Saliva and plasma exosome miR-185 absolute quantification method for early diagnosis of oral cancer

Abstract

The invention relates to an absolute quantification method of saliva and plasma exosome miR-185. The method is mainly used for early diagnosis of oral leukoplakia canceration and oral cancer. According to the method, a complete, reliable and standardized operation process is provided for absolute quantification of exosome miR-185 through extraction of saliva and plasma exosome RNA, design of synthetic primers, synthesis of a miR-185 standard substance, establishment of a miR-185 standard curve, selection of internal reference, RNA reverse transcription and PCR reaction and data statistical analysis; and necessary conditions are provided for early diagnosis of oral leukoplakia canceration and oral cancer.

Description

Technical Field

[0001] The present invention relates to an absolute quantitative method for saliva and exosome miR-185, which is mainly used for the early diagnosis of oral leukoplakia carcinoma and oral cancer. Background Art

[0002] Oral cancer is one of the 10 most common cancers in the world, accounting for 80% of head and neck malignancies. There are more than 350,000 new cases each year, with an incidence rate of 4 / 100,000. Oral squamous cell carcinoma (OSCC) is the most common, with a five-year survival rate of about 35-57%. About 130,000 oral cancer patients die each year. [1-3] In recent years, the incidence of oral cancer has increased and is becoming younger. Despite the continuous improvement of surgical, radiotherapy and chemotherapy techniques for oral cancer, the 5-year survival rate of patients is still less than 50%. Among them, the 5-year survival rate of patients with advanced stage metastasis is only 20%. [4] , if oral cancer is diagnosed early and treated promptly, the 5-year survival rate can be increased to 80%.

[0003] Oral potentially malignant disorders (OPMDs) are a group of diseases that occur before oral squamous cell carcinoma, including oral leukoplakia, erythema, proliferative verrucous leukoplakia, oral submucous fibrosis, oral lichen planus, lupus erythematosus, dyskeratosis congenita, and lichenification. Patients with these diseases have an increased risk of developing oral cancer [5]. Among them, oral leukoplakia is recognized as one of the most typical oral potentially malignant diseases among oral malignant diseases, with a canceration rate as high as 10-36%.

[0004] Oral leukoplakia (OLK), also known as oral mucosal leukoplakia, was first named by Hungarian dermatologist Erno Sohuimmer in 1887. It refers to white or grayish-white abnormal keratinized lesions occurring on the oral mucosa. Oral leukoplakia is common in middle-aged and elderly people, and is prone to occur on the lips, cheeks, tongue, palate and other mucosa. There are generally no subjective symptoms. At the beginning, it appears as a milky white patch with a smooth, flat or slightly higher surface than the normal mucosa. It can take several years to more than ten years for leukoplakia to develop from a precancerous lesion to oral cancer. The process of canceration is also a multi-stage, multi-step process, and it must go through the evolution of hyperplasia → mild, moderate, and severe abnormal hyperplasia → carcinoma in situ → invasive cancer. [6-7] Moreover, most oral leukoplakia can remain in a benign state for a long time without becoming cancerous, and only a small number of the above processes develop into cancer.

[0005] In recent years, the role of exosomes in tumor liquid biopsy has received widespread attention. Exosomes are small extracellular vesicles (EVs) containing proteins, lipids, RNA, metabolites, growth factors and cytokines, with a diameter of 40-160nm, and are multifunctional transporters between cells [8]. Biomolecules are engulfed by early endosomes through plasma membrane invagination. These early endosomes are recycled to the plasma membrane, or located in lysosomes for degradation, or sorted to form late endosomes, called multivesicular bodies (MVBs). When MVBs fuse with the plasma membrane, the intraluminal vesicles in the MVBs enter the extracellular environment through exocytosis, which is the process of exosome secretion. [9] All cells can secrete exosomes in normal and pathological processes, and they can be effectively detected in almost all types of body fluids, such as saliva, blood, urine, breast milk, etc. Exosomes have a special lipid bimolecular structure that can protect their contents from being degraded by proteases, peptidases, and nucleases in body fluids.

