Abnormal expression of TGFβ1 in acute myeloid leukemia and its regulation effect on leukemia cells

CHEN Wen-ting, HUANG Ying, PAN Yan-ping, WANG Shu-wen, TANG Rui-mei

1.Hainan Hospital Affiliated to Hainan Medical University, Haikou 570311, China

2.Department of Hematology, Hainan Provincial People's Hospital, Haikou 570311, China

Keywords:

ABSTRACT Objective: To explore the level of acute myeloid leukemia (COX-2), transforming growth factor β1 (TGFβ1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in acute myeloid leukemia (AML) patients and to investigate the role of TGFβ1 on the proliferation, apoptosis and cycle of AML cells, providing new targets and research bases for the treatment and prognostic evaluation of AML.Methods: Peripheral blood was collected from 80 AML patients and 60 normal people.The levels of COX-2, TGFβ1, bFGF and VEGF in peripheral blood mononuclear cells and serum were determined by using quantitative polymerase chain reaction and enzyme linked immunosorbent assay, respectively.The proliferation, apoptosis and cycle of AML cells affected by overexpression and silencing of TGFβ1 was detected using cell counting kit-8 and flow cytometry.Results: The expression of COX-2, TGFβ1, bFGF and VEGF increased significantly in peripheral blood mononuclear cells and serum in AML patients.TGFβ1 promoted AML cell proliferation, inhibited its apoptosis, and increased stage G2 cell proportion.Conclusion: COX-2, TGFβ1, bFGF and VEGF play important roles in the progression of AML.TGFβ1 is a new potential biomarker for the diagnosis and treatment of AML.

1.Introduction

Acute myeloid leukemia (AML) is a malignant clonal disease caused by abnormal proliferation and differentiation disorders of hematopoietic stem progenitor cells Recurrence is a difficult problem in its treatment[1,2] The etiology and pathogenesis of AML are complex, and molecular and cytogenetic abnormalities are currently considered to be its most important causative and prognostic factors[3]

Studies have shown that cyclooxygenase-2 (COX-2), basic fibroblast growth factor, in serum (basic fibroblast growth factor,bFGF), transforming growth factor β1 (TGFβ1) and levels of factors such as vascular endothelial growth factor (VEGF) are associated with chronic myeloid leukemia (chronic myelogenous leukemia,CML) and acute leukemia (AL) cell load and disease evolution are closely related, Dynamic monitoring of changes in expression levels of these factors can be used as a reference index for judging CML and AML disease and prognostic survival [4-6] Shingai Y et al.have shown that TGFβ signal transduction induces AML progression[7].We have previously studied the relationship between TGFβ1 and VEGF expression in peripheral blood mononuclear cells with clinical prognosis[8] but TGFβ1 The regulation and molecular mechanism of leukemia cell function have not been discussed.The purpose of this study was to explore the levels of COX-2, TGFβ1,bFGF and VEGF in patients with AML, and to explore TGFβ1 versus AML Effects on cell proliferation and apoptosis, as well as COX-2, bFGF, and VEGF expression, to explore TGFβ1 in acute myeloid lines.The cell function and molecular mechanism of leukemia pathogenesis provide a new target and research basis for the treatment and prognosis evaluation of acute myeloid leukemia.

2.Materials and methods

2.1 Subjects of study

From August 2019 to August 2021, 80 patients with AML who were treated in the hematology department for the first time were the subjects of this experimental study, with reference to Zhang Zhinan's " The diagnostic criteria in the Criteria for the Diagnosis and Efficacy of Hematological Diseases"[9] were met by 80 patients with AML.The overall age of the patients was 15 to 75 years, and all included patients were required to agree to participate in the study and sign an informed consent form.Inclusion criteria: age 15-75 years; Diagnosis of acute myeloid leukemia by MIM; No psychiatric disorders.Exclusion criteria: complicated by serious diseases of vital organs such as AIDS; Have received chemotherapy; Recurrent relapses; Those who are allergic to the drugs involved in treatment and have poor tolerance.The 60 normal people in the control group were from the physical examination center, and there was no statistical difference between the age and sex of the AML group.

2.2 Acquisition and treatment of patient serum

Before chemotherapy in all patients, without the use of any drugs related to the mobilization of bone marrow stem cells, local 2%cocaine anesthesia was used on the patient, and 2 mL of bone marrow fluid was extracted from the anterior superior iliac and posterior superior iliac spines using a bone marrow needle and 2 mL DMEM (Gibco, article number) was added 11965092) Mix the cell culture medium.Pipette 10 μL of the mixed solution to the DEPCtreated EP tube, add 10 μL of enzymatic-free water to mix and add 1 mL of PBS solution to resuspend the leukocyte solution 1 000 g at 4 ℃ for 3 min to remove the supernatant.

