New application of FABP5 gene

Abstract

The invention relates to novel application of an FABP5 gene, in particular to application of an FABP5 gene inhibitor in preparation of a medicine for treating skin T cell lymphoma. The novel regulatory factor FABP5 provided by the invention can play a role in promoting proliferation in the skin T cell lymphoma through a targeted FABP5 / PPAR gamma signal channel, plays a key role in generation and development of malignant tumors by regulating lipid metabolism, and further determines that the FABP5 is an effective therapeutic target for reversing lipid metabolism reprogramming of the skin T cell lymphoma. The potential effect of the FABP5 and the skin T cell lymphoma is researched on the gene molecule level, the influence of lipid metabolism related molecules on the skin T cell lymphoma is researched, a theoretical basis is provided for a new target spot for treating the skin T cell lymphoma, and the FABP5 gene inhibitor can be used for preparing a novel targeted drug for treating the skin T cell lymphoma.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a new use of FABP5 gene. Background Art

[0002] Cutaneous T-cell lymphoma (CTCL) is a rare, heterogeneous group of T-cell lymphomas with unclear pathogenesis. In addition, up to 34% of patients experience disease progression to advanced stages, which significantly reduces overall survival, and therefore, in some cases, the rapid progression of cutaneous T-cell lymphoma greatly exacerbates prognostic and survival outcomes. Emerging evidence suggests that FABP5 may contribute to tumor progression. However, the exact biological role of FABP5 in cutaneous T-cell lymphoma remains unexplored.

[0003] Primary cutaneous T-cell lymphoma is a rare non-Hodgkin lymphoma, accounting for 75% to 80% of primary cutaneous lymphomas. Mycosis fungoides (MF) is the most common clinical subtype, characterized by neoplastic proliferation of CD4+ lymphocytes. Compared with other human malignancies, cutaneous T-cell lymphoma has a unique course of development and relatively indolent biological behavior. Clinically, it progresses from the patch stage to the plaque stage, and eventually develops to the tumor stage, which can take several years or even decades, and eventually involves lymph nodes or visceral organs and is fatal. Based on the MF treatment method recommended by the International Society of Lymphoma, a small number of MF patients have treatment resistance; in addition, 20%-50% of MF patients in the tumor stage may undergo large cell transformation, and the 5-year survival rate is less than 20%; when lymphadenopathy, tumors and skin ulcers occur simultaneously, the median survival is only 1 year. It is currently difficult to predict the development of this type of MF with aggressive development and treatment resistance, which also poses a great challenge to the clinical management and treatment options of MF. Therefore, exploring the pathogenesis of MF and early intervention in the malignant evolution and treatment resistance of MF are the keys to improving the quality of life and prognosis of MF patients.

[0004] Metabolic reprogramming has been recognized as one of the hallmarks of cancer and has been explored as an anticancer strategy. Recently, lipid metabolic reprogramming has emerged as a new hallmark of cancer, drawing renewed attention to the important role of fatty acid (FA) metabolism in carcinogenesis. Fatty acids are widely involved in a variety of biological functions, including cell proliferation and survival, and the important roles of lipids in membrane synthesis, energy supply, and cell signaling in cancer cells have been demonstrated. New evidence suggests that fatty acids may be an important energy source for promoting cancer cell proliferation and tumorigenesis. Fatty acids must be bound to fatty acid binding proteins (FABPs) for transport, and they are also an essential energy source for supporting tumor proliferation and metastasis. Fatty acid binding protein 5 (FABP5), also known as epidermal FABP, is expressed in various issues and organs. It belongs to the FABP family and regulates fatty acid binding, transport, and cell growth. As a lipid chaperone, FABP5 promotes the intracellular transport of hydrophobic lipids within specific compartments of the cell and is primarily involved in FAs uptake and metabolism within the cytoplasm. Recent studies have shown that FABP5 is upregulated in several cancers, including liver cancer, colorectal cancer, ovarian cancer, and prostate cancer, and is often associated with poor prognosis due to its role in promoting tumor proliferation, survival, and inflammation. Summary of the invention

