Application of substance for inhibiting expression of deubiquitinating enzyme in preparation of products for treating pancreatic cancer

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of a substance for inhibiting expression of deubiquitinase in preparation of a product for treating pancreatic cancer. The invention provides application of a substance for inhibiting expression of deubiquitinating enzyme USP36 in preparation of a product for treating pancreatic cancer. The amino acid sequence of the deubiquitinating enzyme USP36 is as shown in SEQ ID NO. 1. The silent deubiquitinase USP36 is found to significantly inhibit the killing ability of pancreatic cancer cells to resist CD8 + T cells through deubiquitinase RNA interference library screening for the first time. Moreover, the USP36 is found to promote the deubiquitination of the YAP protein and stabilize the YAP protein, so that more YAP / TEAD is combined to an enhancer region of the PD-L1, the expression of the PD-L1 is promoted, and finally the immunity of pancreatic cancer cells is promoted.

Description

Technical Field

[0001] This invention belongs to the field of drug and disease treatment technology, specifically relating to the application of substances that inhibit the expression of deubiquitinating enzymes in the preparation of products for treating pancreatic cancer. Background Technology

[0002] Pancreatic cancer, commonly known as the "king of cancers," is most commonly seen in pancreatic ductal adenocarcinoma (PDAC), accounting for 95% of all pancreatic cancer cases. Early-stage pancreatic cancer often presents with few or no symptoms, and most patients are diagnosed at an advanced stage, resulting in extremely low post-operative survival rates. KRAS gene mutations (functional activation) are a crucial molecular pathological basis for pancreatic cancer, with mutations found in over 90% of pancreatic cancer tumors. TP53, CDKN2A, and SMAD4 gene mutations (functional inactivation) are found in approximately 50% to 80% of tumors. Currently, treatment options for pancreatic cancer are very limited. Radical surgical resection is the primary treatment for early-stage pancreatic cancer, but over 80% of patients are diagnosed with locally advanced or distant metastases at initial diagnosis, leaving less than 20% of patients eligible for surgery. For patients with advanced pancreatic cancer, chemotherapy is the primary treatment, combined with radiotherapy and targeted therapy. Despite significant increases in research on pancreatic cancer treatments in recent years, the 5-year survival rate remains below 10%. In recent years, with the great success of programmed death receptor-1 (PD-1) monoclonal antibody therapy in melanoma, immunotherapy has become a hot topic in the treatment of malignant tumors. Although immunotherapy may be a new hope for future breakthroughs in pancreatic cancer treatment, current clinical data show that the therapeutic effect of immunotherapy in pancreatic cancer is not ideal, which may be related to the susceptibility of pancreatic cancer cells to immune escape. Therefore, further in-depth research into the mechanisms regulating the occurrence and development of pancreatic cancer and its susceptibility to immune escape, and exploring and intervening in the biological characteristics of pancreatic cancer cells that differ from normal cells, are important directions for exploring effective treatment methods for pancreatic cancer.

[0003] The ubiquitin-proteasome system (UPS) is a crucial pathway regulating protein degradation. Deubiquitinating enzymes (DUBs) regulate ubiquitin signaling by cleaving ubiquitin chains or removing ubiquitin from modified substrates, thereby reversing target protein degradation. Based on their active sites, DUBs can be broadly classified into five families: ubiquitin-specific proteases (USP / UBP), ubiquitin C-terminal hydrolases (UCH), Otubaim (OTU), Josephin domain proteins, and JAMM. The USP family is currently the most studied due to its well-defined functions, making it a novel drug target. Deubiquitinating enzymes play a vital role in tumor immunity. For example, USP22 can directly interact with the C-terminus of PD-L1, inducing its deubiquitination and stabilization, thereby promoting immune escape from liver cancer cells. Recent studies have shown that the deubiquitinating enzyme CSN5 can directly induce PD-L1 deubiquitination, and curcumin can reduce PD-L1 levels in cancer cells by inhibiting CSN5, thereby improving the efficacy of anti-CTLA4 therapy. In recent years, with the emergence of more and more targeted inhibitors of deubiquitinating enzymes, these enzymes may be new potential targets for improving the efficacy of tumor immunotherapy. Therefore, there is an urgent need to develop new applications of deubiquitinating enzymes in tumor immunotherapy. Summary of the Invention

[0004] In order to develop new applications of deubiquitinase in tumor immunotherapy, this invention provides the application of a substance that inhibits the expression of deubiquitinase in the preparation of products for treating pancreatic cancer.

