CN108872578B - Novel method for diagnosing and monitoring oral cancer - Google Patents
Novel method for diagnosing and monitoring oral cancer Download PDFInfo
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Abstract
The invention provides a novel method for monitoring the conversion of oral leukoplakia to oral cancer and diagnosing the oral cancer, which comprises the step of monitoring the concentration change of oral exosomes of an oral leukoplakia subject so as to judge the pathological state of the disease and the canceration risk.
Description
Technical Field
The invention relates to a novel painless, simple and convenient method for monitoring the conversion of oral leukoplakia to oral cancer, which comprises the steps of monitoring the change of the concentration of saliva exosomes of a subject, determining the pathological stage of the oral leukoplakia, finding a cancer-variable risk patient before canceration, carrying out treatment measures on the cancer-variable risk patient in time, and realizing early and effective prevention and treatment of the oral cancer.
Background
Oral cancer is one of the most common 10 cancers worldwide, accounting for 80% of head and neck malignancies, and there are about 5 million oral cancer patients worldwide, with Oral Squamous Cell Carcinoma (OSCC) being the most common, with a five-year survival rate of about 35-57%, and about 13 million oral cancer patients dying each year[1-2]. Oral cancer occurs mainly in the middle-aged and elderly. Despite recent advances in diagnostic techniques, surgery, and chemotherapy and radiotherapy, patients unfortunately have a 5-year survival rate of about 50% in their life. The key to effectively preventing and treating oral cancer is to correctly identify oral diseases with malignant potential, also called precancerous lesions. If the early correct diagnosis, continuous monitoring and timely intervention can be realized, the method has important significance for preventing and treating the oral cancer.
The oral cavity precancerous lesion refers to some clinical or histological changes of the oral cavity and the maxillofacial region and has canceration tendency, and comprises leukoplakia, erythema, lichen planus, discoid lupus erythematosus, submucosal fibrosis, papilloma, chronic ulcer, mucosal black spot, pigmented nevus and the like, wherein the oral cavity leukoplakia is recognized as one of the most typical precancerous lesions in oral cavity speckle diseases, and the canceration rate is as high as 10-36%.
Oral leukoplakia (Oral leukoplakia, OLK), also known as Oral leukoplakia, was first named by Er no Sohummer, the dermatologist Hungari, in 1887, and refers to white or gray-white hyperkeratotic abnormalities occurring on the Oral mucosa. The leukoplakia of the oral mucosa is commonly found in the middle-aged and the elderly, and is better found in the mucous membranes of the lips, cheeks, tongue, palate and the likeOn the membrane, there is generally no subjective symptom, and the membrane initially appears as milky white plaque with a smooth, flat or slightly higher surface than the normal mucous membrane. The progress of the leukoplakia from precancerous lesion to oral cancer can be from several years to ten years, and the canceration process is a multi-stage and multi-step process and is carried out by hyperplasia → squamous metaplasia → mild, moderate and severe abnormal hyperplasia → carcinoma in situ → invasive carcinoma[3-4]Moreover, most oral leukoplakias can be in a benign state for a long time without canceration, and only a small part of oral leukoplakias can be in precancerous state and can be developed into cancer. In recent years, the incidence of oral cancer tends to increase and become significantly younger. Despite the ongoing advances in surgical, radiation and chemotherapy techniques for oral cancer, patients still have less than 50% of their 5-year survival rate. The 5-year survival rate of tumor localizers is about 80%, and the survival rate of tumor localizers is reduced to 20%[5]。
The reasons for the low overall survival rate of oral cancer are manifold, with late patient visit being an important factor and most oral cancers reaching the middle and advanced stages of their development. The key to the early detection of oral cancer is the correct diagnosis, continuous monitoring and early detection of the oral precancerous lesion with malignant potential. If patients with oral cancer can be diagnosed early and treated in time, their prognosis will be greatly improved. Therefore, it becomes a hot spot for many scholars to find early spider-silk traces of oral cancer.
At present, the methods for detecting oral cancer mainly comprise clinical examination, histopathological examination, exfoliative cytology examination, living tissue staining, optical examination, detection of tumor cell molecular markers and the like.
Clinical examination is the most convenient method at present, and a doctor can find lesions in the oral cavity by visual examination or palpation. If the clinical experience of doctors is insufficient and the compliance of patients is poor, the follow-up cannot be performed regularly, and the clinical observation effect is influenced. Whether cancer is prone to be detected cannot be judged only by clinical examination, and further diagnosis by histopathological examination is required.
The exfoliative cytology examination is a simple and convenient detection method, and auxiliary examination means including DNA image quantitative analysis, micronucleus analysis and the like can be used for judging whether lesion tissues are cancerated or not. The success of this technique application depends on the experience of the operator and the number, location, etc. of the cells being brushed.
Biopsy staining is a non-invasive method of diagnosing early stage oral cancer. The principle is that the lesion tissue part is displayed by utilizing the different dyeing degrees of the cancerous tissue to the dyeing agent. The coloring agent is toluidine blue, Lugol liquid, Melan, etc. Currently, toluidine blue is commonly used in stomatology, but the false positive rate is high, and misdiagnosis is easy to occur.
Optical examination methods are a class of examination techniques based on tissue optical characteristics, and are currently studied in the field of tumor screening, including tissue spectroscopy, autofluorescence imaging, and VELscope. The predominant current application in the oral cavity is the VELSCOPE technology, which emits a blue laser with a wavelength between 400-460nm, which can excite green fluorescence in the oral tissue auto-fluorophore. This optical change can reveal metabolic, biochemical and structural information of mucosal cells and can localize regional carcinogenesis. The application of this technique is now based on the fact that pathological biopsy has been used to determine the presence of abnormal hyperplasia or canceration of the oral mucosa, and therefore further clinical validation is needed.
Recently, molecular marker detection of tumor cells and the like have been receiving attention. Biomarkers for detecting oral precancerous lesions malignancy can be broadly divided into four categories: 1. markers related to cell proliferation, such as proliferating cell nuclear antigen, nucleolar constituent zone argentine, Ki 67 Mi b-1, telomerase, etc.; 2. markers associated with apoptosis, such as Bcl-2/Bax, Survivin, epoxide hydrolase 2, and the like. (ii) a 3. Specific genes, such as the p53 gene and its expression products; 4. chromosomal aberrations, e.g. microsatellite markers and loss of heterozygosity, DNA content, etc[6]. Mendez et al found 314 gene expressions in oral cancer with differences compared to normal tissue, with 239 genes up-regulated and 75 genes down-regulated[7]. More and more studies have shown that biomarkers in saliva are associated with the development and progression and prognosis of oral cancer, however, the development of oral cancerMarker markers currently have no one or a set of indices that can achieve a well-recognized reliable effect.
