Introduction

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer deaths worldwide, with an estimated 2.2 million new cases and 1.8 million deaths in 20201. Non-small cell lung cancer (NSCLC) is the most common histologic type of lung cancer and accounts for 80–85% of lung cancer cases. NSCLC tumorigenesis is a multistep process in which normal lung epithelial cells are transformed into cancer cells through a series of genetic or epigenetic events2. Multiple genetic alterations and signalling pathway dysregulation events associated with lung tumorigenesis, metastasis, and chemoresistance have been extensively reported in patients with lung cancer2. However, since most patients do not express “druggable” targets, the 5-year overall survival rate for patients with lung cancer is still low3. Therefore, a better understanding of the aetiological factors that drive NSCLC tumorigenesis is highly important for the development of new therapeutic strategies.

PKA also known as cAMP-dependent kinase, is a serine-threonine kinase that mediates cyclic AMP (cAMP) signal transduction4. cAMP-PKA signalling is involved in the control of a wide variety of cellular processes, from metabolism to ion channel activation, cell growth and differentiation, gene expression, and apoptosis5. PKA regulates the transcription of various target genes mainly through its downstream effector cAMP responsive element binding protein (CREB)6. Dysregulation of PKA signalling is linked to tumour growth, metastasis, and drug resistance in a variety of cancers7,8,9,10. Notably, PKA signalling may have tumour-suppressive or tumour-promoting effects depending on the tumour type and context5,6. Indeed, studies have shown that PKA signalling has paradoxical effects on lung cancer. In the highly malignant small cell lung cancer (SCLC) subtype, PKA acts as an active kinase, and inhibition of its activity suppresses SCLC expansion both in cultured cells and in vivo11,12. In NSCLC cells, cAMP signalling reduces ATM phosphorylation or SIRT6 expression in a PKA-dependent manner and augments radiation-induced apoptosis13,14. The exact reason that PKA plays an oncogenic role in endocrine SCLC but a tumour-suppressive role in NSCLC has not been fully elucidated. Moreover, the functions of PKA in different types of tumours and the precise regulatory mechanisms underlying these functions need to be further explored.

Phosphodiesterase 4D interacting protein (PDE4DIP) is a Golgi/centrosome-associated protein that is ubiquitously expressed in mammalian cells15,16. PDE4DIP was initially discovered as a cyclic nucleotide PDE4DIP; thus, it is considered a putative cAMP-PKA signalling regulator15,17. Indeed, in medulloblastoma cells, Mmg (a variant of PDE4DIP) loss has been shown to mislocalize PDE4D3 from the centrosome, leading to local PKA overactivation18. In mammalian cells, PDE4DIP has been found to play a pivotal role in maintaining endoplasmic reticulum-to-Golgi trafficking, Golgi/centrosome integrity, and microtubule formation15,19,20. Recent genetic studies have provided compelling evidence for an association between PDE4DIP variants and atrial fibrillation, stroke, and heart failure21,22,23, but little is known about the biological functions of PDE4DIP in cancers. PDE4DIP alterations are common in human cancers, and mutations, genomic loss/gain, or gene rearrangements of PDE4DIP have been identified in multiple tumours, including leukaemia, glioma, pineoblastoma, and prostate cancer21,24,25,26,27. A pancancer analysis revealed that the PDE4DIP gene has potential prognostic and immunotherapeutic value in multiple cancers28. Recently, we reported for the first time that PDE4DIP functions as a proto-oncogene in colorectal cancer growth and chemoresistance by regulating the RAS signalling axis in KRAS-mutant colorectal cancer29. In lung cancer, frameshift mutations in PDE4DIP have been detected in the peripheral blood of patients with familial squamous cell lung cancer, and recurrent mutations in PDE4DIP have been found to be associated with brain metastasis of NSCLC30,31. These findings imply that PDE4DIP may be associated with lung cancer progression. However, the biological function of PDE4DIP in lung cancer has yet to be explored.

In the present study, we investigated the biological functions of PDE4DIP in human lung cancer. We found that knockdown of PDE4DIP inhibited NSCLC cell proliferation in vitro and tumorigenicity in vivo. We further demonstrated that knockdown of PDE4DIP promoted apoptosis and cell cycle arrest in NSCLC cells by activating the PKA/CREB signalling pathway. PDE4DIP coordinated with AKAP9 to modulate the Golgi localization and stability of PKA RIIα. Knockdown of PDE4DIP mislocalized PKA RIIα from the Golgi apparatus, leading to its degradation and activation of PKA signalling. Overall, our findings provide novel insights into the roles of PDE4DIP in regulating PKA signalling and lung cancer growth.

Materials and methods

Tissue samples and cell culture

Lung cancer and adjacent tissues were obtained from the First Affiliated Hospital of Wenzhou Medical University with appropriate Institutional Review Board approval and informed consent from the patients. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. All samples were collected with appropriate informed consent from the patients. All ethical regulations relevant to human research participants were followed. H1299 and A549 cell lines were originally obtained from the American Type Culture Collection (USA) and had recently been authenticated using short tandem repeat DNA profiling, and all cell lines tested negative for mycoplasma contamination before use in experiments. H1299 and A549 cells were cultured in RPMI-1640 medium (Gibco, Waltham, MA, USA) and F-12/Ham’s medium (Sigma–Aldrich, St. Louis, MO, USA), respectively, supplemented with 10% foetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified 5% CO2 atmosphere at 37 °C.

Reagents, plasmids and antibodies

8-CPT-cAMP and H89-HCl were purchased from Selleck Chemicals (Houston, Texas, USA). The pCMV6-Entry-PDE4DIP plasmid was purchased from Origene Technologies, Inc. (Rockville, Maryland, USA). Antibodies against human Bim, Cytochrome c (Cyto c), Caspase 9, Caspase 3, P27KIP1, CREB, phospho-CREB (Ser133), phospho-ERK, AKT, phospho-AKT, RB1, phospho-RB1, CDK4, GM130, β-actin, Myc-Tag and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-PDE4DIP antibody was purchased from Sigma‒Aldrich (St. Louis, MO, USA). Antibodies against ERK, PKA RIIα, Bcl-2, Bax, Cyclin D1 and PDE4D were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). The anti-AKAP9 antibody was purchased from Abcam (ab237752, Cambridge, UK).

cAMP activity assay

For the quantification of cAMP in cellular extracts, a cAMP Direct Immunoassay Kit (Cat ab65355; Abcam, Cambridge, MA, USA) was utilized, and the assay was performed according to the manufacturer’s protocol. In brief, cells were harvested and dissociated. One hundred microliters samples were added into microcentrifuge tube and neutralized using neutralizing buffer. After being acetylated, the samples were incubated with anti-cAMP antibody for 1 h. Then, cAMP-HRP and HRP developer were successively added into the samples. The absorbance was measured at a weight length of 450 nm with Microplate Reader (BioRad Model 550).

