WO2018205978A1 - 预防口腔癌的方法 - Google Patents

预防口腔癌的方法 Download PDF

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WO2018205978A1
WO2018205978A1 PCT/CN2018/086347 CN2018086347W WO2018205978A1 WO 2018205978 A1 WO2018205978 A1 WO 2018205978A1 CN 2018086347 W CN2018086347 W CN 2018086347W WO 2018205978 A1 WO2018205978 A1 WO 2018205978A1
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leukoplakia
oral
mir
oral cancer
exosome
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PCT/CN2018/086347
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French (fr)
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周宇
关晓兵
陈莉
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北京捷腾生物科技有限公司
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Priority to CN201880031117.2A priority Critical patent/CN110612127B/zh
Publication of WO2018205978A1 publication Critical patent/WO2018205978A1/zh
Priority to US16/680,374 priority patent/US20200268785A1/en
Priority to US17/371,765 priority patent/US20220016157A1/en

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    • A—HUMAN NECESSITIES
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    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention relates to a method for treating leukoplakia and preventing oral cancer, and in particular to a method for preventing transformation of leukoplakia into oral cancer, comprising administering to a subject an exosome introduced into miR-185.
  • Oral cancer is one of the 10 most common cancers in the world, accounting for 80% of head and neck cancers. There are about 5 million patients with oral cancer worldwide, including oral squamous cell carcinoma (OSCC). More common, its five-year survival rate is about 35-57%, and about 130,000 oral cancer patients die every year [1-2] . Oral cancer mainly occurs in middle-aged and elderly people. Despite advances in diagnostic techniques, surgery, chemotherapy, and radiation therapy, unfortunately, the 5-year survival rate of patients is still hovering around 50%.
  • OSCC oral squamous cell carcinoma
  • Oral precancerous lesions refer to certain clinical and histological changes of the oral and maxillofacial regions, including cancerous tendencies, including leukoplakia, erythema, lichen planus, discoid lupus erythematosus, submucosal fibrosis, papilloma, Chronic ulcers, mucosal dark spots and pigmented nevus, etc., of which oral leukoplakia is recognized as one of the most typical precancerous lesions in oral streak disease, its cancer rate is as high as 10-36%.
  • Oral leukoplakia also known as oral leukoplakia, was first named by the Hungarian dermatologist Er no Sohuimmer in 1887 and refers to white or grayish keratinized abnormal lesions that occur on the oral mucosa.
  • Oral mucosa leukoplakia is common in the middle-aged and elderly people, and it occurs on the mucous membranes such as the lips, cheeks, tongue, and sputum. It usually has no symptoms. It is milky white plaque at the beginning, and the surface is smooth, flat or slightly higher than the normal mucosa. The process of leukoplakia from precancerous lesions to oral cancer can go through several years to ten years.
  • the process of carcinogenesis is also a multi-stage, multi-step process, which must undergo hyperplasia ⁇ squamous metaplasia ⁇ mild, moderate, Severe hyperplasia ⁇ carcinoma in situ ⁇ evolution of invasive carcinoma [3-4] , and most of the oral leukoplakia can be in a benign state for a long time without canceration, and only a small part of the precancerous lesions and precancerous conditions develop into cancer.
  • the incidence of oral cancer has increased and the trend is significantly younger.
  • the surgical, radiotherapy and chemotherapy techniques for oral cancer are progressing, the 5-year survival rate of patients is still less than 50%. Among them, the 5-year survival rate of tumor patients was about 80%, and those who had metastasis dropped to 20% [5] .
  • EMT epithelial-mesenchymal transition
  • angiogenesis apoptosis
  • autophagy are closely related to malignant transformation of oral mucosal leukoplakia [6- 9] .
  • EMT is a phenomenon in which epithelial cells transform into mesenchymal cells under physiological or pathological conditions. During this process, epithelial cells lose cell polarity and cell contact inhibition, and the ability of mesenchymal cells to move is obtained. Tumor cells can acquire the ability of cells to invade and metastasize by activating EMT, including the ability to obtain certain stem cell characteristics and apoptosis escape. EMT is the primary critical step in tumor invasion and metastasis. The mechanism of EMT formation is still unclear and involves multiple signaling pathways. Activation of the PI3K/AKT pathway is the key to activation of EMT [10] .
  • epithelial cells During EMT activation, epithelial cells gradually lose their cell markers such as E-cadherin and ZO-1 [11] , while mesenchymal cells are expressed by vimentin, N-cadherin or fibronectin [12,13] .
  • a series of cytoskeletal reorganization and allosteric, epithelial cells differentiate into fibroblast-like cells and obtain biological properties that facilitate cell migration.
  • EMT is also closely related to the formation of cancer stem cells. Recent studies have found that TGF- ⁇ induces EMT and converts some epithelial cells into mesenchymal stem cells [14] .
  • EMT activation of EMT helps cells to acquire the properties of cancer stem cells, so EMT is closely related to tumor neonatal.
  • breast cancer cells can increase tumorigenicity and tumor cell "stem cell” by high expression of VEGF-A and angiogenesis during EMT [15] .
  • Neonatal cancer stem cells promote malignant metastasis of tumors and make tumor cells lose sensitivity to radiotherapy and chemotherapy.
  • the production of cancer stem cells or the maintenance of stem cell traits is the main cause of treatment failure and tumor recurrence.
  • Autophagy is one of the forms of programmed cell death and has received much attention in recent years. Autophagy is an exogenous stimulus that includes adaptive responses to nutrient deficiencies, cell density loads, hypoxia, oxidative stress, infection, and the like. Autophagy can act as a defense mechanism to remove damaged organelles and metabolites in the cytoplasm, reorganize at the subcellular level, protect damaged cells, and induce cell autonomic death as a cell death program [16] ] . The changes in autophagy activity are related to the occurrence and development of tumors. Autophagy can affect tumor progression from multiple levels, including tumor cell apoptosis, angiogenesis and chemotherapy resistance [17] .
  • EMT can profoundly influence T cell-mediated immune surveillance of cancer cells: during EMT, tumor cells acquire hCD24/CD44+/ALDH-stem cell populations, escaping cytotoxic T cell-mediated autophagy, thereby enabling Tumors acquire chemotherapy resistance.
  • autophagy can regulate the progression of EMT by the expression of adhesion molecules [18] .
  • defects in autophagy can induce the production of EMT and promote the metastasis of gastric cancer cells [19] .
  • activation of autophagy can induce EMT and promote intrahepatic spread of liver cancer cells [20] . It can be seen that the activity of autophagy in cells is completely different in different stages of development of different tumors and even the same tumor.
  • microRNAs are a set of non-coding RNAs consisting of a single strand of 18-25 nucleotides in length, which is complementary to the 3'-non-coding region (3'-UTR) of the target gene ribonucleic acid (mRNA), after transcription.
  • the target gene is modified horizontally to regulate gene expression.
  • MicroRNAs are involved in a variety of biological processes, including growth, differentiation, apoptosis, and proliferation by modulating their target genes [22] .
  • miR-10b and miR-708 were significantly increased in oral leukoplakia with epithelial dysplasia, while the expression of miR-99b, miR-145 and miR-181c was significantly down-regulated [23] .
  • the expression level of microRNA in tissues was correlated with cytopathological features.
  • the expression of miR-21, miR-345 and miR-181b in oral cancer was significantly higher than that in oral leukoplakia and normal mucosa.
  • the expression of miR-21 and miR-181b was increased in oral leukoplakia with increased mitotic figures, high nuclear ratio and deep staining.
  • miR-345 is highly expressed in the nucleus with increased nucleus or increased volume and high nuclear ratio.
  • microRNA is also associated with histopathological progression.
  • the expression levels of miR-21, miR-345 and miR-181b continue to increase with the development of the disease. [23-25] .
  • the clinical treatment of oral leukoplakia mainly uses chemical drugs, traditional Chinese medicine, microwave, freezing and other treatment methods, of which systemic or local drug therapy is applied more, but drug treatment is also only applicable to: 1 large area or multiple lesions; 2 lesions located in the sensitive anatomical site can not be resected; 3 recurrence of lesions after multiple resection; 4 physical condition is not suitable for surgical resection.
  • drug treatment is also only applicable to: 1 large area or multiple lesions; 2 lesions located in the sensitive anatomical site can not be resected; 3 recurrence of lesions after multiple resection; 4 physical condition is not suitable for surgical resection.
  • surgical resection is still the first choice for treatment. Studies have shown that so far, there is no effective clinical method to block the malignant development of oral leukoplakia [33] . Once oral leukoplakia is malignant and converted to oral cancer, its average 5-year survival rate is less than 50% [34-35] , and some treatments may disfigur
  • exosomes carrying miR-185 can effectively treat oral leukoplakia by topical administration, and can prevent the conversion of leukoplakia to abnormal hyperplasia, oral cancer, and prevent the occurrence of oral cancer, and have great clinical development and application value.
  • the present invention finds that by introducing miR-185 into salivary exosomes and then administering it to a subject, it can inhibit inflammatory reaction, inhibit abnormal proliferation of oral mucosal epithelial cells, inhibit mucosal microvascular formation, and block the conversion of oral leukoplakia to oral cancer. .
  • the invention relates to:
  • a method of preventing or preventing the conversion of oral leukoplakia to oral cancer comprising administering to a white spot subject a therapeutically effective amount of an exosome carrying miR-185.
  • the leukoplakia is leukoplakia simple hyperplasia, leukoplakia with abnormal hyperplasia.
  • the oral cancer is oral squamous cell carcinoma.
  • the exosome carrying miR-185 is administered with other drugs or methods that prevent the conversion of oral leukoplakia to oral cancer.
  • the invention also relates to a method of treating oral leukoplaki comprising administering to a white spot subject a therapeutically effective amount of an exosome carrying miR-185.
  • the leukoplakia is leukoplakia simple hyperplasia, leukoplakia with abnormal hyperplasia.
  • the treatment comprises reducing the area of the leukoplakia or eliminating leukoplakia, or reducing leukoplakia with abnormal proliferation, or reversing to simple hyperplasia, or converting leukoplakia to normal.
  • the exosome carrying miR-185 is administered with other drugs or methods of treating oral leukoplakia.
  • the invention relates to the use of an exosome carrying miR-185 for the preparation of a pharmaceutical composition, kit or pharmaceutical preparation for preventing or preventing the conversion of oral leukoplakia to oral cancer in a subject with oral leukoplakia.
  • the leukoplakia is leukoplakia simple hyperplasia, leukoplakia with abnormal hyperplasia.
  • the oral cancer is oral squamous cell carcinoma.
  • the exosome carrying miR-185 is administered with other drugs or methods that prevent or prevent the conversion of oral leukoplakia into oral cancer.
  • the exosome carrying miR-185 described above is administered to the subject by a topical route of administration.
  • the exosome carrying miR-185 is administered to the subject by submucosal injection, topical application or buccal route.
  • the invention also relates to a method of preventing oral cancer comprising administering to a subject a prophylactically effective amount of an exosome carrying miR-185, the exosome preventing the simple mucosa by one or more of the following
  • the invention also relates to the use of an exosome carrying miR-185 for the preparation of a medicament for preventing oral cancer, wherein the exosome prevents leukoplakia from leukoplakia with abnormal proliferation by one or more of the following means And the transformation of oral cancer or prevent leukoplakia with abnormal proliferation to oral cancer: inhibition of inflammation, inhibition of abnormal proliferation of oral mucosal epithelial cells, inhibition of mucosal microvascular formation.
  • the invention relates to an engineered salivary exosome that incorporates a prophylactically or therapeutically effective amount of miR-185.
  • the present invention also relates to a pharmaceutical composition, kit or pharmaceutical preparation comprising the exosomes for preventing or preventing the conversion of oral leukoplakia into oral cancer.
  • the leukoplakia is leukoplakia simple hyperplasia, leukoplakia with abnormal hyperplasia.
  • the oral cancer is oral squamous cell carcinoma.
  • the invention also relates to the use of miR-185 or an exosome carrying miR-185 for the manufacture of a medicament for inhibiting proliferation of oral cancer cells.
