CN114414805A

CN114414805A - Marker related to oral squamous carcinoma cell growth and application thereof

Info

Publication number: CN114414805A
Application number: CN202210023751.XA
Authority: CN
Inventors: 冷卫东; 夏凌云; 余和东; 张天舒; 毛敏; 陈永吉; 解龙川; 严艳; 张丽; 倪小兵; 徐小明; 胡梓秋; 裴钰; 余子敬; 柴红波; 梁琳琳; 刘世琦; 陈越
Original assignee: Shiyan Taihe Hospital
Current assignee: Shiyan Taihe Hospital
Priority date: 2022-01-10
Filing date: 2022-01-10
Publication date: 2022-04-29

Abstract

The invention relates to the technical field of biological medicines, in particular to a marker related to oral squamous cell carcinoma cell growth and application thereof. The invention discloses a fiber regulatory protein as a marker related to the growth of oral squamous cell carcinoma cells and application thereof, wherein the application of the fiber regulatory protein in marking the growth condition of the oral squamous cell carcinoma cells comprises the following steps: a detection kit for oral squamous cell carcinoma; the application of the composition for inhibiting the expression of the fiber regulatory protein in preparing the medicine for inhibiting the proliferation of the oral squamous cell carcinoma cells; the application of the composition for inhibiting the expression of the fiber regulatory protein in preparing the medicine for inhibiting the migration of oral squamous cell carcinoma cells; application of a composition for inhibiting expression of a fiber regulatory protein in preparation of a medicament for inhibiting invasion of oral squamous cell carcinoma cells. The invention discloses that the expression level of the fiber regulatory protein is closely related to the proliferation, migration and invasion of oral squamous cell carcinoma cells, and the fiber regulatory protein is used as a marker to provide a new means for the detection, diagnosis, treatment and prognosis judgment of the oral squamous cell carcinoma.

Description

Marker related to oral squamous carcinoma cell growth and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a marker related to oral squamous cell carcinoma cell growth and application thereof.

Background

Oral cancer is a general term for malignant tumors occurring in the oral cavity, and is a common malignant tumor of the head and neck. The most common cell source in oral cancer is squamous cell carcinoma (oral squamous cell carcinoma, OSCC), which accounts for about 90% of oral cancers and has climbed to the sixth most common malignancy worldwide by 2016. Although the existing surgery and chemoradiotherapy technologies for treating oral squamous cell carcinoma are continuously advanced, the survival rate of a patient in 5 years is still less than 50%, and the treatment effect is poor because the oral squamous cell carcinoma has the characteristics of difficult early discovery, high metastasis and high recurrence rate.

Therefore, the research on the pathogenesis of oral squamous cell carcinoma is carried out deeply, key molecules related to the growth of oral squamous cell carcinoma cells are found to be used as markers, and new diagnosis, treatment and prognosis judgment means of oral squamous cell carcinoma are developed according to the markers, so that the research and treatment effects of oral squamous cell carcinoma are improved, and the survival rate and the life quality of patients are improved.

Disclosure of Invention

The invention mainly aims to disclose a marker related to oral squamous carcinoma cell growth and application thereof.

The technical scheme of the invention is as follows:

in a first aspect, the invention discloses a marker associated with the growth of oral squamous cell carcinoma cells, the marker comprising a fibromodulin.

In a second aspect, the present invention provides the use of a fibromodulin to identify the growth status of oral squamous cell carcinoma cells, comprising:

a detection kit for oral squamous cell carcinoma is used for detecting the expression level of the fiber regulatory protein so as to identify the growth condition of the oral squamous cell carcinoma cells according to the expression level;

use of a composition that inhibits the expression of a fibromodulin in the preparation of a medicament for inhibiting the proliferation of oral squamous carcinoma cells;

use of a composition that inhibits the expression of a fibromodulin in the preparation of a medicament for inhibiting the migration of oral squamous carcinoma cells;

use of a composition for inhibiting the expression of a fibromodulin in the preparation of a medicament for inhibiting the invasion of oral squamous carcinoma cells.

In conclusion, the beneficial effects of the invention are as follows:

the invention discloses the application of the expression level of the fiber regulatory protein in the detection, diagnosis, treatment and prognosis judgment of oral squamous cell carcinoma, which is used as a marker related to the growth of the oral squamous cell carcinoma for the first time. By immunohistochemical staining of clinical tissue specimens and statistical analysis of the expression level of the fiber regulatory protein and clinical staging parameters, the fiber regulatory protein is specifically expressed in oral squamous cell carcinoma cells, and the expression level of the fiber regulatory protein has correlation with the early stage and the middle and late stage of the clinical staging of oral squamous cell carcinoma patients. An EdU staining cell experiment and a nude mouse transplantation tumor experiment show that the inhibition of the expression of the fiber regulatory protein can inhibit the proliferation capacity of oral squamous cell carcinoma cells. The results of the cell scratch healing experiments indicate that inhibition of the expression of the fibromodulin can inhibit the migration ability of the oral squamous cell carcinoma cells. The results of Transwell invasive cell experiments indicate that inhibition of the expression of the fibromodulin can inhibit the invasive ability of oral squamous cell carcinoma cells.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.

FIG. 1 is a graph of immunomodulatory protein immunohistochemical staining of oral squamous carcinoma tissue;

FIG. 2 is a statistical plot of the expression level of the fibromodulin in oral squamous carcinoma tissue versus clinical parameters;

FIG. 3 is a graph showing the result of Western Blot (WB) imaging after transfection of oral squamous carcinoma cells with a fibromodulin-silencing lentivirus;

FIG. 4 is a graph showing the result of EdU staining after transfection of oral squamous cell carcinoma cells with a fibromodulin-silencing lentivirus;

FIG. 5 is a graph of the healing results of cell scratch after transfection of oral squamous carcinoma cells with a fibromodulin-silencing lentivirus;

FIG. 6 is a graph showing the results of a Transwell invasion assay after transfection of an oral squamous cell carcinoma cell with a fibromodulin-silenced lentivirus;

FIG. 7 is a graph showing the results of nude mouse transplantable tumor after oral squamous cell carcinoma cells are transfected with a fibromodulin-silenced lentivirus.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and techniques not described in detail are performed according to standard methods well known to those skilled in the art. The cell lines, reagents and lentiviral vectors mentioned in this application are commercially available or otherwise publicly available, and are by way of example only and not exclusive to the present invention and may be replaced by other suitable means or biomaterials, respectively.

The invention discloses a marker related to oral squamous cell carcinoma cell growth, which comprises fiber regulatory protein.

Further, Fibromodulin (FMOD), a small molecule proteoglycan rich in leucine, is important in extracellular matrix, and its main function is to regulate collagen fiber formation, which is an essential substance for tissue repair of many organs. Fibromodulin plays a very important role in a variety of key biological processes, including regulation of angiogenesis, TGF- β promotion of human fibroblast differentiation into pluripotent cells, inflammation, apoptosis, tumor metastasis, and the like. The tumor microenvironment is one of the important conditions for the occurrence and development of cancer, while proteoglycan in extracellular matrix and stroma is an important component of the tumor microenvironment, and the action mechanism of the fibromodulin in the occurrence and development of different types of tumors is different.