Quantitative reverse transcription–PCR (qRT‒PCR) analysis

Cells were lysed, total RNA was purified using TRIzol Reagent (Invitrogen, 15596018), and reverse transcription was performed using an M-MLV reverse transcriptase kit (Invitrogen, 28025-013). qRT‒PCR was carried out with SYBR Green (Tiangen, China, FP202-02) in biological triplicate in an ABI 7500 Real-Time detection system (Applied Biosystems) according to the manufacturer’s protocol. The primer sequences for amplification of Bim were F: 5′-CAC TAC CAC CAC TTG ATT CTT G-3′ and R: 5′-GGT CAC ACT CAG AAC TTA CAT C-3′; for RB1, F: 5′-ATC ACA GCG ATA CAA ACT TGG AG-3′ and R: 5′-AGC GCA CGC CAA TAA AGA CA-3′; and for GAPDH, F: 5′- ACG GAT TTG GTC GTA TTG GGC-3’ and R: 5’-CTC GCT CCT GGA AGA TGG TGA T-3′. Relative quantification of mRNA expression was performed using the comparative threshold cycle (Ct) method with normalization to GAPDH.

Lentivirus infection and small interfering RNA (siRNA) transfection

Lentiviruses containing short hairpin RNA (shRNA) against PDE4DIP (shP1 or shP2) and negative control shRNA (shNC) were designed and produced by GeneChem Co. (Shanghai, China). The sequences were as follows: shP1, 5′-AAC CTC CAG TGG CTG AAA GAA-3′; shP2, 5′-AAG CAG AGA GAC AGC TCT ATA-3′; and shNC, 5-TTC TCC GAA CGT GTC ACG T-3′. siRNAs targeting Bim and AKAP9 and a nontargeting negative control siRNA (siNC) were synthesized by GenePharma (Shanghai, China). The sequences were as follows: siBim-1, 5′-AUG GUU AUC UUA CGA CUG UUA-3′; siBim-2, 5′-AGC CGA AGA CCA CCC ACG AAU-3′; siAKAP9-1, 5′-AAC UUU GAA GUU AAC UAU CAA-3′; siAKAP9-2, GCA CAA UAA UUA UUG AAU UTT-3′; and siNC, 5′-UUC UCC GAA CGU GUC ACG UTT-3. Cells were grown to a confluence of 50–70%, infected with lentivirus or transfected with 50 nM siRNA using LipofectamineTM RNAiMAX (Invitrogen, CA, USA) according to the manufacturer’s instructions and replated for further experiments. The knockdown efficiency was determined using qRT‒PCR or Western blotting.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, real-time cell analysis (RTCA) and colony formation assay

The MTT assay was performed to determine cell viability as described previously29. In brief, 72 h after transfection with lentivirus, 3 × 103 cells were plated into 96-well plates in triplicate. At the indication time points, 20 µl MTT solution (Sigma–Aldrich, St. Louis, MO, USA) was added into the cells and incubated for 4–6 h. The absorbance was measured at a weight length of 570 nm with Microplate Reader (BioRad Model 550). Cell proliferation was monitored using a RTCA system (ACEA-Biosciences, San Diego, CA, USA). NSCLC cells were seeded in an 8-well E-plate at a density of 4000 cells per well. The cell index was monitored in real time every 15 min for 100 h, and the recorded curve shows the cell index ± standard deviation (SD) values. For the colony formation assay, 1 × 103 cells were seeded in six-well plates in triplicate and cultured in medium for 2 weeks. At the end of the experimental period, the cells were stained with 0.5% crystal violet in 20% methanol, and colonies ( ≥50 cells) were counted.

Cellular apoptosis analysis

An Annexin V-APC/7-AAD Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) was used to analyse cellular apoptosis according to the manufacturer’s instructions. In brief, 96 h after transduction with shRNAs, 1 × 105 cells were harvested and washed twice with cold PBS. Next, the cells were resuspended in 100 ml of 1× binding buffer and incubated with 5 µl of Annexin-APC and 5 µl of 7-aminoactinomycin D (7-AAD) for 15 min at room temperature (RT) in the dark. Cellular apoptosis was measured by flow cytometry (BD FACSCalibur, San Jose, CA, USA) and the results were analysed with FlowJo software (version 10.8.1). The experiment was performed in triplicate.

Cell cycle analysis

Ninety-six hours after transduction with shRNAs, cells were harvested and fixed with 70% cold ethanol overnight at −20 °C. The cell pellets were washed twice with PBS, suspended in 0.5 ml of PI/RNase Staining Buffer and subsequently incubated for 15 min at RT in the dark. The cell cycle distribution was analysed by a BD Accuri C6 flow cytometer (San Jose, CA, USA) and data were analysed with FlowJo software (version 10.8.1). The experiment was performed in triplicate.

