Application of Transmembrane Protein 43 as a Tumor Diagnostic Marker for Pancreatic Cancer

Abstract

The present invention discloses the application of transmembrane protein 43 as a tumor diagnostic marker for pancreatic cancer, belonging to the technical field of biomedicine. It also provides the application of transmembrane protein 43 in the preparation of molecular targeted drugs for pancreatic cancer. Through multivariate analysis, TMEM43 can be used as an independent marker for predicting the prognosis of pancreatic cancer patients. Experimental data show that the expression level of TMEM43 is up-regulated in pancreatic cancer, and TMEM43 may be an independent prognostic marker for pancreatic cancer patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of transmembrane protein 43 as a tumor diagnostic marker for pancreatic cancer. Background Art

[0002] The 5-year survival rate of pancreatic cancer patients is less than 5%, making it one of the most lethal cancers. Due to the highly non-specific symptoms of most patients (such as abdominal pain and weight loss), it is very difficult to diagnose. Only 15-20% of pancreatic cancer patients have the opportunity to undergo surgical treatment. At the same time, due to the drug resistance and low sensitivity of pancreatic tumors, it is very difficult to achieve good therapeutic effects with current radiotherapy, chemotherapy or other combined therapies. Therefore, it is urgent to discover new oncogenes in pancreatic cancer and explore related molecular mechanisms.

[0003] Pancreatic cancer is one of the most malignant tumors in the digestive system. Due to the lack of effective diagnostic markers, most patients are diagnosed at an advanced stage, and pancreatic cancer is also resistant to most radiotherapy and chemotherapy.

[0004] Transmembrane protein 43 (TMEM43) is a member of the TMEM subfamily, which is encoded by a highly conserved gene and is widely expressed in most species. The TMEM43 S358L mutation on chromosome 3p25 is an important cause of arrhythmogenic right ventricular cardiomyopathy (ARVC) because the mutation enhances the NF-κB-TGFβ signaling cascade, leading to early invasive lesions and potentially developing into heart disease later. However, the mechanism of action of TMEM43 in cancer is still unclear. The expression level of TMEM43 is increased in brain tumor cells and accelerates the progression of brain tumors by interacting with the CARD-containing MAGUK protein 3 in the EGFR-induced NF-κB pathway. Currently, the function and molecular mechanism of TMEM43 in pancreatic cancer are still unclear. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems existing in the prior art and provide the application of transmembrane protein 43 as a tumor diagnostic marker for pancreatic cancer.

[0006] In the first aspect of the present invention, there is provided the application of transmembrane protein 43 as a tumor diagnostic marker for pancreatic cancer. Through multivariate analysis, TMEM43 can be used as an independent marker for predicting the prognosis of pancreatic cancer patients. Experimental data show that the expression level of TMEM43 is up-regulated in pancreatic cancer, and TMEM43 may be an independent prognostic marker for pancreatic cancer patients.

[0007] In the second aspect of the present invention, there is provided the application of transmembrane protein 43 in the preparation of molecular targeted drugs for pancreatic cancer. Brief Description of the Drawings

[0008] Figure 1 Upregulation of TMEM43 expression level in pancreatic cancer tumor cells is associated with poor survival;

[0009] (A) Expression levels of TMEM43 mRNA in different tumor tissues and corresponding normal tissues;

[0010] (B) Western blotting and immunohistochemistry (IHC) assays showed the expression levels of TMEM43 in pancreatic cancer tissues and normal pancreatic tissues (n = 5), Scale Bar (left) = 500 μm, Scale Bar (right) = 50 μm;

[0011] (C) Immunohistochemistry showed the expression of TMEM43 in pancreatic cancer tissues and normal tissues 1. Scale Bar (left) = 200 μm, Scale Ba (right) = 50 μm;

[0012] (D) Kaplan–Meier curve analysis using a pancreatic cancer TMA cohort showed the relationship between TMEM43 protein expression level and patient survival;

[0013] (E-F) Kaplan–Meier curve analysis indicated. Univariate and multivariate Cox regression analyses were performed in the TCGA pancreatic cancer dataset (G-H), *p < 0.05;

[0014] Figure 2 TMEM43 promotes the proliferation, migration, and invasion of pancreatic cancer in vitro;