[10] , making exosomes and their contents become stably detectable tumor diagnostic markers.

[0006] The carcinogenesis of oral leukoplakia is associated with precise regulation of molecules including microRNA

[11] MicroRNA is a group of non-coding RNAs consisting of single strands of 18-25 nucleotides in length. It binds to the 3'-untranslated region (3'-UTR) of the target gene RNA (mRNA) by complementary pairing, modifies the target gene at the post-transcriptional level, and thus regulates gene expression. MicroRNA participates in various biological processes, including growth, differentiation, apoptosis, and proliferation, by regulating its target genes.

[12] The study found that the expression of miR-10b and miR-708 was significantly increased in oral leukoplakia tissues with epithelial abnormal hyperplasia, while the expression of miR-99b, miR-145, and miR-181c was significantly downregulated.

[13] . The expression level of microRNA in tissues is correlated with cytopathological characteristics. The expression of miR-21, miR-345 and miR-181b in oral cancer is significantly higher than that in oral leukoplakia and normal mucosal tissues. In oral leukoplakia cells with increased mitotic figures, high nuclear-cytoplasmic ratio and deep staining, the expression of miR-21 and miR-181b is increased. miR-345 is highly expressed in oral leukoplakia with increased nuclei or enlarged volume and high nuclear-cytoplasmic ratio. The expression of microRNA is also associated with the progression of histopathology. In the study of progressive and non-progressive oral leukoplakia, it was found that the expression levels of miR-21, miR-345 and miR-181b continued to increase with the progression of the disease. [13-15]In general, there are obviously abnormally expressed microRNAs in the occurrence and development of oral cancer, and their expression trends and functions are different. [16-22] .

[0007] miR-185 is a microRNA that has been shown to play an inhibitory role in different types of malignant tumors, such as non-small cell lung cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, osteosarcoma, etc. [23-27] We found that not only can exosomes carrying miR-185 effectively treat oral leukoplakia and prevent the transformation of leukoplakia to oral cancer, thereby effectively preventing the occurrence of oral cancer, but also that exosome miR-185 derived from saliva and plasma can also be used as an early diagnostic marker for judging leukoplakia carcinogenesis and oral cancer.

[0008] Accurate quantification of miR-185 in exosomes is a crucial step in the clinical application of salivary and plasma exosome miR-185 as a diagnostic marker. The most common method for microRNA quantification is real-time fluorescence quantitative PCR, which is mainly divided into relative quantification and absolute quantification.

[0009] Relative quantification refers to comparing the expression of a target gene in a treated sample with the expression of the same gene in another untreated sample. The result is the multiple change (increase or decrease) of the expression level of the treated sample relative to the expression level of the untreated sample. This method requires the participation of internal reference genes. The role of internal reference genes is to normalize the expression of target genes so that the target genes are comparable between different samples. However, the ideal internal reference genes that can be used for quantitative analysis of exosomal microRNA are still controversial. In the field of oral diseases, some scholars used miR-16 as an internal reference gene when studying salivary exosomal microRNA in patients with oral lichen planus.

[28] , snRNA U48 was selected for the study of patients with Sjögren's syndrome

[29] In the research on circulating exosomal microRNAs in various diseases, many scholars have chosen snRNAU6 as an internal reference gene.

[30] However, a study of serum exosome samples from patients with hepatitis B or hepatocellular carcinoma and healthy individuals found that miR-221, miR-103, let-7a, miR-181c, miR-181a, and miR-26a were the most stable reference genes, while miR-16, miR-22, and U6 were not reliable.

[31] Recent studies have found that hsa-miR-6835-3p (especially the hsa-miR-6835-3p / hsa-miR-4468-3p combination) is the best tool for analyzing circulating exosomal microRNAs in patients with various tumors (including pancreatic cancer, colorectal cancer, liver cancer, etc.)

