Down-Regulation of SREBP via PI3K/AKT/mTOR Pathway Inhibits the Proliferation and Invasion of Non-Small-Cell Lung Cancer Cells
Received 10 June 2020
Accepted for publication 18 August 2020
Published 8 September 2020 Volume 2020:13 Pages 8951—8961
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Carlos E Vigil
Bo Zhang,1,* Jianchun Wu,1,* Peng Guo,1 Yi Wang,2 Zhihong Fang,1 Jianhui Tian,3 Yongchun Yu,4 Wenjing Teng,1 Yingbin Luo,1 Yan Li1
1Department of Oncology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, People’s Republic of China; 2Department of Pathology, Caner Hospital Affiliated Zhengzhou University, Henan, Zhengzhou 450008, People’s Republic of China; 3Department of Oncology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, People’s Republic of China; 4Institute for Thoracic Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, People’s Republic of China
*These authors contributed equally to this work
Correspondence: Yan Li; Yingbin Luo
Department of Oncology, Shanghai Municipal Hospital of Traditional Chinese Medicine, 274 Middle Zhijiang Rode, Shanghai, People’s Republic of China
Email [email protected]; [email protected]
Background: Lung cancer is one of the most common causes of cancer-related deaths worldwide, metabolic disorders are also a problem that puzzles mankind. SREBP is overexpressed in non-small-cell lung cancer (NSCLC) and is also a key regulator of lipid synthesis. However, the mechanisms by which SREBP regulates the proliferation, migration and invasion in NSCLC remain unclear.
Materials and Methods: CCK-8, colony formation assay, soft agar assay, scratch wound healing assay and transwell assays were performed to detect proliferation, and invasion in NSCLC cells, respectively. In addition, Western blotting assay, qPCR and immunofluorescence were applied to detect the expressions of SREBP1, SREBP2, ki-67, PCNA, Bax, bcl-2, E-cadherin, N-cadherin, Vimentin, PI3K, p-PI3k, AKT, p-AKT, mTOR, p-mTOR in NSCLC cells.
Results: In this study, downregulation of SREBP significantly inhibited the proliferation, migration and invasion of A549 and H1299 cells. Moreover, the method of piecewise inhibition was adopted to prove that SREBP is a downstream molecule of the PI3K/Akt/mTOR signaling pathway.
Conclusion: Our study indicated that downregulation of SREBP inhibited the growth in NSCLC cells via PI3K/AKT/mTOR signaling pathway. Thus, we suggested SREBP may serve as a potential target for the treatment of patients with NSCLC.
Keywords: non-small-cell lung cancer, SREBP, proliferation, invasion, PI3K/AKT/mTOR
Metabolic reprogramming is one of the important features of tumor cells.1 In order to satisfy the material and energy needed for rapid proliferation, tumor cells reprogram their metabolic patterns to promote tumor growth. As one of the three major nutrients in human body, lipids can supply and store energy, which is an important substance in cell life activities and closely related to cell proliferation. As one of the most representative features of tumor disease, abnormal lipid metabolism has become an important research direction in the treatment of tumor in recent years.2 Sterol Regulatory Element-binding Proteins (SREBP) are a key regulator of lipid synthesis,3 the research and development of new drugs targeting SREBP has attracted much attention.
SREBP is a transcription factor that regulates the synthesis of fatty acids, triglycerides and cholesterol. In mammals, SREBP is divided into three subtypes, named SREBP1a, SREBP1c and SREBP2. Although SREBP1a and SREBP1c are produced by different promoter codes, their coding genes are the same, and they are collectively called SREBP1, which regulates the metabolism of fatty acids and triglycerides, while SREBP2 regulates the metabolism of cholesterol.4 Previous studies have focused on its regulatory role in metabolism. Recent studies have found that in addition to its role in regulating metabolism, SREBP also plays a certain role in the occurrence and development of tumors, especially in the proliferation, invasion and migration of tumor cells. Experimental studies have also verified this view. SREBP is highly expressed in prostate cancer.5 In breast cancer, the expression of SREBP1 is related to the metastasis of tumor, and the activation of SREBP can promote the proliferation of breast cancer cells.6 Inhibition of SREBP can promote the apoptosis of endometrial cancer cells.7
The incidence rate and mortality rate of lung cancer are the first in the world,8 metabolic disorders are also a problem that puzzles mankind. We made a reasonable guess as to whether the inhibition of SREBP gene, which regulates metabolism, can inhibit the proliferation, invasion and migration of lung cancer cells and other malignant behaviors.