  • the present invention also relates to a method for inhibiting proliferation of oral cancer cells, comprising administering to a subject an effective amount of miR-185 or an exosome carrying miR-185 to achieve an inhibitory effect on the growth of oral cancer cells.
  • the miR-185 or exosome carrying miR-185 inhibits the growth and proliferation of oral cancer cells by topical administration to a subject.
  • the miR-185 or exosome carrying miR-185 is used in combination with other oral cancer therapeutic drugs or methods.
  • the present application also relates to pharmaceutical compositions, preparations and kits for inhibiting the growth of oral cancer cells comprising miR-185 or exosomes carrying miR-185.
  • the invention relates to the use of miR-185 or an exosome carrying miR-185 for the manufacture of a medicament for the regulation of oral cancer cell-associated protein VEGF and AKT expression in a subject of oral cancer.
  • the present invention relates to a method of regulating oral cancer cell-associated protein VEGF and AKT expression in an oral cancer subject, comprising administering to the subject an effective amount of miR-185 or an exosome carrying miR-185.
  • the modulating comprises inhibiting expression of the oral cancer cell associated protein VEGF and AKT.
  • the miR-185 or exosome carrying miR-185 achieves the modulation by topical administration to a subject.
  • the miR-185 or exosome carrying miR-185 is used in combination with other oral cancer therapeutic drugs or methods. Based on the findings of the present invention, the present application also relates to pharmaceutical compositions, preparations and kits comprising miR-185 or exosomes carrying miR-185, which modulate the expression of oral cancer associated proteins VEGF and AKT.
  • exosomes carrying miR-185 may be exosomes introduced into miR-185 by genetic engineering methods, or may be derived from human tissue cells, blood or body fluids of naturally occurring high-copy miR-185. Exosomes.
  • the miR-185-carrying exosomes of the invention are artificially engineered exosomes that have been introduced into miR-185 by genetic engineering or that have increased the miR-185 copy number by genetic engineering.
  • the miR-185-containing exosomes of the present invention are exosomes obtained by purification, such as stem cells (eg, mesenchymal stem cells), naturally occurring in cells or body fluids of a body tissue, or Other body fluid sources of exosomes carrying high copy miR-185.
  • stem cells eg, mesenchymal stem cells
  • precancerous lesion refers to a type of lesion which is not a cancer itself but is more susceptible to transformation into cancer.
  • oral precancerous/premalignant lesion refers to oral lesions with morphological changes and potential for canceration. Clinically, it is more common for oral epithelial precancerous lesions, such as clinically common white spots. , erythema, lichen planus, discoid lupus erythematosus, submucosal fibrosis, papilloma, chronic ulcers, mucosal dark spots and pigmented nevus.
  • the "oral leukoplakia” is a white-based lesion occurring on the oral mucosa, cannot be wiped off, and cannot be diagnosed as other definable lesions by clinical and histopathological methods, and belongs to precancerous lesions or potential.
  • the category of Potentially Malignant Disorders (PMD) does not include simple hyperkeratosis that can be resolved after local factors such as smoking and local friction are removed.
  • the oral leukoplakia of the present invention is also simply referred to as white spot.
  • Oral leukoplakia can be divided into leukoplakia and leukoplakia with dysplasia according to histopathological manifestations.
  • the former is called leukoplakia (simple hyperplasia), simple hyperplasia leukoplakia or leukoplakia in the present invention.
  • the terms are used interchangeably.
  • the pathological manifestations are: epithelial hyperplasia, excessive keratosis or excessive incomplete keratinization, or both of them appear as mixed keratinization; epithelial simple hyperplasia is benign lesion, manifested as epithelial hyperkeratosis The granules are markedly thickened with spinous layers and there are no atypical cells.
  • the epithelial spikes can be elongated and thickened, but still neat and the basement membrane is clear. Lymphocytes and plasma cells infiltrate in the lamina intestinal and submucosa.
  • the leukoplakia with abnormal proliferation or dysplasia stage called leukoplakia the malignant potential increases with the increase of epithelial abnormal proliferation.
  • epithelial dysplasia The histopathological changes of epithelial dysplasia were: the epithelial basal cell polarity disappeared; more than one basal-like cell appeared; the proportion of nucleoplasm increased; the epithelial nail process was droplet-shaped; the epithelial level was disordered; the mitotic image increased, a few abnormal mitosis were observed; Superficial 1/2 mitosis; cell pleomorphism; nuclear staining; nucleoli enlargement; decreased cell adhesion; keratinization of single or agglomerated cells in the spinous cell layer; Moderate and severe epithelial abnormal hyperplasia.
  • Oral precancerous lesions such as oral leukoplakia
  • oral leukoplakia are not cancers, but if they are not treated promptly and are subjected to various adverse stimuli, they may develop into oral cancer.
  • the histopathological changes of oral cancer are: in well-differentiated squamous cell carcinoma, intercellular bridges can be seen between cells, and layered keratin can be seen in the center of the cancer nest, which is keratinized beads or cancer beads.
  • the poorly differentiated squamous cell carcinoma has no horny bead formation, and even no intercellular bridge.
  • the tumor cells are obviously atypia and see more mitotic figures.
  • the methods currently available for the treatment of oral leukoplakia include: surgical resection, laser, cryotherapy, photodynamic therapy; drug treatments include: vitamin A, 13-cis retinoic acid, isotretinoin, acitretin, lycopene , Fenwei A acid, retinoic acid, vitamin A acid paste and other exfoliating drugs; traditional Chinese medicine treatment methods are still in the exploration stage: such as Gynostemma pentaphyllum, hyperplasia.
  • the method that can be used is currently a conventional treatment for cancer, including surgery, radiation therapy, or chemotherapy.
  • Exosome is a subcellular bilayer membrane vesicle formed by a series of regulatory processes such as "endocytosis-fusion-exclusion” of cells and secreted to the extracellular domain with a diameter of 30-150 nm. It contains proteins, miRNAs, and mRNAs related to cell origin. Exosomes can directly activate receptor cells through plasma membrane receptors, or transport proteins, mRNA, miRNAs and even organelles into recipient cells, and can also carry special "information" contained in cells under different pathological conditions. It enters body fluids (including saliva, blood, etc.) and plays an important role in both physiology and pathology.
  • “Therapeutically effective amount” for oral leukoplakia means that the amount of exosome carrying miR-185 can reduce or eliminate the area of leukoplakia, or reduce leukoplakia with abnormal proliferation, or reverse to simple hyperplasia, or even transform It is normal.
  • a “prophylactically effective amount” for oral cancer means that the administration dose of the exosome carrying miR-185 can achieve any one or more of the following: reducing the number of leukoplakia epithelial cells, reducing or eliminating the white spot area, The leukoplakia inflammatory response is weakened, the mucosal microvascular formation is weakened, the simple leukoplakia to leukoplakia with abnormal proliferation, and even the progression of oral cancer, and the prevention of leukoplakia to oral cancer.
  • FIG. 1A-B show the expression of VEGF and AKT in OSCC cancer cells, and the experimental results show that miR-185 regulates VEGF and AKT transcriptional expression in OSCC cells.
  • Figure 2 shows that miR-185 inhibits cancer cell proliferation.
  • Figure 3 shows the binding site of miR-185 in AKT 3 'UTR, demonstrating that miR-185 has direct regulation of the AKT transcript sequence.
  • Figure 4 shows that miR-185 acts directly on the 3'-UTR region of AKT, regulating the survival of cancer cells.
  • FIG. 5 shows that miR-185 is expressed in exosomes secreted by OSCC cells.
  • Figures 6A-B show the size and concentration of exosomes isolated from OSCC cell lines. Under transmission electron microscopy, the exosomes collected and purified in OSCC cells were uniform in size and uniform in shape, and they were in the form of round or elliptical vesicles. After staining, the vesicles had a complete envelope, which was low. The electron dense material has a diameter of about 100 nm (Fig. 6A). Size analysis of exosomes by NTA technology indicated the presence of exosomes with a diameter of 120 nm (Fig. 6B); insertion: CD81, CD63 and Flotillin are exosome signature proteins.
  • Figure 7 shows PH26 fluorescein-labeled explants carrying miR-185 into OSCC cells.
  • Figure 8A-B shows that exosomes carrying miR-185 alter the expression of VEGF and AKT in OSCC.
  • the results of the experiment showed that the highly expressed miR-185 in the OSCC cell line significantly inhibited the transcriptional expression of VEGF and AKT.
  • Figure 9 shows the results of in situ hybridization of oral mucosal tissue miR-185.
  • the expression level and distribution of miR-185 in oral mucosa were analyzed. It was found that in normal oral mucosa, strong brown-purple reaction appeared in a large number of epithelial nucleus and patina, and miR-185 expression was strongly positive; in oral mucosal leukoplakia, leukoplakia with abnormal hyperplasia and oral cancer, miR- The expression of 185 was significantly attenuated; in oral cancer cases, the expression of miR-185 disappeared in cancerous epithelial tissues.
  • 10A-B show the results of identification of salivary exosomes and blood exosomes, wherein 10A shows the results of identification of salivary exosomes; 10B shows the results of identification of blood exosomes.
  • the size analysis of exosomes by NTA technology indicated the presence of exosomes with a diameter of 110-120 nm. These granules were found to express the exosomal specific structural protein CD81, CD63 or Flotillin by Western blot analysis (Fig. 10A, B).
  • Figures 11A-C show the results of matrix analysis of salivary exosomes carrying small molecule microRNAs.
  • Fig. 11A is a result of matrix analysis of small-sized microRNAs carried by tissue cells of plaque-like hyperplasia relative to salivary exosomes of normal mucosal tissue cells. The results showed that the exosome of oral mucosal saliva had a microRNA content that was significantly different from exosomes from healthy people. Among them, miR-185 from oral mucosal leukoplakia simple hyperplasia salivary exosomes was significantly reduced compared with normal people.
  • Figure 11B-C shows that the concentration of salivary exosomes is significantly elevated in the stage of leukoplakia dysplasia, and the concentration of salivary exosomes is significantly reduced after progression to oral cancer. In contrast, the concentration of blood exosomes is markedly elevated in the stage of oral cancer.
  • Figure 12 is a schematic diagram based on the results of cell level assays showing the effect of exosomes on the intercellular communication of miR-185 and the regulation of transcriptional repression of VEGF and AKT in the oral precancerous lesion signaling pathway.
  • Figure 13A-B shows an experimental experimental route and method map of an animal carrying a miR-185 exosome to delay the progression of precancerous lesions.
  • Figure 14A-H shows changes in lesions and pathological changes of DMBA for 6 weeks after local application of golden hamster cheek pouch mucosa.
  • the cheek pouch was converted from a normal mucosa (Fig. 14A) to an inflammatory state (Fig. 14B-C) and progressed to precancerous lesions (Fig. 14D-E).
  • Pathological changes were converted from normal mucosa (Fig. 14F) to simple hyperplasia (Fig. 14G) and abnormal hyperplasia (Fig. 14H).
  • Figure 15 shows changes in body weight of hamsters. Three groups of hamsters were compared with the negative control group, *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 16 shows the levels of blood biochemical indicators associated with liver and kidney function in hamsters.
  • Figure 17A-B shows the expression and counting of hamster cheek pouch mucositis cells.
  • the expression (A) and count level (B) of three groups of hamster cheek pouchitis cells at different stages were compared with DMBA group, *p ⁇ 0.05, *** p ⁇ 0.001.
  • Figure 18A-B shows the results of simple hyperplasia and abnormal hyperplasia of the buccal mucosa of the hamster.
  • the counts of simple and abnormal hyperplasia of the cheek pouch of the three groups of hamsters were *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figures 19A-D show immunohistochemical staining of hamster cheek pouch mucosa.
  • MMD microvessel density
  • AOD epithelial mean optical density
  • 19D COX2 staining count positive cells
  • Figure 20A-C shows the expression levels of inflammatory factors IL-1 ⁇ , IL-6 and IL-10 in guinea pig serum.
  • Figure 21 shows the expression of inflammatory factor protein in the cheek pouch tissue.
