【作者】 姜春善；

【导师】 张学武；

【作者基本信息】 延边大学 ， 免疫学， 2025， 博士

【摘要】 研究背景和目的:骨肉瘤(osteosarcoma)是一种由间叶细胞恶性增殖并产生类骨基质所形成的骨性肿瘤,主要发生在骨骺生长活跃的部位,如股骨下端、胫骨和肱骨上端,多见于骨骼快速生长发育期的青少年和儿童。由于早期症状并不典型,许多患者在确诊时已进展至晚期,出现明显的肿瘤性病变或骨折,且常伴有远处转移,导致预后极为不良。虽然近年来靶向治疗和免疫治疗等新方法不断涌现,有效降低了整体死亡率,但患者的预后仍然很不理想。因此,寻找新的生物标志物和潜在治疗靶点,对于治疗骨肉瘤具有重要意义。近年来,环状RNA(circular RNAs,circRNAs)作为一种新兴的非编码RNA,逐渐成为研究热点。CircRNA因其独特的环状结构而具有较高的稳定性,不易被RNA酶降解,并能够通过调控基因表达、发挥微小RNA(miRNA)海绵效应等多种机制调控宿主基因转录。已有研究表明,circRNAs在多种恶性肿瘤的发生与发展过程中发挥关键调节作用,涉及细胞增殖、自噬和细胞凋亡等多个生物学过程,其中,circMRPS35是近年来新发现的一种与肿瘤密切相关的circRNA。已有研究显示,其在胃癌和肝细胞癌中表达水平异常,并与患者的临床病理特征及预后密切相关。相关功能研究进一步揭示,circMRPS35通过调控肿瘤抑制因子,如叉头蛋白O1(Forkhead Box O1,FOXO1)和O3a(Forkhead Box O3a,FOXO3a)及其下游靶基因的表达,在调节肿瘤细胞增殖、迁移、侵袭以及耐药等方面发挥重要作用。然而,目前对于circMRPS35在骨肉瘤细胞增殖中的具体作用及其作用机制尚不十分清楚。因此,本研究旨在探讨circMRPS35在调控骨肉瘤细胞增殖中的作用及其分子作用机制,为骨肉瘤的潜在的分子标志物开发及靶点治疗提供理论依据与实验基础。材料和方法:本研究利用荧光定量PCR检测circMRPS35在骨肉瘤组织和邻近正常组织中的差异表达。选择MG63和MNNG/HOS两种骨肉瘤细胞,利用质粒转染实现circMRPS35的过表达,并利用荧光定量PCR检测circMRPS35过表达前后MG63和MNNG/HOS细胞中circMRPS35表达水平的变化。利用CCK-8实验检测circMRPS35过表达对两种骨肉瘤细胞增殖活性的影响。利用EdU染色检测circMRPS35过表达对两种骨肉瘤细胞增殖能力的影响。利用平板克隆形成实验检测circMRPS35过表达对骨肉瘤细胞克隆形成能力的影响。利用PI染色结合流式细胞术分析circMRPS35过表达对两种骨肉瘤细胞周期分布的影响。利用Annexin-V/PI双染并结合流式细胞术检测circMRPS35过表达对两种骨肉瘤细胞凋亡的影响。利用Western Blot检测circMRPS35过表达对两种骨肉瘤细胞自噬相关蛋白微管相关蛋白1轻链3(Microtubule-Associated Protein 1 Light Chain 3,LC3)和Beclin 1表达的影响。构建小鼠皮下异种移植瘤模型,评估circMRPS35过表达对裸鼠体内骨肉瘤生长的影响。为明确circMRPS35调控骨肉瘤细胞增殖的分子机制,本研究利用Ch IP实验检测MG63和MNNG/HOS两种骨肉瘤细胞中H3K23ac对FOXO3启动子的富集。RIP实验验证circMRPS35和赖氨酸乙酰转移酶(Histone Acetyltransferase,KAT6B)的相互作用关系。Western blot检测FOXO1和FOXO3、LC3 II/I比例和Beclin-1蛋白的表达水平,并利用q RT-PCR检测相关基因的表达。利用CCK-8和Annexin-V/PI双染色结合流式细胞术检测细胞增殖和细胞凋亡表型变化。利用ENCORI数据库预测circMRPS35或FOXO1与miR-105-5p的相互作用。荧光素酶报告基因验证circMRPS35或FOXO1和miR-105-5p的靶向关系。免疫组化染色检测肿瘤组织Ki-67表达水平。利用裸鼠成瘤实验验证circMRPS35通过靶向KAT6B对骨肉瘤细胞增殖的影响。结果:1.CircMRPS35参与调控骨肉瘤MG63和MNNG/HOS细胞增殖,过表达circMRPS35显著抑制骨肉瘤细胞增殖,并诱导细胞凋亡、细胞周期阻滞和自噬。过表达circMRPS35显著抑制裸鼠皮下骨肉瘤细胞生长。(1)荧光定量PCR结果显示,circMRPS35在骨肉瘤患者组织中的表达水平显著低于邻近正常组织(P<0.001)。(2)荧光定量PCR结果显示,与对照组相比,转染circMRPS35过表达质粒的MG63和MNNG/HOS细胞中circMRPS35表达水平显著升高(P<0.01),表明circMRPS35过表达质粒转染成功。(3)CCK-8结果显示,与对照组相比,circMRPS35过表达显著抑制了MG63和MNNG/HOS细胞的增殖(P<0.01)。(4)EdU染色实验表明,与对照组相比,circMRPS35过表达显著抑制了MG63和MNNG/HOS中的EdU阳性细胞比例(P<0.01)。(5)平板克隆形成实验结果表明,与对照组相比,circMRPS35过表达显著减少了MG63和MNNG/HOS细胞的克隆数量(P<0.01)。(6)PI染色结合流式细胞术分析结果显示,与对照组相比,circMRPS35过表达显著增加了MG63和MNNG/HOS中G1期细胞的比例(P<0.01)。(7)Annexin-V/PI双染结合流式细胞术分析表明,与对照组相比,circMRPS35过表达后MG63和MNNG/HOS细胞凋亡显著增多(P<0.01)。(8)Western Blot结果显示,与对照组相比,circMRPS35过表达显著提高了MG63和MNNG/HOS细胞中LC3 II/I比例和Beclin-1蛋白的表达水平(P<0.01)。(9)裸鼠皮下移植瘤实验表明,与阴性对照组相比,circMRPS35过表达组小鼠的肿瘤体积更小、重量更轻(P<0.01)。2.CircMRPS35通过两条分子通路调控骨肉瘤细胞增殖:一方面,其通过招募KAT6B促进FOXO3的转录激活;另一方面,通过靶向miR-105-5p的“海绵”解除其对FOXO1的负调控,共同促进骨肉瘤细胞凋亡和自噬,抑制其恶性增殖。(1)通过Ch IP实验结果显示在MG63和MNNG/HOS两种骨肉瘤细胞中circMRPS35的敲除显著降低了FOXO3启动子上组蛋白H3K23ac的富集。