[0015] (A) Protein expression levels of TMEM43 in TMEM43-silenced cells and TMEM43-overexpressing MIAPaCa-2 cells were detected by Western blotting;

[0016] (B-C) Cell counting and colony formation assays showed the effects of silencing and overexpressing TMEM43 on the proliferation of MIAPaCa-2 cells;

[0017] (D) Flow cytometry was used to detect the effects of silencing and overexpressing TMEM43 on the cell cycle of pancreatic cancer cells;

[0018] (E-F) Protein expression levels of TMEM43 in TMEM43 gene knockout SW1990, CAPAN-2 cells and corresponding control cells were detected;

[0019] Cell counting and colony formation assays were performed on TMEM43-silenced SW1990, CAPAN-2 cells and corresponding control cells (G-J);

[0020] Flow cytometry was used to analyze the cell cycle of TMEM43-silenced SW1990, Capan-2 cells and control cells (K-L);

[0021] (M-O) The migratory and invasive abilities of TMEM43-silenced MIAPaCa-2, SW1990, and Capan-2 cells, TMEM43-overexpressing MIAPaCa-2 cells, and corresponding control cells were detected by Transwell assay. Scale bar = 100 μm. Results are shown as the mean ± SD of three independent replicates. *p < 0.05, **p < 0.01, and ***p < 0.001.

[0022] Figure 3 The RAP2B / ERK pathway is essential for TMEM43-mediated pancreatic cancer progression;

[0023] (A) Heatmap showing some differentially expressed proteins in TMEM43-silenced MIAPaCa-2 cells and control cells;

[0024] (B) The protein expression levels of RAP2B, ERK, and p-ERK were detected in TMEM43-silenced, TMEM43-overexpressing cells, and corresponding control cells;

[0025] (C) The indicated protein expression levels were detected in RAP2B-silenced cells and control cells;

[0026] Cell counting was used to detect the proliferation ability of RAP2B-silenced MIAPaCa-2 cells (D), SW1990 cells (E), and corresponding control cells (D-E);

[0027] (F-G) Colony formation assay showed the effect of RAP2B expression level on the growth ability of MIAPaCa-2 cells (F) and SW1990 cells (G);

[0028] (H-I) Flow cytometry was used to detect the cell cycle of RAP2B-silenced MIAPaCa-2 (H) and SW1990 (I) cells and corresponding control cells;

[0029] (J-K) The migratory and invasive abilities of RAP2B gene knockout MIAPaCa-2 (J), SW1990 (K) cells, and control cells were detected;

[0030] (L) Western blot results showed that the protein expression levels in SW1990 cells transfected with control, shTMEM43, or shTMEM43 + RAP2B were stable;

[0031] (M-O) Cell counting, colony formation, and Transwell assays showed that RAP2B could partially eliminate the inhibitory effects of shTMEM43 on the proliferation, migration, and invasion of SW1990 cells. Scale bar = 100 μm. Results are shown as the mean ± SD of three independent replicates. *p < 0.05, **p < 0.01, and ***p < 0.001;

[0032] Figure 4 TMEM43 mediates the stability of PRPF3 protein by directly binding to PRPF3;

[0033] (A) Venn diagram showing proteins regulated by TMEM43 and proteins that directly interact with TMEM43;

[0034] (B) Immunohistochemistry showed a positive correlation between TMEM43 protein expression and PRPF3 protein expression;

[0035] (C) Correlation between PRPF3 mRNA level and RAP2B mRNA level in the GSE71729 dataset;

[0036] (D) Endogenous binding of TMEM43 and PRPF3 was detected by CO-IP and Western blot;

[0037] (E) Exogenous binding of TMEM43 and PRPF3 was analyzed by CO-IP and Western blot;

[0038] (F) Confocal immunofluorescence was used to detect the localization of TMEM43 and PRPF3 in pancreatic cancer cells;

[0039] (G-H) Protein and mRNA levels of PRPF3 were detected in TMEM43-silenced MIAPaCa-2 and SW1990 cells, cells overexpressing TMEM43 in MIAPaCa-2, and corresponding control cells;

