FFAR1 to inhibit malignant progression of glioma by reducing expression of MYBL2 regulated by FoxM1

Abstract

The invention discloses FFAR1 for inhibiting malignant progression of glioma by reducing expression of MYBL2 regulated by FoxM1, and belongs to the field of targeted gene therapy. The invention finds that the expression of the FFAR1 in the brain tissue of a glioma patient is reduced, which indicates that the FFAR1 has obvious correlation with the glioma and can be used as a biomarker of the glioma. The invention finds that glioma cells overexpressing FFAR1 are inhibited in the aspects of proliferation, migration and invasion, and the cell cycle process is slowed down; and glioma cells for silently expressing the FFAR1 can play a reverse effect, which indicates that the FFAR1 can be used as a treatment target of glioma.

Description

Technical Field

[0001] The present invention relates to that FFAR1 inhibits the malignant progression of glioma by reducing the expression of MYBL2 regulated by FoxM1, and belongs to the field of targeted gene therapy. Background Art

[0002] Gliomas are the most malignant tumors in the human brain, accounting for 80% of primary central nervous system tumors. According to the WHO, gliomas have four histological grades, and WHO grade IV glioblastoma remains the most malignant and difficult to treat tumor, with an average survival time of 15.5 months. Currently, invasive surgery combined with radiotherapy and chemotherapy is the main treatment for gliomas. In addition, immune checkpoint blockade has also been adopted, but these methods have not achieved good results. The biggest problem is treatment resistance, including resistance to chemotherapy and radiotherapy. Therefore, there is an urgent need to study the key regulatory targets of glioma development and give effective treatment to improve patient prognosis.

[0003] FFAR1, also known as G protein neuroreceptor 40, belongs to the free fatty acid receptor family (others include FFAR2, FFAR3, and FFAR4), is involved in the transport of MCFAs and LCFAs, and has also been reported to be coupled to Gq proteins. FFAR1 is widely expressed in the pancreas, brain, thyroid, lungs, small intestine, and macrophages. It is reported that FFAR1 has physiological functions such as increasing insulin secretion, anti-inflammatory effects, promoting neural responses, and taste preferences. However, there are few reports on the role of FFAR1 in central nervous system malignancies. FFAR1 is highly expressed in central nervous system tissues, including the cerebral cortex, hippocampus, hypothalamus, and cerebellum, and FFAR1 mainly plays a role in promoting the occurrence of neural responses.

[0004] FoxM1 is widely considered to be a member of the Forkheadbox transcription factor family and plays a crucial regulatory role in cell division and proliferation. FoxM1 has been shown to be highly expressed in a variety of cancer tissues, and there is still a lot of research on the correlation between changes in the FoxM1 signaling pathway and tumorigenesis. In recent years, more and more views have been expressed that FoxM1 is highly expressed in a variety of cancer cells and promotes tumorigenesis in a variety of tissues, including breast cancer, lung cancer, prostate cancer, and colorectal cancer. However, the expression of FoxM1 in different types of cancer cells is unpredictable, and it is difficult to predict whether FoxM1 is related to the development and treatment of glioblastoma.

[0005] The MYB proto-oncogene-like 2 (MYBL2) gene, also known as B-MYB, belongs to the myeloblastosis transcription factor family and was first classified as a cellular homolog of v-myb in 1999. MYBL2 regulates the orderly progress of the cell cycle, actively regulates the cell growth and division process, and reduces the occurrence of cell apoptosis. When studying the role of MYBL2 in cancer, it was found that MYBL2 expression was significantly increased in breast cancer, myeloid leukemia, and hepatocellular carcinoma, suggesting that MYBL2 overexpression may be related to the abnormal activity of tumors.

[0006] The existing technology lacks a glioma treatment method that does not produce drug resistance, and there is an urgent need to find key targets for regulating gliomas in order to provide a safe, effective and side-effect-free glioma treatment method. Summary of the invention

[0007] This study aims to investigate the regulatory role of FFAR1 in the proliferation and invasion of glioblastoma and preliminarily explore its downstream pathways. Database analysis revealed that FFAR1 was downregulated in brain tissues of patients with glioblastoma, and this downregulation was associated with tumor prognosis. In vitro experiments showed that FFAR1 overexpression could inhibit the proliferation and invasion of glioma cell lines, while FFAR1 functional loss had the opposite biological effects on glioblastoma cells. Transcriptome sequencing of human glioma cells with FFAR1 knockdown, differential gene screening, and in vitro experiments showed that MYBL2 regulated by FoxM1 was involved in the inhibitory effect of FFAR1 knockdown on glioblastoma. In addition, the establishment of an orthotopic xenograft tumor model in nude mice showed that FFAR1 overexpression could effectively reduce tumor burden in vivo and prolong the survival time of tumor-bearing animals. In summary, FFAR12 inhibits the progression of glioblastoma by targeting FoxM1 to regulate the expression of MYBL2, providing a new perspective for the potential clinical application of FFAR1 in glioblastoma intervention.