RIP实验结果显示,circMRPS35与KAT6B结合。KAT6B敲除能够减少circMRPS35在FOXO3基因启动子处增加的H3K23ac乙酰化修饰水平。Western blot结果表明,KAT6B的过表达可以逆转circMRPS35缺失对FOXO3的负向调控作用。(2)流式细胞术结果显示,过表达KAT6B显著促进MG63和MNNG/HOS两种骨肉瘤细胞凋亡。(3)Western blot结果表明,过表达KAT6B促进了MG63和MNNG/HOS细胞的自噬相关蛋白LC3 II/I比例和Beclin-1表达。(4)流式细胞术结果显示,抑制FOXO3逆转了circMRPS35对MG63细胞系细胞凋亡的促进作用。(5)Western blot结果表明,抑制FOXO3逆转了circMRPS35对MG63细胞系细胞自噬相关蛋白LC3 II/I比例和Beclin-1表达的促进作用。(6)裸鼠成瘤实验结果表明,circMRPS35组小鼠肿瘤体积及重量较Control组显著降低,circMRPS35+si KAT6B组肿瘤体积及重量较circMRPS35组显著升高,说明抑制KAT6B逆转了circMRPS35对骨肉瘤肿瘤生长的促进作用。(7)免疫组织化学染色检测Ki-67,circMRPS35组Ki-67表达较Control组显著降低,circMRPS35+si KAT6B组Ki-67表达较circMRPS35组显著升高,表明抑制KAT6B逆转了circMRPS35对Ki-67表达的促进作用。(8)利用在线分析网站ENCORI结合荧光素酶报告基因实验,发现circMRPS35在骨肉瘤细胞中发挥miR-105-5p海绵作用,且miR-105-5p靶向骨肉瘤细胞的FOXO1。(9)Western blot结果表明,circMRPS35通过调控miR-105-5p促进FOXO1蛋白的表达。(10)CCK-8和流式细胞凋亡术结果表明,circMRPS35通过靶向miR-105-5p/FOXO1轴调控MG63和MNNG/HOS骨肉瘤细胞增殖和凋亡。结论:1.CircMRPS35参与骨肉瘤细胞的增殖调控。CircMRPS35通过诱导骨肉瘤细胞凋亡、自噬及细胞周期阻滞,负向调控骨肉瘤细胞增殖。2.CircMRPS35通过双通路机制发挥作用。一方面,circMRPS35通过招募KAT6B促进FOXO3的转录激活;另一方面,通过充当miR-105-5p的“海绵”解除其对FOXO1的负向调控。更多还原

【Abstract】 Background and objectives:Osteosarcoma,a bony tumor formed by malignant proliferation of mesenchymal cells and production of osteoid stroma,is the most common primary malignant bone tumor.It occurs mainly in sites with active epiphyseal growth,such as the lower femur,tibia,and upper humerus,and is more common in adolescents and children during rapid bone growth and development.However,early symptoms can be subtle and nonspecific,many patients are diagnosed at an advanced stage when obvious tumor-related changes or fractures have already occurred,often accompanied by distant metastasis.This leads to a challenging prognosis for these individuals.Therefore,the search for new biomarkers and potential therapeutic targets is of great significance for the treatment of osteosarcoma.In recent years,circular RNAs(circRNAs),as an emerging class of non-coding RNAs,have gradually become a research hotspot.Due to their unique circular structure,circRNAs exhibit high stability,making them resistant to degradation by RNAases.They regulate host gene transcription through various mechanisms,including the regulation of gene expression and the sponge effect on micro RNAs(miRNAs).Studies have shown that circRNAs play key regulatory roles in the occurrence and progression of various malignant tumors,involving biological processes such as cell proliferation,autophagy,and apoptosis.Among these,circMRPS35 is a recently identified circRNA closely associated with tumors.Research has shown that circMRPS35 is abnormally expressed in gastric cancer and hepatocellular carcinoma,and its expression levels are closely related to patients’clinical pathological features and prognosis.Functional studies have further revealed that circMRPS35 