[0040] (I) TMEM43-silenced MIAPaCa-2 and SW1990 cells and corresponding control cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 25 μg / mL), and the PRPF3 protein level was detected. Results are shown as the mean ± SD of three independent replicates. *p < 0.05, **p < 0.01, and ***p < 0.001;

[0041] Figure 5 PRPF3 promotes the in vitro proliferation, migration, and invasion of pancreatic cancer through the RAP2B / ERK pathway;

[0042] (A-B) Cell counting was used to detect the effect of PRPF3 on the proliferation of pancreatic cancer cells MIAPaCa-2 (A) and SW1990 (B);

[0043] (C-D) Colony formation assays were performed in MIAPaCa-2 (C) and SW1990 (D) cells;

[0044] (E-F) Effects of PRPF3 on the cell cycle of MIAPaCa-2 (E) and SW1990 (F) cells were detected;

[0045] (G-H) Migration and invasion abilities of PRPF3-silenced MIAPaCa-2 and SW1990 cells and control cells were detected by Transwell assay;

[0046] (I) Protein expression levels of PRPF3-knockout MIAPaCa-2, SW1990 cells and control cells were detected;

[0047] (J) Western blot results showed protein expression levels in SW1990 cells stably transfected with control, shPRPF3 and shPRPF3+RAP2B;

[0048] (K-M) Cell counting, colony formation and Transwell analysis showed that RAP2B could partially cancel the inhibitory effects of shPRPF3 on the proliferation, migration and invasion of SW1990 cells. Scale bar = 100 μm. Results were shown as the mean ± SD of three independent replicates, *p<0.05, **p<0.01 and ***p<0.001;

[0049] Figure 6 TMEM43 promotes pancreatic cancer growth in vivo through the PRPF3 / RAP2B axis;

[0050] (A) Xenografts were taken from nude mice and measured;

[0051] (B) Tumor volumes in nude mice were detected and analyzed every 3 days in the TMEM43-silenced CAPAN-2 cell group and the control group;

[0052] (C) Tumor weights of the TMEM43-silenced Capan-2 cell group and the control group were measured and analyzed;

[0053] (D) Expressions of the above proteins in the two groups were detected by immunohistochemistry;

[0054] (E) Xenograft tumors in nude mice were obtained after different treatments and the sizes of the transplanted tumors were measured;

[0055] (F) Tumor volumes in nude mice were detected and analyzed every 3 days after different treatments;

[0056] (G) Tumor weights after different treatments were measured and analyzed;

[0057] (H) IHC detected the expression of the indicated protein after different treatments. The results are shown as the mean ± SD of three independent replicates. Scale bar = 50 μm, *p < 0.05, **p < 0.01, and ***p < 0.001. Detailed implementation manners

[0058] The present invention will be further described in detail below with reference to the accompanying drawings:

[0059] The present invention proves that the expression level of TMEM43 is closely related to the clinicopathological features of pancreatic cancer patients, which will result in poor survival outcomes. Inhibiting TMEM43 can inhibit the growth, migration, and invasion of pancreatic cancer in vitro and in vivo. At the same time, the research shows that the TMEM43 / PRPF3 / RAP2B / ERK axis plays a crucial role in regulating cancer progression and has potential clinical application value for the treatment of pancreatic cancer.

[0060] Materials and methods

[0061] Patient samples

[0062] All pancreatic cancer samples and adjacent cancer samples were from inpatients in a certain hospital of the Air Force. All patients met the diagnostic criteria of the American Joint Committee on Cancer (AJCC) manual revised in 2018. All pancreatic cancer patients signed informed consent forms. Pancreatic cancer tissue samples were frozen in liquid nitrogen until use.

[0063] Pancreatic cancer cells and lentiviral infection

[0064] MIAPaCa-2 (RRID: CVCL_0428), Capan-2 (RRID: CVCL_0026), and SW1990 cells (RRID: CVCL_1723) were all purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). All pancreatic cancer cells were subjected to STR identification and were mycoplasma-free.

