CN116676392B - Application of USP10-MOF-ANXA2 signal path as drug target in preparation of drug for treating esophageal squamous cell carcinoma - Google Patents
Application of USP10-MOF-ANXA2 signal path as drug target in preparation of drug for treating esophageal squamous cell carcinoma Download PDFInfo
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Abstract
The invention relates to an application of a USP10-MOF-ANXA2 signal pathway as a drug target in preparing a drug for treating esophageal squamous cell carcinoma. The USP10-MOF-ANXA2 signaling pathway includes USP10, MOF and ANXA2 genes. The mRNA nucleotide sequence of the USP10 gene is shown as SEQ ID NO.1, the mRNA nucleotide sequence of the MOF gene is shown as SEQ ID NO.2, and the mRNA nucleotide sequence of the ANXA2 gene is shown as SEQ ID NO. 3. The invention discovers that the proliferation, migration and invasion of esophageal squamous cell carcinoma cells can be effectively inhibited by interfering a USP10-MOF-ANXA2 signal pathway for the first time. The invention also finds that the mixture si-UMA of si-USP10, si-MOF and si-ANXA2 is used for simultaneously and specifically knocking down the mRNA expression levels of USP10, MOF and ANXA2, and the effect of simultaneously knocking down the mRNA expression levels of USP10, MOF and ANXA2 on the inhibition effect of the USP10-MOF-ANXA2 signal path is optimal, so that the effect of inhibiting the proliferation and metastasis of esophageal squamous cell carcinoma is optimal, and the method can be applied to the research and development of targeted therapeutic drugs for esophageal squamous cell carcinoma.
Description
Technical Field
The invention relates to an application of a USP10-MOF-ANXA2 signal pathway serving as a drug target in preparing a drug for treating esophageal squamous cell carcinoma, belonging to the technical field of biomedicine.
Background
Esophageal cancer is a common human digestive tract malignancy, and mainly comprises two pathological types of esophageal squamous cell carcinoma and esophageal adenocarcinoma. The new cases of esophageal cancer account for about 50% of the globally diagnosed cases in China, and mainly include esophageal squamous cell carcinoma. At present, the treatment modes of the esophageal cancer mainly comprise minimally invasive surgical excision, auxiliary or new auxiliary radiotherapy and chemotherapy and emerging immunotherapy, and the development and the application of the treatment modes bring exact clinical benefits to patients with the esophageal cancer. However, the high tumor aggressiveness and heterogeneity of esophageal cancer often leads to treatment failure, which severely restricts the prognosis improvement of esophageal cancer patients, so that the current 5-year survival of esophageal cancer patients is less than 20%. Therefore, the signal path driving the malignant progress of the esophageal cancer is found and clarified, and the signal path is applied to the targeted treatment of the esophageal squamous cell carcinoma, so that the signal path has great clinical significance for improving prognosis of patients with the esophageal cancer squamous cell carcinoma.
The RNA interference technology is mainly applied to the specific knockdown of the expression level of a target gene in cells, and the key of the technology is the specificity and the effectiveness of small interfering RNA (Small interfering RNA, siRNA). siRNA is a double-stranded RNA comprising 20-25 pairs of nucleotides, which enter cells under the action of a transfection reagent, then participate in forming an RNA-induced silencing complex, and bind to target gene mRNA through base complementary pairing, thereby regulating the degradation of target gene mRNA, and finally leading to down-regulation of the level of protein encoded by the target gene. Common problems in siRNA applications include primarily non-ideal target gene knockdown efficiency and interference with expression of non-target genes. Therefore, the siRNA with high knocking-down efficiency and high specificity aiming at the target gene is designed, and has important significance for researching the biological function of the target gene in the malignant progress of esophageal cancer and developing related targeted drugs.
Disclosure of Invention
Aiming at the defects of the prior art, the USP10-MOF-ANXA2 signal pathway is used as a drug target to prepare the drug for treating esophageal squamous cell carcinoma.
The technical scheme of the invention is as follows:
the application of USP10-MOF-ANXA2 signaling pathway as a drug target in preparing drugs for treating esophageal squamous cell carcinoma.
Preferably, according to the present invention, the USP10-MOF-ANXA2 signaling pathway includes USP10, MOF and ANXA2 genes.
According to the invention, preferably, the mRNA nucleotide sequence of the USP10 gene is shown as SEQ ID NO.1, the mRNA nucleotide sequence of the MOF gene is shown as SEQ ID NO.2, and the mRNA nucleotide sequence of the ANXA2 gene is shown as SEQ ID NO. 3.
According to the invention, the medicine for treating esophageal squamous cell carcinoma takes USP10-MOF-ANXA2 signal pathway as an action target point, and can specifically and efficiently inhibit the USP10-MOF-ANXA2 signal pathway based on the interference inhibition of the expression of USP10, MOF and/or ANXA2 genes.