Western blotting

Cells were washed and lysed in protein lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP40) supplemented with protease/phosphatase inhibitors (Cell Signaling Technology, Beverly, MA, USA). After vigorous shaking on ice, the supernatant was collected by centrifugation. Proteins were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS–PAGE) and electrotransferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, CA, USA). The membranes were probed with specific antibodies overnight at 4 °C and were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. Protein expression was measured using an Immun-Star HRP Chemiluminescence Kit (Bio-Rad Laboratories, CA, USA). The antibodies used in this study are summarised in Table S1.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

The TUNEL assay was performed using a DeadEndTM Colorimetric Apoptosis Detection System Kit (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. In brief, tumour tissues were fixed in 10% formalin and embedded in paraffin. After being dewaxed and rehydrated, the sections were incubated with Protein K solution for 15 min at RT. Then, the sections were blocked with 0.3% hydrogen peroxide in PBS for 5 min and incubated with streptavidin HRP solution for 30 min at RT. The sections were incubated with 3,3′-diaminobenzidine (DAB) solution for approximately 10 min until light-brown background staining was visible. The sections were then subjected to haematoxylin counterstaining. For analysis of lung cancer cells, cells were seeded on coverslips and fixed with 4% paraformaldehyde solution for 25 min at RT. The cells were washed and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Next, the cells were treated with 0.3% hydrogen peroxide in PBS for 5 min and incubated with streptavidin HRP solution for 30 min at RT. DAB solution was used to visualize chromatin in the apoptotic cells. The coverslips were mounted with 70% glycerol and sealed with nail polish. Images were acquired using a Zeiss AX10 microscope (Jena, Germany).

Immunohistochemistry (IHC)

IHC was performed as described previously29. In brief, tumour tissues were into 5 μm thick sections and deparaffinized with xylene and rehydrated by graded ethanol. Sections were immersed in citrate buffer for heat-induced antigen retrieval at 100 °C for 5 min. The sections were blocked with 5% BSA at 37 °C for 30 min before incubating with a primary antibody against Ki67 overnight at 4 °C. Slides were washed in PBS and immunoreactions were detected by the Elivision super HRP (Mouse/Rabbit) IHC Kit (Fuzhou MXB Biotechnology Development Co, China). DAB was used as chromogen and counterstained with Mayer’s Hematoxylin. (Abcam, Cambridge, UK). Brown signals in the nuclei indicated positive Ki67 immunostaining. Images were acquired using a Zeiss AX10 microscope (Jena, Germany).

Xenograft model

Five-week-old male BALB/c nude mice were purchased from Vital River Experimental Animal Center (Beijing, China), randomly divided into six groups (8 in each group) and housed under pathogen-free conditions. All in vivo experiments and protocols were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University (No. wydw2019-0842). We have complied with all relevant ethical regulations for animal use. H1299 cells or A549 cells (1 × 107 cells) infected with lentivirus containing shNC, shP1 or shP2 were injected subcutaneously into the dorsal flanks of mice. Tumours were measured every 3 days with a digital calliper, and tumour volumes (mm3) were calculated with the formula V = A × B2 × 0.5326 (A = long axis and B= short axis). The Institutional Animal Care and Use Committee stipulated that the tumours volume can’t exceed 2000 mm3, and in none of the experiments were these limits exceeded. At the end of the study, all the mice were sacrificed, and the tumours were resected and weighed.

Immunofluorescence staining

Cells were washed and fixed with paraformaldehyde for 20 min. Next, the cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and blocked with 2% bovine serum albumin in PBS containing 0.1% Triton X-100. Subsequently, the cells were incubated with primary antibodies (anti-PKA RIIα antibody: 1:100 dilution; anti-Myc-Tag antibody: 1:100; anti-GM130: 1:100 dilution; anti-PDE4DIP antibody: 1:50; anti-PDE4D antibody: 1:50) overnight at 4 °C and then with the corresponding fluorescently labelled secondary antibodies. After nuclei were stained with DAPI (Beyotime Biotechnology, Shanghai, China), the coverslips were mounted on slides and imaged with a Zeiss confocal microscope (Jena, Germany).

Immunoprecipitation

Cells were transfected with 20 µg of PDE4DIP plasmid using Lipofectamine 2000 (Invitrogen) according to the standard protocol. Twenty-four hours after transfection, the cells were lysed in buffer containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40 and protease/phosphatase inhibitors (Cell Signaling Technology, Beverly, MA, USA). The lysates were incubated with an anti-Myc-Tag, anti-PKA IIα or anti-AKAP9 antibody overnight at 4 °C and were subsequently incubated with protein G-Sepharose beads (GE Healthcare, Sweden). The beads were washed 4 times with immunoprecipitation assay buffer and suspended in Laemmli buffer. The samples were analysed by Western blotting.

Ubiquitination assay

The ubiquitination assay was performed as described previously29. In brief, cells were transfected with the HA-Ubiquitin plasmid. Twenty-four hours after transfection, the cells were treated with the proteasome inhibitor MG132 (25 mM) (Sigma, M7449) for 4 h and were subsequently lysed in ubiquitination assay buffer containing protease/phosphatase inhibitors. The cell lysates were clarified and were then incubated with an anti-PKA IIα or anti-AKAP9 antibody overnight at 4 °C. The resulting immunocomplexes were incubated with Protein G-Sepharose (GE Healthcare) for another 3 h at 4 °C, washed four times with wash buffer, and boiled for 5 min in Laemmli buffer before separation by SDS‒PAGE. Western blotting was performed with an anti-HA antibody to detect ubiquitinated PKA IIα or AKAP9.

RNA sequencing (RNA-seq) and data analysis

For RNA-seq, total RNA samples extracted from shPDE4DIP and shNC groups of A549 cells using TRIzol reagent (Invitrogen, 15596018). All the samples were sent to BGI Corporation (Shenzhen, China) for RNA-seq analysis. The RNA-seq was implemented by the DNBSEQ platform (BGI, Shenzhen, China). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by the Dr. Tom network platform of BGI. The RNA-seq data are available under NCBI Bioproject PRJNA1146770 and BioSample SAMN43202506 (A549shNC) and SAMN43202507 (A549shP1).