[0065] MIAPaCa-2 cells were cultured in Dulbecco's modified high-glucose Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin (HyClone, Utah, USA); SW1990 cells were cultured in Leibovitz's L15 medium (Gibco BRL, Rockville, MD); Capan-2 cells were cultured in McCoy's 5A complete medium (HyClone, Utah, USA). pLent-U6 and pLent-EF1a lentiviral plasmids, pCMV adenoviral plasmid, and packaging plasmids (pMD2G and psPAX2) were purchased from Vigene Biosciences (Jinan, China).

[0066] Lentivirus packaging process: 293T cells were cultured in a 100 mm cell culture dish at a concentration of 8×10 6 cells per well. The next day, lentiviral plasmid (12 μg), pMD2G (3.6 μg), psPAX2 (7.2 μg), and 46 μL Lipofectamine 3000 (Invitrogen, California) were added to the 100 mm plate, and the supernatant was collected 48 hours later. Then, the supernatant was filtered through a 0.45 μm filter membrane.

[0067] Cell infection process: Target cells were seeded in a 100 mm cell culture dish at a concentration of 2×10 6 per well. Lentivirus was added to the dish. After 12 hours, the medium containing lentivirus was replaced with fresh complete medium. Three days later, puromycin was used to select positive cells. The sequences of different shRNAs are listed in Table 1.

[0068] Table 1 Sequences of different shRNAs

[0069]

[0070] Cell proliferation and colony formation assays

[0071] Cell proliferation assay: Tumor cells were cultured in triplicate in 6-well plates at a concentration of 3×10 4 per well; after digestion, the cell number in each well was detected using a hemocytometer (QIUJING, Shanghai, China).

[0072] Colony formation assay: Tumor cells (10 3 per well) were seeded in triplicate in 6-well plates. After 14 days of culture, the cells were fixed with 95% ethanol for 20 min and then stained with 1% crystal violet for 30 min; then the culture dish was washed with running water to observe the colony formation ability, and the colonies were counted and the 6-well plate was photographed against a white background.

[0073] Transwell assay

[0074] Matrigel (BD Biosciences) was diluted at a ratio of 1:20 with cell culture medium for invasion assay. The diluted Matrigel was placed at the bottom of the transwell chamber and incubated for 30 minutes. Migration assay was performed without Matrigel. Specifically: Cells were seeded at 10 5The cells / room concentration was directly inoculated into Matrigel in a 24-well plate, and medium with 10% FBS was added to the lower chamber. After 48 h, the cells were fixed with 95% ethanol for 20 min, then stained with 1% crystal violet for 30 min. The cells on the upper surface of the membrane were removed with a cotton swab, and then the chamber was washed with running water and five fields of view were randomly captured to analyze the migration and invasion of pancreatic cancer.

[0075] Flow cytometry analysis

[0076] For flow cytometry, cells (10 6 cells per well) were seeded in triplicate in a 6-well plate and maintained for 24 h, then the cells were digested with trypsin and washed 3 times with PBS, then fixed with 70% ethanol for 20 min, and finally the cells were stained with PI / RNase staining buffer (BD, USA) and flow cytometry was performed to detect the cell cycle (BD, FACSCalibur, USA).

[0077] Co-immunoprecipitation (CO-IP) and Western blotting

[0078] All pancreatic cancer cell and tissue samples were lysed using RIPA lysis buffer (Applygen, Beijing, China) containing phosphatase and protease inhibitors (Roche, USA). All cell lysates (200 μg protein) were incubated with IgG or primary antibody at 4 °C for 3 h, then the solution was incubated overnight with protein A / G or anti-Flag / HA magnetic beads (Bimake, Texas, USA). The CO-IP complex was washed 5 times with PBS and boiled by adding protein buffer, then the proteins were separated by SDS-PAGE gel and then subjected to mass spectrometry (MS) analysis (Mhelix Biotech, Shanghai, China) or immunoblotting.

[0079] Western blotting was performed (Li J, Yang J, Hua L, Wang R, Li H, Zhang C, et al. Ese-3 contributes to colon cancer progression by downregulating EHD2 and transactivating INPP4B. Am J Cancer Res. 2021;11(1):92-107.). ECL Western blotting luminescent reagent (Millipore, USA) was used to detect protein bands in the BIO-RAD ChemiDocTM XRS+ imaging system (California, USA).

