miR-182-5p通过靶向调节KLF7抑制骨肉瘤细胞的增殖、迁移和侵袭

目的:研究miR-182-5p对人骨肉瘤细胞增殖、迁移和侵袭的作用。方法:用qRT-PCR检测法比较人骨肉瘤细胞系（U2OS、MG63、SAOS2）、人正常细胞和人成骨细胞（HFOB1.19）中miR-182-5p的表达水平。转染miRNA模拟物或抑制剂或模拟物+KLF7,以转染miR-182-5p表达的上调或下调。通过EDU、迁移分析和侵袭分析来检测细胞功能。双荧光素酶报告分析检测miR-182-5p与KLF7的关系,蛋白质印迹分析检测KLF7的表达。结果:miR-182-5p在骨肉瘤细胞系中被下调。miR-182-5p过表达抑制肿瘤生长、迁移和侵袭。随后的研究显示,KLF7是骨肉瘤细胞中miR-182-5p的直接和功能靶点。miR-182-5p通过调节KLF7来抑制骨肉瘤细胞的增殖、迁移和侵袭。结论:miR-182-5p通过靶向调节KLF7而起到抑制骨肉瘤细胞增殖、迁移和侵袭的作用。     

MicroRNA 181a improves proliferation and invasion, suppresses apoptosis of osteosarcoma cell

MicroRNA 181a (miR-181a) was found dysregulated in a variety of human cancers and significantly associated with clinical outcome of cancer patients. However, the direct role of miR-181a has not yet been characterized in osteosarcoma progression. This study was aimed at investigating the effects of miR-181a on osteosarcoma cell biological behavior. First, the expression of miR-181a in osteosarcoma cell lines (MG63, HOS, SaOS-2, and U2OS) and a human osteoblastic cell line (hFOB1.19) was detected by qRT-PCR. Results showed that miR-181a was overexpressed in osteosarcoma cell lines compared to human osteoblastic cell line (hFOB1.19). To investigate the effects of miR-181a on proliferation, apoptosis, and invasion of osteosarcoma cells, we generated human osteosarcoma MG63 cells in which miR-181a was either overexpressed or depleted. The MG63 cell viability, cycle, apoptosis, and invasive ability were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining, propidium iodide (PI) staining, Annexin V-FITC/PI double staining, and Transwell invasion experiment, respectively. The results showed that MG63 cell viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in the group with underexpression of miR-181a. The viability, proliferation, and invasive abilities were improved, and the apoptosis was inhibited in the group with overexpression of miR-181a. The results from Western blotting indicated that miR-181a might be associated with the up-regulation of bcl-2 and matrix metalloproteinase 9 and the down-regulation of tissue inhibitor of metalloproteinases-3 and p21 in MG63 cells. Taken together, our results suggested that miR-181a might facilitate proliferation and invasion and suppress apoptosis of osteosarcoma cells, which might be a potential target for the treatment of osteosarcoma.     

微小RNA-101对人骨肉瘤细胞自噬和侵袭性的影响

目的 探讨微小RNA-101（microRNA-101,miR-101）的表达对人骨肉瘤细胞自噬和侵袭性的影响。方法 采用实时荧光定量PCR（qRT-PCR）检测骨肉瘤组织和人骨肉瘤细胞系MG-63细胞以及成骨细胞miR-101的相对表达,蛋白免疫印迹(Western blot)检测两种细胞自噬相关蛋白Beclin1和LC3B的表达;经脂质体转染将miR-101模拟物和miR-101阴性对照转染MG-63细胞,并设立空白对照,通过qRT-PCR、Western blot和侵袭实验（Transwell）检测以上三组细胞miR-101的表达、Beclin1和LC3B的表达以及三组细胞侵袭能力的变化。结果 与癌旁骨组织和正常骨组织或成骨细胞相比,miR-101在骨肉瘤组织和MG-63细胞中表达明显下降（P<0.01）;模拟物转染组miR-101的表达较空白对照组上调了255%,但该组自噬相关蛋白的表达较空白组却显著降低（P<0.01）;Transwell侵袭实验显示,模拟物转染组细胞迁移数目较阴性对照组和空白对照组分别减少了60%和67.67%（P<0.01）。结论 miR-101在骨肉瘤组织和骨肉瘤细胞中呈现低表达,可能与骨肉瘤的发生发展相关。miR-101抑制骨肉瘤细胞侵袭,其机制可能是通过抑制细胞自噬而发挥作用。     

