Transcriptional Regulation of NUPR1 by MYH11 Activates PI3 K/AKT and Promotes Bladder Cancer Progression Through Ferroptosis and M2 Polarization of Macrophages

Abstract

Background

NUPR1 is a small molecule protein that plays an important role in tumor progression and drug resistance. Our previous study found that NUPR1 promotes the progression of bladder cancer, but the specific mechanism is still unclear. MYH11 encodes the smooth muscle myosin heavy chain and belongs to the conventional myosin family. MYH11 has been found to be associated with a variety of malignant tumors.

Methods

We identified MYH11 as an upstream regulator of NUPR1 using a bioinformatics approach and tested this hypothesis by knocking down MYH11 and ChIP-qPCR. Subsequently, we verified the association of MYH11 and NUPR1 with the PI3 K/AKT pathway by WB. In addition, gene enrichment results showed that the effect of NUPR1 on bladder cancer was related to ferroptosis and M2 macrophage polarization. We examined ferroptosis metabolites in bladder cancer cells overexpressing NUPR1 and expression of the M2 macrophage marker CD206 in NUPR1 overexpression or MYH11 knockdown bladder cancer cells.

Results

Bioinformatics results showed that MYH11 was positively correlated with NUPR1, and there may be a mutual binding site at the promoter of NUPR1. Knockdown of MYH11 decreased NUPR1 expression, and ChIP-qPCR showed that MYH11 bound to the promoter of NUPR1. Subsequently, WB results showed that MYH11 knockdown inhibited the PI3 K/AKT pathway, whereas NUPR1 overexpression activated this pathway. After adding ferroptosis activator, the viability of bladder cancer cells decreased, and the content of Fe²⁺ and MDA increased. However, ferroptosis was significantly inhibited after overexpression of NUPR1. Knockdown of MYH11 inhibited M2 macrophage polarization, while overexpression of NUPR1 promoted this process.

Conclusion

This study suggests that MYH11 activates the PI3 K/AKT pathway by up-regulating the expression of NUPR1, and promotes bladder cancer progression by inhibiting ferroptosis and promoting M2 polarization of macrophages.

Keywords

urinary bladder neoplasms gene expression regulation neoplasm protein ferroptosis tumor microenvironment

Introduction

NUPR1(also known as com-1 or p8) is a small nuclear protein that was originally identified as a response to cellular stress in the acute phase of rat pancreatitis.¹ NUPR1 is associated with a variety of benign and malignant diseases. Previous studies have reported that NUPR1 is associated with cardiomyocyte hypertrophy,² participates in glucose metabolism in vivo,³ and regulates neuronal apoptosis and autophagy.⁴ In recent years, the role of NUPR1 in malignant tumors has attracted increasing attention and has been confirmed to play an important role in a variety of cancers. However, there is no consensus on the role of NUPR1 in tumors, and the function of NUPR1 in different tumors is inconsistent. He et al found that NUPR1 was significantly overexpressed in human ccRCC cells compared with adjacent normal tissues, and the expression of NUPR1 gradually increased with the increase of pathological T stage, lymph node metastasis, and clinical stage.⁵ While, NUPR1 is known to be a protective factor in prostate cancer, and its expression is significantly down-regulated in both in vitro⁶ and in vivo⁷ prostate cancer tissues. Our previous study found NUPR1 is closely associated with bladder cancer proliferation, and promotes the migration and invasion of tumor,⁸ but the exact mechanism has not been elucidated.

MYH11 encodes the smooth muscle myosin heavy chain and belongs to the conventional myosin family. The clear biological function of myosin is the ability to use the energy of ATP hydrolysis to move actin filaments and generate muscle force. Recently, myosin has been found to be associated with a variety of other intracellular functions, including cell migration, adhesion, cell shape control, and membrane trafficking,⁹ as well as cell signaling pathways such as interactions with Rho¹⁰ and the pro-apoptotic protein Bmf, a Bcl2-modifying protein.¹¹ In yeast, myosin 5 has been shown to localize the mitotic spindle, so it plays a key role in the establishment of cell polarity.¹²

As reported, MYH11 has been found to be associated with a variety of malignant tumors. Inversion at the MYH11 locus inv(16)(p13q22) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML) and accounts for approximately 8% of all AML cases, especially those of the M4Eo subtype.¹³ In colorectal cancer, MYH11 has been identified as a driver gene, and a somatic frameshift mutation (c.5798delC) in MYH11 has been identified in 55% of colorectal cancers (CRCS) with microsatellite instability (MSI).¹⁴ Although it was reported that somatic mutations in MYH11 are uncommon in prostate cancer,¹⁵ the role for MYH11 in bladder cancer has not been demonstrated.