[0008] The present invention provides the use of FFAR1 protein in preparing medicine for preventing, improving, treating or assisting in treating glioma.

[0009] In one embodiment, the amino acid sequence of the FFAR1 protein is as shown in SEQ ID NO.1.

[0010] In one embodiment, the nucleotide sequence encoding the FFAR1 protein is shown as SEQ ID NO.2.

[0011] In one embodiment, the medicament is used to enhance FFAR1 protein expression.

[0012] In one embodiment, the drug is used for at least one of the following purposes: inhibiting glioma cell proliferation; inhibiting glioma cell migration; inhibiting glioma cell invasion.

[0013] The present invention also provides the use of FFAR1 protein as a biomarker for glioma.

[0014] In one embodiment, the application is a reagent for quantitatively detecting FFAR1 in the preparation of a kit for diagnosing glioma, or assisting in the diagnosis of glioma, or for screening reagents for reducing the occurrence of glioma in patients, or for evaluating the possibility of patients benefiting from treatment with reagents for reducing the risk of glioma.

[0015] In one embodiment, the reagent for quantitatively detecting FFAR1 detects the expression of FFAR1 at the gene or protein level.

[0016] The present invention also provides a glioma diagnosis kit, which contains a detection reagent for FFAR1 protein.

[0017] The present invention also provides a drug for preventing, improving, treating or assisting in treating glioma, wherein the active ingredient of the drug is a preparation for enhancing the expression of FFAR1.

[0018] In one embodiment, the enhancing FFAR1 expression comprises enhancing the expression of FFAR1 at the gene or protein level.

[0019] Beneficial effects:

[0020] 1. The present invention found that FFAR1 expression was decreased in the brain tissue of glioma patients, indicating that there was a significant correlation between FFAR1 and glioma, and that FFAR1 could be used as a biomarker for glioma.

[0021] 2. The present invention found that the proliferation, migration and invasion of glioma cells overexpressing FFAR1 were inhibited, and the cell cycle process was slowed down; while glioma cells silencing the expression of FFAR1 had the opposite effect, indicating that FFAR1 can be used as a therapeutic target for glioma.

[0022] 3. The present invention has been verified by animal experiments, and after increasing the expression level of FFAR1 in glioma cells in individuals, the tumor volume of glioma is effectively reduced, which proves the effectiveness of FFAR1 as a target for glioma treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :The experimental process of this patent.

[0024] Figure 2 : FFAR1 is downregulated in glioma tissues and correlated with patient prognosis. (A) Expression difference of FFAR1 in normal brain tissues and glioma tissues in TCGA and GEO databases. (BC) WB and IHC analysis of FFAR1 in 6 normal brain tissues and 12 glioma tissues. (D) Overall survival difference of FFAR1 expression level in glioma patients.

[0025] Figure 3 : Downregulation of FFAR1 promotes the progression of glioma cells, while overexpression inhibits the progression of glioma cells. (AB) Expression of FFAR1 in HEB, U251, T98G, LN229, A172 and U87 cell lines. (CD) WB analysis of FFAR1 gene knockdown in U251 and LN229 cell lines and FFAR1 gene overexpression in A172 and U87 cell lines.

[0026] Figure 4 : CCK-8 detection results in FFAR1 knockdown U251 and LN229 cell lines and FFAR1 overexpression A172 and U87 cell lines; NC represents the siRNA empty sequence group, siFFAR1 represents the FFAR1 knockdown group, vector group represents the FFAR1 plasmid vector group, and FFAR1 represents the FFAR1 overexpression group.

[0027] Figure 5 : Colony formation results in FFAR1 knockdown U251 and LN229 cell lines and FFAR1 overexpression A172 and U87 cell lines; NC represents the siRNA empty sequence group, siFFAR1 represents the FFAR1 knockdown group, vector group represents the FFAR1 plasmid vector group, and FFAR1 represents the FFAR1 overexpression group.

[0028] Figure 6 : Cell migration rates of FFAR1 knockdown U251 and LN229 cell lines (A) and FFAR1 overexpression A172 and U87 cell lines (B); NC represents the siRNA empty sequence group, siFFAR1 represents the FFAR1 knockdown group, vector group represents the FFAR1 plasmid vector group, and FFAR1 represents the FFAR1 overexpression group.

[0029] Figure 7 : Invasion ability of FFAR1 knockdown U251, LN229 cell lines (A) and FFAR1 overexpression A172, U87 cell lines (B); NC represents the siRNA empty sequence group, siFFAR1 represents the FFAR1 knockdown group, vector group represents the FFAR1 plasmid vector group, and FFAR1 represents the FFAR1 overexpression group.