  • OSCC cell line Take frozen OSCC cells (acquired by ATCC) CRL-1623 TM , Manassas, VA, USA), rapidly melted in a constant temperature water bath at 37 ° C; inject into a centrifuge tube and centrifuge at 1000 rpm / min for 5 minutes; discard the supernatant and add the culture solution, place The cells were cultured at 37 ° C, 5% CO 2 , and saturated humidity; after 24 hours, the cells were observed under an inverted microscope, and the culture solution was changed.
  • OSCC cell line Take frozen OSCC cells (acquired by ATCC) CRL-1623 TM , Manassas, VA, USA), rapidly melted in a constant temperature water bath at 37 ° C; inject into a centrifuge tube and centrifuge at 1000 rpm / min for 5 minutes; discard the supernatant and add the culture solution, place The cells were cultured at 37 ° C, 5% CO 2 , and saturated humidity; after 24 hours, the cells were observed under
  • Cell culture and passage cells are passaged when they are over 80% to 90%; cells are digested with 0.25% trypsin, pipetted, transferred to a test tube, centrifuged, and the supernatant is discarded; the culture solution is added, 1:2 or 1 :3 After passage, the cells were cultured under the conditions of 37 ° C, 5% CO 2 and saturated humidity (in order to ensure the stability of the cells, experiments were carried out using cells within 10 generations).
  • miR-185 mimetic purchased by Qiagen, miScript miRNA Mimic-219600
  • inhibitor purchased by Qiagen, miScript miRNA Inhibitor-219300, and negative control (random sequence or by liposome-encapsulated transfection reagent) random inhibitor, Qiagen purchased miScript inhibitor Neg.Control-102727
  • OSCC cell lines LiNza Nucleofector TM system
  • the cells were collected after 48 hours for extraction of total mRNA (Qiagen purchase, RNeasy Mini Kit-74104 Then, the expression level of VEGF or AKT was analyzed by qRT-PCR technique.
  • Detection of cancer cell proliferation index by MTT assay Collect (4) transfected cells, inoculate in 96-well plates, and culture for 48 hours, using Abcam MTT assay kit (Burlingame, CA, USA) to MTT method. The cancer cell proliferation index was measured.
  • AKT luciferase reporter plasmid The potential miR-185 binding site on the AKT gene was screened by the miRBase data analysis system (microRNA.org). The full-length 3'-non-coding translational region of AKT (3'-UTR) was amplified from genomic DNA and cloned into the plasmid vector Fire-Ctx sensor lentivector (miR-selection Fire-Ctx lentivector, purchased by SBI, Palo Alto, CA, In the USA, it carries a firefly luciferase reporter gene and a cytotoxin (CTX) drug-sensitive gene downstream. In the experiment, the Fire-Ctx sensor lentivector vector was used as an experimental control.
  • Fire-Ctx sensor lentivector vector was used as an experimental control.
  • (7) was transfected plasmid and cytotoxicity assays: The constructed plasmids using electroporation transfected into OSCC cell lines (Lonza Nucleofector TM system, Walkersville, MD, USA), while cells cotransfected front miR-185 body (pre-miR-185, purchased from Exiqon, Woburn, MA, USA). To control transfection efficiency, cells were also transfected with the pRL-CMV vector plasmid (purchased by Promega Corporation - E2261, San Luis Obispo, CA, USA), including the luciferase reporter gene. In response to the experiments, cells were treated with cytotoxin (CTX) for 3-4 days after 24 hours of transfection as described above, after which the degree of cell viability was measured.
  • CTX cytotoxin
  • miR-185 was screened for a direct regulatory site on the AKT transcript (see Figure 3).
  • Isolation and purification of exosomes in cell culture medium cultured OSCC cells (as described above), after 48 hours of serum starvation, the cell culture medium was collected and centrifuged at 2000 ⁇ g for 20 minutes, 10000 ⁇ at 4°C. g was centrifuged for 30 minutes to remove cell debris, and exosomes were obtained by step purification using an exosome isolation kit (Exosome isolation Kit, Cat. NO: GET301-10, Genexosome Technologies Inc., Freehold, New Jersey, USA). Resuspend in a volume of sterile PBS buffer and dilute.
  • CD63 (1:250) and Flotillin (1:1000) monoclonal antibody purchased from Abcam
  • Flotillin (1:1000) monoclonal antibody purchased from Abcam
  • horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added (1) : 2500, Sigma St. Louis, MO, USA), shaking gently for 1 h at room temperature.
  • detection was carried out using a chemiluminescent substrate (ECL, purchased from Thermo Fisher Scientific, Carlsbad, CA, USA).
  • exosome carrying high-copy miR-185 in cell-to-cell transmission and transcriptional repressor regulation of signaling signals in oral precancerous lesion signaling pathway: culture of OSCC in medium carrying exosome with high copy of miR-185 Target cells, by detecting the expression levels of VEGF and AKT in the target cells, determine whether exosomes carry miR-185 into the recipient target cells, whether it is effective in reversing the cancer.
  • the exosomes collected and purified in OSCC cells have uniform size and uniform morphology, and they are in the form of round or elliptical double lipid vesicles. After staining, the vesicles are intact.
  • FIG. 12 is a schematic diagram based on the results of cell level assays showing the effect of exosomes on the intercellular communication of miR-185 and the regulation of transcriptional repression of VEGF and AKT in the oral precancerous lesion signaling pathway.
  • the miR-185 or control sequence probe (Exiqon Inc.) was hybridized with fixed tissue sections at 55 ° C for 60 minutes in 1X in situ hybridization (ISH) buffer (purchased from Exiqon Inc., Woburn, MA USA). Wash at 55 °C using different concentrations of SSC buffer. As described below, the probe was incubated with monoclonal anti-digoxigenin alkaline phosphatase antibody (1:800) (Roche, Indianapolis, IN USA) for 60 minutes, followed by nitroblue tetrazolium at 30 °C and 5-Bromo-4-chloro-3'-polyphosphate substrate (Roche, Pleasanton, CA, USA) was incubated for 2 hours. Finally, using Nuclear Fast Red TM double staining, using The medium (VWR, Radnor, PA, USA) was mounted and examined by confocal microscopy.
  • ISH in situ hybridization
  • miR-185 expression was strongly positive (purple) in the normal group; miR-185 expression was significantly attenuated in the leukoplakia samples, but was seen in the abnormal hyperplasia group and the oral cancer group samples.
  • the experiment found that from the simple proliferation of oral leukoplakia to oral mucosal leukoplakia with abnormal proliferation, oral cancer transformation, PI3K / AKT-mTOR pathway activation, EMT occurred, while miR-185 expression decreased or even absent.
  • Example 4 Oral salivary exosomes or blood exosomes carry miR-185 associated with disease states
  • Exosomes collected from the clinical and pathological diagnosis of oral mucosal leukoplakia, plaque hyperplasia, oral cancer (oral squamous cell carcinoma), normal oral saliva, and blood, as described in Example 3 above, Purify oral salivary exosomes or blood exosomes.
  • the above patient or normal person does not gargle before taking saliva, and fasts water for 1 hour.
  • the head is naturally lowered, and the saliva in the mouth is naturally spit out into a disposable tray, about 2 ml, do not cough.
  • the collected saliva was immediately placed in a small centrifuge tube.
  • the sample was centrifuged at 10,000 x g for 20 minutes to remove impurities, and the sample supernatant was filtered twice through a 0.22 ⁇ m filter using an exosome isolation kit (Exosome isolation Kit, Cat. NO: GET200-10, Genexosome Technologies). Inc., Freehold, New Jersey, USA) Purification of the exosomes by step, resuspended in a volume of sterile PBS buffer, and diluted.
  • Exosome isolation Kit Cat. NO: GET200-10, Genexosome Technologies
  • the size and concentration of exosomes were detected by NTA technology.
  • CD63 (1:250) and Flotillin (1;1000) (Abeam) monoclonal antibody were reacted at 4 ° C overnight, and after elution again, horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added at room temperature. Shake gently for 1 hour. After washing the membrane 3 times with 1X TBST buffer, it was detected with a chemiluminescent substrate (ECL, Thermo Fisher Scientific).
  • Salivary exosomes carry small molecular microRNA matrix analysis
  • Salivary-derived exosomes or blood-derived exosomes were detected by NTA technology to be between 110-120 nm (Fig. 10A, B). These granules were found to express the exosomal specific structural protein CD81, CD63 or Flotillin by Western blot analysis, see Figure 10A, B.
  • Example 5 Exosomes carrying miR-185 block the progression of precancerous lesions
  • the reagents used in this experiment and the following experiments were configured and stored as follows: 0.5 g of dimethylbenzindole (DMBA) was dissolved in 50 ml of acetone and 50 ml of liquid paraffin to prepare a 0.5% DMBA solution, which was stored at room temperature in the dark.
  • the exosomes carrying miR-185 are exosomes derived from mesenchymal stem cells containing high copy miR-185, available from GenExsome Technology under the trade name GET MSCEXO 101-1ug.
  • the exosome solution of the exosome solution carrying miR-185 was 2 ⁇ 10 11 particles/ml, stored at -80 ° C, and transferred to 4 ° C for 24 h before use.
  • the other two groups were coated with 0.5% dimethylbenzopyrene (DMBA) solution in the left cheek pouch from the first week, 3 times a week, until the end of the experiment, 25 Only the positive control group was no longer treated; the 20 treatment groups applied the exosome solution 3 times a week to the same portion of the DMBA application from the 3rd week until the end of the 6th week. From the end of the third weekend, 6 hamsters in the DMBA group, 5 hamsters in the DMBA+EXO group, and the remaining hamsters were sacrificed at the end of the 6th week. During the experiment, the health and disease status of the hamster were observed and recorded, and the body weight was recorded weekly. The experiment was carried out in accordance with the technical route of Fig. 13A and the experimental method of Fig. 13B.
  • DMBA dimethylbenzopyrene
  • Liver and kidney function The kit was tested by commercial kit.
  • the kits were purchased from Yingke Xinchuang (Xiamen) Technology Co., Ltd., and the alanine aminotransferase (ALT) was UV-lactate dehydrogenase method.
  • Aspartate aminotransferase (AST) uses UV-malate dehydrogenase method, creatinine (Scr) enzymatic method, urea nitrogen (BUN) using UV-glutamate dehydrogenase method, the experiment strictly follows the kit instructions operating.
  • Embedding slice The squirrel cheek pouch tissue was fixed in 10% formalin solution for 24 hours, then taken out, cut into strips of about 3-5 mm, rolled into a tube shape, fixed with a steel needle, and automatically dehydrated. The machine was dehydrated, and the steel needle was placed and embedded in paraffin. Each sample was continuously cut into 21 5 ⁇ m sections, and the first, ten, and 20 sheets were taken for HE staining, and the second, eleven, and 21 sheets were subjected to immunohistochemical staining.
  • HE staining slides were baked in a oven at 65 °C for 1 h, routinely dewaxed to water, rinsed with tap water for 2 min, rinsed with hematoxylin for 4 min, rinsed with tap water, differentiated for 2 s, returned to blue for 4 s, soaked in tap water for 5 min.
  • Into the red dye solution for 40s rinsed with tap water for 30s, dehydrated into xylene, sealed with neutral resin. 400 times under the microscope to observe the lamina limbalin for 4 min, rinsed with tap water, differentiated for 2 s, returned to blue for 4 s, soaked in tap water for 5 min.
  • red dye solution for 40s rinsed with tap water for 30s, dehydrated into xylene, sealed with neutral resin. 400 times under the microscope to observe the lamina limbalin cells (according to the morphology can be determined as lymphocytes and neutrophils) infiltration, each slide selected 3-10 inflammatory cells more field of view, 200 times under the microscope Count.
  • Simple hyperplasia showed increased cell number, epithelial granule layer and acanthosis, no atypical cells; abnormal proliferation according to WHO diagnostic criteria, including epithelial basal cell polarity disappeared, more than one basal-like cell appeared, the proportion of nucleoplasm increased
  • the epithelial nails are droplet-shaped, the epithelial level is disordered, and the nucleus fission is increased. A few abnormal mitotic phases are observed.