Materials and Methods

Bioinformatics Analysis

We used the R language to analyze the differential expression of the bladder cancer dataset GSE65635 in the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Then, the hTFtarget database (http://bioinfo.life.hust.edu.cn/hTFtarget) was used to predict transcription factors of NUPR1. Further, we used data from TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) to analyze the expression of MYH11 in pan-cancers and its expression levels in different ethnic groups. We used the GEPIA database (http://gepia.cancer-pku.cn/) to analyze the correlation between the expression of MYH11 and the survival cycle of patients with bladder cancer. Starbase database (http://starbase.sysu.edu.cn) was used to analyze the correlation between MYH11 and NUPR1 expression in bladder cancer. The string database (https://cn.string-db.org/) was used to construct the protein-protein interaction network of NUPR1 and performed the enrichment analysis. TIMER 2.0 database (http://timer.cistrome.org/) was used to analyze the correlation between MYH11 and NUPR1 and M2 macrophage infiltration in bladder cancer.

Cell Culture

The human BLCA cell lines, T24 and 5637, were purchased from the Chinese Academy of Sciences (Shanghai, China). T24 cell line wasderived from a female patient with grade III BLCA. 5637 cell line was from a grade II BLCA male patient. The cells were cultured in Roswell Park Memorial Institute-1640 medium (Gibco, USA), including 10% fetal calf serum, 100U/ml penicillin and streptomycin in a 5% carbon dioxide (CO2) incubator at 37 °C. The culture media of these two cell lines were changed every 3 days. The cells were harvested when they reached about 80% confluency.

RT-qPCR and Transfection of Liposome

Total RNA was isolated from tissues and cells with the help of TRIzol reagent (Takara Holdings Inc, Japan). The concentration and purity of RNA samples were measured by Nanodrop 2000 (Thermo Fisher Scientific, USA). Total RNA was reversely transcribed to cDNA using the PrimeScript RT kit (Takara, Japan). Then, RT-qPCR was performed using TB Green Premix Ex Taq II (Takara, Japan) and ABI Prism 7500 Sequence Detection System (Applied Biosystems, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as a housekeeping gene, and the comparative cycle threshold (Ct) method (2-ΔΔCt) was used to analyze differences in each transcript. Liposome-mediated shRNA was adopted to knockdown the expression of MYH11 in two human bladder cancer cell lines. The experiment adopted liposome-mediated delivery of MYH11-specific shRNA(5'-GAGGACGTAGAGTTATTGAAA-3’) to knockdown its expression in two human bladder cancer cell lines. The shRNA was encapsulated in liposomes for efficient cellular uptake, and the cells were transfected with the complexes. Following transfection, quantitative methods were used to assess the extent of MYH11 knockdown at the mRNA or protein level.

ChIP-qPCR

Chromatin immunoprecipitation (ChIP) was performed using the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Merck Millipore, USA) with an antibody specific for MYH11, or normal rabbit IgG (Santa Cruz Biotechnology, USA). For ChIP‒qPCR, immunoprecipitation was performed at 4 °C overnight with anti-MYH11 or normal rabbit IgG antibody. Then, Rt-qPCR was utilized to quantify the immunoprecipitated DNA, and the data were normalized to the input.

Western Blotting

Western blotting for the determination of protein expression was conducted thus: Cells were lysed using Beyotime Biotechnology IP lysis buffer (P0013, China). Protein concentration was determined using BCA protein assay kits (Beyotime, China). Absorbance was taken at 562 nm using a microplate spectrophotometer (Fisher Scientific, USA). Protein (10 μg) was separated on 10% SDS–polyacrylamide gel and then transferred onto nitrocellulose membranes at 200 mA for 85 min. Membranes were incubated with primary antibodies overnight at 4 °C. Finally, the protein bands were visualized by the Enhanced Chemiluminescent Western Blotting Substrate Kit (Abcam). Relative expression of proteins was analyzed using Quantity One software with GAPDH as an internal reference.

