Application of lncRNA MIR181A2HG in the preparation of drugs for inhibiting gastric cancer lymph node metastasis

Abstract

The present invention provides the use of lncRNA MIR181A2HG in the preparation of a drug for inhibiting gastric cancer lymph node metastasis, which relates to the field of biomedical technology. This application discovered that a new lncRNA MIR181A2HG is differentially expressed between gastric cancer cells and normal cells, and is particularly highly expressed in gastric cancer cells. Furthermore, through validation experiments, this application found that knocking down the expression of lncRNA MIR181A2HG significantly inhibited gastric cancer lymph node metastasis in nude mice. This helps provide more accurate diagnosis and treatment for gastric cancer patients, improving their prognosis and quality of life.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of lncRNA MIR181A2HG in preparing a drug for inhibiting gastric cancer lymph node metastasis. Background Art

[0002] Gastric cancer (GC) is the most common malignant tumor of the digestive system and ranks third in mortality among malignant tumors worldwide. Lymph node metastasis is one of the main causes of poor prognosis. Lymph node metastasis not only increases the risk of recurrence and distant metastasis but also significantly reduces patient survival.

[0003] Noncoding RNA (ncRNA) plays a crucial role in the study of gastric cancer lymph node metastasis, with the regulatory role of long noncoding RNA (lncRNA) attracting particular attention. lncRNAs are a class of RNA molecules exceeding 200 bases in length. While they do not encode proteins, they can regulate gene expression at multiple levels, including epigenetic, transcriptional, and post-transcriptional levels, through their RNA form. As research deepens, researchers have discovered that many lncRNAs play a crucial role in the process of gastric cancer lymph node metastasis.

[0004] Studies have shown that there are significant differences in lncRNA expression between gastric cancer tissues and lymph node metastasis tissues. Certain lncRNAs have been shown to be significantly upregulated in gastric cancer lymph node metastasis. These lncRNAs can promote the invasion and migration ability of gastric cancer cells, thereby promoting the occurrence and development of lymph node metastasis. For example, certain lncRNAs, such as NEAT1, STARD13-AS, MT1JP, and LINC01089, play an important regulatory role in gastric cancer lymph node metastasis. The high expression of these lncRNAs is closely related to the invasion and metastasis ability of gastric cancer cells. At the same time, some newly discovered lncRNAs (such as HOAX-AS1, LINC00942, CASC11, and MACC1-AS1) are enriched in human gastric cancer tissues and significantly increased in the plasma of gastric cancer patients, and are associated with gastric cancer lymph node metastasis.

[0005] The treatment of gastric cancer lymph node metastasis has always been a difficult problem in gastric cancer treatment. Although surgical resection is the main treatment for gastric cancer, its effect is not ideal for gastric cancer that has already metastasized to the lymph nodes, and it may lead to increased surgical trauma and postoperative complications. Although chemotherapy is one of the conventional treatment methods, it may cause a series of side effects and tumor cells to develop drug resistance. Radiotherapy has a certain effect on controlling the local progression of gastric cancer, but its therapeutic effect on lymph node metastasis is limited. Although targeted therapy and immunotherapy provide new treatment directions, they still face problems such as tumor heterogeneity, drug resistance and high treatment costs. In addition, although the research on long non-coding RNA (lncRNA) has gradually increased in recent years, its specific mechanism in gastric cancer lymph node metastasis is still not fully understood, which limits the development of therapeutic strategies targeting lncRNA. Therefore, the treatment of gastric cancer lymph node metastasis still faces many challenges, and new treatment strategies and methods are urgently needed. Summary of the Invention

[0006] The purpose of this invention is to discover a new lncRNA, MIR181A2HG, which has the potential to be applied in the study of gastric cancer lymph node metastasis. This will help provide more accurate diagnosis and treatment for gastric cancer patients, and improve their prognosis and quality of life.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] Application of lncRNA MIR181A2HG in the preparation of drugs for inhibiting gastric cancer lymph node metastasis.

[0009] Preferably, the sequence number of the lncRNA MIR181A2HG is as shown in SEQ ID NO: 1.

[0010] The present application also provides the use of a lncRNA MIR181A2HG inhibitor in the preparation of a drug for inhibiting gastric cancer lymph node metastasis.

[0011] The present application also provides a drug for inhibiting gastric cancer lymph node metastasis, characterized in that the drug is used to knock down the expression of lncRNA MIR181A2HG.

[0012] Preferably, the drug comprises an inhibitor of lncRNA MIR181A2HG with a sequence number as described in SEQ ID NO: 1.