[0030] Figure 8: Flow cytometry was used to count the cell cycle of FFAR1 knockdown U251 (A), LN229 (B) cell lines and FFAR1 overexpression A172 (C), U87 (D) cell lines; NC represents the siRNA empty sequence group, siFFAR1 represents the FFAR1 knockdown group, vector group represents the FFAR1 plasmid vector group, and FFAR1 represents the FFAR1 overexpression group.

[0031] Fig. 9 : Flow cytometry was used to count the apoptosis of FFAR1 knockdown U251 and LN229 cell lines (A) and FFAR1 overexpression A172 and U87 cell lines (B); NC represents the siRNA empty sequence group, siFFAR1 represents the FFAR1 knockdown group, vector group represents the FFAR1 plasmid vector group, and FFAR1 represents the FFAR1 overexpression group.

[0032] Fig.10 :Knockdown of FFAR1 increases the expression of FoxM1 / MYBL2. (AB) Knockdown and volcano plot of differentially expressed genes in U251 cell line after FFAR1 treatment. (C) GO analysis of differentially expressed genes in cell components. (D) KEEG pathway analysis of pathways involved in FFAR1 regulation.

[0033] Fig.11 :(A) Expression levels of FoxM1 and MYBL2 after changing FFAR1 expression. (B) GEPIA analysis of the correlation between FoxM1 and MYBL2. (C) WB analysis of FoxM1 and MYBL2 expression levels in FFAR1-silenced U251 and LN229 cell lines and FFAR1-overexpressing A172 and U87 cell lines.

[0034] Fig.12 :(A) WB analysis of the changes in FoxM1 expression. (B) WB analysis of FoxM1 regulating MYBL2 expression.

[0035] Fig.13 : FFAR1 affects the progression of glioma cells by changing the expression of FoxM1 / MYBL2. (A) Western blot analysis of MYBL2 expression changes. (BC) Western blot analysis of FFAR1, FoxM1 and MYBL2 proteins.

[0036] Fig.14 : CCK-8 assay results of U251 (A), LN229 (B) cell lines and A172 (C), U87 (D) cell lines after regulation.

[0037] Fig.15: Colony formation results of U251, LN229 cell lines (A) and A172, U87 cell lines (B) after regulation.

[0038] Fig.16 : Wounds and cell migration of U251 (A), LN229 (B) cell lines and A172 (C), U87 (D) cell lines after regulation.

[0039] Fig.17 : Invasion experiment results of U251 (A), LN229 (B) cell lines and A172 (C), U87 (D) cell lines after regulation.

[0040] Fig.18 : Cell cycle analysis of U251 (A), LN229 (B), A172 (C), and U87 (D) cell lines by flow cytometry.

[0041] Fig.19 : In animal models, FFAR1 overexpression can inhibit the progression of glioma. (A, B) Imaging evidence and quantification of tumor volume. (C) HE staining and microscopic observation of brain tissue. (D) Immunohistochemical staining of Ki-67 protein expression in mouse glioblastoma. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0043] The experimental materials involved in the following embodiments are as follows:

[0044] (1) Human samples

[0045] All human samples and their corresponding clinical pathological data used in this study were obtained from the Department of Neurosurgery, Wuxi Second People's Hospital, Nanjing Medical University in 2024 with the patients' preoperative informed consent. The pathological identification of all specimens was confirmed by the Department of Pathology, Wuxi Second People's Hospital, and all were first-line patients who did not undergo radiotherapy or chemotherapy. The relevant ethics were approved by the Institute of Ethics Committee of Wuxi Second People's Hospital Affiliated to Nanjing Medical University.

[0046] (2) Cell culture

[0047] Human glioma cells were purchased from the cell bank of the Shanghai Branch of the Chinese Academy of Sciences and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Before the experiment, all cell cultures were kept in an incubator at 37°C with 5% CO2.

[0048] (3) Experimental animals

[0049] 6-8 weeks old clean grade nude mice were purchased from Gempharma. Mice were maintained in a constant temperature and humidity environment. Animal experiments were approved and supervised by the Animal Ethics Committee of Wuxi Second People's Hospital. Animal care and use were strictly in accordance with the guidelines of the National Institutes of Health.

[0050] The gene silencing methods involved in the following embodiments are as follows:

[0051] Four different small interfering RNAs (siRNAs) specifically silencing FFAR1, FoxM1, and MYBL2 gene expression and negative control siRNA were provided by GenePharma (Shanghai, China). Transfection was performed when the cell confluency was 70-90%. After liquid exchange, appropriate concentrations of FFAR1, FoxM1, or MYBL2 siRNA were transfected using P3000TM reagent (Invitrogen, USA). When green fluorescent spots were visible in the cells, the cells were captured for further analysis.

[0052] The gene overexpression method involved in the following embodiments is as follows:

[0053] The FFAR1 gene (as shown in SEQ ID NO.2), FoxM1 gene (as shown in SEQ ID NO.3) and MYBL2 gene (as shown in SEQ ID NO.4) were introduced into the pcDNA3.1+ vector to synthesize the overexpression plasmid by Shanghai GenePharma Co. According to the instruction manual (Invitrogen, USA), the plasmid was transfected into U251, LN229, A172 and U87 cells using Lipo3000 reagent.