[0005] The present invention is intended to overcome the problems in the prior art that the role of FABP5 in promoting the progression of cutaneous T-cell lymphoma is still unclear and the use of the FABP5 gene in preparing drugs for treating cutaneous T-cell lymphoma has not been studied. The present invention discloses that the FABP5 gene has the role of promoting the progression of cutaneous T-cell lymphoma, further supplements and expands the treatment methods for cutaneous T-cell lymphoma, and discloses the role of FABP5 gene inhibitors in inhibiting fatty acid uptake and de novo synthesis of fatty acids in cutaneous T-cell lymphoma, providing evidence for FABP5 becoming a therapeutic target.

[0006] In order to achieve the purpose of the above invention, the present invention is implemented by the following technical solutions: In a first aspect, the present invention provides a use of a FABP5 gene inhibitor in the preparation of a drug for treating T-cell lymphoma.

[0007] Furthermore, the cutaneous T-cell lymphoma is mycosis fungoides.

[0008] Further, the FABP5 gene inhibitor includes any one of BMS-309403, SBFI-26, ART26.12, and Resveratrol. BMS-309403 is an effective selective adipocyte fatty acid binding protein and is an inhibitor specifically designed to target FABP4. The present invention discloses the effect of BMS-309403 in inhibiting the FABP5 gene in cutaneous T-cell lymphoma.

[0009] In a second aspect, the present invention also discloses the use of a FABP5 gene inhibitor in the preparation of a drug for inhibiting proliferation, invasion and inducing apoptosis of cutaneous T-cell lymphoma cells.

[0010] In a third aspect, the present invention also discloses the use of a FABP5 gene inhibitor in the preparation of a drug for blocking epithelial-mesenchymal transition in cutaneous T-cell lymphoma.

[0011] In a fourth aspect, the present invention also discloses the use of a FABP5 gene inhibitor in the preparation of a drug for inhibiting fatty acid synthesis and uptake in cutaneous T-cell lymphoma.

[0012] In a fifth aspect, the present invention also discloses the use of a FABP5 gene inhibitor in a drug for regulating lipid metabolism of cutaneous T-cell lymphoma via the FABP5 / PPARγ signaling pathway.

[0013] In a sixth aspect, the present invention further discloses a pharmaceutical composition for cutaneous T-cell lymphoma, wherein the pharmaceutical composition comprises a FABP5 gene inhibitor.

[0014] Therefore, the present invention has the following beneficial effects: (1) This invention proposes a new mechanism by which a new regulatory factor FABP5 promotes the progression of cutaneous T-cell lymphoma by reprogramming lipid metabolism. By targeting the expression pattern of this factor, the mechanism of action of cutaneous T-cell lymphoma can be further studied, revealing the potential association between FABP5 and the occurrence and development of cutaneous T-cell lymphoma.

[0015] (2) The present invention inhibits the uptake of fatty acids by tumor cells by inhibiting the FABP5 gene, thereby hindering the energy supply of tumor cells and affecting the de novo synthesis of fatty acids, thereby exerting an anti-tumor effect.

