Medicine for preventing and treating melanoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a reagent for early diagnosis of melanoma and a prevention and treatment medicine. According to the reagent for early diagnosis of melanoma provided by the invention, ADAM19, LINC00511 and / or miR-150-5p are taken as molecular markers for clinical diagnosis, so that early accurate diagnosis of melanoma can be realized, and the survival rate and life quality of patients can be improved. The medicine for preventing and treating melanoma, provided by the invention, comprises a medicine effect component for inhibiting the expression of ADAM19 and / or LINC00511 and a medicine effect component for promoting the expression of miR-150-5p. Living cell experiment results show that the medicinal components can effectively inhibit proliferation, migration and invasion of melanoma cells, and animal experiment results show that the medicinal components can reduce the weight of a melanoma body, reduce the volume of the melanoma body and slow down the growth speed of the melanoma body.

Description

[0001] This application is a divisional application with application date of August 2, 2022, application number 202210923564.7, and invention name “A reagent and preventive and therapeutic drug for early diagnosis of melanoma”. Technical Field

[0002] The present invention belongs to the technical field of biomedicine, and specifically relates to a reagent for early diagnosis of melanoma, and a medicine for preventing and treating melanoma. Background Art

[0003] Melanoma is a highly invasive, highly malignant, highly metastatic and difficult to cure tumor. As the most invasive skin tumor, the incidence of malignant melanoma has been increasing year by year in the world in recent years. Melanoma is a highly malignant tumor originating from ectoderm neural crest melanocytes, which is very prone to invasion and metastasis, with poor clinical prognosis and high mortality. About 90% of melanomas occur on the skin, such as the head and neck, chest, back and abdomen of the trunk, and limbs. Most of them develop from pigmented nevi. In addition, they may also occur in mucosal tissues such as the nasal cavity, digestive tract, urogenital tract and choroid. The survival rate of early melanoma can reach 90% through surgical treatment, but malignant melanoma has a very strong ability to invade and metastasize, and it is very easy to metastasize early. Once metastasis occurs, the cure rate of patients will be very low.

[0004] In recent years, melanoma has become one of the fastest growing malignant tumors, with an annual growth rate of 3-5%. Data show that the male-female ratio of malignant melanoma in my country is 1.12:1, and the median age of diagnosis is 53, of which 17.8% are elderly patients. The increasing incidence and mortality of malignant melanoma have seriously threatened human health. Clinically, breakthrough progress has been made in related targeted therapy and biological immunotherapy methods such as mitogen-activated protein kinase (MAPK) pathway inhibitors, anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) / programmed cell death ligand 1 (PD-L1) pathway blockers, which have broken the limitations of traditional treatment to a certain extent and greatly improved the prognosis of melanoma patients, but the treatment effect on patients with recurrent and metastatic melanoma is still poor. Therefore, in-depth exploration of the pathogenesis and regulatory mechanisms of melanoma and the discovery of key molecules that can effectively intervene in the invasion and metastasis of melanoma can provide new strategies for clinical treatment and are of great significance for finding more effective, durable and personalized treatment plans.

[0005] ADAM19, also known as meltrin-β, is a catalytically active metalloprotease that can cleave many typical ADAM substrates in vitro, including KL1 and TNFα. ADAM19 has also been found to be involved in the progression of tumors such as glioma and colorectal cancer (Bin Qi et al., 2009). In addition, ADAM19 has been reported to be associated with the invasion and proliferation of retinoblastoma (Zhe Sun et al., 2015).

[0006] As we all know, most human genes can be transcribed, but most of the products are non-coding RNA (ncRNA). Some ncRNAs can regulate life activities by affecting genes and epigenetics. Long non-coding RNA (lncRNA) and microRNA (miRNA) are the two most important types, and they play important roles in many life activities.

[0007] lncRNA is a type of RNA molecule with a transcript length greater than 200 nt, lacking an effective open reading frame, and not encoding proteins. It is transcribed and synthesized by RNA polymerase II, mainly distributed in the cell nucleus, and a small amount exists in the cytoplasm. Studies have found that lncRNAs tend to fold into thermodynamically stable secondary or higher structures to perform their functions. Some lncRNAs are spliced ​​into structures with polyA tails and promoters similar to mRNAs, and they can be dynamically expressed and spliced ​​in different ways during the differentiation of tissues and organs. Therefore, lncRNAs have temporal and spatial specificity. At present, lncRNAs can be divided into five categories according to their transcriptional locations: lncRNAs located in intergenic regions, natural antisense lncRNAs, intronic lncRNAs, sense lncRNAs, and bidirectional lncRNAs.

[0008] miRNA is a type of non-coding short sequence RNA with a length of 18-25 nt and no open reading frame. It is widely present in eukaryotes. Mature miRNA has a phosphate group at the 5′ end and a hydroxyl group at the 3′ end, which distinguishes it from most oligonucleotides and degradation fragments of functional RNA. The vast majority of miRNAs are highly conservative, sequential, and organizational. Many studies have shown that miRNA is an important link in the function of lncRNA. In the pathological cells of many diseases, it is found that a certain lncRNA has a certain quantitative relationship with certain miRNAs, and their quantitative changes are closely related to the occurrence and development of related diseases (Je-HyunYoon et al., 2014). Both lncRNA and miRNA have their own regulatory networks, but the regulatory networks of the two do not exist independently. In many cases, the regulation of lncRNA and miRNA in the occurrence and development of a disease is interdependent and intertwined, forming a complex regulatory network together.

