【作者】 刘敏；

【导师】 徐又佳；

【作者基本信息】 苏州大学 ， 骨外科（专业学位）， 2020， 博士

【摘要】 第一部分miRNA在唑来膦酸抑制骨肉瘤中的表达谱特征及生物信息学分析目的通过对miRNA在唑来膦酸抑制骨肉瘤中的表达谱特征及生物信息学分析,寻找其潜在miRNA靶基因。方法用CCK8细胞增殖实验分析不同的浓度条件下唑来膦酸对于骨肉瘤U-2OS细胞和MG-63细胞增殖能力的影响。同时采用Transwell实验探讨唑来膦酸对骨肉瘤U-2OS细胞和MG-63细胞侵袭能力的影响。用有效浓度唑来膦酸处理骨肉瘤细胞后,设置相同条件下的未处理对照组,然后分别收集细胞,运用BGISeq-500测序仪进行RNA-seq转录组学高通量测序,获得差异表达的miRNA和m RNA表达谱数据,然后进行生物信息学分析。结果CCK8实验的结果表明唑来膦酸对骨肉瘤U-2OS细胞和MG-63细胞的增殖可以起到明显的抑制作用,且这种作用存在浓度依赖性,其抑制U-2OS细胞、MG-63细胞的IC50分别为20μM、50μM处理24h。而采用IC50的处理浓度时间进行Transwell实验结果表明,骨肉瘤U-2 OS、MG63细胞的侵袭也被唑来膦酸明显抑制,较未处理对照组相比分别下降43.0%和37.7%(P<0.05)。在唑来膦酸抑制骨肉瘤细胞的过程中,RNA-seq转录组学高通量测序发现了一些差异表达的miRNA及其相对应的靶基因。而进一步进行的生物信息学分析提示,相关的miRNA可能有miRNA-423-5p、miR-663a、miR-1237、miR-1247-5p、miR-187、miR-3194,潜在的靶基因可能包括FJX1,KLF2、MAPK7、RHOB。结论唑来膦酸可以抑制骨肉瘤细胞的增殖和侵袭,且在其抑制过程中伴随着一些差异表达的miRNA,以及与之相对应的差异表达的m RNA。第二部分唑来膦酸处理骨肉瘤细胞后差异表达miRNA的初步验证和大数据分析目的对唑来膦酸处理骨肉瘤细胞后差异表达潜在miRNA进行初步试验验证和大数据分析,明确其后续研究价值。方法荧光定量PCR检测唑来膦酸作用骨肉瘤细胞后潜在靶基因的表达情况,验证其研究价值。利用TCGA数据库获得相关潜在靶基因的表达数据及临床预后数据,采用Kaplan-Meier对FJX1、KLF2、RHOB、MRGPRF进行5年生存率的生存分析。结果PCR检测显示,与对照组相比,有效浓度的唑来膦酸处理骨肉瘤(U-2OS和MG63)细胞后,miR-1237、miR-187、miR-663a、KLF2 m RNA、RHOB m RNA的表达量均明显上升(P<0.05)。而FJX1 m RNA在U-2OS细胞中表达量下降了75%,在MG63细胞中表达量下降40%,表达量的差异具有统计学意义(P<0.05)。而进一步的Kaplan-Meier生存分析结果显示,患者的5年生存率在FJX1 m RNA高表达组中要较低表达组明显降低,这种差异存在统计学意义(P<0.05),提示其作为致癌基因可能;而在KLF2、RHOB和MRGPRF m RNA高表达组中,5年生存率较低表达组要明显提高,差异均有统计学意义(P<0.05),提示其作为抑癌基因可能。结论miR-1237、miR-187、miR-663a以及相关的m RNA(包括FJX1、KLF2、RHOB、MRGPRF)在唑来膦酸抑制骨肉瘤细胞中的差异变化,可能是其潜在调控基因。第三部分miRNA-187通过靶向调节MAPK7调控骨肉瘤增殖和迁移的作用机制研究目的探讨miRNA-187通过靶向调节MAPK7调控骨肉瘤增殖和迁移的作用机制研究。方法采用q RT-PCR检测骨肉瘤多种细胞株中miR-187的表达量。将miR-187模拟物或NC转染到骨肉瘤细胞中,通过WST-1分析监测SAOS-2细胞的增殖速率和集落形成测定。采用FACS流式细胞仪评估细胞在不同细胞周期阶段的分布。利用划痕实验和Transwell实验评估miR-187过表达后,对骨肉瘤SAOS-2细胞迁移和侵袭的影响。运用双荧光报告实验和Western blot实验来证实,miR-187通过靶向调节MAPK7调控骨肉瘤细胞。结果q RT-PCR检测显示miR-187的在骨肉瘤多种细胞株中表达均显著下调,而其中以SAOS-2细胞表达下调最明显,约为8倍。WST-1分析监测结果显示,miR-187的过表达导致了SAOS-2细胞的增殖速率显著下降,细胞的集落形成被抑制了62%(P<0.05)。细胞周期分析显示miR-187的过表达引起G2/M期细胞的积累,同时还伴随细胞周期蛋白B1表达的抑制。划痕实验中,在24h时,miR-187 mimics组细胞愈合宽度明显要比miR-NC组大,彼此间的差异具有统计学意义(P<0.05)。而Transwell实验结果显示,miR-187过表达后,骨肉瘤SAOS-2细胞的侵袭数目较miR-NC转染组减少了60%,差异有统计学意义(P<0.05)。双荧光素酶测定结果显示,在转染质粒p GL3-MAPK7′-UTR-WT同时加入miR-187质粒的实验组与加入空质粒的对照组相比,荧光素酶活性显著下调(P<0.05)。而在转染p GL3-MAPK7′-UTR-MUT组质粒同时加入miR-187质粒的实验组与加入空质粒的对照组相比,荧光素酶活性下降无显著差异。蛋白质印迹结果表明:MAPK7在骨肉瘤细胞中的表达水平显著高于对照组人成骨细胞(h FOB 1.19),差异有统计学意义(P<0.05)。而miR-187的过表达导致SAOS-2细胞中的MAPK7表达被显著抑制。结论miRNA-187是通过靶向调节MAPK7调控骨肉瘤增殖和迁移。更多还原

【Abstract】 Part I Expression profile and bioinformatics analysis of zoledronic acid-based osteosarcoma based on miRNAObjectives In order to find the potential miRNA target gene,the expression profile and bioinformatics of miRNA in zoledronic acid inhibited osteosarcoma was analyzed.Methods Different concentrations of zoledronic acid acted on osteosarcoma U-2OS cells and MG-63 cells,and the effect of proliferation was analyzed by CCK8 cell proliferation experiments.At the same time,the effect of zoledronic acid on the invasion ability of osteosarcoma U-2OS cells and MG-63 cells was investigated using Transwell