[32] .

[0010] Absolute quantification refers to the process of amplifying a known amount of a sample after serial dilution in a real-time fluorescence quantitative PCR experiment to generate a standard curve, which is applicable to studies that require determination of the number of copies of a target gene, such as the microRNA molecular markers for diagnosing oral leukoplakia and oral cancer involved in the present invention. Therefore, the present invention has discovered an absolute quantification method for exosome miR-185, which is used for the early diagnosis of oral leukoplakia and oral cancer, thereby improving the early diagnosis rate of oral cancer and helping to improve the prognosis of patients. Summary of the invention

[0011] The purpose of the present invention is to quickly and accurately quantify exosomal miR-185 through the absolute quantitative method we established, thereby achieving early diagnosis of patients with oral leukoplakia and oral cancer.

[0012] Therefore, the present invention relates to:

[0013] 1. An absolute quantitative method for exosomal miR-185, mainly used for the early diagnosis of oral leukoplakia and oral cancer.

[0014] 2. The method of item 1, wherein the leukoplakia is simple hyperplasia of leukoplakia, leukoplakia with mild epithelial hyperplasia, leukoplakia with moderate epithelial hyperplasia, leukoplakia with severe epithelial hyperplasia

[0015] 3. The method of item 1 or 2, wherein the oral cancer is oral squamous cell carcinoma.

[0016] 4. The method of items 1-3, wherein the exosomes are derived from saliva or plasma of patients with oral leukoplakia and oral cancer.

[0017] 5. The methods of items 1-4 mainly involve the following processes: extraction of saliva and plasma exosome RNA, primer design and synthesis, synthesis of miR-185 standard substances, establishment of standard curve, selection of internal reference, reverse transcription and PCR reaction of miRNA, and statistical analysis of data. It provides a complete, reliable and standardized operating procedure for the absolute quantification of exosome miR-185, and provides the necessary conditions for the early diagnosis of oral leukoplakia and oral cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the melting curve of miR-185SYBR Green fluorescence quantitative PCR;

[0019] Figure 2 This is the standard curve of miR-185SYBR Green fluorescence quantitative PCR;

[0020] Figure 3 Scatter plot of the mean distribution of Ct values ​​of saliva and plasma exosomes

[0021] Figure 4 The scatter plots are for the expression levels of salivary exosome miR-185 in healthy people (N), leukoplakia simple hyperplasia (OLK), leukoplakia with mild-moderate epithelial dysplasia (LD, including mild and moderate), leukoplakia with severe epithelial dysplasia (HD), and oral squamous cell carcinoma (OSCC) patients;

[0022] Figure 5 This is a scatter plot of the expression levels of plasma exosomal miR-185 in patients with leukoplakia with mild to moderate epithelial dysplasia (LD, including mild and moderate), leukoplakia with severe epithelial dysplasia (HD), and oral squamous cell carcinoma without metastasis (OSCC). DETAILED DESCRIPTION

[0023] The present invention is described in conjunction with specific examples, but the embodiments do not limit the present invention in any form.