[0080] Immunohistochemistry (IHC) analysis

[0081] One tissue microarray of cohort 1 and two tissue microarrays of cohort 2 were purchased from Outdo Biotech Company (Shanghai, China).

[0082] IHC and TMA experiments were performed, and the results were analyzed according to the staining intensity (0, no staining; 1, light yellow; 2, light red; 3, brown) and the percentage of stained cells (0 - 5%, 0; 6 - 25%, 1; 26–50%, 2; 51–75%, 3; and 76–100%, 4). At the same time, the overall score of immunostaining was obtained by multiplying the intensity score by the percentage of stained cells. A staining score < 6 was described as low expression, while a staining score > 6 was described as high expression.

[0083] Digital images were analyzed using Panoramic viewer 1.15.3 (3DHistech Ltd, Hungary).

[0084] Quantitative RT-PCR

[0085] According to the reagent instructions, total RNA was obtained from pancreatic cancer cell samples using Trizol reagent (Ambion, Texas, USA). The contents of TMEM43, PRPF3, and GAPDH genes were measured using SYBR Green qPCR Master Mix (Servicebio, Wuhan, China) reagent on a PCR machine (Bio-Rad, California, USA). The primer sequences are listed in Table 2.

[0086] Table 2 Primer sequences

[0087]

[0088] Immunofluorescence assay

[0089] Tumor cells were washed twice with PBS (HyClone, Utah, USA) and fixed with 4% paraformaldehyde (Biosharp, Shanghai, China) for 15 minutes, then permeabilized with 0.3% Triton X-100 for 5 minutes and blocked with 5% BSA for 45 minutes. The primary antibodies were rabbit anti-PRPF3 (A5482, Proteintech, China) and mouse anti-TMEM43 (SC-365298, Santa, USA); the secondary antibodies were Alexa Fluor 647-labeled anti-rabbit (ab150083, abcam, USA) and FITC-labeled anti-mouse (ab6785, abcam, USA). Cell images were obtained on a Nikon Eclipse Ti-SR system.

[0090] Label-free quantitative LC / MS proteomic analysis

[0091] MIAPaCa-2 cells with silenced TMEM43 and control cells were cultured in triplicate in 100 mm culture dishes. When the cell density reached 90%, the cells were digested with trypsin, washed 3 times with PBS, and finally subjected to label-free quantitative LC / MS proteomic analysis by Mhelix Biotech Company (Shanghai, China).

[0092] Animal studies

[0093] Male nude mice (3 - 5 weeks old, body weight: 16 - 20 g) were selected. All nude mice were housed in an SPF environment. 5×10 6 cells were injected into the right back of the nude mice (n = 6 per group). The tumor volume was measured every three days (volume = longest diameter × shortest diameter 2 ×0.5). After the experiment, the nude mice were anesthetized and sacrificed with 2% sodium pentobarbital (0.5 mL). Then, the subcutaneous tumors were excised, weighed, and photographed. Finally, the tumors were fixed with 10% neutralized formalin until use.

[0094] Statistical analysis

[0095] The experimental data were analyzed using SPSS 19.0, and the experimental data were described as mean ± SD.

[0096] The Student’s t-test was used to analyze the differences between two unpaired samples; the χ2 test was used to analyze the correlations between mRNA / protein levels and clinicopathological parameters. Cox regression analysis was used to evaluate the prognostic value. Kaplan-Meier curves were used to evaluate the relationships between mRNA / protein levels and OS and DFS.

[0097] All experiments were performed with at least three independent replicates, and *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.

[0098] Results

[0099] (I) Increased TMEM43 expression levels are associated with poor survival rates in pancreatic cancer patients

[0100] First, the GEPIA database was used to evaluate the TMEM43 mRNA levels in different tumor samples and corresponding control samples. The TMEM43 mRNA levels were significantly increased in cholangiocarcinoma (CHOL), glioblastoma multiforme (GBM), kidney renal papillary cell carcinoma (KIRP), low-grade glioma (LGG), pancreatic adenocarcinoma (PAAD), and stomach adenocarcinoma (STAD) samples (*p<0.05, Figure 1A ). Analysis by Western blotting in five pairs of pancreatic cancer tissue samples and peritumoral samples revealed that the TMEM43 expression level was also upregulated, and this was further confirmed by IHC staining in one of the pairs of samples ( Figure 1B ). Analysis of the TMA cohort 1 samples from pancreatic cancer patients further confirmed that the TMEM43 expression level was significantly increased in pancreatic cancer samples compared with control samples (p<0.0001, Figure 1C ).