experiment.After osteosarcoma cells were treated with effective zoledronic acid concentration,a control group was set up under the same conditions.Then the cells were collected respectively.The high-throughput RNA SEQ transcriptase sequencing was carried out with Bgiseq-500 sequencer to obtain the data of miRNA and m RNA expression profiles of differential expression,and then biological informatics analysis was carried out.Results The results of the CCK8 experiment suggest that zoledronic acid can significantly inhibit the proliferation of osteosarcoma U-2OS cells and MG-63 cells in a concentration-dependent manner.The IC50 of zoledronic acid inhibited u-2os cells and MG-63 cells was 20 μM and 50 μM respectively for 24 hours.The results of Transwell experiment with IC50 concentration time show that the invasion of osteosarcoma u-2os and MG63 cells was also inhibited by zoledronic acid,which decreased by 43.0% and 37.7% respectively compared with untreated control group(P<0.05).In the process of zoledronic acid inhibiting osteosarcoma cells,a large number of differentially expressed miRNA and corresponding differentially expressed m RNA were found by RNA SEQ high-throughput sequencing.Bioinformatics analysis suggested that miR-423-5p,miR-663 a,miR-1237,miR-1247-5p,miR-187 and miR-3194 might be potential miRNA The potential target genes include FJX1,KLF2,MAPK7 and Rho B.Conclusions Zoledronic acid can inhibit the proliferation and invasion of osteosarcoma cells,accompanied by a large number of differentially expressed miRNA and corresponding differentially expressed m RNA.Part II Preliminary validation and big data analysis of miRNA differentially expressed in osteosarcoma cells treated with zoledronic acidObjectives The potential miRNA differential expression of osteosarcoma cells treated with zoledronic acid was verified by preliminary experiments and big data analysis to clarify the value of its follow-up study.Methods Quantitative real-time PCR was used to detect the expression of potential target genes after zoledronic acid treatment of osteosarcoma cells,confirming its research value.TCGA database was used to obtain the expression data of related potential target genes and clinical prognosis data.The 5-year survival rate of FJX1,KLF2,Rho A and MRGPRD was analyzed by Kaplan Meier.Results PCR detection showed that the expression of miR-1237,miR-187,miR-663 a,KLF2 m RNA and Rho B m RNA in osteosarcoma u-2os and MG63 cells treated with zoledronic acid increased significantly compared with the control group(P < 0.05).The expression of FJX1 m RNA in u-2os cells decreased by 75% and MG63 cells by 40%,the difference was statistically significant(P <0.05).Further Kaplan Meier survival analysis showed that the 5-year survival rate of patients in the high expression group of FJX1 m RNA was significantly lower than that in the low expression group,which was statistically significant(P < 0.05),suggesting that it might be a carcinogenic gene;while in the high expression group of KLF2,Rho