The invention provides application of fiber regulatory protein in identifying growth conditions of oral squamous cell carcinoma cells.

Optionally, the kit is used for detecting the expression level of the fiber regulatory protein so as to identify the growth condition of oral squamous cell carcinoma cells according to the expression level.

Further, in one embodiment, the reagent for detecting the expression level of the fibromodulin includes xylene, absolute ethyl alcohol, 75% alcohol, concentrated hydrochloric acid, triple distilled water, an EDTA antigen retrieval solution, a phosphate buffer solution, a 30% hydrogen peroxide solution, TritonX-100, a primary anti-diluent, goat serum (C0265, Biyun), a murine anti-human fibromodulin monoclonal antibody (bs-123362R, Boaosen), a high-sensitivity enzyme-labeled anti-mouse/rabbit IgG polymer (KIT-9921, Mixin), a reaction amplifier, a DAB color development solution (KIT-9921, Mixin) and a hematoxylin dyeing solution.

Optionally, the kit detects the expression level of the fiber regulatory protein in the biological sample by an immunohistochemical staining method;

the immunohistochemical staining method comprises the following steps:

staining the biological sample;

and if the cell nucleus and/or the cytoplasm in the biological sample show homogeneous staining, the staining result is positive, otherwise, the staining result is negative.

Furthermore, the immunohistochemical staining method refers to a method for qualitatively, positioning and quantitatively determining corresponding antigens by the specific antibodies marked by the color developing agents in situ in tissue cells through antigen-antibody reaction and histochemical color development reaction. The immunohistochemical staining method combines the specificity of immunoreaction and the histochemical visibility skillfully, and detects various antigen substances such as proteins, polypeptides, enzymes, hormones, pathogens, receptors and the like at a cellular level and a subcellular level by virtue of the development and amplification of a microscope. In one embodiment, the process of the immunohistochemical staining method comprises: dewaxing; rehydration; antigen retrieval; blocking endogenous peroxidase; penetrating the core; blocking non-specific antigens; incubating the primary antibody; incubating a reaction amplifier; incubating the enzyme-labeled IgG polymer; DAB color development; dyeing the core; returning blue; baking slices; sealing a sheet; microscopic examination; photographing collection and data analysis.

Optionally, the kit comprises an antibody having a function of specifically recognizing a fibromodulin.

Further, an antibody that specifically recognizes fibromodulin includes an antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., for specifically recognizing binding fibromodulin. The antibodies or fragments thereof included in the kit include monoclonal or polyclonal, and the antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens; after immunizing an animal with the antigen, obtaining immune cells from the immunized animal and fusing myeloma cells to obtain hybridomas; the antibody is then collected from the hybridoma culture; finally, a monoclonal antibody that specifically recognizes fibromodulin can be obtained by performing antigen-specific purification of the obtained antibody using fibromodulin or a portion thereof used as an antigen. The antibody fragment is obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.

Optionally, the biological sample is processed by paraffin embedding.

Further, the biological sample comprises an oral squamous carcinoma pathological tissue, and in one embodiment, the oral squamous carcinoma pathological tissue is formalin-fixed, and the fixed tissue block is dehydrated and transparent, and then is immersed in molten paraffin for embedding to prepare paraffin-embedded 5-micron sections.

Optionally, counting the total number of cancer cells and the number of cancer cells with positive staining reaction in the selected area under a microscope to calculate the positive expression rate of the fiber regulatory protein of the oral squamous carcinoma cells.

Further, counting the total number of cancer cells and the number of cancer cells with positive staining reaction in the selected area under a microscope, and calculating the positive expression rate of the fiber regulatory protein, wherein the higher the expression level of the fiber regulatory protein is, the higher the malignancy degree of the oral squamous cell carcinoma is. The malignancy degree of oral squamous cell carcinoma is represented by tumor stage, which is determined according to the degree of tumor tissue change, including differentiation degree, arrangement mode, nuclear division number, local infiltration degree, etc., and can provide reference basis for clinical treatment and prognosis estimation. The TNM staging system is the most widely used tumor staging system, and most medical institutions use the TNM system as the primary method for cancer staging reporting. T in the TNM staging system refers to the primary tumor, N to the spread and number of nearby lymph nodes, M to metastatic tumors, and the number of each letter suffix indicates the size and/or extent of the primary tumor, and the extent of cancer spread. Wherein Tx in the primary tumor (T) indicates that the primary tumor cannot be evaluated, T0 indicates no evidence of the presence of the primary tumor, TIS indicates carcinoma in situ, T1, T2, T3, T4 indicate the size and/or extent of the primary tumor; in the regional lymph node (N), Nx represents that the regional lymph node cannot be evaluated, N0 represents that the regional lymph node is not affected, N1, N2 and N3 represent the degree of regional lymph node affected and the position and the number of affected lymph nodes; mx in distant metastasis (M) indicates that distant metastasis cannot be assessed, M0 indicates no distant metastasis, and M1 indicates the presence of distant metastasis. For example, oral squamous cell carcinoma of stage T3N2M0 indicates that the primary tumor is large, has spread to adjacent lymph nodes, but has not metastasized to other distant organs. The five stage staging system is to stage the cancer into five stages, i.e., stage 0, stage I, stage II, stage III and stage IV. TNM staging necessarily corresponds to one of the five stages, but this correspondence is not completely consistent across different types of cancer.

Optionally, the application of the composition for inhibiting the expression of the fiber regulatory protein in preparing the medicament for inhibiting the proliferation of the oral squamous cell carcinoma cell.

Furthermore, the composition for inhibiting the expression of the fibrous regulating protein can reduce the expression level of the fibrous regulating protein of the oral squamous carcinoma cell, and is a potential medicament for inhibiting the proliferation of the oral squamous carcinoma cell. The composition comprises an inhibitor for the functional expression of the fibrous regulatory protein, medicines compatible with the inhibitor, and a pharmaceutically acceptable carrier and/or an auxiliary material, wherein the inhibitor comprises a small molecular substance and a specific antibody which specifically inhibit the expression or activity of the fibrous regulatory protein.

Optionally, the application of the composition for inhibiting the expression of the fiber regulatory protein in preparing the medicament for inhibiting the migration of oral squamous cell carcinoma cells.

Furthermore, the composition for inhibiting the expression of the fibrous regulating protein can reduce the expression level of the fibrous regulating protein of oral squamous carcinoma cells, and is a potential medicament for inhibiting the migration of the oral squamous carcinoma cells. The composition comprises an inhibitor for the functional expression of the fibrous regulatory protein, medicines compatible with the inhibitor, and a pharmaceutically acceptable carrier and/or an auxiliary material, wherein the inhibitor comprises a small molecular substance and a specific antibody which specifically inhibit the expression or activity of the fibrous regulatory protein.

Optionally, the application of the composition for inhibiting the expression of the fiber regulatory protein in preparing a medicament for inhibiting the invasion of oral squamous cell carcinoma cells.