Statistical and reproducibility

Unless stated otherwise, the data are expressed as the mean ± SD values. Differences between two groups were analysed using Student’s t test. P < 0.05 was considered to indicate statistical significance. Correlations between the PDE4DIP level and clinicopathological features were analysed using the chi-square test. Kaplan–Meier survival analyses were performed using Kaplan–Meier Plotter database (www.kmplot.com) and R2 Genomics Analysis (http://r2.amc.nl). All in vitro experiments were performed with three independent biological replicates and triple technical replicates. For in vivo experiments, the nude mice were randomly grouped into eight per group using a blinding method. All other materials and methods used in this study are described in the Supplemental Materials.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Results

PDE4DIP expression is upregulated in human lung cancer

To explore the dysregulation of PDE4DIP in lung cancer, we analysed PDE4DIP protein expression in primary human NSCLC samples. In samples from 51 of the 60 (85%) patients, the PDE4DIP protein level was greater in the tumour tissues than in the matched adjacent normal tissues (Fig. 1A, B). Correlation analysis of our lung cancer cohort revealed significant associations between upregulated PDE4DIP expression and larger tumour size (P < 0.01), higher TNM stage (P < 0.05), and positive lymph node metastasis (P < 0.01) (Table S2). To further determine the clinical significance of PDE4DIP, we mined datasets from Kaplan–Meier Plotter database and the Gene Expression Omnibus (GEO; GSE3141). Kaplan–Meier survival analysis revealed that patients with higher PDE4DIP mRNA expression had significantly worse overall survival and disease-free survival than did patients with lower PDE4DIP mRNA expression (Fig. 1C, D). Taken together, the results from both our database analysis and our analysis of clinical specimens indicated that upregulation of PDE4DIP is associated with poor clinical outcomes in patients, suggesting that PDE4DIP may play an important role in NSCLC.

Fig. 1: PDE4DIP expression and prognostic analysis in lung cancer.
figure 1

A Representative Western blot of PDE4DIP expression in NSCLC tissues. β-Actin was used as a control. N, adjacent tissues; T, tumour tissues. B Semiquantitative analysis of PDE4DIP protein levels in human NSCLC tumours. PDE4DIP protein expression in 60 paired NSCLC tumour tissues and adjacent tissues was assessed via Western blotting and quantified via Image J software. The optical density of the PDE4DIP band was quantified and normalized to that of the corresponding loading control, β-actin. C Kaplan–Meier survival analysis of patients with high vs. low PDE4DIP expression in lung tumour tissues in the GSE3141 cohort. The data were obtained using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). The optimal cut-off value for classification was determined by Kaplan–Meier scanning approach. D Kaplan–Meier disease-free survival analysis of patients with PDE4DIP-high vs. PDE4DIP-low NSCLC tumors obtained from Kaplan–Meier Plotter database (www.kmplot.com). The optimal cut-off value for classification was median.

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Knockdown of PDE4DIP inhibits NSCLC growth

To explore the potential functional role of PDE4DIP in lung cancer, we generated PDE4DIP-knockdown (PDE4DIP-KD) cell lines by transduction of lentiviral vectors containing one of two distinct shRNAs targeting PDE4DIP (shP1 and shP2) into H1299 and A549 cells. Successful shRNA transduction was confirmed by the significant decrease in PDE4DIP protein expression compared to that in the corresponding cells transduced with shNC (Fig. 2A). Next, we examined the effect of PDE4DIP-KD on cell viability in vitro. The MTT assay and RTCA showed that knockdown of PDE4DIP significantly suppressed cell proliferation compared with that of shNC cells (Fig. 2B, C). Knocking down PDE4DIP also dramatically decreased the colony-forming capacity of H1299, A549 and PC9 cells (Fig. 2D and Supplementary Fig. 1). Taken together, these results suggest that PDE4DIP functions as an oncogenic factor in lung cancer cell proliferation. To further verify the oncogenic role of PDE4DIP in vivo, we established a xenograft model and measured tumour volumes at 3-day intervals. The growth curve showed that PDE4DIP KD significantly delayed the growth of tumours (Fig. 2E). At the termination of the experiment, we sacrificed the mice and harvested the tumours (Fig. 2F). Knockdown of PDE4DIP led to a marked decrease in tumour weight (Fig. 2G). In addition, overexpression of PDE4DIP in H1299 and A549 cells enhanced their growth capability markedly (Supplementary Fig. 2). Taken together, these results demonstrate that knockdown of PDE4DIP has an inhibitory effect on lung cancer cell growth both in vitro and in vivo, suggesting that PDE4DIP plays an oncogenic role in NSCLC tumorigenesis.

Fig. 2: Knockdown of PDE4DIP inhibits lung cancer cell proliferation in vitro and tumorigenicity in vivo.
figure 2

A shRNA-mediated knockdown of PDE4DIP expression was evaluated via Western blotting. H1299 and A549 cells were infected with lentivirus carrying either control shRNA (shNC) or a PDE4DIP-specific shRNA (shP1 or shP2) to generate PDE4DIP-KD cell lines. GAPDH was used as a control. B MTT assay in control (shNC) and PDE4DIP-KD H1299 and A549 cells. The data are representative of at least three independent experiments performed in triplicate. C Growth curves of PDE4DIP-KD H1299 and A549 cells generated via RTCA. The data were obtained from triplicate samples. D Colony formation assay in PDE4DIP-KD lung cancer cells. Left, representative images of the assay; right, data representative of at least three independent experiments performed in triplicate. Scale bar: 1.0 cm. E Growth curve of xenografts generated from control and PDE4DIP-KD NSCLC cells (n = 8). F Representative images of half of the tumours in the control and PDE4DIP shRNA-transfected groups are shown at 42 (H1299) and 25 (A549) days after cell injection. G Weights of xenograft tumours derived from control and PD4DIP shRNA-transfected NSCLC cells (n = 8 mice). ***P < 0.001. The data are presented as the means ± SD, and the P values were determined by Student’s t test. **P < 0.01; ***P < 0.001 vs. shNC.