Despite the many oral cancer detection methods described above, histopathological examination remains the gold standard for oral leukoplakia and oral cancer diagnosis. The operator determines whether to excise or excise the biopsy after comprehensively considering the size, the position and the definition of the boundary of the lesion. The high-risk phases of the lesions are not necessarily uniformly distributed, and suspicious spots and possibly multi-spot biopsy need to be observed.
Since the examination procedure itself is traumatic and irritating to the lesion, post-operative scarring can also affect the observation, further limiting the reproducibility of the examination. For monitoring the transformation of leukoplakia into oral cancer at a stage of simple or abnormal hyperplasia, histopathological examination is difficult to use as a conventional monitoring means because it requires repeated acquisition of living tissues.
The monitoring of the leukoplakia canceration needs to regularly check the pathological changes and record the changes, so as to make a proper evaluation on the canceration risk, strive for finding a high-risk patient before the leukoplakia is converted into oral cancer and take proper treatment on the patient in time, and once the patient is converted from the oral leukoplakia into the oral squamous cell carcinoma, the 5-year survival rate of the patient is less than 50 percent[8]. Therefore, there is a need to find a method for accurately and timely monitoring the transformation of leukoplakia to oral cancer, which not only needs to reflect the dynamic process of lesion transformation, but also needs to be easy to implement and acceptable to patients.
The inventor of the application finds that the conversion of the simply hyperplastic oral leukoplakia to leukoplakia accompanied with abnormal hyperplasia and oral cancer causes the concentration of saliva exosomes of a patient to be obviously changed, namely, when the leukoplakia is in a simply hyperplastic state, the concentration of the saliva exosomes is not obviously different from that of normal people; but when the leukoplakia progresses to the abnormal hyperplasia stage, the concentration of saliva exosomes is obviously increased relative to normal people, and the difference is obvious; once the abnormal hyperplasia turns into oral cancer, the concentration of saliva exosomes is rather significantly reduced and different compared to normal humans. With pathological changes from simple hyperplasia and abnormal hyperplasia to canceration of white spots, the concentration of saliva exosomes of patients is subjected to a dynamic change process of increasing firstly and then reducing. This finding suggests that the conversion of leukoplakia into oral cancer can be monitored by measuring the concentration of saliva exosomes in the patient, and that the pathological course of leukoplakia conversion into oral cancer can be monitored by monitoring the concentration of saliva exosomes in the patient on a regular basis. The concentration of saliva exosomes is expected to become a new method for diagnosing leukoplakia and oral cancer, predicting the risk of leukoplakia canceration, monitoring the leukoplakia canceration process and the like.
Disclosure of Invention
The invention provides a method for auxiliary diagnosis of leukoplakia and oral cancer, which can be repeatedly implemented and is simple and easy to implement. Meanwhile, the invention also provides a method for monitoring the conversion of the oral leukoplakia to the oral cancer, which comprises the steps of regularly monitoring the concentration of saliva exosomes of an oral leukoplakia subject, judging the state (stage) of a lesion, and making an adaptive follow-up plan and treatment scheme. Meanwhile, the canceration trend of the oral cavity white spot is judged by monitoring the concentration of saliva exosomes of the testee, and the high-risk oral cavity cancer patient is found in time and is treated appropriately.
Accordingly, one aspect of the present invention relates to:
1. an auxiliary diagnostic method for oral cancer, comprising detecting the concentration of saliva exosomes of an oral leukoplakia subject, wherein a significant decrease in the concentration of saliva exosomes of the subject compared to a normal person indicates that the oral leukoplakia of the subject is transformed into oral cancer.
2. The aided diagnosis method of item 1, wherein the concentration of saliva exosomes of the subject is significantly increased compared to normal persons, suggesting that oral leukoplakia is accompanied by abnormal hyperplasia in the subject, the development of which needs to be closely monitored.
3. A method of monitoring the conversion of an oral leukoplakia to oral cancer comprising monitoring the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significantly elevated concentration of salivary exosomes relative to normal indicates that the leukoplakia is associated with abnormal proliferation and requires rigorous monitoring, but a significantly reduced concentration of salivary exosomes relative to normal indicates that the oral leukoplakia has been converted from abnormal proliferation to oral cancer.
4. A method of predicting the risk of conversion of oral leukoplakia to oral cancer comprising monitoring the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significantly elevated concentration of salivary exosomes relative to normal human indicates an increased risk of conversion of oral leukoplakia to oral cancer.
5. The method of item 4, wherein a transition from a significantly elevated concentration of salivary exosomes relative to normal to a significantly reduced concentration relative to normal indicates a likelihood that an oral leukoplakia has been converted from abnormal hyperplasia to oral cancer.
6. A method for screening for oral cancer, comprising detecting a concentration of salivary exosomes in a subject, wherein a significant decrease in the concentration of salivary exosomes in the subject compared to a normal human indicates a likelihood of acquiring oral cancer in the subject.
7. The method of any one of items 1-6, wherein the oral cancer is oral squamous cell carcinoma.
8. A method for assisting in diagnosing pathological stages of oral leukoplakia comprises the steps of detecting the concentration of saliva exosomes of an oral leukoplakia subject, wherein the concentration of the saliva exosomes is not obviously different from that of normal people, so that the oral leukoplakia is in a simple hyperplasia stage, the concentration of the saliva exosomes is obviously increased relative to that of the normal people, so that the oral leukoplakia is accompanied with abnormal hyperplasia, and the concentration of the saliva exosomes of the subject is obviously reduced compared with that of the normal people, so that the oral leukoplakia of the subject is converted into oral cancer.
9. The method of item 8, wherein the oral cancer is oral squamous cell carcinoma.
10. An auxiliary method for identifying suspected oral leukoplakia comprising determining the level of miRNA185 in saliva exosomes, wherein a significant decrease in the level compared to normal indicates a likelihood of the subject suffering from oral leukoplakia.