[0005] To achieve the above objectives, the present invention adopts the following technical solution.

[0006] The purpose of this invention is to provide the use of a substance that inhibits the expression of deubiquitinase USP36 in the preparation of products for treating pancreatic cancer. The amino acid sequence of said deubiquitinase USP36 is shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the nucleic acid encoding the deubiquitinase USP36 is shown in SEQ ID NO.2.

[0008] Preferably, the substance that inhibits the expression of deubiquitinase USP36 includes siRNA.

[0009] Preferably, the siRNA transfection concentration is 20 nM to 50 nM.

[0010] Preferably, the siRNA is selected from any one or more of siUSP36#1, siUSP36#2 and siUSP36#3.

[0011] The justice chain of siUSP36#1 is shown in SEQ ID NO.3.

[0012] The justice chain of siUSP36#2 is shown in SEQ ID NO.5.

[0013] The justice chain of siUSP36#3 is shown in SEQ ID NO.7.

[0014] Preferably, the product uses the substance that inhibits the expression of deubiquitinase USP36 as the sole active ingredient or one of the active ingredients.

[0015] Preferably, when the product uses the substance that inhibits the expression of deubiquitinase USP36 as one of the active ingredients, the active ingredient also includes a chemotherapy drug.

[0016] Preferably, the chemotherapy drug is selected from any one or more of gemcitabine, paclitaxel, and 5-fluorouracil.

[0017] The present invention also provides a medicament for treating pancreatic cancer, the medicament comprising the substance that inhibits the expression of deubiquitinase USP36.

[0018] Preferably, the substance that inhibits the expression of deubiquitinase USP36 includes siRNA.

[0019] Preferably, the siRNA is selected from any one or more of siUSP36#1, siUSP36#2 and siUSP36#3.

[0020] The justice chain of siUSP36#1 is shown in SEQ ID NO.3.

[0021] The justice chain of siUSP36#2 is shown in SEQ ID NO.5.

[0022] The justice chain of siUSP36#3 is shown in SEQ ID NO.7.

[0023] Compared with the prior art, the present invention has the following beneficial effects: To develop new applications of deubiquitinases in tumor immunotherapy, this invention provides the application of a substance that inhibits deubiquitinase expression in the preparation of products for treating pancreatic cancer, offering a new strategy for treating pancreatic cancer and expanding the scope of application of deubiquitinases in tumor immunotherapy. The substance provided by this invention that inhibits deubiquitinase expression has a significant effect on promoting CD8 expression. +T cells kill cancer cells and inhibit the mRNA and protein levels of PD-L1 in cancer cells. Further research found that overexpression of USP36 can inhibit the ubiquitination level of YAP protein, while YAP can promote the transcription of PD-L1, thus enabling research on the mechanism by which pancreatic cancer progression is regulated, and providing new insights for the development of therapeutic drugs for pancreatic cancer. Attached Figure Description

[0024] Figure 1 The silencing of the deubiquitinating enzyme USP36 in this invention significantly promotes CD8. + T cell killing; among which: A is a flowchart for screening deubiquitinase RNA interference libraries; B is a quantitative graph showing the screening results of the deubiquitinase RNA interference library; C~D represent the detection of protein and mRNA levels of deubiquitinase USP36 after silencing by Western blot and RT-qPCR. E is the silencing of the deubiquitinating enzyme USP36, CD8 + Quantitative graph of T cell killing experiment.

[0025] Figure 2 In this invention, the deubiquitinase USP36 promotes the transcriptional level of PD-L1 in pancreatic cancer cells, thereby enhancing the resistance of pancreatic cancer cells to CD8. + The killing ability of T cells; among which: A represents the expression level of the deubiquitinating enzyme USP36 in normal pancreatic tissue and pancreatic cancer, analyzed using the TCGA database. B represents the analysis of RNA-seq data from the siControl and siUSP36 groups (thresholds: p < 0.05 and fold change > 1.5). C represents a heatmap analysis of the deubiquitinase USP36 and PD-L1 genes; D represents the expression of PD-L1 on the cell membrane surface after silencing the deubiquitinating enzyme USP36, as determined by flow cytometry. E~F represent the results of silencing the deubiquitinating enzyme USP36 in SW1990 cells and then overexpressing PD-L1. The protein and mRNA levels of PD-L1 were detected by Western blot and RT-qPCR. G is CD8 + T-cell killing assays were performed after silencing deubiquitinase USP36 and after PD-L1 overexpression of CD8. + The ability of T cells to kill cancer cells; H represents the Co-IP assay used to detect the binding of the deubiquitinase USP36 protein to the PD-L1 protein.