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Knockdown of PDE4DIP induces apoptosis and cell cycle arrest

To explore the mechanism by which PDE4DIP affects lung cancer growth, we first performed gene set enrichment analysis (GSEA) to screen for potential involved pathways by dividing the patient samples from the TCGA-LUAD dataset into “high” and “low” PDE4DIP expression groups. PDE4DIP expression was negatively correlated with the expression of apoptosis-related genes but was positively correlated with the expression of cell cycle-related genes (Supplementary Fig. 3). Next, we compared the gene expression profiles of parental and PDE4DIP KD A549 cells through transcriptomic sequencing. Similar to the findings from the KEGG analysis using a TCGA cohort, the dysregulated genes were enriched in cell cycle and p53 signalling—related signatures (Fig. 3A). Consistent with these transcriptome analysis results, fluorescence-activated cell sorting (FACS) analysis of H1299 and A549 cells revealed that knockdown of PDE4DIP led to a marked increase in the number of apoptotic cells compared with that in the shNC cell group (Fig. 3B). TUNEL assays also showed greater numbers of TUNEL-positive cells among PDE4DIP-KD cells and in PDE4DIP-KD xenograft tumour tissues than in corresponding control groups (Fig. 3C and Supplementary Fig. 4A). FACS analysis also revealed that silencing PDE4DIP expression led to a significant increase in the proportion of cells in G1 phase and a concomitant decrease in the proportion of cells in S phase (Fig. 3D). These data indicate that knockdown of PDE4DIP induces apoptosis and cell cycle arrest in NSCLC cells. Consistent with the apoptotic phenotypes, silencing PDE4DIP increased the levels of Bim, Bax, Cytochrome c and cleaved Caspase 3/9 and concurrently decreased Bcl-2 expression in the two tested cell lines (Fig. 3E). Functionally, Bim knockdown alone inhibited apoptosis in A549 and H1299 cells (Supplementary Fig. 4B, C). Furthermore, silencing of Bim strongly suppressed the apoptosis triggered by PDE4DIP interference in both NSCLC cell lines (Fig. 3F and Supplementary Fig. 4D). These data suggest that PDE4DIP-KD-induced apoptosis in NSCLC cells is Bim dependent. To further understand the mechanism through which PDE4DIP modulates cell cycle progression, we examined the expression of proteins that regulate cell cycle progression. Knockdown of PDE4DIP prominently reduced the expression of Cyclin D1 and CDK4 but increased p27KIP1 and RB1 expression compared with that in shNC cells (Fig. 3G). Furthermore, there were fewer Ki67-positive cells in PDE4DIP-KD cell-derived tumour xenograft tissues than in shNC cell-derived tissues (Supplementary Fig. 5). These characteristics are hallmarks of the inhibitory effect of PDE4DIP on cell proliferation. Taken together, these data indicate that PDE4DIP has an inhibitory effect on apoptosis while promoting cell cycle progression, thus playing a tumour growth-promoting role in NSCLC.

Fig. 3: Effect of PDE4DIP knockdown on apoptosis and cell cycle progression.
figure 3

A KEGG enrichment analysis of the pathways affected by knockdown of PDE4DIP in A549 cells. B PDE4DIP-KD-induced apoptosis was analysed via flow cytometry. Cells were infected with nontargeting control shRNA (shNC) or PDE4DIP shRNA (shP1 or shP2) for 84 h. Left panel, representative images of the apoptosis assay results; right panel, calculated numbers of apoptotic cells (%, Q4). C Apoptosis was examined by a TUNEL assay in PDE4DIP-KD NSCLC tissues from xenograft. Upper panel, representative images of TUNEL positivity, with the arrow indicating TUNEL-positive cells; lower panel, histograms showing the quantification of TUNEL-positive cells. Scale bar: 100 μm. D Flow cytometric analysis of the cell cycle in PDE4DIP-KD NSCLC cells. Cells were infected with lentivirus containing shP1, shP2 or the nontargeting shNC for 84 h. Upper panel, representative images of the cell cycle analysis results; lower panel, histograms showing the quantification of the cell cycle distribution. E Western blot analysis of apoptosis-related proteins in control and PDE4DIP-KD cells. Cl Casp-3/9, cleaved caspase-3/9. F Silencing Bim abolished PDE4DIP-KD-induced apoptosis. Left panel, evaluation of interference with Bim expression by Western blotting; right panel, histograms showing the quantification of apoptosis. G Western blot analysis of cell cycle-related proteins in control and PDE4DIP-KD cells (B–D and F). The data are shown as the means ± SD of a representative experiment performed in triplicate. **P < 0.01; ***P < 0.001 vs. shNC.

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Knockdown of PDE4DIP inhibits NSCLC growth by activating the PKA-CREB signalling pathway

Next, we sought to explore the mechanisms involved in the promoting effects of PDE4DIP on NSCLC tumorigenesis. PDE4DIP is considered a putative PKA signalling modulator due to its interaction with PDE4D. Therefore, we first examined whether PDE4DIP affects PKA signal transduction. Knockdown of PDE4DIP in both of the tested NSCLC cell lines increased the phosphorylation of CREB, a direct target of PKA, while having minimal effects on ERK and AKT phosphorylation (Fig. 4A and Supplementary Fig. 6A, B). In contrast, ectopic overexpression of PDE4DIP resulted in a reduction in CREB phosphorylation (Fig. 4B). Furthermore, pretreatment with H89, a specific PKA inhibitor that binds to the PKA catalytic subunit to inhibit its activation, completely abrogated the PDE4DIP KD-mediated increase in CREB phosphorylation (Fig. 4C), whereas treatment with a cAMP analogue, 8-CPT cAMP, completely abolished the inhibitory effect of PDE4DIP on CREB phosphorylation (Supplementary Fig. 6C). These results suggest that PDE4DIP regulates the activation of PKA-CREB signalling in NSCLC. Overexpression of PDE4DIP significantly reduced the protein expression of Bim, which is a key proapoptotic protein (Fig. 4B). Moreover, overexpression of PDE4DIP reduced the levels of the cell cycle inhibitory proteins p27KIP1 and RB1 (Fig. 4B). Furthermore, inhibition of PKA activity by H89 abolished the PDE4DIP KD-mediated changes in the protein levels of Bim, p27KIP1, RB1, Cyclin D1, and CDK4 (Fig. 4C). In addition, treatment with the PKA activator 8-CPT cAMP led to increases in Bim, p27KIP1 and RB1 protein levels in both cell lines (Supplementary Fig. 6D). These data suggest that PDE4DIP inhibits the expression of Bim, RB1, and p27KIP1 by suppressing PKA signalling activity. Both Bim and RB1 are known downstream target genes of CREB and contain CREB binding sites in their promoter regions32,33. qRT‒PCR analysis showed that overexpression of PDE4DIP decreased the mRNA levels of Bim and RB1 in both NSCLC cell lines (Fig. 4D). Moreover, while PDE4DIP KD increased the mRNA expression of Bim and RB1, the addition of H89 completely blocked the PDE4DIP-KD-mediated increase in the transcription of these two genes (Fig. 4E). These data suggest that PDE4DIP KD triggers apoptosis through PKA-CREB-mediated transcriptional induction of the proapoptotic factor Bim while also inducing cell cycle arrest by increasing RB1 transcription. Taken together, these data suggest that transcriptional induction of Bim and RB1 expression through the PKA-CREB cascade is a key mechanism by which PDE4DIP-KD inhibits NSCLC growth.