To test this hypothesis, we knocked down SREBP1 and SREBP2 genes of lung cancer cells A549 and H1299 by lentivirus infection, and then observed the proliferation, apoptosis, invasion and migration of lung cancer cells. Our aim was to determine whether SREBP plays a role in promoting the development of lung cancer.
Materials and Methods
Cell Culture and Tissue Source
The human NSCLC cell lines A549 and H1299 were from the Shanghai cell bank of the Chinese Academy of Sciences (Shanghai, China). DMEM high-sugar medium containing 10% FBS ((Thermo Fisher Scientific, Waltham, MA, USA)) and 1% penicillin streptomycin mixture was used for culture. The conditions of CO2 incubator were 37 °C, 5% CO2 and 95% air. Experiments were performed when the cells were in the logarithmic growth phase. At the Cancer Hospital affiliated Zhengzhou University, 4 fresh cases of human non-small cell lung cancer were obtained and paired with normal tissue. All samples were collected with the patient’s informed consent.
The cells were digested and resuspended and diluted to a certain concentration to ensure that the cell density was not less than 104 cells/mL. Draw 10 µl cell suspension and add the cell suspension along the side of the cover slide. Under a microscope, the number of cells in four large squares at the edge of the cell count plate is calculated. A cluster of cells is counted as a cell. After counting, the number of cells per mL of suspension was calculated. Cell suspension number/mL = (total number of 4 large cells n/4)×104× dilution factor; Take the average after three repetitions.
Lentivirus and Plasmid Transfection
Short hairpin RNA (shRNAs) targeting human LINRIS or non-specific oligonucleotides attached to LV-3 vectors can down-regulate the expression of SREBP1 and SREBP2. The A549 and H1299 cells were transfected with lentivirus according to the manufacturer’s instructions. Stable transfected cell lines were obtained after treating with puromycin for 2 weeks (2–3 g/mL). After the knockdown efficiency was verified by quantitative PCR (qPCR) and Western blotting, the cells were used for subsequent experiments. shRNA targeting sequences for SREBP1 are as the following: 5′-GCCATCGACTACATTCGCTTT-3′ (a) and 5′-CCAGAAACTCAAGCAGGAGAA-3′ (b); for SREBP2: 5′-CCTCAGATCATCAAGACAGAT-3′ (a) and 5′-GACCTGAAGATCGAGGACTT3′ (b).
After the cell suspension and cells counted, they were inoculated into a 96-well plate. Each well contained 200μL media supplemented with 10% FBS and 1×104 cells. Each group was given 3 double holes and another blank hole with media supplemented with 10% FBS. The 96-well plate was placed in an incubator at 37°C for 24h. Replace the media and add 10μL CCK8 reagent (CCK-8, Dojindo, Tokyo, Japan) to 100μL media. After 2 hours of continuous culture, absorbance at 450nm was determined with a micrometer (Thermo Scientific, USA). Cell activity inhibition rate (%) = [(average OD value of the control group – average OD value of the experimental group)/(average OD value of the control group – average OD value of the blank group)]×100%.
RNA Purification and q-PCR Assay
Total RNA was extracted from A549 and H1299 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc). The first strand cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis kit (Takara Bio, Inc.) following the manufacturer’s protocol. PCRs were performed using the rTaq DNA Polymerase (Takara Bio, Inc.) in a total volume of 20 µL, and amplification protocol consisted of an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation for 45 sec at 94°C, annealing for 45 sec at 60°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 10 min.