2.3 Peripheral blood mononuclear cells (PBMCs) isolated

Carefully mix 3 mL of peripheral blood with an equal volume of pre-chilled PBS and carefully add the mixed solution along the wall of the test tube to a volume of 3 mL lymphocyte isolate.Set the centrifuge lift speed to 0 and centrifuge at 20 rpm at 20 ℃ for 20 min.Carefully transfer the intermediate mononuclear cell layer into pre-cooled PBS with a pipette and centrifuge at 1 700 rpm at 4 ℃for 4 min.Discard the supernatant after centrifugation.Resuspend the cells using 3 mL FACS buffer and centrifuge at 1 700 rpm at 4 ℃ for 4 min, and repeat the above steps once.Resuspend cells using 2 mL of pre-chilled PBS, accurately aspirate 10 μL of cell resuspension using a micropipette for counting, centrifuge the remaining cells at 1 700 rpm at 4 ℃ for 4 min, discard the supernatant and set aside.

2.4 Total RNA extraction and reverse transcription

1 mL volume of Trizol (Invitrogen, article number 15596026)added about 5 106 cells, extracted cell total RNA, detected and extracted using Thermo NanoDrop 2000 The concentration and purity of R NA, absorbance (A)A260/A280are 1.8～2.0.

The extracted RNA is then reversed-transcripted into cDNA using a reverse transcription kit (Takara, catalog number RR037A) following the instructions steps.

2.5 qPCR measures mRNA expression levels of COX-2,TGFβ1, bFGF, and VEGF

q-PCR detection with ThermoFisher QuantStudio 5 real-time quantitative cycler and Applied Biosystems? PowerUp? SYBR?Green master mix And use GAPDH as an internal reference.Shanghai Sangong Biotechnology Co., Ltd designed and synthesized the gene primer of interest, and the primer list is shown in Table 1.The data for qPCR were statistically processed using the 2-ΔΔCTmethod.Set up 3 complex wells per sample with reaction mixture(total volume 20 μL): cDNA template 2 μL, 1 μL each of the positive and negative primers of SYBR Green II.enzyme 10 μL, ddH2O 7 μL.Set the reaction conditions according to the instructions.

2.6 ELISA detects the expression of COX-2, TGFβ1, bFGF,and VEGF in serum

COX-2, TGFβ1, bFGF, and VEGF enzyme-linked immunoassay kits were purchased at Shanghai Lianzu Biotechnology Co., Ltd.,and 80 patients with AML and 6 were detected according to the kit procedures The expression levels of COX-2, TGFβ1, bFGF, and VEGF in serum in 0 control groups.

2.7 Recombinant overexpression and construction of RNAi vectors

PCR amplification of TGFβ1, bFGF, VEGF genes: 95 ℃prevarications for 5 min; 95 ℃ denaturation 30 s, 52 ℃ annealings 30 s, 72 ℃ extensions 30 s, 30 cycles, 72 ℃ extensions 10 min, the product is detected by agarose gel electrophoresis, followed by the recovery of the gel fraction containing the PCR product using the DNA gel recovery kit.The recovered TGFβ1, bFGF, and VEGF gene fragments were linked to the pMD18-T cloning vector and transformed into competent DH5 in E.coli.Send the recombinant clone vector to the sequencing company for sequencing comparison.The plasmid of the bacterial liquid is extracted, enzymatic identification is performed, the identification and observation are performed using agarose gel electrophoresis, and the TGFβ1, bFGF,and VEGF genes recovered by the DNA gel recovery kit are used.The recovered fragments are ligated with linear fragments of the pEGFP-N1 overexpression vector after digestion and transformed into Enterobacter competent DH5.

The RNAi vector of the TGFβ1 gene was constructed by Guangzhou Anno Biologics.

Tab 1 Primer sequence

2.8 Recombinant overexpression and transfection of RNAi vectors

Select OCI-AML2 and OCI-AML5 cells in a good growth state and seeded the cells to 6 at a concentration of 6×105cells/well In the well-plate, the transfection step is carried out according to the LipofectamineTM2000 product instructions, and 50 μL of recombinant overexpression vector or RNAi vector solution is added to the EP tube for 250 μL RPMI-1640 medium (Gibco, carton number 22400089) to another E-P tube Add 250 μL of RPMI-1640 and 10 μL of lip2000 solution and let stand at room temperature for 5 min.Mix the solution in two EP tubes, let stand at room temperature for 20 min and then add to the corresponding cells, add 1.5 mL of RPMI-1640 medium per well and culture After 12 h, replace with 2 mL of LRPMI-1640 complete culture solution.