Preferably, according to the present invention, the medicament for treating esophageal squamous cell carcinoma is siRNA, shRNA, microRNA or a USP10-MOF-ANXA2 signaling pathway chemical inhibitor.
Further preferred, the siRNA is si-USP10, si-MOF, si-ANXA2 or si-UMA;
the nucleotide sequence of the si-USP10 is shown as SEQ ID NO.4, the nucleotide sequence of the si-MOF is shown as SEQ ID NO.5, and the nucleotide sequence of the si-ANXA2 is shown as SEQ ID NO. 6;
the si-UMA is a mixture of si-USP10, si-MOF and si-ANXA2 mixed in a molar ratio of 1:1:1.
A medicament for treating esophageal squamous cell carcinoma comprising an inhibitor of USP10-MOF-ANXA2 signaling pathway.
The beneficial effects are that:
1. the invention discovers that the proliferation, migration and invasion of esophageal squamous cell carcinoma cells can be effectively inhibited by interfering a USP10-MOF-ANXA2 signal pathway for the first time. The invention designs si-USP10, si-MOF and si-ANXA2 which specifically knock down the expression level of USP10, MOF and ANXA2mRNA, and the USP10-MOF-ANXA2 signal pathway is inhibited by knocking down the expression level of the USP10, MOF and ANXA2mRNA, so that the proliferation, migration and invasion of esophageal squamous cell carcinoma cells are successfully inhibited, and therefore, the USP10-MOF-ANXA2 signal pathway can be used as a treatment target for preparing a medicament for treating esophageal squamous cell carcinoma.
2. The invention also finds that the mixture si-UMA of si-USP10, si-MOF and si-ANXA2 is used for specifically knocking down the mRNA expression level of USP10, MOF and ANXA2, compared with the single knocking down of the mRNA expression level of USP10, MOF or ANXA2 by using the same dosage si-USP10, si-MOF or si-ANXA2, the inhibition effect on the proliferation, migration and invasion capacity of esophageal squamous cell carcinoma cells is more obvious, so that the inhibition effect on the signaling pathway of USP10-MOF-ANXA2 by knocking down the mRNA expression level of USP10, MOF and ANXA2 is optimal, the proliferation and metastasis inhibition effect of esophageal squamous cell carcinoma is optimal, and the method can be applied to the research and development of esophageal squamous cell carcinoma targeted therapeutic drugs.
Drawings
FIG. 1 shows the statistical significance of the differences between groups as determined by transfection of si-UMA and the same dose of si-NC into KYSE-150 cells, respectively, detection of USP10, MOF and ANXA2mRNA levels using reverse transcription-real time fluorescent quantitative PCR, and detection using Student's t.
FIG. 2 shows the statistical significance of differences between groups determined by the use of a CCK-8 assay to detect KYSE-150 cell proliferation using si-USP10, si-MOF, si-ANXA2 and si-NC transfected into KYSE-150 cells, respectively, at the same dose for each siRNA.
FIG. 3 shows the statistical significance of the differences between groups determined by selecting KYSE-150 cells, transfecting si-UMA, si-USP10, si-MOF, si-ANXA2 and si-NC, respectively, using the same amount of transfection agent for each siRNA, using Transwell experiments to detect changes in KYSE-150 cell migration and invasion capacity, and using one-way variance tests.
Detailed Description
The technical scheme of the present invention will be further described with reference to specific experimental examples, but the scope of the present invention is not limited thereto. The reagents and materials referred to in the examples are all commercially available products unless otherwise specified.
Human esophageal squamous cell carcinoma cell line KYSE-150 cells, available from Shanghai Fuchong biosciences Inc.
Example 1
Previous studies by the inventors found that MOF protein levels were abnormally up-regulated in esophageal squamous cell carcinoma and correlated closely with poor prognosis for patients; after further studies, deubiquitinase USP10 was found to be a key upstream regulatory molecule for abnormal up-regulation of MOF proteins, and abnormal up-regulated USP10 also suggested shorter survival in patients with esophageal squamous cell carcinoma; in the process of exploring a downstream molecular mechanism of MOF for regulating malignant progress of esophageal cancer, the inventor performs transcriptome sequencing on MOF knockdown esophageal squamous cell carcinoma cells, screens ANXA2 as a potential downstream target of MOF, and verifies that MOF plays a role in promoting cancer in esophageal squamous cell carcinoma by up-regulating ANXA2 expression. Thus, the inventors have for the first time clarified the biological role of the abnormally activated USP10-MOF-ANXA2 signaling pathway in driving malignant progression of esophageal squamous cell carcinoma, and thought that the USP10-MOF-ANXA2 signaling pathway could be targeted for use in the preparation of a medicament for treating esophageal squamous cell carcinoma.