  • the epithelial superficial 1/2 has mitosis, the pleomorphism of the cells, the nuclear staining, the nucleoli enlargement, and the cell adhesion. Decreased, keratinized in a single or agglomerated cell in the spinous cell layer. Strictly follow the standard to record the total number of simple and abnormal proliferation of the sample.
  • the concentrations were: anti-CD31 Antibody 1:200, anti-PCNA antibody 1:30000, anti-COX2 antibody 1:1000, these antibodies were purchased from Abcam, and the blank was replaced with PBS instead of primary antibody, overnight at 4 °C. After the next day, the temperature was rewarmed for 1 h at room temperature, washed with PBS buffer, and the secondary antibody was added dropwise, and incubated at 37 ° C for 0.5 h. After washing the slide, DAB (diaminobenzidine) was added dropwise, and the observation under the microscope showed that the condition was long and the washing was terminated. Color development, hematoxylin counterstaining, anti-blue, dehydrated, transparent, neutral resin sealing.
  • PCNA protein was located in the nucleus.
  • the epithelium was harvested at 3-5 epithelial hyperplasia (plaque hyperplasia, dysplasia) per 100-fold microscope. Image density was analyzed by Image pro plus software, and the average optical density (IOD) was recorded.
  • the expression of COX2 protein is located in the nuclear membrane. Two to five inflammatory cells in each cell are selected under the microscope of 100 times. The positive expression of the nuclear membrane is brown or brown, and the positive cell count is performed.
  • CD31 protein is expressed in the endothelial cell membrane, and the Weidner method is used.
  • the microvessels (less than 8 red blood cells in diameter) were selected in a 100-fold microscope under each microscope. The number of CD31-labeled microvessels was counted under a 400-fold microscope. The mean value was the MVD value (microvessel density), and the above data were recorded and compared between groups.
  • Enzyme-linked immunosorbent assay (9) Enzyme-linked immunosorbent assay (ELISA): These cytokines were detected using a commercial hamster IL-6, IL-1 ⁇ , IL-10 ELISA kit under the brand name MyBioSource (San Diego, CA, USA). Remove the required strips from the aluminum foil bag after 20 minutes of equilibration at room temperature, set blank control wells, standard wells and sample wells, add 50 ⁇ l of sample dilution to the blank wells, add 50 ⁇ l of standard concentration to standard wells, and add 50 ⁇ l to the sample wells.
  • HRP horseradish peroxidase
  • the protein was adjusted to a volume of 1.5 ml by dilution, 15 ⁇ l of the dissolved antibody was added to each sample, mixed, and incubated for 1 h at room temperature.
  • the blocking solution in the four-well plate was removed, and the sample antibody mixture was added and incubated overnight at 4 ° C on a shaker.
  • the next day the membrane was removed and washed on a shaker for 10 min, repeated three times, and the four-well plate was washed. 2 ml of diluted streptavidin-HRP was added to each well, and four membranes were placed in a four-well plate and incubated at room temperature for 30 min. Wash the membrane, place the membrane number up in the cassette, evenly drop 1 ml of the developer, incubate for 1 min, develop, and expose. Observation scans and gray value analysis were performed on different protein sites of each membrane for comparison between groups.
  • the fifth week to the sixth week was the precancerous lesion.
  • the mucosa was rough, whitish, slightly thickened, and some white spots were visible. Block lesions (see Figure 14E).
  • the histopathological changes observed under light microscopy were gradually changed from normal mucosa (see Figure 14F) to simple hyperplasia (see Figure 14G) and abnormal hyperplasia (see Figure 14H).
  • CD31, PCNA, COX2 expression analysis CD31, PCNA, COX2 expression is shown in Figure 19A.
  • the results of calculating the microvessel density (MVD) values of CD31-labeled vascular endothelial cells showed that the DMBA+EXO group was lower than the DMBA group at each stage, and the difference was statistically significant at the fifth and sixth weeks (see Fig. 19B), confirming that the topical application carried miR-185. Exosomes have a good effect on inhibiting mucosal microvascular formation.
  • the calculated epithelial AOD values after PCNA staining showed that the DMBA+EXO group was significantly lower than the DMBA group at each stage (see Figure 19C), confirming that topical application of exosome carrying miR-185 has a good effect on inhibiting epithelial proliferation, and also with abnormal proliferation count.
  • the results are consistent.
  • the COX2 staining positive cells showed that the DMBA+EXO group was basically the same as the DMBA group in the three weekends, and the treatment group was lower than the DMBA group in the fourth, fifth and sixth weekends. The difference was not significant (see Fig. 19D), and it was confirmed that the local application carried miR-185.
  • the body has a certain inhibitory effect on mucosal inflammation.
  • Serum IL-1 ⁇ , IL-6, IL-10 enzyme-linked immunosorbent assay the expression levels of pro-inflammatory factors IL-1 ⁇ and IL-6 were highly consistent at each stage and in each group, and the acute inflammatory phase was observed at the end of the third week.
  • the DMBA+EXO group was significantly lower than the DMBA group at the 6th week of precancerous lesions, and the expression levels were slightly fluctuated at 4 and 5 weeks, no statistical difference (see Figure 20A, 20B).