[0016] (3) The present invention provides new evidence that FABP5 is a candidate protein for therapeutic targets and also provides new ideas for the treatment of cutaneous T-cell lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure shows the expression results of FABP5 in CTCL cells and tissues; Figure 2 Pseudo-time analysis diagram of FABP5; Figure 3 This is a comparison of the results of FABP5 knockout inhibiting cell proliferation, migration and invasion; Figure 4 This is a comparison of the contents of free FA, TC, and TG in cells after knocking out FABP5; Figure 5 This is a graph of CTCL cell viability with increasing concentrations of FABP5 gene inhibitor; Figure 6 This is a comparison chart of TC and TG ELISA before and after FABP5 gene inhibitor treatment; Figure 7 This is a comparison chart of the expression of downstream protein p-PARγ before and after treatment with FABP5 gene inhibitor. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0019] To evaluate the expression of FABP5 in CTCL, the present invention performed single-cell transcriptome analysis on 8 samples from 2 advanced MF patients and 1 matched healthy control (HC). The experimental process is as follows: Expression of FABP5 in CTCL cells and tissues: Skin tissues, adjacent skin tissues and peripheral blood samples from two patients with MF at different stages of tumor stage and skin samples from one HC were collected, and a total of 8 samples were subjected to single-cell transcriptome analysis. The collected samples were made into single-cell suspensions, and the cell concentration was adjusted to 700-1200 cells / μl. The library construction, sequencing and data analysis were completed by Shanghai Ouyi Biomedical Technology Co., Ltd. The raw data (raw reads) generated in high-throughput sequencing are fastq format sequences. The 10x genomics official software CellRanger was used to perform data quality statistics on the raw data and compare it to the reference genome. The software quantified the high-throughput single-cell transcriptome data by identifying the Barcode markers that distinguish cells in the sequence and the UMI markers of different mRNA molecules in each cell, and obtained quality control statistical information such as high-quality cell number, gene median, and sequencing saturation.

[0020] Cell clustering and data preprocessing: Cell clustering was performed first. The output of the CellRanger command was loaded into R using v3 and analyzed by modified integration. According to the gene expression in the single-cell transcriptome data, the copy number variation value of each chromosome region was evaluated using the infer CNV (v1.0.4) package. T lymphocytes were selected as cancer cells and the remaining cells were selected as normal cells. Genes were sorted by chromosomal position, and the average gene expression was calculated with 101 genes as the sliding window size. The expression of normal cells was then used as a control to remove noise and generate the final CNV result file. In addition, the Cell Ranger package v3.1 was used to perform data analysis on the raw sequence data of the 10X Chromium experiment.

[0021] Cell culture: Hut-78, HH, and Hut-102 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a cell culture incubator set at 5% CO2 and 37°C.

[0022] RT-PCR detection: RT-PCR was used to detect the expression levels of FABP5 mRNA in three groups of skin T lymphoma cells, and the target cell line with the highest FABP5 expression level and meeting the requirements of gene knockdown experiment was selected.

[0023] Lentivirus infection of cells in each group: randomly selected target cells in the logarithmic growth phase were digested with trypsin 12 hours before transfection, prepared into cell suspension, and inoculated into 6-well plates. Cultured in a 5% CO2 and 37°C incubator until the cell confluence reached 20%-30% for transfection. Polybrene and infection enhancement solution were added to each well, and the infection multiple MOI was 10. Three replicate wells were set up in each group, and the results were finally observed under a fluorescent inverted microscope. Stably transfected cells were obtained after 4-5 days. Lentivirus recombinant vector LV-shRNA-FABP5 (1.2.3) was added to the three experimental groups, respectively, with KD1.2.3. LV-shRNA-NC was added to the negative control group, and the blank control group was cultured conventionally. Cultured in RPMI-1640 medium with 10% fetal bovine serum. 72 hours after the negative control group (LV-shRNA-NC) and the three experimental groups (LV-shRNA-FABP5) were transfected into the target cells, 3ug / ml of puromycin was added for treatment, and the cell transfection efficiency was observed under a fluorescence microscope 96 hours later.

[0024] RT-PCR was used to detect the expression of FABP5 in each group of cells: 72 hours after transfection, denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 60 seconds, for a total of 40 cycles. The PCR products were subjected to 1.2% agarose gel electrophoresis, and the results were observed using a gel imager. The UVI gel imaging system was used to take pictures. The gray value was analyzed using Image Pro Plus 7.0 software, and the FABP-5 / GAPDH ratio was used as the relative expression level of FABP-5 mRNA. WB was used to detect the expression of FABP5 in each group of cells: 72 hours after transfection, the total protein of each experimental group was extracted, and the protein concentration was determined using a BCA kit. The total protein (50 μg) was subjected to 8% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane by wet transfer. After membrane transfer, the membrane was blocked with 10% skim dry milk for 2 hours at room temperature. Subsequently, rabbit polyclonal FABP-5 antibody and rabbit polyclonal GAPDH antibody were incubated at 4°C. The next day, the membrane was washed three times with (triethanolamine buffered saline solution) TBS-T for 10 min each, and then a secondary goat anti-rabbit (HRP) IgG antibody was added.

