LncRNA XLOC₀05950 Mediated Hsa-miR-542-3p Regulated The Energy Metabolism And Proliferation In Osteosarcoma Cells

BackgroundOsteosarcoma（OS）is the most common primary malignant bone cancer,whichcomes of osteoblast.OS could occur in the metaphysis of the long limbs of the extremities at any age,the most typical is the lower end of the femur and the upper end of the tibia of adolescents.Osteosarcoma cells are characterized by the formation of osteoid substrate,but their tissue forms are very different from those produced by osteoblasts.In recent years,the focus of research on osteosarcoma has been transferred to the level of biological macromolecule and gene.A variety of factors such as the over expression or lack of expression of biological macromolecules,and even gene mutation have jointly promoted the occurrence and development of osteosarcoma.People want to make breakthroughs in the biology of osteosarcoma and eventually apply it to prevention and treatment of the disease in clinical.The clinical goal of osteosarcoma research is to identify factors that affect prognosis,which can not only serve as a theoretical basis for individualized treatment,but also help to prioritize the clinical trials of new therapeutic drugs.LncRNA is a noncoding RNA with a length greater than 200 nucleotides.It is widely distributed in the human genome,and has a wide range of biological functions,such as playing as molecular bait,regulated the expression of mi RNA and mRNA as competitive endogenous RNA.In the metabolism of tumor,the metabolic reprogramming of tumor can meet the demand of energy for tumor,and also provide the basis of macromolecules for the rapid proliferation of tumor cells,and provide the acidic microenvironment to help tumor cells evade the immune system.LncRNA XLOC₀05950,which is found in the analysis of gene chips by Moran,and in previous preliminary experiments,we found that lncRNA was highly expressed in osteosarcoma.Therefore,it is of great value and significance to study the regulation and molecular mechanism of non-coding RNA（lncRNA XLOC₀05950）on the energy metabolism pathway of osteosarcoma and to provide new targets and new ideas for exploring the development,diagnosis,treatment and prognosis of osteosarcoma.PurposeThis study investigated the expression levels of lncRNA XLOC₀05950 and PFKM in OS tissues and the matched adjacent tissues,and in human osteosarcoma cell line MG63 and human osteoblast cell line hFOB1.19.CRISPR/Cas9 was used to knock out LncRNA XLOC₀05950 in MG63,and the expression of PFKM and lactate content,glucose content and cell proliferation activity and apoptosis rate in cells were observed.In addition,bioinformatics was used to predict the miRNA molecule and regulation mode of LncRNA XLOC₀05950 regulating PFKM,and the regulation mode was verified after transfection.Therefore,the aim of this study is to investigate the effect of lncRNA XLOC₀05950 on the regulation of aerobic glycolysis energy metabolism and cell proliferation and apoptosis in osteosarcoma cells.This result will be of great significance for the biological behavior of tumor and clinical research in later stage.Methods1.In the First Affiliated Hospital of Zhengzhou University from September 2015 to March 2017,25 cases of osteosarcoma and matched adjacent tissues were collected.The expression of LncRNA XLOC₀05950 and PFKM mRNA in osteosarcoma tissues and matched adjacent tissues were detected by quantitative real-time PCR（qRT-PCR）.The expression of LncRNA XLOC₀05950 and PFKM mRNA in osteoblast cells hFOB1.19 and osteosarcoma cells MG63 were detected,too.2.The method of CRISPR/Cas9 gene editing was used to knock out LncRNA XLOC₀05950 in MG63 cells.3.The osteosarcoma cells MG63 knocked out LncRNA XLOC₀05950 was labeled MG63^-/-,and the change of MG63^-/-index was detected as follow:（1）We used qRT-PCR to detecte the expression of PFKM mRNA in the osteosarcoma cells MG63^-/-.（2）The PFKM activity,glucose and lactic acid content in MG63^-/-cells were measured,and the glycolysis efficiency of osteosarcoma cells MG63^-/-was observed.（3）CCK-8 experiment was used to evaluate the changes of cell viability and proliferation in MG63^-/-.（4）Flow cytometry were used to evaluate the apoptosis effect of MG63^-/-.（5）We used western blot to investigate the expressions of PFKM protein of MG63^-/-.4.Bioinformatics software such as Diana Tools,TargetScan,miRBase was used to predicte the miRNA which is binding to PFKM and LncRNA XLOC₀05950.5.We used qRT-PCR to detecte the expression of hsa-miR-542-3p in osteosarcoma tissues and matched adjacent tissues,and osteosarcoma cells MG63^-/-and in osteoblast cells hFOB1.19.6.We used the liposomes to transfect synthetic hsa-miR-542-3p mimics and hsa-miR-542-3p negative control into human osteosarcoma cell line MG63respectively.The experiment was grouped as follow:（1）miR-542 group:cells were transfected with liposomes and hsa-miR-542-3p mimics.