Application of ESRP1 in preparation of medicine for treating gastric cancer

Abstract

The invention relates to application of ESRP1 in preparation of a medicine for treating gastric cancer. Relative expression, clinical significance and biological functions of ESRP1 in in-vitro and in-vivo GC cells are analyzed. Researches find that ESRP1 is overexpressed in GC formalin-fixed paraffin embedded tissues and is related to poor prognosis of GC patients. The ESRP1 can inhibit ferroptosis of GC cells in vitro and in vivo. In addition, the invention also finds that the ESRP1 can effectively splice PLA2G4C by regulating exon jump. Therefore, ESRP1 may be a new potential therapeutic target for resisting GC.

Description

Technical Field

[0001] The present invention relates to the field of tumor molecular biology, and in particular to an application of ESRP1 in preparing a drug for treating gastric cancer. Background Art

[0002] Gastric cancer (GC) remains an important cancer worldwide, with more than 1 million new cases in 2020 and an estimated 769,000 deaths from GC (equivalent to 1 in 13 people worldwide), ranking fifth in the world in incidence and fourth in mortality. The incidence rate in men is twice that in women. [1] , East Asia, Eastern Europe, and South America are high-incidence areas for gastric cancer and mortality. Risk factors for gastric cancer include Helicobacter pylori infection, age, high salt intake, and low intake of fruits and vegetables. [2] Early gastric cancer without lymph node metastasis can be treated with endoscopic mucosal resection or endoscopic submucosal resection, and even completely cured. The 5-year survival rate of early gastric cancer is over 90%. However, once gastric cancer enters the late stage, treatment can only rely on surgical resection or chemotherapy and radiotherapy. Patients often face a higher risk of distant metastasis and recurrence, and the 5-year survival rate is less than 30%. [3] Although the emergence of targeted therapy and immunotherapy has made many advances in the comprehensive treatment of gastric cancer, the overall long-term prognosis of patients has not been significantly improved. In recent years, with the continuous development of high-throughput genomics, transcriptomics, proteomics and metabolomics technologies, more and more gastric cancer dysregulation markers have been gradually discovered. [2] .

[0003] Since 2012, ferroptosis has been recognized as a new form of regulated cell death (RCD), distinct from necrosis, apoptosis, and autophagy. [3] Ferroptosis is accompanied by the accumulation of lipid reactive oxygen species (ROS) generated by iron metabolism. The main characteristics of ferroptosis are mitochondrial condensation and increased bilayer membrane density. [4] Recently, a large body of evidence has shown that ferroptosis is regulated by multiple signals from different organelles, such as lysosomes, mitochondria, and endoplasmic reticulum. [5] In short, iron accumulation induces lipid peroxidation, which in turn triggers plasma membrane disruption. [6] Existing evidence shows that ferroptosis is involved in regulating a variety of diseases in different organs, such as the liver, lung, breast, pancreas, and stomach. [7] .

[0004] Alternative splicing of pre-messenger RNA (pre-mRNA) provides a pathway for a single gene to generate multiple mRNA transcripts and protein isoforms with different functions. [8,9] Dysregulation of alternative splicing provides a potentially rich source of novel tumor-specific and subtype-specific therapeutic targets

[10] The alternative splicing process is catalyzed by the spliceosome, which is composed of five small nuclear ribonucleoproteins [11,12] Alternative splicing regulators have emerged as oncogenes and tumor suppressors that contribute to the initiation and progression of many human cancers.

[13] .

[0005] Many splicing factors reportedly affect hallmarks of cancer

[14] Aberrant splicing in cancer, including gastric cancer, is an emerging area that may affect genes and proteins at both the expression and functional levels. These events are regulated by a complex process involving the core spliceosomal machinery and multiple regulatory factors.

[15] Epithelial splicing regulatory protein 1 (ESRP1) and its closely related paralogous relative, epithelial splicing regulatory protein 2 (ESRP2), are known as the "splicing masterminds" of epithelial cells.

[16] ESRP1 and ESRP2 are involved in cancer invasion and metastasis and in the regulation of splicing programs involved in epithelial to mesenchymal transition (EMT) [17,18] .

[0006] Phospholipase A2 (PLA2) is an enzyme that hydrolyzes the sn-2 fatty acid of phospholipids. There are more than 50 enzymes in the PLA2 superfamily.

[19] PLA is mainly divided into secretory PLA2 (sPLA2), cytoplasmic PLA (cPLA2), Ca 2+ Independent PLA2 (iPLA2) and lipoprotein-associated phospholipase A2 (Lp-PLA2; encoded by PLA2G7, also known as plasma platelet-activating factor acetylhydrolase (PAF-AH)), both of which have different sequences and structures [20,21] As a member of the PLA2 enzyme family, phospholipase A2 IVC group (PLA2G4C) hydrolyzes glycerophospholipids to produce free fatty acids and lysophospholipids, both of which are precursors for the production of signaling molecules. Summary of the invention

[0007] The purpose of the present invention is to provide an application of ESRP1 in the preparation of a drug for treating gastric cancer. The present invention obtains target genes by integrating the Cancer Therapeutics Response Portal (CTRP) database, The Cancer Genome Atlas (TCGA) data, and RNA seq combined with non-targeted metabolomics. Through multiple in vitro and in vivo experiments, the role of ESRP1 and PLA2G4C in GC ferroptosis response was discovered. ESRP1 inhibits ferroptosis of gastric cancer by regulating exon skipping of PLA2G4C. Disrupting the ESRP1 / PLA2G4C axis may be a promising strategy for treating gastric cancer.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The first object of the present invention is to provide an application of ESRP1 in the preparation of a drug for treating gastric cancer.

[0010] Furthermore, the ESRP1 is used in the preparation of a drug for inhibiting ferroptosis of gastric cancer cells.

[0011] The second object of the present invention is to provide a use of PLA2G4C in preparing a drug for treating gastric cancer, where PLA2G4C is a target gene of ESRP1.