[0024] 1. Materials

[0025] Constant temperature high speed centrifuge Eppendorf, Germany

[0026] OptimaMAX-XP benchtop ultracentrifuge Beckman Coulter, USA

[0027] Ultracentrifuge tubes (343778) Beckman Coulter, USA

[0028] Ultra-micro-volume spectrophotometer ThermoFish, USA

[0029] T100 Thermal Cycler Bio-Rad, USA

[0030] Real-time quantitative PCR instrument Bio-Rad, USA

[0031] Bulge-Loop miRNAqRT-PCR StarterKit RiboBio, Guangzhou, China

[0032] Primer RiboBio, Guangzhou, China

[0033] miR-185 Reference Material, RiboBio, Guangzhou, China

[0034] TRIzon Reagen Kangwei Century, Beijing, China

[0035] PBS Biosharp, Beijing, China

[0036] Chloroform, isopropanol, anhydrous ethanol

[0037] 2. Sample Collection

[0038] Saliva collection: including the collection of saliva from healthy volunteers, and the collection of saliva from patients clinically diagnosed with leukoplakia or oral cancer before biopsy surgery. Eating, smoking, brushing teeth and gargling are prohibited within one hour before collecting saliva. The patient's body is slightly forward, allowing the saliva in the mouth to naturally gather and flow into the sample cup, and 2-3ml of non-irritating whole saliva is collected. Centrifuge at 3000rpm at 4℃ for 10 minutes, aspirate the supernatant, transfer to a 1.5mL centrifuge tube (1mL / tube), label the sample name, and store in a -80℃ refrigerator.

[0039] Plasma collection: Before biopsy surgery, plasma was collected from patients clinically diagnosed with leukoplakia or oral cancer. Blood was collected from the upper arm vein using a 5 ml plasma tube, centrifuged at 3000 rpm for 10 min at 4 °C, and the upper light yellow supernatant was taken and transferred to a 1.5 ml centrifuge tube (1 ml / tube), and stored in a -80 °C refrigerator.

[0040] Note: Biopsy was performed on the patients. If the histopathological diagnosis was leukoplakia or oral squamous cell carcinoma, the following operation was performed and the patients were divided into groups according to the histopathological results: simple hyperplasia of leukoplakia (OLK group), leukoplakia with mild to moderate epithelial dysplasia (LD group, including mild and moderate), leukoplakia with severe epithelial dysplasia (HD group), oral squamous cell carcinoma (OSCC group); in addition, healthy volunteers were group N.

[0041] 3. Extraction of Exosomes

[0042] Take out the saliva or plasma sample from the -80℃ refrigerator, slowly dissolve it on ice, centrifuge it at 2000×g for 30min at 4℃, discard the precipitate, take the supernatant and transfer it to a new centrifuge tube; centrifuge it at 12000×g for 45min at 4℃, discard the precipitate, take the supernatant and transfer it to an ultracentrifuge tube (1mL / tube); place the ultracentrifuge tube in an ultracentrifuge, centrifuge it at 110000×g for 70min at 4℃, discard the supernatant and keep the precipitate; add 1mL PBS to the precipitate and resuspend it by pipetting, and then ultracentrifuge it at 110000×g for 70min at 4℃, discard the supernatant, and the precipitate obtained is the exosome. Add 100μL PBS and resuspend it by pipetting, and transfer it to a new 1.5mL centrifuge tube. Use it immediately or freeze it at -80℃ for later use.

[0043] 4. Extraction of Exosomal RNA

[0044] The exosome solution was taken out from -80℃, slowly dissolved on ice, 500μL TRIzol was added, mixed, and allowed to stand at 15-30℃ for 5 min; then 100μL chloroform was added, mixed thoroughly by inverting, allowed to stand at 15-30℃ for 5 min, and centrifuged at 12000×g for 15 min at 2-8℃. After centrifugation, the solution was separated into three layers; the upper aqueous phase was transferred to a new centrifuge tube, 250μL isopropanol was added, mixed, and allowed to stand at 15-30℃ for 10 min. Centrifuge at 12000×g for 10 minutes at 2-8°C; remove the supernatant, add 500μL 80% ethanol, shake and mix, and centrifuge at 7500×g for 10 minutes at 2-8°C; remove the supernatant, place the centrifuge tube on ice, and dry the RNA in air; resuspend the RNA by pipetting with 10-20μL RNase-free water; measure the RNA concentration by ultra-micro spectrophotometer.

[0045] 5. Primer Design

[0046] Reverse transcription primers, upstream primers, and downstream universal primers were all designed and synthesized by RiboBio.

[0047] 6. Synthesis of miR-185 (hsa-miR-185-5p, human) standard material

[0048] The miR-185 standard material was synthesized by Ruibo Biotechnology, and the gene sequence was UGGAGAGAAAGGCAGUUCCUGA.

