| Objective: Heart failure is an important cause of non-cancer-related death for patients suffering from lung cancer.Current studies mainly focus on the cardiotoxicity of anti-cancer treatment,but it has been found that myocardium remodeling coexists with lung cancer before anti-cancer treatment,indicating that lung cancer induces direct adverse effects on the heart.However,the mechanism is not clear yet.Exosomes are heterogeneous membranous vesicles with a diameter of about 40-160 nm secreted by almost all living cells.Exosomes are able to stably carry a variety of active substances such as mi RNAs and transport them to recipient cells to complete intercellular communication.The role of exosomes in non-metastatic effects of tumors on remote organs has been increasingly valued.It has been reported that lung cancer may rewire the circadian rhythm of other organs through STAT3 signaling pathway.Rev-erbα,as a negative regulator in the circadian timedelayed transcription-translation feedback loop,is essential for the normal cardial structure and function.This study aims to investigate whether lung tumor derived-exosomal mi R-221-3p induces myocardium remodeling through STAT3/Rev-erbα signaling pathway.Methods: C57BL/6J mice were injected subcutaneously with LLC cells to establish transplant lung tumor bearing models.Heart function was evaluated by cardiac echo,and structures were assessed by cardiac weighing and tissue section staining.Western blot and PCR were used to detect the expression of biomarkers of myocardial hypertrophy and fibrosis.In vitro,HL-1 cardiomyocytes and NIH3T3 fibroblasts were treated with cultured medium of LLC cells.The phosphorylation of STAT3,the expression of Rev-erbα and biomarkers of myocardial hypertrophy and fibrosis were detected by Western blot and PCR.Phosphorylated STAT3 nuclear translocation was showed by immunofluorescence.The section areas of cardiomyocytes were evaluated by crystal violet and phalloidin staining.The proliferation and migration of fibroblasts were assessed by CCK8 and scratch test.STAT3 phosphorylation inhibitor Stattic and Rev-erbα agonist SR8278 were used to investigate the changes of cardiomyocyte and fibroblast phenotype and biomarkers.To explore the regulation of STAT3 on Rev-erbα,transient transfection of si RNA and plasmid were applied to silence and overexpress STAT3,respectively.Exosomes in the cultured medium of LLC lung cancer cells and MLE-12 lung epithelial cells were extracted by ultracentrifugation.Immunofluorescence was used to detect whether exosomes with staining label were uptaken into cells.LLC derived-exosomes were added into cardiomyocytes and fibroblasts,and Western blot and PCR were used to identify the expression levels of STAT3/Rev-erbα signaling pathway,as well as the expression levels of myocardial hypertrophy and fibrosis-related markers.SOCS1 repressing STAT3 phosphorylation was presumed as the target gene,and mi R-221-3p was selected through bioinformatics analysis.The levels of mi R-221-3p in the heart and subcutaneous tumor tissues of tumor-bearing animals were compared by PCR,as well as among cardiomyocytes,fibroblasts,lung cancer cells and lung epithelial cells.The expression of mi R-221-3p in exosomes from lung cancer cells and lung epithelial cells was also quantified.The levels of the mi R-221-3p synthesis precursor pri-mi R-221-3p in cardiomyocytes and fibroblasts given LLC cultured medium were detected to verify the source of mi R-221-3p.mi R-221-3p mimics and inhibitors were transfected to overexpress and inhibit mi R-221-3p,and the expression of myocardial hypertrophy and fibrosis-related biomarkers and SOCS1/STAT3/Rev-erbα signaling pathway were detected in cardiomyocytes and fibroblasts,respectively.Results: In vivo,mice with subcutaneous tumors had larger heart weight/tibial length,thick ventricular wall,and significant myocardial fibrosis.The expression of phosphorylation STAT3 and Rev-erbα increased in left ventricular tissue,as well as myocardial hypertrophy markers ANP and BNP,and fibrosis biomarkers COL3A1.In vitro,the culture medium of LLC cells promoted STAT3 phosphorylation and nuclear transposition,and upregulated Rev-erbα expression in both cardiomyocytes and fibroblasts.LLC culture medium also induced cardiomyocytes hypertrophy with elevated levels of ANP,BNP,MYH7 B and fibroblasts