Cell Viability and Colony Formation Assay

Viable cells were measured using Cell Counting Kit-8 (Dojindo, Japan). In summary, cells were seeded onto 96-well plates at a density of 1×10⁴ cells per well. 10 μL of CCK-8 reagent (in 100 μL of medium per well) were incubated for 1 h after indicated treatment and time. Absorbance was then taken at 450 nm using a microplate spectrophotometer (Fisher Scientific, USA). Cells were seeded on 6-well and 12-well plates and incubated for 14 days after relevant treatment. Cell colonies were dyed with methylene blue for 10 min. Pictures of adherent cells were taken using Bio-rad Gel Doc XR imaging system (Bio-Rad, USA).

ROS Assay

Intracellular ROS was measured using oxidation-sensitive fluorescent probe DHE. Briefly, 4 × 10⁴ cells were treated with DHE (Invitrogen, USA). They were then incubated for 45 min and then washed, and treated with 5 μM Hoechst for 10 min. The fluorescence intensity of DHE was quantitatively determined using TECAN Infinite M200 PRO microplate reader (Tecan, Switzerland). The fluorescence intensity of oxidized DHE was normalized to that of Hoechst. In order to obtain images showing the fluorescence intensities of oxidized DHE, 4×10⁵ cells were incubated with DHE for 45 min. Fluorescent images were then taken using Nikon fluorescent microscope (Nikon Instruments Inc, USA).

Malondialdehyde and Iron Assay

A lipid peroxidation MDA assay kit (Beyotime, Shanghai, China) was used to determine the relative MDA concentrations following the manufacturer's protocol.

Intracellular ferrous iron (Fe²⁺) levels were determined using an Iron Assay Kit (ab86633, Abcam, United Kingdom) according to the manufacturer's instructions. The absorbance at 593 nm was measured using a VARIOSKAN LUX microplate reader (Thermo Fisher Scientific, USA).

Statistical Analysis

Cell experimental data in this study were expressed as mean ± standard deviation (SD). R was used for statistical analysis, and GraphPad Prism software was used to draw statistical graphs. When p was less than 0.05, the difference was considered statistically significant.

Results

MYH11 May Be an Upstream Factor Regulating NUPR1 Expression

To explore the reasons for the differential expression of NUPR1 in bladder cancer, we downloaded the list of transcription factors predicted to regulate NUPR1 expression from the hTFtarget database, and analyzed the differentially expressed genes between bladder cancer tissues and normal bladder tissues in the GEO database analysis dataset GSE65635 (Figure 1A). After cross screening, we found a total of 26 overlapping genes (Figure 1B). The analysis of the differential expression of intersection genes in bladder cancer shows that MYH11 has the most significant differential expression (adj.p.value = 1.03E-10), and the specific role of MYH11 in promoting or inhibiting cancer in bladder cancer is unclear. In GEPIA database, we analyzed the relationship between the expression of MYH11 and the prognosis of bladder cancer. The patient survival curve showed that the clinical patients with low expression of MYH11 had better prognosis (Figure 1C and S1C). Subsequently, we verified through cell viability experiments that the proliferation capacity of bladder cancer cells was improved after MYH11 was overexpressed, and the proliferation capacity of tumor was decreased after MYH11 expression was knocked down (Figure S1A and S1B). It indicates that MYH11 may have a cancer promoting effect. In the Starbase database, the analysis of the expression correlation between MYH11 and NUPR1 shows that MYH11 and NUPR1 are significantly positively correlated in bladder cancer (Figure 1D). We obtained the promoter sequence information of NUPR1 in the UCSC database, and analyzed the binding sites between MYH11 and NUPR1 in hTFtarget. It was found that MYH11 has multiple binding sites at the NUPR1 promoter (Figure 1E). MYH11 may be a potential upstream regulatory factor for NUPR1 expression changes.

Figure 1.

Using bioinformatics methods to screen the upstream molecules regulating NUPR1 expression. (A) Analysis of differentially expressed genes in bladder cancer tissues using dataset GSE65635; (B) Intersection of transcription factors predicted to regulate NUPR1 transcription from hTFtarget database and differentially expressed genes in bladder cancer from dataset GSE65635; (C) GEPIA database was used to analyze the correlation between the high and low expression of MYH11 and the survival cycle of patients; (D) Starbase database was used to analyze the correlation between MYH11 and NUPR1 expression in bladder cancer; (E) hTFtarget database was used to analyze the binding sites of MYH11 and NUPR1 promoter.