Western Blot Assay
The cells were homogenized in a cool tissue lysis buffer, which was centrifuged at 4°C at 12,000 r/min for 15 minutes. The supernatant reflects the extraction of total cellular proteins, the protein concentrations of which were determined using BCA protein assay (Beyotime, China). The samples were boiled in Laemmli buffer at 100°C for 8 minutes. The samples were examined by Western blot. The primary antibody was incubated overnight at 4°C. Antibodies were diluted to 1:1000 for SREBP1 (Thermo Fisher Scientific, USA), SREBP2 (Thermo), Ki-67 (Cell Signalling Technology, USA), p53 (CST), PCNA (CST), bax (CST), bcl-2 (SCST), E-cadherin (CST), N-cadherin (CST), Vimentin (CST), PI3K (CST), p-PI3K (CST), AKT (CST), p-AKT (CST), mTOR (CST), p-mTOR (CST), GAPDH (CST), β-actin (CST). After washing, use a secondary antibody; Leave at room temperature for 1 hour. The ECL color rendering solution was prepared, mixed and dropped onto the film surface, exposed and photographed.
The A549 and H1299 cells were 4%PFA fixed (15min) and then incubated in 1%BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with antibody overnight at 4°C. The secondary antibody (red) was Alexa Fluor 555 goat anti-rabbit lgG used at a 1/1000 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43Um.
Colony Formation Assay
After the cells were digested and resuspended, the cells were counted. According to the density of 500 cells per well, the cells were inoculated into 6-well plates, and 3 double holes were set up in each group. Replenish the medium to 2mL per well; The six-well plate was cultured in an incubator of 5%CO2 and 37°C for 7 days. When visible clones appeared in petri dishes, the culture medium was discarded and washed twice with PBS. Fixed with 4% paraformaldehyde for 15min and then dried; Dye with 0.1% crystal violet for 15min and rinse with tap water for 3 times. Take photos after air drying. Clone formation rate = number of clones/number of inoculated cells ×100%.
Soft Agar Assay
For soft agar assay, 5×103 cells were fully mixed with DMEM with 10% agarose and 0.3% agarose, and they were quickly placed on the DMEM curing layer with 0.75% agarose and 10% agarose. DMEM was preheated at 37°C in advance. 0.5mL DMEM containing 10% FBS was added weekly to each well to fed Cells. After 3 weeks, colonies were scored in 5 random field which were larger than 50 μm.
Scratch Wound Healing Assay
In the scratch wound healing assay, cells were plated in a 6-well plate. When the cells were fused to about 90%, a 200μL pipette tip was used to scrape the monolayers of cells in a straight line to create a “scratch”. After the exfoliated cells and cell fragments were washed, fresh medium containing 1% fetal bovine serum was added and the cells were photographed with a microscope at a predetermined time. Use ImageJ software to evaluate migration distances. The relative mobility of each treatment was calculated by cell relative migration area.
Transwell Migration Assay
The Transwell migration assay was carried out by 8μm Transwell Chambers (Corning Costar). The upper chambers were filled with 5×103 cells in 200μL media supplemented without FBS. Five hundred microliter DMEM supplemented with 10%FBS were plated in the lower chambers. After 24 hours of culture at 37°C, residual cells on the upper surface of the mem membrane were removed. The cells on the submembrane surface were fixed with 4% paraformaldehyde, and stained with crystal violet, and then take pictures under a microscope.
Male nude mice aged 6 weeks were injected subcutaneously with 105 A549, Sh-Control, Sh-SREBP1, Sh-SREBP2 cells. Tumor growth was followed for 28 days. Tumor volume was determined using the ellipsoidal volume formula. The expression of related proteins was detected by immunohistochemistry (IHC).
Data were presented as Mean±SEM and analyzed using GraphPad Prism 8.0. Each experiment was independently repeated three times. P < 0.05 was considered statistically significant.
SREBP1 and SREBP2 are Highly Expressed in Lung Cancer Cells and Tissues
In order to clarify the expression of SREBP protein in lung cancer cells, A549 and H1299 cell lines were selected, and human bronchial epithelial cells (BEAS-2B) were used as the control. Western Blot assay was used to detect the expression levels of SREBP1 and SREBP2 proteins. Compared with HBE, SREBP1 and SREBP2 were both highly expressed (Figure 1A). Then, we detected the expression of SREBP1 and SREBP2 proteins in the matched para-carcinoma tissues of 4 lung cancer specimens, and the results showed that SREBP was highly expressed in cancer tissues. (Figure 1B).