Effects of Kruppel-like factor 6 on osteosarcoma cell biological behavior

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene frequently downregulated in a number of human cancers, including osteosarcoma. However, the role of KLF6 in osteosarcoma remains unclear. This study was aimed at investigating the effects of KLF6 on osteosarcoma cell biological behavior. First, the expression of KLF6 in osteosarcoma cell lines (MG63, SaOS-2, U2OS, and HOS) and a human osteoblastic cell line (hFOB1.19) was detected by Western blotting. Results showed that KLF6 displayed a significant downregulation in osteosarcoma cell lines (MG63, SaOS-2, U2OS, and HOS) compared with human osteoblastic cell line (hFOB1.19). To investigate the role of KLF6 in osteosarcoma cell proliferation, apoptosis, and invasion, we generated human osteosarcoma MG63 cells in which KLF6 was either overexpressed or depleted. The MG63 cell viability, cycle, apoptosis, and invasive ability were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining, propidium iodide (PI) staining, Annexin-V-FITC/PI double staining, and Transwell invasion experiment, respectively. Results showed that the viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in MG63 cells with overexpression of KLF6. The viability, proliferation, and invasive abilities were improved, and the apoptosis was inhibited in MG63 cells with knockdown of KLF6. At the same time, these molecules, including p21, bcl-2, and MMP-9, associated with the events about cell cycle, apoptosis, and invasion, were detected. Results showed that the expressions of bcl-2 and MMP-9 were downregulated, and the expressions of p21 were upregulated in the MG-63 cells with overexpression of KLF6. Taken together, our results suggested that KLF6 could inhibit proliferation and invasion, and facilitate apoptosis of osteosarcoma cells, which might be a potential target for the treatment of osteosarcoma.     

lncRNA HOXA11-AS对骨肉瘤细胞增殖、迁移和侵袭能力的影响

目的:探究长链非编码RNA(long-nocoding RNA,lncRNA) HOXA11-AS在骨肉瘤中的表达及其对骨肉瘤细胞增殖、迁移和侵袭的影响及其作用机制.方法:采用2012年1月至2015年12月浙江省人民医院骨外科13例骨肉瘤组织及癌旁正常组织,以及骨肉瘤细胞系MG63、U20S和人成骨细胞株hFOB1.19,用实时荧光定量PCR检测骨肉瘤组织和骨肉瘤细胞系MG63和U20S中lncRNA HOXA11-AS的表达水平.用慢病毒载体构建稳定过表达lncRNA HOXA11-AS的骨肉瘤MG63及U20S细胞,用CCK-8和克隆形成实验、Transwell小室法分别检测过表达lncRNA HOXA11-AS对骨肉瘤细胞增殖、迁移和侵袭的影响.建立过表达lncRNA HOXA11-AS MG63骨肉瘤细胞裸鼠移植瘤模型,以空载体慢病毒Lv-NC转染的细胞作为对照,观察过表达lncRNA HOXA11-AS对裸鼠移植瘤生长的影响.结果:lncRNA在骨肉瘤组织和MG63及U20S细胞中表达水平显著下调(P＜0.01).与hFOB1.19细胞比,过表达lncRNA HOXA11-AS细胞后,(1)显著抑制骨肉瘤MG63及U20S细胞的增殖[(4.03 ±0.98) vs(6.96 ±0.54),(4.68±0.77) vs (8.87±1.23),均P＜0.01]、迁移细胞数[(83.00 ±6.03) vs(168±12.54),(96.00 ±8.77)vs(184.00±14.63)个,均P＜0.01]和侵袭细胞数[(35.00±3.48) vs (97.00±8.32),(38.00±1.73) vs (87.00±6.37)个,均P＜0.01];(2)显著抑制骨肉瘤裸鼠皮下移植瘤的生长(P＜0.01).结论:lncRNA HOXA11-AS在骨肉瘤细胞中低表达,且lncRNA HOXA11-AS过表达对骨肉瘤的发生发展具有抑制作用,可以作为骨肉瘤治疗潜在的分子靶点.     