[0026] Figure 3 In this invention, the deubiquitinating enzyme USP36 promotes the ability of pancreatic cancer cells to resist CD8+ T cell killing by regulating YAP; wherein: A represents the gene set enrichment analysis (GSEA) of deubiquitinase USP36 and YAP-related genes in RNA-seq data analysis. B~C represents the correlation analysis between the deubiquitinase USP36 and the classic YAP downstream genes CYR61 and CTGF in the TCGA database. D~F represent Western blot experiments, RT-qPCR, and CD8 assays. + T-cell killing assays were performed to examine the effects of silencing the deubiquitinase USP36 followed by overexpression of YAP on the transcriptional levels of YAP protein and downstream target genes, as well as CD8+. + The impact of T's lethality.

[0027] Figure 4 In this invention, the deubiquitinating enzyme USP36 regulates the mRNA and protein levels of PD-L1 via YAP; wherein: A represents the correlation analysis between PD-L1 and YAP in the TCGA database; B~C represent Western blot and RT-qPCR experiments, which were conducted to detect the effects of silencing the deubiquitinase USP36 followed by overexpression of YAP on PD-L1 protein and mRNA levels. D represents the expression level of PD-L1 on the surface of SW1990 cell membranes as detected by flow cytometry. E~F represent the analysis of the relationship between YAP and PD-L1 and progression-free survival in pancreatic cancer patients using the KMplot database.

[0028] Figure 5 In this invention, the deubiquitinating enzyme USP36 regulates the stability of the YAP protein by promoting the deubiquitination level of YAP; wherein: A represents the mRNA level of YAP after silencing the deubiquitinase USP36 using an RT-qPCR experiment. B is an immunofluorescence assay used to detect the cellular localization of deubiquitinases USP36 and YAP. C represents a protein immunoprecipitation experiment showing that the deubiquitinating enzyme USP36 protein can bind to the YAP protein. After blocking the proteasome degradation pathway with MG132, the downregulation of YAP protein caused by silencing the deubiquitinating enzyme USP36 can be eliminated. Overexpression of the deubiquitinating enzyme USP36 (E) can significantly inhibit the polyubiquitination of YAP protein.

[0029] Figure 6 This is a schematic diagram illustrating how the deubiquitinating enzyme USP36 promotes immune escape from pancreatic cancer cells via YAP / PD-L1 in this invention. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] The following are the experimental cells and materials used in the examples: 1. Experimental cells Pancreatic cancer SW1990 cells, PANC-1 cells, and human embryonic kidney HEK293T cells were purchased from the American Type Culture Collection (ATCC). Pancreatic cancer SW1990 cells are referred to simply as SW1990 cells.

[0032] 2. Experimental Materials The lactate dehydrogenase cytotoxicity kit was purchased from Beyotime, catalog number: C0016.

[0033] The deubiquitinase USP36 antibody was purchased from Huaan Biotechnology, catalog number: ER1706-23.

[0034] YAP antibody was purchased from Cell Signaling Technology, catalog number: #14074.

[0035] The PD-L1 antibody was purchased from Cell Signaling Technology, catalog number: #13684.

[0036] The β-Actin antibody was purchased from Cell Signaling Technology, catalog number 3700.

[0037] The siRNA was purchased from Sigma, and each of its 3' ends was connected to a TT end overhang.

[0038] siControl sequences: 5'-UUCUCCGAACGUGUCACGU-3' (as shown in SEQ ID NO.9) and 5'-ACGUGACACGUUCGGAGAA-3' (as shown in SEQ ID NO.10).

[0039] The siUSP36#1 sequence is 5'-GCAAAUAUGUGUUGCUCAA-3' (as shown in SEQ ID NO.3) and 5'-UUGAGCAACACAUAUUUGC-3' (as shown in SEQ ID NO.4).