Lentivirus Infection Resulted in the Stable Transformation of Lung Cancer Cells with Reduced SREBP1 and SREBP2
Using lentivirus infection technique, gene knockout, screening and expanding culture were carried out for lung cancer cell lines A549 and H1299, respectively. Western Blot, qPCR and Immunofluorescence (Figure 2A–C) were used to verify the success of gene knockout, and the next experiment could be carried out after successful knockout. In this experiment, the cell infection rate was more than 90%.
Down-Regulation of SREBP Gene Inhibits the Proliferation of Lung Cancer Cells and Promotes Apoptosis
In the lung cancer cell lines knocked down by SREBP, the expressions of proliferation-related proteins ki-67 and PCNA were reduced (Figure 3A), and the expressions of apoptosis-related proteins P53 and Bax/bcl-2 were increased. The immunofluorescence results were consistent with Western Blot and qPCR (Figure 3B). In terms of functional experiment, CCK8 detected the proliferation ability of cells, and the results showed that the proliferation ability of gene knockout cells was significantly decreased (Figure 3C). The results of the cloning experiments showed that the number and size of the cloned cells were lower than that of the normal lung cancer cell lines (Figure 3D–E). The above results showed that SREBP gene could affect the proliferation ability of lung cancer cells A549 and H1299, and inhibit this gene, which could inhibit the proliferation of lung cancer cells.
Down-Regulation of SREBP Gene Can Inhibit the Migration and Invasion of Lung Cancer Cells
In the lung cancer cell lines knocked down by SREBP, the expression of epithelial marker protein E-cadherin was increased, and the expression of mesenchymal marker proteins N-cadherin and Vimentin were decreased (Figure 4A). This result was verified by immunofluorescence results (Figure 4B). Epithelial-mesenchymal transition (EMT) enables tumors to acquire the ability to move and migrate, and invasion and metastasis, which is a key step for the migration and invasion of tumor cells.9 Therefore, we used scratch test and transwell test to explore the influence of SREBP gene on the migration and invasion of lung cancer cells. Twenty-four hours after the scratch culture, the normal lung cancer cells healed nearly 50%, while the knockdown cells healed less than 20%, which was significantly inhibited (Figure 4C). In the transwell transfer experiment, the number of cells of A549 and H1299 cells crossing the compartment PET membrane was significantly reduced. Matrigel was added to the upper compartment to verify the invasion ability of cells, and the same result was obtained (Figure 4D).
SREBP Can Be Considered as a Downstream Factor of PI3K/AKT/mTOR
The PI3K/AKT/mTOR signaling pathway is overactivated in a variety of cancers, plays an important role in cell growth and survival, and can maintain the malignant behavior of tumor cells.10 In order to investigate whether SREBP gene is a downstream factor of this pathway, we adopted the method of block inhibition, respectively using PI3K inhibitor PDK-1 (Thermo), AKT inhibitor MK-2206 (Thermo) and mTOR inhibitor Rapamycin (Thermo) to inhibit this pathway, and obtained the IC50 concentration of each inhibitor by CCK8 assay. The inhibitor was added and cultured for 24 hours, and the protein was extracted. The levels of PI3K, p-PI3k, AKT, p-AKT, mTOR, p-mTOR and SREBP1 and SREBP2 were detected by Western Blot. The results showed that with the decrease of the phosphorylation level of the pathway, the expression of SREBP1 and SREBP2 also decreased (Figure 5).
In vivo Experiments of Sh-SREBP A549 Cells
Normal cells, Sh-Control cells, Sh-SEREBP1 and Sh-SREBP2 A549 cells were subcutaneously injected into BALB/C nude mice (6 weeks) and tumor growth was monitored within 28 days. We found that Sh-SREBP1 and Sh-SREBP2 tumor growth rate and average tumor weight (Figure 6A–C). Immunohistochemistry was performed on subcutaneous transplanted tumor of mice, and the expressions of ki-67, PCNA, E-cadherin, N-cadherin were detected. The results were consistent with in vitro (Figure 6D).
