【作者】 王亚东；

【导师】 李月白；

【作者基本信息】 郑州大学 ， 生物化学与分子生物学， 2018， 硕士

【摘要】 研究背景骨肉瘤（osteosarcoma,OS）是一种起源于骨母细胞,最常见的原发性恶性骨肿瘤,可发生在任何年龄,好发于四肢长骨干骺端,最为典型的是青少年的股骨下端与胫骨上端。骨肉瘤细胞以形成骨样基质为特征,但其组织形态与成骨细胞产生的骨质存在着很大的差异。近年来,针对骨肉瘤的研究重点已经转移到生物大分子与基因层次。生物大分子的异常高表达或低表达,基因突变等多种因素促进了骨肉瘤的发生与发展。人们希望从骨肉瘤的生物学研究方面取得突破并最终用于临床达到对该病的防治。骨肉瘤研究的临床目标是找到影响预后的因子,其不仅可以作为个体化治疗的理论依据,还有助于新治疗药物的临床试验的优选。长链非编码RNA（long non-coding RNA,lncRNA）是一种长度大于200核苷酸（nucleotide,nt）的非编码RNA,其在人体基因组中分布非常广泛,且具有广泛的生物学功能,如发挥分子诱饵作用,作为ceRNA调控miRNA与mRNA的表达。在肿瘤的代谢中,肿瘤的代谢重编程既满足了肿瘤对能量的需求,又提供了肿瘤细胞快速增殖的大分子物质基础,提供的酸性微环境有助于肿瘤细胞逃避免疫系统。LncRNA XLOC₀05950由Moran等在基因芯片的分析中发现,我们在前期预实验中发现此lncRNA在骨肉瘤组织中高表达。因此,研究非编码RNA（lncRNA XLOC₀05950）对骨肉瘤能量代谢途径的调节及其作用的分子机制,为探讨骨肉瘤的发生发展、诊治及预后提供新靶点和新思路具有重要价值和意义。研究目的本实验首先检测骨肉瘤患者组织标本及其配对的癌旁组织与正常成骨细胞hFOB1.19和骨肉瘤细胞MG63中LncRNA XLOC₀05950及磷酸果糖激酶（Phosphofructokinase Muscle,PFKM）的表达差异。采用CRISPR/Cas9基因编辑方法敲除MG63中LncRNA XLOC₀05950,观察细胞中PFKM的表达、乳酸含量、葡萄糖含量和细胞增殖活性及凋亡率的变化。并使用生物信息学方法预测LncRNA XLOC₀05950调控PFKM的微小RNA（MicroRNA,miRNA）分子及调控方式,转染后验证其调控方式。因此,本研究旨在探讨lncRNAXLOC₀05950对骨肉瘤细胞有氧糖酵解能量代谢途径的调节及细胞增殖与凋亡的影响,此结果将对肿瘤生物学行为和后期的临床研究具有重大意义。研究方法1.在郑州大学第一附属医院（2015年9月至2017年3月期间）收集25例骨肉瘤患者的癌组织及其配对的癌旁组织,使用荧光实时定量PCR（quantitative real-time PCR,qRT-PCR）检测已收集组织标本中LncRNA XLOC₀05950与PFKM mRNA的表达。并检测成骨细胞hFOB1.19和骨肉瘤细胞MG63中LncRNA XLOC₀05950与PFKM mRNA的表达。2.使用CRISPR/Cas9基因编辑方法敲除骨肉瘤MG63细胞中LncRNA XLOC₀05950。3.敲除LncRNA XLOC₀05950后的骨肉瘤细胞MG63标为MG63^-/-,检测细胞MG63^-/-指标的变化:（1）qRT-PCR检测MG63^-/-中PFKM mRNA的表达变化。（2）检测MG63^-/-中PFKM活性、葡萄糖和乳酸含量,观察骨肉瘤细胞MG63的糖酵解效率变化。（3）CCK-8检测MG63^-/-的细胞活性和增殖变化。（4）流式细胞术检测MG63^-/-细胞凋亡率的变化。（5）Western Blot检测MG63^-/-中PFKM蛋白表达的变化。4.采用Diana Tools,TargetScan,miRbase等生物信息学软件预测与LncRNA XLOC₀05950及PFKM结合的miRNA。5.qRT-PCR检测骨肉瘤组织和其癌旁组织及骨肉瘤细胞MG63、MG63^-/-和成骨细胞hFOB1.19中hsa-miR-542-3p的表达。6.