activation with increased expression of COL1A1,COL3A1,αSMA.Stattic and SR8278 could reverse these effects.The expression of Rev-erbα decreased with STAT3 inhibition,and increased with STAT3 overexpression and activation.Exosomes isolated from LLC and MLE-12 culture medium could be uptaken into cardiomyocytes and fibroblasts.Compared with MLE-12-derived exosomes,LLC-derived exosomes significantly activated STAT3/Rev-erbα signaling pathway,and induced cardiomyocytes hypertrophy and fibroblasts transformation.The levels of mi R-221-3p in subcutaneous tumor were significantly higher than in normal lung tissues,and were also higher in heart tissues of tumor-bearing mice.PCR detected more mi R-221-3p in LLC cells than in MLE-12 lung epithelial cells,HL-1 cardiomyocytes,and NIH3T3 fibroblasts,but the expression of precursor pri-mi R-221-3p did not change significantly.The expression of SOCS1 reduced with mi R-221-3p mimic transfection,and the expression of phosphorylated STAT3,Rev-erbα,ANP,BNP and MYH7 B in cardiomyocytes,and COL1A1,COL3A1 and αSMA in fibroblasts was increased.mi R-221-3p inhibitors could ameliorate such effects.Conclusions: Myocardium remodeling occurred in mice with Lewis lung adenocarcinoma subcutaneous transplanted tumor.LLC cells culture medium induced STAT3 phosphorylation activation and Rev-erbα upregulation,resulting in cardiomyocyte hypertrophy and fibroblast proliferation and activation.Lung tumor-derived exosomal mi R-221-3p could be responsible for such adverse effects by repressing SOCS1 expression and activating the STAT3/Rev-erbα signaling pathway improperly.Part Ⅰ: STAT3/Rev-erbα Signaling Pathway in Myocardium Remodeling of Subcutaneous Transplanted Lung Tumor Mice ModelObjective: Heart failure is an important cause of long-term non-cancer-related death in adult patients with lung cancer,but the mechanism of myocardium remodeling in early stage is still unclear.Studies have reported that lung cancer may influence circadian rhythm of remote organs through STAT3 signaling pathway.Cardiac Rev-erbα-knockout mice showed myocardial remodeling and fatal heart failure.However,the relationship between STAT3 and Rev-erbα in the heart and the impact of lung cancer on them have not been reported.It would be explored that the role of STAT3 phosphorylation in regulating Rev-erbα expression during myocardial remodeling in mice with subcutaneous transplanted lung tumors.Methods: HL-1 cardiomyocytes and NIH3T3 fibroblasts were treated with LLC lung cancer cells cultured medium.The expression of STAT3 phosphorylation,Rev-erbα and biomarkers of myocardial hypertrophy and fibrosis was detected by Western blot and PCR.Immunofluorescence showed phosphorylated STAT3 nuclear translocation.Crystal violet and phalloidin staining were used to calculate section area of cardiomyocytes.Proliferation and migration of fibroblasts were estimated by CCK8 and scratch test.STAT3 phosphorylation inhibitor Stattic and Rev-erbα activity inhibitor SR8278 were co-treated with LLC cultured medium,and indicators of cardiomyocyte hypertrophy and fibroblast activation were detected.Transient transfection of si RNA and plasmid were applied to investigate regulation of Rev-erbα through STAT3.C57BL/6J mice were subcutaneously injected with LLC lung cancer cells to construct lung cancer model.After 10 days of tumor bearing,echocardiography was performed.Heart weighing and tissue section staining were conducted after sacrifice,and left ventricular tissues were grinded for Western blot and PCR.The extent of myocardial hypertrophy and fibrosis was compared between tumor-bearing mice and control individuals.Results: In vitro,LLC culture medium induced STAT3 phosphorylation activation in cardiomyocytes and fibroblasts,promoted nuclear transposition,increased expression of Rev-erbα and myocardial hypertrophy and fibrosis biomarkers.Cardiomyocyte hypertrophy and fibroblast activation could be alleviated by Stattic and SR8278.When the expression level of STAT3 is reduced by si RNA,the expression of Rev-erbα also decreased,which was upregulated with STAT3 overactivation by plasmid.In vivo,mice with transplanted lung tumor displayed elevated heart weight/tibial length and ventricular wall thickening.Sirius red staining showed significant myocardial fibrosis.Western blot and PCR indicated increased expression of