficiency diseases, infectious diseases, severe infections, and severe medical complications.

4) Doctor-patient communication disorder.

5) Distant metastasis was found preoperatively.

6) There are history of drug abuse, history of drug inhalation, and history of facial surgery.

7) With periodontal and mucosal diseases.

8) Coagulation dysfunction.

9) Gestation and lactation.

2) Differential expression analysis

Test set: the sequencing data of FGF6 in the test set is subjected to differential expression analysis by using SPSS software, and the differential expression of genes between healthy people and oral mucosa precancerous lesions, oral mucosa precancerous lesions and oral mucosa cancers and healthy people and oral mucosa cancers in the screening set is compared.

Verification set: (1) RNA extraction

Total RNA was extracted from the samples using the RNeasy kit (Beyotide, shanghai,456 China, R0027). The method comprises the following steps:

a. sample preparation: 20mg of the sample was placed in a 1.5ml centrifuge tube, about 6 zirconia beads were added, 600. Mu.l of ice-bath pre-chilled lysate was rapidly added, and homogenized with a micro-electric homogenizer. After grinding or homogenizing, gently blowing the homogenate for 8-10 times and standing at room temperature for 3-5 minutes. The supernatant was then removed to a new centrifuge tube by centrifugation at about 14,000g for 2 minutes.

b. Adding the equal volume of the binding solution into the lysate, and gently reversing and mixing for 3-5 times.

c. The mixture of lysate and conjugate was transferred to a purification column, placed in a centrifuge for centrifugation at 16,000g for 1 min, and the lower layer of liquid was discarded.

d. 600. Mu.L of washing solution I was added to the column, centrifuged at 16,000g for 1 min, and the lower layer was discarded. 600. Mu.L of washing solution II was added to the column, centrifuged at 16,000g for 1 min, and the lower layer was discarded. This step was repeated twice.

e.16,000g for 2 min to remove residual liquid from the purification column.

f. The collection tube was discarded, the RNA purification column was placed in an RNA elution tube, 50. Mu.L of eluent was added to the center of the purification column, and the column was left at room temperature for 3 minutes and centrifuged at 16,000g for 30 seconds. The resulting eluate was again added to the center of the purification column and eluted repeatedly, and centrifuged at 16,000g for 30 seconds to obtain purified RNA.

(2) Reverse transcription:

a. determining the concentration and quality of the sample RNA:

reverse transcription was performed on sample RNA at concentrations above 143 ng/. Mu.L and with standard OD230/260 and OD 280/260.

b. Removing DNA from RNA:

a mixed solution of gDNA Eraser and 5X gDNA Eraser Buffer was prepared on ice and dispensed into each reaction tube, followed by the addition of 1. Mu.g total RNA and enzyme-free water.

The above reaction system was heated at 42℃for two minutes to remove DNA.

c. Reverse transcription reaction:

firstly preparing a mixed solution except the first-step reaction solution on ice, and then adding the first-step reaction solution.

The above reaction system was heated at 37℃for 15 minutes and at 85℃for 30 seconds. 180 mu LRNase Free dH was added per tube ₂ O dilution.

③PCR

a, preparing a reaction system:

TB Green Premix Ex Taq II reagent is protected from light, and PCR forward and reverse primers are mixed in advance.

Preparing a mixed solution except cDNA on ice, subpackaging to eight rows, and then adding cDNA.

b. Eight rows of centrifugation and loading.

The real-time quantitative PCR detection analysis uses a two-step PCR standard amplification procedure, reaction conditions: using cDNA as template, performing qRT-PCR, using GAPDH as internal reference, and performing amplification under the reaction conditions: the first step of pre-denaturation (30 seconds at 98 ℃) and the second step of PCR amplification (5 seconds at 95 ℃, 30 seconds at 60 ℃) for 40 cycles.

Recording and analyzing the detection data results by a qRT-PCR instrument specific software program according to the formula multiple = 2 ^-ΔΔCt The relative expression level of each target gene was calculated.

3) Diagnostic efficacy analysis

Drawing a working curve (ROC) of a subject by adopting R package pROC, and analyzing the screened FGF6 markers which are remarkably and differentially expressed between healthy people and oral mucosa precancerous lesions and oral mucosa cancers, and respectively judging the diagnostic efficacy of the FGF6 markers on the oral mucosa precancerous lesions and the oral mucosa cancers by using AUC values, sensitivity and specificity in a test set and a verification set.