MALAT1靶向调控miR-205与骨肉瘤侵袭的实验研究

目的   探讨人肺腺癌转移相关转录本1（MALAT1）与miR-205的关系,揭示MALAT1促进骨肉瘤发生发展的分子机制。方法   实时荧光定量PCR（QPCR）法检测骨肉瘤组织、癌旁正常组织、人成骨细胞（hFOB）以及骨肉瘤MG63、Sao-2细胞株中MALAT1和miR-205的表达;生物信息学及荧光素酶实验观察MALAT1对miR-205的靶向调控;miR-205 mimics转染Sao-2和MG63细胞后QPCR检测MALAT1表达, si-MALAT1转染Sao-2和MG63细胞后QPCR检测miR-205表达;miR-205 mimics和si-MALAT1分别转染MG63细胞,Transwell小室实验检测细胞侵袭能力,Western blotting检测MMP-2、MMP-9表达。结果   骨肉瘤组织、癌旁正常组织中MALAT1和miR-205表达量分别为4.7±0.6、2.6±0.08和2.2±0.09、3.7±0.3,差异有统计学意义（P＜0.05）;MG63、Sao-2细胞株中MALAT1和miR-205表达量分别为2.4±0.7、2.1±0.05和0.53±0.04、0.47±0.02,与hFOB中的0.9±0.01、0.82±0.04比较,差异有统计学意义（P＜0.05）;生物信息学及荧光素酶检测显示MALAT1序列中存在miR-205的3个互补序列,且两者存在靶向调控作用;转染si-MALAT1的Sao-2和MG63细胞中miR-205的表达量分别为3.4±0.7、3.8±0.6,对照组为1.4±0.5、1.0±0.1,差异有统计学意义（P＜0.05）;转染miR-205 mimics的Sao-2、MG63细胞中MALAT1的表达量分别为0.51±0.05、0.42±0.03,而对照组为1.5±0.7、1.28±0.4,差异有统计学意义（P＜0.05）;Transwell实验结果 显示miR-205 mimics组MG63细胞侵袭数为（28.52±3.68）个,低于miR-205 mimics与pMALAT1共转染组的（42.63±5.67）个,差异具有统计学意义（P＜0.05）;Western blotting结果 显示miR-205 mimics组MMP-2、MMP-9的表达量分别为0.41±0.02、0.49±0.04,显著低于miR-205与pMALAT1共转染组的0.73±0.01、0.80±0.05,差异具有统计学意义（P＜0.05）。结论   MALAT1与miR-205之间存在双向抑制关系,MALAT1通过抑制miR-205的表达促进骨肉瘤细胞的侵袭。     

miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1抑制人骨肉瘤细胞MG-63侵袭转移的实验研究

背景与目的：微小RNA(microRNA,miRNA)是一类小分子内源性RNA,主要在转录后水平调节靶基因的表达。microRNA-335(miR-335)作为一种肿瘤抑制因子,参与了多种人类肿瘤的发生、发展过程。本研究旨在探讨miR-335是否靶向抑制Rho相关卷曲螺旋形成蛋白激酶1(Rho associated coiled-coil formingprotein kinase,ROCK1)基因的表达,并以此调控人骨肉瘤细胞MG-63侵袭及转移。方法：理论预测并通过荧光素酶基因报告验证miR-335与ROCK1基因的3'-非翻译区(untranslated region,UTR)的特异性结合作用;real-time PCR和蛋白质印迹法(Western blot)分别从基因和蛋白水平检测miR-335对ROCK1表达的负性调控作用;Transwell小室法检测miR-335过表达及下调ROCK1表达后MG-63侵袭及转移能力的变化。结果：Targetscan预测显示,miR-335与ROCK1 3'-UTR存在结合位点。荧光素酶基因报告实验结果显示,miR-335 mimic和ROCK1 3'-UTR能够靶向结合;miR-335在MG-63细胞中低表达,ROCK1则呈高表达。Western blot检测结果显示,转染miR-335 mimic或转染ROCK1 siRNA后ROCK1的蛋白表达减少。Transwell小室法检测结果显示,过表达miR-335或下调ROCK1后穿过基膜的细胞数目明显下降。结论：miR-335能特异性结合于ROCK1基因的3'-UTR并下调ROCK1的表达,抑制人骨肉瘤细胞MG-63侵袭转移。     