  • the anti-inflammatory factor IL-10 was acutely inflammatory at three weeks.
  • the DMBA+EXO group was significantly higher than the DMBA group.
  • the level of the fourth week was slightly fluctuating.
  • the DMBA+EXO group was still higher than the DMBA group in the 5th and 6th week. There was no statistical difference (see Figure 20C).

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Abstract

携带miR-185的外泌体预防和治疗口腔癌的方法，以及包含经过改造的唾液外泌体的、用于预防和治疗口腔癌的药物组合物。

Description

预防口腔癌的方法 技术领域
本发明涉及一种治疗白斑和预防口腔癌的方法，具体涉及预防白斑向口腔癌转化的方法，包括向受试者给药导入miR-185的外泌体。
背景技术
口腔癌是世界上最常见的10种癌症之一，占头颈部恶性肿瘤的80％，全球范围内约有5百万口腔癌患者，其中以口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)多见，其五年生存率约为35-57％，每年约有13万口腔癌患者死亡 [1-2]。口腔癌主要发生于中老年人。尽管近年来在诊断技术、外科手术以及化疗、放疗方面取得了进步，但不幸的是患者的5年存活率仍然徘徊在50％左右。
口腔癌前病变是指口腔颌面部的某些临床即组织学有改变，并具有癌变倾向的病变，包括白斑、红斑、扁平苔藓、盘状红斑狼疮、黏膜下纤维性变、乳头状瘤、慢性溃疡、黏膜黑斑及色素痣等，其中口腔白斑被公认为口腔斑纹疾病中最典型的癌前病变之一，其癌变率高达10-36％。
口腔白斑(Oral leukoplakia，OLK)也称为口腔黏膜白斑，是由匈牙利皮肤科医生Er no Sohuimmer于1887年首先命名，是指发生于口腔黏膜上的白色或灰白色角化异常性病变。口腔黏膜白斑常见于中老年人群,好发于唇、颊、舌、腭等黏膜上,一般无自觉症状,初起时呈乳白色斑块,表面光滑、平或稍高出正常黏膜。白斑从癌前病变到发展为口腔癌可以经历几年到十几年,其癌变的过程也是一个多阶段、多步骤的过程,都要经历增生→鳞状上皮化生→轻度、中度、重度异常增生→原位癌→浸润癌的演变 [3-4],而且大部分口腔白斑可长期处于良性状态不发生癌变，只有少部分经历癌前病变、癌前状态发展成癌。近年来，口腔癌发病率有增高及明显年轻化的趋势。尽管对口腔癌的外科手术、放疗及化疗技术在不断进步，但患者的5年存活率仍然不到50％。其中肿瘤局限者5年生存率大约为80％，而发生转移者则 下降到20％ [5]。
白斑向癌症转化的分子生物学机制尚不十分清楚。研究表明,上皮间质转化(epithelial-mesenchymal transition,EMT)，血管新生(angiogenesis),细胞增殖凋亡(apoptosis)和细胞自噬功能(autophagy)的异常与口腔黏膜白斑的恶变密切相关 [6-9]。
EMT是上皮细胞在生理或病理状态下向间充质细胞转化的现象。在此过程中，上皮细胞失去细胞极性和细胞接触性抑制，获得间充质细胞的移动能力。肿瘤细胞可通过激活EMT获得细胞侵袭转移的能力,包括获得一定的干细胞特性和凋亡逃逸的能力。EMT是肿瘤侵袭和转移首要的关键步骤。EMT形成机理目前还不清楚,涉及多个信号通路，其中，PI3K/AKT通路的活化是激活EMT的关键 [10]。EMT激活过程中，上皮细胞逐渐失去其细胞标记物如E-cadherin和ZO-1等 [11],而表达间充质细胞的标记物vimentin，N-cadherin或fibronectin等 [12,13]，通过一系列的细胞骨架重组和变构，上皮细胞分化成类成纤维细胞，并获得有利于细胞移动的生物学特性。除了获得细胞转移侵袭能力外，EMT还与肿瘤干细胞的形成有着密切的关系。近期研究发现,TGF-β诱导EMT，可使部分上皮细胞转化成间充质干细胞 [14]。可见EMT的激活，帮助细胞获得肿瘤干细胞的属性，因此EMT与肿瘤的新生密切相关。研究还证实乳腺癌细胞可在EMT过程中通过VEGF-A的高表达和血管新生来增加致瘤性和肿瘤细胞"干细胞化" [15]。新生的肿瘤干细胞促进肿瘤的恶性转移并使得肿瘤细胞失去对放﹑化疗的敏感性。由此可见，肿瘤干细胞的产生或者干细胞性状的维持是导致治疗失败和肿瘤复发的主要原因。
细胞自噬(autography)是程序性细胞死亡形式之一,近年来备受关注。自噬是对外源性刺激,包括营养缺乏﹑细胞密度负荷﹑低氧﹑氧化应激﹑感染等的适应性反应。自噬既可以作为一种防御机制清除胞质内受损的细胞器和代谢产物,进行亚细胞水平上的重组,保护受损的细胞；又可作为一种细胞死亡程序诱导细胞自主性死亡 [16]。自噬活性的变化与肿瘤的发生和发展有关,自噬可从多个层面影响肿瘤进程,包括肿瘤细胞凋亡﹑血管生成及化疗抗性等方面 [17]。研究显示,EMT可以深度影响T细胞介导的对癌细胞的免疫监控：在EMT过程中,肿瘤细胞获得hCD24/CD44+/ALDH-干细胞群，逃脱细胞毒T细胞介导的细胞自噬,从而使肿瘤获得化疗抗性。反之,细胞自噬能通过黏附分子的表达来调节EMT的进程 [18]。研究发现,细胞自噬能力缺 陷可以诱导EMT的产生,而促进胃癌细胞的转移 [19]。然而,有报道显示细胞自噬的激活可以诱导EMT并促进肝癌细胞的肝内扩散 [20]。由此可见，细胞自噬的活性在不同的肿瘤甚至同一肿瘤的不同发展阶段是完全不同的。
口腔黏膜白斑的EMT和恶变过程与包括microRNA在内的分子的精确调节相关 [21]。microRNA是一组长度在18-25个核苷酸单链组成的非编码RNA,通过与靶基因核糖核酸(mRNA)的3’-非编码区(3’-UTR)互补配对结合,在转录后水平修饰靶基因,从而调控基因表达。microRNA通过调节其靶基因参与各种生物过程，包括生长、分化、凋亡和增殖 [22]。研究发现miR-10b、miR-708在伴有上皮异常增生的口腔黏膜白斑组织中表达显著升高,而miR-99b、miR-145、miR-181c的表达显著下调 [23]。组织中microRNA的表达水平与细胞病理学特征相关，miR-21、miR-345和miR-181b在口腔癌中的表达显著高于口腔黏膜白斑和正常黏膜组织。而在有丝分裂象增多、核质比高、染色深的口腔黏膜白斑细胞中，miR-21、miR-181b的表达增高。在细胞核增多或体积增大，核质比高的口腔黏膜白斑中miR-345高表达。microRNA的表达还与组织病理学进展相关，在对进行性和非进行性发展口腔黏膜白斑的研究中发现，miR-21、miR-345和miR-181b的表达水平随着疾病的发展持续升高 [23-25]。
综合来看，口腔癌的发生发展过程中出现明显异常表达的microRNA，且表达趋势不同、作用各不相同 [26-32]。
目前对口腔白斑的临床治疗主要采用化学药物、中药、微波、冷冻等治疗方式，其中全身或局部的药物治疗应用较多，但药物治疗也仅适用于：①大面积或多发性的病损；②病损位于敏感解剖部位不能切除者；③多次切除后复发的病损；④身体状况不适于手术切除者。对于有较高癌变风险的，如果病损局限且手术操作可行，手术切除依然是治疗的第一选择。研究表明到目前为止，尚无有效的临床方法以阻断口腔白斑的恶性发展 [33]。而一旦口腔白斑发生恶变，转化为口腔癌，其平均5年生存率不足50％ [34-35]，而某些治疗可能会毁容或造成残障。
因此，人们渴望找到治疗白斑和阻止白斑向口腔癌转化的有效方法，从而从根本上预防口腔癌的发生。本发明发现携带miR-185的外泌体可以通过局部用药方式有效治疗口腔白斑，并且能够阻止白斑向异常增生、口腔癌的转化，预防口腔癌的发生，有极大的临床开发和应用价值。
发明内容
发明概述
本发明发现通过将miR-185导入唾液外泌体，然后将其施用于受试者，可以抑制炎症反应、抑制口腔黏膜上皮细胞异常增生、抑制黏膜微血管形成、阻断口腔白斑向口腔癌的转化。
因此，一方面，本发明涉及：
一种预防或阻止口腔白斑向口腔癌转化的方法，包括给药白斑受试者治疗有效量的携带miR-185的外泌体。在一个优选实施方案中，所述白斑为白斑单纯增生、白斑伴异常增生。在一个优选实施方案中，所述口腔癌为口腔鳞状细胞癌。在一个优选实施方案中，所述携带miR-185的外泌体与阻止口腔白斑向口腔癌转化的其它药物或方法一起施用。
一方面，本发明还涉及一种治疗口腔白斑的方法，包括给药白斑受试者治疗有效量的携带miR-185的外泌体。在一个优选实施方案中，所述白斑为白斑单纯增生、白斑伴异常增生。在一个优选实施方案中，其中所述治疗包括减小白斑的面积或消除白斑，或使白斑伴异常增生减轻，或向单纯增生逆转，或使白斑转化为正常。在一个优选实施方案中，所述携带miR-185的外泌体与治疗口腔白斑的其它药物或方法一起施用。
一方面，本发明涉及携带miR-185的外泌体在制备预防或阻止口腔白斑受试者口腔白斑向口腔癌转化的药物组合物、药盒或药物制品中的用途。在一个优选实施方案中，所述白斑为白斑单纯增生、白斑伴异常增生。在一个优选实施方案中，所述口腔癌为口腔鳞状细胞癌。在一个优选实施方案中，所述携带miR-185的外泌体与预防或阻止口腔白斑向口腔癌转化的其它药物或方法一起施用。
在一个优选实施方案中，上述携带miR-185的外泌体通过局部给药途径给药受试者。在一个优选实施方案中，所述携带miR-185的外泌体通过黏膜下注射、局部涂抹或口腔含服途径给药受试者。
一方面，本发明还涉及一种预防口腔癌的方法，包括给药受试者预防有效量的携带miR-185的外泌体，该外泌体通过如下的一种或多种方式阻止单纯黏膜白斑向白斑伴异常增生和口腔癌的转化或阻止白斑伴异常增生向口腔癌的转化：抑制炎症反应、抑制口腔黏膜上皮细胞异常增生、抑制黏膜微 血管形成。
一方面，本发明还涉及携带miR-185的外泌体在制备预防口腔癌的药物中的用途，其中所述外泌体通过如下的一种或多种方式阻止单纯黏膜白斑向白斑伴异常增生和口腔癌的转化或阻止白斑伴异常增生向口腔癌的转化：抑制炎症反应、抑制口腔黏膜上皮细胞异常增生、抑制黏膜微血管形成。
一方面，本发明涉及一种经过改造的唾液外泌体，该外泌体导入了预防或治疗有效量的miR-185。本发明还涉及包含该外泌体的用于预防或阻止口腔白斑向口腔癌转化的药物组合物、药盒或药物制品。在一个优选实施方案中，所述白斑为白斑单纯增生、白斑伴异常增生。在一个优选实施方案中，所述口腔癌为口腔鳞状细胞癌。
一方面，本发明还涉及miR-185或携带miR-185的外泌体在制备抑制口腔癌细胞增殖的药物中的用途。同时，本发明还涉及一种抑制口腔癌细胞增殖的方法，包括给药受试者有效量的miR-185或携带miR-185的外泌体来实现对口腔癌细胞生长的抑制作用。在一个优选实施方案中，所述miR-185或携带miR-185的外泌体通过局部给药受试者来抑制口腔癌细胞的生长和增殖。在一个优选实施方案中，所述miR-185或携带miR-185的外泌体和其它口腔癌治疗药物或方法联合使用。基于本发明的发现，本申请还涉及包含miR-185或携带miR-185的外泌体的抑制口腔癌细胞生长的药物组合物、制品和试剂盒。
一方面，本发明涉及miR-185或携带miR-185的外泌体在制备调控口腔癌受试者中口腔癌细胞相关蛋白VEGF以及AKT表达的药物中的用途。同时，本发明还涉及一种调控口腔癌受试者中口腔癌细胞相关蛋白VEGF以及AKT表达的方法，包括给药受试者有效量的miR-185或携带miR-185的外泌体。在一个优选实施方案中，所述调控包括抑制口腔癌细胞相关蛋白VEGF以及AKT的表达。在一个优选实施方案中，所述miR-185或携带miR-185的外泌体通过局部给药受试者来实现所述调控作用。在一个优选实施方案中，所述miR-185或携带miR-185的外泌体和其它口腔癌治疗药物或方法联合使用。基于本发明的发现，本申请还涉及包含miR-185或携带miR-185的外泌体的、调控口腔癌相关蛋白VEGF以及AKT的表达的药物组合物、制品和试剂盒。