[0054] The H&E staining and immunohistochemical staining methods involved in the following embodiments are as follows:

[0055] Take 20 ml of 4% paraformaldehyde cardiac perfusion fixed mouse brain tissue, place it in 4% paraformaldehyde, and fix it in a 4°C refrigerator for 12-24 hours. H&E staining of paraffin-embedded tissue sections was performed by Servicebio biotech.Com (Wuhan, China).

[0056] The amino acid sequence of the FFAR1 protein involved in the following examples is shown in SEQ ID NO.1; the nucleotide sequence encoding the FFAR1 protein is shown in SEQ ID NO.2; the nucleotide sequence of the FoxM1 gene is shown in SEQ ID NO.3; and the nucleotide sequence of the MYBL2 gene is shown in SEQ ID NO.4.

[0057] Example 1 FFAR1 is downregulated in glioma tissue and correlated with patient prognosis

[0058] 1. Database analysis

[0059] To determine whether FFAR1 is significantly expressed in normal brain tissues and glioma tissues, the TCGA and GEO databases were used to describe the differences in FFAR1 expression profiles in glioma tissues and normal tissues in human samples.

[0060] FFAR1 gene expression data and clinical pathological data were downloaded from the TCGA database, and a total of 662 glioma tissues and 1157 normal tissue data were used for FFAR1 differential expression analysis. Kaplan-Meier analysis was used to plot the overall survival (OS) of glioma patient cohorts with different target gene expression levels in the TCGA and GEO databases. GEPIA was used to analyze the correlation between FFAR1 and FoxM1, FFAR1 and MYBL2, and FoxM1 and MYBL2 genes.

[0061] like Figure 2 As shown in A, the expression of FFAR1 was significantly decreased when glioma was diagnosed compared with normal brain.

[0062] 2. Verification of FFAR1 differential expression

[0063] RT-qPCR and WB were used in 18 brain tissue samples (including 6 normal brain tissues and 12 glioma brain tissues). Figure 2 B) Detection of FFAR1 mRNA and protein levels. Western Blot analysis: Protein samples were separated by 10% SDS-PAGE. They were transferred to nitrocellulose membranes, blocked in 5% skim milk, incubated with primary antibody (bs-10738R, 1:2000) at 4°C overnight, and then incubated with secondary antibody (CST-7074, 1:5000) with yamagen peroxidase and tetramethylbenzidine for 1 hour at room temperature, and the bands were detected using an electroluminescent substrate in an imaging system.

[0064] The results showed that the protein expression level of FFAR1 in glioma brain tissue was reduced compared with normal brain tissue.

[0065] In addition, IHC also confirmed this result ( Figure 2 C) Gepia was used to predict the relationship between FFAR1 expression and prognosis in 781 glioma patients. Figure 2 As shown in D, the overall survival (OS) of patients with high FFAR1 expression was more significant than that of patients with low FFAR1 expression (P<0.001, HR=0.69), and their prognosis was also better.

[0066] Example 2 Downregulation of FFAR1 promotes the progression of glioma cells, whereas overexpression inhibits the progression of glioma cells

[0067] 1. Construction of FFAR1 high-expression and silent-expression cells

[0068] Six cell lines were screened by RT-qPCR and WB, and U251 and LN229 were selected as FFAR1 high-expressing cell lines, and U87 and A172 were selected as two low-expressing cell lines ( Figure 3 A,3B).

[0069] Small interfering RNA (siRNA) specifically silencing FFAR1 gene expression and negative control siRNA (obtained by HPLC purification) were provided by GenePharma (Shanghai, China). Transfection was performed when the cell confluency was 70-90%. After the culture medium was replaced, 8ul 3.3mmol / ul FFAR1 siRNA was transfected using P3000TM reagent (Invitrogen, USA). When green fluorescent spots were seen in the cells, the cells were captured for WB verification of the target gene knockout. The above methods were used to construct U251 and LN229 cell lines that silenced FFAR1, respectively.

[0070] The FFAR1 gene (as shown in SEQ ID NO.2) was introduced into the pcDNA3.1+ vector by Shanghai GenePharma Company to synthesize an overexpression plasmid with a CMV binding site. According to the instructions, the FFAR1 overexpression plasmid was transfected into A172 and U87 cells using Lipo3000 reagent (Invitrogen, USA). When the cells grew to a confluence of 80%-90%, the original cell culture medium was discarded, washed twice with PBS (Gibco, USA), and 2 ml of reduced serum medium opti-MEM (Gibco, USA) was added. 5 ug of plasmid was added. After 8 hours of transfection, the cell culture medium was discarded, washed twice with PBS (Gibco, USA), and cultured for 36 hours with 2 ml of complete medium. The expression of the target gene was verified by WB. The U251 and LN229 cell lines with silencing FFAR1 constructed above, as well as the U87 and A172 cell lines with explosive expression of FFAR1 were verified by WB. The results showed that the expression of FFAR1 reached the expected level ( Figure 3 CD).