[0049] 7. Establishment of miR-185 quantitative standard curve

[0050] (1) Dissolution and packaging: The dry powder of miR-185 standard substance was centrifuged instantly, 1 nmol was added with 100 μL RNase-free water, and the mixture was shaken to fully dissolve, and a final concentration of 10 μM Standard miR-185 storage solution was prepared and stored in aliquots;

[0051] (2) Gradient dilution: Take 5uL of 10uM Standard miR-185 storage solution, add 45uL RNase-free Water, prepare gradient 1 sample with a concentration of 1uM, take 5uL of gradient 1 sample and add 45uL RNase-free Water to prepare gradient 2 sample with a concentration of 100nM, take 5uL of gradient N sample and add 45uL RNase-free Water to prepare gradient N+1 sample with a concentration one order of magnitude lower, select 5 gradients from 1fM to 100nM for miRNA qRT-PCR experiment;

[0052] (3) Reverse transcription reaction: Take 5 μL of the Standard miR-185 stock solution diluted in equal steps and mix with 2 μL of 5× Reverse Transtcription Buffer, 2 μL of RTase Mix, and 1 μL of Bulge-loop miR-185RT Primer (5 μM) to make a 10 μL system. Mix well, centrifuge briefly, and perform reverse transcription reaction according to the following procedure: 42°C for 60 min, 70°C for 10 min.

[0053] (4) Fluorescence quantitative PCR: 10 μL 2×SYBR Green Mix, 0.8 μL miR-185 upstream primer (5 μM), 0.8 μL downstream universal primer (5 μM), 2 μL reverse transcription product (cDNA), 6.4 μL ddH2O were prepared into a 20 μL PCR reaction system, and 3 replicate wells were set for each gradient; reaction conditions: pre-denaturation at 95°C for 10 min → amplification reaction at 95°C for 2 s, 60°C for 20 s, 70°C for 10 s, 40 cycles → melting curve analysis. Observe the melting curve of the primers to determine the specificity of the primers. See attached. Figure 1 As shown, all amplified products were single peaks, indicating that the primers had good specificity.

[0054] (5) Standard curve drawing:

[0055] Copy number calculation formula:

[0056] NA is Avogadro's constant: 6.02×1023 copies / mol, and the average molecular weight of miR-185 is 7144.39.

[0057] The calculated initial copy numbers for 100nM, 10nM, 1pM, 100pM, and 10pM were 6.02×10 10 copies / μL, 6.02×10 9 copies / μL, 6.02×10 8 copies / μL, 6.02×10 7 copies / μL, 6.02×10 6 The standard curve was drawn with the log value of the initial copy number of StandardmiR-185 storage solution as the horizontal axis (x) and the Ct value as the vertical axis (y). Figure 2 The calculation formula is: Ct mean = -2.977*initial copy number log value + 39.85, R 2 =0.9837, indicating that in 6.02×10 6 ~6.02×10 10The copies / μL had a good linear relationship.

[0058] 8. Reverse transcription reaction of RNA to be tested

[0059] (1) Take a certain amount (100 ng) of total RNA of the sample to be tested, mix it with 2 μL of 5× Reverse Transfection Buffer, 2 μL of RTase Mix, and 1 μL of Bulge-loop miR-185RT Primer (5 μM) to make a 10 μL system, mix well, and centrifuge briefly;

[0060] (2) Similarly, take an equal amount of total RNA from the sample to be tested, mix it with 2 μL of 5× Reverse Transfection Buffer, 2 μL of RTase Mix, and 1 μL of Bulge-loop U6 RT Primer (5 μM) to make a 10 μL system, mix well, and centrifuge briefly;

[0061] (3) Perform reverse transcription reaction according to the following procedure: 42°C for 60 min, 70°C for 10 min.