本发明所述的携带miR-185的外泌体可以是通过基因工程方法导入了 miR-185的外泌体，也可以是天然存在高拷贝miR-185的来源于机体组织细胞、血液或体液的外泌体。在一些实施方案中，本发明所述的携带miR-185的外泌体是通过基因工程方法导入了miR-185或通过基因工程方法增加了miR-185拷贝数的人工改造的外泌体。在一些实施方案中，本发明所述的携带miR-185的外泌体是天然存在于机体组织细胞或体液中、通过纯化获得的外泌体，例如干细胞(例如间充质干细胞)来源的或其他体液来源的携带高拷贝miR-185的外泌体。本领域技术人员可以理解，通过常规的基因工程方法，本领域技术人员即可以实现将miR-185导入外泌体或提高外泌体中miR-185的拷贝数的目的。
发明详述
定义：
本发明所述的“癌前病变(precancerous lesion)”是指本身不是癌，但更易转变为癌的一类病损。其中，“口腔癌前病变”(oral precancerous/premalignant lesion,OPL)是指有形态学变化并具有癌变潜在可能性的口腔病变，临床上多见为口腔上皮性癌前病变，例如临床常见的白斑、红斑、扁平苔藓、盘状红斑狼疮、黏膜下纤维性变、乳头状瘤、慢性溃疡、黏膜黑斑及色素痣等。
本发明所述的“口腔白斑”是发生于口腔黏膜上以白色为主的损害，不能擦去，也不能以临床和组织病理学的方法诊断为其他可定义的损害，属于癌前病变或潜在的恶性疾患(Potentially Malignant Disorders,PMD)范畴，不包括吸烟、局部摩擦等局部因素去除后可以消退的单纯性过角化病。本发明的口腔白斑也简称为白斑。
口腔白斑依据其组织病理学表现可分为单纯增生状态的白斑和白斑伴(有)异常增生，前者在本发明中称为白斑(单纯增生)、单纯增生的白斑或白斑的单纯增生阶段(这些术语可互换使用)，病理表现为：上皮增生，有过度正角化或过度不全角化，或者两者同时出现为混合角化；上皮单纯性增生为良性病变，表现为上皮过度正角化，粒层明显和棘层增厚，没有非典型细胞。上皮钉突可伸长且变粗，但仍整齐且基底膜清晰。固有层和黏膜下层中有淋巴细胞、浆细胞浸润。白斑伴异常增生或称为白斑的异常增生阶段，恶变潜能随上皮异常增生程度的增加而增加。上皮异常增生的 组织病理变化为：上皮基底细胞极性消失；出现一层以上基底样细胞；核浆比例增加；上皮钉突呈滴状；上皮层次紊乱；有丝分裂象增加，可见少数异常有丝分裂；上皮浅表1/2出现有丝分裂；细胞多形性；细胞核浓染；核仁增大；细胞黏着力下降；在棘细胞层中单个或成团细胞角化；根据上述项目出现的数目分为轻、中、重度上皮异常增生。
口腔癌前病变，例如口腔白斑，不是癌，但如果没有及时治疗，又受到各种不良刺激，便可能发展成口腔癌。口腔癌的组织病理变化为：分化好的鳞状细胞癌中，细胞间可见细胞间桥，在癌巢的中央可出现层状角化物，为角化珠或癌珠。分化较差的鳞状细胞癌无角化珠形成，甚至也无细胞间桥，瘤细胞呈明显的异型性并见较多的核分裂象。
口腔白斑的治疗目前可以采用的方法有：手术切除、激光、冷冻、光动力治疗；药物治疗包括：维生素A、13-顺式维A酸、异维A酸、阿维A酸、番茄红素、芬维A酸、维胺酸、维A酸糊剂等去角化药物；中医中药的治疗方法仍处于探索阶段：如绞股蓝、增生平等。当白斑转化为口腔癌时，可以采用的方法是目前对癌症治疗常规的治疗方法，包括手术、放射治疗或化疗。
“外泌体(exosome)”是由细胞经过“内吞－融合－外排”等一系列调控过程而形成，并可分泌至胞外的分子直径为30～150nm的亚细胞双层膜囊泡，其内含有与细胞来源相关的蛋白质、miRNA及mRNA等物质。外泌体既可以通过质膜受体直接激活受体细胞，也可以转运蛋白质、mRNA、miRNA甚至细胞器进入受体细胞内，同时也可以携带处于不同病理状态下的细胞所含有的特殊“信息”，进入体液(包括唾液，血液等)，从而在生理学和病理学上都发挥着重要作用。
针对口腔白斑而言的“治疗有效量”是指携带miR-185的外泌体的施用量能使白斑的面积减小或消除，或使白斑伴异常增生减轻，或向单纯增生逆转，甚至转化为正常。
针对口腔癌而言的“预防有效量”是指携带miR-185的外泌体的施用剂量能够实现以下的任一项或多项：使白斑异型上皮细胞数减少、白斑面积减小或消失、白斑炎症反应减弱、黏膜微血管形成减弱、阻止单纯黏膜白斑向白斑伴异常增生、甚至口腔癌的进展、阻止白斑向口腔癌的转化。
附图说明
图1A-B显示OSCC癌细胞中VEGF和AKT的表达，实验结果显示miR-185在OSCC细胞中调控VEGF以及AKT转录表达。
图2显示miR-185抑制癌细胞增殖作用。
图3显示miR-185在AKT 3‘UTR中的结合位点，证明miR-185对AKT转录序列存在着直接调控。
图4显示miR-185直接作用于AKT的3’-UTR区域，调控癌细胞的存
活。
图5显示miR-185表达在OSCC细胞分泌的外泌体中。
图6A-B显示OSCC细胞株分离外泌体的大小和浓度。透射电镜下可见，OSCC细胞中收集、纯化的外泌体颗粒大小均匀、形态一致，呈圆形或椭圆形膜性小囊泡形态，染色后可见囊泡有完整的包膜，其内有低电子致密物，其直径大小约在100纳米左右(图6A)。NTA技术针对外泌体的大小分析表明存在有直径为120nm的外泌体(图6B)；插入:CD81，CD63以及Flotillin为外泌体特征标记蛋白。
图7显示PH26荧光素标记的携带miR-185外泌体进入OSCC细胞。
图8A-B显示外泌体携带miR-185改变OSCC中VEGF和AKT的表达。实验结果显示OSCC细胞株中高表达的miR-185明显地抑制VEGF和AKT的转录表达。
图9显示口腔黏膜组织miR-185原位杂交结果。实验分析了miR-185在口腔黏膜组织中的表达水平及分布。结果发现，在正常口腔黏膜中，可见大量上皮细胞核和包浆中出现强棕紫色反应，miR-185表达呈现强阳性；在口腔黏膜白斑单纯增生、白斑伴异常增生以及口腔癌病例中，miR-185表达显著减弱；在口腔癌病例中，miR-185在癌上皮组织中表达消失。
图10A-B显示唾液外泌体和血液外泌体鉴定结果，其中10A显示对唾液外泌体的鉴定结果；10B显示对血液外泌体的鉴定结果。NTA技术针对外泌体的大小分析表明存在有直径为110-120nm的外泌体。通过蛋白免疫印迹检测发现这些颗粒表达外泌体特异性结构蛋白CD81、CD63或Flotillin(图10A，B)。
图11A-C显示唾液外泌体携带小分子微小RNA矩阵分析结果。图11A为白斑单纯增生的组织细胞相对于正常粘膜组织细胞的唾液外泌体携带小 分子微小RNA的矩阵分析结果。结果显示，口腔黏膜白斑唾液的外泌体所具有的微小RNA含量明显不同于来自健康人的外泌体。其中来自口腔黏膜白斑单纯增生唾液外泌体的miR-185相对正常人显著减少。图11B-C显示唾液外泌体浓度在白斑异常增生阶段明显升高，而发展成为口腔癌后唾液外泌体浓度显著降低。相反，血液外泌体浓度在口腔癌阶段明显升高。
图12为基于细胞水平测定结果绘制的模式图，该图显示外泌体介导miR-185在细胞间的传递作用以及对口腔癌前病变信号通路中VEGF和AKT的转录抑制调节的影响。
图13A-B显示携带miR-185的外泌体延迟癌前病变进展的动物实验技术路线图及方法图。
图14A-H显示金黄地鼠颊囊黏膜局部涂抹DMBA 6周的病损变化情况及病理改变。颊囊由正常黏膜(图14A)转变为炎症状态(图14B-C)，并发展至癌前病变(图14D-E)。病理变化由正常黏膜(图14F)向单纯增生(图14G)及异常增生(图14H)转变。
图15显示地鼠的体重变化情况，三组地鼠与阴性对照组比较，*p<0.05,**p<0.01。
图16显示地鼠肝肾功能相关的血生化指标水平。
图17A-B显示地鼠颊囊黏膜炎细胞表达及计数，不同阶段三组地鼠颊囊炎细胞表达(A)及计数水平(B)，与DMBA组比较，*p<0.05,***p<0.001。
图18A-B显示地鼠颊囊黏膜单纯增生及异常增生计数结果，三组地鼠颊囊单纯及异常增生计数，*p<0.05,**p<0.01,***p<0.001。
图19A-D显示地鼠颊囊黏膜免疫组化染色。CD31、PCNA、COX2在组间的表达(19A)；CD31标记血管内皮细胞计算微血管密度(MVD)(19B)、PCNA染色计算上皮平均光密度值(AOD)(19C)、COX2染色计数阳性细胞(19D)的结果。不同阶段三组地鼠颊囊COX2、PCNA、CD31免疫组化染色，与DMBA组比较，*p<0.05,**p<0.01，***p<0.001。
图20A-C显示地鼠血清中炎性因子IL-1β、IL-6、IL-10的表达水平，不同阶段三组地鼠血清中细胞因子IL-6、IL-1β、IL-10表达水平，与DMBA组比较，*p<0.05,**p<0.01。
图21显示颊囊组织炎性因子蛋白的表达情况。
实施例
实施例1 miR-185在癌前病变向口腔癌转化过程中的调控作用
方法
(1)永生化口腔鳞状细胞癌(OSCC细胞系)复苏：取冻存的OSCC细胞(ATCC公司购买
Figure PCTCN2018086347-appb-000001
CRL-1623 TM，Manassas,VA,USA)，迅速置于37℃恒温水浴箱中快速融化；注入离心管并滴加培养液1000rpm/min离心5分钟；弃去上清液加入培养液，置于37℃、5％CO2、饱和湿度条件下培养；24小时后倒置显微镜下观察细胞，更换培养液。
(2)细胞培养与传代：细胞长满至80％～90％时传代；0.25％胰蛋白酶消化细胞，吹打后移入试管中，离心，弃上清液；加入培养液，按1:2或1:3传代后置于37℃、5％CO2、饱和湿度条件下培养(为保证细胞性质的稳定，实验均采用10代以内的细胞进行实验)。
(3)应用实时荧光定量PCR(qRT-PCR)技术分析miR-185表达水平：将细胞中miRNAs反转录成单链cDNA(Qiagen公司购买，Omniscript RT Kit—205111,Germantown,MD,USA)。SYBR Green嵌合荧光法进行qRT-PCR检测miR-185表达水平，小片段RNA U6作为内参照(Qiagen公司购买，miScript
Figure PCTCN2018086347-appb-000002
Green PCR Kit—218073)。
(4)采用脂质体包裹转染试剂将miR-185拟似物(Qiagen公司购买，miScript miRNA Mimic-219600)或者抑制物(Qiagen公司购买，miScript miRNA Inhibitor-219300、以及阴性对照(随机序列或随机抑制物,Qiagen公司购买miScript Inhibitor Neg.Control-102727)分别转染入OSCC细胞系中(Lonza Nucleofector TM系统)，48小时后收集细胞用于提取总mRNA(Qiagen公司购买，RNeasy Mini Kit-74104)。之后运用qRT-PCR技术方法分析VEGF或AKT表达水平。
(5)MTT法检测癌细胞增殖指数：收集(4)中转染细胞，接种于96孔板内，培养48小时后，采用Abcam公司MTT检测试剂盒(Burlingame,CA,USA),以MTT法检测癌细胞增殖指数。
(6)构建AKT荧光素酶报告基因质粒：通过miRBase数据分析系统(microRNA.org)，筛选出AKT基因上潜在的miR-185结合位点。AKT的全长3’-非编码翻译区(3’-UTR)从基因组DNA中扩增并克隆到质粒载体Fire-Ctx sensor lentivector(miR-selection Fire-Ctx lentivector,SBI公司购买Palo  Alto,CA,USA)中，其下游携带荧火虫荧光素酶(Firefly luciferase)报告基因以及细胞毒素(CTX)药物敏感基因。实验中，Fire-Ctx sensor lentivector载体作为实验对照。
(7)质粒的转染和细胞毒性检测：将构建的质粒采用电穿孔技术转染入OSCC细胞株中(Lonza Nucleofector TM系统,Walkersville,MD,USA)，同时细胞中共转染miR-185前体(pre-miR-185,购买于Exiqon公司，Woburn,MA,USA)。为了控制转染效率，细胞还同时转染了pRL-CMV载体质粒(Promega公司购买—E2261,San Luis Obispo,CA,USA)，其中包括荧光素酶(Renilla luciferase)报告基因。相对应实验，细胞如上述转染24小时后，以细胞毒素(CTX)药物处理3－4天，之后测量细胞存活程度。
结果
实验发现，在OSCC细胞株中转染miR-185(核苷酸序列：5’uggagagaaaggcaguuccuga 3’)拟似物可以明显降低VEGF以及AKT的转录表达。相反，在OSCC中共转染miR-185的抑制序列有效地抑制了miR-185拟似物的作用效果。而对照随机序列(scramble)不能发挥作用。本实验说明，miR-185明显地调控口腔癌细胞相关蛋白VEGF以及AKT的表达(见图1A-B)。本实验还发现OSCC转染miR-185后有效地抑制了癌细胞增殖作用(见图2)。
利用miRBase数据分析系统(microRNA.org)，筛选出miR-185在AKT转录序列上存在着直接调控位点(见图3)。