Sterol Regulatory Element Binding Protein (SREBP) has the structure of basic helix- loop- helix-leucine zipper (Bhlh-ZIP), which plays an important role in the regulation of cholesterol metabolism and fatty acid metabolism in mammals.11 New synthesis precursor SREBP is inactive, which attached to the endoplasmic reticulum membrane, when cells with lipid needs, endoplasmic reticulum of SREBP precursor proteins are transferred to the golgi apparatus, through two pyrolysis activation and release properties of N (nSREBPs), and then be transferred to the nucleus, together with the corresponding target gene promoter, promote its downstream proteins ACC, ACLY, FASN, SCD1, LDLR, this enzyme gene transcription, which regulates cholesterol and fatty acid synthesis.12 SREBP1 mature body is mainly involved in fatty acid and triglyceride metabolism, while SREBP2 mature body is the main regulator of cholesterol metabolism13.
Under normal physiological conditions, lipid dynamic equilibrium is strictly maintained. However, under certain pathological conditions, the expression level of SREBP is often maladjusted. Lipid metabolism plays an important role in regulating cell growth, proliferation, differentiation, survival, apoptosis, and membrane homeostasis.14,15 Studies have shown that abnormal lipid metabolism is involved in the occurrence and development of various tumor diseases, such as prostate cancer, breast cancer, and colorectal cancer.16 Studies have also found that SREBP is overexpressed in many cancers, and it can regulate the expression of genes needed to maintain the lipid homeostasis of cells,14 which has a great effect on the proliferation and metastasis of tumors. For example, SREBP1 and its downstream target genes ACC and FASN were highly expressed in human glioblastoma tissues.17 The expression level of SREBP1 is related to tumor size, histological grade and invasion, and metastasis.18 The functionally deficient SREBP1 inhibits cell proliferation, invasion and metastasis and induces apoptosis, which has been found in liver cancer, ovarian cancer and endometrial cancer.19,20 Inhibition of SREBP1 expression at the gene level or through drugs will cause the death of tumor cells and significantly inhibit the proliferation of tumor cells.21–23 This study found that SREBP was also overexpressed in lung cancer cells and tissues, and affected the proliferation, migration and invasion of lung cancer cells. Therefore, using SREBP as a tumor treatment target and drug target has a little research potential and application prospect. For example, in the process of ripening, folding and post-transcriptional modification of SREBP, protein phosphorylation, acetylation, saponization and ubiquitination are modified to regulate the expression and stability of SREBP,24 so as to prevent and treat diseases.
Ki-67 is a nuclear antigen expressed in proliferating cells and is one of the most reliable indicators to detect the proliferating activity of tumor cells.25 PCNA exists in various stages of the proliferation cycle of tumor cells and normal proliferating cells, and is closely related to the synthesis of cell DNA and it is also involved in the initiation process of cell proliferation and can reflect the state of cell proliferation.26 Members of the bcl-2 family include Bax, which promotes apoptosis, and bcl-2, which fights apoptosis.27 The ratio of Bax to bcl-2 determines the state of apoptosis.28 When the balance of Bax/bcl-2 in vivo is destroyed, its inhibitory effect on tumor cell proliferation is weakened.29 P53 is a tumor suppressor gene and has an inhibitory effect on tumor growth.30 In this study, the expression of proliferation-related genes ki-67 and PCNA decreased and the expression of apoptosis-related genes P53 and Bax/bcl-2 increased in the lung cancer cell lines with SREBP knockdown, indicating that the proliferation of lung cancer cells was inhibited and the apoptosis of lung cancer cells was promoted. EMT refers to the enhancement of cell movement and invasion after phenotypic change, weakened adhesion ability and loss of polarity of epithelial cells, which is considered as the initial stage of tumor metastasis.31 EMT not only promotes tumor development and invasion and metastasis, but also enhances the anti-apoptotic ability of tumor cells.32 The occurrence of EMT is often accompanied by the change of cell morphology (fusing/fibroblast morphology), the decrease of epithelial marker proteins such as E-cadherin, and the increase of mesenchymal marker proteins such as N-cadherin and Vimentin.33 In this study, it was found that the expression of e-cadherin decreased and the expression of n-cadherin and vimentin increased in the lung cancer cell lines with the reduction of SREBP, indicating that the lung cancer cells had invasion and metastasis initiated by epithelial mesenchymal transformation.