11. The method of item 10, comprising further determining the concentration of salivary exosomes of the subject as determined by a significant decrease in miRNA185 level in the subject to determine the pathological stage of oral leukoplakia, wherein no significant difference in the concentration of exosomes compared to normal indicates that the oral leukoplakia of the subject is in a simple hyperplastic stage, a significant increase in the concentration of salivary exosomes relative to normal indicates that the oral leukoplakia of the subject is accompanied by abnormal hyperplasia, and a significant decrease in the concentration of salivary exosomes relative to normal indicates the likelihood that the oral leukoplakia of the subject has been converted into oral cancer.
12. The method of item 11, wherein the oral cancer is oral squamous cell carcinoma.
In another aspect, the invention relates to:
1. use of a reagent or an apparatus for detecting salivary exosomes in the preparation of a reagent or a kit, or an apparatus for use in a method for the assisted diagnosis of oral cancer, the method comprising detecting the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significant decrease in the concentration of salivary exosomes in the subject compared to normal persons is indicative of conversion of oral leukoplakia in the subject to oral cancer.
2. The adjunctive use of item 1, wherein a significant increase in the concentration of saliva exosomes in the subject compared to normal persons is indicative of oral leukoplakia with abnormal proliferation in the subject, the development of which needs to be closely monitored.
3. Use of a reagent or apparatus for detecting salivary exosomes in the manufacture of a reagent or kit, or apparatus, for use in a method of monitoring the conversion of an oral exudate to an oral cancer, the method comprising monitoring the concentration of salivary exosomes in an oral exudate subject, wherein a significantly increased concentration of salivary exosomes relative to normal is indicative of an exudate with abnormal hyperplasia, requiring close monitoring, but a significantly decreased concentration of salivary exosomes relative to normal, is indicative of an oral exudate having been converted from abnormal hyperplasia to an oral cancer.
4. Use of a reagent or an apparatus for detecting salivary exosomes in the preparation of a reagent or kit, or an apparatus, for use in a method of predicting risk of oral leukoplakia to oral cancer, the method comprising monitoring the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significantly increased concentration of salivary exosomes relative to normal persons is indicative of an increased risk of oral leukoplakia to oral cancer conversion.
5. The use of item 4, wherein a transition from a significant increase in saliva exosome concentration relative to normal to a significant decrease relative to normal indicates the likelihood that an oral leukoplakia has been converted from abnormal hyperplasia to oral cancer.
6. Use of a reagent or apparatus for detecting salivary exosomes in the preparation of a reagent or kit, or apparatus, for use in a method of screening for oral cancer, the method comprising detecting the concentration of salivary exosomes in a subject, wherein a significant decrease in the concentration of salivary exosomes in the subject compared to normal is indicative of a likelihood of the subject suffering from oral cancer.
7. The use of any one of items 1-6, wherein the oral cancer is oral squamous cell carcinoma.
8. Use of a reagent or an apparatus for detecting oral exudate in the preparation of a reagent or a kit or an apparatus for use in a method for assisting in diagnosing pathological stages where oral leukoplakia is located, the method comprising detecting the concentration of the oral leukoplakia subject's oral exudate, wherein the concentration of the oral leukoplakia is not significantly different from that of normal persons, suggesting that the oral leukoplakia is in a simple proliferative stage, the concentration of the oral leukoplakia is significantly increased from that of normal persons, suggesting that the oral leukoplakia is accompanied by abnormal proliferation, and the concentration of the subject's oral exudate is significantly decreased from that of normal persons, suggesting that the subject's oral leukoplakia is transformed into oral cancer.
9. The use of item 8, wherein the oral cancer is oral squamous cell carcinoma.
10. Use of an agent for detecting the level of miRNA185 in saliva exosomes in the preparation of a reagent or kit for use in a method of aiding in the identification of a suspected oral leukoplakia, the method comprising determining the level of miRNA185 in saliva exosomes, wherein a significant decrease in the level compared to normal is indicative of a subject's potential for developing an oral leukoplakia.
11. The use of item 10, which comprises further determining the concentration of salivary exosomes of the subject as determined by a significant decrease in miRNA185 level in a subject to determine the pathological stage of oral leukoplakia, wherein no significant difference in the concentration of exosomes compared with normal persons indicates that the oral leukoplakia of the subject is in a simple hyperplastic stage, a significant increase in the concentration of salivary exosomes relative to normal persons indicates that the oral leukoplakia of the subject is accompanied by abnormal hyperplasia, and a significant decrease in the concentration of salivary exosomes relative to normal persons indicates the possibility that the oral leukoplakia of the subject has been converted into oral cancer.
12. The use of item 11, wherein the oral cancer is oral squamous cell carcinoma.
In another aspect, the present invention also relates to:
1. use of a method for aiding in the diagnosis of oral cancer by detecting the concentration of salivary exosomes in an oral leukoplakia subject in the manufacture of a reagent or kit for use in the method for aiding in the diagnosis of oral cancer, the reagent or kit having a package insert with instructions for a method for aiding in the diagnosis of oral cancer by detecting the concentration of salivary exosomes in an oral leukoplakia subject, the method comprising detecting the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significant decrease in the concentration of salivary exosomes in the subject compared to a normal person is indicative of conversion of oral leukoplakia in the subject to oral cancer.
2. The use of item 1 for aiding diagnosis, wherein the concentration of saliva exosomes of the subject is significantly increased compared to normal persons, suggesting that oral leukoplakia is accompanied by abnormal hyperplasia in the subject, the development of which needs to be closely monitored.
3. Use of a method of monitoring the conversion of oral leukoplakia to oral cancer by monitoring the concentration of salivary exosomes in an oral leukoplakia subject in the manufacture of a reagent or kit for use in the method, the reagent or kit having a package insert with instructions for a method of monitoring the conversion of oral leukoplakia to oral cancer by monitoring the concentration of salivary exosomes in an oral leukoplakia subject, the method comprising monitoring the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significantly elevated concentration of salivary exosomes relative to normal persons indicates that the leukoplakia is accompanied by abnormal proliferation, requiring tight monitoring, but a significantly reduced concentration of salivary exosomes relative to normal persons indicates that the oral leukoplakia has been converted from abnormal proliferation to oral cancer.
4. Use of a method of predicting the risk of conversion of oral leukoplakia to oral cancer by monitoring the concentration of salivary exosomes in an oral leukoplakia subject in the manufacture of a reagent or kit for use in the method, the reagent or kit having a package insert with instructions for a method of predicting the risk of conversion of oral leukoplakia to oral cancer by monitoring the concentration of salivary exosomes in an oral leukoplakia subject, the method comprising monitoring the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significantly increased concentration of salivary exosomes relative to normal humans is indicative of an increased risk of conversion of oral leukoplakia to oral cancer.