实验发现，在OSCC细胞株中转染Fire-Ctx AKT 3’-UTR质粒，其质粒下游携带细胞毒素(CTX)药物敏感基因。实验结果发现，细胞培养基中加入CTX毒性药物时明显导致大量细胞死亡，但高表达pre-miR-185的OSCC细胞株存活率明显升高，和对照组无明显差异(见图4)。此实验提示miR-185特异性地作用AKT的3’-UTR区域，抑制了细胞毒素(CTX)药物敏感基因的表达，从而降低了细胞毒性反应，提升了细胞的存活率。
实施例2 外泌体投递miR-185对受体细胞癌变信号通路分子转录的抑制
方法
(1)细胞培养液中分离、纯化外泌体：培养的OSCC细胞(如前所述)，血清饥饿48小时后，收集细胞培养基，分别在4℃下2000×g离心20分钟、10000×g离心30分钟以去除细胞碎片，使用外泌体分离试剂盒(Exosome isolation Kit,Cat.NO:GET301-10,Genexosome Technologies Inc.,Freehold,New Jersey,USA)按步骤纯化得到外泌体后，以一定体积的无菌PBS缓冲液重悬，稀释。
(2)外泌体性状鉴定：通过透射电子显微镜对收获的外泌体进行形态学观察；通过NTA(Nano-tracking analysis,ParticleMetrix GmbH,Meerbusch,Germany)分析技术对外泌体的大小和浓度进行测定。
通过Western blot对外泌体携带的蛋白标记物进行特征性分析。配制15％分离胶和5％浓缩胶，取外泌体悬液40μl与5X SDS上样缓冲液10μl混合煮沸5分钟，加于凝胶上样孔内，浓缩胶恒压80V，分离胶恒压120V，200mA恒流1.5小时。将凝胶中的蛋白质通过湿转法转移至硝酸纤维素膜上，室温下用含5％脱脂牛奶的封闭液封闭处理1h，经1X TBST缓冲液洗脱后，加入CD81(1：400)、CD63(1：250)以及Flotillin(1：1000)单克隆抗体(Abcam公司购买)于4℃条件下反应过夜，再次洗脱后，加入辣根过氧化物酶标记的羊抗兔二抗(1：2500，Sigma St.Louis,MO,USA)，于室温下平缓摇动1h。经1X TBST缓冲液洗膜3次后，用化学发光底物(ECL,购买于Thermo Fisher Scientific,Carlsbad,CA,USA)进行检测。
(3)应用实时荧光定量PCR(qRT-PCR)技术分析OSCC细胞及其分泌外泌体中miR-185表达水平：将细胞及外泌体中miRNAs反转录成单链cDNA。SYBR Green嵌合荧光法进行qRT-PCR检测miR-185表达水平，小片段RNA U6作为内参照。
(4)外泌体在细胞间的传递：提取携带高拷贝miR-185的OSCC细胞条件培养基中的外泌体。PKH26荧光标记外泌体后(PKH26 Red Fluorescent Cell Linker Kit，购买于Sigma公司)，加入到OSCC细胞培养液中，24小时后观测PKH26荧光标记的外泌体被OSCC细胞吸收。
(5)外泌体携带高拷贝miR-185在细胞间的传递作用以及对口腔癌前病变信号通路信号分子的转录抑制调节影响:将携带高拷贝miR-185的外泌体的培养基培养OSCC靶细胞，通过检测靶细胞内VEGF及AKT表达水平，确定外泌体携带miR-185进入受体靶细胞后，是否有效逆转癌变。
结果
(1)1.透射电镜下可见，OSCC细胞中收集、纯化的外泌体颗粒大小均匀、形态一致，呈圆形或椭圆形双脂膜性小囊泡形态，染色后可见囊泡有完整的双层脂质膜结构，其内有低电子致密物，其直径大小约在100纳米左右，参见图5。
实验结果显示miR-185携带于外泌体中，抑制VEGF和AKT转录表达。文献报道，miRNAs被外泌体(Exosome)包裹，释放到细胞外基质中去。本实验前期结果发现，miR-185表达在OSCC细胞分泌的外泌体中(见图5)。通过NTA技术分析发现，OSCC外泌体直径在120nm(见图6)。Western blot鉴定结果确定OSCC外泌体高表达CD81，CD63以及Flotillin等外泌体标志物(见图6)。
(2)采用PKH26红色荧光标记物标记携带miR-185外泌体，之后加入OSCC细胞培养基中，48小时后观察可见外泌体被OSCC细胞摄取(见图7)。QRT-PCR结果显示摄取miR-185后的OSCC高表达miR-185(未显示结果)，并且明显地抑制VEGF和AKT的转录表达(见图8A-B)。图12为基于细胞水平测定结果绘制的模式图，该图显示外泌体介导miR-185在细胞间的传递作用以及对口腔癌前病变信号通路中VEGF和AKT的转录抑制调节的影响。
实施例3 口腔白斑向癌变过程中miR-185表达变化
方法
选取临床和病理诊断为口腔黏膜白斑单纯增生、白斑伴异常增生以及白斑癌变(口腔鳞状细胞癌)患者的组织标本以及正常组织标本作为研究对象。
根据病理诊断结果进行分组：分为白斑单纯增生组(N＝15)；白斑异常增生组(N＝10)、癌变组，也称为口腔癌组(N＝15)。
正常对照组(N＝8)组织标本选自排除口腔黏膜病、因手术治疗需要切除部分正常组织并且愿意提供该组织用于研究的患者。
原位杂交定位miR-185表达
在1X原位杂交(ISH)缓冲液(购买于Exiqon Inc.,Woburn,MA USA)中将miR-185或对照序列探针(Exiqon Inc.)与固定的组织切片在55℃杂交60 分钟，随后使用不同浓度的SSC缓冲液在55℃洗涤。如下述检测探针：使用单克隆抗地高辛碱性磷酸酶抗体(1:800)(Roche,Indianapolis,IN USA)温育60分钟，随后在30℃条件下，使用硝基蓝四唑和5-溴-4-氯-3'-多聚磷酸盐底物(Roche,Pleasanton,CA,USA)温育2小时。最终，使用Nuclear Fast Red TM复染色切片，使用
Figure PCTCN2018086347-appb-000003
培养基(VWR,Radnor,PA,USA)封固，并通过共聚焦显微镜检查。
结果
原位杂交定位miR-185表达实验发现正常组样品中miR-185表达呈现强阳性(紫色)；在白斑组样品中，miR-185表达明显减弱，而在异常增生组和口腔癌组样品中可见少部分上皮细胞核和包浆中出现轻微棕紫色反应，miR-185表达呈现轻微阳性，或者miR-185表达几乎消失，见图9。
近来，已报道了多种直接靶向EMT转录因子和细胞结构组分的miRNA。上述实验结果发现，白斑单纯增生组，白斑伴异常增生组和口腔癌组患者样品中miR-185的水平相比正常对照显著下降。
综上所述，实验发现从口腔白斑单纯增生向口腔黏膜白斑伴异常增生、口腔癌转化过程中，PI3K/AKT-mTOR通路激活，EMT发生，同时miR-185表达下降甚至缺失。
实施例4 口腔唾液外泌体或者血液外泌体携带与疾病状态相关的miR-185
方法
外泌体：从上述实施例3的临床和病理诊断患有口腔黏膜白斑单纯增生、白斑异常增生、口腔癌(口腔鳞状细胞癌)的患者以及正常人口腔唾液以及血液中按如下方法收集、纯化口腔唾液外泌体或者血液外泌体。
上述患者或正常人在取唾液前不漱口，禁食水1小时。取唾液时坐位，头自然低下，口内唾液自然吐出至一次性托盘中，约2毫升左右，不要咳嗽。将收集的唾液立即放入小离心管中。
将样品4℃，10,000×g离心20分钟除去杂质，样本上清液通过0.22μm的滤膜过滤二次，使用外泌体分离试剂盒(Exosome isolation Kit,Cat.NO:GET200-10,Genexosome Technologies Inc.,Freehold,New Jersey,USA)按步骤纯化得到外泌体后，以一定体积的无菌PBS缓冲液重悬，稀释。
1.唾液外泌体以及血液外泌体鉴定
(1)外泌体的形态特征观察
取外泌体悬液10μl滴于孔径2nm的载样铜网上，室温下静置10分钟，用滤纸从滤网侧边吸干液体，滴加3％磷钨酸溶液30μl，室温环境下复染5分钟，用滤纸吸干复染液，并在室温下干燥后，将此铜网置于透射电镜的样品室内，观察外泌体形态并拍摄电镜照片。
(2)唾液外泌体以及血液外泌体性状鉴定
通过NTA技术对外泌体的大小和浓度进行检测。
(3)外泌体特异性结构蛋白的分析
配制15％分离胶和5％浓缩胶，取外泌体悬液40μl与5X SDS上样缓冲液10μl混合煮沸5分钟，加于凝胶上样孔内，浓缩胶恒压80V，分离胶恒压120V，200mA恒流1.5小时。将凝胶中的蛋白质通过湿转法转移至硝酸纤维素膜上，室温下用含5％脱脂牛奶的封闭液封闭处理1h，经1X TBST缓冲液洗脱后，加入CD81(1:400)、CD63(1:250)以及Flotillin(1；1000)(Abcam)单克隆抗体于4℃条件下反应过夜，再次洗脱后，加入辣根过氧化物酶标记的羊抗兔二抗，于室温下平缓摇动1h。经1X TBST缓冲液洗膜3次后，用化学发光底物(ECL,Thermo Fisher Scientific)进行检测。
2.唾液外泌体携带小分子微小RNA矩阵分析
使用microRNeasy Plus试剂盒(Qiagen,Valencia,CA USA)从唾液外泌体提取总RNA，并根据制造商的说明书使用miScript II RT试剂盒(Qiagen)进行逆转录。根据制造商的说明书通过微小RNA矩阵分析获得的转录物并通过qRT-PCR进行验证。将qRT-PCR标准化至U6snRNA引物。
3.通过NTA技术检测口腔白斑发展至癌变过程中唾液携带外泌体以及血液外泌体浓度变化
结果
通过NTA技术检测到唾液来源的外泌体或者血液来源的外泌体大小均在110-120nm之间(图10A,B)。通过蛋白免疫印迹检测发现这些颗粒表达外泌体特异性结构蛋白CD81、CD63或Flotillin，参见图10A,B。
2.通过微小RNA矩阵，我们首次发现来自口腔黏膜白斑单纯增生患者唾液的外泌体所具有的miR-185,其含量明显低于来自正常人的外泌体，参见图11A。
3.口腔黏膜白斑单纯增生、白斑异常增生、口腔癌患者唾液外泌体浓度明显不同。口腔黏膜白斑伴有异常增生患者唾液外泌体浓度明显升高，而出现癌变后其浓度显著下降，参见图11B。相反，癌变患者血液外泌体的浓度则明显升高。此发现表明唾液外泌体浓度与疾病发展状况紧密相关，而血液外泌体和唾液外泌体呈相反分泌趋势(图11B,C)。
实施例5 携带miR-185的外泌体阻断癌前病变的进展
方法
(1)试剂与保存
本实验和以下实验中使用的试剂如下配置和保存：0.5克二甲基苯并蒽(DMBA)溶解于50ml丙酮和50ml液体石蜡中，制备成0.5％DMBA溶液，室温下避光保存。携带miR-185的外泌体均为间充质干细胞来源的包含高拷贝miR-185的外泌体，购自GenExsome Technology公司，商品名为GET MSCEXO101-1ug。该携带miR-185的外泌体溶液外泌体颗粒浓度为2×10 11粒子/ml，保存于-80℃，使用前24h转移至4℃保存。
(2)选择SPF级7周龄雄性叙利亚金黄地鼠(北京维通利华实验动物技术公司)，平均体重115g。饲养条件为温度24-26℃，湿度40-60％，12-14小时光照。适应性饲养一周后将53只地鼠随机分为3组。阴性对照组(NC)8只，阳性对照组(即给予二甲基苯并蒽的阳性对照组，简写DMBA组)25只，局部涂抹携带高拷贝miR-185外泌体溶液组(DMBA+EXO组,也称治疗组)20只。阴性对照组整个实验过程中不做药物处理，其余两组自第一周起以0.5％二甲基苯并蒽(DMBA)溶液涂于左侧颊囊，每周3次，至实验结束，25只阳性对照组不再做其他处理；20只治疗组自第3周起在DMBA涂抹的相同部位涂抹外泌体溶液每周3次，至第6周实验结束。自第3周末起，每周末各断髓处死DMBA组6只地鼠，DMBA+EXO组5只地鼠，6周末处死剩余地鼠。实验过程中，观察并记录地鼠的健康及病损状况，每周记录体重。实验按图13A的技术路线、图13B的实验方法进行。
(3)涂抹方法：4号画笔蘸取液体，瓶口挤掉多余液体，涂于地鼠左侧颊囊黏膜中央，同一方向圆周式运动涂布，经定量试验调整画笔刷毛长度及形状，确定每次涂布量约为100μl，涂抹载有miR-185的外泌体溶液与DMBA溶液时间间隔4h，涂药后禁食水2小时。
(4)血清的提取与保存：地鼠处死前收集全血并保存于EP管中，常温静置30min后于4℃，3000×g离心10min后分离血浆及血清，提取血清并储存于-80℃。
(5)肝肾功能：采用商品化试剂盒进行检测，试剂盒均购自英科新创(厦门)科技有限公司，丙氨酸氨基转移酶(ALT)采用紫外-乳酸脱氢酶法、天冬氨酸氨基转移酶(AST)采用紫外-苹果酸脱氢酶法、肌酐(Scr)采用酶法、尿素氮(BUN)采用紫外-谷氨酸脱氢酶法，实验严格遵照试剂盒说明进行操作。
(6)包埋切片：将地鼠颊囊组织于10％福尔马林溶液内固定24小时后取出，分切成约3-5mm条状，卷成筒状，用钢针固定，自动脱水机脱水，去钢针，常规石蜡包埋，每个样本连续切21张5μm切片，取第1、10、20张进行HE染色，第2、11、21张进行免疫组化染色。