[0009] Studies have shown that certain genes are associated with melanoma, such as SPINK5, which is differentially expressed in clinical tissue samples of skin malignant melanoma. Therefore, finding more new targets for melanoma is of great significance for the biological diagnosis and treatment of melanoma. Summary of the invention

[0010] The main technical problem solved by the present invention is to improve the accuracy of early diagnosis of melanoma. At the same time, a medicine for preventing and treating melanoma is provided.

[0011] In order to solve the above technical problems, the present invention provides a reagent for early diagnosis of melanoma, comprising one or more of the following:

[0012] (1) Reagents for detecting the expression level of the ADAM19 gene;

[0013] (2) Reagents for detecting the expression level of lncRNA that regulates ADAM19 gene expression;

[0014] (3) Reagents for detecting the expression level of miRNA that regulates ADAM19 gene expression;

[0015] The lncRNA is ENSG00000227036 (LINC00511);

[0016] The miRNA is miR-150-5p.

[0017] Specifically, the accession number of the ADAM19 gene is ENSG00000135074 (Ensembl database); the accession number of the LINC00511 is ENSG00000227036 (Ensembl database); the accession number of the miR-150-5p is ENSG00000207782 (Ensembl database).

[0018] As a preferred embodiment of the present invention, the reagent for detecting the expression level of the ADAM19 gene is a quantitative PCR reagent and / or an immunodetection reagent. The quantitative PCR reagent includes primers for specific amplification of the ADAM19 gene. The immunodetection reagent includes an antibody or polypeptide that specifically binds to the ADAM19 protein.

[0019] Preferably, the primer sequences for specifically amplifying the ADAM19 gene are shown in SEQ ID NOs. 1 to 2.

[0020] As a preferred embodiment of the present invention, the reagent for detecting the expression level of lncRNA regulating the expression of ADAM19 gene is a quantitative PCR reagent. The quantitative PCR reagent includes primers for specific amplification of ENSG00000227036 (LINC00511).

[0021] Preferably, the primer sequences for specific amplification of ENSG00000227036 (LINC00511) are shown in SEQ ID NOs. 3 to 4.

[0022] As a preferred embodiment of the present invention, the reagent for detecting the expression level of miRNA regulating ADAM19 gene expression is a quantitative PCR reagent. The quantitative PCR reagent includes primers for specific amplification of miR-150-5p.

[0023] Preferably, the primer sequences for specifically amplifying miR-150-5p are shown in SEQ ID NOs. 23 to 24.

[0024] As a preferred embodiment of the present invention, the reagent for early diagnosis of melanoma also includes a reagent for detecting the expression level of an internal reference gene (such as GAPDH, etc.) The reagent is a quantitative PCR reagent, including primers for specific amplification of the internal reference gene.

[0025] Preferably, the primer sequences for specifically amplifying the internal reference gene GAPDH are shown in SEQ ID NOs. 5 to 6.

[0026] The present invention provides a reagent for early diagnosis of melanoma, which uses ADAM19, LINC00511 and / or miR-150-5p as molecular markers for clinical diagnosis, can achieve accurate early diagnosis of melanoma, and is beneficial to improving the survival rate and quality of life of patients. When ADAM19, LINC00511 and miR-150-5p are used as diagnostic markers together, it is also beneficial to solve the problem of false positive or false negative in the single detection of ADAM19, LINC00511 or miR-150-5p.

[0027] The present invention also provides a drug for preventing and treating melanoma, comprising one or more of the following:

[0028] (1) Drugs that inhibit ADAM19 expression;

[0029] (2) drugs that inhibit LINC00511 expression;

[0030] (3) Drugs that promote the expression of miR-150-5p.

[0031] As a preferred embodiment of the present invention, the drug for inhibiting the expression of ADAM19 is siRNA, interference vector and the like.

[0032] Preferably, the drug for inhibiting ADAM19 expression is Si-ADAM19-1 and / or Si-ADAM19-2, the sequence of Si-ADAM19-1 is shown in SEQ ID NOs.7 to 8, and the sequence of Si-ADAM19-2 is shown in SEQ ID NOs.9 to 10.

[0033] As a preferred embodiment of the present invention, the drug for inhibiting the expression of LINC00511 is siRNA, interference vector, etc.

[0034] Preferably, the drug for inhibiting the expression of LINC00511 is Si-LINC00511-1 and / or Si-LINC00511-2, the sequence of Si-LINC00511-1 is shown in SEQ ID NOs.13 to 14, and the sequence of Si-LINC00511-2 is shown in SEQ ID NOs.15 to 16.

[0035] As a preferred embodiment of the present invention, the drug that promotes the expression of miR-150-5p is miRNA mimics, overexpression vector and the like.

[0036] Preferably, the drug that promotes the expression of miR-150-5p is miR-150-5p mimics, and the sequence of the miR-150-5pmimics is shown in SEQ ID NOs. 19-20.