5. The use of item 4, wherein a transition from a significant increase in saliva exosome concentration relative to normal to a significant decrease relative to normal indicates the likelihood that an oral leukoplakia has been converted from abnormal hyperplasia to oral cancer.
6. Use of a method of screening for oral cancer by detecting the concentration of saliva exosomes in a subject in the manufacture of a reagent or kit for use in the method, the reagent or kit having a package insert with instructions for a method of screening for oral cancer by detecting the concentration of saliva exosomes in a subject, the method comprising detecting the concentration of saliva exosomes in a subject, wherein a significant decrease in the concentration of saliva exosomes in the subject compared to normal is indicative of a subject's potential to suffer from oral cancer.
7. The use of any one of items 1-6, wherein the oral cancer is oral squamous cell carcinoma.
8. The use of a method for the auxiliary diagnosis of pathological stages of oral leukoplakia in the preparation of a reagent or a kit used in the method by detecting the concentration of salivary exosomes of an oral leukoplakia subject, the reagent or the kit is provided with a package insert, and the package insert is provided with instructions for a method for assisting in diagnosing pathological stages of oral leukoplakia by detecting the concentration of saliva exosomes of an oral leukoplakia subject, wherein the method comprises detecting the concentration of the saliva exosomes of the oral leukoplakia subject, wherein the concentration of the saliva exosomes has no significant difference relative to normal persons, which indicates that oral leukoplakia is in a simple proliferation stage, the concentration of the saliva exosomes is obviously increased relative to normal people, which indicates that oral leukoplakia is accompanied with abnormal hyperplasia, the subject's significantly reduced concentration of salivary exosomes compared to normal persons is indicative of conversion of oral leukoplakia to oral cancer in the subject.
9. The use of item 8, wherein the oral cancer is oral squamous cell carcinoma.
10. Use of a method for aiding in the identification of a suspected oral leukoplakia by determining the level of miRNA185 in a saliva exosome in the preparation of a reagent or kit for use in the method, the reagent or kit having a package insert with instructions for a method of aiding in the identification of a suspected oral leukoplakia by determining the level of miRNA185 in a saliva exosome, the method comprising determining the level of miRNA185 in a saliva exosome, wherein a significant decrease in the level compared to a normal person is indicative of a subject's potential to suffer from an oral leukoplakia.
11. The use of item 10, which comprises further determining the concentration of salivary exosomes of the subject as determined by a significant decrease in miRNA185 level in the subject to determine the pathological stage of oral leukoplakia, wherein no significant difference in the concentration of exosomes compared with normal persons indicates that the oral leukoplakia of the subject is in a simple hyperplastic stage, a significant increase in the concentration of salivary exosomes relative to normal persons indicates that the oral leukoplakia of the subject is accompanied by abnormal hyperplasia, and a significant decrease in the concentration of salivary exosomes relative to normal persons indicates the possibility that the oral leukoplakia of the subject has been converted into oral cancer.
12. The use of item 11, wherein the oral cancer is oral squamous cell carcinoma.
Definition of
The "precancerous lesion" as used herein refers to a lesion which is not a cancer itself but is more likely to be converted into a cancer. The "oral precancerous lesion" (OPL) refers to an oral lesion with morphological changes and potential canceration, and is clinically often an oral epithelial precancerous lesion, such as clinically common leukoplakia, erythema, lichen planus, discoid lupus erythematosus, submucosal fibrosis, papilloma, chronic ulcer, mucosal black spot, pigmented nevus, and the like.
The oral leukoplakia is mainly white lesion on oral mucosa, can not be wiped off, and can not be diagnosed as other definable lesions by clinical and histopathological methods, belongs to the category of precancerous lesion or Potential Malignant Disease (PMD), and does not include simple hyperkeratosis which can be resolved after local factors such as smoking, local friction and the like are removed. The oral leukoplakia of the present invention is also referred to simply as leukoplakia.
Oral leukoplakia can be divided into simply hyperplastic leukoplakia and leukoplakia associated with (with) abnormal hyperplasia according to histopathological manifestations, the former is called as leukoplakia (simple hyperplasia), simply hyperplastic leukoplakia or simply hyperplastic stage of leukoplakia in the invention, and the pathological manifestations are as follows: epithelial hyperplasia with hyperkeratosis or hyperkeratosis, or both, occurring as a mixed keratosis; simple epithelial hyperplasia is a benign condition, manifested by hyperkeratosis of the epithelium, marked granular layer and thickened spinous layer, with no atypical cells. The epithelial spikes were elongated and thickened but still neat and the base film was clear. The lamina propria and submucosa are infiltrated with lymphocytes and plasma cells. Leukoplakia with abnormal hyperplasia, or abnormal hyperplastic stage known as leukoplakia, the malignant potential increases with increasing degree of abnormal hyperplasia of the epithelium. The histopathological changes of epithelial dysplasia are: epithelial basal cell polarity disappeared; presenting more than one layer of basal-like cells; the proportion of nuclear pulp is increased; the epithelial nail is drop-shaped; disorders of the epithelial layer; mitotic picture increases, with few abnormal mitoses visible; epithelial superficial 1/2 exhibits mitosis; (ii) cellular polymorphism; carrying out nuclear thick dyeing; enlargement of nucleolus; decreased cell adhesion; keratinization of single or clumped cells in the echinocyte layer; according to the number of the above items, the epithelial dysplasia is divided into light, medium and heavy epithelial hyperplasia.
Precancerous lesions of the oral cavity, such as oral leukoplakia, are not cancerous, but may develop into oral cancer if not treated promptly, but also subject to various adverse stimuli. The histopathological changes of oral cancer are: in well-differentiated squamous cell carcinoma, intercellular bridges are visible between cells, and lamellar keratotic substances, either keratotic beads or carcinomatous beads, appear in the center of the cancer nests. The squamous cell carcinoma with poor differentiation has no keratinized bead formation and even no intercellular bridge, and the tumor cells have obvious abnormal shapes and are seen in more nuclear division images.