(7)HE染色：玻片于65℃烤箱烤1h，常规脱蜡至水，自来水冲洗2min，入苏木素染液4min后自来水冲洗，分化液分化2s，返蓝液返蓝4s，自来水浸泡5min，入伊红染液40s，自来水冲洗30s，脱水入二甲苯，中性树脂封片。400倍镜下观察黏膜固有层和黏膜下层有炎细胞(根据形态可判定为淋巴细胞和中性粒细胞)浸润，每张玻片选取3-10处炎细胞较多的视野，200倍镜下进行计数。单纯增生表现为细胞数目增多，上皮颗粒层明显和棘层增生，没有非典型性细胞；异常增生根据WHO诊断标准，包括上皮基底细胞极性消失，出现一层以上基底样细胞，核浆比例增加，上皮钉突呈滴状，上皮层次紊乱，有丝核分裂增加，可见少数异常核分裂相，上皮浅表1/2出现有丝分裂，细胞的多形性，细胞核浓染，核仁增大，细胞黏着力下降，在棘细胞层中单个或成团细胞角化。严格遵照该标准记录样本全部单纯及异常增生数目。
(8)免疫组化实验：切片于65℃烤箱中烤1.5h，常规脱蜡至水，PBS缓冲液清洗，0.01mol/L柠檬酸钠缓冲液微波修复，放置至室温，清洗后，将玻片置于湿盒中，3％过氧化氢避光室温封闭15min后清洗玻片，10％山羊血清37℃孵育1h以封闭抗原，弃去多余血清，滴加一抗，浓度分别为：抗CD31抗体1:200，抗PCNA抗体1:30000，抗COX2抗体1:1000，这些抗体均购自Abcam公司，空白对照用PBS代替一抗，4℃过夜。第二天取出后室温复温1h，PBS缓冲液清洗，滴加二抗，37℃孵育0.5h，清洗玻片后滴加DAB (二氨基联苯胺)，显微镜下观察显示情况及时长，水洗终止显色，苏木素复染，反蓝，脱水，透明，中性树脂封片。PCNA蛋白表达位于细胞核，每玻片100倍镜下选取3-5处上皮增生(单纯增生、异常增生)处截取上皮，应用Image pro plus软件进行光密度分析，记录平均光密度值(IOD)；COX2蛋白表达位于核膜，每样本100倍镜下选取2-5处炎细胞密集区，以核膜出现棕黄色或棕褐色为阳性，进行阳性细胞计数；CD31蛋白表达于内皮细胞膜，参照Weidner方法,每样本100倍镜下选取微血管(直径小于8个红细胞)密集处，400倍镜下计数CD31标记的微血管数量，平均值即为MVD值(微血管密度)，记录上述数据并进行组间比较。
(9)酶联免疫吸附实验(ELISA):使用商品化地鼠IL-6、IL-1β、IL-10ELISA试剂盒检测这些细胞因子，品牌为MyBioSource(San Diego,CA，USA)。从室温平衡20min后的铝箔袋中取出所需板条，设置空白对照孔、标准品孔和样本孔，空白孔加50μl样本稀释液，标准品孔加不同浓度的标准品50μl，样本孔加50μl待测血清，每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μl，用封板膜封住反应孔，37℃恒温箱温育60min，弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，甩去洗涤液，吸水纸上拍干，重复洗板5次，每孔加入底物A、B各50μl，37℃避光孵育15min，每孔加入终止液50μl，15min内，在450nm波长处测定各孔的OD值，应用公式计算得出R 2值及细胞因子的蛋白浓度并进行组间比较。
(10)黏膜蛋白检测：提取三周末(急性炎症期)三组地鼠全部颊囊黏膜总蛋白，六周末DMBA+EXO组全部颊囊黏膜总蛋白，共计四组，按Proteome Profiler Array Mouse Cytokine Array Panel A试剂盒(品牌R&D)说明书要求，在四孔板中每孔中各滴入封闭液2ml进行封闭，四张膜置于四孔板中，摇床孵育1h，准备样本，每试管中加入蛋白，通过稀释液调整体积为1.5ml，每样本加入15μl溶解的抗体，混匀，室温孵育1h，移除四孔板中封闭液，加入样本抗体混合液，4℃摇床孵育过夜。第二天取出膜于摇床清洗10min，重复三次，清洗四孔板，每孔中滴入2ml稀释的链霉亲和素-HRP，将四张膜放入四孔板，室温摇床孵育30min，洗膜，将膜数字朝上放于暗盒中，均匀滴显色剂1ml，孵育1min，显影，曝光。对每张膜的不同蛋白位点进行观察扫描与灰度值分析，进行组间比较。
(11)统计学方法：应用SPSS 20.0统计软件进行统计分析，数据用平 均值±SD表示，采用单因素方差分析(one-way ANOVA)参数检验，LSD进行两两比较；单纯及异常增生计数用中值(IQR)表示，采用秩和检验，Mann-Whitney进行两两比较，均以α＝0.05作为检验水准，*p<0.05，**p<0.01，***p＜0.001。
结果
(1)病损变化情况及病理改变：健康黏膜为淡粉色，光滑、菲薄、连续，黏膜下血管清晰可见(见图14A)，第二周初至第三周末为炎症期，黏膜充血、水肿，有黄色液体样炎性渗出(见图14B)，随渗出不断增多，凝结成块状，可擦去，擦去时易出血(见图14C)，后期黏膜逐渐结痂，弹性良好。第四周始黏膜弹性逐渐下降，部分出现角化发白(见图14D)，第五周初至第六周末为癌前病变期，黏膜粗糙、发白，轻度增厚，部分可见白色斑块状病损(见图14E)。光镜下观察组织病理变化由正常黏膜(见图14F)向单纯增生(见图14G)、异常增生(见图14H)逐步转变。
(2)体重：实验初始各组体重接近，第二、三、四周，DMBA组、DMBA+EXO组因颊囊受DMBA持续作用均处于急性炎症期，影响进食，体重增长慢，与阴性对照组比较差异显著，四周后进入癌前病变期，黏膜增厚、粗糙，对进食无影响，三组体重逐渐接近(见图15)。
(3)肝肾功能：经统计学分析，四组地鼠血清中肝肾功能相关的生化指标中ALT、AST、BUN无组间差异，Scr在DMBA组水平较高，差异具有统计学意义，推测DMBA组地鼠可能存在一定程度肾功能损伤，DMBA+EXO组四项指标与阴性对照组无统计学差异，进一步证实外泌体作为一种天然脂质体，在体内有良好的耐受性，稳定、无毒，是理想的药物载体(见图16)。
(4)炎细胞表达以及计数：实验第二至三周末为急性炎症期，四周始因DMBA持续作用逐渐向癌前病损转变并同时伴有慢性炎症(见图17A)，经统计学分析DMBA+EXO组较DMBA组在各个阶段炎细胞数量均显示减少，四、五周末差异显著(见图17B)，证实局部涂抹携带miR-185的外泌体对黏膜局部炎症具有抑制作用。
(5)单纯增生及异常增生计数：单纯增生计数经统计学分析显示DMBA组、DMBA+EXO组无组间差异，治疗未有效减少单纯增生的数量。异常增生计数显示DMBA+EXO组显著低于DMBA组，治疗延缓病变由单 纯向异常增生的转化，有效减少异常增生的数量，阻断癌前病变的发展进程，结果参见下表及图18A-B。
Figure PCTCN2018086347-appb-000004
(6)CD31、PCNA、COX2表达分析：CD31、PCNA、COX2表达如图19A。CD31标记血管内皮细胞计算微血管密度(MVD)值的结果显示各阶段DMBA+EXO组均低于DMBA组，五、六周末差异有统计学意义(见图19B)，证实局部涂抹携带miR-185的外泌体对抑制黏膜微血管形成具有良好作用。PCNA染色后计算上皮AOD值显示各阶段DMBA+EXO组均显著低于DMBA组(见图19C)，证实局部涂抹携带miR-185的外泌体对抑制上皮增殖具有良好作用，也与异常增生计数的结果相一致。COX2染色计数阳性细胞显示三周末DMBA+EXO组与DMBA组基本相同，四、五、六周末治疗组均低于DMBA组，差异不显著(见图19D)，证实局部涂抹携带miR-185的外泌体对黏膜炎症具有一定的抑制作用。
(7)血清IL-1β、IL-6、IL-10酶联免疫吸附实验：促炎因子IL-1β、IL-6在各阶段及各组中的表达水平高度一致，三周末急性炎症期、六周末癌前病变期DMBA+EXO组均显著低于DMBA组，四、五周表达水平稍有波动，无统计学差异(见图20A、20B)，抗炎因子IL-10在三周末急性炎症期DMBA+EXO组显著高于DMBA组，第四周水平稍有波动，五、六周DMBA+EXO组仍高于DMBA组，无统计学差异(见图20C)，我们发现炎性前体因子IL-10与炎性因子IL-1β、IL-6的表达互相协同，证实通过局部涂抹携带miR-185的外泌体对血清中的炎性因子具有明显地抑制作用。
(8)蛋白质组轮廓仪-小鼠细胞因子阵列板A(Proteome Profiler Array Mouse Cytokine Array Panel A)检测：结果显示三周末DMBA组较DMBA+EXO组及阴性对照组在IL-1β、IL-16、TREM-1等位点蛋白表达量增加，DMBA+EXO组三周末与六周末炎性因子的蛋白表达未见明显变化，与阴性对照组也无明显差异(见图21)，可证实通过局部涂抹携带miR-185的外泌体对黏膜炎症具有良好的抑制作用。
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Claims (27)

  1. 一种预防或阻止口腔白斑向口腔癌转化的方法，包括给药白斑受试者治疗有效量的携带miR-185的外泌体。
  2. 权利要求1的方法，其中所述白斑为白斑单纯增生、白斑伴异常增生。
  3. 权利要求1或2的方法，其中所述口腔癌为口腔鳞状细胞癌。
  4. 权利要求1至3任一项的方法，其中所述携带miR-185的外泌体与阻止口腔白斑向口腔癌转化的其它药物或方法一起施用。
  5. 一种治疗口腔白斑的方法，包括给药白斑受试者治疗有效量的携带miR-185的外泌体。
  6. 权利要求5的方法，其中所述白斑为白斑单纯增生、白斑伴异常增生。
  7. 权利要求5或6的方法，其中所述治疗包括减小白斑的面积或消除白斑，或使白斑伴异常增生减轻，或向单纯增生逆转，或使白斑转化为正常。
  8. 权利要求5至7任一项的方法，其中所述携带miR-185的外泌体与治疗口腔白斑的其它药物或方法一起施用。
  9. 携带miR-185的外泌体在制备预防或阻止口腔白斑受试者口腔白斑向口腔癌转化的药物组合物、药盒或药物制品中的用途。
  10. 权利要求9的用途，其中所述白斑为白斑单纯增生、白斑伴异常增生。
  11. 权利要求9或10的用途，其中所述口腔癌为口腔鳞状细胞癌。
  12. 权利要求9至11任一项的用途，其中所述携带miR-185的外泌体与预防或阻止口腔白斑向口腔癌转化的其它药物或方法一起施用。
  13. 权利要求1-8任一项的方法、权利要求9-12任一项的用途，其中所述携带miR-185的外泌体通过局部给药途径给药受试者。
  14. 权利要求1-8任一项的方法、权利要求9-12任一项的用途，其中所述携带miR-185的外泌体通过黏膜下注射、局部涂抹或口腔含服途径给药受试者。
  15. 一种预防口腔癌的方法，包括给药受试者预防有效量的携带 miR-185的外泌体，该外泌体通过如下的一种或多种方式阻止单纯黏膜白斑向白斑伴异常增生和口腔癌的转化或阻止白斑伴异常增生向口腔癌的转化：抑制炎症反应、抑制口腔黏膜上皮细胞异常增生、抑制黏膜微血管形成。
  16. 携带miR-185的外泌体在制备预防口腔癌的药物中的用途，其中所述外泌体通过如下的一种或多种方式阻止单纯黏膜白斑向白斑伴异常增生和口腔癌的转化或阻止白斑伴异常增生向口腔癌的转化：抑制炎症反应、抑制口腔黏膜上皮细胞异常增生、抑制黏膜微血管形成。
  17. 一种经过改造的唾液外泌体，该外泌体导入了预防或治疗有效量的miR-185。
  18. 一种用于预防或阻止口腔白斑向口腔癌转化的药物组合物，包含权利要求17的经过改造的唾液外泌体。
  19. 权利要求18的组合物，其中所述白斑为白斑单纯增生、白斑伴异常增生。
  20. 权利要求18或19的组合物，其中所述口腔癌为口腔鳞状细胞癌。
  21. 包含权利要求17的携带miR-185的外泌体、或权利要求18-20任一项的药物组合物的药盒或药物制品。
  22. miR-185或携带miR-185的外泌体在制备抑制口腔癌细胞增殖的药物中的用途。
  23. miR-185或携带miR-185的外泌体在制备调控口腔癌受试者中口腔癌细胞相关蛋白VEGF以及AKT表达的药物中的用途。
  24. 权利要求23的用途，其中所述调控包括抑制口腔癌细胞相关蛋白VEGF以及AKT的表达。
  25. 一种抑制口腔癌细胞增殖的方法，包括给药受试者有效量的miR-185或携带miR-185的外泌体。
  26. 一种调控口腔癌受试者中口腔癌细胞相关蛋白VEGF以及AKT表达的方法，包括给药受试者有效量的miR-185或携带miR-185的外泌体。
  27. 权利要求26的用途，其中所述调控包括抑制口腔癌细胞相关蛋白VEGF以及AKT的表达。
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Citations (1)

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