regulates the expression of tumor suppressors,such as Forkhead Box O1(FOXO1)and Forkhead Box O3a(FOXO3a),and their downstream target genes,playing a significant role in regulating tumor cell proliferation,migration,invasion,and drug resistance.However,the specific role and underlying mechanisms of circMRPS35 in osteosarcoma cell proliferation remain unclear.Therefore,this study aims to investigate the role of circMRPS35 in regulating osteosarcoma cell proliferation and its molecular mechanisms,providing a theoretical basis and experimental foundation for the development of potential molecular markers and targeted therapies for osteosarcoma.Materials and Methods:The expression of circMRPS35 in osteosarcoma tissues and adjacent normal tissues was detected by quantitative real-time PCR.The circMRPS35-overexpressing plasmids were transfected into MG63 and MNNG/HOS osteosarcoma cells,and the expression of circMRPS35 was detected by quantitative real-time PCR.The effect of circMRPS35 overexpression on the viability of MG63 and MNNG/HOS cells was detected by CCK-8 assay.The effect of circMRPS35 overexpression on the proliferation of MG63 and MNNG/HOS cells was measured by EdU assay.The effect of circMRPS35 overexpression on the colony formation ability of MG63 and MNNG/HOS cells was analyzed by colony formation assay.The effect of circMRPS35 overexpression on cell cycle of MG63 and MNNG/HOS cells was detected by PI staining combined with flow cytometry.The effect of circMRPS35overexpression on the apoptosis of MG63 and MNNG/HOS cells was detected by Annexin V/PI double staining combined with flow cytometry.The effect of circMRPS35 overexpression on the autophagy-related proteins LC3 and Beclin 1 of MG63 and MNNG/HOS cells was detected by Western Blot.The effect of circMRPS35 overexpression on tumor growth was evaluated in subcutaneous xenograft tumor models.To elucidate the molecular mechanisms through which circMRPS35 regulates osteosarcoma cell proliferation,this study employed the following experimental approaches:Ch IP assays were used to detect the enrichment of H3K23ac on the FOXO3 promoter in MG63 and MNNG/HOS osteosarcoma cells.RIP assays were performed to verify the interaction between circMRPS35 and histone acetyltransferase(KAT6B).Western blotting was used to measure the expression levels of FOXO1,FOXO3,the LC3 II/I ratio,and Beclin-1 protein.q RT-PCR was employed to assess the expression of relevant genes.Cell proliferation and apoptosis phenotypic changes were analyzed using CCK-8 assays and Annexin-V/PI double staining followed by flow cytometry.The ENCORI database was used to predict the interaction between circMRPS35 or FOXO1 and miR-105-5p.Luciferase reporter assays were performed to validate the targeting relationship between