Furthermore, the composition for inhibiting the expression of the fibrous regulating protein can reduce the expression level of the fibrous regulating protein of oral squamous carcinoma cells, and is a potential medicament for inhibiting the invasion of the oral squamous carcinoma cells. The composition comprises an inhibitor for the functional expression of the fibrous regulatory protein, medicines compatible with the inhibitor, and a pharmaceutically acceptable carrier and/or an auxiliary material, wherein the inhibitor comprises a small molecular substance and a specific antibody which specifically inhibit the expression or activity of the fibrous regulatory protein.

Example 1 correlation between Fibromodulin expression levels and parameters of clinical characteristics of patients with oral squamous cell carcinoma

Clinical samples of oral squamous cell carcinoma patients were prepared as paraffin tissue sections and immunohistochemical staining was performed. Meanwhile, clinical data such as age, sex, smoking, drinking, tumor stage and the like of the patient are collected and analyzed to study the correlation between the expression of the fiber regulatory protein and clinical characteristic parameters of the oral squamous cell carcinoma patient.

The materials and methods for immunohistochemical staining were as follows:

obtaining a clinical tissue specimen: the pathological oral squamous carcinoma tissue 77 and the pathological normal cancer adjacent tissue 10 are obtained from the Pathology department of Hospital, and the clinical pathological information is obtained from the medical record of patients. Paraffin-embedded, formalin-fixed 5 micron sections were prepared for immunohistochemical staining.

Preparing a detection reagent: mixing 20mL of EDTA (50 x) and 980mL of three-distilled water uniformly to prepare EDTA antigen repairing solution which is used as it is; mixing 95mL of absolute ethyl alcohol and 5mL of triple distilled water uniformly to prepare 95% ethyl alcohol, and using the mixture immediately; 2, completely dissolving the phosphate powder in 2L of triple distilled water to prepare a phosphate buffer solution, and uniformly mixing; fully and uniformly mixing 20mL of 30% hydrogen peroxide solution and 180mL of distilled water, diluting into 3% hydrogen peroxide solution with final concentration, and refrigerating at 4 ℃ for later use; dissolving 1 XTritonX-100 (50 μ L) in 1 Xphosphate buffer (10mL) to obtain 0.5% cell-penetrating solution, and refrigerating at 4 deg.C; the primary anti-dilution ratio is found out to be 1:500 according to the pre-experimental result, and the primary anti-dilution solution is diluted to prepare the fibrin antibody dilution solution; preparing DAB working solution according to the kit specification, sequentially taking 850 mu L of double distilled water, 50 mu L of DAB buffer solution (20 x), DAB substrate (20 x) and DAB chromogen (20 x) 3 tubes of liquid, and fully and uniformly mixing with triple distilled water to prepare DAB color developing solution for use at present; slowly adding 2mL of concentrated hydrochloric acid into absolute ethyl alcohol along the wall of the beaker to prepare 1% hydrochloric acid alcohol, operating on ice to prevent high-temperature hydrochloric acid from being sprayed out, uniformly mixing and storing in a shade place.

Dewaxing: placing the paraffin section in a constant temperature oven at 65 ℃ for baking for 30 minutes; the dewaxed slices were immersed in a dye vat containing xylene solution for 10 minutes, repeated 2 times; the immersion was continued for 10 minutes with replacement of fresh xylene solution.

Rehydration: soaking in anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 min.

Antigen retrieval: gently rinsing with phosphate buffer solution for 5 minutes, repeating for 3 times, removing the phosphate buffer solution, and placing the prepared EDTA antigen repairing solution into a pressure cooker without heating; soaking the slices in a repairing solution for 1 minute; covering the cover, opening a power supply, heating the repairing liquid until the gas spraying valve ascends and the gas is sprayed out, and timing for 1.5 minutes; and (3) turning off the power supply, removing the heat source, and after the solution in the pot is naturally cooled for 1 hour, directly opening the valve to reduce the pressure so as to prevent the secondary boiling of the liquid in the pot from causing the flaking.

Blocking endogenous peroxidase: the cells were gently rinsed with phosphate buffer for 5 minutes, repeated 3 times, excess water was aspirated from the periphery of the sliced tissue, and the slices were immersed in 3% hydrogen peroxide solution for 25 minutes at room temperature.

Core penetration: after 3 repetitions, excess water was aspirated from the surrounding tissue sections, and the tissue was covered with 200. mu.L of 0.5% TrionX-100 solution at room temperature for 3 minutes.

Blocking of non-specific antigens: and (3) rinsing the tissue gently for 5 minutes by using a phosphate buffer solution, repeating the rinsing for 3 times, removing the phosphate buffer solution, dropwise adding goat serum to cover the whole tissue area and exceed the edge of the tissue by about 2-3 mm, placing the section in a wet box, and incubating the section in a constant-temperature water bath kettle at 37 ℃ for 1 hour.

Incubating the primary antibody: gently rinsing with phosphate buffer solution for 5 minutes, repeating for 3 times, and then circling around the sliced tissue by using an immunohistochemical pen or a crayon 2-3 mm away from the tissue to ensure that the tissue is fully covered by the antibody; dripping primary anti-dilution liquid to cover the whole tissue and properly exceed the edge of the tissue, placing the slices in a wet box, and incubating overnight in a refrigerator at 4 ℃; the next day, the wet box was placed in a 37 ℃ thermostat water bath for 1 hour of rewarming.

Incubation reaction amplifier: phosphate buffer was gently rinsed for 5 minutes, repeated 3 times, then removed, reaction amplifier was added dropwise to cover the entire tissue, and incubated at room temperature for 15 minutes.

Incubation of enzyme-labeled IgG polymer: and (3) gently rinsing with phosphate buffer for 5 minutes, repeating the rinsing for 3 times, removing the phosphate buffer, dropwise adding a high-sensitivity enzyme-labeled IgG polymer, covering the whole tissue and properly exceeding the edge of the tissue, and incubating for 15 minutes at room temperature.

DAB color development: and (3) dropwise adding DAB working solution, observing the dyeing degree under a microscope, generating a positive reaction when the tissue turns into light brown, slightly flushing the dyeing solution by using phosphate buffer solution, and washing the section by using running water to stop the color development. If the color development time is too long, the background is too high, and false positives appear.

Dyeing the core: gently rinsing with phosphate buffer solution for 5 minutes, repeating for 3 times, removing the phosphate buffer solution, carrying out hematoxylin counterstaining for 1.5 minutes at room temperature, immediately and gently rinsing with tap water, and stopping staining; and (3) rapidly soaking the slices in 1% hydrochloric acid alcohol for 1-2 seconds to eliminate background staining, so that the slices are not suitable for being decolorized due to too long time.

Returning blue: the sections were placed in tap water and rewetted for 2 minutes.

Baking slices: the mixture was placed in an oven at a constant temperature of 60 ℃ for 2 hours.

Sealing: appropriate neutral gum was dropped onto the tissue mass, covered with a glass coverslip, and gently worked to prevent the formation of air bubbles.

Microscopic examination: inviting the pathologist expert to interpret the organized result, wherein the pathologist expert is unknown about the clinical medical record data of the specimen; the judgment standard of the result of the expression and organization of the fiber regulatory protein under the microscope is determined as positive staining by the homogeneous brown yellow of the cell nucleus and/or cytoplasm, otherwise, the result is negative.