[0025] CCK-8 method to detect cell proliferation: The cells in each experimental group in the logarithmic growth phase were digested with trypsin and inoculated in 96-well culture medium at 4×103 / well. 3-5 duplicate wells were set for each group, and a separate blank well was set as a control. CCK-8 (20µl) was added to each well. After incubation for 4 hours, the absorbance at 490nm was detected with an ELISA reader, the average value of the five wells was calculated, and the growth curve was drawn.

[0026] Transwell invasion assay: 50 mg of artificial matrix gel was added to each well and cultured in 10% fetal bovine serum. The cell suspensions of the three groups were added to the upper chamber and placed in an incubator for 24 h. They were fixed with 4% paraformaldehyde for 10 min and then stained with hematoxylin for 20 min. The cells were counted under an optical microscope.

[0027] Flow cytometry detection of cell cycle: When the cell growth rate of each group was about 80%, the cell culture supernatant was discarded, the cells were washed once with PBS, the cells were digested with trypsin, and the cells were collected in a centrifuge tube. Three replicate wells were set for each group. The supernatant was discarded after centrifugation. The cell pellet was washed with PBS and the cells were collected after centrifugation. 70% ethanol was fixed; the fixative was discarded after centrifugation, and the cell pellet was washed with PBS; 100ul RNase was added to each tube, and the cells were incubated at 37℃ for 30min, and 500ul PI dye solution was added to each tube; incubated at room temperature and away from light for 30min, and then tested on the machine. The flow cytometer was used to analyze the DNA content, and the software was used to analyze the number and proportion of cells in the G0 / G1, S, and G2 / M phases. .

[0028] Flow cytometry for apoptosis: 72 hours after transfection, cells from each group were collected and digested with EDTA-free trypsin, centrifuged at 4˚C, 1650 xg for 5 min, the supernatant was discarded, the cells were washed twice with PBS, and 500µl PBS was added to resuspend the cells. Annexin V-FITC (5µl) and PI (5µl) were added in the dark at room temperature for 5-15 minutes, and the cells were detected by flow cytometry within 1 hour.

[0029] Detection of the expression level of apoptosis-related factors caspase-3 / 9: Nile red solution was stored in a refrigerator at -20°C. Cells were seeded at 3.0×103 / cm2 in 96-well black-walled / transparent-bottom plates. After incubation in DMEM / F12 medium containing 1% BSA for 24 hours, the cells were incubated in DMEM / V12 medium supplemented with 0.2 and 0.4 mM oleic acid BSA (Sigma) for 24 hours. The cells were incubated with 100ul 1uM Nile red and 1% Pluronic F-127 (Sigma) at room temperature in the dark for 4 hours, then washed and incubated with HBSS for 16 hours. Fluorescence was detected using a Synergy HT microplate reader and KC4 software. Neutral lipid deposition in these cells was observed by a Zeiss Axiovert 200 microscope.

[0030] Measurement of free fatty acids, cholesterol, cholesterol esters and triglycerides: FFA quantification kit, cholesterol / cholesterol ester quantification kit and triglyceride colorimetric analysis kit were used to detect the levels of FFA, cholesterol, cholesterol ester and diglyceride, respectively.