Fig. 4: Knockdown of PDE4DIP induces PKA-CREB signalling activation in NSCLC cells.
figure 4

A Western blot analysis of PDE4DIP-mediated signalling pathway components in control and PDE4DIP-KD NSCLC cells. B Western blot analysis of CREB phosphorylation and downstream target gene expression in NSCLC cells overexpressing PDE4DIP (PI). Cells were transfected with the PDE4DIP expression plasmid for 48 h, and the pCDNA3.1 empty vector was used as a control (Vec). C Western blot showing that treatment with the PKA inhibitor H89 blocked PDE4DIP KD-induced PKA signalling activation and alterations in downstream target gene expression. Cells were transduced with shRNAs for 72 h and were then treated with either 10 µM H89 or DMSO for 24 h. (D) qRT‒PCR analysis of Bim and RB1 mRNA expression in control and PDE4DIP overexpressing H1299 and A549 cells. ***P < 0.001 vs. Control (PI). E qRT‒PCR analysis results showing that treatment with H89 blocked PDE4DIP KD-induced upregulation of Bim and RB1 mRNA expression. ***P < 0.001 vs. shNC.

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PDE4DIP inhibits PKA signalling activation by stabilizing PKA RIIα

PDE4DIP has been reported to interact with PDE4D variants to inhibit PKA signalling in certain cells18. Surprisingly, knockdown of PDE4DIP neither affected PDE4D expression nor increased cAMP production in A549 and H1299 cells (Fig. 5A, B). Moreover, PDE4DIP and PDE4D were not colocalized in either NSCLC cell line (Supplementary Fig. 7A). Although silencing PDE4D expression notably increased the levels of phosphorylated CREB in both cell lines, it had no substantial effect on PDE4DIP interference-induced CREB activation (Supplementary Fig. 7B, C). Taken together, these data suggest that in NSCLC cells, PDE4DIP affects PKA signalling in a PDE4D/cAMP-independent manner. PKA RIIα is a key negative regulatory subunit of the PKA holoenzyme, and several cellular studies have reported that it is prominently localized at the Golgi apparatus34. Consistent with these reports, our confocal microscopy images showed that the dominant signal intensity profile of endogenous PKA RIIα was consistent with that of PDE4DIP at the Golgi apparatus in NSCLC cells (Fig. 5C). This finding prompted us to consider whether PDE4DIP may modulate PKA signal transduction by interacting with PKA RIIα. Indeed, coimmunoprecipitation experiments demonstrated a reciprocal interaction between the PDE4DIP and PKA RIIα proteins in both NSCLC cell lines (Fig. 5D, E). Furthermore, knockdown of PDE4DIP in NSCLC cells impaired the accumulation of PKA RIIα at the Golgi, as shown by decreased colocalization with the Golgi marker GM130 and a more diffuse staining pattern in the cytoplasm (Fig. 5F). These results suggest that the PDE4DIP-PKA RIIα interaction is essential for the localization of PKA RIIα to the Golgi. Notably, the total protein level of PKA RIIα was also markedly decreased in PDE4DIP-KD cells, as shown by Western blotting and immunofluorescence staining (Fig. 5B and F). Moreover, the ubiquitination assay revealed that PDE4DIP KD promoted the ubiquitination of PKA RIIα in both NSCLC cell lines (Fig. 5G). These results indicate that the interaction of PDE4DIP with PKA RIIα at the Golgi apparatus stabilizes PKA RIIα by preventing its ubiquitination and degradation. In addition, we observed that the Golgi apparatus was dispersed and partially fragmented in a minority of PDE4DIP KD cells (Supplementary Fig. 8), suggesting that maintenance of Golgi integrity may also contribute to the promoting effect of PDE4DIP on NSCLC cell growth. Taken together, our results suggest that the binding of PDE4DIP to PKA RIIα at the Golgi apparatus increases the stability and cellular accumulation of PKA RIIα, thereby leading to an inhibition of PKA signalling activation.

Fig. 5: Knockdown of PDE4DIP mislocalizes PKA RIIα from the Golgi apparatus and promotes its degradation.
figure 5

A The production of cAMP was not increased in PDE4DIP-KD cells, as determined by a cAMP assay. Forskolin was used as a positive control. NS not statistically significant. B Western blot analysis of PDE4D and PKA RIIα (RIIα) expression in control and PDE4DIP-KD NSCLC cells. C Immunofluorescence staining showing the colocalization of PKA RIIα (RIIα, red) and PDE4DIP (green) in NSCLC cells. Scale bars, 10 μm. D, E Coimmunoprecipitation of PDE4DIP and PKA RIIα in PDE4DIP-overexpressing cells. The PDE4DIP-Myc plasmid was transfected into H1299 and A549 cells. Immunoprecipitation was performed with either an anti-PKA RIIα (RIIα) or an anti-Myc (PDE4DIP) antibody. TCL total cell lysate. F Immunofluorescence analysis of PKA RIIα (red) and GM130 (green) colocalization at the Golgi apparatus in control (shNC) and PDE4DIP-KD (shP1) NSCLC cells. Scale bars, 10 μm. Representative images (left) and colocalization values (Rcoloc) of PKA RIIα and GM130 are shown (right). The average co-localization was calculated using the JACoP plug in of the ImageJ program in 10–15 images obtained for each condition. The data are presented as the means ± SD of three biological replicates, and the P values were determined by Student’s t test. ***P < 0.001 vs. control. G Western blot analysis of PKA RIIα ubiquitination in control and PDE4DIP-KD NSCLC cells.