circMRPS35 or FOXO1 and miR-105-5p.Immunohistochemical staining was used to assess the expression levels of Ki-67 in tumor tissues.Finally,a xenograft mouse model was employed to verify the effect of circMRPS35 targeting KAT6B on osteosarcoma cell proliferation.Results:1.CircMRPS35 is involved in regulating the proliferation of osteosarcoma MG63 and MNNG/HOS cells.Overexpression of circMRPS35 significantly inhibits osteosarcoma cell proliferation and induces apoptosis,cell cycle arrest,and autophagy.Additionally,overexpression of circMRPS35 notably suppresses tumor growth in xenograft mice.(1)The results of quantitative PCR showed that the expression level of circMRPS35 in osteosarcoma tissues was significantly lower than that in adjacent normal tissues(P<0.001).(2)The results of Quantitative PCR showed that,the transfection of circMRPS35-overexpressing plasmids significantly increased the expression levels of circMRPS35 in the MG63 and MNNG/HOS cells,respectively(P<0.01),indicating that circMRPS35-expressing transfection was successful.(3)The results of CCK-8 showed that,compared with the control group,circMRPS35 overexpression significantly decreased the proliferation ability of MG63and MNNG/HOS cells(P<0.01).(4)The results of EdU staining showed that,compared with the control group,circMRPS35 overexpression significantly reduced the proportion of EdU-positive cells in MG63 and MNNG/HOS cells(P<0.01).(5)The results of clonal formation experiment showed that,compared with the control group,circMRPS35 overexpression significantly reduced the number of clones of MG63 and MNNG/HOS cells(P<0.01).(6)The results of PI staining and flow cytometry analysis showed that,compared with the control group,the proportion of G1 phase cells was increased in the circMRPS35 overexpression group(P<0.01).(7)The results of Annexin-V/PI double staining and flow cytometry analysis showed that,compared with the control group,circMRPS35 overexpression increased the proportion of apoptotic cells in MG63 and MNNG/HOS cells(P<0.01).(8)The results of Western Blot showed that,compared with the control group,LC3II/I ratio and Beclin 1 expression in the circMRPS35 overexpression group of MG63and MNNG/HOS cells were significantly increased(P<0.01).(9)The results of subcutaneous tumor transplantation showed that,compared with the control group,circMRPS35-overexpressed mice had smaller tumor size and lighter weight(P<0.01).2.CircMRPS35 regulates osteosarcoma cell proliferation through two molecular pathways:on one hand,it recruits KAT6B to promote the transcriptional activation of FOXO3;on the other hand,it acts as a"sponge"for miR-105-5p,relieving its negative regulation on FOXO1.These mechanisms collectively promote osteosarcoma cell apoptosis and autophagy,inhibiting their malignant progression.