2.9 CCK8 detected the proliferation of cell lines in each group after transfection

The transfected OCI-AML2 and OCI-AML5 cells of each group were inserted into 96-well plates at concentrations of 2×103cells,and cultured for 24 h, 48 h and 72 h.Add 10 μL of CCK-8 solution(Guangzhou Jingxin Biologics, catalog no.180306) After 4 h of incubation, discard the culture medium, add DMSO solution, mix the reaction at 50 rpm on a horizontal shaker for 10 min, and measure the absorbance of each well using an enzyme-linked immunoassay.

2.10 Western blot to detect the expression of COX-2, TGFβ1,bFGF, and VEGF in AML cells

The total protein of OCI-AML2 and OCI-AML5 cells after transfection was extracted using cell lysate, and the concentration of the protein was determined using BCA kit, and a 5 × Loading buffer was added to denature in a metal bath at 100 ℃ for 15 min.When loading, calculate the amount of protein loaded per group of 20 μg of protein, transfer the protein to a PVDF membrane of 0.22 μm after SDS-PAGE.5% of BSAs are blocked at 37 ℃ for 1 h,COX2 (Abcam, ab179800), TGFβ1 (Abcam, ab92486 ), bFGF(CST,20102S), VEGF(Abcam, ab32152) and GAPDH (Proteintech,60004-1-Ig) primary antibody 1: 1 000 dilutions and incubation at 4 ℃ overnight, after washing the membrane 6 times 5 min, 1:5 000 dilution of goat anti-rabbit secondary antibody, incubation under greenhouse for 60 min.Wash the film for 4×5 min.The chemiluminescent agent developing protein was added, the image was collected in the gel imaging system, the grayscale expression of the protein band was analyzed by Image J, and the relative expression of COX-2, TGFβ1, bFGF and VEGF was analyzed with GAPDH as the internal control.

2.11 Apoptosis detection of each group of cells after transfection

Each group of transfected OCI-AML2 and OCI-AML5 cells was inserted into the 6-well plate at a concentration of 106cells/mL.Apoptosis in each group was detected using the Keygen BioAnnexin V-FITC/PI (KGA104) apoptosis assay kit, in the following steps Collect cells with EDTA-free pancreatic enzyme digestion, wash cells with pre-chilled PBS, centrifuge to collect 15×105cells, use 500 μL volume Binding Buffer resuspend; First stained with 5 μL of Annexin V-FITC, followed by 5 μL of Propidium Iodide; Leave at room temperature protected from light for 20 min.Finally, the number of apoptosis cells in each group was measured by BD FACSCanto flow cytometry.

2.12 Cell cycle detection of each group of cell lines after transfection

Digest the transfected cells of each group with EDTA-free pancreatic enzymes and prepare a cell suspension at a concentration of 106cells/mL.Detect the cell cycles of each group using the KGA512 Keygen Bio Cell Cycle Assay Kit as follows: PBS washes the collected cells once, centrifuges the cells and adjusts the concentration to 1×106/mL, after which it is aspirated up to 1mL single cell suspension; Centrifuge and pour supernatant and add 500 uL of 70% cold ethanol to freeze for 2 h.After PBS cleaning, it was screened at 200 mesh and centrifuged at 1 000 rpm for 3 min; Add 500 μL PI/RNase A staining working solution and incubate at room temperature protected from light for 40 min; Detection using BD FACSCanto flow cytometry records red fluorescence at excitation wavelength 488 nm.

2.13 Statistical processing

Adopted separately SPSS 23.0 and GraphPad Prism8.0.2 Software for data disposed of graphing, Conforming to the normal distribution The measurement data is measured using the mean±standard deviation(±s).Comparison between two groups adopt inspection.P＜0.05 deemed to have statistics difference.

3.Results

3.1 COX-2, TGFβ1, bFGF, VEGF are highly expressed in AML patients

q-PCR test results show that TGFβ1 The expression level of PBMCs in AML patients (1.661 ± 0.067) was significantly higher than that in the control group (1.008 ± 0.030, P＜0.001, t =7.985,Figure 1A); The expression of bFGF in PBMCs of AML patients(2.962 ± 0.057) was significantly higher than that of the control group (1.226 ± 0.048, P＜0.001, t=22, Figure 1B); The expression of COX-2 in PBMCs of AML patients (1.552 ± 0.060) was significantly higher than that in the control group (1.226 ± 0.048,P＜0.001, t=6.135, Figure 1C); The expression of VEGF in PBMCs of AML patients (2.447 ± 0.076) was significantly higher than that of the control group (1.049 ± 0.022, P＜0.001, t=15.75, Figure 1D).