[0012] The third object of the present invention is to provide an ESRP1 inhibitor for use as a ferroptosis regulator in the preparation of a gastric cancer targeted drug, wherein the ESRP1 inhibitor downregulates ESRP1 expression and promotes ferroptosis of gastric cancer cells.

[0013] Furthermore, the gastric cancer targeted drug is in the form of tablets, capsules, granules, injections or sprays.

[0014] The fourth object of the present invention is to provide a use of ESRP1 in preparing a kit for predicting the prognosis of gastric cancer.

[0015] Furthermore, ESRP1 was highly expressed in tissue samples of gastric cancer patients, suggesting a poor prognosis for gastric cancer patients.

[0016] The fifth object of the present invention is to provide an application of PLA2G4C in preparing a kit for predicting the prognosis of gastric cancer.

[0017] The sixth object of the present invention is to provide a kit for determining ferroptosis of gastric cancer cells, wherein the kit can detect the content of the marker ESRP1 in gastric cancer tissue.

[0018] Furthermore, the kit includes an upstream primer sequence and a downstream primer sequence of Human ESRP1, the upstream primer sequence is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQ ID NO.10.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention analyzes the relative expression, clinical significance and biological function of ESRP1 in GC cells in vitro and in vivo. The study found that ESRP1 is overexpressed in GC formalin-fixed paraffin-embedded tissue and is associated with poor prognosis in GC patients. ESRP1 can inhibit ferroptosis of GC cells both in vitro and in vivo. In addition, the present invention also found that ESRP1 effectively splices PLA2G4C by regulating exon skipping. Therefore, ESRP1 may be a new potential therapeutic target for combating GC.

[0021] Through mechanism exploration and multiple verification methods, the present invention confirms that ESRP1 regulates ferroptosis and lipid metabolism in gastric cancer by acting on PLA2G4C, ultimately leading to the malignant biological behavior of gastric cancer cells.

[0022] The present invention found that epithelial splicing regulatory protein 1 (ESRP1) regulates lipid metabolism-related gene phospholipase A2 group IVC (PLA2G4C) and gastric cancer ferroptosis response through alternative splicing, and GC epithelial splicing regulatory protein 1 (ESRP1) regulates lipid metabolism-related gene phospholipase A2 group IVC (PLA2G4C) and gastric cancer (GC) ferroptosis response through alternative splicing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 ESRP1 is associated with ferroptosis and is upregulated during human gastric cell carcinoma. A is a schematic diagram of the research results showing the correlation between the Pearson correlation Z score and the five ferroxidase inhibitors (Erastin, RSL3, ML162, ML210, and lapatinib) associated with ESRP1 in the CTRP database. B is that in the TCGA database, compared with adjacent tissues, the expression levels of ESRP1 and ESRP2 in gastric cancer tissues are higher, and ESRP1 is more significant. C is compared with ESRP2, ESRP1 is more significantly correlated with the prognosis and survival rate of gastric cancer patients. D is an immunohistochemical analysis of clinical samples from 10 gastric cancer patients, which found that the expression of ESRP1 in gastric cancer tissues was higher than that in adjacent tissues.

[0024] Figure 2 Schematic diagram of the research results of the biological role of ESRP1 in gastric cancer. ESRP1 does not promote the development of gastric cancer by affecting cell proliferation, cell cycle and EMT progression. A is qPCR to prove that ESRP1 is effectively knocked down, B is CCK8 flow cytometry to prove that there is no difference between the si-ESRP1 group and the NC group, C is the comparison of the cell cycle of ESRP1 knockout and the control group by flow cytometry, and the results show no difference, and D is protein blotting to prove that after ESRP1 is knocked out in gastric cancer cells, there is no significant difference between the two specifically expressed proteins: e-cadherin and vimentin;

[0025] Figure 3 Schematic diagram of the detection of ferroptosis-related indicators in the control group and ESRP1 knockout group. ESRP1 inhibits ferroptosis of GC cells. A shows that the luminescence intensity of CellTiter Glo in the control group is lower than that in the ESRP1 knockout group. B shows that the MDA content analysis results show that the MDA level in the ESRP1 knockdown group is significantly increased. C shows that the fluorescence intensity of C11Bodypy 581 / 591 in the control group is higher than that in the ESRP1 knockout group.

[0026] Figure 4 The figure is a schematic diagram of the results of RNA sequencing of GC cells between ESRP1 siRNA knockout cells and control cells, wherein A is a schematic diagram of gene up-regulation and down-regulation in GC cells knocked out by ESRP1 siRNA, B is a schematic diagram of the five alternative splicing events that occurred after knocking out ESRP1, C is a schematic diagram of the correlation between high and low expression groups of ESRP1 in gastric cancer analyzed by the TCGA database, and D is a schematic diagram of the gene PLA2G4C obtained by cross-combination of RNA sequencing data in the TCGA database;

[0027] Figure 5 Schematic diagram of the metabolite mass spectrometry analysis of gastric cancer cells with simultaneous knockout of ESRP1 and PLA2G4C;

[0028] Figure 6 Schematic diagram of 4HNE staining of tumor samples;

[0029] Figure 7 Schematic diagram of the RNA and protein expression levels of PLA2G4C in gastric cancer cells after knocking out ESRP1;

[0030] Figure 8 It is a mechanism diagram of the present invention. DETAILED DESCRIPTION

[0031] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0032] In the following embodiments, the material purchasing channels are as follows:

[0033] The gastric cancer cell line SNU-216 was purchased from Procell, and the AGS, HGC27, MKN45, and MKN28 cell lines were purchased from Fuheng Biotechnology. All cell lines were regularly tested for mycoplasma.

[0034] RPMI-1640 medium and DMEM medium for cell culture were purchased from Biosharp, F12K medium for cell culture was purchased from Fuheng Biotechnology, and fetal bovine serum (FBS) and penicillin / streptomycin were purchased from BIOEXPLORER Life Sciences.

[0035] BALB / c male nude mice, 4 weeks old, weighing 20-25 g, were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0036] Lipofectamine TM 3000 and BODIPY-581 / 591 C11 transfection kits were purchased from Thermo Fisher Scientific.