(1)The results of Ch IP assay showed that,knockdown of circMRPS35 in MG63 and MNNG/HOS cells significantly reduced the enrichment of histone H3K23ac at the FOXO3 promoter.The results of RIP experiment showed that circMRPS35 could bind to histone acetyltransferase KAT6B,and KAT6B knockout could weaken the H3K23ac level at the FOXO3 gene promoter increased by circMRPS35.The results of Western blot showed that,overexpression of KAT6B reversed the negative regulatory effect of circMRPS35 deletion on FOXO3.(2)Flow cytometry results showed that,overexpression of KAT6B significantly promoted apoptosis in two types of osteosarcoma cells,MG63 and MNNG/HOS.(3)Western blot results showed that,overexpression of KAT6B promoted autophagy in MG63 and MNNG/HOS cells.(4)Flow cytometry results showed that,inhibition of FOXO3 reversed the promoting effect of circMRPS35 on apoptosis of MG63 cell line.(5)Western blot results showed that,inhibition of FOXO3 reversed the promoting effect of circMRPS35 on autophagy in MG63 cell line.(6)The tumor formation experiment in nude mice showed that,the tumor volume and weight of circMRPS35 group were significantly lower than those of the control group,and the tumor volume and weight of circMRPS35+si KAT6B group were significantly higher than those of circMRPS35 group.This indicated that inhibition of KAT6B reversed the promoting effect of circMRPS35 on osteosarcoma tumor growth.(7)Immunohistochemical staining of Ki-67 showed that,the expression of Ki-67 in circMRPS35 group was significantly lower than that in the control group,and the expression of Ki-67 in circMRPS35+si KAT6B group was significantly higher than that in circMRPS35 group.This indicated that inhibition of KAT6B reversed the promoting effect of circMRPS35 on Ki-67 expression.(8)Using the online analysis tool ENCORI in combination with luciferase reporter assays,it was found that circMRPS35 functions as a"sponge"for miR-105-5p in osteosarcoma cells,and miR-105-5p targets FOXO1 in these cells.(9)Western blot results indicate that,circMRPS35 promotes the expression of FOXO1 protein by regulating miR-105-5p.(10)CCK-8 and flow cytometry apoptosis assays demonstrate that circMRPS35regulates the proliferation and apoptosis of MG63 and MNNG/HOS osteosarcoma cells through the miR-105-5p/FOXO1 axis.Conclusion:1.CircMRPS35 participates in the regulation of osteosarcoma cell proliferation by negatively modulating cell growth through the induction of apoptosis,autophagy,and cell cycle arrest.2.CircMRPS35 exerts its effects through a dual-pathway mechanism.On one hand,circMRPS35 recruits KAT6B to promote the transcriptional activation of FOXO3;on the other hand,it acts as a"sponge"for miR-105-5p,relieving its negative regulation of FOXO1.更多还原