phosphorylated STAT3,Rev-erbα,COL3A1,ANP and BNP in left ventricular tissues.Conclusions: LLC cells cultured medium induced STAT3 phosphorylation activation in cardiomyocytes and fibroblasts,upregulated Rev-erbα expression,and led to cardiomyocyte hypertrophy and fibroblast transformation.Myocardial remodeling was also observed in mice with Lewis lung adenocarcinoma subcutaneous transplanted tumor.Part Ⅱ: Lung Tumor-Derived Exosomal mi R-221-3p Regulates STAT3/ Rev-erbα Signaling Pathway in Myocardium RemodelingObjective: Lung cancer cells cultured medium can induce cardiomyocyte hypertrophy and fibroblast activation,but the factors exerted the effects are uncertain.Exosomes are heterogeneous membranous vesicles secreted by living cells with a diameter of about 40-160 nm,which can stably carry a variety of active substances such as mi RNA and transport them to recipient cells for intercellular communication.At present,exosomes are valued in tumorigenesis and development.Exosomes contact tumor cells and non-tumors for tumor microenvironment and macroenvironment,which contribute to the adverse effects of tumors on remote organs and systems.This section aims to explore whether lung tumor-derived exosomes and exosomal mi R-221-3p have an impact on myocardial hypertrophy and fibrosis through STAT3/Rev-erbα signaling pathway.Methods: The exosomes from LLC lung cancer cells and MLE-12 lung epithelial cells cultured medium were extracted by ultracentrifugation.Immunofluorescence detected whether PKH26 stained exosomes were uptaken into the cells.Co-cultured exosomes with HL-1 cardiomyocytes and NIH3T3 fibroblasts,Western blot and PCR were used to detect the expression of STAT3/Rev-erbα signaling pathway and biomarkers related to cardiac hypertrophy and fibrosis.The possible effector mi R-221-3p in exosomes was screened by bioinformatics analysis,and its target gene was predicted to be STAT3 phosphorylation negative feedback factor SOCS1.The levels of mi R-221-3p in heart and subcutaneous tumor tissues were compared between tumor-bearing and control group.PCR detected mi R-221-3p in cardiomyocytes,fibroblasts,lung cancer cells and lung epithelial cells,as well as exosomes from lung cancer cells and lung epithelial cells.pri-mi R-221-3p,the synthesis precursor of mi R-221-3p,was also tested in exosomes treated cardiomyocytes and fibroblasts.mi R-221-3p mimic and inhibitor were transfected to overexpress and inhibit mi R-221-3p,respectively,and the expression level of SOCS1/STAT3/Rev-erbα signaling pathway was detected to investigate whether exosomal mi R-221-3p mediates LLC cultured medium-induced cardiomyocyte hypertrophy and fibroblast phenotype transformation.Results: Exosomes were effectively isolated and stained,and immunofluorescence showed high fluorescence intensity in cardiomyocytes and fibroblasts,proving that exosomes could be uptaken into cells.Compared with MLE-12 lung epithelial cell-derived exosomes,LLC lung cancer cell-derived exosomes showed significant activation of STAT3/Rev-erbα signaling pathway in HL-1 cardiomyocytes and NIH3T3 fibroblasts,and the expression of cardiomyocyte hypertrophy markers and fibroblast phenotypic transformation markers also increased.The level of mi R-221-3p in subcutaneous tumor tissues was significantly higher than that in lung tissues,and the level of mi R-221-3p in the heart tissues of mice in the tumor-bearing group was significantly higher than that in the control group.The expression of mi R-221-3p in LLC was much higher than in MLE-12 lung epithelial cells,HL-1 cardiomyocytes,and NIH3T3 fibroblasts.Stimulated by LLC cultured medium,the expression of mi R-221-3p in NIH3T3 fibroblasts and HL-1 cardiomyocytes increased significantly without precursor pri-mi R-221-3p ascending.The expression of SOCS1 decreased after mi R-221-3p mimic transfection,with STAT3 activation and Rev-erbα upregulation.The expression of ANP,BNP and MYH7 B in cardiomyocytes and the expression of COL1A1,COL3A1 and αSMA in fibroblasts were all increased.mi R-221-3p inhibitor reversed these effects by antagonizing downstream target genes binding sites with mi R-221-3p.Conclusions: mi R-221-3p carried by LLC lung cancer cells-derived exosomes inhibited SOCS1 expression.STAT3/Rev-erbα signaling pathway was then activated and finally led to myocardial hypertrophy and fibrosis. |