Fig 1 The level of COX-2(A), TGFβ1(B), bFGF(C) and VEGF(D) in PBMCs in peripheral blood examined by qPCR (***P＜0.001)

The results of ELISA showed that The expression level of TGFβ1 in serum of AML patients (480.81 ± 20.1) was significantly higher than that in the control group (256.1 ± 13.51, P＜0.001, t=8.641,Figure 2A); The expression of bFGF in the serum of AML patients(281.3 ± 9.723) was significantly higher than that of the control group (159.4 ± 7.683, P＜0.001, t=9.302, Figure 2B); The expression of COX-2 in the serum of AML patients (331.4 ± 11.17) was significantly higher than that in the control group (151.4 ± 12.36,P＜0.001, t=10.74, Figure 2C); The expression of VEGF in the serum of AML patients (711.3 ± 21.83) was significantly higher than that in the control group (508.1 ± 21.10, P＜0.001, t=6.525, Figure 2D).

Fig 2 The level of COX-2(A), TGFβ1(B), bFGF(C) and VEGF(D) examined by ELISA (***P＜0.001)

3.2 TGFβ1 regulates the proliferation, apoptosis and cycle of AML cells

CCK8 test results showed that OCI-AML2 and OCI-AML5 cells OE- TGFβ1 on the fifth day OD450 in plasmid group 1 was 2.589± 0.044 and 2.882 ± 0.029, respectively, which was significantly higher than 2.103 ± 0.048 (P＜0.001, t=12.90) and 2.302 ± 0.056(P＜0.001, t=15.97) in OCI-AML2 and OCI-AML5 OE-NC cells,indicating that TGFβ1 Overexpression significantly promoted the proliferation of OCI-AML2 and OCI-AML5 cells; On the fifth day,OCI-AML2 and OCI-AML5 cells with sh-TGFβ1 OD450 of plasmid group was 1.553 ± 0.014 and 1.689 ± 0.034 respectively, which was significantly lower than 2.367 ± 0.069 (P＜0.001, t=20.10) and 2.424± 0.074 (P＜0.001, t=15.54) in OCI-AML2 and OCI-AML5 KD-NC groups, indicating that TGF β 1 Silencing significantly inhibited the proliferation of OCI-AML2 and OCI-AML5 cells (Figure 3A).

Flow cytometry showed that OCI-AML2 and OCI-AML5 cells were transfected with OE-TGFβ1 The apoptotic rate after plasmid was 7.787 ± 0.516% and 5.477 ± 0.065%, respectively, which was significantly lower than that in OE-NC group, which was 13.75 ± 0.486% (P=0.007＜0.01, t=12.10) and 9.003 ± 0.335%(P=0.002＜0.01, t=22.86); after Sh-TGFβ1 transfected with OCIAML2 and OCI-AML5 cells Apoptosis rate was 21.33 ± 0.934% and 15.11 ± 0.461% respectively, which was significantly higher than that of KD-NC group (14.78 ± 0.446%, P=0.002＜0.01, t=22.86) and 10.84 ± 0.350% (P＜0.001, t=55.63), Figure 3B.

Fig 3 Effect of TGFβ1 expression on the proliferation(A), apoptosis(B) and cell cycle(C) of AML cells (***P＜0.001)

4.Discussion

AML is the most common adult leukemia, the incidence of which increases with age, and is characterized by the uncontrolled proliferation of low-differentiated myeloid cells in the bone marrow[10].It is a heterogeneous disease with a high recurrence rate and poor overall survival[1] In recent decades, we have made progress in understanding the mechanism and biological characteristics of AML, but the pathogenesis of AML is still unclear.

The effect of COX-2 on angiogenesis and tumor has become one of the research focuses in the field of anti-tumor.COX-2 can induce the expression of VEGF, bFGF and other angiogenic factors,and promote the migration of stimulated endothelial cells[11].The expression rate and level of COX-2 in CML patients were significantly higher than those in the control group, suggesting that COX-2 is related to the pathogenesis of CML[12].In this study,we found that the expression of COX-2 in PBMCs and serum of AML patients was significantly higher than that of normal controls,suggesting that COX-2 is also closely related to the pathogenesis of AML.Many studies have shown that COX-2 inhibitors can significantly inhibit the malignant progression of AML cells[13-15],which is consistent with our findings that COX-2 overexpression in AML promotes AML.