[0013] This application discovered a novel lncRNA, MIR181A2HG, that is differentially expressed between gastric cancer cells and normal cells, with particular high expression in gastric cancer cells. Furthermore, validation experiments in this application revealed that knocking down the expression of lncRNA MIR181A2HG significantly inhibited popliteal lymph node metastasis in nude mice with gastric cancer. This could help provide more accurate diagnosis and treatment for gastric cancer patients, improving their prognosis and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a comparison diagram of the expression of MIR181A2HG in GES-1 and GC cell lines detected by qRT-PCR experiment in one embodiment of the present invention;

[0015] Figure 2 This is a comparison chart of the knockdown efficiency of MIR181A2HG in the GC cell line detected by qRT-PCR in one embodiment of the present invention (*P<0.05, **P<0.01, #P>0.05).

[0016] Figure 3 This is a photograph of a nude mouse popliteal LN metastasis model in one embodiment of the present invention;

[0017] Figure 4 This is a bioluminescence image of popliteal metastatic LN after MIR181A2HG knockdown in one embodiment of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below with reference to specific embodiments.

[0019] Application of lncRNA MIR181A2HG in the preparation of drugs for inhibiting gastric cancer lymph node metastasis.

[0020] Specifically, in one embodiment, the drug is used to inhibit gastric cancer lymph node metastasis, and the sequence number of the lncRNAMIR181A2HG is as shown in SEQ ID NO: 1.

[0021] The present application also provides the use of a lncRNA MIR181A2HG inhibitor in the preparation of a drug for inhibiting gastric cancer lymph node metastasis.

[0022] The present application also provides a drug for inhibiting gastric cancer lymph node metastasis, which comprises an inhibitor of lncRNA MIR181A2HG with a sequence number as SEQ ID NO: 1.

[0023] The application of lncRNA MIR181A2HG in gastric cancer cells is described below with reference to specific examples.

[0024] Example 1:

[0025] RNA extraction

[0026] S1: Take well-growing gastric cancer cell lines (HGC-27, BGC-823, AGS, MKN-45, SGC-7901) and normal gastric mucosal epithelial cells GES-1, wash the cells twice with 2 ml PBS, digest with trypsin, add complete culture medium and centrifuge to obtain the lower cell pellet, add 1 ml Trizol and pipette to mix, place in an ice box for 10 minutes, and then transfer to an enzyme-free 1.5 ml enzyme-free EP tube.

[0027] S2: Add 200 μL of chloroform to a 1.5 ml enzyme-free EP tube, shake well for 10 seconds, and let stand on ice for 3 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes. Slowly aspirate the topmost layer of clear liquid with a pipette and transfer to a new 1.5 ml enzyme-free EP tube. Add approximately 500 μL of isopropanol and let stand on ice for 15 minutes. During this time, prepare a 75% ethanol solution: 75% anhydrous ethanol + 25% DEPC water. Centrifuge at 12,000 rpm at 4°C for 10 minutes.

[0028] S3: Discard the supernatant and add 1 ml of the previously prepared 75% ethanol solution to the white precipitate at the bottom of the RNA tube. Wash the RNA tube until the white precipitate is visible. Centrifuge at 7500 rpm at 4°C for 7 minutes. Discard the supernatant and incubate at 50°C for 10 minutes until the RNA precipitate becomes transparent. Add 20-30 μl of DEPC water and incubate at 60°C for 5-10 minutes to obtain the final RNA stock solution.

[0029] S4: Use a UV spectrophotometer to check the concentration and purity. Label and record the freezing time. Seal with parafilm and quickly store in a -80°C freezer.

[0030] Example 2: Reverse transcription

[0031] Prepare an ice box in advance. Remove the RNA stock solution obtained in Example 1 from a -80°C freezer and shake for 10 seconds. Check the concentration and purity again using a UV spectrophotometer. Add the reagents in the order shown in Table 1. Add the mixture to an EP tube and mix thoroughly. Reverse transcription is then performed: first reaction at 42°C for 60 minutes; second reaction at 75°C for 10 minutes; and third reaction at 4°C for 8 minutes. After the reaction, obtain cDNA and store it in a -80°C freezer.

[0032] Table 1 Reverse transcription system (20ul)

[0033]

[0034] Example 3: PCR

[0035] Prepare an ice box, take out the cDNA stock solution in Example 2 from the -80°C refrigerator, shake and mix for 10 seconds, and add the PCR system reagents to the eight tube strips according to Table 2.

[0036] The PCR primer sequences are

[0037] Forward:5'-CGCGGTTCAATACCTCGTCT-3';

[0038] Reverse:5'-TGCTGTGGCTAGAGGACAA-3';

[0039] GAPDH was used as a control, and the primer sequences were:

[0040] Forward:5′-GAGTCAACGGATTTGGTCGT-3′;

[0041] Reverse: 5′-TGGGTGGAATCATATTGGAA-3′, and three replicate wells were set for each sample.