3. Experimental results

1) Differential expression analysis

Test set 1: the analysis results of the differential expression of FGF6 in healthy and oral mucosa cancer patients in TCGA database tissue samples in test set 1 are shown in fig. 2, and the expression level of FGF6 in the tissues of oral mucosa cancer patients is significantly reduced compared with healthy people.

Test set 2: the analysis results of the differential expression of FGF6 in the serum samples collected clinically in the test set 2 are shown in fig. 14, wherein the expression level of FGF6 in the serum of the patients with oral mucosa cancer is obviously up-regulated compared with the serum samples of the patients with oral mucosa precancerous lesions; FGF6 was significantly upregulated in serum from patients with oral mucosal cancer compared to healthy humans.

Verification set: in order to verify the functions of FGF6 in the test set, a serum sample is clinically collected as a verification set, and through WB experiments, the differential expression of FGF6 in each group of serum of oral mucosa canceration is verified, and the result is shown in FIG. 15, compared with a healthy person, the expression level of FGF6 in the serum of a patient with oral mucosa precancerous lesions is obviously up-regulated; compared with the patients with the precancerous lesions of the oral mucosa, the expression level of FGF6 in the serum of the patients with the oral mucosa is obviously up-regulated; FGF6 was significantly upregulated in serum from patients with oral mucosal cancer compared to healthy humans.

Clinical tissue samples are collected as a verification set, and differential expression of FGF6 in various groups of tissues of oral mucosa canceration is verified through WB experiments, and the result is shown in figure 16, wherein the expression level of FGF6 in the tissues of the oral mucosa cancer patients is obviously reduced compared with those of the oral mucosa precancerous lesion patients; the expression level of FGF6 in the tissues of patients with oral mucosa cancer was significantly down-regulated compared to healthy humans.

Clinical tissue samples are collected as a verification set, and through immunohistochemical experiments, differential expression of FGF6 in various groups of tissues of oral mucosa canceration is verified, and the result is shown in figure 17, and compared with a patient with oral mucosa precancerous lesions, the expression level of FGF6 in the tissues of the patient with oral mucosa canceration is obviously reduced; the expression level of FGF6 in the tissues of patients with oral mucosa cancer was significantly down-regulated compared to healthy humans.

2) ROC curve analysis of marker FGF6 for diagnosing precancerous lesions and cancers of healthy people and oral mucosa, and healthy people and cancers of oral mucosa

The result of drawing ROC curves of healthy people and oral mucosa cancer patients by using a TCGA database tissue sample as a test set and using a marker FGF6 as a variable is shown in fig. 18, and the result shows that the AUC value in the ROC curve drawn by the TCGA database sample is 0.662, so that the marker FGF6 can be used as a marker for diagnosing the healthy people and oral mucosa cancer.

The serum sample is used as a test set by taking the marker FGF6 as a variable, and a ROC curve of a healthy person and an oral mucosa cancer patient, an oral mucosa precancerous lesion patient and an oral mucosa cancer patient is drawn, the result is shown in a graph 19, in the ROC curve drawn by the serum sample, the AUC value of the healthy person and the oral mucosa cancer patient is 1.000, and the AUC value of the oral mucosa precancerous lesion patient and the oral mucosa cancer patient is 0.972, so that the marker FGF6 can be used as a marker for diagnosing the healthy person and the oral mucosa cancer, and the oral mucosa precancerous lesion and the oral mucosa cancer.

In order to verify the reliability of the above results, serum samples are collected by using a marker FGF6 as a variable, and ROC curves of healthy people and precancerous lesions of oral mucosa, precancerous lesions of oral mucosa and oral mucosa cancer are drawn, and the results are shown in fig. 20, wherein in the ROC curves drawn by collecting the serum samples in the real world, the AUC values of the healthy people and precancerous lesions of oral mucosa are 0.750 (Cl: 0.350-1.000), the AUC values of the precancerous lesions of oral mucosa and oral mucosa cancer are 0.750 (Cl: 0.530-0.970), and the AUC values of the healthy people and oral mucosa cancer are 0.868, which indicates that the marker FGF6 can be used as a marker for diagnosing the precancerous lesions of oral mucosa, precancerous lesions of oral mucosa and oral mucosa cancer of healthy people.