[0040] The siUSP36#2 sequence is 5'-CCGGCAAGCUGCGAAUAUU-3' (as shown in SEQ ID NO.5) and 5'-AAUAUUCGCAGCUUGCCGG-3' (as shown in SEQ ID NO.6).

[0041] The siUSP36#3 sequence is 5'-GCACACCACUGAAGAGAUU-3' (as shown in SEQ ID NO.7) and 5'-AAUCUCUUCAGUGGUGUGC-3' (as shown in SEQ ID NO.8).

[0042] 0. Deubiquitinase USP36 The deubiquitinase USP36 used in this invention is a key regulatory protein primarily located in the nucleolar. Its core function is to stabilize substrate proteins and regulate their activity by removing ubiquitin chains from them. USP36 is best known for regulating ribosome biosynthesis. By deubiquitinizing and stabilizing the transcription factor c-Myc and the nucleolar protein fibrillarin, it positively regulates the transcription and processing of ribosomal RNA, thereby driving cell growth and proliferation. Due to its crucial role in the cell cycle, USP36 has been found to be frequently amplified and overexpressed in various cancers, particularly by stabilizing the oncogene c-Myc, forming an important USP36-c-Myc oncogenic axis and promoting tumor development. Therefore, USP36 is considered an important oncogene and a highly promising target for novel anticancer drugs. It has also been suggested that it may be involved in the pathological process of neurodegenerative diseases such as Alzheimer's disease; based on its physiological function and disease association, USP36 is not only an important research object for improving the deubiquitination regulatory network, but also has the potential to become a new anti-cancer target. The development of related specific inhibitors has also provided a new direction for tumor treatment.

[0043] The deubiquitinase USP36, abbreviated as USP36, has the amino acid sequence shown in SEQ ID NO.1;

[0044] The base sequence encoding the deubiquitinase USP36 is shown in SEQ ID NO.2:

[0045] Example 1 1. Silencing the deubiquitinating enzyme USP36 significantly promotes CD8. + T cell killing This invention, through screening a deubiquitinase RNA interference library, discovered that silencing the deubiquitinase USP36 significantly inhibited pancreatic cancer cells' resistance to CD8. + The killing ability of T cells. Specific methods are as follows;

[0046] First, a systematic study was conducted using an siRNA library containing approximately 100 deubiquitinase families (DUBs) to investigate deubiquitinating enzymes associated with immune evasion in pancreatic cancer cells SW1990. This experiment aimed to systematically screen key deubiquitinating enzymes regulating CD8⁺T cell immune evasion in pancreatic cancer SW1990 cells using a cytotoxicity assay based on lactate dehydrogenase (LDH) release. The experimental principle is that CD8⁺T cell-mediated target cell killing leads to increased membrane permeability, resulting in the leakage of intracellular LDH into the culture supernatant. The LDH activity in the supernatant is directly proportional to the target cell death rate. This invention accurately assesses the killing efficacy of CD8⁺T cells by calculating the percentage of specific cytotoxicity. Based on this, a high-throughput screening of SW1990 cells was performed using an siRNA library covering nearly 100 deubiquitinating enzymes. By analyzing the changes in the percentage of cytotoxicity after knocking down specific deubiquitinating enzymes, deubiquitinating enzymes that negatively regulate CD8⁺T cell killing function under basal conditions and thus promote tumor immune escape were identified. In pancreatic cancer SW1990 cells, genome-wide functional screening was conducted, with a 2-fold Fold change and an adjusted p-value less than 0.05 as the screening criterion. This indicates that compared to the control group, CD8⁺T cells significantly increased the specific killing rate of SW1990 cells. Furthermore, this result has been corrected for multiple hypothesis testing, greatly reducing the probability of false positives and ensuring the reproducibility and reliability of the results. Finally, statistical analysis revealed that 15 deubiquitinating enzymes, including the deubiquitinating enzyme USP36, are involved in resisting CD8⁺T cells. + The cytotoxic function of T cells, including those such as USP22, USP7, and USP9X, has been proven by research. Figure 1 A~ Figure 1 (B in the middle).

[0047] To further verify the effect of deubiquitinase USP36 on CD8 +The effect of T cell killing function: In pancreatic cancer cells SW1990, this invention silenced the deubiquitinase USP36. Western blot and RT-qPCR were used to detect the protein and mRNA levels of deubiquitinase USP36 after silencing. The results showed that deubiquitinase USP36 was successfully silenced in SW1990 cells. Figure 1 C~ Figure 1 (D in the middle).