[0037] siRNA was purchased from Shanghai Gene Pharmaceutical Co., Ltd.

[0038] DAB was purchased from Wuhan Servicebio Technology, Wuhan, China.

[0039] Chemiluminescent detection reagents were purchased from Promega, Beijing, China.

[0040] TRIzol reagent was purchased from Invitrogen, Carlsbad, CA, USA.

[0041] Primers were provided by Shanghai Sangon Biotechnology Co., Ltd.

[0042] PE Annexin V Cell Apoptosis Detection Kit I was purchased from BD Biosciences.

[0043] CCK-8 solution was purchased from Bio-Medical, Shanghai, China.

[0044] CytoFLEX cell counter and CytExpert software were from Beckman Coμlter.

[0045] The nuclear and cytoplasmic protein extraction kit (P0028) was purchased from BEYOTIME.

[0046] The primary antibodies included 1. Anti-Vimentin Rabbit pAb, Sevier Biotechnology GB111308;

[0047] 2. beta-Tubulin Ab-T0023 Affinity;

[0048] 3. GAPDH Ab-AF7021 Affinity;

[0049] 4. ESRP1 Polyclonal antibody proteintech;

[0050] 5. Anti-PLA2G4C 3115308 SIGEMA;

[0051] The secondary antibody was from Goat Anti-Rabbit lgG(H+L)HRP-S0001 Affinity.

[0052] In the following examples, the nucleotide sequences are as follows:

[0053] The nucleotide sequence of Human ESRP1 (262) is shown in SEQ ID NO.1:

[0054] 5′-GAGAAGGUAGUGAUCCUGCUGUUCU-3′;

[0055] The nucleotide sequence of Human ESRP1 (981) is shown in SEQ ID NO.2:

[0056] 5′-CGGAGAAGCUUCGUGUAGGUUGUUGUA-3′;

[0057] The nucleotide sequence of Human ESRP2 (862) is shown in SEQ ID NO.3:

[0058] 5′-CCGAGGUGAUAAAGCAGAAAUACGA-3′;

[0059] The nucleotide sequence of Human ESRP2 (1126) is shown in SEQ ID NO.4:

[0060] 5′-GCGUCCUGCUAUAUGUGUAUAA-3′;

[0061] The nucleotide sequence of Human PLA2G4C (369) is shown in SEQ ID NO.5:

[0062] 5′-GCUCUGAAGUUCCAUAAUTT-3′;

[0063] The nucleotide sequence of Human PLA2G4C (1165) is shown in SEQ ID NO.6:

[0064] 5′-GGCUGUGCUAAUGCUAAATT-3′;

[0065] The nucleotide sequence of Human PLA2G4C (608) is shown in SEQ ID NO.7:

[0066] 5′-GGAUCCACUGGGCAAUUTT-3′;

[0067] The nucleotide sequence of Human PLA2G4C (1367) is shown in SEQ ID NO.8:

[0068] 5′-GGCUCACUCAGUAACCUGATT-3′;

[0069] The upstream primer sequence of Human ESRP1 is shown in SEQ ID NO.9:

[0070] 5′-CAATATTGCCAAGGGAGGTG-3′;

[0071] The downstream primer sequence of Human ESRP1 is shown in SEQ ID NO.10:

[0072] 5′-GTCCCCATGTGATGTTTGTG-3′;

[0073] The upstream primer sequence of Human ESRP2 is shown in SEQ ID NO.11:

[0074] 5′-TGCCACAGAGGATGACTTTG-3′;

[0075] The downstream primer sequence of Human ESRP2 is shown in SEQ ID NO.12:

[0076] 5′-ATTGACTGCTGGCTTTG-3′;

[0077] The upstream primer sequence of Human PLA2G4C is shown in SEQ ID NO.13:

[0078] 5′-CCGCAGTGCACCATGGGAAGCTC-3′;

[0079] The downstream primer sequence of Human PLA2G4C is shown in SEQ ID NO.14:

[0080] 5′-CTCCCAGCTACCCGGCCAGTTC-3′.

[0081] The TT at the end of the above sequences is mainly to strengthen the sequence viscosity, improve the sequence stability, and enhance the interference effect without affecting the sequence itself.

[0082] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0083] Example 1

[0084] 1 Method

[0085] 1.1 The Cancer Genome Atlas (TCGA) analysis method

[0086] Normalized expression analysis of ESRP1 and ESRP2 was performed in 449 gastric adenocarcinoma patients enrolled in the Cancer Genome Atlas (TCGA) database gastric adenocarcinoma (STAD) (https: / / portal.gdc.cancer.gov / projects / TCGA-STAD) subtype classification study.

[0087] For the purpose of survival modeling, gene expression levels below or equal to the median were considered low expression, and those above the median were considered high expression. Overall survival was calculated from the date of initial diagnosis to disease-specific death (patients whose vital status was referred to as dead) and to the month of last follow-up (patients who survived). Statistical analysis (log-rank test) and generation of Kaplan-Meier curves were performed using the "survival" package in R (R Foundation for Statistical Computing).

[0088] 1.2 Clinical samples

[0089] A total of 10 paired tumor samples and adjacent healthy tissue samples were collected from GC patients who underwent surgery at Shanghai Ninth People's Hospital in 2020. All 10 GC patients included in the present invention were pathologically diagnosed as moderately to poorly differentiated adenocarcinoma, pathologically stage III, and required conventional chemotherapy after surgery. Written consent was obtained from all relevant patients, and the study was approved by the Institutional Ethics Review Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, and GC tissues and normal tissues were obtained.

[0090] 1.3 Cell culture

[0091] SNU-216, MKN45 and MKN28 cell lines were cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2; AGS cell line was cultured in F12K medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2; HGC27 cell line was cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2.