The exosome is formed by cells through a series of regulation processes of endocytosis, fusion, efflux and the like, can be secreted to the outside of the cells, is a subcellular bilayer membrane vesicle with the molecular diameter of 40-100 nm, and contains substances such as protein, miRNA, mRNA and the like related to cell sources. Exosomes can directly activate receptor cells through plasma membrane receptors, can also transport proteins, mRNA, miRNA and even organelles into the receptor cells, and can also carry special 'information' contained in cells in different pathological states to enter body fluids (including saliva, blood and the like), thereby playing important roles in physiology and pathology.
Methods for isolating and extracting exosomes from bodily fluids are known in the art [9]The exosomes are usually separated in the previous stage by ultracentrifugation, magnetic bead immunocapture, precipitation or filtration. Analyzing the size and shape of the RNA by using an electron microscope, analyzing cell surface markers by using a flow cytometer, analyzing proteins by using methods such as Western blot (Western blot) and ELISA (enzyme-linked immunosorbent assay), or analyzing RNA by using qPCR (quantitative polymerase chain reaction) and Next Generation Sequencing (NGS), wherein the analysis comprises the detection of miRNA, mRNA and other substances[10]。
"significantly increased" or "significantly decreased (decreased)" means that the degree of increase or decrease (decrease) is statistically significant compared to normal humans.
Drawings
FIG. 1 shows typical pathological findings of H & E stained white spots at different stages.
FIG. 2 shows the expression levels of E-cadherin (E-cadherin) and Vimentin (Vimentin) in the pathological tissues of normal population, white spot simple hyperplasia, white spot associated abnormal hyperplasia, and oral cancer patients. The figure shows that the expression of E-cadherin and vimentin in oral cancer tissues is abnormal, statistical analysis shows that the expression level of E-cadherin in normal oral mucosa, oral leukoplakia simple hyperplasia, leukoplakia with abnormal hyperplasia and oral cancer tissues (early infiltration) is obviously reduced, the expression level of vimentin is increased gradually, the staining intensity of the vimentin is increased gradually along with the development of the normal mucosa transferred to cancer, and the difference among groups has statistical significance (P < 0.01).
FIG. 3 shows the expression of PI3K/AKT pathway and EMT-related proteins in normal population, white spot simple hyperplasia, white spot with abnormal hyperplasia, and pathological tissues of oral cancer patients. During the progression from normal mucosal carcinogenesis, the phosphorylation degree of PI3K was significantly increased, and the AKT expression level was gradually increased to 500% of the normal mucosal group. The literature reports that activation of the PI3K/AKT signaling pathway induces the development of EMT. The experimental result proves that the expression level of the E-cadherin is gradually reduced along with the development of the normal mucosa metastasis to the oral cancer, and on the contrary, the expression level of the vimentin is obviously increased (P < 0.001).
Fig. 4 shows the results of in situ hybridization of miRNA185 to oral mucosal tissue. As shown in example 1, the expression level and the expression site of miR-185 in oral mucosal tissues are analyzed experimentally. The result shows that in normal oral mucosa, a large amount of epithelial cell nucleuses and plasma are seen to have strong brown-purple reaction, and miR-185 expression shows strong positive; in cases of simple hyperplasia of oral leukoplakia, abnormal hyperplasia of leukoplakia and oral cancer, miR-185 expression is obviously weakened; in the case of oral cancer, miR-185 expression disappears in cancer epithelial tissue.
FIGS. 5A-C show saliva exosome identification results. It can be seen from the transmission electron microscope that the samples collected and purified from the normal group, the white spot group, the hyperplastic group and the oral cancer group have uniform particle size and consistent shape, and are in the form of round or oval membrane vesicles, and after dyeing, the vesicles have complete envelopes, low electron density substances are contained in the vesicles, and the diameter of the vesicles is about 100 nanometers (fig. 5A). The collected, purified sample was found to be 108nm in size by dynamic light scattering and was found to have a high net negative charge by zeta potential analysis (fig. 5B). These particles were found to express the exosome-specific structural proteins CD81, CD63 or Foltillin-1 by western blot detection (fig. 5C).
FIGS. 6A-B show the results of matrix analysis of small molecule microRNA carried by salivary exosomes. The results in fig. 6A show that exosomes of oral leukoplakia saliva have microrna content significantly different from exosomes from healthy humans. FIG. 6B shows that miR-185 from saliva exosomes of oral cancer patients with simple hyperplasia of oral leukoplakia, abnormal hyperplasia of leukoplakia, and significant reduction compared to normal persons.
Figure 7A shows the results of nanosight analysis showing that high frequency of oral saliva exosomes occurs in the region of particles about 100nm in diameter, indicating that exosomes are about 100nm in size. Figure 7B shows the dynamic change in oral saliva exosome concentration during oral cancer transformation.
FIG. 8 shows the comparison of saliva exosome concentrations in different stages of development of white spots.
Examples
Example 1 alteration of important signaling pathways during precancerous lesions in the oral cavity
Method
Tissue specimens of patients clinically and pathologically diagnosed as oral leukoplakia (simple hyperplasia), leukoplakia with abnormal hyperplasia and leukoplakia canceration (oral squamous cell carcinoma) are selected as study objects. An exemplary H & E staining pathology of the tissue specimens is shown in fig. 1.
Grouping according to pathological diagnosis results: dividing into white spot simple hyperplasia group (N15); leukoplakia with dysplasia (N ═ 10), and cancerization, also known as oral cancer (N ═ 15).
The normal control (N ═ 5) tissue specimens were selected from patients who excluded oral mucosal disease, required removal of a portion of normal tissue for surgical treatment, and were willing to provide this tissue for study.
1. Immunohistochemical detection
Fixing tissue samples of a normal group, a white spot group, an abnormal hyperplasia group and an oral cancer group in a buffered formalin solution, slicing the tissue samples into sections with the thickness of 4 mu m, and performing immunohistochemical detection on the tissue sections according to the following steps for E-cadherin (E-cadherin) and Vimentin (Vimentin): anti-E-cadherin antibody (5. mu.g/ml, Abcam, Cambridge, MA) or anti-vimentin antibody (1:500, Abcam, Cambridge, MA) was added, reacted at 4 ℃ overnight, and then reacted with Alexa at room temperature
568 goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, Calif.) and Alexa
488 goat anti-mouse IgG (1:1000, Invitrogen, Carlsbad, Calif.) was incubated for 1 hour. Next, the cells were mounted in Vectashield mounting solution (Vector Laboratories, Burlingame, CA) containing DAPI and examined by confocal laser microscopy (LSM510, Carl Zeiss co.ltd., Jena, Germany).