[0048] Through the above CD8 + T-cell killing experiments further demonstrated that after silencing the deubiquitinase USP36, pancreatic cancer cells were more susceptible to CD8+ cytotoxicity. + T cells kill ( Figure 1 (E).

[0049] 2. The deubiquitinating enzyme USP36 enhances the transcriptional level of PD-L1 in pancreatic cancer cells, thereby increasing the resistance of pancreatic cancer cells to CD8. + T cell killing ability To further investigate how the deubiquitinase USP36 promotes pancreatic cancer cells' resistance to CD8. + This invention investigates the killing ability of T cells and the mechanisms by which they promote cellular immune escape. Clinical data from pancreatic cancer patients were downloaded from the TCGA database, and then analyzed statistically and graphically using Graphpad Prism9 software. The expression of USP36 in normal pancreatic tissue and pancreatic cancer was studied. TCGA database analysis demonstrated high expression of USP36 in pancreatic cancer. The correlation between USP36 and PD-L1 was analyzed. RNA-Seq sequencing was performed on the siControl and siUSP36 groups in SW1990 cells, with three samples from each group. The sequencing results were then analyzed using the Limma package in R language for FPKM (Fragments per kilo base per million mapped reads), adjusted for a 1.5-fold fold change. p Values ​​less than 0.05 were used as screening criteria. Finally, gene enrichment analysis of immune responses was performed on the siControl and siUSP36 groups using GSEA 4.1.0 software. Simultaneously, heatmap analysis of differentially expressed genes from sequencing results was used to explore the effect of USP36 on biomarkers such as PD-L1 involved in regulating immune escape in pancreatic cancer. RNA-seq data analysis revealed that silencing USP36 promoted the immune response of pancreatic cancer cells, and silencing USP36 significantly inhibited the transcriptional level of PD-L1. Figure 2 B in Figure 2(C) Flow cytometry was used to detect the expression of PD-L1 on the cell membrane surface after silencing the deubiquitinating enzyme USP36. The results showed that silencing the deubiquitinating enzyme USP36 inhibited the expression level of PD-L1 on the cell membrane (C). Figure 2 (D in the middle).

[0050] The siControl group refers to the control group, which did not have USP36 silenced compared to the experimental group. The treatment method involved transfecting SW1990 cells with siControl siRNA. The siControl nucleotide sequence is shown in SEQ ID NO. 9 and SEQ ID NO. 10. The siRNA transfection concentration was 35 nM.

[0051] The siUSP36 group refers to the experimental group, in which USP36 was silenced in SW1990 cells. The treatment method was to transfect SW1990 cells with siUSP36 siRNA. The siRNA transfection concentration was 35 nM.

[0052] The siRNAs include siUSP36#1, siUSP36#2, and siUSP36#3. The nucleotide sequences of siUSP36#1 are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequences of siUSP36#2 are shown in SEQ ID NO.5 and SEQ ID NO.6; and the nucleotide sequences of siUSP36#3 are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0053] Next, siRNA containing the deubiquitinase USP36 was transfected into SW1990 cells. The siRNA transfection concentration was 35 nM.

[0054] The siRNAs include siUSP36#1 and siUSP36#2. The nucleotide sequences of siUSP36#1 are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequences of siUSP36#2 are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0055] After silencing USP36, PD-L1 was overexpressed, and the protein and mRNA levels of PD-L1, as well as CD8, were detected by Western blot and RT-qPCR. + The T-cell killing assays further validated that the deubiquitinating enzyme USP36 enhances the pancreatic cancer cells' resistance to CD8 by promoting the transcriptional level of PD-L1. + The killing ability of T cells ( Figure 2 E~ Figure 2 (G in the middle).