[0092] 1.4 Small interfering RNA (siRNA) transfection

[0093] Use of Lipofectamine TM GC cells were transfected with siRNA specifically targeting ESRP1 or ESRP2 (Shanghai Gene Pharmaceutical Co., Ltd.) using the 3000 transfection kit (Thermo Fisher Scientific, L3000008) according to the manufacturer's instructions and the specified time. The sequences used in the small interfering RNA (siRNA) transfection are shown in SEQ ID NO.1 to SEQ ID NO.8.

[0094] 1.5 Immunohistochemistry (IHC)

[0095] Immunostaining was performed on paraffin-embedded GC tissues and paired normal tissues obtained in step 1.2. The sections were incubated in 3% H2O2 at 37°C for 30 min to quench endogenous peroxidase activity, followed by antigen retrieval in a microwave oven and then blocked with 5% BSA for 60 min at room temperature. Primary antibodies were used to detect the expression of the specified proteins. After incubation with secondary antibodies conjugated to horseradish peroxidase (HRP), the sections were developed with DAB (Wuhan Servicebio Technology, Wuhan, China). The sections were stained with hematoxylin, dehydrated, and mounted for bright field microscopy.

[0096] 1.6 Protein extraction and Western blotting

[0097] The cells were rinsed twice with cold PBS and then lysed with RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors and phosphatase inhibitor cocktails (5 mM sodium fluoride, 1 mM sodium vanadate, and 1 mM PMSF). The blots were incubated with rabbit polyclonal anti-ESRP1, rabbit polyclonal anti-vimentin, and mouse monoclonal anti-E-cadherin antibodies. Proteins were developed using enhanced chemiluminescence detection reagents according to the manufacturer's instructions and densitometry was analyzed using Image J software.

[0098] 1.7q-PCR

[0099] Total RNA was extracted from GC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA content was determined according to the instructions of the RNA extraction kit from Invitrogen. Secreted RNA was then reverse transcribed into cDNA using a reverse transcription kit. PCR amplification was performed using a 20 μl reaction system and detected using BIO-RAD fluorescent real-time PCR.

[0100] The sequences of the gene-specific primers are shown in SEQ ID NO.9 to SEQ ID NO.14.

[0101] 1.8 Cell apoptosis detection

[0102] Flow cytometry was performed using the PE Annexin V Cell Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer's instructions. ESRP1 siRNA and control siRNA cells (uninterfered siRNA in normal tissues) (5 × 10 5 cells / ml), washed with PBS and centrifuged, the cells were suspended in 1× binding buffer, and then 5μl Annexin V fluorescein isothiocyanate was added. After 15 minutes, 10μl propidium iodide was added, and the suspension was incubated in the dark at room temperature for 15 minutes. Binding buffer (1×400μl) was added to the suspension and vortexed gently. Cells were analyzed using an EXFLOW flow cytometer. The numbers indicate the percentage of cells in each quadrant (LL-lower left: intact cells; LR-lower right: early apoptotic cells; ΜL-upper left: necrotic cells; UR-upper right: late apoptotic or necrotic cells).

[0103] 1.9 Cell counting kit-8 (CCK-8) assay

[0104] GC cells were transfected with the corresponding oligonucleotides (si-ESRP1 and si-NC). 3 GC cells at 10 cells / ml were seeded into a 96-well plate and cultured at 37°C for 96 h. CCK-8 solution (10 μl, Bious, Shanghai, China) was added to 100 μl of culture medium containing 10% FBS every 24 h.

[0105] 1.10 Colony formation test

[0106] In the colony formation assay, GC cells were cultured at 1 × 10 3 The cells were inoculated into 6-well plates at a density of 1:1 / well and cultured at 37°C for 14 days. The colonies were stained with 0.1% Giemsa and 20% methanol solution. The cells were then counted and analyzed.

[0107] 1.11 Lipid ROS Detection

[0108] In a 12-well plate, 1 × 10 5 cells. 10 μM BODIPY-581 / 591C11 (D3861, Thermo Fisher Scientific) was added to the culture medium and cultured at 37°C, 5% CO2 for 30 minutes. The cells were then collected with EDTA-free trypsin, rinsed twice with PBS, and resuspended in 500 μl PBS. ROS levels were analyzed using a CytoFLEX cell counter (Beckman Coμlter), and data were analyzed using CytExpert software (Beckman Coμlter).

[0109] 1.12 RNA sequencing

[0110] Total RNA from transfected GC cells was extracted by TRIzol (Invitrogen, Carlsbad, CA, USA), and RNA sequence analysis was performed by Shanghai Shupu Biotechnology Co., Ltd.

[0111] 1.13 Non-targeted metabolomics

[0112] The nuclear and cytoplasmic protein extraction kit (P0028) of BEYOTIME was used to extract the protein of gastric cancer cells.

[0113] 1.14 Tumor xenograft models

[0114] BALB / c male nude mice, 4 weeks old, weighing 20-25 g, were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. All animals were housed in an environment with a temperature of 22±1°C, a relative humidity of 50±1%, and a light / dark cycle of 12 / 12 h. All animal studies (including mouse euthanasia procedures) followed the regulations and guidelines for institutional animal care at Shanghai Jiao Tong University and were performed in accordance with AAALAC and IACUC guidelines.

[0115] 1.15 Statistical analysis

[0116] Statistical data were analyzed using SPSS 25.0 (IBM, Chicago, IL, USA). Results are presented as mean ± standard deviation. Chi-square test, Student's t test, and analysis of variance were used to assess the statistical significance of differences between two or more groups. Pearson correlation analysis was used to analyze correlations. P values ​​less than 0.05 were considered statistically significant.

[0117] 2 Conclusion

[0118] ESRP1 is associated with ferroptosis and is upregulated during human gastric cell carcinoma

[0119] Research shows that ferritin deposition plays an important role in the development of GC

[22] . To identify the relevant key genes, the present invention used the Cancer Therapeutics Response Portal (CTRP) database. Among all the genes associated with iron oxidase inhibitors, ESRP1 was found to be associated with five iron oxidase inhibitors, including Erastin (Pearson correlation z-score 5.65; correlation 0.222), RSL3 (Pearson correlation z-score 6.78; correlation 0.264), ML162 (Pearson correlation z-score 7.34; correlation 0.286), ML210 (Pearson correlation z-score 8.01; correlation 0.314) and lapatinib (Pearson correlation z-score 10.7; correlation 0.415) ( Figure 1 A), therefore, it is speculated that ESRP1 may play an important role in ferroptosis in gastric cancer.