Example 2
According to mRNA nucleotide sequences of USP10, MOF and ANXA2 genes, designing si-USP10, si-MOF and si-ANXA2 which specifically knock down expression levels of the USP10, MOF and ANXA2mRNA, wherein the nucleotide sequence of the si-USP10 is shown as SEQ ID NO.4, the nucleotide sequence of the si-MOF is shown as SEQ ID NO.5, the nucleotide sequence of the si-ANXA2 is shown as SEQ ID NO.6, and then mixing the si-USP10, the si-MOF and the si-ANXA2 according to a molar ratio of 1:1:1 to obtain si-UMA. The same doses of si-UMA and si-NC were transfected into KYSE-150 cells, respectively, and USP10, MOF and ANXA2mRNA levels were detected using reverse transcription-real time fluorescent quantitative PCR, the specific results are shown in FIG. 1.
As can be seen from FIG. 1, si-UMA is effective in knocking down USP10, MOF and ANXA2mRNA levels in KYSE-150 cells, interfering with the inhibition of the USP10-MOF-ANXA2 signaling pathway.
The specific implementation process is as follows:
(1) KYSE-150 cells were cultured in 6-well plates, and when the cell growth density exceeded 60%, the same dose (volume 3. Mu.L, concentration 10. Mu.M) of siUMA and siNC were transfected into KYSE-150 cells, respectively, using transfection reagent Lipofectamine RNAiMAX Reagent (Life technologies, 13778-150), and the cells were cultured for a further 48 hours;
(2) Extracting RNA from the cells obtained in the step (1) by using an RNA-Quick Purification Kit (China fir, RN 001) kit, detecting the concentration of the extracted RNA by using a NanoDrop2000, and evaluating the integrity of the extracted RNA by using agarose gel electrophoresis;
(3) Using the RNA extracted in the step (2) as a template and applying a reverse transcription kit LunaScript TM cDNA products were synthesized from RT Supermix Kit (NEB, E3010) using PowerGreen Master Mix (Thermo Fisher Scientific, 4367659) kit and Quantum studio TM Fluorescent quantitative PCR was performed by a 5System (Thermo Fisher Scientific) PCR apparatus using ACTB as a reference, according to 2 -ΔΔCT The formulas calculate the relative expression levels of USP10, MOF, ANXA2 mRNA.
Example 3
KYSE-150 cells were selected and transfected with si-UMA, si-USP10, si-MOF, si-ANXA2 and si-NC, respectively, and the effect of the above siRNA on the proliferation potency of KYSE-150 cells was examined by CCK-8 assay, and the specific results are shown in FIG. 2.
As can be seen from FIG. 2, transfection of si-UMA significantly attenuated KYSE-150 cells in proliferation capacity compared to transfection of the same dose of si-USP10, si-MOF, si-ANXA2 alone. And it was also found that the use of si-USP10, si-MOF, si-ANXA2 mixture si-UMA simultaneously and specifically knockdown USP10, MOF and ANXA2mRNA expression levels, showed more significant inhibition of KYSE-150 cell proliferation capacity than the use of the same dosage of si-USP10, si-MOF or si-ANXA2 alone knockdown USP10, MOF or ANXA2mRNA expression levels, indicating that the simultaneous knockdown of USP10, MOF and ANXA2mRNA expression levels gave the best inhibition of USP10-MOF-ANXA2 signaling pathway, with the best inhibition of esophageal squamous cell carcinoma proliferation.
The specific implementation process is as follows:
(1) The same doses (volume 3. Mu.L, concentration 10. Mu.M) of si-UMA, si-USP10, si-MOF, si-ANXA2 and si-NC were transfected into KYSE-150 cells, respectively, as detailed in example 2, transfected cells were collected and the different transfected cells were re-cultured in 96-well plates at a concentration of 2,000 cells/well, 4 groups each comprising five transfected cells si-UMA, si-USP10, si-MOF, si-ANXA2, si-NC, 5 multiple wells each;
(2) After the cells are attached (about 2-4 hours), adding CCK-8 reagent (TargetMol, C0005) (10 mu L/hole) into the 1 st group, placing in a 37 ℃ incubator for 1 hour, and measuring absorbance value (450 nm) in an enzyme-labeled instrument after simple shaking, namely the absorbance value of the initial planted cells;
(3) The absorbance values of the remaining three groups of cell well plates at 450nm were measured by using CCK-8 reagent at 1, 2 and 3 days after completion of the measurement of the absorbance values of the initial cells, respectively, to thereby calculate the cell viability of the different transfected cells in each group.