BFGF is one of the positive regulators of angiogenesis with a strong effect and high specificity, plays an important role in the occurrence, development and metastasis of solid tumors, and is also an important indicator to judge the prognosis of solid tumors[16,17].The content of bFGF in the serum of CLL patients was significantly increased and was associated with a poor prognosis[18].BFGF is also highly expressed in the serum of AL patients, which is reduced after treatment[19].Because of the possible pathogenic effect of bFGF in AML, drug development for bFGF is very promising for AML patients[20].In this study, we found that the expression of bFGF in PBMCs and serum of AML patients was significantly higher than that of normal controls, suggesting that bFGF is also closely related to the pathogenesis of AML.

VEGF is a multifunctional cytokine specifically acting on vascular endothelial cells, which can promote the angiogenesis of vascular endothelial cells in vivo and in vitro[21].VEGF and its receptor are highly expressed in the bone marrow and peripheral blood leukemia cells of patients with AML, CML, ALL (acute lymphoblastic leukemia) and CLL (chronic lymphocytic leukemia).They can induce the proliferation of leukemia cells and inhibit their apoptosis through autocrine and paracrine[22-24].In this study, we found that the expression of VEGF in PBMCs and serum of AML patients was significantly higher than that of normal controls, suggesting that VEGF is also closely related to the pathogenesis of AML.

TGFβ1 is one of the strong blood cell inhibitors discovered at present.TGF β 1 is highly expressed in the serum of AML patients[25].In addition, TGFβ1 can regulate the growth of AML,B-ALL and other cells[26,27].TGFβ1 in the serum of CML patients which level decreased significantly and returned to normal after treatment[28].Megakaryocytes produce excessive TGFβ1 and Inhibit the proliferation of AML normal hematopoietic stem cells[29].TGFβ1 in serum of untreated AML patients.It is significantly expressed,and its expression level is related to the prognosis of patients[30].In this study, we found that The expression of TGFβ1 in PBMCs and serum of AML patients was significantly higher than that of normal controls, It is closely related to the pathogenesis of AML.

Research shows that TGFβ1 promotes the migration and invasion of breast cancer cells[31], promotes the proliferation and migration of endothelial cells and smooth muscle cells by activating JAK / STAT pathway[4], and mediates the inhibition of apoptosis of liver cancer cells by sulf2.In this study, we found that TGFβ1 was overexpressed and it can promote the proliferation of AML cells and inhibit their apoptosis.The proportion of cells in the G2 phase is significantly increased while silencing the expression of TGFβ1 has the opposite effect, which is consistent with the previous research conclusion of A on cell proliferation and migration.

TGFβ1 in serum of CML patients level was negatively correlated with COX-2 and bFGF levels, suggesting TGFβ1 is a negative hematopoietic regulator, it inhibits the expression of COX-2 and bFGF [32].TGFβ1 in serum of AL patients was negatively correlated with COX-2, bFGF and VEGF levels [5,6].Therefore, TGFβ1 is monitored dynamically.The changes of COX-2, bFGF and VEGF expression levels can be used as a reference index to judge the condition, prognosis and survival of AL and CML.

However, due to the small sample size of this study, the research is not detailed enough, and the conclusions drawn should be handled with caution, which needs further research from large samples and multiple centers to confirm.In addition, we only explored overexpression and silence of TGFβ1 in this study.The specific effects of regulating the expression of COX2, bFGF and VEGF on the proliferation, apoptosis and cycle of AML cells were not fully explored, which should be further discussed in detail.In addition,TGFβ1 should be further explored.The relationship between the expression of COX2, bFGF and VEGF and the prognosis of AML patients to further confirm TGFβ1.COX2, bFGF and VEGF play an important role in the progression of AML disease, and also provide a new potential target for the diagnosis and treatment of AML.

To sum up, TGFβ1.COX-2, bFGF and VEGF were significantly overexpressed in PBMCs and serum of AML patients, and TGFβ1 regulates the proliferation, apoptosis and cycle of AML cells.Our research aims to reveal TGFβ1.The role of COX-2, bFGF and VEGF in the development of AML provides new evidence.

Author's Contribution:

Chen Wen-ting: Financial support and article writing; Huang Ying:Clinical sample collection; Pan Yan-ping: Clinical sample testing;Wang Shu-wen: Cell experiments and assays; Tang Rui-mei: Cell experiments and assays.

All authors declare no conflict of interest.