And (3) photographing: taking a picture under 40-fold magnification, 100-fold magnification and 200-fold magnification of an inverted microscope for observation, selecting a certain area, counting the total number of cancer cells and the number of positive reaction cancer cells in the area, and calculating the positive expression rate of the fiber regulatory protein in the oral squamous cell carcinoma tissue cells.

During collection and data analysis, an immunohistochemical image is collected by using an Olympus DP70 inverted microscope camera system; statistical analysis of the data using IBM SPSS 23.0, correlation analysis between fibromodulin expression and parameters of clinical characteristics of oral squamous cell carcinoma patients using Fisher's exact test, differences in P <0.05 were statistically significant.

The results of immunohistochemical staining of the tumor tissues of 77 patients with oral squamous cell carcinoma are shown in fig. 1, and the results show that the fibromodulin is specifically and highly expressed in the tumor tissues and is not expressed in the tumor stroma, which indicates that the fibromodulin plays an important role in the growth of the oral squamous cell carcinoma cells.

The results of statistical analysis of the expression levels of the fibromodulin in clinical tissue specimens of 77 patients and clinical parameters such as the TNM stage of the patients are shown in fig. 2, and the results show that the expression levels of the fibromodulin in the early TNM stage patients are different from those in the middle and late stage patients, and the later the stage of development of oral squamous cell carcinoma is, the higher the expression level of the fibromodulin is. This result suggests that the fibromodulin may promote malignant progression of oral squamous cell carcinoma, and that the detection of the expression level of the fibromodulin in a biological sample by an immunohistochemical staining method may be useful for the detection of oral squamous cell carcinoma.

Example 2 Effect of Sirtuin silencing expression on proliferation, migration and invasion of oral squamous cell carcinoma cells

In this example, several oral squamous cell carcinoma cell lines were selected and cultured under appropriate conditions, fiber regulatory proteins were silenced by lentiviruses, expression of fiber regulatory proteins in lentivirus-transfected oral squamous cell carcinoma cell lines was detected by Western Blot (WB) experiment, proliferation of lentivirus-transfected oral squamous cell carcinoma cell lines was detected by EdU staining experiment, migration of lentivirus-transfected oral squamous cell carcinoma cell lines was detected by cell scratch healing experiment, and invasion of lentivirus-transfected oral squamous cell carcinoma cell lines was detected by Transwell experiment.

The DMEM/F-12 complete culture medium required by cell culture is prepared by uniformly mixing Gibco/BI fetal bovine serum (50mL), DMEM/F-12 culture medium (445mL) and penicillin-streptomycin solution (100X, 5mL) to prepare a complete culture medium containing 10% fetal bovine serum, and subpackaging the complete culture medium into a 50mL sterile centrifuge tube for refrigeration at 4 ℃ for later use.

The human oral squamous carcinoma cell line CAL-27 of the embodiment is purchased from Shanghai Hongshun technology Co., Ltd and is identified and confirmed to be correct by STR of Suzhou Jianda technology Co., Ltd. Cells purchased by the bio-company may require shipment after revival or may revive themselves after shipment of the cryopreserved cells in the cold chain. The cells purchased in the embodiment are recovered by a biological company to a T25 sterile breathable cell culture bottle, the density of the cells is more than 75%, and then the cells are delivered, the T25 sterile breathable cell culture bottle is required to be filled with the culture medium during delivery, the phenomenon that the wall is separated due to the scouring effect of the shaking culture medium on adherent cells in the transportation process is prevented, and the bottle mouth is sealed by a sealing film. After the cells reached the destination, the T25 sterile air-permeable cell culture bottle was sprayed with 75% alcohol for sterilization, the state of the cells was observed under a microscope, and the cells were taken for use if they were floating and dead on the wall. If the cells are more floating, the cells are communicated with the biological company. When the cell state is good, the cells are left in a 5% carbon dioxide incubator at 37 ℃ for 24 hours until they are stable. After stabilization, the medium in the T25 sterile gas-permeable cell culture flask was aspirated, only 4mL of medium was left to continue culturing cells, and the excess medium was filled into a 50mL centrifuge tube for subsequent cell culture.

Reagents and consumables required for lentivirus transfection and tumor transplantation models in this example include sterile enzyme-free EP tubes (0.2mL and 1.5mL), RIPA cell tissue universal strong lysis Solution, phenylmethylsulfonyl fluoride (PMSP), 5 xSDS protein loading buffer Solution, pre-stained protein Marker, ECL chemiluminescence kit, PAGE gel rapid preparation kit (Yazyme), electrophoresis Solution, transfer membrane Solution, protein-free rapid blocking Solution (5X), washing Solution (P0023C3, Biyunyan), primary anti-diluent, secondary anti-diluent, methanol, Serum-free DMEM medium, RNAi-mate, culture dishes (10cm and 15cm), DMEM medium + 10% FBS Fetal Bovine Serum (Fetal Bovine Serum, trypsin digestion Solution (containing EDTA), D-Hank's Solution, FBS-free sterile centrifuge tubes (15mL and 50mL), 40 Balb/C-nu mice (3-4 weeks old), and, 0.9% sterile physiological saline, distilled water, phosphate buffered saline (PBS solution), DMEM/F12, count plate.

(1) Constructing a fibromodulin-knockdown recombinant lentivirus comprises:

mRNA sequence and shRNA sequence of fiber regulatory protein (FMOD) in recombinant lentivirus: after acquiring the mRNA full-length sequence of human fiber regulatory protein from a PubMed organ network database, (https:// www.ncbi.nlm.nih.gov/gene/2331), 4 shFMOD fragments are designed to be used as corresponding shRNA sequences and recombined into an LV10N lentiviral vector for knocking down the fiber regulatory protein expression in CAL-27 cells. The shFMOD sequence is as follows: LV 10N-FMOD-Homo-361: 5'-GGCCATGTACTGTGACAATCG-3', respectively; LV 10N-FMOD-Homo-460: 5'-GGAAGGCGTCTTTGACAATGC-3', respectively; LV 10N-FMOD-Homo-952: 5'-GGCCTCCAACACCTTCAATTC-3', respectively; LV 10N-FMOD-Homo-1042: 5'-GGAGAACCTCTACCTCCAAGG-3' are provided. Wherein LV10N-FMOD-Homo-1042 is a preparation fragment, 1 nonsense sequence is simultaneously designed and recombined into LV10N lentiviral vector as a negative control, and the sequence is LV 10N-FMOD-NC: 5'-TTCTCCGAACGTGTCACGT-3' are provided.