[0037] The present invention provides a drug for preventing and treating melanoma, including a pharmacological component that inhibits the expression of ADAM19 and / or LINC00511 and a pharmacological component that promotes the expression of miR-150-5p. The results of in vivo cell experiments show that the above-mentioned pharmacological components can effectively inhibit the proliferation, migration and invasion of melanoma cells, and the results of animal experiments show that the above-mentioned pharmacological components can reduce the tumor weight of melanoma, reduce the tumor volume, and slow down the growth rate of the tumor. In summary, the pharmacological components that inhibit the expression of ADAM19 and / or LINC00511 and the pharmacological components that promote the expression of miR-150-5p can effectively inhibit the further development of melanoma, and may play a certain therapeutic effect on the treatment of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the mRNA expression level of ADAM19 in different melanoma cells in Experimental Example 1;

[0039] Figure 2The mRNA expression level of long noncoding RNA LINC00511 in different melanoma cells in Experimental Example 1;

[0040] Figure 3 The effect of silencing LINC00511 on the expression level of LINC00511 in Experimental Example 2;

[0041] Figure 4 This is the effect of silencing LINC00511 on the expression level of ADAM19 in Experimental Example 2;

[0042] Figure 5 This is the effect of silencing LINC00511 on the expression level of miR-150-5p in Experimental Example 2;

[0043] Figure 6 The effect of overexpression of LINC00511 on the expression level of LINC00511 in Experimental Example 2;

[0044] Figure 7 The effect of overexpression of LINC00511 on the expression level of ADAM19 in Experimental Example 2;

[0045] Figure 8 The effect of overexpression of LINC00511 on the expression level of miR-150-5p in Experimental Example 2;

[0046] Fig. 9 The effect of silencing ADAM19 on the expression level of ADAM19 in Experimental Example 2;

[0047] Fig.10 The effect of silencing ADAM19 on the expression level of miR-150-5p in Experimental Example 2;

[0048] Fig.11 The effect of overexpression of ADAM19 on the expression level of miR-150-5p in Experimental Example 2;

[0049] Fig.12 The effect of inhibiting or overexpressing miR-150-5p on the expression level of miR-150-5p in Experimental Example 2;

[0050] Fig.13 The effect of inhibiting miR-150-5p on the expression level of ADAM19 in Experimental Example 2;

[0051] Fig.14 The effect of overexpression of miR-150-5p on the expression level of ADAM19 in Experimental Example 2;

[0052] Fig.15This is the effect of silencing LINC00511 and overexpressing ADAM19 on the expression level of ADAM19 in Experiment 2;

[0053] Fig.16 The effect of silencing ADAM19 and inhibiting miR-150-5p on the expression level of ADAM19 in Experimental Example 2;

[0054] Fig.17 This is the effect of silencing LINC00511 and inhibiting miR-150-5p on the expression level of ADAM19 in Experimental Example 2;

[0055] Fig.18 The effect of silencing LINC00511 on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0056] Fig.19 The effect of overexpression of LINC00511 on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0057] Fig. 20 The effect of silencing ADAM19 on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0058] Fig.21 The effect of overexpression of ADAM19 on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0059] Fig. 22 The effect of simultaneous overexpression of ADAM19 and miR-150-5p on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0060] Fig.23 The effect of silencing ADAM19 and inhibiting miR-150-5p on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0061] Fig.24 The effect of simultaneous overexpression of LINC00511 and miR-150-5p on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0062] Fig.25 This is the effect of silencing LINC00511 and inhibiting miR-150-5p on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0063] Fig.26The effect of silencing LINC00511 and overexpressing ADAM19 on the proliferation of human melanoma cells A375 and SK-Mel-28 in Experimental Example 3;

[0064] Fig. 27 The effect of silencing ADAM19 on the migration of human melanoma cells A375 and SK-Mel-28 in Experimental Example 4;

[0065] Fig.28 This is the effect of silencing LINC00511 on the migration of human melanoma cells A375 and SK-Mel-28 in Experimental Example 4.

[0066] Fig.29 The results of the analysis of the differential expression of ADAM19 and LINC00511 in melanoma patients and normal people in Experimental Example 5;

[0067] Fig.30 The effect of silencing ADAM19 on the tumor weight, volume and growth rate of tumor-bearing mice in Experimental Example 6;

[0068] Fig.31 This is the effect of silencing LINC00511 on tumor weight, volume and growth rate of tumor-bearing mice in Experimental Example 6. DETAILED DESCRIPTION

[0069] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the embodiments described below are only used to illustrate the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, such as the embodiments obtained after modification or replacement, belong to the scope of protection of the present invention.

[0070] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the raw materials, reagents, instruments, etc. used are all available through commercial channels unless otherwise specified.

[0071] Example 1

[0072] This embodiment provides a reagent for early diagnosis of melanoma, comprising:

[0073] (1) A quantitative PCR reagent for detecting the expression level of the ADAM19 gene, comprising primers for specifically amplifying the ADAM19 gene, the sequences of which are shown in SEQ ID NOs. 1 to 2;

[0074] (2) A quantitative PCR reagent for detecting the expression level of ENSG00000227036 (LINC00511) that regulates the expression of the ADAM19 gene, including primers for specific amplification of LINC00511, the sequences of which are shown in SEQ ID NOs. 3 to 4;

[0075] (3) A quantitative PCR reagent for detecting the expression level of miR-150-5p that regulates ADAM19 gene expression, including primers for specific amplification of miR-150-5p, the sequences of which are shown in SEQ ID NOs. 23 to 24;

[0076] (4) A quantitative PCR reagent for detecting the expression level of the internal reference gene GAPDH, comprising primers for specifically amplifying the internal reference gene GAPDH, the sequences of which are shown in SEQ ID NOs. 5 to 6.