[0062] 9. Fluorescence quantitative PCR

[0063] (1) 10 μL 2×SYBR Green Mix, 0.8 μL miR-185 upstream primer (5 μM), 0.8 μL downstream universal primer (5 μM), 2 μL reverse transcription product (cDNA), and 6.4 μL ddH2O were prepared into a 20 μL PCR reaction system, and three replicate wells were set for each sample;

[0064] (2) 10 μL 2×SYBR Green Mix, 0.8 μL U6 upstream primer (5 μM), 0.8 μL U6 downstream primer (5 μM), 2 μL reverse transcription product (cDNA), and 6.4 μL ddH2O were prepared into a 20 μL PCR reaction system, and three replicate wells were set up for each sample;

[0065] (3) Reaction conditions: pre-denaturation at 95°C for 10 min → amplification at 95°C for 2 s, 60°C for 20 s, and 70°C for 10 s, 40 cycles → melting curve analysis.

[0066] 10. Absolute quantification

[0067] Compare the Ct value of the PCR reaction with the standard curve to accurately calculate the copy number or concentration of miRNA.

[0068] 11. Internal reference selection

[0069] According to previous literature, U6, miR-16, and hsa-let-7a-5p were selected as internal references, and reverse transcription and PCR reactions were performed respectively (same as steps 8 and 9). The average Ct value of each sample to be tested was statistically analyzed, and the most stable internal reference was selected for data correction.

[0070] 12. Data Correction and Statistical Analysis

[0071] (1) Calculate the copy number of miR-185 in total exosomal RNA of the same mass (100 ng) according to the standard curve formula:

[0072] Initial copy number log value = (Ct mean -39.85) / (-2.977)

[0073] Initial copy number = 10 (初始拷贝数log值)

[0074] (2) Data correction: mean Ct value of different sample internal references ( Figure 3 ) showed that the mean Ct values ​​of salivary exosomes U6, miR-16, and hsa-let-7a-5p (expressed in the form of Mean±SD) were 32.24±3.00, 37.4794±0.96, and 36.75±0.69, respectively, and the mean Ct value of plasma exosomes hsa-let-7a-5p was 36.57±0.64, indicating that hsa-let-7a-5p has better stability than U6 and miR-16 as an internal reference of salivary and plasma exosome miRNA. The present invention selects hsa-let-7a-5p as an internal reference for data correction. The average value of the hsa-let-7a-5pCt value of each sample was calculated, and the initial copy number obtained by the standard curve formula was corrected. The calculation formula is as follows:

[0075] Correction factor = 1 / 2 [(average of all hsa-let-7a-5p Ct values) - (average of the Ct values ​​of the samples to be tested)]

[0076] The absolute copy number of miR-185 in the same mass (100 ng) of total exosomal RNA = correction factor × 10 (初始拷贝数log值)

[0077] (3) Statistical analysis:

[0078] The corrected absolute copy numbers of salivary exosomal miR-185 were: 1154111-4550992 copies / μL in the N group, 1123199-17826525 copies / μL in the OLK group, 861791-21683577 copies / μL in the LD group, 411709-4025327 copies / μL in the HD group, and 148948-7677804 copies / μL in the OSCC group.

[0079] The absolute copy number of plasma exosomal miR-185 after correction: 813785-19624060 copies / μL in the LD group, 144145-2381163 copies / μL in the HD group, and 85329-5620309 copies / μL in the OSCC group. (Note: Due to the lack of clinical samples in the white spot simple hyperplasia group, this example only shows the plasma exosomal miR-185 results of the LD, HD, and OSCC groups. This part of the data will be supplemented when the number of samples increases in the future.)

[0080] For data that conformed to normal distribution and had homogeneous variance, one-way analysis of variance was used, and for data that did not conform to normal distribution, non-parametric tests such as rank sum test and Kruskal-Wallis test were performed. SPSS.20 software was used for statistical analysis, and the test level α = 0.05.