MYH11 is Lowly Expressed in Bladder Cancer Patients

Bioinformatics analysis showed that MYH11 was differentially expressed in a variety of cancers, and its expression was low in bladder cancer patients (Figure 2). Further results showed that MYH11 expression was significantly reduced in Asian, African, and Caucasian bladder cancer patients (Figure 3A), and the low expression of MYH11 was positively correlated with the prognosis of bladder cancer patients (Figure 3B-3D).

Figure 2.

Expression of MYH11 in pan-tumor.

Figure 3.

The expression of MYH11 in bladder cancer was analyzed by bioinformatics. (A) Bioinformatics analysis showed that MYH11 was lowly expressed in Asian, African, and white bladder cancers; (B) The ROC curve of the Asian bladder cancer subgroup (AUC = 0.987). (C) The ROC curve for the African American subgroup (AUC = 0.945), (D) The ROC curve for the white bladder cancer subgroup (AUC = 0.929).

The Binding of MYH11 and NUPR1 Promotes the Transcription Activation of NUPR1 and the PI3 K/AKT Signaling Pathway

It is well known that the phosphatidylinositol 3-kinase/protein kinase B(PI3 K/AKT) signaling pathway is one of the most common oncogenic pathways in human tumors by promoting tumor cell survival, proliferation, metabolism, invasion, and angiogenesis.^16,17 Therefore, in this study, we evaluated changes in the expression of this signaling protein. Rt-qPCR showed that the expression of MYH11 was significantly down-regulated in sh-MYH11 cells compared with control cells, while the expression of NUPR1 was also significantly reduced (Figure 4A and 4B). Furthermore, ChIP-qPCR assay confirmed that NUPR1 promoter was enriched in the MYH11 antibody pull-down DNA fragment (Figure 4C), indicating that MYH11 binds to NUPR1 promoter. Subsequently, cells overexpressing NUPR1 were constructed and the efficiency of overexpression was verified (Figure 4D). Analysis of the activation of PI3 K/AKT signaling pathway in cells showed that overexpression of NUPR1 promoted the activation of PI3 K/AKT signaling pathway, while knockdown of MYH11 inhibited the activation of PI3 K/AKT signaling pathway (Figure 4E). Subsequently, we overexpressed MYH11 in bladder cancer cell lines and found that NUPR1 expression was increased and effectively activated the PI3 K/AKT signaling pathway (Figure S2B).

Figure 4.

Cell experiments confirmed that MYH11 binds to NUPR1 activating the PI3 K/AKT signaling pathway. (A) Rt-qPCR was used to detect the expression of MYH11 in 5637 and T24 cells transfected with MYH11 knockdown plasmid. (B) To detect NUPR1 expression in 5637 and T24 cells transfected with MYH11 knockdown plasmid by Rt-qPCR. (C) MYH11 enrichment at the NUPR1 promoter in 5637 and T24 cells was determined by ChIP-qPCR. (D) Rt-qPCR was used to detect NUPR1 expression in 5637 and T24 cells transfected with NUPR1 overexpression plasmid. (E) Western blot analysis of PI3 K/AKT signaling activation in 5637 and T24 cells with MYH11 knockdown or NUPR1 overexpression. (Two-factor analysis was used for comparison among multiple groups, * indicates p < 0.05, and the experiment was repeated three times independently).

NUPR1 Expression Inhibits Ferroptosis in Bladder Cancer Cells

To explore the possible roles of NUPR1 in the development and progression of bladder cancer, we predicted the interacting proteins of NUPR1 in the String database and constructed a protein interaction network (Figure 5A). Further analysis of the enrichment of the protein interaction network on the KEGG pathway revealed that the interaction network was significantly enriched on the ferroptosis pathway (Figure 5B). Ferroptosis, an iron-dependent form of regulated cell death, has garnered significant attention in the context of urothelial carcinoma (UC) due to its unique mechanisms and potential therapeutic implications. Meanwhile, ferroptosis holds paramount importance in many carcinomas as a novel therapeutic target with the potential to develop combination treatment strategies, overcome drug resistance, and improve patient outcomes.^18–20 Furthermore, combined with the identification of NUPR1 as a ferroptosis repressor in other cancers,²¹ we examined whether NUPR1 could be involved in affecting ferroptosis in bladder cancer cells. We treated 5637 and T24 cells overexpressing NUPR1 with ferroptosis inducer Erastin. CCK-8 assay showed that the production of ferroptosis led to a decrease in cell viability, which was alleviated by overexpression of NUPR1 (Figure 5C). Meanwhile, overexpression of NUPR1 also inhibited the increase of ROS associated with ferroptosis in the cells (Figure 5D). Fe²⁺ and lipid peroxide MDA in cells also significantly decreased with overexpression of NUPR1 (Figure 5E-5F).

