| Background:Thyroid cancer is one of the most common malignancies worldwide with a sharp increase rate in the last two decades,among which,papillary thyroid cancer(PTC)accounts for up to 80%.Although the overall prognosis of PTC is good,there are still 10%-15% of patients with recurrence,5% of patients have distant metastasis to lung and bone tissue,and some patients have PTC-specific death.In recent years,the use of tyrosinase inhibitors has prolonged median progression-free survival in a subset of patients with refractory PTC,but overall survival has not increased and the occurrence of serious side effects and drug resistance still exist.Therefore,it is urgent to explore the mechanisms of PTC development at the molecular level to find potential therapeutic targets and explore safer and more effective treatments.Reprogramming of lipid metabolism is currently a hot research issue in oncology.As energy substances,cell membrane components,and signalling molecules,lipids play important roles in tumor development.Studies on the reprogramming of lipid metabolism in PTC are still lacking,so further exploration is needed to deepen the understanding of it.In this study,we finally identified PDE5 A,a key gene related to lipid metabolism in PTC,by screening lipid metabolism-related genes from public databases and performing bioinformatics analysis of differentially expressed genes of PTC in the TCGA database.PDE5A is a member of the phosphodiesterase family,which can affect the nucleotide signaling pathway by specifically hydrolyzing the intracellular second messenger c GMP.In recent years,many studies have found that PDE5 A is closely related to the development of tumors such as Hodgkin’s lymphoma,melanoma and colorectal cancer,but its role in PTC has not been studied in depth.In this study,we intend to analyze the relationship between lipids and lymph node metastasis in the central region of PTC patients,and explore the mechanism by which PDE5 A regulates the development of PTC through lipid metabolism,in order to provide a theoretical basis for the diagnosis and treatment of PTC targeting lipid metabolism.Methods:1.A retrospective analysis was performed on 308 patients with PTC who underwent thyroid lobectomy with central lymph node dissection in the clinical center between March2021 and September 2021.Histopathological characteristics of the tumor were collected and analyzed.After adjusting for confounding factors by propensity score matching,the correlations between serum triglycerides,total cholesterol,low density lipoprotein,high density lipoprotein,and central lymph node metastasis were examined separately by the Logistic regression analysis.2.RNA-Sequencing data and clinicopathological data of the THCA dataset were downloaded from the Cancer Genome Atlas database.Differentially expressed genes in PTC tumor tissues and paracancerous tissues were analyzed.The genes related to lipid metabolism phenotype were downloaded through the Var Elect tool in the Genecards database.The intersecting genes were taken and genes associated with PTC lymph node metastasis were screened using the Logistic regression model and the least absolute shrinkage and selection operator.PDE5 A was then confirmed as the target gene from candidate genes by the Human Protein Atlas database and literature research.We collected the tumor tissues and corresponding paraneoplastic tissues of 24 PTC patients.RT-qPCR and Western Blot were performed to compare the expression levels of PDE5 A between them,and the correlations between the relative expression of PDE5 A and clinicopathological characteristics of the patients were analyzed.3.The expression of PDE5 A in Nthy-ori 3-1,BCPAP,KTC-1,K1 and TPC-1 cell lines were detected by RT-qPCR and Western Blot.The KTC-1 was selected to knock down PDE5 A through si RNA,and the BCPAP was selected to establish LV-PDE5 A with stable overexpression of PDE5 A and negative control cell line LV-NC using lentiviral vectors.The effects of knockdown and overexpression of PDE5 A on cell proliferation,apoptosis,migration and invasion ability were verified by CCK8 cell proliferation assay,clone formation assay,flow cytometry Annexin V/PI double-staining apoptosis assay,scratch assay and Transwell invasion assay,respectively.Finally,the effect of overexpression of PDE5 A on cell tumorigenic ability was examined in vitro by establishing a Patient-derived xenografts model of LV-PDE5 A cell line,and the expression of related proteins was detected by immunohistochemistry.4.PDE5A-related lipid metabolism pathway and its downstream target HSL were predicted by protein-protein interaction analysis and pathway enrichment.Changes in the contents of triglycerides,glycerol and free fatty acids in cells after knockdown or overexpression of PDE5 A were examined respectively,and changes in the contents of lipid droplets in cells were observed using Oil Red O staining and BODIPY fluorescence staining.The expression of p HSL(Ser660)was detected using Western Blot,and the effects of HSL inhibitor on the lipid composition and cell proliferation,migration and invasion ability in LV-PDE5 A cells were observed in the rescue experiment using HSL inhibitor.Finally,the changes in the AKT/mTOR pathway by PDE5 A and HSL inhibitors were detected using Western Blot.Results:1.After adjusting for confounding factors by propensity score in our retrospective cohort study,multivariate logistic regression showed that male,tumor diameter >10 mm,and TG ≤1.7 mmol/L were risk factors for central lymph node metastasis of PTC.The levels of serum cholesterol,low density lipoprotein and high density lipoprotein were not statistically related to the occurrence of central lymph node metastasis.2.Differentially expressed genes between paired papillary thyroid cancer and normal thyroid tissues in the TCGA database were analyzed,and a total of 60 differentially expressed genes related to lipid metabolism were identified.Logistic regression showed that 15 of them were associated with lymph node metastasis.PDE5 A was then confirmed as the study target through the Human Protein Atlas database and literature.The expression of PDE5 A in PTC tumor tissues was significantly higher than that in paracancerous tissues,and a strong positive correlation existed between the PDE5 A expression was significantly associated with lymph node metastasis and capsule invasion.3.Knockdown of PDE5 A significantly impeded the proliferation,migration and invasion ability and increased the apoptosis rate of KTC-1 cells;overexpression of PDE5 A significantly promote the proliferation,migration and invasion ability of BCPAP cells,but had no effect on the apoptosis rate.In in vitro experiments,the patient-derived xenografts of LV-PDE5 A cells in nude mice resulted in higher tumor growth rate and final tumor volume and weight than those of LV-NC cells.Immunohistochemistry results showed that the expressions of PDE5 A and Ki67 in the tumors of nude mice in the overexpression group were significantly higher than those of the control group.4.The content of triglyceride was increased and the contents of glycerol and free fatty acid decreased with the knockdown of PDE5 A in KTC-1 cells.The intracellular lipid droplets were also increased in KTC-1 cells,and the expression of phospho-HSL was decreased.Overexpression of PDE5 A induced the decrease of triglyceride,while the contents of glycerol and free fatty acid were increased,and intracellular lipid droplets were decreased in BCPAP cells.Western Blot showed that the expression of phospho-HSL was increased.HSL inhibitors reduced the proliferation,migration and invasion ability of LVPDE5 A cells,and Western Blot assay showed that overexpression of PDE5 A could activate the AKT/mTOR pathway.HSL inhibitors reversed the activation of this pathway.Conclusions:1.Serum TG ≤1.7 mmol/L was significantly associated with central lymph node metastasis in patients with PTC.2.PDE5 A is an oncogene in PTC,and its high expression is associated with the capsule invasion and central lymph node metastasis of PTC.3.PDE5 A can regulate the proliferation,migration and invasion ability of PTC cells in vitro,and overexpression of PDE5 A can improve the tumorigenic ability of BCPAP cells in nude mice.4.Overexpression of PDE5 A could promote intracellular lipolysis by upregulating the expression of p HSL(Ser660)and promoted the proliferation,migration and invasion ability of PTC cells;inhibition of p HSL(Ser660)could rescue the enhancement of the malignant phenotype of cells caused by overexpression of PDE5 A,a process closely related to the activation and inhibition of AKT/mTOR pathway. |
PDF Full Text Request