[0101] To evaluate the correlation between the TMEM43 expression level and the clinical characteristics of pancreatic cancer patients, the relationship between the TMEM43 expression level and clinical parameters was analyzed in 64 pancreatic cancer patients from TMA cohort 2. The results showed that the TMEM43 expression level was significantly correlated with T stage (p = 0.047). The Kaplan-Meier curve analysis was used to evaluate the relationship between the TMEM43 level and the survival rate of pancreatic cancer patients. The results found that the overall survival OS of pancreatic cancer patients with high TMEM43 expression level in TMA cohort 2 (p = 0.0354, Figure 1D ) was poor, and patients with high TMEM43 expression level in the Cancer Genome Atlas (TCGA) database also had poor OS (p = 0.0092) and disease-free survival DFS (p = 0.0003) ( Figure 1E 、 1F ).

[0102] Using the TCGA database, univariate and multivariate analyses were performed to analyze TMEM43 as a biomarker for prognostic value. Univariate analysis showed that T stage and TMEM43 expression level may have significant prognostic value in pancreatic cancer patients (p = 0.011, p = 0.015), Figure 1G ). Through multivariate analysis, TMEM43 can be used as an independent biomarker for predicting the prognosis of pancreatic cancer patients (p = 0.016, Figure 1H ). The experimental data indicate that the TMEM43 expression level is upregulated in pancreatic cancer, and TMEM43 may be an independent prognostic biomarker for pancreatic cancer patients.

[0103] (II) TMEM43 promotes the proliferation, migration, and invasion of pancreatic cancer cells in vitro

[0104] To evaluate the function of TMEM43 in pancreatic cancer, a stable cell line with knockdown of TMEM43 was established in MIAPaCa-2 cells, and TMEM43 was overexpressed in MIAPaCa-2 cells with silenced TMEM43( Figure 2A ). Downregulation of TMEM43 significantly reduced the tumor cell proliferation and colony formation ability in MIAPaCa-2 cells, while upregulation of TMEM43 in MIAPaCa-2 cells with silenced TMEM43 significantly promoted the cell proliferation and colony formation ability( Figure 2B , 2C). Flow cytometry showed that compared with control cells, the percentages of G0 / 1 phase and G2 / M phase in MIAPaCa-2 cells with knockdown of TMEM43 were significantly increased, and the percentage of S phase was decreased; overexpression of TMEM43 in MIAPaCa-2 cells with silenced TMEM43 had the opposite results( Figure 2D ). In addition, the same experiments were performed by silencing TMEM43 in SW1990 and Capan-2 cells( Figure 2E , 2F), and the results also confirmed that downregulation of TMEM43 significantly reduced the growth and colony formation ability of pancreatic cancer cells( Figures 2G to 2J ). Flow cytometry analysis showed that knockdown of TMEM43 significantly decreased the percentage of OS phase in SW1990 and Capan-2 cells( Figure 2K , 2L). In addition, transwell assays were used to explore the effect of TMEM43 on metastasis, and the results showed that compared with control cells, knockdown of TMEM43 significantly reduced the migration and invasion ability of tumor cells in MIAPaCa-2, SW1990 and Capan-2 cells( Figure 2M , 2N, 2O). Overexpression of TMEM43 significantly promoted the migration and invasion of MIAPaCa-2 cells compared with the control group( Figure 2M ). Overall, these results indicate that TMEM43 can promote the growth, migration and invasion of pancreatic cancer in vitro.