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PDE4DIP coordinates with AKAP9 to stabilize PKA RIIα at the Golgi

A-kinase anchoring proteins (AKAPs) determine the spatial and temporal aspects of PKA activation by tethering the PKA holoenzyme to distinct cellular compartments35. Previously, we and others reported that PDE4DIP localizes predominantly to Golgi networks by interacting with AKAP9 and that the expression of these two proteins is mutually dependent19,29. In NSCLC cells, silencing of PDE4DIP resulted in a significant decrease in the AKAP9 protein level, and knocking down AKAP9 reduced the protein level of PDE4DIP (Fig. 6A, B). AKAP9 also coimmunoprecipitated with PDE4DIP (Fig. 6C). These results indicate that the expression of PDE4DIP and AKAP9 in NSCLC is interdependent and that their interaction is required for the stability of both proteins. The AKAP9 and PKA RIIα proteins also coimmunoprecipitated (Fig. 6C). Knocking down AKAP9 not only reduced the protein level of PKA RIIα but also significantly reduced the abundance of PDE4DIP-colocalized PKA RIIα on the Golgi apparatus (Fig. 6B and D). These results indicate that the formation of the PDE4DIP-AKAP9 complex is essential for tethering PKA-RII to the Golgi apparatus. In addition, knocking down AKAP9 promoted the ubiquitination of PKA RIIα in both tested tumour cell lines (Fig. 6E). Together, these data suggest that PDE4DIP coordinates with AKAP9 at the Golgi to increase both the stability and the accumulation of PKA RIIα, thereby negatively regulating PKA activation.

Fig. 6: The PDE4DIP-AKAP9 interaction is essential for the Golgi localization and stability of PKA RIIα.
figure 6

A Western blot analysis of AKAP9 expression in control and PDE4DIP-KD NSCLC cells. B Western blot analysis of PDE4DIP and PKA RIIα (RIIα) expression in control and AKAP9-silenced NSCLC cells. Cells were transfected with control siNC or siRNA targeting AKAP9 (siA1, siA2) for 48 h. C Coimmunoprecipitation of endogenous AKAP9 with PDE4DIP or PKA RIIα in H1299 and A549 cells. Cell lysates were immunoprecipitated with an anti-AKAP9 antibody and subjected to Western blotting with anti-AKAP9, anti-PDE4DIP, and anti-PKA RIIα antibodies. TCL, total cell lysate. D Immunofluorescence analysis of PDE4DIP (green) and PKA RIIα (red) colocalization at the Golgi apparatus in control (siNC) and AKAP9-silenced (siAKAP9/siA1) NSCLC cells. Scale bars, 10 μm. Representative images (left) and colocalization values (Rcoloc) of PDE4DIP and PKA RIIα are shown (right). The data are presented as the means ± SD of three biological replicates, and the P values were determined by Student’s t test. ***P < 0.001 vs. control. E Western blot analysis of PKA RIIα ubiquitination in control (siNC) and AKAP9-silenced (siA1) NSCLC cells.

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Discussion

Mutations in the PDE4DIP gene have been detected in lung cancers and are thought to be associated with cancer metastasis30,31. However, the biological function of PDE4DIP in lung cancer is still unknown. Here, we found that PDE4DIP was highly expressed in clinical NSCLC tissues and that its upregulation was correlated with poor survival in patients. We showed that knocking down PDE4DIP expression significantly reduced NSCLC cell viability in vitro and tumorigenesis in vivo. Mechanistically, we found that PDE4DIP mainly regulates apoptosis and cell cycle progression via PKA/CREB signalling, thereby affecting NSCLC tumour growth. PDE4DIP is predominantly localized at the Golgi apparatus, and the PDE4DIP-AKAP9 complex serves as a docking platform for PKA RIIα. Loss of PDE4DIP mislocalizes PKA RIIα from the Golgi, leading to its degradation and the activation of PKA-CREB signalling in a cAMP-independent manner. Based on our study, we propose a novel PDE4DIP-mediated PKA signal transduction mechanism that plays an important role in NSCLC tumorigenesis (Fig. 7).

Fig. 7: Schematic of the proposed mechanism by which PDE4DIP promotes lung cancer cell growth.
figure 7

In NSCLC cells expressing high levels of PDE4DIP, the formation of the PDE4DIP-AKAP9-PKA RIIα complex at the Golgi apparatus increases the stability of PKA RIIα, resulting in PKA inactivation. Knockdown of PDE4DIP promotes the ubiquitination-mediated degradation of PKA RIIα, thereby releasing active PKA catalytic subunits, enhancing CREB phosphorylation, and leading to the expression of downstream target genes related to apoptosis and cell cycle arrest.

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In the present study, we demonstrated that PDE4DIP plays an oncogenic role in NSCLC and that PDE4DIP-KD inhibits tumour growth through the induction of apoptosis and cell cycle arrest. Our data showed that PDE4DIP-KD transcriptionally induced the expression of Bim, a well-established CREB target gene. Upregulation of Bim has been shown to be an important determinant of PKA-CREB-mediated mitochondrial apoptosis in acute myeloid leukaemia and immature T cells32,36,37. Consistent with these findings, we found that PDE4DIP KD reduced the expression of Bcl-2 and increased the expression of Bax and Cyto c, indicating that PDE4DIP KD promotes apoptosis in NSCLC cells through a mitochondria-dependent pathway. Our study also revealed that PDE4DIP KD increased the expression of genes encoding the RB1 and p27KIP1 tumour suppressors, and this dual inactivation event may result in dysregulation of the G1/S transition, as observed in our experiments. Previous studies have reported that the promoters of the Bim and RB1 genes contain a CREB binding site32,33, suggesting that in NSCLC cells, PDE4DIP KD may directly promote the transcription of Bim and RB1 through CREB binding. Moreover, disruption of PKA activation completely counteracted the PDE4DIP KD-induced upregulation of Bim, RB1 and p27KIP1 expression. Taken together, the results of our study suggest that PDE4DIP KD-induced activation of PKA signalling inhibits NSCLC growth through CREB-mediated upregulation of Bim, RB1, and p27KIP1 expression. Notably, mutations activating PKA are associated with the development of a spectrum of neuroendocrine tumours, and PKA signalling activation promotes the growth of SCLC11,38. However, studies have shown that PKA promotes cell proliferation and the cancer stem cell status in SCLC mainly by regulating pathways independent of CREB and its downstream targets11. Taken together, the findings of our study may provide a reasonable explanation for the contrasting effects of PKA on the growth of SCLC and NSCLC tumours.