[0077] Example 2

[0078] This embodiment provides a reagent for early diagnosis of melanoma, comprising:

[0079] (1) A quantitative PCR reagent for detecting the expression level of the ADAM19 gene, comprising primers for specifically amplifying the ADAM19 gene, the sequences of which are shown in SEQ ID NOs. 1 to 2;

[0080] (2) A quantitative PCR reagent for detecting the expression level of ENSG00000227036 (LINC00511) that regulates the expression of the ADAM19 gene, including primers for specific amplification of LINC00511, the sequences of which are shown in SEQ ID NOs. 3 to 4;

[0081] (3) A quantitative PCR reagent for detecting the expression level of the internal reference gene GAPDH, comprising primers for specifically amplifying the internal reference gene GAPDH, the sequences of which are shown in SEQ ID NOs. 5 to 6.

[0082] In other embodiments of the present invention, the reagent for early diagnosis of melanoma may include one or more of the following:

[0083] (1) A quantitative PCR reagent for detecting the expression level of the ADAM19 gene, comprising primers for specifically amplifying the ADAM19 gene, the sequences of which are shown in SEQ ID NOs. 1 to 2;

[0084] (2) A quantitative PCR reagent for detecting the expression level of ENSG00000227036 (LINC00511) that regulates the expression of the ADAM19 gene, including primers for specific amplification of LINC00511, the sequences of which are shown in SEQ ID NOs. 3 to 4;

[0085] (3) A quantitative PCR reagent for detecting the expression level of miR-150-5p that regulates ADAM19 gene expression, including primers for specific amplification of miR-150-5p, the sequences of which are shown in SEQ ID NOs. 23 to 24;

[0086] (4) A quantitative PCR reagent for detecting the expression level of the internal reference gene GAPDH, comprising primers for specifically amplifying the internal reference gene GAPDH, the sequences of which are shown in SEQ ID NOs. 5 to 6.

[0087] Example 3

[0088] This embodiment provides a drug for preventing and treating melanoma, comprising:

[0089] (1) A drug for inhibiting the expression of ADAM19, specifically Si-ADAM19-1, the sequence of which is shown in SEQ ID NOs. 7 to 8;

[0090] (2) a drug that inhibits the expression of LINC00511, specifically Si-LINC00511-1, the sequence of which is shown in SEQ ID NOs. 13 to 14;

[0091] (3) A drug that promotes the expression of miR-150-5p, specifically miR-150-5p mimics, wherein the sequence of the miR-150-5p mimics is shown in SEQ ID NOs. 19 to 20.

[0092] Example 4

[0093] This embodiment provides a drug for preventing and treating melanoma, comprising:

[0094] (1) A drug for inhibiting the expression of ADAM19, specifically Si-ADAM19-2, the sequence of which is shown in SEQ ID NOs. 9 to 10;

[0095] (2) a drug that inhibits the expression of LINC00511, specifically Si-LINC00511-2, the sequence of which is shown in SEQ ID NOs. 15 to 16;

[0096] (3) A drug that promotes the expression of miR-150-5p, specifically miR-150-5p mimics, wherein the sequence of the miR-150-5p mimics is shown in SEQ ID NOs. 19 to 20.

[0097] Example 5

[0098] This embodiment provides a drug for preventing and treating melanoma, comprising:

[0099] (1) A drug for inhibiting the expression of ADAM19, specifically Si-ADAM19-1, the sequence of which is shown in SEQ ID NOs. 7 to 8;

[0100] (2) A drug that inhibits the expression of LINC00511, specifically Si-LINC00511-2, the sequence of which is shown in SEQ ID NOs. 15 to 16.

[0101] In other embodiments of the present invention, the drug for preventing and treating melanoma may include one or more of the following:

[0102] (1) A drug that inhibits the expression of ADAM19, specifically Si-ADAM19-1 and / or Si-ADAM19-2, wherein the sequence of Si-ADAM19-1 is shown in SEQ ID NOs. 7 to 8, and the sequence of Si-ADAM19-2 is shown in SEQ ID NOs. 9 to 10;

[0103] (2) a drug that inhibits the expression of LINC00511, specifically Si-LINC00511-1 and / or Si-LINC00511-2, wherein the sequence of Si-LINC00511-1 is shown in SEQ ID NOs. 13 to 14, and the sequence of Si-LINC00511-2 is shown in SEQ ID NOs. 15 to 16;

[0104] (3) A drug that promotes the expression of miR-150-5p, specifically miR-150-5p mimics, wherein the sequence of the miR-150-5p mimics is shown in SEQ ID NOs. 19 to 20.

[0105] Experimental Example 1

[0106] The expression of ADAM19 gene and the non-coding RNA LINC00511 that regulates its expression were detected in human melanoma cells A375, SK-Mel-28 and human normal skin melanocytes PIG1.

[0107] 1. Experimental subjects

[0108] The experiment was conducted on human melanoma cells A375, SK-Mel-28 and human skin normal melanocytes PIG1.

[0109] 2. Extract cellular RNA using Trizol method

[0110] (1) Preparation: Precool the high-speed centrifuge to 4°C in advance; pipette tips and EP tubes must be sterile and enzyme-free.

[0111] (2) Trizol treatment: Take the treated 6-well plate, aspirate the cell culture medium, wash twice with PBS, add 1 mL Trizol / well, lyse at room temperature for 1 min, then use a pipette to blow the cells off, collect them in EP tubes, and place them at room temperature for 5 min (be careful to blow off the cells at the edge when blowing).

[0112] Tissue samples can be homogenized by adding Trizol, and then ground with a glass homogenizer until the tissue blocks are fully lysed: 1200rpm, 5 cycles, 40s per cycle, 10s interval (the specific steps can be adjusted according to the difficulty of tissue lysis). The subsequent steps are the same as for cell samples. The amount of Trizol used is generally 1mL, which can process at least 25cm long. 2 Cells or 200 mg of tissue per cell culture flask.