2. Protein immunoblotting (Western blot)
A sample lysate of the Western immunoblot (10. mu.g) was isolated from tissue sections and immunoprecipitated on a 10-12% SDS-PAGE gel, followed by immunoblotting using an anti-E-cadherin (1: 500; Abcam, Cambridge, MA) antibody, an anti-vimentin (1: 500; Abcam) antibody, an anti-phosphorylated AKT (1: 5000; Abcam) antibody or an anti-phosphorylated PI3K (1: 500; Abcam) antibody, and dried and exposed to X-ray film (Amersham Biosciences, Piscataway, NJ, USA). The blots were stripped and incubated with anti-beta-actin (1:2,000; Abcam) to confirm that the same amount of sample was loaded in each experiment.
3. In situ hybridization localization miR-185 expression
miR-185 or a control sequence probe (Exiqon Inc.) was hybridized to fixed tissue sections in 1X In Situ Hybridization (ISH) buffer (Exiqon Inc., Woburn, MA USA) for 60 minutes at 55 ℃, followed by washing at 55 ℃ using different concentrations of SSC buffer. The probes were detected as follows: the incubation was performed for 60 minutes using monoclonal anti-digoxin alkaline phosphatase antibody (1:800) (Roche, Indianapolis, IN USA), followed by 2 hours at 30 ℃ using nitroblue tetrazolium and 5-bromo-4-chloro-3' -polyphosphate substrate (Roche). Finally, the sections were counterstained with Nuclear Fast RedTM, using
The medium (VWR, Radnor, PA) was blocked and examined by confocal microscopy.
Results
1. The immunohistochemical detection results show that the staining intensity of E-cadherin in sample tissues is gradually reduced, the staining intensity of vimentin is gradually enhanced and the difference between groups has statistical significance (P <0.05) from a normal group, an exudate simple hyperplasia group, an exudate accompanied abnormal hyperplasia group to an oral cancer group, and refer to fig. 2.
Oral leukoplakia is the most common premalignant lesion of the oral mucosa. Leukoplakia in the mouth undergoes a process of abnormal proliferation of the mucosa during the transition to oral cancer, with the pathology being represented by the transition of epithelial to mesenchymal morphology (EMT). In this process epithelial cells acquire mesenchymal, fibroblast-like characteristics and intercellular adhesion is reduced and motility is enhanced.
Important molecular events of EMT are the down-regulation of E-cadherin and the up-regulation of vimentin. Experiments show that the staining intensity of E-cadherin in sample tissues is gradually reduced and the staining intensity of vimentin is gradually enhanced from the white spot group, the abnormal hyperplasia group to the oral cancer group, and the occurrence of an EMT event is proved from the molecular level in the process of converting the white spot into the oral cancer. The molecular level findings of these test samples are consistent with the clinical and pathological diagnosis of the patients from which such test samples are derived.
2. Protein immunoblotting (Western blot) experiments show that the phosphorylation degree of PI3K is obviously increased from the normal group, the white spot group and the abnormal hyperplasia group to the oral cancer group, the AKT phosphorylation level is gradually increased to 500% of that of the normal mucosa group (P <0.01), the E-cadherin expression level is gradually reduced, and on the contrary, the vimentin expression level is obviously increased (P <0.001), which is shown in figure 3.
The expression of E-cadherin and vimentin is found to be abnormal in oral cancer tissues, statistical analysis shows that the expression level of E-cadherin in normal oral mucosa, oral leukoplakia simple hyperplasia, leukoplakia complicated abnormal hyperplasia and oral cancer tissues (early infiltration) is obviously reduced, on the contrary, the expression level of vimentin is gradually increased, the dyeing intensity of the vimentin is gradually enhanced along with the development of normal mucosa metastasis to cancer, and the difference among groups has statistical significance (P < 0.01).
Activation of the PI3K/AKT axis is a major feature in the EMT signaling pathway. The western blot experiment proves that the phosphorylation degree of PI3K and AKT is obviously increased in the process of progressing from white spots to oral cancer, the PI3K/AKT axis is activated, E-cadherin is down-regulated, vimentin is up-regulated, and EMT is accompanied. The molecular level findings of these test samples are consistent with the clinical and pathological diagnosis of the patients from which such test samples are derived.
3. In-situ hybridization positioning miR-185 expression experiments show that miR-185 expression in normal group samples shows strong positive (purple); in the white spot group samples, the miR-185 expression is obviously weakened, while in the abnormal hyperplasia group and oral cancer group samples, a small part of epithelial cell nuclei and plasma are seen to have slight brown-purple reaction, the miR-185 expression is slightly positive, or the miR-185 expression is almost disappeared, as shown in figure 4.
Recently, a variety of mirnas have been reported that directly target EMT transcription factors and cellular structural components. The experimental results show that the miR-185 level in the samples of the patients in the white spot simple hyperplasia group, the white spot accompanied abnormal hyperplasia group and the oral cancer group is obviously reduced compared with the normal control.
In conclusion, experiments show that in the process of transforming oral leukoplakia from simple hyperplasia of oral leukoplakia to oral leukoplakia with abnormal hyperplasia and oral cancer, a 3K/AKT-mTOR pathway is activated, EMT occurs, and meanwhile miR-185 expression is reduced or even lost.
Example 2 oral saliva exosomes carrying miR-185 consistently reflect oral precancerous lesion process
Method
Exosomes: oral saliva exosomes were collected and purified from oral saliva of patients with oral leukoplakia (simple hyperplasia), leukoplakia with (abnormal hyperplasia), oral cancer (oral squamous cell carcinoma) and normal persons clinically and pathologically diagnosed in example 1 above, as follows.
The patients or normal people do not gargle before taking saliva, and are prohibited to eat water for 1 hour. When the saliva is taken, the head is naturally low, and the saliva in the mouth is naturally spitted into the disposable tray, about 2 ml, without cough. The collected saliva was immediately placed in a small centrifuge tube and stored at 4 ℃.
The samples were centrifuged at 4 ℃ and 410,000 Xg for 20 min to remove microvesicles (microviscles), the supernatant was filtered twice through a 0.22 μm filter, centrifuged at 100,000 Xg using a Ti70 fixed angle ultracentrifuge (Beckman Coulter, Brea, CA, US) to give an exosome pool, washed once with PBS, centrifuged at 100,000 Xg for 1h at 4 ℃ and then immediately used for the experiment or stored at-80 ℃ for future use.