PDE4DIP is considered a negative regulator of the PKA signalling pathway because it has been found to interact with PDE4D, a cAMP hydrolase phosphodiesterase17. Here, we demonstrated that PDE4DIP indeed had an inhibitory effect on the PKA signalling pathway in NSCLC, albeit via a PDE4D-independent mechanism. Our data indicated that PDE4DIP KD had no effect on the expression or cellular distribution of PDE4D but did significantly decrease the protein level and Golgi localization of PKA RIIα. PKA is a tetrameric holoenzyme composed of two regulatory subunits (PKA R) and two catalytic subunits (PKA C). The PKA R subunit has four isoforms (RIα/RIβ and RIIα/RIIβ) that exhibit distinct subcellular localizations and nonredundant biological functions4,39. cAMP-dependent PKA activation is typically achieved by the binding of two cAMP molecules to each regulatory subunit, which removes the autoinhibitory contact of PKA and allows dissociation of the catalytic subunits, resulting in PKA activation. Intriguingly, whereas we did not observe a change in the cellular cAMP level, PDE4DIP KD reduced the PKA RIIα protein level in NSCLC cells by promoting its ubiquitination and degradation. Our study thus suggested that PDE4DIP regulates PKA signalling in a cAMP-independent but PKA RIIα-associated manner. In HEK293 cells, depletion of PKA RIIα has been shown to cause the redistribution of dissociated PKA C to the cytoplasm and its subsequent translocation to the nucleus, thereby inducing robust and persistent activation of PKA34. While germline and somatic mutations in the genes encoding the PKA R subunits have been characterized, little is known about the biological functions of aberrant PKA RIIα expression in human tumours40. Overexpression of PKA RIIα has been shown to increase the survival of prostate cancer cells treated with Taxol41. In HCC cells, anchoring of PKA RIIα at the Golgi is required for efficient and coordinated delivery of proteins and sphingolipids to the apical membrane42. Our results demonstrated that PDE4DIP is essential for the Golgi localization and stability of PKA RIIα, suggesting that PDE4DIP-mediated PKA signalling may also play a role in cellular trafficking and the tumour response to chemotherapy.

In mammalian cells, PKA exists as two isoforms, type I and type II, which are distinguished by their different regulatory subunits, namely, RI and RII. PKA type I has a primarily cytoplasmic localization, and most PKA type II proteins are anchored to specific organelles and cellular structures via their binding to AKAPs43. In this study, our data demonstrated that PDE4DIP regulates type II PKA signalling in NSCLC via coordination with AKAP9. AKAP9, also known as AKAP450 or CG-NAP, is a PKA scaffolding protein that localizes predominantly to the Golgi and regulates microtubule dynamics and nucleation44. In NSCLC cells, we observed prominent colocalization of PDE4DIP and AKAP9 at the Golgi and verified that their physical interaction was required for the stability of both proteins, as reported in other cells19,29. Moreover, we showed that knocking down AKAP9 reduced the protein level of PKA RIIα and simultaneously impaired its Golgi localization. These results suggest that PDE4DIP coordinates with AKAP9 to regulate the stability and subcellular localization of PKA RIIα. Overall, our study supports the hypothesis that PDE4DIP forms a binary complex with AKAP9 on the Golgi apparatus, thereby providing a docking platform to anchor PKA RIIα, which can both prevent PKA RIIα degradation and tether additional PKA holoenzymes to the Golgi, leading to sequestration and inactivation of PKA. In addition, our study indicates that AKAP9 KD also promotes PKA RIIα ubiquitination. Praja2 is another AKAP that has been shown to control the activation of PKA by promoting the ubiquitination and degradation of PKA RIIα in HEK293 cells34,45. However, further work is needed to elucidate the mechanism by which AKAPs lead to different ubiquitination fates of PKA RIIα.

In this study, we found that the Golgi apparatus was dispersed and fragmented upon PDE4DIP depletion in a subset of NSCLC cells. PDE4DIP has been shown to play a role in the organization and function of the Golgi apparatus by recruiting the γ-tubulin complex to participate in Golgi microtubule nucleation17. This finding suggests that the Golgi integrity maintained by PDE4DIP is more likely to be attributed to its function as a Golgi structural protein. Whether PDE4DIP-mediated PKA signaling is involved in maintaining the integrity of the Golgi apparatus requires further study to clarify. Intriguingly, it has also been reported that overexpression of MMG8, a variant of PDE4DIP, can cause disorganization of the Golgi19. These studies suggest that ensuring proper structural organization of the Golgi requires maintenance of the homeostasis of Golgi-localized PDE4DIP. The Golgi apparatus is a membrane organelle that has essential impacts on protein processing, sorting and transport and on signal transmission46. Recent studies have reported that Golgi dispersion can induce the transduction of apoptotic signals and that cleavage of the Golgi structural proteins P115 and Golgin-160 can lead to Golgi disassembly, thereby promoting apoptosis47,48. We showed that PDE4DIP-KD-induced both Golgi dispersion and cellular apoptosis, suggesting that PDE4DIP may also affect apoptosis through disruption of the Golgi structure in NSCLC. Damage to or structural abnormalities in the Golgi apparatus can result in dysfunction of signalling pathways; thus, therapeutically targeting Golgi apparatus proteins is an antitumour strategy49,50. Indeed, the findings of our study suggest that targeting the Golgi protein PDE4DIP may be a therapeutic approach for NSCLC.

In summary, we report that PDE4DIP plays an oncogenic role in NSCLC tumorigenesis through modulation of the PKA-CREB signalling pathway. Our study reveals a novel PDE4DIP/AKAP9/PKA RIIα-mediated PKA signal transduction axis that is critical for apoptosis and cell cycle progression and highlights PDE4DIP as a promising therapeutic target for NSCLC.

Study approval

All human studies received ethical approval of the Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. Appropriate informed consent was obtained from the patients. All ethical regulations relevant to human research participants were followed. All animal experiments and protocols were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University (No. wydw2019-0842). We have complied with all relevant ethical regulations for animal use.