[0113] Note: If RNA is not to be extracted temporarily, it can be stored at -80℃.

[0114] (3) Add 0.2 mL of chloroform, mix by inversion 10 times, shake vigorously for 30 seconds, and leave at room temperature for 2 minutes.

[0115] (4) Centrifuge at 4°C and 12,000 rpm for 15 min. At this point, stratification will occur. Transfer the upper aqueous phase to a new EP tube, taking care not to touch the white material.

[0116] (5) Add 0.5 mL of isopropanol, invert to mix, and let stand at room temperature for 10 min.

[0117] (6) Centrifuge at 4°C and 12,000 rpm for 10 min. A white precipitate will appear.

[0118] (7) Discard the supernatant, but be careful not to discard the precipitate. Then add 0.5 mL of 75% ethanol (pre-cooled with DEPC water) and shake the tube upside down until the white precipitate floats.

[0119] (8) Centrifuge at 4°C, 7500 rpm for 5 min.

[0120] (9) Repeat steps (7) and (8).

[0121] (10) Discard the supernatant, turn the EP tube upside down on filter paper, and use the filter paper strip to absorb the water around the precipitate and the remaining water on the tube wall.

[0122] (11) Add 20 μL DEPC water to dissolve. Then measure the RNA concentration and proceed to the next step or store in a -80°C refrigerator.

[0123] 3. Reverse transcription to synthesize cDNA

[0124] (1) Removal of genomic DNA

[0125] The reaction system is shown in Table 1.

[0126] Table 1 Reaction system for removing genomic DNA

[0127] Reagents Usage 5×gDNAEraserBuffer 2.0μL gDNAEraser 1.0μL RNA 1.0 μg <![CDATA[RNaseFreedH2O]]> Add to 10 μL

[0128] Reaction conditions: 42°C, 2 min; stored at 4°C to obtain the first step reaction solution.

[0129] (2) Reverse transcription

[0130] The reaction system is shown in Table 2.

[0131] Table 2 Reaction system for reverse transcription synthesis of cDNA

[0132] Reagents Usage First step reaction solution 10μL PrimeScriptRTEnzymeMixI 1.0μL RTPrimerMix 1.0μL 5×PrimeScriptBuffer2 4.0μL <![CDATA[RNaseFreedH2O]]> 4.0μL Total volume 20μL

[0133] Reaction conditions: 37°C, 15 min; 85°C, 5 s; stored at 4°C.

[0134] 4. Fluorescence quantitative PCR detection

[0135] The corresponding primers were used for fluorescence quantitative PCR detection, and the △△Ct method was used to process the fluorescence quantitative PCR data to calculate the expression changes of ADAM19 and Linc00511. The results are as follows Figures 1-2 shown.

[0136] The primer sequences are shown in SEQ ID NOs. 1 to 6.

[0137] Primers for ADAM19 are:

[0138] Forward: 5′-atggcgagtgtagtgctgc-3′; Reverse: 5′-gatcactgtcacgtctttcgt-3′.

[0139] The primers for Linc00511 are:

[0140] Forward: 5′-ctgcttgggtgtgggactga-3′; Reverse: 5′-cgatgaggtcatacgccgtaa-3′.

[0141] The primers for GAPDH are:

[0142] Forward: 5′-tgaaggtcggagtcaacggatt-3′; Reverse: 5′-cctggaagatggtgatgggatt-3′.

[0143] 5. Results Analysis

[0144] from Figures 1-2 It can be seen that compared with normal human skin melanocytes PIG1, the mRNA expression levels of Linc00511 and ADAM19 in human melanoma cells A375 and SK-Mel-28 were significantly upregulated, and the differences were statistically significant.

[0145] Experimental Example 2

[0146] 1. Experimental subjects

[0147] The experiment was conducted on human melanoma cells A375 and SK-Mel-28.

[0148] 2. Experimental Materials

[0149] a. Cell transfection reagent:

[0150] Lipofectamine TM 2000 Transfection Reagent was purchased from Invitrogen.

[0151] b. Main solution:

[0152] Cell culture medium: DMEM medium + 10% standard fetal bovine serum.

[0153] PBS (balanced salt solution): Dissolve 8 g NaCl, 0.25 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water, adjust the pH of the solution to 7.4 with HCl, add water to make up to 1 L, sterilize by high pressure, and store at room temperature.

[0154] 0.25% trypsin digestion solution: Add 0.25 g trypsin to 100 mL deionized water, filter sterilize, and aliquot for later use.

[0155] c. Sequence design and vector synthesis:

[0156] The Linc00511, ADAM19, and miR-150-5p sequences were sent to Jima Genetics, which chemically synthesized Si-LINC00511, Si-ADAM19, miR-150-5p mimics, miR-150-5p inhibitor, and nonspecific controls; Jiutian Genetics synthesized ADAM19 overexpression vector and LINC00511 overexpression vector.

[0157] The designed and synthesized siRNA, miRNAmimics, and miRNAinhibitor sequences are shown in SEQ ID NOs. 7 to 22.

[0158] (1) ADAM19

[0159] Si-ADAM19-1: sense strand: 5′-gcaagggccaacaccuuautt-3′; antisense strand: 5′-auaagguguuggcccuugctt-3′.

[0160] Si-ADAM19-2: sense strand: 5′-gcuccuuccuacacagaaatt-3′; antisense strand: 5′-uuucuguguaggaaggacctt-3′.