[0071] 2. Effect of FFAR1 on glioma cell proliferation

[0072] Through CCK-8 test ( Figure 4 ) and colony formation assay ( Figure 5 ) to investigate whether FFAR1 affects the proliferation or viability of glioma cells.

[0073] CCK-8 assay: When cells were cultured to exponential growth, they were digested with trypsin, centrifuged, suspended and counted, and diluted to an appropriate density. The target cells were inoculated on a culture plate (initial inoculation density was 3000 cells / 2 mL / well), 0.1 mL CCK-8 reagent (Biosharp, Shanghai, China) was added, and then cultured for 5 consecutive days. The absorbance at 450 nm was detected every day with an ELISA reader (Invitrogen, USA), and the cell proliferation rate was calculated as (absorbance on day x - absorbance on day 1) / absorbance on day 1.

[0074] Colony formation assay: Add 100ul of culture medium to each well of a six-well plate and inoculate at a density of 600 cells / well. Spread the plate evenly and change the medium every 3 days. Observe the size of the cell colony under a fluorescence microscope (Olympus, Japan). Wash with PBS, fix with 4% paraformaldehyde (Sigma, USA), count the number of colonies after Giemsa staining, wash several times with ddH2O, and count the number of colonies on the 8th day.

[0075] The results showed that in U251 and LN229 cell lines, silencing of FFAR1 significantly promoted active cell proliferation. In the CCK8 experiment, after 5 days of culture, the proliferation rates of U251 and LN229 cells with FFAR1 silencing increased by 1.5 and 1.8 times, respectively, compared with the control NC without genetic manipulation; in the colony formation experiment, the number of colonies of U251 and LN229 cells with FFAR1 silencing increased by 1.9 and 2.1 times, respectively, compared with the control NC without genetic manipulation. In the other two cell lines, the cell viability of the FFAR1 group was significantly lower than that of the vector group. In the CCK8 experiment, after 5 days of culture, the proliferation rates of U251 and LN229 cells overexpressing FFAR1 decreased by 2.5 and 3.1 times, respectively, compared with the control NC without genetic manipulation; in the colony formation experiment, the number of colonies of U251 and LN229 cells overexpressing FFAR1 decreased by 3.8 and 4.0 times, respectively, compared with the control NC without genetic manipulation.

[0076] These results indicate that FFAR1 overexpression weakens the proliferation of glioma cells.

[0077] 3. Effect of FFAR1 on glioma cell migration

[0078] Through wound healing experiment ( Figure 6 ) to investigate the effect of FFAR1 on migration. The transfected cells were inoculated into 6-well plates and cultured for a period of time until the cell confluence reached 90-100%. The artificial wound was gently scratched with the tip of a micropipette, and the floating cells and debris were gently washed with PBS (Gibco, USA). The wound was placed in the incubator again for 24 hours, and photographed under a microscope at 0h and 24h to record the wound healing. The results were expressed as migration rate (migration rate (%) = width difference / initial width).

[0079] The results showed that in U251 and LN229 cell lines, the healing rate of the si-FFAR1 group was significantly faster than that of the NC group, and in U87 and A172 groups, the rate of the FFAR1 group was lower than that of the vehicle group, indicating that FFAR1 mediates cell migration in glioma cells.

[0080] 4. Effect of FFAR1 on glioma cell invasion

[0081] Concentrated matrix gel (Invitrogen, USA) was thawed at 4°C, and then the matrix gel was diluted to an appropriate concentration and formed into a membrane in a 37°C incubator. Cells cultured to the logarithmic growth phase were diluted to 2.5*10 4 Cells / mL were inoculated on the top of the hydrated membrane, and serum-containing medium was added to the other side. After the cells passed through the membrane, they were fixed, stained, washed, and photographed under a fluorescence microscope (Olympus, Japan).

[0082] Transwell invasion assay ( Figure 7 ) showed that after FFAR1 knockdown, the invasion ability of cells was enhanced, while overexpressed FFAR1 occupied the cells.

[0083] 5. Effect of FFAR1 on glioma cell cycle and apoptosis

[0084] Cell cycle detection was performed on U251 cells, LN229 cells transfected with FFAR1 siRNA, and A172 and U87 cells with FFAR1 overexpression plasmids using a cell cycle detection kit (Beyotime biotechnology, Nanjing, China). The transfected cells were centrifuged, washed with PBS, fixed with 75% alcohol, washed again by centrifugation, and 500ul of working solution was added. The cells were incubated at 37°C without light for half an hour, and the number of cells in different cycles was counted by flow cytometry. Apoptotic cells were labeled with Annexin V-FITC / PI cell apoptosis detection kit (Beyotime biotechnology, Nanjing, China). After centrifugation and washing, binding solution, Annexin V-FITC and propyl iodide staining reagent (PI) were added and incubated in the dark for 20 minutes. The stained cells were counted by flow cytometry.