[0120] like Figure 1 As shown in Figure B, the expression levels of ESRP1 and ESRP2 in gastric cancer tissues were higher than those in adjacent tissues, and the expression level of ESRP1 in gastric cancer tissues was more significantly different from that in adjacent tissues. Figure 1 As shown in C, ESRP1 is strongly correlated with the prognosis and survival of gastric cancer patients. Figure 1 As shown in D, ten pairs of clinical samples from gastric cancer patients were used for immunohistochemical staining to detect the differential expression of ESRP1 in gastric cancer and adjacent tissues. The results showed that the expression of ESRP1 in gastric cancer tissues was higher than that in adjacent tissues.

[0121] 2.2ESRP1 does not promote the development of gastric cancer by affecting cell proliferation and cell cycle

[0122] Changes in cell cycle and cell proliferation are responsible for the progression of many tumors. To determine the biological role of ESRP1 in gastric cancer, we first knocked out the expression of ESRP1 in gastric cancer cells, and then verified the knockout by q-PCR and WB. The results showed that ESRP1 was effectively knocked out ( Figure 2 A). The results of CCK and clone formation assays after si-ESRP1 were not different from those of the control group ( Figure 2 B), indicating that ESRP1 does not affect gastric cancer by promoting cell proliferation. In addition, there was no difference in the cell cycle of si-ESRP1 and the control group by flow cytometry ( Figure 2 C), indicating that ESRP1 does not affect the cell cycle of gastric cancer.

[0123] 2.3ESRP1 has no significant effect on the EMT process of gastric cancer cells

[0124] Epithelial-mesenchymal transition (EMT) is a cellular process in which cells lose epithelial characteristics and acquire mesenchymal characteristics. EMT is associated with a variety of tumor functions, including tumor initiation, malignant progression, tumor stemness, tumor cell migration, infiltration into the blood, metastasis, and drug resistance. [23-25] For a long time, EMT was considered a binary process with two distinct cell populations, epithelial and mesenchymal cells. [23,26] EMT is usually defined by the loss of the epithelial marker E-cadherin and the gain of the mesenchymal marker vimentin.

[27] . Increasing evidence suggests that epithelial splicing regulatory protein 1 (ESRP1) can inhibit epithelial to mesenchymal transition (EMT), thereby playing a key role in regulating tumor metastasis and progression. In order to study whether ESRP1 plays a similar role in gastric cancer, the present invention knocked out the expression of ESRP1 in gastric cancer cells and compared the specific proteins e-cadherin and vimentin ( Figure 2 D), it was found that there was no significant difference in the protein expression between the two groups. This indicates that the promotion of ESRP1 on gastric cancer cells is not achieved by affecting the EMT process.

[0125] 2.4ESRP1 can inhibit ferroptosis of GC cells

[0126] Ferroptosis is driven by iron-dependent phospholipid peroxidation. In order to further study the relationship between ESRP1 and gastric cancer ferroptosis response, the present invention detected the key markers of ferroptosis response, ROS, lipid peroxidation level and cell viability. In vivo, free radicals act on lipids to cause peroxidation, and the final product of oxidation is malondialdehyde. Malondialdehyde (MDA) is a common indicator of membrane lipid peroxidation. Under high temperature (95 degrees Celsius) and acidic environment, malondialdehyde (MDA) can condense with thiobarbituric acid (TBA) to form a red MDA-TBA adduct. Malondialdehyde (MDA) has a maximum absorption peak at 532nm, and the content of lipid peroxidation in the sample can be estimated after colorimetry; the absorbance at 600nm is measured at the same time, and the absorbance difference between 532nm and 600nm is used to calculate the content of MDA.

[0127] BODIPY 581 / 591C11 is a ratiometric fluorescence probe for lipid oxidation that can be used to detect reactive oxygen species (ROS) in cells and cell membranes. Upon oxidation of the polyunsaturated butadiene groups in the dye, the fluorescence emission peak shifts from approximately 590 nm to approximately 510 nm. The ratio of fluorescence readings measured at 590 nm / 510 nm emission light can reflect the extent of cellular lipid peroxidation.

[0128] Compared with the control group and the ESRP1 knockout group, the ferroptosis-related indicators showed significant changes. The present invention used an MDA detection kit to detect the MDA content, and the results were as follows Figure 3 The results showed that compared with the control group, the MDA level in the ESRP1 knockout group was significantly increased ( Figure 3 B). Flow cytometry and laser confocal microscopy showed that the fluorescence intensity of C11Bodypy 581 / 591 in the control group was higher than that in the ESRP1 knockout group ( Figure 3 C). CellTiter Glo (CTG) is a rapid cell viability assay based on ATP detection. The luminescence intensity of CellTiter Glo in the control group was lower than that in the ESRP1 knockout group ( Figure 3 A). Therefore, ESRP1 promotes GC development by affecting ferroptosis of GC cells.

[0129] 2.5 ESRP1 and the epithelial splicing program

[0130] Alternative splicing (AS) is a highly regulated gene expression process that can result in the production of multiple protein isoforms from a single gene. Almost all human pre-RNA transcripts (greater than 90%) undergo alternative splicing, with an average of 7-8 alternative splicing events per multi-exon gene.

[28] Although recent studies strongly suggest that a large number of AS events lead to the production of protein isozymes, the functional consequences of AS at the level of protein function remain unknown for the vast majority of alternatively spliced ​​transcripts.

[29] Dysregulation of AS has been shown to contribute to a variety of human diseases, thus requiring a better understanding of how inappropriate expression of specific protein isozymes is associated with specific disease phenotypes.

[30] However, it is not easy to link global changes in splicing to the functions of specific protein isozymes.