The result is shown in fig. 21, in the ROC curve drawn by the real world collected tissue sample, the AUC value of the precancerous lesion of the oral mucosa and the oral mucosa cancer is 0.750, and the AUC value of the healthy person and the oral mucosa cancer is 0.654, which indicates that the marker FGF6 can be used as a marker for diagnosing the precancerous lesion of the oral mucosa and the oral mucosa cancer, and the healthy person and the oral mucosa cancer.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. Application of a reagent for detecting FGF6 markers in samples in preparation of products for diagnosing oral mucosa precancerous lesions.

2. The use of claim 1, wherein the detection reagent for the marker comprises a reagent for specifically detecting the expression level of FGF6 gene, or a reagent for specifically binding to a protein encoded by FGF6 gene;

Preferably, the oral mucosa precancerous lesions include oral potential malignant diseases, oral epithelial dysplasia, and proliferative wart leukoplakia;

preferably, the oral pre-mucosal lesions include oral leukoplakia, oral lichen planus, oral erythema, oral submucosal fibrotic lesions, discoid lupus erythematosus, oral chronic candidiasis, and actinic cheilitis;

preferably, the sample comprises bone marrow, blood cells, serum, peripheral blood, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, irrigation fluid, lavage, aspirate, scratch, bone marrow sample, tissue biopsy sample, surgical sample, secretion and/or fecal matter;

preferably, the sample comprises serum, peripheral blood.

3. Application of a reagent for detecting FGF6 markers in samples in preparation of products for oral mucosa precancerous lesions and oral mucosa cancer diagnosis;

preferably, the detection reagent of the marker comprises a reagent for specifically detecting the expression level of the FGF6 gene, or a reagent for specifically binding to a protein encoded by the FGF6 gene;

preferably, the oral pre-cancerous lesions are oral leukoplakia;

preferably, the sample comprises serum, tissue, peripheral blood.

4. Application of a reagent for detecting FGF6 markers in samples in preparation of products for diagnosing oral mucosa cancers;

preferably, the sample comprises serum, tissue, peripheral blood.

5. A product for diagnosing whether a subject is a precancerous condition of the oral mucosa or is an oral mucosa cancer, the product comprising an agent that detects the level of gene or protein expression of an FGF6 marker in a sample;

preferably, the product comprises a kit, a primer, a test paper, a nucleic acid membrane strip, a probe and a chip.

Use of an inhibitor of fgf6 for the preparation of a pharmaceutical composition for treating and/or preventing a precancerous lesion of the oral mucosa of a subject, or for treating and/or preventing a cancer of the oral mucosa of a subject;

preferably, the oral pre-cancerous lesions are oral leukoplakia;

preferably, the individual comprises a human or non-human mammal;

preferably, the individual is a human.

7. A pharmaceutical composition for treating and/or preventing a precancerous lesion of the oral mucosa, or for treating and/or preventing a cancer of the oral mucosa, the pharmaceutical composition comprising a therapeutically effective amount and/or a prophylactically effective amount of an inhibitor of FGF 6;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Application of FGF6 marker in constructing computer model for diagnosing precancerous lesion of oral mucosa or oral mucosa cancer.

9. A system for diagnosing a precancerous condition of or diagnosing a cancer of the oral mucosa using the marker FGF6, comprising: the result judging unit is used for comparing the critical value of the marker obtained by the data processing unit with a set diagnostic value;

preferably, the system comprises a nucleic acid or protein sample separation unit for separating a nucleic acid or protein sample from a sample provided by a test subject;

Preferably, the system comprises a sequencing unit for sequencing a nucleic acid or protein sample to obtain sequencing results;

preferably, the system comprises a data processing unit for detecting the expression level of the marker according to the sequencing result, and analyzing the obtained expression level to obtain the critical value of the marker.

10. Use of a marker FGF6 for screening a drug candidate for the treatment of a precancerous lesion or an oral mucosa cancer of an oral mucosa by in vitro tissue or cells of the precancerous lesion or the oral mucosa cancer of the oral mucosa.