[0105] (III) TMEM43 accelerates the progression of pancreatic cancer through the RAP2B / ERK signaling pathway

[0106] To further determine the molecular mechanism by which TMEM43 promotes the progression of pancreatic cancer, label-free quantitative proteomics was used to identify differentially expressed proteins mediated by TMEM43. The results showed that knockdown of TMEM43 led to downregulation of 243 proteins (fold change < 0.77) and upregulation of 648 proteins (fold change > 1.3). The expression differences of proteins are as Figure 3AAs shown, compared with the control cells, the protein expression level of RAP2B decreased in MIAPaCa-2 cells with knockdown of TMEM43. Compared with the corresponding control cells, knockdown of TMEM43 decreased the expression levels of RAP2B and phosphorylated ERK in MIAPaCa-2, SW1990 and Capan-2 cells ( Figure 3B ). In addition, compared with the control cells, overexpression of TMEM43 also increased the expression levels of RAP2B and phosphorylated ERK in TMEM43-silenced MIAPaCa-2 cells ( Figure 3B ).

[0107] To demonstrate the role of RAP2B in pancreatic cancer, RAP2B was knocked down in SW1990 and MIAPaCa-2 cells respectively. It was found that compared with the corresponding controls, the expression level of phosphorylated ERK decreased in MIAPaCa-2 and SW1990 cells with knockdown of RAP2B ( Figure 3C ). In addition, cell counting and colony formation assays showed that compared with the control cells, knockdown of RAP2B decreased the growth and colony formation ability of MIAPaCa-2 cells ( Figure 3D , 3F) and SW1990 cells ( Figure 3E , 3G). In MIAPaCa-2 cells with knockdown of RAP2B, the percentage of G0 / 1 phase increased and the percentage of S phase decreased ( Figure 3H ). Knockdown of RAP2B in SW1990 cells significantly increased the percentages of G0 / 1 phase and G2 / M phase and decreased the percentage of S phase ( Figure 3I ). Transwell assays confirmed that knockdown of RAP2B inhibited the migration and invasion of SW1990 and MIAPaCa-2 cells ( Figure 3J , 3K).

[0108] To confirm that TMEM43 promotes the progression of pancreatic cancer through RAP2B, RAP2B was overexpressed in SW1990 cells with silenced TMEM43. The results showed that overexpression of RAP2B restored the protein expression of RAP2B and p-ERK ( Figure 3L ). Cell counting, colony formation and transwell assays showed that the reduction of proliferation, migration and invasion induced by silenced TMEM43 in SW1990 cells could be partially restored by overexpression of RAP2B ( Figures 3M to 3O ). In summary, the results showed that TMEM43 promotes the progression of pancreatic cancer through the RAP2B / ERK axis.

[0109] (IV) TMEM43 mediates the stability of PRPF3 protein by directly binding to PRPF3

[0110] To explore the potential mechanism by which TMEM43 promotes the progression of pancreatic cancer through the RAP2B / ERK signaling pathway, the present invention conducted CO-IP experiments and protein MS to explore the proteins that bind to TMEM43 in MIAPaCa-2 cells. Label-free quantitative proteomics and CO-IP protein MS found that there may be 6 proteins binding to the TMEM43 protein ( Figure 4A ). The association between the expression level of TMEM43 protein and the expression level of PRPF3 protein was analyzed using IHC, and the results showed that the TMEM43 protein level was significantly positively correlated with the PRPF3 protein level in TMA cohort 2 ( Figure 4B ). It was found that the PRPF3 mRNA level was significantly positively correlated with the RAP2B mRNA level in the GSE71729 dataset ( Figure 4C ). To further confirm the interaction between TMEM43 and PRPF3, it was found that immunoprecipitation of total protein could be performed using anti-TMEM43 or anti-PRPF3 antibodies. TMEM43 and PRPF3 were co-immunoprecipitated in SW1990 and MIAPaCa-2 cells ( Figure 4D ). In addition, the binding of exogenous TMEM43 and PRPF3 was demonstrated using IP / Western blotting in HEK293 cells ( Figure 4E ). Then, immunofluorescence assays were performed to determine the expression and localization of TMEM43 and PRPF3 in SW1990 and MIAPaCa-2 cells, and confocal microscopy was used to find that TMEM43 and PRPF3 co-localized in the cytoplasm ( Figure 4F ).

[0111] To further understand the mechanism by which TMEM43 regulates PRPF3, the protein level was detected. It was found that compared with control cells, PRPF3 was significantly downregulated in TMEM43-silenced MIAPaCa-2 and SW1990 cells, while overexpression of TMEM43 increased the PRPF3 protein level ( Figure 4G ). However, the PRPF3 mRNA level did not change after overexpression or silencing of TMEM43 ( Figure 4H ). Based on the above results, it is speculated that TMEM43 may regulate the stability of PRPF3 in pancreatic cancer.