Figure 5.

NUPR1 expression inhibits the ferroptosis of bladder cancer cells. (A) The protein-protein interaction network of NUPR1 was analyzed using string database; (B) KEGG pathway enrichment analysis of NUPR1 protein interaction network; (C) CCK-8 was used to detect the viability of 5637 and T24 cells overexpressing NUPR1 treated with ferroptosis inducer Erastin. (D) DHE fluorescent probe labeled ROS to investigate the effect of NUPR1 overexpression on ferroptosis-related hydrogen peroxide production. (E) Iron content detection kit was used to detect the change of iron content in 5637 and T24 cells overexpressing NUPR1 after treatment with ferroptosis inducer Erastin. (F) NUPR1 overexpressed 5637 and T24 cells were treated with Erastin, and MDA content was detected by MDA content detection kit. (Two-factor analysis was used for comparison among multiple groups, * indicates p < 0.05, and the experiment was repeated three times independently).

MYH11 and NUPR1 are Related to M2 Polarization of Macrophages in Bladder Cancer

In the previous study, we found that NUPR1 has the highest correlation with macrophage infiltration in bladder cancer.⁸ We then analyzed its correlation with macrophage polarization in TIMER 2.0 database(http://timer.cistrome.org/) and found that NUPR1 expression was significantly positively correlated with M2 macrophage infiltration in bladder cancer (Figure 6A). Further analysis of MYH11 also showed a positive correlation with M2 macrophage infiltration in bladder cancer (Figure 6B). To this end, we constructed a co-culture system of bladder cancer cells and monocytes. WB and Rt-qPCR analysis of the effect of MYH11 and NUPR1 expression changes on macrophage polarization confirmed that MYH11 knockdown inhibited M2 polarization of macrophages and significantly reduced CD206 expression, and CD206 is an important mark of the M2 macrophages. While overexpression of NUPR1 promoted M2 polarization of macrophages and promoted CD206 expression (Figure 6C-6D). This confirmed the effect of MYH11 and NUPR1 on macrophage M2 polarization.

Figure 6.

MYH11 and NUPR1 are associated with M2 polarization of macrophages in bladder cancer. (A-B) TIMER 2.0 database was used to analyze the correlation between MYH11 and NUPR1 and M2 macrophage infiltration in bladder cancer. (C) WB analysis of the expression of M2 polarization marker CD206 in macrophages co-cultured with MYH11 knockdown and NUPR1 overexpression 5637 and T24 cells. (D) Expression of M2 polarization marker CD206 in macrophages co-cultured with MYH11 knockdown or NUPR1 overexpression 5637 and T24 cells by Rt-qPCR. (Two-factor analysis was used for comparison among multiple groups, * indicates p < 0.05, and the experiment was repeated three times independently).

Discussion

Bladder cancer is one of the most common cancers worldwide, with about half a million new cases each year,²² which imposes a huge burden on society, especially on men and the elderly.²³ Bladder cancer can be divided into non-muscle invasive bladder cancer and muscle invasive bladder cancer according to whether the muscle layer is invaded. Among them, NMIBC accounts for about 70% of new bladder cancer. Bladder cancer is also one of the most expensive tumors to treat given the usual need for repeated use of cystoscopy for tumor removal and review.²⁴ For MIBC, conventional radical resection and bladder preservation surgery are also difficult to achieve the best treatment effect, and the prognosis of patients is often poor.²⁵

Recently, the discovery of many DNA, RNA, and protein biomarkers of bladder cancer due to sequencing and gene expression studies^26–28 has provided an opportunity for a structural shift in the diagnosis and detection of recurrence of bladder cancer. In addition, the success of immunotherapy regimens and renewed attention to the tumor microenvironment (TME) in recent years have expanded the range of potential targeted therapies.