In a variety of human tumor tissues, the PI3K/Akt phosphorylation pathway is highly activated,34 and the activation of Akt promotes the transcriptional activation of SREBP1, thus promoting the intracellular lipid synthesis of tumor cells.35 MTOR regulated in the downstream of PI3K/Akt pathway can also regulate the expression of SREBP1 gene level to a certain extent and promote the intracellular localization of SREBP1 protein maturation.36 Studies have shown that SREBPs is an important component in the downstream of the metabolic regulation network of PI3K/Akt/mTOR signaling pathway.37 MTOR is an important downstream effector of AKT. Although the activity of mTOR is regulated by special amino acids, it also requires the enrichment of mature karyotype SREBP-1 in the nucleus.38
mTOR pathway is the key regulator of cell proliferation, metabolism and survival, and there is abnormal regulation of mTOR signal in most cancers. Rapamycin is the inhibitor of mTOR pathway. Different doses of rapamycin affect the effect of action.39 AKT phosphorylates ACLY and activates the expression of several genes involved in the synthesis of cholesterol and fatty acids.40 mTOR also regulates the expression of sterol regulatory element binding protein gene 1 (SREBP1) and stimulates the production of lipids in the liver.41 PI3K/AKT signal can not only up-regulate glucose uptake and glycolysis, but also promote the synthesis of fatty acids.42 In this study, the method of piecewise inhibition was adopted to prove that SREBP is a downstream molecule of the PI3K/Akt/mTOR signaling pathway. Therefore, SREBP can be regarded as an important metabolic integrator in the process of regulating glucose production of fatty acids by PI3K/AKT/mTOR signaling.
Abnormal tumor metabolism is considered to be an important feature of tumors. In-depth study of the regulatory mechanism and biological functions of SREBP can help us have a deeper understanding of metabolic diseases and tumors, and at the same time will have a far-reaching impact on the guidance of clinical treatment. In the anticancer research targeted at lipid metabolism, it is worth further research to find and develop effective SREBP inhibitors, which can not only inhibit the synthesis of lipids at the source, but also inhibit the growth of tumor cells.
Regulation of SREBP through PI3K/AKT/mTOR pathway can affect the proliferation and invasion of lung cancer cells. Whether the changes in the expression of fatty acids or cholesterol downstream of SREBP affect the function of cells, or affect the expression of some genes, such as the bypass of advanced G1 glutamine (G1n),43 is the subject of further study.
In this study, we explored the mechanisms underlying the role of SREBP in NSCLC. Downregulation of SREBP inhibited cell proliferation, migration and invasion via PI3K/AKT/mTOR signaling pathway. These studies indicated an important role for SREBP as an oncogene in the NSCLC.
All human studies have been approved by the Research Ethical Committee from Municipal Hospital of Traditional Chinese Medicine Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China (2016SHL-KYYS-16). Informed consent was obtained from all study subjects.
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Municipal Hospital of Traditional Chinese Medicine Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China. The maximal tumor measurements/volumes are in accordance with the IACUC. All procedures involving animal treatment followed the guidelines of the National Standardization Administration of China institutional committee of animal care and use (GB/T1.2-2016).
This work is supported by grants to Yan Li from the National Natural Science Foundation of China (81973795, 81673947), the grant to Yingbin Luo from the Natural Science Foundation of Shanghai (17ZR1428000), the grant to Peng Guo from Innovation project of Shanghai University of Traditional Chinese Medicine (2019LK094), the grant to Yi Wang from Science and technology project of Henan province (192102310374), and the grant to Jianchun Wu from Outstanding youth project of Shanghai Municipal Health Committee (2017YQ049).
Yan Li, Bo Zhang, Yingbin Luo and Jianchun Wu designed the experiments and interpreted data. Yi Wang provided tumor tissue. Peng Guo, Zhihong Fang, Jianhui Tian, Yongchun Yu gave advice on experimental design and paper writing. Bo Zhang, Peng Guo and Wenjing Teng performed the experiments. Bo Zhang, Jianchun Wu analyzed data. Bo Zhang and Yan Li wrote the paper. All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
The authors report no conflicts of interest for this work.
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