[0112] To prove our hypothesis, cycloheximide (CHX), a protein synthesis inhibitor, was used to treat TMEM43-silenced MIAPaCa-2 and SW1990 cells and the corresponding control cells, and the results showed that PRPF3 was more unstable in TMEM43-knockdown SW1990 and MIAPaCa-2 cells ( Figure 4I)。In addition, IP analysis showed that in SW1990 cells with knocked-down TMEM43, the polyubiquitination of the PRPF3 band was enhanced after MG132 treatment. Collectively, these results indicate that TMEM43 regulates the protein stability of PRPF3 by affecting its ubiquitination.

[0113] (V) PRPF3 promotes the progression of pancreatic cancer through the RAP2B / ERK signaling pathway

[0114] To explore the function of PRPF3 in pancreatic cancer, stable cell lines with knocked-down PRPF3 were first established in the MIAPaCa-2 and SW1990 cell lines. Cell counting and colony formation assays showed that knocking down PRPF3 significantly inhibited the growth and colony formation ability in MIAPaCa-2 and SW1990 cells ( Figures 5A to 5D ). Transwell analysis showed that knocking down PRPF3 inhibited the migration and invasion ability of MIAPaCa-2 and SW1990 cells ( Figure 5G , 5H). In addition, by flow cytometry analysis, compared with control cells, the percentage of cells in the G0 / 1 phase increased and the percentage of cells in the S phase decreased in MIAPaCa-2 and SW1990 cells with knocked-down PRPF3 ( Figure 5E , 5F). In addition, it was demonstrated that the protein expression levels of RAP2B and phosphorylated ERK were decreased in pancreatic cancer cells with silenced PRPF3 ( Figure 5I ). To confirm that PRPF3 promotes the progression of pancreatic cancer through RAP2B, RAP2B was overexpressed in SW1990 cells with silenced PRPF3, and it was found that overexpressing RAP2B restored the protein levels of RAP2B and p-ERK ( Figure 5J ). Cell counting, colony formation, and transwell assays showed that the reduction in proliferation, migration, and invasion induced by knocking down PRPF3 in SW1990 cells was partially restored by overexpressed RAP2B ( Figures 5K to 5M ). The research results indicate that PRPF3 promotes the progression of pancreatic cancer through the RAP2B / ERK axis.

[0115] (VI) TMEM43 promotes the growth of pancreatic cancer through the PRPF3 / RAP2B axis in vivo

[0116] First, Capan-2 cells with knocked-down TMEM43 and control cells were injected into the backs of nude mice, and the results showed that the tumor volume and tumor weight of Capan-2 cells with silenced TMEM43 were significantly reduced compared with control cells ( Figures 6A to 6C ). IHC staining confirmed that the expression level of TMEM43 in xenografts of Capan-2 cells with knocked-down TMEM43 was downregulated compared with the control group ( Figure 6D) The expressions of PRPF3, RAP2B, p-ERK, and Ki67 were significantly decreased in TMEM43-silenced Capan-2 cells ( Figure 6D ). In addition, tumor xenografts were performed to confirm the functions of PRPF3 and RAP2B in vivo, and the results showed that the downregulation of TMEM43, PRPF3, and RAP2B significantly reduced tumor growth in MIAPaCa-2 cells ( Figures 6E to 6G ). The Ki67 protein level was significantly decreased in MIAPaCa-2 cells with TMEM43 knockdown, PRPF3 knockdown, and RAP2B knockdown compared with control cells ( Figure 6H ).

[0117] Finally, it should be noted that the above technical solutions are only one implementation manner of the present invention. For those skilled in the art, based on the disclosed application methods and principles of the present invention, various types of improvements or deformations can be easily made, not limited to the methods described in the above specific implementation manners of the present invention. Therefore, the above-described manner is only preferred and does not have a restrictive meaning.

Claims

1. Use of an antibody that detects transmembrane protein 43 in the preparation of a pancreatic cancer tumor diagnostic reagent.

Images