（2）NC group:cells were transfected with liposomes and hsa-miR-542-3p negative control.（3）Blank group:cells were transfected with only liposomes.7.The effect of hsa-miR-542-3p on osteosarcoma cells MG63 was detected,after transfection of hsa-miR-542-3p mimics.（1）We used qRT-PCR to detecte the expression of PFKM mRNA in the osteosarcoma cells MG63.（2）The PFKM activity,glucose and lactic acid content in MG63 cells were measured,and the glycolysis efficiency was observed.（3）CCK-8 experiment was used to evaluate the changes of cell viability and proliferation of MG63 cells.（4）Flow cytometry were used to evaluate the apoptosis effect of MG63 cells.（5）We used western blot to investigate the expressions of PFKM of MG63 cells.8.We used Luciferase reporter assay to confirm that the PFKM was a target gene of hsa-miR-542-3p,and the XLOC₀05950 was also a target of hsa-miR-542-3p.Results1.Compared to the adjacent tissues,the expression level of LncRNA XLOC₀05950and PFKM mRNA in OS tissues were significantly higher（P<0.01）.Beside,the expression of LncRNA XLOC₀05950 and PFKM mRNA in osteosarcoma cells MG63 was increased compared with osteoblast cells hFOB1.19（P<0.01）.2.The sequencing results showed that compared with the osteosarcoma cell MG63,the LncRNA XLOC₀05950 of MG63^-/-cells had been edited and there was a large fragment deletion,indicating that the gene editing the osteosarcoma cells MG63 of knock-out LncRNA XLOC₀05950 was successfully constructed.3.The changes of metabolism and traits in osteosarcoma cells MG63^-/-as follow:（1）PFKM mRNA expression in osteosarcoma cells MG63^-/-was decreased compared with MG63（P<0.05）.（2）PFKM activity,glucose and lactic acid content in MG63^-/-cells were all decreased（P<0.05）.The results means that the efficiency of glycolysis in MG63（-）cells is reduced.（3）CCK-8 experiment showed that the OD450 value in MG63^-/-was decreased（P<0.05）.（4）Flow cytometry showed that the apoptotic capacity was increased（P<0.05）in MG63^-/-cells.（5）Western Blot showed that the protein expression of PFKM was decreased in MG63^-/-cells.4.In bioinformatics analysis,hsa-miR-542-3p has a matching binding region with LncRNA XLOC₀05950 and PFKM.5.Compared to the adjacent tissues,the expression level of hsa-miR-542-3p in OS tissues were lower（P<0.05）.Beside,hsa-miR-542-3p expression in osteosarcoma cells MG63 was decreased compared with osteoblast cells hFOB1.19（P<0.01）.When the gene editing of knock-out LncRNA XLOC₀05950 in MG63 cells,the expression of hsa-miR-542-3p in the MG63^-/-group was significantly upregulated compared with the MG63 group,and the difference was statistically significant（P<0.05）.Knocked-out LncRNA XLOC₀05950 can increase the expression of hsa-mi R-542-3p in MG63 cells,indicating that LncRNA XLOC₀05950 can regulate the expression of hsa-mi R-542-3p in MG63 cells.6.The changes in the osteosarcoma cells MG63 after transfection of hsa-miR-542-3p mimics as follows:（1）The expression level of PFKM mRNA in transfected cells was decreased（P<0.05）.（2）PFKM activity,glucose and lactic acid content in mi R-542 group were all decreased（P<0.05）.The result means that hsa-miR-542-3p could decrease glycolysis efficiency.（3）CCK-8 experiment showed that the OD450 values in miR-542 group were decreased（P<0.05）.（4）Apoptosis of MG63 cells was significantly increased and the rate of apoptosis increased in transfected cells（P<0.05）.（5）Western blot assay showed that the expression level of PFKM was reduced in miR-542 group.7.Luciferase reporter assay showed that PFKM and LncRNA XLOC₀05950 both act as the targets of hsa-miR-542-3p.Conclusions1.In osteosarcoma tissues and MG63 cells,LncRNA XLOC₀05950 and PFKM shows high expression,while hsa-miR-542-3p is low expression.2.After knocking out LncRNA XLOC₀05950 by gene editing or transfecting hsa-miR-542-3p mimics in MG63 cells,the expression of PFKM which is the key enzyme of glucolysis pathway decreased,the intracellular glucose and lactate content decreased.Meanwhile,the proliferation of osteosarcoma cells was inhibited,and the apoptosis was promoted.3.LncRNA XLOC₀05950 can regulate the expression of PFKM by hsa-miR-542-3p,prohibit the aerobic glycolysis pathway of osteosarcoma cells,inhibit the main energy supply of tumor cells,and then regulate the proliferation and apoptosis of osteosarcoma cells.