lncRNA RHPN1-AS1靶向miR-485-5p调控骨肉瘤细胞增殖、凋亡、迁移、侵袭及其分子机制

目的:探讨lncRNA RHPN1反义RNA1（RHPN1-AS1）靶向miR-485-5p对骨肉瘤细胞增殖、凋亡、迁移、侵袭的影响和机制。方法:实时荧光定量PCR（qRT-PCR）检测20例骨肉瘤组织与其对应的癌旁组织、人正常成骨细胞hFOB1.19以及3种骨肉瘤细胞（U-2OS、SAOS-2、HOS）中RHPN1-AS1和miR-485-5p的表达水平。利用脂质体转染法将RHPN1-AS1小干扰RNA（si-RHPN1-AS1）、小干扰RNA阴性对照（si-NC）、miR-485-5p模拟物（miR-485-5p mimics）、miRNA阴性对照（miR-NC）分别转染U-2OS细胞,四甲基偶氮唑蓝（MTT）法检测细胞活力,流式细胞术检测细胞凋亡,Transwell实验检测细胞迁移和侵袭能力,蛋白质印记（Western blot）检测细胞周期蛋白D1（Cyclin D1）、p21、p27、B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）和基质金属蛋白酶14（MMP-14）蛋白的表达。双荧光素酶报告基因实验和qRT-PCR验证RHPN1-AS1和miR-485-5p的靶向调控关系。结果:与癌旁组织比较,骨肉瘤组织中RHPN1-AS1的表达水平显著升高,miR-485-5p的表达水平显著降低（P＜0.05）;与hFOB1.19细胞比较,3种骨肉瘤细胞中RHPN1-AS1的表达水平显著升高,miR-485-5p的表达水平显著降低（P＜0.05）。与si-NC组比较,si-RHPN1-AS1组U-2OS细胞的活力显著降低,迁移和侵袭能力显著降低,细胞凋亡率显著升高,Cyclin D1、Bcl-2、MMP-2、MMP-9和MMP-14蛋白的表达水平显著降低,p21、p27和Bax蛋白的表达水平显著升高（P＜0.05）;与miR-NC组比较,miR-485-5p组U-2OS细胞的活力显著降低,迁移和侵袭能力显著降低,细胞凋亡率显著升高,Cyclin D1、Bcl-2、MMP-2和MMP-9蛋白的表达水平显著降低,p21和Bax蛋白的表达水平显著升高（P＜0.05）。RHPN1-AS1靶向负性调控miR-485-5p表达。干扰miR-485-5p表达逆转了抑制lncRNA RHPN1-AS1表达对骨肉瘤U-2OS细胞增殖、凋亡、迁移和侵袭的影响。结论:抑制RHPN1-AS1通过上调miR-485-5p抑制骨肉瘤细胞增殖、迁移和侵袭,诱导细胞凋亡。     

MicroRNA-320 inhibits osteosarcoma cells proliferation by directly targeting fatty acid synthase

Increasing evidence has demonstrated that small noncoding microRNAs (miRNAs) could contribute to cancer development and progression. Besides, they are differentially expressed in human tumor tissues. In the current study, we found that miR-320 was significantly downregulated in human osteosarcoma tissues, compared with adjacent normal tissues. Introduction of miR-320 mimics into U2OS and MG63 cells inhibited cell proliferation, while cell apoptosis rate remained unaltered. Additionally, miR-320 overexpression could also suppress tumor growth in the nude mice. At the molecular level, our results further revealed that the expression of fatty acid synthase (FASN), a key enzyme for de novo biosynthesis of fatty acids, was negatively regulated by miR-320. Therefore, our results suggest that miR-320 may act as a tumor suppressor for osteosarcoma.     

miR-520b靶向DAD1调控肾癌细胞增殖、迁移和侵袭的作用机制

目的:探讨miR-520b和抗细胞凋亡因子（DAD1）对肾癌细胞增殖、迁移和侵袭的影响及分子机制。方法:qRT-PCR和Western blot实验检测A498人肾癌细胞中miR-520b和DAD1表达。将miR-520b mimics或DAD1 siRNA转染至A498细胞，MTT和Transwell实验检测细胞活力、迁移和侵袭。Targetscan在线预测、双荧光素酶报告基因和Western blot实验验证miR-520b和DAD1的靶向关系。将miR-520b mimics和pcDNA-DAD1共转染至A498细胞，检测细胞活力、迁移和侵袭。结果:在A498细胞中，miR-520b表达下调而DAD1表达上调。在A498细胞中过表达miR-520b或敲减DAD1，细胞活力下降，迁移和侵袭细胞数减少。Targetscan在线预测、双荧光素酶报告基因和Western blot实验结果表明，miR-520b可负调控DAD1蛋白表达。过表达DAD1可逆转miR-520b对A498细胞增殖、迁移和侵袭的抑制作用。结论:miR-520b能够通过靶向调节DAD1表达抑制A498细胞增殖、迁移和侵袭。     