B and MRGPRD m RNA,the 5-year survival rate was significantly higher than that in the low expression group,and the difference was statistically significant(P< 0.05),suggesting that they may be used as tumor suppressor genes.Conclusions miR-1237,miR-187,miR-663 a and related m RNA(including FJX1,KLF2,Rho B and MRGPRF)in zoledronic acid inhibited the differential changes in osteosarcoma cells,which may be the potential regulatory genes.Part III Study on the mechanism of mirna-187 targeting MAPK7 toregulate the proliferation and migration of osteosarcoma.Objectives Study on the mechanism of mirna-187 regulating the proliferation and migration of osteosarcoma by targeting MAPK7Methods q RT-PCR was used to detect the expression of miR-187 in various osteosarcoma cell lines.miR-187 minics or NC was transected into osteosarcoma cells,and the proliferation rate and colony assay of Saos-2 cells were monitored by WST-1 analysis.The distribution of cells in different cell cycle stages was evaluated by FACS flow cytometry.Wound-healing and Transwell test was used to determine the effect of over expression of miR-187 on the migration and invasion of Saos-2 cells.Double fluorescence report and Western blot were used to verify the regulation of miR-187 targeting mapk7 on osteosarcoma cells.Results q RT-PCR showed that the expression of miR-187 was significantly down regulated in a variety of osteosarcoma cell lines,and Saos-2 cell line was the most significantly down regulated,about 8 times down regulated.WST-1 analysis showed that the overexpression of miR-187 led to a significant decrease in the proliferation rate of Saos-2 cells,and the colony formation of Saos-2 cells was inhibited by 62%(P < 0.05).Cell cycle analysis showed that the overexpression of miR-187 led to the accumulation of G2 / M phase cells and the inhibition of cyclin B1 expression.In the wound-healing test,the cell healing width of mir-187 mimics group was significantly larger than that of miR-NC group at 24 hours,and the difference was statistically significant(P < 0.05).Transwell test showed that the number of Saos-2 cells invading after over expression of miR-187 was 60% lower than that of miR-NC group(P < 0.05).The results of double luciferase assay showed that the luciferase activity of the experimental group transfected with plasmid p GL3-MAPK7′-UTR-WT and mir-187 was significantly lower than that of the control group(P < 0.05).However,the luciferase activity between the experimental group(transfected with plasmid p GL3-MAPK7′-UTR-MUT and miR-187)and the control group was proved no significant difference.The result of Western blot showed that,the expression level of MAPK7 in osteosarcoma cells was significantly higher than that in human osteoblasts(h FOB 1.19).The difference was statistically significant(P < 0.05).The overexpression of miR-187 resulted in the inhibition of mapk7 expression in Saos-2 cells.Conclusions miR-187 regulates the proliferation and migration of osteosarcoma by targeting MAPK7.更多还原