1. Saliva exosome identification
(1) Morphological feature observation of exosomes
And (3) dripping 20 mu l of the exosome suspension on a sample-carrying copper net with the aperture of 2nm, standing for 10 minutes at room temperature, sucking liquid from the side edge of a filter screen by using filter paper, dripping 30 mu l of 3% phosphotungstic acid solution, re-dyeing for 5 minutes at room temperature, sucking the re-dyeing liquid by using the filter paper, drying at room temperature, placing the copper net in a sample chamber of a transmission electron microscope, observing the exosome form and taking an electron microscope picture.
(2) Saliva exosome character identification
The size of exosomes and membrane potential were measured by dynamic light scattering and electrokinetic potential analyzers.
(3) Analysis of exosome-specific structural proteins
Preparing 15% separation gel and 5% concentration gel, mixing and boiling 40 μ l of exosome suspension and 10 μ l of 5XSDS loading buffer solution for 5 minutes, adding into gel loading holes, keeping the pressure of the concentration gel constant at 80V, keeping the pressure of the separation gel constant at 120V, and keeping the flow constant at 200mA for 1.5 hours. Transferring the protein in the gel to a nitrocellulose membrane by a wet transfer method, sealing the nitrocellulose membrane with a sealing solution containing 5% skimmed milk at room temperature for 1h, eluting the nitrocellulose membrane by a 1XTBST buffer solution, adding CD81, CD63 and Flottilin 1 monoclonal antibodies, reacting at 4 ℃ overnight, eluting again, adding horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and gently shaking at room temperature for 1 h. After washing the membrane 3 times with 1XTBST buffer, detection was performed with chemiluminescent substrate (ECL, Thermo Fisher Scientific.).
2. Matrix analysis of small molecule micro RNA carried by saliva exosome
Total RNA was extracted from saliva exosomes using microRNeasy Plus kit (Qiagen, Valencia, CA USA) and reverse transcribed using the miScript II RT kit (Qiagen) according to the manufacturer's instructions. The obtained transcripts were analyzed by microrna matrix according to the manufacturer's instructions and verified by qRT-PCR. qRT-PCR was normalized to U6snRNA primers.
Results
1. It can be seen from the transmission electron microscope that the samples collected and purified from the normal group, the white spot group, the abnormal proliferation group and the oral cancer group have uniform particle size and consistent shape, and are in the shape of round or oval double lipid membrane vesicles, and the dyed vesicles have complete double lipid membrane structures, low electron density substances are contained in the vesicles, and the diameter of the vesicles is about 100 nanometers, as shown in fig. 5A.
The collected, purified sample was found to be 108 nanometers in size by dynamic light scattering and was found to have a high net negative charge by zeta potential analysis, see fig. 5B.
These particles were found to express the exosome-specific structural proteins CD81, CD63 or Foltillin-1 by western blot detection, see fig. 5C.
2. Through the micro RNA matrix, the exosomes from oral mucosa leukoplakia (simple hyperplasia), leukoplakia with abnormal hyperplasia and saliva of oral cancer patients are found to have the micro RNA content obviously different from the exosomes from normal people for the first time, and see fig. 6A. miR185 (nucleotide sequence: 5 'uggagagaaaggcaguuccuga 3') from oral leukoplakia (simple hyperplasia), leukoplakia with abnormal hyperplasia, and salivary exosomes of oral cancer patients is significantly reduced compared with normal people, see FIG. 6B. The finding shows that the exosome carries miR-185 disease information and exists in saliva, so that the miR-185 carried by the saliva exosome can be detected to distinguish normal patients from patients suffering from oral diseases, such as patients suffering from oral leukoplakia (simple hyperplasia), leukoplakia with abnormal hyperplasia and oral cancer.
Example 3 dynamic changes in oral saliva exosomes during oral cancer transformation
Method
The saliva exosomes collected and purified in the above example 2 were quantitatively analyzed by the following method.
Purified saliva exosome samples were evaluated for exosome size and frequency by NanoSight nanoparticle tracking analysis (Malvern Instruments, Westborough, MA).
Nanoparticle Tracking Analysis was performed by using a NanoSight NS300 instrument (NanoSight NTA 2.3 Nanoparticle Tracking and Analysis Release Version Build 0025). The size distribution of the exosome preparations was analyzed and quantified by measuring the rate of brownian motion using a NanoSight LM10 system (NanoSight, Wiltshire, United Kingdom) equipped with fast video capture and particle tracking software. The purified exosomes were diluted in 400 μ l of 1 × PBS/5mM EDTA solution before samples were injected into the NanoSigt sample chamber and the mean + -SD size distribution of exosomes was determined.
Results
It was confirmed that exosomes carrying micro RNAs were released into saliva and the content dynamically changed with the occurrence and development of disease, as shown in fig. 7. Compared with normal people, the saliva of a white spot (simple hyperplasia) patient has no significant difference in carrying exosome content; when the disease progresses to the stage of white spot with abnormal hyperplasia, the content of exosome carried in saliva is obviously increased, and the exosome is obviously different from normal people; once malignant change occurs and the cancer is transformed into oral cancer, the content of exosome carried in saliva is reduced, and the reduction degree is remarkably different from that of normal people, so that the method has statistical significance. The content of carried exosomes in saliva shows a dynamic change process of increasing and then decreasing along with the development of white spots (simple hyperplasia) to abnormal hyperplasia and canceration, and the dynamic change process is shown in figure 7. The content of saliva exosomes changes along with the change from simple hyperplasia of leukoplakia to abnormal hyperplasia and canceration, which can be undoubtedly used for indicating the pathological development and change of diseases and assisting the diagnosis of oral leukoplakia (simple hyperplasia), leukoplakia with abnormal hyperplasia and oral cancer.
In addition, the content of saliva exosome changes with the pathological change of the oral leukoplakia disease, so that the saliva exosome can be used for dynamically monitoring the pathological development process of the disease and early discovering patients with oral canceration.
On the other hand, because the saliva carries a dynamic change process that the content of exosomes is increased firstly (see fig. 8A) and then is decreased (see fig. 8B) along with the simple hyperplasia of the white spot to the abnormal hyperplasia and the canceration, the risk of the oral cancer can be predicted by monitoring and recording the content of exosomes in the saliva of the patient, if the content of exosomes is continuously increased, the risk of the oral cancer is predicted to be gradually increased (see fig. 8A), and if the content of exosomes is changed from increasing to decreasing (see fig. 8B), the possibility that the white spot lesion is cancerated is suggested.