[0131] cell type or disease-related phenotype remains a major challenge, as definition of the biological function of even a single AS event requires years of detailed study.

[29] Recent studies have shown that the main effect of AS at the protein level is to alter protein-protein interactions. Protein regions encoded by alternatively spliced ​​exons are highly enriched in protein disorder and post-translationally modified regions, both of which are involved in protein interactions.

[31] Alternative exons with cell- or tissue-specific splicing differences were more highly enriched for these signatures, suggesting that a major function of tissue-specific AS is to “rewire” protein-protein interaction networks in different cell types, thereby having a global impact on the functions and properties of different cells.

[0132] Next, the present invention performed RNA sequencing (RNA-Seq) on GC cells between ESRP1 siRNA knockout cells and control cells. The results showed that there were significant differences in the RNA profiles of these gene knockout clones. Based on the 2-fold change and P < 0.05, the differentially regulated genes and commonly overlapping genes in each group were determined. Among the 409 significant genes, 263 genes were upregulated and 146 genes were downregulated in ESRP1 siRNA knockout GC cells ( Figure 4 A).

[0133] ESRP1 is an important alternative splicing factor that also regulates alternative splicing events in gastric cancer. Therefore, we further analyzed the role of ESRP1 in alternative splicing and found that five alternative splicing events occurred after ESRP1 knockout: in SE events, 1562 genes were upregulated and 1488 genes were downregulated; in RI events, 401 genes were upregulated and 183 genes were downregulated; in MXE events, 274 genes were upregulated and 194 genes were downregulated; in A5SS events, 260 genes were upregulated and 158 genes were downregulated; in A3SS events, 287 genes were upregulated and 239 genes were downregulated ( Figure 4 B).

[0134] The present invention uses the TCGA database to analyze the correlation between high and low expression groups of ESRP1 in gastric cancer, and a total of 101 differentially expressed genes ( Figure 4 C). Then, the RNA sequencing data in the TCGA database were cross-combined and a gene with special significance, PLA2G4C ( Figure 4 D).

[0135] 2.6PLA2G4C expression levels are significantly correlated with ESRP1 in human gastric cancer cell lines

[0136] In order to verify the synergistic effect of PLA2G4C and ESRP1 in GC, the present invention detected ferroptosis-related indicators in GC cells with ESRP1 and PLA2G4C knockout and GC cells with ESRP1 knockout and PLA2G4C overexpression.

[0137] The RNA and protein expression levels of PLA2G4C were detected by WB and q-PCR to investigate whether ESRP1 is a target of PLA2G4C. Figure 7 As shown in the figure, after knocking out ESRP1, the RNA and protein expression levels of PLA2G4C in gastric cancer cells were decreased compared with the control group, indicating that PLA2G4C is a target gene of ESRP1.

[0138] 2.7 New functional effects of ESRP1 gene knockout on gastric cancer: Effects of ESRP1 on metabolic pathways

[0139] PLA2G4C, as a member of the PLA2 family, is also closely related to lipid metabolism. In order to study which metabolites of gastric cancer cells are most affected by PLA2G4C, mass spectrometry analysis was performed. The present invention simultaneously knocked out ESRP1 and overexpressed PLA2G4C gastric cancer cells, as well as simultaneously knocked out ESRP1 and PLA2G4C gastric cancer cells. The content of the overexpressed pla2 group was significantly higher than that of the Nc group and the overexpressed PLA2G4C group ( Figure 5 ). Comparing the differences in metabolites between the two groups, it was found that LysoPC (16:0) changed most significantly between the two groups.

[0140] ESRP1 / PLA2G4C promotes tumor growth in vitro and in vivo

[0141] The role of the ESRP1 / PLA2G4C axis in GC cell growth in vitro and in vivo was studied. The study found that low expression of ESRP1 and PLA2G4C promoted ferroptosis of GC cells. When ESRP1 and PLA2G4C were at high expression levels, ferroptosis of GC cells was significantly inhibited.

[0142] 4-Hydroxynonenal (4-HNE) is an α, β-unsaturated hydroxyalkenal, a biomarker of lipid peroxidation, and plays an important biological role in cells. Ferroptosis is a regulated cell necrosis caused by lipid peroxidation induced by iron ions and reactive oxygen species. Lipid peroxidation produces a series of oxidation products, among which 4-HNE is one of the important oxidation products, with strong biological activity and toxicity, and is one of the indicators of ferroptosis. Tumor samples of nude mice were stained with 4HNE, and it was found that the fluorescent staining level of the knockout group was higher than that of the control group ( Figure 6 ), indicating that ESRP1 inhibited the ferroptosis process of gastric cancer cells in vivo by targeting PLA2G4C.

[0143] In the verification of this part, it was further clarified that ESRP1 regulates the malignant biological behavior of GC cells through PLA2G4C, and the activation of PLA2G4C is an important step for ESRP1 to promote the progression of GC.

[0144] 3 Discussions

[0145] Most GC patients are diagnosed at an advanced stage, which is the main reason for the overall poor prognosis. Although the pathophysiological reasons for the rapid deterioration of GC cells from appearance to rapid deterioration remain unclear. It is a huge challenge to explore diagnostic GC biomarkers and elucidate the mechanism of GC progression. Increasing evidence shows that splicing factors play a key role in cancers including gastric cancer. [14,32]Splicing factors are RNA-binding proteins that interact with specific RNA sequences or motifs and serve as master regulators of gene expression and cell behavior. Ferroptosis is an iron-dependent, regulated form of cell death that results from the accumulation of lipid peroxidation products.

[0146] These results are consistent with previous reports by Zhang et al.

[33] In line with this, this report analyzed that splicing factors inhibit tumor ferritin deposition by regulating alternative splicing events in endometrial cancer, thereby playing a pro-cancer role. The study by Liu et al.