[0085] Flow cytometry ( Figure 8 ) showed that knockdown of FFAR1 increased the proportion of S / G2 phase (from 40% to 60% in U251 cells; from 25% to 40% in LN229 cells), while overexpression of FFAR1 decreased this proportion (from 60% to 30% in A172 cells; from 55% to 35% in U87 cells). At the same time, the apoptotic cells counted by flow cytometry ( Fig. 9 ) showed that altering FFAR1 expression had almost no common effect on cell apoptosis, regardless of the number of NC and si-FFAR1 groups, vehicle and FFAR1 groups.

[0086] Example 3 FFAR1 affects the progression of glioma cells by changing the expression of FoxM1 / MYBL2

[0087] 1. Knockdown of FFAR1 increases the expression of FoxM1 / MYBL2

[0088] To determine how FFAR1 inhibits the malignant progression of glioma cells, the relative gene expression profile induced by FFAR1 silencing was determined by RNA-seq analysis. The above primary cultures of glioma cells and U251 cells (including FFAR1 knockdown and negative controls) were maintained and cultured using Total RNA was extracted using 2100 Bioanalyser (Agilent) and NanoDrop (Thermo Fisher Scientific) to quantify the integrity of total RNA. About 500 ng of high-quality RNA sample was obtained for the construction of sequencing library. rRNADepletion Kit(H / M / R)(Vazyme#N406) and The Universal V6 RNA-seq Library Prep Kit for Illumina (#N401-NR604) generates sequencing libraries and then adds index codes to the attribute sequences of each sample. The libraries are sequenced on the Illumina platform to generate 150 bp paired-end reads.

[0089] Expression profiles ( Fig.10 AB) showed that a total of 222 related genes were expressed differently, and silencing FFAR1 upregulated 115 related genes and downregulated 107 related genes. GO enrichment was used to further classify the genes with changed expression into Figure 3 C in Biological Process (BP), Cellular components (CC) and Molecular Function (MF). For BP, FFAR1 mainly responds to external stimuli and participates in cell progression. Regarding the CC category, participation in the composition of the nuclear cavity is the most prioritized category. The following are Golgi apparatus, cell processes, plasma membrane parts and vesicle membranes. In the MF category, FFAR1 mainly regulates genes involved in binding proteins, activating transcriptional regulatory factors, structural molecular activity, signal receptor binding and purine nucleotide binding. In addition, through KEGG database analysis (3D), it was found that FFAR1 mainly plays a role in the following aspects: cancer pathways, cancer micro-RNAs, cell cycle, cell senescence and viral infection.

[0090] Confirmation of altered expression genes associated with glioma progression by RT-qPCR:

[0091] (1) Changes in FFAR1 expression trigger changes in FoxM1 and MYBL2 expression

[0092] like Fig.11 As shown in AB, when FFAR1 expression was changed, FoxM1 and MYBL2 showed changes consistent with RNA sequence analysis. Knockdown of FFAR1 expression upregulated the expression of FoxM1 and MYBL2, while overexpression of FFAR1 downregulated the expression of FoxM1 and MYBL2. WB results ( Fig.11 C) also confirmed this.

[0093] (2) Changes in FoxM1 expression trigger changes in MYBL2 expression

[0094] The expression of FoxM1 was changed by referring to the method described in Example 2. FoxM1 siRNA was transfected into U251 and LN229 cells to construct cells silencing the expression of FoxM1. A172 and U87 cells were transfected with a pcDNA3.1+ vector (the gene connection site on the vector was CMV) containing the FoxM1 gene (as shown in SEQ ID NO.3) to construct cells overexpressing FoxM1.

[0095] WB( Fig.12 ) The results showed that when FoxM1 expression was upregulated, MYBL2 was also upregulated, and when FoxM1 expression was downregulated, MYBL2 was downregulated. This suggests that FoxM1 regulates the expression of MYBL2.

[0096] 2. FFAR1 affects the progression of glioma cells by changing the expression of FoxM1 / MYBL2.

[0097] First, by changing the expression of FFAR1, FoxM1 and MYBL2 were up-regulated or down-regulated at the same time, as follows: FFAR1 siRNA + FoxM1 siRNA co-transfected U251 and LN229 cells, FFAR1 plasmid + FoxM1 plasmid co-transfected A172 and U87 cells, FFAR1 siRNA + FoxM1 siRNA + MYBL2 plasmid co-transfected U251 and LN229 cells, FFAR1 plasmid + FoxM1 plasmid + MYBL2 siRNA co-transfected A172 and U87 cells. To determine whether FFAR1 affects the progression of glioma cells by reducing the expression of FoxM1 and MYBL2.