And (3) slow virus packaging: culturing 293T cells in a 10cm culture dish until 80-90% of cells are fused, and inoculating the cells to a 15cm culture dish; adding 1mL of 0.25% trypsin cell digestive juice (containing EDTA), mixing, and standing at 37 deg.C for 1 min; adding 2mL of DMEM culture solution containing 10% FBS serum to stop digestion, and blowing to make cells form single cell suspension; inoculating the cell suspension into a 15cm culture dish, adding 18mL of DMEM culture solution containing 10% FBS, uniformly mixing, and then standing overnight in a 5% carbon dioxide incubator at 37 ℃; adding 1.5mL of serum-free DMEM, shuttle plasmid and packaging plasmid (pGag/Pol, pRev and pVSV-G) into one sterile 5mL of centrifuge tube, uniformly mixing, taking the other sterile 5mL of centrifuge tube, adding 1.5mL of serum-free DMEM, adding 300 mu L of RNAi-Mate, uniformly mixing, standing at room temperature for 5 minutes, mixing the two tubes, and standing at room temperature for 20-25 minutes; removing the culture solution in a 15cm culture dish, and adding 8mL of serum-free DMEM culture solution; dropwise adding the transfection mixture into a 15cm culture dish, slightly shaking the culture dish back and forth to mix the compound uniformly, and incubating for 4-6 hours in a 5% carbon dioxide incubator at 37 ℃; the transfection solution was aspirated, 18mL of DMEM medium containing 10% FBS was added, and the culture was continued at 37 ℃ in a 5% carbon dioxide incubator for 72 hours.

Collecting viruses: the cell supernatant in the dish was pipetted into a 50mL centrifuge tube and centrifuged (4 ℃, 4000rpm, 4 minutes); after low-speed centrifugation, pouring supernatant of the centrifuge tube into a 50mL syringe, and filtering by using a 0.45-micrometer filter; the filtrate was ultracentrifuged in a centrifuge (4 ℃, 20000rpm, 2 hours), the concentrate was collected and dispensed into 1mL freezer tubes, labeled, and stored in a-80 ℃ freezer.

And (3) detecting the titer of the lentivirus: 293T cells were arranged at 3X 10⁴Cells/100. mu.L/well were seeded into 96-well cell culture plates and cells platedThen placing the mixture in a 5% carbon dioxide incubator at 37 ℃ for 24 hours; diluting the lentivirus stock solution by 10 times of complete culture solution for 5 concentration gradients; as Polybrene has certain cytotoxicity, the Polybrene with the final concentration of 5 mug/muL can be properly considered according to the cell state so as to improve the transfection efficiency; removing culture solution from 96-well plate, adding 100 μ L virus solution diluted in gradient into each well, and culturing in 5% carbon dioxide incubator at 37 deg.C for 24 hr; the virus-containing culture solution (4/5-containing solution if necessary depending on the cell state) in the 96-well plate was aspirated off, 100. mu.L of complete DMEM was added to each well, and the culture was continued for 72 hours in a 5% carbon dioxide incubator at 37 ℃; fluorescent cells were counted using a fluorescence microscope and the virus titer MOI was calculated in combination with the lentivirus dilution factor. Viral titer (TU/mL) fluorescence cell rate (%) × total cells × dilution of virus fluid/volume of virus fluid (mL).

Target cell infection: the first day, target cells were seeded into 24-well cell culture plates (0.5X 10)⁵One cell/hole), culturing overnight in a 5% carbon dioxide incubator at 37 ℃ until the cells adhere to the wall; the following day, after mixing the diluted virus, target cell maintenance medium and Polybrene (final concentration 5. mu.g/mL), different volumes of lentiviral stock solutions (20, 40, 60, 80, 100. mu.L) were added to 500. mu.L of the mixture, respectively; removing the culture solution in the 24-well cell culture plate, adding the diluted virus solution, establishing a blank control without adding virus, and standing overnight in a 5% carbon dioxide incubator at 37 ℃; on the fifth day, the fluorescence expression rate was observed under a fluorescence microscope, the culture medium was removed, 0.5mL of complete culture medium was added, and the mixture was cultured overnight in a 5% carbon dioxide incubator at 37 ℃; on the sixth day, according to the cell state and type, if necessary, 4/5 old culture solution is left, 0.5mL of complete culture solution is added, and the culture time is properly prolonged for 24-48 hours; on the seventh day, the fluorescence intensity was observed under a fluorescence microscope, and the old culture medium was replaced with a fresh complete culture medium containing puromycin at a high concentration of 2 μ g/mL to select the positive cells that were successfully transfected; when the cell is changed for the next time, the cell is replaced by a puromycin culture medium with the final concentration of 1 mug/mL to maintain the cells which are successfully transfected by culture; subsequent protein immunoblotting experiment is adopted to detect the expression of the fiber regulatory protein of the slow virus transfected cellsThe situation is.

(2) Western Blot (WB) assay for the expression of Fibromodulin in lentiviral-transfected CAL-27 included:

cell lysis: from the carbon dioxide incubator, T25 sterile air-permeable cell culture bottles with good growth (in logarithmic growth phase) and adherence rate of about 90 percent were taken, and CAL-27-shCON (blank control group), CAL-27-shFMOD 361, CAL-27-shFMOD 460 and CAL-27-shFMOD 952 were used for total protein extraction. Adherent cells were washed 3 times with phosphate buffer, and excess liquid was aspirated as far as possible. After preparing the whole cell lysate (ready for use) by RIPA strong lysate and PMSF according to the proportion of 1:100, uniformly dripping 1mL of the whole cell lysate onto a cell growth surface to ensure that all cells can contact the lysate, then flatly placing a culture bottle on ice, and standing and cracking for 30 minutes.

Protein sample extraction: the cells were scraped off with a clean cell scraper, and the liquid was observed to be light milky semifluid, and the cell lysate was pipetted into a sterile EP tube with a sterile enzyme-free Tip and the cell name was marked. After centrifugation at 4 ℃ for 30 minutes (12000rpm), a white precipitate was observed at the bottom of the EP tube. The supernatant (protein sample fluid) was pipetted into a new EP sample tube and the cell name was labeled.

Protein sample concentration determination and denaturation: before the protein concentration is measured by adopting a Nanodrop ND-2000 ultramicro spectrophotometer, a probe is cleaned by 2.5 mu L double distilled water for three times, and blank calibration is carried out on a measurement baseline by using a protein lysate; sequentially taking 1 mu L of protein samples to a probe, and detecting the protein concentration; adopting a whole protein lysate to adjust each protein sample with different concentrations into consistent concentrations according to the lowest concentration, wherein the calculation formula is as follows: v₂(volume of lysate added) [ c ]₁(actual concentration)/c₂(minimum concentration) -1]*V₁(self volume). To achieve a final concentration of 1 x protein loading buffer, 5 x protein loading buffer corresponding to 1/4 volume after sample adjustment was added. c. C₃(final actual sample concentration) ═ c₂(minimum concentration) × 0.8. The loading amount of each sample is 30 mug, and the loading volume V of each sample is calculated₃(final sample volume) 30/c₃(ii) a All samples were mixed well with the protein loading buffer,heating in boiling water for 10 min after instantaneous centrifugation, and storing the denatured protein sample in refrigerator at-20 deg.C for use (6 months).