[34] It was also shown that alternative splicing events inhibit ferroptosis of liver cancer cells, thereby promoting their resistance to chemotherapeutic drugs. This invention first demonstrated that the increase in the level of splicing factor ESRP1 regulates the expression of PLA2G4C, thereby reducing the level of lipid peroxidation in GC cells, thereby inhibiting the ferritin deposition in GC cells, and ultimately leading to GC deterioration ( Figure 8 ).

[0147] ESRP1 and its closely related homolog ESRP2 are known as the "splicing masterminds" of epithelial cells.

[35] , ESRP1 and ESRP2 proteins are mainly studied as splicing regulators in the cell nucleus (ESRP1 is on human chromosome 8 and ESRP2 is on human chromosome 16). The correlation between inhibitors and genes in the CTRP database reflects the close relationship between the ESRP1 gene and the ferroptosis response. The TCGA database analyzed a large number of cases, which may be the reason for the differences observed in gastric cancer. This prompted the present invention to analyze the relevant indicators of ESRP1 and overall survival of gastric cancer. The correlation of ESRP1 high expression in gastric cancer tissue was verified by immunohistochemistry. Although ESRP1's related protein ESRP2 (epithelial splicing regulatory protein 2) is also elevated in gastric cancer, its expression is not correlated with the results in the TCGA database. The present invention concludes that ESRP1 (but not ESRP2) is an important player in ferroptosis in gastric cancer. Unlike previous studies on the role of ESRP1 in other tumors, ESRP1 did not change the proliferation and cell cycle of gastric cancer cells. Moreover, in the present invention, no correlation was observed between the high expression of ESRP1 and the development of EMT in gastric cancer cells. However, by detecting three key indicators of ferroptosis response (MDA, BODIPY 581 / 591C11, CellTiter-Glo), the present invention found that high expression of ESRP1 can reduce the lipid peroxidation level of gastric cancer cells, thereby inhibiting tumor ferroptosis response.

[0148] PLA2G4C hydrolyzes glycerophospholipids to produce free fatty acids and lysophospholipids, both of which are precursors for the production of signal molecules. In the present invention, PLA2G4C, as a downstream gene regulated by ESRP1 variable splicing, participates in the inhibition of gastric cancer ferroptosis together with ESRP1. PLA2G4C, as an important enzyme, is also closely related to lipid metabolism. The present invention found that the expression of PLA2G4C and LysoPC (16:0) in gastric cancer cells is closely related. Therefore, the ESRP1 / PLA2G4C axis (upstream and downstream gene axis) inhibits the ferroptosis process of gastric cancer by inhibiting the level of lipid peroxidation represented by LysoPC (16:0).

[0149] In summary, the present invention has demonstrated through in vivo and in vitro experiments that ESRP1 can inhibit ferroptosis of tumor cells. ESRP1 targets PLA2G4C by regulating alternative splicing of a series of lipid metabolism-related genes. The newly discovered ESRP1 / PLA2G4C axis may be a promising therapeutic target for gastric cancer, and it is expected that potential cancer therapies will be further explored in the future by intervening in the biological functions of the ESRP1 / PLA2G4C axis.

[0150] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

[0151] References are as follows:

[0152] [1]Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F: Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36Cancers in 185Countries.CA Cancer J Clin 2021,71(3):209-249.

[0153] [2]Smyth EC,Nilsson M,Grabsch HI,van Grieken NC,Lordick F:Gastric Cancer.Lancet 2020,396(10251):635-648.

[0154] [3]Nishida T,Sugimoto A,Tomita R,Higaki Y,Osugi N,Takahashi K,MukaiK,Matsubara T,Nakamatsu D,Hayashi S et al:Impact of time from diagnosis tochemotherapy in advanced gastric cancer:A Propensity Score Matching Study toBalance Prognostic Factors.World J Gastrointest Oncol 2019,11(1):28-3

[0155] [4]Yagoda N,von Rechenberg M,Zaganjor E,Bauer AJ,Yang WS,Fridman DJ,Wolpaw AJ,Smukste I,Peltier JM,Boniface JJ et al:RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.Nature 2007,447(7146):864-868.

[0156] [5]Chen X,Kang R,Kroemer G,Tang D:Organelle-specific regµlation offerroptosis.Cell Death Differ 2021,28(10):2843-2856.

[0157] [6]Chen X,Li J,Kang R,Klionsky DJ,Tang D:Ferroptosis:machinery andregµlation.Autophagy 2021,17(9):2054-2081.

[0158] [7]Xu C,Liu Z,Xiao J:Ferroptosis:A Double-Edged Sword inGastrointestinal Disease.Int J Mol Sci 2021,22(22).

[0159] [8]Hoyos LE,Abdel-Wahab O:Cancer-Specific Splicing Changes and thePotential for Splicing-Derived Neoantigens.Cancer Cell 2018,34(2):181-183.

[0160] [9]Marzese DM,Manughian-Peter AO,Orozco JIJ,Hoon DSB:Alternativesplicing and cancer metastasis:prognostic and therapeutic applications.ClinExp Metastasis 2018,35(5-6):393-402.

[0161]

[10] Wang E,Aifantis I:RNA Splicing and Cancer.Trends Cancer 2020,6(8):631-644.

[0162]

[11] Rappsilber J,Ryder U,Lamond AI,Mann M:Large-scale proteomicanalysis of the human spliceosome.Genome Res 2002,12(8):1231-1245.

[0163]

[12] Wahl MC,Will CL,Lührmann R:The spliceosome:design principles of adynamic RNP machine.Cell 2009,136(4):701-718.

[0164]

[13] Urbanski LM,Leclair N,Anczuków O:Alternative-splicing defects incancer:Splicing regμlators and their downstream targets,guiding the way tonovel cancer therapeutics.Wiley Interdiscip Rev RNA 2018,9(4):e1476.

[0165]

[14] Oltean S,Bates DO:Hallmarks of alternative splicing incancer.Oncogene2014,33(46):5311-5318.

[0166]

[15] Irimia M,Blencowe BJ:Alternative splicing:decoding an expansiveregμlatory layer.Curr Opin Cell Biol 2012,24(3):323-332.