[0098] Experimental groups:

[0099] NC+vector: U251 and LN229 cells were co-transfected with NCsiRNA and plasmid vector;

[0100] siFFAR1+NC+vector: U251 and LN229 cells were co-transfected with FFAR1 siRNA+NC siRNA and plasmid vector;

[0101] siFFAR1+siFoxM1+vector: U251 and LN229 cells were co-transfected with FFAR1siRNA+FoxM1siRNA and plasmid vector;

[0102] siFFAR1+siFoxM1+MYBL2: U251 and LN229 cells were co-transfected with FFAR1siRNA+FoxM1siRNA+MYBL2 plasmids;

[0103] Vector+NC: A172 and U87 cells were co-transfected with plasmid vector and NCsiRNA;

[0104] FFAR1+vector+NC: A172 and U87 cells were co-transfected with FFAR1 plasmid+plasmid vector and NC siRNA;

[0105] FFAR1+FoxM1+NC: A172 and U87 cells were co-transfected with FFAR1 plasmid + FoxM1 plasmid and NC siRNA;

[0106] FFAR1+FoxM1+siMYBL2: A172 and U87 cells were co-transfected with FFAR1 plasmid+FoxM1 plasmid+MYBL2 siRNA;

[0107] The levels of FFAR1, FoxM1 or MYBL2 in the Control, NC and NC+vector groups were detected by WB (12B), and it was found that there was basically no difference in their levels, indicating that si-RNA and empty plasmid vector did not affect their basic expression levels.

[0108] like Fig.13 As shown in A, it shows the effectiveness of MYBL2 knockdown sequence and overexpression sequence. Experimental cells were constructed by transfecting gene sequences, and the expression of target genes in each experimental group was verified by WB ( Fig.13 BC), the results showed that the target protein levels in each group reached the expected level.

[0109] CCK-8 assay and colony formation assay were performed according to the method in Example 2. CCK-8 assay of four cell lines ( Fig.14 ) and colony formation assay ( Fig.15 ) showed that knocking down FoxM1 significantly weakened cell viability after silencing FFAR1, while overexpression of MYBL2 abolished this effect. It was confirmed that FoxM1 upregulation could alleviate the impaired cell proliferation induced by FFAR1 overexpression, but with silencing of MYBL2, cell proliferation was blocked again.

[0110] like Fig.16 As shown, when FFAR1 was silenced, further silencing of FoxM1 (si-FFAR1+si-FoxM1+vector) resulted in a decrease in the cell healing rate; while further overexpression of MYBL2 (si-FFAR1+si-FoxM1+MYBL2) increased the healing rate, and in U87 and A172 cell lines, the healing rate of the FFAR1+FoxM1+NC group was higher than that of the FFAR1+vector+NC group, and the healing rate of the FFAR1+FoxM1+si-MYBL2 group was lower than that of the FFAR1+FoxM1+NC group.

[0111] Transwell invasion assay results ( Fig.17) showed that FoxM1 silencing inhibited the increased cell invasion induced by FFAR1 knockdown, while FoxM1 overexpression reversed the invasion inhibition induced by FFAR1 overexpression. Changing the expression of MYBL2 can weaken the reversal effect of FoxM1 on FFAR1.

[0112] Flow cytometry ( Fig.18 ) The results showed that after FFAR1 was knocked down, MYBL2 upregulation could eliminate the decrease in the S / G2 phase ratio caused by FoxM1 silencing. Overexpression of FoxM1 could reverse the decrease in the S / G2 phase ratio caused by FFAR1 upregulation, and on this basis, downregulation of MYBL2 weakened this ratio.

[0113] In summary, FFAR1 can change the expression of FoxM1, thereby affecting MYBL2 and further changing the progression of glioma cells.

[0114] Example 4 Overexpression of FFAR1 can reduce tumor burden by altering the expression of FoxM1 / MYBL2 in vivo

[0115] Experimental groups:

[0116] (1) U87 cells transfected with only the empty vector sequence served as the control and were named the NC group.

[0117] (2) The FFAR1 gene was introduced into the CMV site of the pcDNA3.1+ vector by Shanghai GenePharma Company to synthesize an overexpression plasmid. According to the instructions (Invitrogen, USA), the transfection plasmid was transfected into U87 cells using Lipo3000 reagent to construct U87 cells overexpressing FFAR1, which was used as the FFAR1 group.

[0118] (3) Shanghai GenePharma Company introduced the FFAR1 gene and FoxM1 gene into the CMV sites of two pcDNA3.1+ vectors to synthesize overexpression plasmids. According to the instructions (Invitrogen, USA), the transfection plasmids were transfected into U87 cells using Lipo3000 reagent to construct U87 cells overexpressing FFAR1 and FoxM1, which were used as the FFAR1+FoxM1 group.