Preparing electrophoresis related reagents: preparing PAGE gel comprises selecting appropriate separation gel concentration according to protein molecule size, and preparing electrophoresis PAGE gel. The ingredients are as follows:

TABLE 1 PAGE gel kit Components

Component name	Volume and quantity
		Upper glue solution (2X)	80mL
Color supernatant buffer (2X)	80mL
		Lower layer glue solution (2X)	250mL
Lower layer gel buffer (2X)	250mL
		Improved accelerator	8mL

Taking a piece of mini glue with the thickness of 0.75/1.0/1.5mm as an example, taking equal volumes of lower glue solution and lower glue buffer solution, respectively 2.0/2.7/4.0mL, and uniformly mixing; adding 40/60/80 μ L of improved coagulant into the mixed solution, and mixing; injecting the mixed solution into a glass plate for making glue until the distance between the liquid level and the upper edge of the short glass plate is 0.5cm longer than the comb teeth (note: the solution is excessive, and is not injected completely, and can be remained a little in a glue preparation cup to judge the glue solidification state), and adding appropriate amount of water or alcohol (such as isopropanol, n-butanol, etc.) to cover the lower layer of glue; after the lower layer glue is solidified (about 10 minutes), pouring out the upper layer water or alcohol; note that gelatin is said to have solidified when there is a line of refraction between the water (alcohol) and the glue; taking the supernatant solution and the color supernatant buffer solution which are equal in volume and are respectively 0.5/0.75/1.0mL, and uniformly mixing; note that: the dye is shaken up before use due to the special physicochemical property of the dye; adding 10/15/20 μ L of improved coagulant into the mixed solution, and mixing; injecting the mixed solution into a glue-making glass plate, and inserting comb teeth (note that bubbles cannot be formed at the bottom of the comb teeth so as to avoid forming waste holes due to small pits after gelation); after the upper layer is solidified (about 10-15 minutes), the comb teeth are pulled out for electrophoresis. The formulas of the lower layer glue and the upper layer glue are as follows:

TABLE 2 formulation of lower and upper layer glues

Preparing an electrophoretic solution: pouring the electrophoretic solution powder into a beaker filled with 1L of distilled water, putting a magnet, and stirring for 5 minutes in a magnetic stirrer at 1000 rpm; preparing a membrane transferring solution: uniformly mixing 800mL of distilled water and 200mL of methanol, pouring the electrophoretic solution powder into the mixture, putting the mixture into a magnet, and stirring the mixture in a magnetic stirrer at 1000rpm for 5 minutes; preparing a quick sealing liquid: the rapid blocking solution and PBST (PBS: Tween 1000:1) were prepared in a ratio of 1: 4.

Loading: and (3) freeze-thawing the protein sample and the Marker after being taken out from the refrigerator, wherein the maximum amount of the protein sample added into each hole is 25 mu L and the amount of the Marker is 5 mu L. And (3) after the glass plate is clamped (the short glass plate is arranged on the inner side), adding the electrophoresis liquid, spreading the upper edge of the inner short glass plate, pulling out the comb, and observing whether the liquid leaks or not, wherein if the liquid leaks more, the glass plate needs to be clamped again. And (3) preparing for sample loading after clamping, uniformly blowing the protein sample and the Marker by using a trace sample loader, sucking the sample by adherence, and taking care that air bubbles cannot be sucked. The applicator tip is inserted to the bottom of the well and slowly added with the sample while moving up slowly (loading may not be too fast to avoid the sample rushing out of the well or forming bubbles).

Electrophoresis: after the sample is added, the inner groove black electrode is opposite to the outer groove black electrode, the inner groove red electrode is opposite to the outer groove black electrode, and the cover is black-to-black and red-to-red. Turning on a direct current power supply, performing electrophoresis at a constant voltage of 150V for 50 minutes (the electrophoresis condition is specific to gel preparation, generally 80V for 30 minutes, and 120V for 90 minutes, and stopping electrophoresis when the blue dye reaches the position near the bottom of the gel.

Cutting the glue: according to the size of the Marker indication strip, the PAGE glue where the target strip is located is cut to be wide as much as possible, and the length and the width are measured for subsequent film transfer.

Film transfer: soaking sponge and filter paper in a film transferring liquid in advance, cutting a PVDF film according to the length and width of the cut glue, subtracting the upper right corner after cutting to be used as a mark, placing a film transferring clamp according to the sequence of a black clamping surface, the sponge, the filter paper, the PAGE glue, the PVDF film, the filter paper, the sponge and a white clamping surface, wherein the black clamp corresponds to the black surface of the inner groove, the white clamp corresponds to the white surface of the inner groove (black glue and white film, black is black to black, and red is white), the black side of the inner groove corresponds to the black electrode of the outer groove when the inner groove is provided with a film transferring device, and the red side of the inner groove corresponds to the red electrode of the outer groove (black is black to black, and red is red to red). Constant current of 260mA is kept at low temperature (ice water is added around the transfer printing groove for cooling), and the film is transferred within 2 hours.

Blocking antigen: and taking out the transferred PVDF membrane, putting the PVDF membrane into an antibody incubation box, adding a sealing liquid, shaking the PVDF membrane on a shaker at room temperature, and slightly shaking the PVDF membrane for sealing for 30 minutes.

Incubating the primary antibody: after removing the blocking solution and washing 1 time (5 min), add primary antibody (diluted according to the instructions) in 3mL primary antibody diluent formulation and incubate overnight in a shaker at 4 ℃.

Incubation of secondary antibody: after removing the primary antibody and washing 3 times (15 min/time) the secondary antibody formulated with secondary antibody diluent was added and incubated with gentle shaking for 1 hour at room temperature. Care was taken that the wash was thorough to avoid background interference.

And (3) developing: the secondary antibody was removed and the wash washed 3 times (15 min/time) with a shaker. Care must be taken to keep the PVDF membrane in a wet state. According to the following steps of 1: ECL chemiluminescent reagents A and B are mixed according to a proportion of 1 to prepare an ECL chemiluminescent reagent, the ECL chemiluminescent reagent is uniformly coated on a PVDF membrane, and the PVDF membrane is developed and photographed. Note that: the amount of the developer is not so large that the developer is easily overexposed and the developing band is not visible. The chemiluminescent liquid needs to be uniformly coated to avoid signal non-uniformity.

In this example, lentiviruses with down-expression of fibromodulin were transfected into oral squamous cell carcinoma CAL-27 to inhibit the expression level of fibromodulin, and the expression level of fibromodulin was detected by Western Blot (WB) experiment to verify the down-expression effect of lentiviruses, and the development results are shown in the left panel of fig. 3, and compared with a blank control group CAL 27-shCON without the down-expression effect of fibromodulin, two lentiviruses had significant down-expression of fibromodulin, which are CAL27-shFMOD 952 and CAL27-shFMOD 361. WB quantification and statistical analysis showed that Cal27-shFMOD 952 and Cal27-shFMOD 361 showed the best Fibromodulin inhibition as shown in the right panel of FIG. 3. Results of subsequent experiments Cal27-shFMOD 952 and Cal27-shFMOD 361 were abbreviated as Cal27-shFMOD-1 and Cal27-shFMOD-2, and subsequent experiments were performed with Cal27-shFMOD-1 and Cal 27-shFMOD-2.