[0161] Si-NC: sense strand: 5′-uucuccgaacgugucacgutt-3′; antisense strand: 5′-acgugacacguucggagaatt-3′.

[0162] (2) Linc00511

[0163] Si-LINC00511-1:

[0164] Sense strand: 5′-ccaucgaucgaccuacaaatt-3′; antisense strand: 5′-uuuguaggucgaucgauggtt-3′.

[0165] Si-LINC00511-2:

[0166] Sense strand: 5′-gcuugugcccuuggaauuatt-3′; antisense strand: 5′-uaauuccaagggcacaagctt-3′.

[0167] Si-NC: sense strand: 5′-uucuccgaacgugucacgutt-3′; antisense strand: 5′-acgugacacguucggagaatt-3′.

[0168] (3) miR-150-5p

[0169] miR-150-5p mimics:

[0170] Sense strand: 5′-ucucccaacccuuguaccagug-3′; antisense strand: 5′-cugguacaaggguugggagauu-3′.

[0171] miR-150-5p inhibintor: 5′-cacugguacaaggguugggaga-3′.

[0172] miR-150-5p NC: 5′-caguacuuuuguguaguacaa-3′.

[0173] 3. Experimental methods

[0174] (1) Cell passaging

[0175] a. Discard the original cell culture medium in the culture flasks filled with A375 and SK-Mel-28 cells, add 1 mL of 0.25% trypsin digestion solution to cover the cell layer, disinfect the flask mouth, and cover it.

[0176] b. Observe the changes of cells under an inverted microscope. As time goes by, the originally attached cells gradually become round, the intercellular matrix shrinks, and the intercellular space increases. Before the cells float up, discard the trypsin digestion solution and add 5 mL of cell culture medium containing 10% fetal bovine serum to terminate the digestion.

[0177] c. Cell counting: Take 0.5 mL of the above cell suspension, dilute it appropriately and drop it into a blood cell counting plate. Count the total number of cells in the four corners of the four grids according to the white blood cell counting method. Only count the cells with intact nuclei and cytoplasm. Cells in piles are counted as one cell. Convert the total number of cells in the four grids into the number of cells per milliliter of cell suspension according to the following formula:

[0178] Total number of cells / mL = total number of cells in four compartments / 4×10 4 × dilution factor.

[0179] d. According to the cell counting results, further dilute with cell culture medium to contain 3×10 5 The cells were divided into culture bottles (8 mL / bottle) and cultured in a 37°C, 5% CO2 incubator.

[0180] (2) Transient transfection of miRNA

[0181] Cationic liposome method was used for transient transfection. The operation was carried out according to Lipofectamin TM 2000 reagent instructions. 24h before transfection, cells with good growth status were inoculated into 12-well plates, and the cell count was about 2×10 4 , routinely cultured until the day of transfection, and the experiment was performed when the cell confluency was 50% to 60%. 20nM miRNAmimics was added to 100μL DMEM medium and gently mixed; 2μL Lipofectamin was diluted with 100μL DMEM medium.TM 2000 liposomes, gently mix, and incubate at room temperature for 5 minutes; mix DMEM-lipids and DMEM-miRNAs, incubate at room temperature for 20 minutes to form a transfection complex; then add the above transfection complex to the cell culture medium, gently mix, and replace the complete culture medium after 6 hours of culture; after 48 hours of culture, extract the total RNA of the cells (the method is the same as that of Experimental Example 1) and proceed to the next step of the experiment. Among them, the non-specific sequence (Si-NC or miRNC) is used as a negative control, and the blank control is only transfected with liposomes.

[0182] siRNA transfection was the same as above.

[0183] (3) Fluorescence quantitative PCR detection

[0184] Corresponding primers were used for fluorescence quantitative PCR detection, and the △△Ct method was used to process the fluorescence quantitative PCR data to calculate the expression changes of ADAM19, Linc00511, and miR-150-5p. The results are shown in Figures 3 to 17 shown.

[0185] The primer sequences of ADAM19, Linc00511, and GAPDH are the same as above, and the primer sequence of miR-150-5p is shown in SEQ ID NOs. 23 to 24.

[0186] The primers for miR-150-5p are:

[0187] Forward: 5′-tgaaggtcggagtcaacggatt-3′; Reverse: 5′-gtcgtatccagtgcagggtccgaggt-3′.

[0188] (4) Results analysis

[0189] from Figures 3 to 5 It can be seen that after silencing LINC00511, LINC00511 and ADAM19 were downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, while miR-150-5p was upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, and the differences were statistically significant.

[0190] from Figures 6 to 8 It can be seen that overexpression of LINC00511, LINC00511 and ADAM19 were upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, while miR-150-5p was downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, and the differences were statistically significant.

[0191] In summary, ADAM19 and LIN00511 are both highly expressed in human melanoma. Detecting LINC00511 while knocking down ADAM19 will also produce different degrees of downregulation, and vice versa, indicating that simultaneous detection of both is more reliable and accurate than detecting one alone.

[0192] from Figures 9-10 It can be seen that after silencing ADAM19, ADAM19 was downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, while miR-150-5p was upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, and the differences were statistically significant.

[0193] from Fig.11 It can be seen that overexpression of ADAM19 and miR-150-5p were downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, and the differences were statistically significant.

[0194] from Fig.12 It can be seen that when miR-150-5p was inhibited, miR-150-5p was downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28; while when miR-150-5p was overexpressed, miR-150-5p was upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, and the differences were statistically significant.

