Study On The Mechanism Of HIF-1α Regulating The Lipid Metabolism Of Tumor-Associated Fibroblasts And The Progression Of Lung Cancer

Background:Malignant tumors,or cancers,are one of the leading causes of human death.Among all the malignant cancers,lung cancer has been the highest mortality rate in the world and the second worldwide incidence rate.Lung adenocarcinoma is the most common pathological type of lung cancer,it is of great significance to study of the development mechanism of lung cancer in-depth for the diagnosis and treatment.Recent studies have found that tumorigenesis and development are closely related to tumor microenvironment(TME).Among them,cancer-associated fibroblasts(CAFs)are the main non-hematopoietic stromal cells of tumor stroma,participate in the tumor progression.Changes in cell energy metabolism process will lead to changes of cell function.Under the influence of TME,CAFs promote the growth of tumor through its metabolic reprogramming.It has been reported that the reprogramming of aerobic glycolysis or amino acid metabolism in CAFs can promote tumor progression.However,there are few studies on lipid metabolic reprogramming,which is another important energy pathway of CAFs.Elucidating the mechanism of lipid metabolic reprogramming of CAFs may provide a new strategy for regulating the function of CAFs.The hypoxia characteristic of TME is an important pathological feature in many malignant tumors,including lung cancer,which induce the expression of hypoxia-inducible factors-1α(HIF-1α).In recent years,it has been found that HIF-1αparticipates in the proliferation and survival of tumor cells by reprogramming glucose metabolism and amino acid metabolism.In addition,HIF-1α-mediated lipid metabolism is also an important factor to maintain the rapid proliferation of tumor cells.However,whether HIF-1α has an effect on lipid metabolism in CAFs is unclear.To better understand the biological characteristics of CAFs,we investigated the regulation of HIF-la on lipid metabolic reprogramming in lung adenocarcinoma CAFs and its related molecular mechanism in this study.Purpose:The purpose of this study is to investigate the expression of HIF-1α in lung cancer-associated fibroblasts,and its effect on lipid metabolism and function of fibroblasts.On this basis,the mechanism and clinical significance of HIF-1α in regulating lipid metabolism of fibroblasts are further studied to provide a new theoretical basis for the mechanism of lung cancer progression,it also provides a new strategy and target for future therapeutic intervention of lung cancer.Methods:1)Immunofluorescence(IF)was used to detect the expression of HIF-1α on lung cancer fibroblasts and normal lung fibroblasts in lung cancer patients and mice lung cancer spontaneous tumor models,as well as the clinical prognosis of lung cancer was analyzed.2)Real-time quantification-polymerase chain reaction(RT-PCR)and western blot(WB)were used to detect the fibroblast activation-related proteins in normal fibroblasts(MEF/3T3),which were treated with hypoxia,transforming growth factor-β1(TGF-β1),Lewis cell-cultured medium(LLC-CM).3)Flow cytometry was used to detect the effects of hypoxia,TGF-β1,LLC-CM on lipid droplets in normal fibroblasts(MEF/3T3).4)CRISPR-cas9 technology was used to knock out the expression of HIF-1α in fibroblasts.5)RNA-sequence was used to detect the change of lipid droplet synthesis related genes after knockout the expression of HIF-1α in fibroblasts.6)Dual luciferase report experiment and DNA pull-down experiment were taken to detect the regulation of HIF-1α on the downstream molecule stearoyl-CoA desaturase 1(SCD1)expression.7)Confocal microscopy and flow cytometry were used to detect the lipid droplets in lewis lung cancer cell(LLC)after co-cultured with fibroblasts.8)Detecting the tumor growth and the expression of PCNA in three co-injection groups of mice:single tumor cell group,tumor cell and fibroblasts co-injection group,tumor cell and SCD1 overexpressing fibroblasts co-injection group.9)IF co-staining was used to detect the expression of SCD1 in CAFs and normal fibroblasts(NFs)of lung cancer patients,and the correlation between SCD1 and HIF-la in clinical samples of lung cancer patients and the clinical prognosis of lung cancer patients were analyzed.Results:1)The expression of HIF-1Ⅰ in CAFs of lung cancer and mouse spontaneous lung cancer were higher than that in the corresponding NFs,and the higher expression level of HIF-1α in lung cancer CAFs,the shorter of total survival time.2)Hypoxia,TGF-β1 and tumor cell condition medium could up-regulate HIF-1αto promote the activation of fibroblasts and the accumulation of lipid droplets.3)HIF-1α knockout could significantly inhibit the expression of α-SMA and COL1A2 and the content of lipid droplets in fibroblasts.4)In the mouse spontaneous tumor model,the content of lipid droplets in CAFs was significantly higher than that in NFs.5)The intervention of oleic acid increased the lipid droplet in fibroblasts,thereby up-regulating the expression of fibroblast activation markers like α-SMA and COL1A2.6)RNA-sequence detection found that the knockout of HIF-1α in fibroblasts could down-regulate the expression of SCD1.DNA Pull-down was used and found that HIF-1α regulated the expression of SCD1 by binding to the promoter region of SCD1.7)Up-regulating the expression of SCD1 could promote the increase of lipid droplet in fibroblasts and strengthen the expression of α-SMA and COL1A2.8)Co-culture experiments of fibroblasts and tumor cells showed that fibroblasts with increased lipid droplets had more contact with tumor cells,and the lipid droplets content of tumor cells increased significantly.9)The co-injection of SCD1 overexpressing fibroblasts and tumor cells can significantly promote the growth of lung cancer.10)The higher expression of SCD1 in lung cancer fibroblasts,the shorter of overall survival in lung cancer patients.The expression of HIF-1α and SCD1 on fibroblasts in lung cancer tissues is positively correlated.Conclusion1)HIF-1α is highly expressed in lung CAFs and predicts a poor prognosis of patients.2)HIF-1α promotes the activation of fibroblasts by increasing the synthesis of lipid droplets in fibroblasts.3)HIF-la regulates the synthesis of lipid droplets in fibroblasts by up-regulating the expression of SCD1.4)SCD1 regulates lipid metabolism in fibroblasts and promotes the progression of lung cancer.