[0081] like Figure 4 As shown in the figure, the expression level of salivary exosome miR-185 in the N, OLK and LD groups was higher than that in the HD and OSCC groups, among which the LD group was significantly higher than that in the HD group (P < 0.05); Figure 5 As shown in the figure, the expression level of plasma exosomal miR-185 in the LD group was higher than that in the HD and OSCC groups, among which the LD group was significantly higher than that in the HD group (P<0.05).

[0082] References:

[0083] 1. Noguti J, De Moura CF, De Jesus GP, et al. Metastasis from oral cancer: an overview. Cancer Genomics Proteomics 2012;9:329-35.

[0084] 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.

[0085] 3.Sarode G,Maniyar N,Sarode SC,et al.Epidemiologic aspeCts of oral cancer[J].Dis Mon,2020,66(12):100988.

[0086] 4. Lin Mei. Clinical detection and risk assessment of oral precancerous lesions [J / CD]. Chinese Journal of Stomatological Research (Electronic Edition), 2008, 2(5): 427-430.

[0087] 5. Warnakulasuriya S. Oral potentially malignant disorders: Acomprehensive review on clinical aspeCts and management[J]. Oral Oncol, 2020,102:104550.

[0088] 6. 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 proliferativeaCtivity. ACta Otolaryngol 2003;123:767-75.

[0089] 7. Thompson L. World Health Organization classification of tumours: pathology and genetics of head and neck tumours. EarNose Throat J 2006;85:74.

[0090] 8.Weng Z,Zhang B,Wu C,et al.Therapeutic roles of mesenchymalstemcell-derived extracellular vesicles in cancer[J].J Hematol Oncol,2021,14(1):136.

[0091] 9.Salimi L,Akbari A,Jabbari N,et al.Synergies in exosomes andautophagy pathways for cellular homeostasis and metastasis of tumor cells[J].Cell Biosci,2020,10:64.

[0092] 10.Zhang J,Li S,Li L,et al.Exosome and exosomal microRNA:trafficking,sorting,and funCtion[J].Genomics Proteomics Bioinformatics,2015,13(1):17-24.

[0093] 11.Zhu G,He Y,Yang S,et al.Identification of Gene and MicroRNASignatures for Oral Cancer Developed from Oral Leukoplakia.Biomed ResInt2015;2015:841956.

[0094] 12.Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,andfunCtion.Cell 2004;116:281-97.

[0095] 13.Cervigne NK,Reis PP,Machado J,et al.Identification ofa microRNAsignature associated with progression ofleukoplakia to oral carcinoma.Hum MolGenet 2009;18:4818-29.

[0096] 14.Gorenchtein M,Poh CF,Saini R,et al.MicroRNAs in an oralcancercontext-from basic biology to clinical utility.J Dent Res 2012:91:440-6.

[0097] 15.Wu BH,Xiong XP,Jia J,et al.MicroRNAs:new actors in the oralcancerscene.Oral Oncol 2011:47:314-9.

[0098] 16.Li J,Huang H,Sun L,et al.MiR-21 indicates poorprognosis of tongue squamous cell carcinomas as an inhibitor of apoptosis[J].ClinCancer Res,2009,15(12):3998-4008.doi:10.1158 / 1078-0432.CCR-08-3053.

[0099] 17.Liu X,Wang A,Heidbreder CE,et al.MicroRNA-24 targetingRNAbinding protein DND1 in tongue squamous cell carcinoma[J].FEBS Lett,2010,584(18):4115-4120.doi:10.1016 / j.febslet.2010.08.040.

[0100] [ PubMed ] 18. Lu YC, Chang JT, Liao Ct, et al.OncomiR-196 promotes noninvasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway[J].Mol Cancer,2014,13:218.doi:10.1186 / 1476-4598-13-218.

[0101] 19.Tiwari A,Shivananda S,Gopinath KS,et al.MicroRNA-125areducesproliferation and invasion of oral squamous cell carcinoma cells bytargetingestrogen-related receptor alpha:IMPLICATIONS FOR CANCERTHERAPEUTICS[J].J Biol Chem,2014,289(46):32276-32290.doi:10.1074 / jbc.M114.584136.