[0167]

[16] Aaron L,Franco OE,Hayward SW:Review of Prostate Anatomy andEmbryology and the Etiology of Benign Prostatic Hyperplasia.Urol Clin NorthAm 2016,43(3):279-288.

[0168]

[17] Warzecha CC,Sato TK,Nabet B,Hogenesch JB,Carstens RP:ESRP1 andESRP2 are epithelial cell-type-specific regμlators of FGFR2 splicing.Mol Cell2009,33(5):591-601.

[0169]

[18] Warzecha CC,Jiang P,Amirikian K,Dittmar KA,Lu H,Shen S,Guo W,XingY,Carstens RP:An ESRP-regμlated splicing programme is abrogated during theepithelial-mesenchymal transition.Embo j 2010,29(19):3286-3300.

[0170]

[19] Peng Z,Chang Y,Fan J,Ji W,Su C:Phospholipase A2 superfamily incancer.Cancer Lett 2021,497:165-177.

[0171]

[20] Burke JE,Dennis EA:Phospholipase A2 structure / function,mechanism,and signaling.J Lipid Res 2009,50 Suppl(Suppl):S237-242.

[0172]

[21] Murakami M,Sato H,Taketomi Y:Updating Phospholipase A(2)Biology.Biomolecμles 2020,10(10).

[0173]

[22] Yang P,Yang W,Wei Z,Li Y,Yang Y,Wang J:Novel targets for gastriccancer:The tumor microenvironment(TME),N6-methyladenosine(m6A),pyroptosis,autophagy,ferroptosis and cuproptosis.Biomed Pharmacother 2023,163:114883.

[0174]

[23] Nieto MA,Huang RY,Jackson RA,Thiery JP:EMT:2016.Cell 2016,166(1):21-45.

[0175]

[24] .Brabletz T:To differentiate or not--routes towardsmetastasis.Nat Rev Cancer2012,12(6):425-436.

[0176]

[25] De Craene B,Berx G:Regμlatory networks defining EMT during cancerinitiation and progression.Nat Rev Cancer 2013,13(2):97-110.

[0177]

[26] Puisieux A,Brabletz T,Caramel J:Oncogenic roles of EMT-inducingtranscription factors.Nat Cell Biol 2014,16(6):488-494.

[0178]

[27] Pastushenko I,Blanpain C:EMT Transition States during TumorProgression and Metastasis.Trends Cell Biol 2019,29(3):212-226.

[0179]

[28] Pan Q,Shai O,Lee LJ,Frey BJ,Blencowe BJ:Deep surveying ofalternative splicing complexity in the human transcriptome by high-throughputsequencing.Nat Genet 2008,40(12):1413-1415.

[0180]

[29] Blencowe BJ:The Relationship between Alternative Splicing andProteomic Complexity.Trends Biochem Sci 2017,42(6):407-408.

[0181]

[30] Cieply B,Carstens RP:Functional roles of alternative splicingfactors in human disease.Wiley Interdiscip Rev RNA 2015,6(3):311-326.

[0182]

[31] Bμljan M,Chalancon G,Eustermann S,Wagner GP,Fuxreiter M,BatemanA,Babu MM:Tissue-specific splicing of disordered segments that embed bindingmotifs rewires protein interaction networks.Mol Cell 2012,46(6):871-883.

[0183]

[32] Feng H,Jin Z,Liu K,Peng Y,Jiang S,Wang C,Hu J,Shen X,Qiu W,ChengX et al:Identification and validation of critical alternative splicing eventsand splicing factors in gastric cancer progression.J Cell Mol Med 2020,24(21):12667-12680.

[0184]

[33] CircRAPGEF5 interacts with RBFOX2 to confer ferroptosisresistance by modulating alternative splicing of TFRC in endometrialcancer.doi:10.1016 / j.redox.2022.102493.PMID:36182807;PMCID:PMC9526237.

[0185]

[34] HBV Enhances Sorafenib Resistance in Hepatocellular Carcinoma byReducing Ferroptosis via SRSF2-Mediated Abnormal PCLAF Splicing.doi:10.3390 / ijms24043263.PMID:36834680;PMCID:PMC9967099.

[0186]

[35] Tavanez JP,Valcárcel J.A splicing mastermind for EMT.EMBO J.2010;29:3217–8.

Claims

1. An application of ESRP1 in the preparation of a drug for treating gastric cancer.

2. The use of ESRP1 in the preparation of a drug for treating gastric cancer according to claim 1, characterized in that: The ESRP1 is used in preparing a drug for inhibiting ferroptosis of gastric cancer cells.

3. A use of PLA2G4C in preparing a drug for treating gastric cancer, characterized in that: PLA2G4C is a target gene of ESRP1.

4. Use of an ESRP1 inhibitor as a ferroptosis regulator in the preparation of a gastric cancer targeted drug, characterized in that: The ESRP1 inhibitor downregulates ESRP1 expression and promotes ferroptosis of gastric cancer cells.

5. Use of an ESRP1 inhibitor as a ferroptosis regulator in the preparation of a gastric cancer targeted drug according to claim 4, characterized in that: The dosage form of the gastric cancer targeted drug is tablets, capsules, granules, injections or sprays.

6. An application of ESRP1 in preparing a kit for predicting the prognosis of gastric cancer.

7. The use of ESRP1 in preparing a kit for predicting the prognosis of gastric cancer according to claim 6, characterized in that: High expression of ESRP1 in gastric cancer patient tissue samples indicates a poor prognosis for gastric cancer patients.

8. An application of PLA2G4C in preparing a kit for predicting the prognosis of gastric cancer.

9. A kit for determining ferroptosis of gastric cancer cells, characterized in that: The kit can detect the content of the marker ESRP1 in gastric cancer tissue.

10. A kit for determining ferroptosis of gastric cancer cells according to claim 9, characterized in that: The kit includes an upstream primer sequence and a downstream primer sequence of Human ESRP1, the upstream primer sequence is shown as SEQ ID NO.9, and the downstream primer sequence is shown as SEQ ID NO.10.

Images