[0031] Enzyme-linked immunosorbent assay (ELISA): After the cell specimens of each group were treated accordingly, the cell supernatant was collected using an ELISA kit according to the manufacturer's recommendations to determine the levels of TC and TG. Cell culture supernatant: Collected in a sterile tube. Centrifuged for about 20 minutes (2000-3000 rpm). Sample addition: Set 10 wells with 5 concentration points for the standard, set parallel wells for each concentration, add 50 μl of standard at different concentrations, set 1 well for the blank well and add 50 μl of distilled water (the blank control well does not add samples and enzyme labeling reagents, and the rest of the steps are the same), the wells for the samples to be tested, first add 40 μl of sample diluent to the wells for the samples to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Sample addition: Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the wall of the well, and gently shake to mix. Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes. Preparation: Dilute the 30 (20 times of 48T) times concentrated washing solution with 30 (20 times of 48T) times distilled water and set aside. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it, repeat this 5 times, and pat dry. Add enzyme: Add 50μl of enzyme labeling reagent to each well, except for the blank well. Incubation: The operation is the same as 2. Washing: The operation is the same as 4. Color development: First add 50μl of color developer A to each well, then add 50μl of color developer B, gently shake and mix, and color development at 37℃ in the dark for 15 minutes. Stop: Add 50μl of stop solution to each well to stop the reaction (the blue color immediately turns yellow). Determination: Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0032] like Figure 1 As shown, the samples were clustered into 16 major clusters in an unsupervised manner and visualized by UMAP. We also performed large-scale CNVs analysis to identify malignant cells. The results showed that FABP5 expression was significantly increased in malignant T cell subtypes, especially in C02, C11, C13, and C16.

[0033] like Figure 2As shown, pseudo-time analysis showed that the expression of FABP5, a lipid metabolism-related marker, changed with the differentiation trajectory of T cells. This finding indicates that the metabolic pattern changes during T cell differentiation, with a particular emphasis on the activation of lipid metabolism in T cells. In addition, the expression of FASN and SREBP1, which are closely related to lipid metabolism, was also increased in several CTCL malignant T cells. The upregulation of FABP5 and FASN indicates that lipid metabolism is activated during T cell differentiation. We further performed qRT-PCR detection, and the results showed that FABP5 was expressed at a higher mRNA level in CTCL cell lines compared with healthy peripheral blood mononuclear cells (PBMCs). Together, these findings indicate that FABP5 is upregulated in CTCL and that elevated FABP5 is associated with the malignant progression of CTCL.

[0034] like Figure 3 As shown in Figure 2, to investigate the potential function of FABP5 in CTCL, we performed loss-of-function experiments in CTCL cells. We constructed a CTCL cell line with stable knockout of FABP5 using siRNA lentivirus and verified the efficiency of transfection using qRT-PCR. After these cells were stably transfected, the FABP5 mRNA level was significantly lower than that of other groups. Since FA is one of the main energy sources supporting tumor cell proliferation and anti-apoptosis, we examined the effect of FABP5 expression on CTCL cell viability and survival. CCK-8 and Transwell assays were used to assess cell proliferation and invasion. The results showed that knockout of FABP5 significantly inhibited cell proliferation, migration and invasion compared with the sh-NC group and the control group. Taken together, these results indicate that knockout of FABP5 significantly inhibited the proliferation, colony formation and migration ability of CTCL cells in vitro, indicating that FABP5 may play an important role as a pro-oncogenic protein in the proliferation and invasion of CTCL. We further evaluated the cell cycle distribution and apoptosis levels using flow cytometry. The results showed that knocking down FABP5 significantly promoted cell apoptosis, reduced the proportion of cells in the S phase, and increased the proportion of cells in the G1 phase. In addition, the number of cells in the G0 / G1 phase was higher in the FABP5 shRNA transfection group compared with the other two groups, indicating that knocking down FABP5 induced cell cycle arrest in the G0 / G1 phase. Therefore, these data indicate that FABP5 increases tumor cell proliferation by promoting cell division and inhibiting apoptosis.

[0035] like Figure 4As shown, to investigate the effect of knocking out FABP5 on fatty acid (FA) synthesis in CTCL, we measured the intracellular levels of free FA (FFA), cholesterol (TC), and triglyceride (TG) to assess lipolytic activity. FABP5 is a key protein in FA metabolism, which affects differentiation and growth, regulates gene expression or signal transduction. The results showed that inhibition of FABP5 resulted in a decrease in free FAs, and the levels of free TG and TC also showed similar trends. These results suggest that FABP5 promotes lipolysis and FA synthesis, and may promote tumor progression by reprogramming lipid metabolism, including promoting lipid droplet decomposition, activating signaling pathways, and expressing FA synthases.