Reagents for cell phenotype experiments (proliferation, migration, invasion) in this example include phosphate buffered saline (PBS, cell culture grade PH 7.4), DMEM/F-12 medium, Fetal Bovine Serum (FBS), 100 × cyan-streptomycin solution, 0.25% trypsin digestion solution, 6-well plate, 24-well plate, 96-well plate, cell EdU DNA staining kit, fixative solution, washing solution, permeant solution, matrigel, Transwell chamber, crystal violet solution, Annexin V-FITC/PI apoptosis kit, cisplatin solution, phalloidin.

(3) The detection of CAL-27-shFMOD and CAL-27shCON cell proliferation rates by EdU staining experiments comprises the following steps:

cell preparation: conventional cell culture, digestion, counting, resuspension, adjusting cell density to 1 × 10⁵after/mL, 100. mu.L of cell suspension was added to the corresponding well of a 96-well cell culture plate (i.e., 3X 10 cells were seeded per well)⁴One), the plates were then placed in an incubator for 48 hours.

Preparation of reagents: preparing 2 × EdU working solution: because the EdU working solution and the culture solution are added into the pore plate in equal volume, 2 multiplied working solution needs to be prepared; the recommended final EdU concentration is 10. mu.M (1X), and 2X EdU working solution (20. mu.M) can be obtained by diluting EdU (10mM) with 1:500 cell culture solution. Considering the EdU cytotoxicity, if long-time incubation (> 24 hours) is selected, a low-concentration working medium of 1-5 μ M is preferably adopted; if a short incubation time is chosen (< 6 hours), 100. mu.L of high concentration EdU (50. mu.M) medium can be added directly per well to incubate the cells for 2 hours. The EdU kit package contents are as follows:

TABLE 3 EdU reagent formula

Product name	Volume and quantity
		EdU(10mM)	200μL
Azide 488	55μL
		Click Reaction Buffer	30mL
CuSO₄	1.1mL
		Click Additive
	2 tube
		Hoechst 33342(1000×)	50μL

And (3) washing the cells: the medium was discarded and the washing solution washed the cells 3 times (5 min/time). The purpose of the wash was to elute EdU that did not penetrate into the DNA. The washing time and times are set according to the cell types, and the washing strength can be properly reduced by the cells which are not firmly adhered.

Cell fixation: add 100. mu.L of 4% paraformaldehyde to each well and incubate for 30 minutes at room temperature before removing the fixative.

Cell washing and permeabilization: add 100. mu.L of washing solution to each well, wash with decolorizing shaker for 5 min, discard the washing solution, add 100. mu.L of penetrant (PBS containing 0.5% Triton X-100) to each well, incubate with decolorizing shaker for 10 min with gentle shaking, wash 2 times (5 min/time).

Click dyeing: mu.L of a staining reaction solution (Click reaction solution, prepared according to the table composition) was added to each well. After incubating for 30 minutes at room temperature in a dark decolorizing shaker, the staining reaction solution was discarded, 100. mu.L of washing solution was added, and the decolorizing shaker was washed 3 times (10 min/time). For cells with high dye adsorption, an enhanced elution mode is adopted to reduce the dyeing background. The Click staining solution formula is as follows:

TABLE 4 Click staining solution formula

DNA staining: the reagent is diluted by deionized water according to the proportion of 1:1000 to prepare 1 Xhoechst 33342 reaction solution which is stored in dark. mu.L of 1 × Hoechst 33342 reaction solution was added to each well, incubated for 30 minutes in the dark at room temperature with slow shaking in a decolorization shaker, and then the reaction solution was discarded and washed 3 times (5 minutes/time).

And (3) photographing: 100 μ L of PBS was added to each well, and photographed under a fluorescent microscope.

Calculating cell proliferation: the proportion of proliferating cells was counted by ImageJ software and histogram of proliferation rate was plotted on GraphPad prism 8.0.

The EdU compound can be inserted into a replicating DNA molecule when a cell proliferates, and efficient and rapid cell proliferation detection analysis can be carried out based on the conjugation reaction of the EdU and a dye, so that the percentage of cells in the S phase can be effectively detected.

The proliferation capacity of the oral squamous carcinoma cell line transfected by the lentivirus is detected by an EdU staining experiment, and the result shows that the proliferation rate of Cal27-shFMOD-1 and Cal27-shFMOD-2 is reduced (as shown in the left picture of figure 4) and has statistical difference (as shown in the right picture of figure 4) compared with a blank control group Cal 27-shCON. Experiments show that the inhibition of the expression of the fiber regulatory protein can inhibit the proliferation capacity of oral squamous cell carcinoma cells.

(4) Cell scratch healing experiments to test the migration capacity of CAL-27-shFMOD and CAL-27shCON cells include:

cell plating: the cells were cultured at 1X 10⁶The density of each well was uniformly inoculated into 6-well plates, and the plates were incubated at 37 ℃ in a 5% carbon dioxide incubator for 48 hours.

Manufacturing "wound surface" (scratch): after the cell monolayer fusion rate reaches 100%, pressing the pore plate by using a ruler on an ultraclean workbench, taking a 200-mu-L sterile Tip head, uniformly and forcefully scratching the cell surface in the pore by clinging to the edge of the ruler (ensuring the scratch width of each cell to be consistent), and slightly washing the scratched floating cells in the pore plate by using sterile PBS for 5 times to ensure that the photographing field of vision is clean and clear.

And (4) photographing and recording: taking a picture under a microscope to record scratches for 0 hour, replacing a serum-free fresh culture medium, and continuously culturing in a 5% carbon dioxide incubator at 37 ℃. After the dead cells were rinsed with PBS at 24 and 48 hours, the healing of the cell scar at the same site was observed under a microscope and photographed, and a mobility bar graph was plotted on GraphPad prism 8.0.

The results of the cell scratch healing experiments show that the rates of scratch healing of Cal27-shFMOD-1 and Cal27-shFMOD-2 are significantly slower (as shown in the left panel of FIG. 5) and statistically different (as shown in the right panel of FIG. 5) compared with the blank control group Cal 27-shCON. Experiments show that the inhibition of the expression of the fiber regulatory protein can inhibit the migration capability of oral squamous cell carcinoma cells.

(5) The detection of CAL-27-shFMOD and CAL-27shCON cell invasiveness by Transwell chamber invasion assay comprises:

matrix glue plating cells: taking out the matrigel from a refrigerator at the temperature of-20 ℃, and dissolving the matrigel on ice; taking a sterile 1.5mL centrifuge tube, diluting matrigel with serum-free DMEM/F12 culture medium according to the ratio of 9:1, and then gently mixing uniformly; opening a Transwell chamber in the biological safety cabinet, adding 100 mu L matrigel into a groove on the upper layer of the chamber, and gently flowing into the bottom along the wall of the chamber during adding to avoid generating bubbles to influence subsequent cell membrane penetration and photographing; the chamber was placed in a 24-well cell culture plate, air-dried naturally, or procoagulant in an incubator at 37 ℃ (this step is simulated by artificial extracellular matrix barrier).