[0195] from Figures 13-14 It can be seen that when miR-150-5p was inhibited, ADAM19 was upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28; while when miR-150-5p was overexpressed, ADAM19 was downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, and the difference was statistically significant.

[0196] from Fig.15 It can be seen that ADAM19 was downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28 when LINC00511 was silenced, but ADAM19 was upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28 when ADAM19 was overexpressed while LINC00511 was silenced, indicating that overexpression of ADAM19 had a certain rescue effect.

[0197] from Fig.16It can be seen that when ADAM19 was silenced, ADAM19 was downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, but when ADAM19 was silenced and miR-150-5p was inhibited at the same time, ADAM19 was upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, indicating that inhibition of miR-150-5p has a certain rescue effect.

[0198] from Fig.17 It can be seen that silencing LINC00511 caused LINC00511 to be downregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, but silencing LINC00511 and inhibiting miR-150-5p simultaneously caused LINC00511 to be upregulated to varying degrees in human melanoma cells A375 and SK-Mel-28, indicating that inhibiting miR-150-5p has a certain rescue effect.

[0199] Experimental Example 3

[0200] The specific steps of the cell proliferation experiment are as follows:

[0201] (1) Human melanoma cells A375 and SK-Mel-28 were inoculated into 96-well plates at a cell density of 5000 cells / mL, 100 μL per well, and three replicate wells were set up in each group. The plates were placed in a 37° C., 5% CO 2 incubator for static culture.

[0202] (2) Human melanoma cells A375 and SK-Mel-28 were transfected with si-NC, si-LINC00511, si-ADAM19, si-LINC00511+ADAM19, etc., respectively. 4 h before the end of culture, 10 μL of 5 mg / mL CCK-8 solution was added to each well and the cells were placed in a 37°C, 5% CO2 incubator for static culture until the end.

[0203] (3) Use an ELISA reader to detect the absorbance at 490 nm and draw a growth curve.

[0204] (4) SPSS 19.0 statistical software was used to establish a database and perform statistical analysis. The measurement data were expressed as mean ± standard deviation. The independent sample t-test was used to compare the means of the two groups. The difference was considered statistically significant when p < 0.05. The results are as follows: Figures 18 to 26 shown.

[0205] from Figures 18-19It can be seen that silencing LINC00511 has different degrees of inhibitory effects on the proliferation of human melanoma cells A375 and SK-Mel-28, while overexpressing LINC00511 has a certain promoting effect on the proliferation of human melanoma cells A375 and SK-Mel-28, and the difference is statistically significant.

[0206] from Figures 20-21 It can be seen that silencing ADAM19 has different degrees of inhibitory effect on the proliferation of human melanoma cells A375 and SK-Mel-28, while overexpressing ADAM19 has a certain promoting effect on the proliferation of human melanoma cells A375 and SK-Mel-28, and the difference is statistically significant.

[0207] from Figures 22-23 It can be seen that the simultaneous overexpression of ADAM19 and miR-150-5p has different degrees of inhibitory effects on the proliferation of human melanoma cells A375 and SK-Mel-28 compared with the overexpression of ADAM19 alone, which weakens the effect of overexpression of ADAM19 alone on cell proliferation; silencing ADAM19 and inhibiting miR-150-5p have different degrees of promoting effects on the proliferation of human melanoma cells A375 and SK-Mel-28 compared with the silencing of ADAM19 alone, which weakens the effect of silencing ADAM19 alone on cell proliferation.

[0208] from Figures 24-25 It can be seen that the simultaneous overexpression of LINC00511 and miR-150-5p had different degrees of inhibitory effects on the proliferation of human melanoma cells A375 and SK-Mel-28 compared with the overexpression of LINC00511 alone, which weakened the effect of overexpression of LINC00511 alone on cell proliferation; silencing LINC00511 and inhibiting miR-150-5p had different degrees of promoting effects on the proliferation of human melanoma cells A375 and SK-Mel-28 compared with the silencing of LINC00511 alone, which weakened the effect of silencing LINC00511 alone on cell proliferation.

[0209] from Fig.26 It can be seen that silencing LINC00511 and overexpressing ADAM19 weakened the effect of silencing LINC00511 alone on the proliferation of human melanoma cells A375 and SK-Mel-28.

[0210] Experimental Example 4

[0211] The specific steps of the cell migration experiment are as follows:

[0212] (1) According to 10×10 4Human melanoma cells A375 and SK-Mel-28 were inoculated into 24-well plates at a cell density of cells / mL, 200 μL per well, 3 replicate wells per group, and placed in a 37°C, 5% CO2 incubator for static culture.

[0213] (2) Human melanoma cells A375 and SK-Mel-28 were transfected with si-NC, si-LINC00511, and si-ADAM19, respectively, and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0214] (3) Take photos using an inverted microscope. Figures 27-28 shown.

[0215] from Figures 27-28 It can be seen that silencing ADAM19 and LINC00511 has different degrees of inhibitory effects on the migration of human melanoma cells A375 and SK-Mel-28.

[0216] Experimental Example 5

[0217] The melanoma TCGA sample library was selected from the GEPIA database (https: / / gepia.nih.gov / ), and the ADAM19 and LINC00511 gene names were entered for T-test analysis. A P value less than 0.05 indicated that there was a statistically significant difference between the two groups. The results are as follows: Fig.29 As shown, A: ADAM19, B: LINC00511.