[0056] As a deubiquitinating enzyme, USP36 is questionable whether it can participate in regulating PD-L1 ubiquitination levels by binding to PD-L1. This invention demonstrates this through protein immunoprecipitation (Co-IP) experiments. HEK293T cells were co-transfected with Flag-USP36 overexpression plasmid and Flag-PD-L1 overexpression plasmid and cultured for 40 hours until protein expression was sufficient. Cell lysis: After discarding the supernatant, the cells were washed twice with pre-cooled PBS (containing protease inhibitors), incubated with IP lysis buffer for 30 minutes on ice, centrifuged, and the supernatant (total protein) was collected, with 10 μL reserved as input. Pre-cleansing: 50 μL of protein A / G liposuction beads were added to the supernatant, incubated at 4°C for 1 hour, and centrifuged to collect the supernatant. Immunoprecipitation: The supernatant was divided into three groups, with Flag antibody, PDL1 antibody, and normal host IgG (negative control) added respectively, and incubated overnight at 4°C; the next day, agarose beads were added and incubated for 3 hours. Washing and elution: Centrifuge and discard supernatant, wash 5 times with lysis buffer, add 2×SDS loading buffer, heat at 95℃ for 10 min, centrifuge and collect supernatant. Western blot detection: Input and IP samples are electrophoresed and transferred to a membrane, and detected with the corresponding antibody. The results show that the deubiquitinase USP36 protein does not interact with PD-L1. Figure 2 (H in the text) Preliminary evidence shows that the deubiquitinase USP36 promotes PD-L1 expression by increasing the transcriptional level of PD-L1.

[0057] The above experimental results indicate that the deubiquitinase USP36 enhances the transcriptional level of PD-L1 in pancreatic cancer cells, thereby increasing the resistance of pancreatic cancer cells to CD8. + The killing ability of T cells.

[0058] 3. Deubiquitinase USP36 promotes pancreatic cancer cells' resistance to CD8 by regulating YAP. + T cell killing ability Gene set enrichment analysis (GSEA) of RNA-seq data was performed as follows: RNA-Seq sequencing was performed on the siControl and siUSP36 groups in SW1990 cells, with three samples from each group. The sequencing results were then analyzed using the Limma package in R to measure FPKM (Fragments per kilo base per million mapped reads), adjusted to a Foldchange of 1.5-fold. pValues ​​less than 0.05 were used as screening criteria. Finally, gene enrichment analysis of YAP was performed on the siControl and siUSP36 groups using GSEA 4.1.0 software. The results showed that YAP-related genes were mainly enriched in the siControl group, indicating that the expression level of YAP-related genes decreased after silencing the deubiquitinase USP36. Figure 3 (A in the middle).

[0059] This invention further analyzes the relationship between the deubiquitinase USP36 and the classic downstream genes of YAP, CYR61 and CTGF, from the Tumor Genome Atlas (TCGA) database. The analysis results show that there is a significant positive correlation between the deubiquitinase USP36 and the classic downstream genes of YAP, such as CYR61 and CTGF. Figure 3 B in Figure 3 (C in the middle).

[0060] By silencing the deubiquitinase USP36 through transfection of siUSP36 in SW1990 cells and simultaneously overexpressing Flag-YAP, it was confirmed at the cellular level that silencing the deubiquitinase USP36 significantly downregulated YAP protein and its downstream target genes CTGF and CYR61. Figure 3 D~ Figure 3 Furthermore, in SW1990 cells, transfecting siUSP36 to silence the deubiquitinase USP36, and simultaneously overexpressing YAP, the results showed that overexpressing YAP on the basis of silencing the deubiquitinase USP36 significantly promoted the resistance of pancreatic cancer cells to CD8. + The killing ability of T cells ( Figure 3 (F in the middle).

[0061] The experimental results above indicate that the deubiquitinating enzyme USP36 promotes pancreatic cancer cells' resistance to CD8 by regulating YAP. + The killing ability of T cells.

[0062] 4. The deubiquitinating enzyme USP36 regulates the mRNA and protein levels of PD-L1 via YAP. To further investigate the regulatory role of the deubiquitinating enzyme USP36 on PD-L1 in pancreatic cancer, this invention analyzed clinical data of pancreatic cancer patients downloaded from the TCGA database. Graphpad Prism 9 software was then used for data analysis, statistical analysis, and graphing. The correlation between PD-L1 and YAP was analyzed, and the results showed a significant positive correlation between PD-L1 and YAP in pancreatic cancer. Figure 4In SW1990 cells, overexpression of YAP after silencing the deubiquitinase USP36 significantly promoted the protein and mRNA levels of PD-L1, as detected by Western blot and RT-qPCR. Figure 4 B in Figure 4 (D in the original text). Furthermore, this invention analyzes the KMplot database, which categorizes pancreatic cancer patient samples into high-expression and low-expression groups based on different quantiles of YAP and PD-L1 gene expression levels. Then, survival curves are plotted using the Kaplan-Meier method to visually demonstrate the changing trends of progression-free survival (PFS) over time in different groups. Simultaneously, the log-rank test is used to calculate the significance of differences between the two groups, yielding the log-rank P-value. In addition, the hazard ratio (HR) and its 95% confidence interval (CI) are calculated to assess the impact of YAP and PD-L1 gene expression levels on PFS in pancreatic cancer patients. It was found that patients with high YAP expression in pancreatic cancer have shorter PFS (progression-free survival). Figure 4 In addition to E), the progression-free survival (PFS) of pancreatic cancer patients with high PD-L1 expression was also significantly shortened. Figure 4 (F in the middle).