Cell counting and plating: the cells were resuspended in fresh serum-free medium (cell concentration: 3X 10)⁴One cell/100 μ L), then 100 μ L of the cell suspension was seeded into the upper well of the chamber in a 24-well plate (the front side of the well bottom was matrigel that had been air dried before); gently lifting the edge of the chamber (taking care not to pour the cells inside) with forceps, adding 500. mu.L of complete medium per well, then lowering the chamber to immerse the lower layer of the chamber in the complete medium, if any, lifting the edge of the chamber again to remove the bubbles; immediately, the cells were dispersed by shaking the plate horizontally in the "8" format and cultured in a 5% carbon dioxide incubator at 37 ℃ for 24 hours.

Cell fixation and staining: taking out the 96-well plate, after rinsing the back of the chamber once with PBS, fixing and transferring the cell to the back of the membrane of the Transwell chamber for 20 minutes (room temperature) with 4% paraformaldehyde, and rinsing with PBS 3 times; soaking the small chamber in 0.4% crystal violet solution for dyeing for 30 minutes, and washing for 3 times by using PBS solution; gently wiping the cells which do not invade and penetrate through the Transwell membrane and the residual staining solution in the upper layer of the chamber by using a cotton swab (the cells are not easy to be too forcefully rubbed so as not to damage the membrane of the chamber and influence the photographing); the chamber was placed in a new 24-well plate and observed under an optical microscope, photographed, and counted. Counting photos once by averaging five visual fields, and ensuring the representativeness of the photos; statistical analysis: the number of stained cells in the pictures was counted using ImageJ software and the invasiveness bar chart was plotted on GraphPad prism 8.0.

The results of the Transwell invasion experiment show that Cal27-shFMOD-1 and Cal27-shFMOD-2 have obviously reduced numbers of invaded cells compared with the blank control group Cal 27-shCON (upper panel in FIG. 6 and left panel in lower panel in FIG. 6), and have statistical difference (right panel in lower panel in FIG. 6). Experiments show that the inhibition of the expression of the fiber regulatory protein can inhibit the invasion capacity of oral squamous cell carcinoma cells.

Example 3 establishment of nude mouse transplantable tumor model to verify the Effect of inhibition of Fibromodulin on the growth of oral squamous cell carcinoma tumor tissue

In the embodiment, the influence of inhibiting the fibroblast on the growth of oral squamous cell carcinoma tumor tissues is verified by injecting the cell for silencing the fibroblast into a nude mouse and recording the weight change and the growth condition of transplanted tumors of grouped mice. The specific experimental method is as follows:

(1) animal purchasing and raising: 12 healthy female Balb/c-nu nude mice of 3-4 weeks old were purchased from Beijing Wintonli Hua, and were bred in SPF-level animal houses in the center of laboratory animals in Hubei Hospital, pharmaceutical institutes, and 6 nude mice per group were planned.

(2) Cell culture: cell 2 groups were CAL-27shCON group and CAL-27shFMOD group. Adherent cells were expanded to T75 cell flasks after conventional subculture. Cells with good growth state in logarithmic growth phase are taken for preparing cell suspension, and each nude mouse is inoculated with 10⁶And (4) cells.

(3) And (3) centrifugal resuspension: after digesting the cells with conventional trypsin digestion solution, the cells were suspended in a sterile 15mL centrifuge tube, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and 2mL serum-free DMEM/F12 medium was added to resuspend the cells for further use. In order to ensure the cell activity, it is preferable that many people assist in shortening the cell processing time.

(4) Cell counting: adding 10 μ L cell suspension into the pore channel of the cell counting plate, counting under microscope, adjusting cell density to 1 × 10⁶One/100. mu.L.

(5) Subcutaneous inoculation: (2) the time to (4) is controlled to be compact, and excessive number of inactivated cells is avoided. Enter SPF animal house according to standard procedure. Nude mice were housed, near the forelimb, subcutaneously tilted 45 degrees and injected with 100 μ L of cell suspension. The action needs to be accurate, soft and stable, the suspension is prevented from leaking from the needle hole, and the suspension can stay for 3 seconds slightly before the needle is pulled out. After the injection is finished, the injection port is disinfected by alcohol cotton balls and is put back into the cage for feeding.

(6) And (4) observing and recording: the subcutaneous tumor formation of the nude mice was observed every 3 days from the day of inoculation, the longest diameter a and the shortest diameter b of the tumor were measured with a vernier caliper, and the tumor volume was calculated (c ═ a ═ b-²)/2). The weight of the nude mouse is weighed by an electronic balance, and the mental state of the nude mouse is observed.

(7) And (4) counting results: after 21 days of growth of the transplanted tumors, CAL-27shCON group and CAL-27shFMOD group were removed, the subcutaneous transplanted tumors were removed using surgical scissors and forceps. After the blood water of the transplanted tumor is cleaned by PBS solution, the nude mouse and the corresponding transplanted tumor are placed on white paper, a straight ruler is placed beside the white paper for taking a picture, the weight of the tumor is weighed, and the weight is recorded and counted.

Cal27-shFMOD-1 was selected for nude mouse transplantation tumor experiments because the proliferation inhibition rate of Cal27-shFMOD-1 was the most significant in cell experiments. The results showed that the tumor volume of Cal27-shFMOD-1 was significantly smaller than that of the blank control group Cal 27-shCON (FIG. 7, left panel and FIG. 7, middle panel). Statistical analysis of the transplanted tumors in nude mice revealed that the growth rate of the Cal27-shFMOD-1 transplanted tumors was significantly slower (FIG. 7, right panel). The EdU staining experiment and the nude mouse transplantation tumor experiment show that the inhibition of the expression of the fiber regulatory protein can inhibit the proliferation of oral squamous cell carcinoma cells and the growth of tumor tissues from the in vitro level and the in vivo level.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A marker associated with oral squamous carcinoma cell growth, wherein said marker comprises a fibromodulin.

2. Application of fiber regulatory protein in identifying growth condition of oral squamous cell carcinoma cells.

3. The kit is used for detecting the expression level of the fiber regulatory protein so as to identify the growth condition of oral squamous cell carcinoma cells according to the expression level.

4. The kit of claim 3, wherein the kit detects the expression level of the fibromodulin in the biological sample by an immunohistochemical staining method;

the immunohistochemical staining method comprises the following steps:

staining the biological sample;

5. The kit according to claim 3, wherein the kit comprises an antibody having a function of specifically recognizing a fibromodulin.

6. The kit of claim 4, wherein the biological sample is processed by paraffin embedding.

7. The kit according to claim 4, wherein the total number of cancer cells and the number of cancer cells that positively react with staining in the selected region are counted under a microscope to calculate the positive expression rate of the fibromodulin in of the oral squamous carcinoma cells.

8. Application of a composition for inhibiting expression of a fiber regulatory protein in preparation of a medicament for inhibiting proliferation of oral squamous cell carcinoma cells.

9. Application of a composition for inhibiting expression of a fiber regulatory protein in preparation of a medicament for inhibiting migration of oral squamous cell carcinoma cells.

10. Application of a composition for inhibiting expression of a fiber regulatory protein in preparation of a medicament for inhibiting invasion of oral squamous cell carcinoma cells.