[0119] (4) Shanghai GenePharma Company introduced FFAR1 gene and FoxM1 gene into CMV and CMV sites of pcDNA3.1+ vector to synthesize overexpression plasmids. According to the instruction manual (Invitrogen, USA), Lipo3000 reagent was used to transfect U87 cells with plasmids to construct U87 cells overexpressing FFAR1 and FoxM1. U87 cells overexpressing FFAR1 and FoxM1 were cultured until the cell confluence was 70-90% and then transfected. After liquid exchange, P3000TM reagent (Invitrogen, USA) was used to transfect MYBL2 siRNA at an appropriate concentration. When green fluorescent spots were seen in the cells, the cells were captured for further analysis, and U87 cells overexpressing FFAR1 and FoxM1 and silencing MYBL2 were obtained as FFAR1+FoxM1+si-MYBL2 group.

[0120] U87 cells transfected with the target gene were cultured at 5*10 5 The number of si-MYBL2 cells was orthotopically implanted into the frontal lobe of mice (6 per group, NC, FFAR1, FFAR1+FoxM1, and FFAR1+FoxM1+si-MYBL2) and placed under 24°C light / dark cycle control for 12 h. After 12 days, the mice were subjected to MRI (Siemens, Germany) to observe the tumor size, record the length, width (L) and width (W) of the largest cross section of the tumor, and the number of imaging layers (H) (0.7 mm per layer), and calculate the tumor volume using the following formula: volume (mm3) = 4 / 3×π×L×W×H. After the animal experiment, the mice were euthanized and their brain tissues were obtained for hematoxylin-eosin staining and immunohistochemistry. All animal experiments were in accordance with the European Parliament Directive (2010 / 63 / EU) and approved by the Institutional Animal Care and Use Committee of Jiangnan University School of Medicine.

[0121] The transfection efficiency of the sequences has been verified before, indicating that the downregulation of FoxM1 induced by overexpression of FFAR1+FoxM1 was alleviated in the FFAR1+FoxM1 group compared with the FFAR1 group, and the upregulation of MYBL2 induced by overexpression of FFAR1+FoxM1+si-MYBL2 was significantly attenuated in the FFAR1+FoxM1 group compared with the FFAR1+FoxM1 group. Fig.19 As shown in A, the MRI images of in situ glioma formation in the brains of different nude mice showed that the tumor volume of the brains of nude mice overexpressing FFAR1 was significantly reduced, which was reduced by 3 times ( Fig.19B), indicating that FFAR1 overexpression can inhibit the growth of glioma. However, upregulating the expression of FoxM1 can alleviate this inhibitory effect. At the same time, comparing the FFAR1+FoxM1 and FFAR1+FoxM1+si-MYBL2 groups, MYBL2 knockdown significantly inhibited the occurrence of glioma promoted by FoxM1 overexpression. The results are consistent with the volume statistical expression.

[0122] Representative hematoxylin-eosin stained areas in the model brain tissue ( Fig.19 C) (determined by the cross-sectional area occupied by the tumor) showed the same results as the MRI images, FoxM1 overexpression alleviated the inhibitory effect of FFAR1 overexpression, and MYBL2 knockdown could hinder the promotion effect of FoxM1 overexpression in glioma. Fig.19 As shown in D, immunohistochemical analysis of Ki-67 in paraffin-embedded specimens of the four groups of tumor burden models revealed that Ki-67 decreased with upregulation of FFAR1, increased with upregulation of FoxM1, and decreased with silencing of MYBL2. These indicate that FoxM1 and MYBL2 increase tumor burden in vivo by inhibiting the expression of MYBL2 caused by FoxM1, while FFAR1 reduces tumor burden by inhibiting the expression of MYBL2 caused by FoxM1.

[0123] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of FFAR1 protein in the preparation of drugs for preventing, improving, treating or assisting in the treatment of glioma.

2. The use according to claim 1, characterized in that The amino acid sequence of the FFAR1 protein is shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that: The drug is used for enhancing the expression of FFAR1 protein.

4. The use according to claim 3, characterized in that: The drug is used for at least one of the following purposes: inhibiting the proliferation of glioma cells; inhibiting the migration of glioma cells; inhibiting the invasion of glioma cells.

5. Application of FFAR1 protein as a biomarker for glioma.

6. The use according to claim 5, characterized in that The application is a reagent for quantitatively detecting FFAR1 in the preparation of a kit for diagnosing glioma, or assisting in the diagnosis of glioma, or for screening a reagent for reducing the occurrence of glioma in patients, or for evaluating the possibility of a patient benefiting from treatment with a reagent for reducing the risk of glioma.

7. The use according to claim 6, characterized in that The reagent for quantitatively detecting FFAR1 is used to detect the expression of FFAR1 at the gene or protein level.

8. A glioma diagnostic kit, characterized in that: A detection reagent containing FFAR1 protein.

9. A drug for preventing, improving, treating or assisting in the treatment of glioma, characterized in that: The active ingredient of the drug is a preparation that enhances the expression of FFAR1.

10. The drug according to claim 9, characterized in that The enhancing of FFAR1 expression includes enhancing the expression of FFAR1 at the gene or protein level.

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