[0036] like Figure 5 To determine whether pharmacological inhibition of FABP5 would affect cell viability, we used the competitive inhibitor of FABP5, BMS-309403, to inhibit the function of FABP5. Cell viability was measured by CCK-8 after treatment with BMS-309403 at concentrations of 25, 50, 75, 100, 125, and 150 µM. The results showed that BMS-309403 significantly inhibited the viability of CTCL cells in a concentration-dependent manner, with a maximum inhibitory effect of approximately 62% observed at a concentration of 100 µM. In addition, we also studied the effects of different concentrations (50 µM and 100 µM) of BMS-309403 on the induction of cell apoptosis. The results showed that BMS-309408 could significantly increase the percentage of apoptosis in CTCL cells, and the apoptotic effect was enhanced with increasing drug concentrations and prolonged treatment time. In conclusion, the FABP5 inhibitor BMS-309403 impeded the proliferation of CTCL cells and promoted their apoptosis.

[0037] like Figure 6 As shown in Figure 2, since FABP5 has been shown to regulate new FA synthesis and FA uptake, we further investigated the effect of FABP5 on FA metabolism in CTCL cells and its potential role in tumor suppression. In order to quantitatively detect the levels of cholesterol (TC) and triglyceride (TG) in CTCL cells after BMS-309403 treatment compared with the untreated control group, we used an enzyme-linked immunosorbent assay (ELISA) method. Compared with other groups, the TC and TG levels in the BMS-309403 treatment group were significantly reduced, while those in the FABP5 group were significantly increased. This is because FABP5 can promote the absorption of extracellular FAs and increase intracellular FAs, thereby stimulating PPARγ-mediated lipid metabolism.

[0038] like Figure 7As shown in Figure 2, we detected the expression of p-PARγ, a downstream protein of FABP5, and found that its expression level was significantly decreased compared with the control group. This suggests that FABP5 may regulate the lipid metabolism of CTCL cells through PPARγ, and BMS-309403 may inhibit the malignant progression of CTCL cells by inhibiting FABP5 / PPARγ signal transduction.

[0039] The present invention proposes a new mechanism by which a new regulatory factor FABP5 promotes the progression of cutaneous T-cell lymphoma by reprogramming lipid metabolism. By targeting the expression pattern of this factor, the mechanism of action of cutaneous T-cell lymphoma can be further studied, revealing a potential association between FABP5 and the occurrence and development of cutaneous T-cell lymphoma. This factor may exert a potential tumor-suppressing effect by inhibiting the uptake of fatty acids by tumor cells, hindering the energy supply of tumor cells, and affecting the de novo synthesis of fatty acids. This provides new evidence for FABP5 to become a candidate protein for therapeutic targets and also provides new ideas for the treatment of cutaneous T-cell lymphoma.

Claims

1. Use of a FABP5 gene inhibitor in the preparation of a drug for treating cutaneous T-cell lymphoma.

2. The use according to claim 1, characterized in that: The cutaneous T-cell lymphoma is mycosis fungoides.

3. The use according to claim 1, characterized in that: The FABP5 gene inhibitor includes any one of BMS-309403, SBFI-26, ART26.12, and Resveratrol.

4. Use of a FABP5 gene inhibitor in the preparation of a drug for inhibiting proliferation, invasion and inducing apoptosis of cutaneous T-cell lymphoma cells.

5. Use of a FABP5 gene inhibitor in the preparation of a drug for blocking epithelial-mesenchymal transition in cutaneous T-cell lymphoma.

6. Use of a FABP5 gene inhibitor in the preparation of a drug for inhibiting fatty acid synthesis and uptake in cutaneous T-cell lymphoma.

7. Application of a FABP5 gene inhibitor in a lipid metabolism drug for regulating cutaneous T-cell lymphoma via the FABP5 / PPARγ signaling pathway.

8. A pharmaceutical composition for skin T-cell lymphoma, characterized in that: Including FABP5 gene inhibitors.

Images