[0102] 20.Chang CJ,Hsu CC,Chang CH,et al.Let-7d funCtions as novelregulatorof epithe lial-mesenchymal transition and chemoresistant property inoralcancer[J].Oncol Rep,2011,26(4):1003-1010.doi:10.3892 / or.2011.1360.

[0103] 21.Kim JS,Park SY,Lee SA,et al.MicroRNA-205 suppressestheoralcarcinoma oncogenic aCtivity via down-regulation of Axin-2 in KBhumanoral cancer cell[J].Mol Cell Biochem,2014,387(1-2):71-79.doi:10.1007 / s11010-013-1872-7.

[0104] 22.Momen-Heravi F,TrachtenbergAJ,Kuo WP,et al.Genomewide studyofsalivary MicroRNAs for deteCtion of oral cancer[J].J Dent Res,2014,93(7suppl):86S-93S.doi:10.1177 / 0022034514531018.

[0105] 23.MiR-185 inhibits cell proliferation and invasion ofnon-small celllungcancerby targeting KLF7.[J].Oncology Research E-pub,01 May 2018.

[0106] 24.miR-185 inhibits cell migration and invasion ofhepatocellularcarcinoma through CDC42[J].Oncology Letters,2018,16(3):3101-3107.

[0107] 25.MicroRNA-185 inhibits proliferation,migration and invasion inhuman osteosarcoma MG63 cells by targeting vesicle-associated membraneprotein 2[J] Gene. 2019,696:80-87.

[0108] 26.XIST promotes gastric cancer(GC)progression through TGF-β1 viatargeting miR-185[J].Journal ofCellular Biochemistry,2017,119(3):2787-2796.

[0109] 27.miRNA-185 serves as a prognostic faCtor and suppresses migrationand invasion through Wnt1 in colon cancer[J].European Journal ofPharmacology,2018:S0014299918300979.

[0110] 28.Byun JS,Hong SH,Choi JK,Jung JK,Lee HJ.Diagnostic profiling ofsalivary exosomal microRNAs in oral lichen planus patients.Oral Dis.2015;21:987–93.

[0111] 29.Michael A,Bajracharya SD,Yuen PS,Zhou H,Star RA,Illei GG,AlevizosI.Exosomes from human saliva as a source of microRNA biomarkers.OralDis.2010;16:34–8

[0112] 30.Occhipinti,G.,et al.,The choice of endogenous controls in exosomalmicroRNA assessments from biofluids.Tumour Biol,2016.37(9):p.11657-11665.

[0113] 31.Li Y,Zhang L,Liu F,Xiang G,Jiang D,Pu X.Identification ofendogenous controls for analyzing serum exosomal miRNA in patients withhepatitis B orhepatocellular carcinoma.Dis Markers.2015;2015:893594.

[0114] 32.Dai,Y.,et al.,Unbiased RNA-Seq-driven identification andvalidation of reference genes for quantitative RT-PCR analyses ofpooledcancer exosomes.BMC Genomics,2021.22(1):p.27.

Claims

1. An absolute quantitative method for exosomal miR-185, which is mainly used for the early diagnosis of oral leukoplakia and oral cancer.

2. The method of claim 1, wherein the leukoplakia is simple hyperplasia of leukoplakia, leukoplakia with mild epithelial dysplasia, leukoplakia with moderate epithelial dysplasia, or leukoplakia with severe epithelial dysplasia.

3. The method of claim 1 or 2, wherein the oral cancer is oral squamous cell carcinoma.

4. The method of claims 1-3, wherein the exosomes are derived from saliva or plasma of patients with oral leukoplakia and oral cancer.

5. The method of claims 1-4 mainly involves the following processes: extraction of saliva and plasma exosome RNA, primer design and synthesis, synthesis of miR-185 standard substance, establishment of standard curve, selection of internal reference, reverse transcription and PCR reaction of miRNA, and data statistical analysis.

Images