| PURPOSE:Lung cancer remains the leading malignant tumor in terms of incidence and mortality worldwide.Bone is one of the common metastatic sites of lung cancer,and once bone metastasis occurs,lung cancer is prone to bone-related events,which seriously affects patients’quality of life and survival.The mechanism of bone metastasis of lung cancer is still not well understood.It was found that tumor debulking treatment,ovarian removal and parathyroid hormone treatment could accelerate tumor bone metastasis;the use of inhibitors that inhibit osteoclasts in bone metastasis treatment,such as bisphosphonates and denosumab,could delay the progression of tumor bone metastasis,suggesting that active osteoclasts have a facilitating role in the progression of tumor bone metastasis.Therefore,the aim of this study was to investigate the mechanism by which osteoclast activity promotes bone metastasis in lung cancer.METHODS:(1)In vitro dormant lung adenocarcinoma cell model was constructed:dormancy was induced by serum deprivation method in lung adenocarcinoma cell lines(H1975 and A549),and cell activity was detected by CCK8,cell cycle and apoptosis changes by flow cytometry,cell senescence changes byβ-galactosidase staining,and the expression of dormancy-related markers Nanog,NR2F1,p27 and p21 was examined by Western Blot to verify whether the dormant lung adenocarcinoma cell model was successfully constructed.(2)In vitro experiments demonstrated the activation effect of activated osteoclasts on dormant lung adenocarcinoma cells:human peripheral blood mononuclear cells(PBMC)were extracted and isolated in vitro,and PBMC were induced to differentiate into mature osteoclasts(OC)by human-derived M-CSF and h-RANKL recombinant factor induction method,and the morphological characteristics,activity and specific markers of OC were identified according to cellular immunofluorescence staining,anti-tartrate acid phosphatase(TARP)staining,and bone trap resorption assay,respectively,and the culture supernatant identified as OC was collected;the collected OC supernatant was co-cultured with dormant H1975 cells for 24h,and the co-cultured cells were collected for staining with PI to detect cell cycle changes,CFAD-SE staining to detect cell proliferation changes,Transwell to detect cell migration ability,and Western Blot to detect expression changes of Epithelial-Mesenchymal Transition(EMT)markers E-cadherin,N-cadherin and dormancy-related markers Nanog,NR2F1,p27,p21 to verify whether OC could activate dormant lung adenocarcinoma cells.(3)In vivo experiments to verify that osteoclast activation promotes lung adenocarcinoma cell proliferation:experimental group(s RANKL injection group)and control group(PBS injection group)were set up.s RANKL group was injected intraperitoneally with murine-derived RANKL(1 mg/kg per nude mouse)in BALB/C nude mice from 6 to 8 weeks old for 3consecutive days,and the control group was injected intraperitoneally with the same dose of PBS on day 4 with GFP~+-DID~+-labeled A549 cells(1×106cell/each)were injected into the tibia of the nude mice,and the distribution of lung adenocarcinoma cells was observed 96 h after cell injection using a biological live imaging system,and the proportion of proliferating lung adenocarcinoma cells in the tibia was isolated for flow cytometry detection.(4)Gene expression differences analyzed by transcriptome sequencing before and after OC supernatant treatment of dormant lung adenocarcinoma cells and explore the potential mechanism of action:total RNA from OC supernatant treated dormant A549 cells was extracted for transcriptome sequencing after 24h,and the data were filtered and differentially analyzed by R software,etc.GO and KEGG enrichment analyses were performed to screen for changes in cellular functions and signaling pathways involved in the differential genes,and by Western Blot preliminary validation of the potential molecular mechanism of OC supernatant activation of dormant lung adenocarcinoma cells.RESULTS:(1)Compared with normal culture,lung adenocarcinoma cells in the serum deprivation group showed reduced activity,cell cycle arrest in G0/G1 phase,cell proliferation arrest,no apoptosis and senescence,and upregulated expression of dormancy-related markers,suggesting that the serum deprivation method can successfully construct a dormant lung adenocarcinoma cell model;(2)Osteoclasts induced by human-derived M-CSF and h-RANKL recombinant factor induction method.Immunofluorescence results showed that OCs were giant cells containing multiple nuclei,and TRAP staining showed positive cells with purplish-red cytoplasm and a significant increase in the number and area of bone resorption traps,suggesting that human peripheral blood mononuclear cells could be successfully induced as osteoclasts;the cell cycle of dormant lung adenocarcinoma cells after OC supernatant treatment resumed progression,and proliferation,migration ability and EMT were significantly enhanced.Meanwhile,the expression of dormancy-related markers was down-regulated,suggesting that OC could activate dormant lung adenocarcinoma cells;(3)The results of in vivo imaging of animals in the s-RANKL-injected group showed that the fluorescence intensity of lung adenocarcinoma cells in bone was significantly higher than that in the control group,and the results of flow cytometry detection indicated that the number of proliferating lung adenocarcinoma cells in the bone of nude mice in the s-RANKL-injected group was significantly higher than that in the control group,suggesting that the activation of osteoclasts could promote the proliferation of lung adenocarcinoma cells;(4)Transcriptome sequencing data analysis of A549 treated with OC supernatant for 24h yielded 3144 differential genes,of which1592 genes were expressed up-regulated and 1552 genes were expressed down-regulated;GO functional enrichment of differential genes showed that differential genes were significantly enriched in the biological function of extracellular matrix,suggesting that OC supernatant may mediate extracellular matrix changes affecting dormant lung adenocarcinoma cell growth,proliferation,migration and metabolism.The enrichment analysis of KEGG signaling pathway showed that the up-regulated genes in OC supernatant-treated lung adenocarcinoma cells were enriched in cell cycle,p53,extracellular matrix composition and other signaling pathways,while the down-regulated differential genes were enriched in cell autophagy,extracellular junctions,mTOR and other signaling pathways;this suggested that OC supernatant could activate cell cycle,p53,mTOR and other signaling pathways through either up-regulated genes or down-regulated genes,Western Blot results showed that AMPK dephosphorylation and mTOR phosphorylation increased after OC culture supernatant treatment,indicating that AMPK/mTOR signaling pathway was activated in lung adenocarcinoma cells,suggesting that OC promotes proliferation and migration of dormant lung adenocarcinoma cells through activation of AMPK/mTOR signaling,etc.CONCLUSION:Osteoclasts can promote the progression of lung adenocarcinoma bone metastasis by activating dormant lung adenocarcinoma cells,and the molecular mechanism is related to the AMPK/mTOR signaling pathway. |
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