[0042] Transfer to the qRT-PCR instrument and set the following parameters: pre-denaturation, 95°C, 10 min; amplification reaction, 95°C, 2 s; 60°C, 20 s; 70°C, 10 s; 45 cycles. For melting curve analysis, please refer to Figure 1 ,According to the experimental results, compared with the normal gastric mucosal epithelial cell GES-1, lncRNA MIR181A2HG was highly expressed in gastric cancer cell lines, and SGC-7901 had the highest expression level.

[0043] Table 2 Fluorescence quantitative PCR reaction system: (20ul)

[0044]

[0045] Example 4: Transfection of siRNA

[0046] Gastric cancer cells SGC-7901 with good growth status were collected and 3×10 5 Culture 100 cells / well in a 6-well plate and incubate for 24 hours. After the cells have attached to the wall and their morphology has returned to normal, the experiment is performed when the cell density reaches 70%. According to the PolyplusjetPRIME siRNA assay instructions, add 200ul jetPRIME buffer, 4ul jetPRIME, 5.5ul siRNA, and 2ml of incubation. After 24 hours of incubation, remove the experimental group cells and wash with PBS. Figure 2 According to the PCR results, compared with the control group, si-1 and si-2 could significantly reduce the expression of MIR181A2HG in the gastric cancer cell line SGC-7901 (***P<0.001).

[0047] The knockdown sequence is:

[0048] Si1:S:GAUGCAGAAUCUACCUACATT;

[0049] AS:UGUAGGUAGAUUCUGCAUCTT;

[0050] Si2:S:AUGGAGUAGAUAAUAAAATT;

[0051] AS:UUUAUUAUCUACUCCAUGCTT;

[0052] Si3: S:GGUUCCAGUAUCUAAUTT;

[0053] AS:AUUAGAUACUGGAACCCAGTT

[0054] Example 5: Construction of a footpad-popliteal lymph node metastasis model

[0055] Specific procedures are as follows: 6-week-old female BALB / c nude mice were purchased from the Experimental Animal Center of Nantong University (Nantong, China). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Nantong University.

[0056] The mice were divided into three groups, with 10 mice in each group; 50ul PBS gastric cancer cell suspension was inoculated into the footpad, and these suspended cells were transfected with si-NC-luc, si-MIR181A2HG#1-luc, and si-MIR181A2HG#2-luc.

[0057] Three weeks later, the mice were anesthetized with isoflurane and intraperitoneally injected with D-Luciferin sodium solution. Lymphatic metastasis was monitored and imaged using a bioluminescence imaging system (PerkinElmer, IVIS Spectrum Imaging System). Figure 3 、 4 As shown: It was found that knockdown of MIR181A2HG could significantly inhibit the popliteal lymph node metastasis of gastric cancer cell line SGC-7901.

[0058] This application discovered a novel lncRNA, MIR181A2HG, that is differentially expressed between gastric cancer cells and normal cells. Specifically, it is highly expressed in gastric cancer cells. This differential expression may be associated with the development and progression of gastric cancer. Furthermore, validation experiments in this application revealed that knocking down the expression of lncRNA MIR181A2HG significantly inhibited popliteal lymph node metastasis in nude mice with gastric cancer. This may provide a new therapeutic target and potentially lead to more precise treatment strategies for gastric cancer patients. Furthermore, the high expression of lncRNA MIR181A2HG may also provide new clues for the early diagnosis of gastric cancer. By detecting its expression, we may be able to detect gastric cancer earlier, allowing for preemptive treatment and improving patient survival and quality of life. In the future, targeted drugs targeting MIR181A2HG could be designed to inhibit lymph node metastasis of gastric cancer and improve patient survival. In summary, the discovery of lncRNA MIR181A2HG provides a new perspective, helping us gain a deeper understanding of the mechanisms of gastric cancer development and progression. By intervening in this disease, we can provide more precise and effective treatments for gastric cancer patients, thereby improving their prognosis and quality of life.

Claims

1. Use of a lncRNA MIR181A2HG inhibitor in the preparation of a drug for inhibiting gastric cancer lymph node metastasis, characterized in that: The lncRNA MIR181A2HG inhibitor includes one of the following knockdown sequences: Si1:S:GAUGCAGAAUCUACCUACATT; AS:UGUAGGUAGAUUCUGCAUCTT; Si2: S: AUGGAGUAGAUAAUAAATT; AS: UUUAUUAUCUACUCCAUGCTT.

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