[0218] from Fig.29 It can be seen that ADAM19 and LINC00511 are differentially expressed in melanoma patients and normal people, and the expression levels are upregulated in melanoma patients, and the differences are statistically significant.

[0219] Experimental Example 6

[0220] (1) Establishment of stable transgenic lines

[0221] Human melanoma A375 cells were plated in 6-well plates, with 5×10 5 Different concentrations of target gene ADAM19 and LINC00511 lentiviral vector suspension (synthesized by Shanghai Jima) were added to each well, and the medium was changed after 24 hours. Fluorescence observation was performed after 72 hours. The screened cell lines were identified to obtain A375 cell lines with stable low expression of ADAM19 and LINC00511.

[0222] (2) Establishment of xenogeneic mouse tumor model

[0223] A375 cell lines with stable low expression of ADAM19 and LINC00511 were placed in DMEM culture medium containing 10% fetal bovine serum (FBS) and cultured in a 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase were taken to prepare a 1×10 7 / mL of cell suspension was placed in 4℃ for precooling and set aside; BALB / c-Nude nude mice, six males and six females were selected and divided into control and experimental groups, and 0.2mL of cell suspension was taken per mouse and inoculated into the groin of the nude mice; three days later, subcutaneous nodules were observed at the inoculation site, proving that the subcutaneous transplant tumor model had been successfully established; then volume measurement was performed: the tumor growth was observed every 3 days, and the tumor length (L) and width (W) were measured with a vernier caliper, the formula: tumor volume = (L×W2) / 2, and the tumor growth curve was drawn; 30 days later, the nude mice were killed by cervical dislocation, the tumor was peeled off and placed under a ruler for photography, weighed and the data were recorded, and the results were as follows Figures 30-31 shown.

[0224] from Figures 30-31 It can be seen that stable low expression of ADAM19 and LINC00511 can reduce tumor weight, reduce tumor volume, and slow down tumor growth. Compared with the control group LV-si-Ctrl, the growth rate of tumors infected with LV-si-ADAM19 / LV-si-LINC00511 was significantly reduced (p<0.05).

[0225] The present invention conducts a multi-dimensional study on melanoma cells from mRNA to miRNA to lncRNA, and provides the ADAM19 gene closely related to melanoma, and the lncRNA and miRNA that regulate the expression of the ADAM19 gene, which provides a good idea for the study of the mechanism of melanoma, and also provides a new molecular marker for the clinical diagnosis of melanoma, which is of great significance.

[0226] The present invention shows through in vivo cell experiments that inhibiting the expression of ADAM19 and LINC00511 or overexpressing miR-150-5p can effectively inhibit the proliferation, migration and invasion of melanoma cells. At the same time, animal experiments show that interfering with the expression of ADAM19 and LINC00511 can reduce tumor weight, reduce tumor volume, and slow down tumor growth rate.

[0227] The present invention silences ADAM19, LINC00511 and miR-150-5p or overexpresses them. Experimental results show that they have a certain inhibitory effect on melanoma cells, and it is found that ADAM19 and LINC00511 are consistent and reverse with miR-150-5p. Animal experiments also prove that silencing ADAM19 and LINC00511 has a significant inhibitory effect on the formation of mouse tumors. Comprehensively prove that ADAM19, LINC00511 and miR-150-5p have a certain regulatory effect on the treatment of melanoma.

[0228] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A drug for preventing and treating melanoma, characterized in that: The drugs include one or more of the following: (1) Drugs that inhibit ADAM19 expression; (2) drugs that inhibit LINC00511 expression; (3) Drugs that promote the expression of miR-150-5p.

2. The drug for preventing and treating melanoma according to claim 1, characterized in that: The drug for inhibiting ADAM19 expression is selected from siRNA and interference vector.

3. The drug for preventing and treating melanoma according to claim 2, characterized in that: The drug for inhibiting ADAM19 expression is Si-ADAM19-1 and / or Si-ADAM19-2; The sequence of Si-ADAM19-1 is shown in SEQ ID NO.7-8; The sequence of the Si-ADAM19-2 is shown in SEQ ID NOs. 9 to 10.

4. The drug for preventing and treating melanoma according to claim 1, characterized in that: The drug for inhibiting the expression of LINC00511 is selected from siRNA and interference vector.

5. The drug for preventing and treating melanoma according to claim 4, characterized in that: The drug for inhibiting the expression of LINC00511 is Si-LINC00511-1 and / or Si-LINC00511-2; The sequence of Si-LINC00511-1 is shown in SEQ ID NOs. 13 to 14; The sequence of Si-LINC00511-2 is shown in SEQ ID NOs. 15 to 16.

6. The drug for preventing and treating melanoma according to claim 1, characterized in that: The drugs for promoting the expression of miR-150-5p are miRNA mimics and overexpression vectors.

7. The drug for preventing and treating melanoma according to claim 6, characterized in that: The drug that promotes the expression of miR-150-5p is miR-150-5p mimics; The sequences of the miR-150-5p mimics are shown in SEQ ID NOs. 19 to 20.

8. The drug for preventing and treating melanoma according to any one of claims 1 to 7, characterized in that: The drug is used to inhibit the development of melanoma.

9. The drug for preventing and treating melanoma according to claim 8, characterized in that: The drug is used to inhibit the proliferation, migration and invasion of melanoma cells.

10. The drug for preventing and treating melanoma according to claim 8, characterized in that: The drug is used to reduce the weight of melanoma tumors, reduce the volume of tumors, and slow down the growth rate of tumors.

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