[0063] The experimental results above show that the deubiquitinating enzyme USP36 regulates the mRNA and protein levels of PD-L1 through YAP.

[0064] 5. The deubiquitination enzyme USP36 regulates the stability of YAP protein by promoting the deubiquitination level of YAP. The above results indicate that transfecting siUSP36 into SW1990 cells to silence the deubiquitinating enzyme USP36 significantly inhibits the protein expression level of YAP. The specific method for real-time quantitative PCR is as follows: First, RNA is extracted. Cells transfected with siUSP36 in a 12-well plate are lysed using a Trizol extraction kit to remove impurities such as protein and DNA. Pure total RNA is obtained by elution, and RNA purity (A260 / A280≈2.0) and concentration are detected using Nanodrop. Reverse transcription to synthesize cDNA: Following the reverse transcription kit instructions, total RNA is used as a template. Reverse transcriptase, primers (Oligo dT or random primers), and other reagents are added. Reverse transcription is performed on a PCR instrument (usually incubated at 37℃ for 45 min, then enzyme activity is inactivated at 85℃) to obtain cDNA. qPCR reaction system preparation: In the qPCR reaction tube, add cDNA template, specific primers (target gene and internal reference gene, YAP and 36B4), fluorescent dye (SYBR Green), and qPCR Mix in proportion. Add enzyme-free water to the total volume and mix gently. qPCR amplification and fluorescence acquisition: Place the reaction tube in the qPCR instrument, set the reaction program (pre-denaturation: 95℃ 30s; cycling: 95℃ 5s, 60℃ 30s, 40 cycles, fluorescence signal acquired at the end of each cycle; melting curve: 95℃ 15s, 60℃ 1min, 95℃ 15s), and start the reaction. Result analysis: After the reaction, analyze the melting curve using the qPCR instrument's built-in software (to verify primer specificity; a single peak indicates acceptable results). A 2^ (-ΔΔCt) The relative expression level of the target gene was calculated using a method similar to the standard method, or Ct values ​​were directly read for inter-group comparisons. The results showed that the mRNA level of YAP was not statistically different from that of the control group. Figure 5 (A) Based on the above results, the present invention can preliminarily determine that the deubiquitinase USP36 does not affect the mRNA level of YAP, but may regulate the post-translational modification of the YAP protein.