In this study, we took the newly discovered oncogenic factor nuclear protein 1(NUPR1) as the object of study and further explored its mechanism in bladder cancer based on the evidence that NUPR1 promotes bladder cancer progression.⁸ We identified MYH11 as a regulator of NUPR1 expression and further validated the role of MYH11 in the regulation of NUPR1 expression and MYH11 can bind to NUPR1 promoter regions using cellular experiments. Then, we investigated the regulation of NUPR1 expression by MYH11 and the role of it in bladder cancer. Taken together with our previous studies,^8,29 our results suggest that NUPR1 promotes the proliferation, migration and invasion of bladder cancer, and the high expression of it leads to the poor prognosis of bladder cancer patients. In addition, upregulation of NUPR1 promoted the activation of PI3 K/AKT pathway, while knockdown of MYH11 significantly inhibited this process. MYH11 up-regulates the expression of NUPR1 and activates the PI3 K/AKT pathway, which plays an important role in promoting the progression of bladder cancer.

Subsequently, by enrichment analysis, we found that the promoting effect of NUPR1 on bladder cancer was associated with the inhibition of ferroptosis. Overexpression of NUPR1 significantly reduced the content of Fe²⁺ and ROS in bladder cancer cells. Previous studies have also shown that NUPR1-mediated lipocalin-2(LCN2) expression prevents ferroptosis of cells by reducing iron accumulation and subsequent oxidative damage,³⁰ which is consistent with our results. However, whether there is a direct correlation between MYH11 and ferroptosis requires further investigation.

Tumor microenvironment (TME) refers to the non-cancer cells and components present in a tumor, including the molecules they produce and release. The continuous interaction between tumor cells and tumor microenvironment plays a decisive role in the occurrence, progression, metastasis and treatment response of tumors.³¹ Macrophages present in the tumor microenvironment are called tumor-associated macrophages (TAM). As the major infiltrating immune cells in the tumor microenvironment,^32–34 the pro-tumor effects of Tams have been widely studied, such as tumorigenesis, angiogenesis, metastasis, drug resistance and anti-tumor immunosuppression.³⁵ There are two states of TAM, namely the classical state (M1) and the alternative state (M2), in which M2 macrophages mainly perform immunosuppression and promote tumor progression.³⁶ Our results showed that MYH11 knockdown inhibited the activation of M2 macrophages, while NUPR1 overexpression promoted the activation of M2 macrophages, confirming that MYH11-mediated NUPR1 promoted bladder cancer progression by altering the tumor microenvironment.

Conclusion

In conclusion, NUPR1 is highly expressed in bladder cancer, especially in high-grade bladder cancer. MYH11 is a potential upstream regulator of NUPR1. MYH11 activates the PI3 K/AKT pathway by up-regulating the expression of NUPR1 and promotes the progression of bladder cancer by inhibiting ferroptosis and promoting M2 polarization of macrophages. The results of this study will provide new ideas for the targeted therapy of bladder cancer.

Supplemental Material

sj-docx-1-tct-10.1177_15330338241305434 - Supplemental material for Transcriptional Regulation of NUPR1 by MYH11 Activates PI3 K/AKT and Promotes Bladder Cancer Progression Through Ferroptosis and M2 Polarization of Macrophages

Supplemental material, sj-docx-1-tct-10.1177_15330338241305434 for Transcriptional Regulation of NUPR1 by MYH11 Activates PI3 K/AKT and Promotes Bladder Cancer Progression Through Ferroptosis and M2 Polarization of Macrophages by Lifeng Zhang, Li Zhang, Zebin Shi, Yuanyuan Mi, Lei Zhang, Xiaokai Shi, Shenglin Gao and Li Zuo in Technology in Cancer Research & Treatment

Footnotes

Abbreviations

Acknowledgments

We thank all the members who participated in this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Disclosure

The current study has been approved by Ethics Committee of the Changzhou No.2 People's Hospital (Approval number: [2020]KY223-01).

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Basic research project of Changzhou Medical Center, Nanjing Medical University (CMCB202319, CMCB202313). Jiangsu Province 333 Project (No. RC202202), Top Talent of Changzhou “The 14th Five-Year Plan” High-Level Health Talents Training Project (2022CZBJ057, 2022CZBJ058), Jiangsu Province Postdoctoral Research Foundation (2021K588C), Changzhou Sci&Tech Program (CJ20220146), Changzhou Health Commission Young Talent Project (No. CZQM2020065), Postdoctoral Science Startup Foundation (BSH202009, BSH202214).

ORCID iD

Li Zhang

Supplemental Material

Supplemental material for this article is available online.

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