The content of saliva exosomes can also be applied to clinical diagnosis and differential diagnosis by combining the detection of miR-185. As described above, miRNAs are carried by exosomes released into saliva, and it is found through research that miR-185 carried by saliva exosomes in patients with white spots (simple hyperplasia), patients with white spots accompanied with abnormal hyperplasia and oral cancer patients is significantly lower than that in normal persons, and the research has statistical significance. This finding can be used to separate normal persons from patients with leukoplakia, abnormal hyperplasia or oral cancer. For example, in the process of physical examination or oral disease screening, when the mucous membrane of a subject cannot be judged to be normal or suffering from leukoplakia, the miR-185 level in the saliva exosome of the subject can be detected, and if the miR-185 level in the saliva exosome of the subject is remarkably reduced compared with that of a normal person, the subject is prompted to suffer from leukoplakia, so that the auxiliary diagnosis significance is provided for a suspected leukoplakia patient.
If the level of miR-185 in saliva exosomes of a subject is significantly reduced compared with that of normal people, the pathological stage of the white spot needs to be further determined, and the canceration risk assessment is carried out on the pathological stage, the saliva exosome content of the subject can be further determined as described above. If the content of saliva exosomes is not obviously different from that of normal people, the patient is indicated to suffer from leukoplakia (simple hyperplasia); if the saliva exosome content is obviously increased relative to normal people, the leukoplakia is accompanied with abnormal hyperplasia, and is obviously reduced relative to normal people, which indicates that the leukoplakia is cancerated.
Reference documents:
1.Noguti J,De Moura CF,De Jesus GP,et al.Metastasis from oral cancer:an overview.Cancer Genomics Proteomics 2012；9:329-35.
2.Lingen MW,Pinto A,Mendes RA,et al.Genetics/epigenetics of oral premalignancy:current status and future research.Oral Dis 2011；17Suppl 1:7-22.
3.Nogami T,Kuyama K,Yamamoto H.Histopathological and immunohistochemical study of malignant transformation of oral leukoplakia,with special reference to apoptosis-related gene products and proliferative activity.Acta Otolaryngol 2003；123:767-75.
4.Thompson L.World Health Organization classification of tumours:pathology and genetics of head and neck tumours.Ear Nose Throat J 2006；85:74.
5. forest plum clinical screening and risk assessment of oral precancerous lesions [ J/CD ]. chinese journal of oral medical research (electronic), 2008, 2 (5): 427-430.
6. current state of the study of Jiangjingang, Najo, oral precancerous lesion markers [ J ]. journal of clinical military and medical sciences, 2006, 34 (2): 24-227.2006, vol 4, month 34, No. 2: 224-227.
7.Mendez E，Cheng C，Farwell DG，et al.Transcriptional expressionprofiles of oral squamous cell carcinomas[J].Cancer，2002，95(7)：1482-1494.
8. forest plum clinical screening and risk assessment of oral precancerous lesions [ J/CD ]. chinese journal of oral medical research (electronic), 2008, 2 (5): 427-430.
9.Witwer KW,Buzás EI,Bemis LT,et al.Standardization of sample collection,isolation and analysis methods in extracellular vesicle research.[J].J Extracell Vesicles,2013,27:2.
10.Kim KM,Abdelmohsen K,Mustapic M,et al.RNA in extracellular vesicles.[J].Wiley Interdiscip Rev RNA.2017,doi:10.1002/wrna.1413.
Claims (8)
1. use of an agent for detecting salivary exosomes in the preparation of an agent or kit for use in a method of monitoring the conversion of oral leukoplakia to oral cancer, the method comprising monitoring the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significantly increased concentration of salivary exosomes relative to normal is indicative of leukoplakia with abnormal proliferation, requiring close monitoring, but a significantly decreased concentration of salivary exosomes relative to normal is indicative of oral leukoplakia having been converted from abnormal proliferation to oral cancer;
the oral cancer is oral squamous cell carcinoma.
2. Use of an agent for detecting salivary exosomes in the preparation of an agent or kit for use in a method of predicting risk of conversion of oral leukoplakia to oral cancer, the method comprising monitoring the concentration of salivary exosomes in an oral leukoplakia subject, wherein a significantly elevated concentration of salivary exosomes relative to normal persons is indicative of an increased risk of conversion of oral leukoplakia to oral cancer;
the oral cancer is oral squamous cell carcinoma.
3. The use of claim 2 wherein a shift from a significantly elevated concentration of salivary exosomes relative to normal to a significantly reduced concentration relative to normal is indicative of a likelihood that an oral leukoplakia has been converted from abnormal hyperplasia to oral cancer.
4. Use of a reagent for detecting saliva exosomes in preparation of a reagent or a kit for use in a method for assisting in diagnosing pathological stages of oral leukoplakia, wherein the method comprises detecting the concentration of saliva exosomes of an oral leukoplakia subject, wherein the concentration of the saliva exosomes is not significantly different from that of normal persons, indicating that the oral leukoplakia is in a simple proliferation stage, the concentration of the saliva exosomes is significantly increased relative to that of the normal persons, indicating that the oral leukoplakia is accompanied by abnormal proliferation, and the concentration of the saliva exosomes of the subject is significantly decreased compared with that of the normal persons, indicating that the oral leukoplakia of the subject is converted into oral cancer.
5. The use of claim 4, wherein the oral cancer is oral squamous cell carcinoma.
6. Use of an agent for detecting the level of miRNA185 in saliva exosomes in the preparation of a reagent or kit for use in a method of assisted identification of suspected oral leukoplakia, the method comprising determining the level of miRNA185 in saliva exosomes, wherein a significant decrease in the level compared to normal indicates a likelihood of a subject suffering from oral leukoplakia.
7. The use of claim 6, comprising determining the pathological stage of oral leukoplakia in a subject whose miRNA185 level is significantly reduced by further determining the concentration of salivary exosomes in said subject, wherein no significant difference in the concentration of exosomes compared with normal persons indicates that the oral leukoplakia in said subject is in a simple hyperplastic stage, a significant increase in the concentration of salivary exosomes relative to normal persons indicates that the oral leukoplakia in the subject is accompanied by abnormal hyperplasia, and a significant decrease in the concentration of salivary exosomes relative to normal persons indicates the possibility that the oral leukoplakia in the subject has been converted into oral cancer.
8. The use of claim 7, wherein the oral cancer is oral squamous cell carcinoma.
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