| ObjectiveThis study focused on the clinical characteristics of the rapid growth and metastasis of OS.The “Warburg effect-energy metabolism” was used as the breakthrough point,combing with the current hot spot lncRNA playing an important role in the progression of OS.The scientific hypothesis was proposed and validated:By adsorption of mi R-34a-5p,lncRNA C1QTNF1-AS1 simultaneously promoted the expression of LDHA catalyzing pyruvate to produce lactic acid and PDK inhibited pyruvate aerobic oxidation,regulating the Warburg effect in a multi-targeted interaction to promote the development of tumors.Methods(一)To construct a prognostic model based on co-expression network prediction and energy metabolism characteristic genes of OS1.By downloading the data of energy metabolism genes from the TARGET database and GSE21257 Gene Chip,we applied Nonnegative matrix factorization(NMF)to performed cluster analysis according to mortality and prognosis.Finally,molecular subtypes were determined for the prognosis of energy metabolism of OS.2.Based on the Weighted Gene Co-expressed Network Analysis(WGCNA),Univariate Cox proportional analysis,the LASSO Cox algorithm,and Multivariate Cox survival analysis,a prognostic model related to energy metabolism of OS was determined.3.Univariate and multivariate Cox regressions were used to analyze prognostic models related to energy metabolism in clinical information contained in the TARGET training set,TARGET validation set,and GSE21257 dataset.(二)Identification of key proteins involved in the Warburg Effect of energy metabolism of OS by lncRNA C1QTNF1-AS1 adsorption of mi R-34a-5p1.The Ch IPBase,NONCODE,star Base v2.0,and Lnc Base Predicted v.2 and other databases were applied to predict mi RNAs bound to the above 7 lncRNAs and predicted the mi RNAs of LDHA/PDK1-4,then,taking the two parts of mi RNA intersection.lncRNA and mi RNAs were finally determined.2.Expression levels of lncRNA C1QTNF1-AS1 and mi R-34a-5p,LDHA,PDK1,and PDK3 were detected in OS cell lines(MG63)and normal osteoblast cell line(h FOB1.19)to clarify the correlation of the expression in OS.(三)Molecular mechanism of lncRNA C1QTNF1-AS1 absorbing mi R-34a-5p multi-target linkage to regulate the Warburg Effect of OS energy metabolism1.We constructed the lncRNA C1QTNF1-AS1 overexpressed/knockdown plasmid,transfected into MG63 cells,screened out stably expressed cell lines,dividing them into NC group,lncRNA C1QTNF1-AS1 overexpressed group,and sh-C1QTNF1-AS1 group.Then,transwell chamber experiments were detected cell invasion ability,ATP contents were measured to evaluate cell energy metabolism,the scratch test was used to detect cell proliferation,and glucose(GO)is used to detect the consumption of glucose.2.RT-PCR,Western Blot,ELISA,and Dual-Luciferase Reporter Assay were applied to detect the expression levels of LDHA,PDK1,and PDK3 in the NC group,lncRNA C1QTNF1-AS1 overexpressed group,and sh-C1QTNF1-AS1 group.Results(一)To construct a prognostic model based on co-expression network prediction and energy metabolism characteristic genes of OS1.The NMF algorithm was performed cluster analysis on the energy metabolism genes downloaded from the TARGET database and lncRNA osteosarcoma Chip data of GSE21257 according to mortality and prognosis,selecting the standard“Brunet” to perform 50 cycles.Finally,two lncRNA subtypes were determined for the prognosis of energy metabolism of OS.2.Based on the WGCNA algorithm,the significant modules corresponding to the clinical characteristics of lncRNA subtypes related to energy metabolism were screened out.Obtained 492 lncRNAs in the significant modules were applied Univariate Cox proportional analysis,and 65 lncRNAs were found to have significant expression(p < 0.05).The LASSO Cox algorithm was applied to analyze the identified 65 lncRNA,and 3-fold cross-validation was used to construct the model.When lambda = 0.101608,the model reached the optimum,and 13 lncRNAs were obtained as the final genes.Finally,13 lncRNAs obtained were analyzed by multivariate Cox survival analysis,and 7 lncRNAs with the lowest AIC value(AIC = 156.28)were retained as the prognostic model of energy metabolism of OS.3.In the TARGET training set,TARGET validation set,and GSE21257 dataset,it was shown that the high-risk group was significantly associated with survival,indicating that the established 7-gene signature prognostic model is a prognostic indicator independent of other clinical factors,with a good predictive performance in clinical application.(二)Identification of key proteins involved in the Warburg Effect of energy metabolism of OS by lncRNA C1QTNF1-AS1 adsorption of mi R-34a-5p1.7 lncRNAs targeted mi RNAs and LDHA / PDK1-4 targeted mi RNAs were predicted by databases.Finally,mi RNAs binding to lncRNA C1QTNF1-AS1 were determined,including mi R-34a-5p,mi R-346,mi R-147 a,mi R-484,mi R-212-5p,and other mi RNAs,which were all targeted LDHA,and PDK.Mi R-34a-5p was selected as the research object of this study.2.Compared with the normal osteoblast cell line(h FOB 1.19),lncRNA C1QTNF1-AS1 and mi R-34a-5p were significantly low expressed in MG63 osteosarcoma cells(**p < 0.01),while LDHA,PDK1,and PDK3 were significantly high expressed(**p < 0.01).(三)Molecular mechanism of lncRNA C1QTNF1-AS1 absorbing mi R-34a-5p multi-target linkage to regulate the Warburg Effect of OS energy metabolism1.Compared with the lncRNA C1QTNF1-AS1 overexpression group and NC group,the constructed sh-C1QTNF1-AS1 group has higher ATP consumption,enhanced energy metabolism,and enhanced migration and invasion capabilities,which were beneficial to the further metastasis and spread of tumor cells;2.The expression levels of LDHA,PDK1,and PDK3 in the lncRNA C1QTNF1-AS1 overexpression group were significantly reduced.The mi R-34a-5p could bind to the PDK3 and LDHA promoter regions,and the mi R-34a-5p plasmid transfections had reversal effects on the genes transcription of PDK3 and LDHA in the shC1QTNF1-AS1 group,further confirming the molecular mechanism of lncRNA C1QTNF1-AS1 absorption on mi R-34a-5p multi-targeted PDK3 and LDHA linkage in regulating the Warburg effect of energy metabolism of OS.ConclusionlncRNA C1QTNF1-AS1 is a molecular target related to energy metabolism in osteosarcoma,and the targeted binding of mi R-34a-5p affects the Warburg effect by regulating LDHA and PDK3.Results clarified the role and molecular mechanism of lncRNA C1QTNF1-AS1 in regulating the Warburg effect through mi R-34a-5p multitargeting linkage,providing a more powerful theoretical basis for the application of lncRNA C1QTNF1-AS1 in the diagnosis and targeted therapy of osteosarcoma. |
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