[0065] To verify whether the deubiquitinase USP36 is involved in the ubiquitination and degradation of YAP protein, this invention first used immunofluorescence experiments. SW1990 cells were seeded on cell slides and cultured to a suitable state. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. YAP (mouse anti) and deubiquitinase USP36 (rabbit anti) specific antibodies were added for incubation. After washing with PBS, fluorescently labeled secondary antibodies were added for incubation, followed by nucleus staining with DAPI. Finally, the slides were mounted, and the expression and localization of YAP and deubiquitinase USP36 proteins were observed under a fluorescence microscope. Co-localization of deubiquitinase USP36 and YAP proteins was found. Simultaneously, a protein immunoprecipitation experiment was conducted. HEK293T cells were co-transfected with Flag-USP36 overexpression plasmid and Myc-YAP overexpression plasmid and cultured for 30 hours until the protein was fully expressed. Cell lysis: After discarding the supernatant, wash twice with pre-cooled PBS (containing protease inhibitors), add IP lysis buffer and incubate on ice for 30 min, centrifuge to collect the supernatant (total protein), and reserve 10 μL for input. Pre-cleansing: Add 50 μL of protein A / G liposuction beads to the supernatant, incubate at 4℃ for 1 h, centrifuge to collect the supernatant. Immunoprecipitation: Divide the supernatant into 3 groups, add Flag antibody, PDL1 antibody, and normal host IgG (negative control) respectively, and incubate at 4℃ overnight; the next day, add agarose beads and incubate for 3 h. Washing and elution: Centrifuge to discard the supernatant, wash 5 times with lysis buffer, finally add 2×SDS loading buffer, heat at 95℃ for 10 min, centrifuge to collect the supernatant. Western blot detection: Input and IP samples are electrophoretically transferred to a membrane and detected with the corresponding antibodies. The results further illustrate the interaction between the deubiquitinase USP36 and YAP protein ( Figure 5 B in Figure 5 (C) In SW199 cells, siControl or siUSP36 was transfected, and after 48 hours, the cells were treated with DMSO or 10 μM MG132 for 6 hours. Western blot was used to detect the protein level of YAP. If MG132 treatment eliminated the downregulation of YAP protein caused by silencing the deubiquitinase USP36, it indicates that the deubiquitinase USP36 affects the ubiquitin-proteasome degradation pathway of YAP protein. The results show that after MG132 blocked the proteasome degradation pathway, the downregulation of YAP protein caused by silencing the deubiquitinase USP36 could be eliminated. The above experimental results preliminarily demonstrate that the deubiquitinase USP36 affects the stability of YAP protein by regulating the proteasome pathway. Figure 5(D in the original text). Furthermore, in HEK293T cells, this invention overexpressed Flag-USP36 and Myc-YAP, and after treatment with 10 μM MG132 for 6 h, the effect of the deubiquitinating enzyme USP36 on the ubiquitination level of YAP protein was observed by Western blot. This invention used Western blot to detect the ubiquitination level of YAP protein and found that overexpression of the deubiquitinating enzyme USP36 significantly inhibited the ubiquitination level of YAP protein, that is, the deubiquitinating enzyme USP36 promoted the deubiquitination of YAP protein (D in the original text). Figure 5 (E in the text).

[0066] The experimental results above show that the deubiquitination enzyme USP36 regulates the stability of YAP protein by promoting the deubiquitination level of YAP.

[0067] In summary, the deubiquitinase USP36 stabilizes YAP protein by promoting its deubiquitination, thereby enabling more YAP / TEAD to bind to the enhancer region of PD-L1, promoting PD-L1 expression, and ultimately facilitating immune escape from pancreatic cancer cells (see [link to article]). Figure 6 ).

[0068] This invention provides the application of deubiquitinating enzymes in the preparation of pancreatic cancer drugs, and provides a new method for treating pancreatic cancer.

[0069] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0070] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0071] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a substance that inhibits the expression of deubiquitinase USP36 in the preparation of products for treating pancreatic cancer, characterized in that, The amino acid sequence of the deubiquitinase USP36 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid encoding the deubiquitinase USP36 is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The substance that inhibits the expression of deubiquitinase USP36 includes siRNA.

4. The application according to claim 3, characterized in that, The siRNA is selected from any one or more of siUSP36#1, siUSP36#2 and siUSP36#3. The justice chain of siUSP36#1 is shown in SEQ ID NO.3; The justice chain of siUSP36#2 is shown in SEQ ID NO.5; The justice chain of siUSP36#3 is shown in SEQ ID NO.

7.

5. The application according to claim 1, characterized in that, The product uses the substance that inhibits the expression of deubiquitinase USP36 as the sole active ingredient or one of the active ingredients.

6. The application according to claim 5, characterized in that, When the product uses the substance that inhibits the expression of the deubiquitinase USP36 as one of its active ingredients, the active ingredient also includes a chemotherapy drug.

7. The application according to claim 6, characterized in that, The chemotherapy drug is selected from any one or more of gemcitabine, paclitaxel, and 5-fluorouracil.

8. A drug for treating pancreatic cancer, characterized in that, The drug includes the substance of claim 1 that inhibits the expression of deubiquitinase USP36.

9. The medicament according to claim 8, characterized in that, The substance that inhibits the expression of deubiquitinase USP36 includes siRNA.

10. The medicament according to claim 8, characterized in that, The siRNA is selected from any one or more of siUSP36#1, siUSP36#2 and siUSP36#3. The justice chain of siUSP36#1 is shown in SEQ ID NO.3; The justice chain of siUSP36#2 is shown in SEQ ID NO.5; The justice chain of siUSP36#3 is shown in SEQ ID NO.7.

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