合成hsa-miR-542-3p mimics和hsa-miR-542-3p negative control,采用脂质体转染法将其分别转染入MG63细胞中,实验分组:（1）miR-542组:转染试剂脂质体及hsa-miR-542-3p mimics;（2）NC组:转染试剂脂质体及hsa-miR-542-3p negative control;（3）Blank组:转染试剂脂质体。7.骨肉瘤细胞MG63转染hsa-miR-542-3p mimics后,检测hsa-miR-542-3p对其细胞影响:（1）qRT-PCR检测细胞中PFKM的mRNA表达水平。（2）检测细胞中PFKM活性、葡萄糖和乳酸含量,观察MG63细胞糖酵解效率改变。（3）CCK-8检测MG63细胞的活性和增殖。（4）流式细胞术观察MG63细胞凋亡率的变化。（5）Western Blot检测MG63细胞中PFKM蛋白的表达。8.双荧光素酶报告实验证实:PFKM是hsa-mi R-542-3p作用的靶基因,LncRNA XLOC₀05950是hsa-miR-542-3p作用的靶标。研究结果1.与癌旁组织比较,骨肉瘤组织中LncRNA XLOC₀05950及PFKM mRNA的表达水平均显著升高（P<0.01）;与成骨细胞hFOB1.19比较,骨肉瘤细胞MG63中LncRNA XLOC₀05950及PFKM mRNA的表达水平显著升高（P<0.01）。2.测序结果显示,与正常骨肉瘤细胞MG63相比,MG63^-/-细胞的lncRNA XLOC₀05950已被编辑,出现大片段缺失,表明成功构建了基因编辑敲除lncRNA XLOC₀05950的骨肉瘤细胞MG63^-/-。3.MG63^-/-细胞中代谢及性状的改变:（1）MG63^-/-中PFKM mRNA表达明显降低（P<0.05）。（2）MG63^-/-中PFKM活性下降（P<0.05）、葡萄糖含量下降（P<0.01）及细胞中乳酸含量下降（P<0.05）。表明细胞中糖酵解效率降低。（3）MG63^-/-的增殖活性下降（P<0.05）。（4）MG63^-/-的细胞凋亡显著增加,凋亡率上升（P<0.05）。（5）MG63^-/-中PFKM蛋白表达下降。4.生物信息学分析,hsa-mi R-542-3p与LncRNA XLOC₀05950及PFKM有匹配结合区域。5.与癌旁组织比较,骨肉瘤组织中hsa-miR-542-3p的表达水平降低（P<0.05）;与成骨细胞hFOB1.19比较,骨肉瘤MG63细胞中hsa-miR-542-3p的表达水平显著降低,差异有显著统计学意义（P<0.01）。而当MG63细胞基因编辑敲除LncRNA XLOC₀05950,MG63^-/-组中hsa-miR-542-3p的表达与MG63组比较其表达显著上调,差异具有统计学意义（P<0.05）。6.骨肉瘤细胞MG63转染hsa-miR-542-3p mimics后,转染后细胞变化:（1）转染细胞中PFKM的mRNA表达降低（P<0.05）。（2）转染细胞中PFKM活性下降（P<0.05）、葡萄糖含量及乳酸含量均下降（P<0.05）。表明转染hsa-miR-542-3p mimics的细胞中糖酵解效率降低。（3）转染细胞的增殖活性下降（P<0.05）。（4）转染细胞MG63细胞凋亡明显增加,凋亡率上升（P<0.05）。（5）转染细胞中PFKM蛋白表达下降。7.双荧光素酶报告实验结果显示,LncRNA XLOC₀05950及PFKM均为hsa-miR-542-3p的作用靶标。结论1.骨肉瘤组织和骨肉瘤细胞MG63中,LncRNA XLOC₀05950和PFKM均高表达,hsa-mi R-542-3p低表达。2.基因编辑敲除lncRNA XLOC₀05950或转染hsa-miR-542-3p后,骨肉瘤细胞MG63糖酵解关键酶PFKM表达下降、细胞内葡萄糖及乳酸含量下降,骨肉瘤细胞的主要供能方式有氧糖酵解受抑制。同时,骨肉瘤细胞的增殖活性受到抑制,凋亡率增加。3.lncRNA XLOC₀05950可以通过hsa-miR-542-3p调控PFKM的表达,阻止骨肉瘤细胞的有氧糖酵解途径,抑制肿瘤细胞的主要能量供应,进而调控骨肉瘤细胞的增殖与凋亡。更多还原

【Abstract】 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.更多还原