Example 4
The same doses of si-UMA, si-USP10, si-MOF, si-ANXA2, si-NC were transfected into KYSE-150 cells and the change in cell migration and invasiveness was examined using a Transwell experiment, and the specific results are shown in FIG. 3.
As can be seen from FIG. 3, the ability of si-UMA to significantly inhibit migration and invasion of KYSE-150 cells compared to si-USP10, si-MOF, si-ANXA2 alone at the same transfection agent amount. And it was also found that the use of si-USP10, si-MOF, si-ANXA2 mixture si-UMA simultaneously and specifically knockdown USP10, MOF and ANXA2mRNA expression levels, the inhibition of KYSE-150 cell migration and invasiveness was more pronounced, indicating that the simultaneous knockdown of USP10, MOF and ANXA2mRNA expression levels had the best effect on USP10-MOF-ANXA2 signaling pathway inhibition of esophageal squamous cell carcinoma migration and invasion compared to the use of the same dose of si-USP10, si-MOF or si-ANXA2 alone knockdown USP10, MOF or ANXA2mRNA expression levels.
The specific implementation process is as follows:
(1) KYSE-150 cells were selected and transfected with si-UMA, si-USP10, si-MOF, si-ANXA2 and si-NC to KYSE-150 cells, respectively, and all doses of each siRNA were the same (volume 3. Mu.L, concentration 10. Mu.M) as described in example 2;
(2) Selecting a Transwell chamber (Corning, 3422), pre-spreading matrigel for detecting the invasive capacity of cells, and not spreading matrigel for detecting the migration capacity of cells, placing the Transwell chamber into a 24-well plate (600 mu L of complete culture medium is added in advance to each well);
(3) Transfected cells were collected, resuspended in serum-free medium to a cell concentration of 2 ten thousand cells/200. Mu.L, 200. Mu.L of the cell suspension was added to a transwell chamber at 37℃with 5% CO 2 Culturing cells in a incubator for 48 hours;
(4) The transwell cells were removed and washed clean, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, then stained with 0.1% crystal violet overnight, the non-migrated, invasive cells on the inside of the cells were carefully removed with a cotton swab, and the cells on the outside of the cells were observed and counted using an inverted microscope.
Claims (2)
1. The application of the siRNA of specific interference inhibition USP10, the siRNA of specific interference inhibition MOF and the siRNA of specific interference inhibition ANXA2 in preparing medicines for treating esophageal squamous cell carcinoma; the method is characterized in that the nucleotide sequence of the siRNA of the specific interference suppression USP10 is shown as SEQ ID NO.4, the nucleotide sequence of the siRNA of the specific interference suppression MOF is shown as SEQ ID NO.5, and the nucleotide sequence of the siRNA of the specific interference suppression ANXA2 is shown as SEQ ID NO. 6.
2. The use according to claim 1, wherein the molar ratio of the siRNA of specific interference suppression USP10, the siRNA of specific interference suppression MOF and the siRNA of specific interference suppression ANXA2 is 1:1:1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009044793A1 (en) * | 2007-10-02 | 2009-04-09 | Alphagen Co., Ltd. | siRNA TARGETING ONCOGENE |
| CN114075562A (en) * | 2021-03-10 | 2022-02-22 | 北京肿瘤医院(北京大学肿瘤医院) | MAGE-C3 inhibitor and application thereof in preparation of drugs for treating and/or preventing esophageal squamous cell carcinoma |
| CN114807372A (en) * | 2022-05-11 | 2022-07-29 | 山东大学第二医院 | Application of human HHIPL2mRNA in targeted therapy and prognosis evaluation of esophageal squamous cell carcinoma and kit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009044793A1 (en) * | 2007-10-02 | 2009-04-09 | Alphagen Co., Ltd. | siRNA TARGETING ONCOGENE |
| CN114075562A (en) * | 2021-03-10 | 2022-02-22 | 北京肿瘤医院(北京大学肿瘤医院) | MAGE-C3 inhibitor and application thereof in preparation of drugs for treating and/or preventing esophageal squamous cell carcinoma |
| CN114807372A (en) * | 2022-05-11 | 2022-07-29 | 山东大学第二医院 | Application of human HHIPL2mRNA in targeted therapy and prognosis evaluation of esophageal squamous cell carcinoma and kit |
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| Title |
|---|
| ANXA2 promotes esophageal cancer progression by activating MYC-HIF1A-VEGF axis;Sai Ma等;J Exp Clin Cancer Res;第37卷(第1期);第1-13页 * |
| Status of epigenetic chromatin modification enzymes and esophageal squamous cell carcinoma risk in northeast Indian population;Virendra Singh等;Am J Cancer Res;第5卷(第3期);第979-999页 * |
| Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma;Yu-Fei Cao等;Cell Death Differ;第30卷(第2期);第527-543页 * |
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