长链非编码RNA TUG1调控miR-138-5p对骨肉瘤细胞增殖、凋亡、侵袭和迁移的影响

目的:探讨长链非编码RNA(LncRNA)牛磺酸调节基因1(TUG1)调控miR-138-5p对骨肉瘤细胞增殖、凋亡、侵袭和迁移的影响。方法:在骨肉瘤细胞U-2OS中转染TUG1 siRNA和siRNA control,qRT-PCR测定干扰效果,MTT测定增殖,流式细胞术测定凋亡,Transwell小室测定细胞侵袭和迁移。starBase预测TUG1与miR-138-5p有结合位点,双荧光素酶报告载体鉴定靶向调控关系。将miR-138-5p抑制物和TUG1 siRNA共转染至骨肉瘤细胞中,测定干扰miR-138-5p对下调TUG1的骨肉瘤细胞增殖、凋亡、侵袭和迁移的影响。结果:转染TUG1 siRNA后的骨肉瘤细胞中TUG1表达水平明显低于转染siRNA control后的细胞（P<0.05）。下调TUG1后的骨肉瘤细胞增殖、侵袭和迁移能力降低,细胞凋亡率升高（P<0.05）。野生型TUG1荧光素酶报告载体与miR-138-5p共转染后细胞荧光素酶活性降低（P<0.05）。与转染TUG1 siRNA的细胞比较,miR-138-5p抑制物和TUG1 siRNA共转染后的细胞增殖、侵袭和迁移能力升高,细胞凋亡率降低（P<0.05）。结论:下调LncRNA TUG1表达通过调控miR-138-5p表达抑制骨肉瘤细胞增殖、侵袭、迁移能力并诱导细胞凋亡。     

Knockdown of Long Noncoding RNA CAT104 Inhibits the Proliferation,Migration, and Invasion of Human Osteosarcoma Cells by Regulating MicroRNA-381

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. This study aimed to explore the effects of long noncoding RNA CAT104 and microRNA-381 (miR-381) on osteosarcoma cell proliferation, migration, invasion, and apoptosis, as well as the underlying potential mechanism. We found that CAT104 was highly expressed in osteosarcoma MG63 and OS-732 cells. Knockdown of CAT104 significantly inhibited OS-732 cell proliferation, migration, and invasion, but promoted cell apoptosis. CAT104 regulated the expression of miR-381, and miR-381 participated in the effects of CAT104 on OS-732 cells. Zinc finger E-box-binding homeobox 1 (ZEB1) was a direct target gene of miR-381, which was involved in the regulatory roles of miR-381 in OS-732 cell proliferation, migration, invasion, and apoptosis, as well as c-Jun N-terminal kinase (JNK) and Wnt/ -catenin pathways. In conclusion, our research verified that suppression of CAT104 exerted significant inhibitory effects on osteosarcoma cell proliferation, migration, and invasion by regulating the expression of miR-381 and downstream ZEB1, as well as JNK and Wnt/ -catenin pathways.     

MiR-29c模拟物对骨肉瘤细胞侵袭和迁移能力的影响

目的:观察miR-29c在具有不同转移能力的F4和F5M2细胞系中的表达差异;研究miR-29c模拟物对骨肉瘤细胞侵袭和迁移能力的影响。方法:利用qRT-PCR技术检测F4与F5M2间miR-29c的表达差异;将骨肉瘤细胞F5M2分为3组,运用细胞转染技术分别对两组转染miR-29c模拟物、模拟物阴性对照,第3组不作处理,随后用qRT-PCR技术检测3组间miR-29c的表达差异。Transwell法检测3组不同处理的F5M2细胞的侵袭和迁移能力是否存在差异。结果:MiR-29c在F4和F5M2细胞系中表达差异显著,miR-29c在高转移性的F5M2细胞中表达水平显著下降。与阴性对照组和空白组相比,转染miR-29c模拟物的F5M2细胞侵袭和迁移能力均明显减弱,且差异具有统计学意义（P<0.05）。结论:MiR-29c在高转移性骨肉瘤细胞中低表达,且miR-29c模拟物具有抑制骨肉瘤细胞侵袭和转移的作用。MiR-29c有可能作为骨肉瘤转移基因治疗的一个新靶点。     

柴胡皂苷D通过逆转EMT抑制人成骨肉瘤细胞MG-63的迁移和侵袭

目的:研究柴胡皂苷D对人成骨肉瘤细胞MG-63的迁移和侵袭能力的影响并初步探讨其潜在分子机制.方法:运用MTT试验检测柴胡皂苷D对细胞增殖的影响,划痕试验和Transwell迁移试验检测柴胡皂苷D细胞迁移能力的影响,Transwell侵袭试验研究柴胡皂苷D对细胞侵袭能力的影响,Western-blot检测柴胡皂苷D对EMT相关蛋白的影响.结果:对MG-63增殖无明显影响浓度的柴胡皂苷D显著抑制细胞的迁移和侵袭能力(P<0.001),柴胡皂苷D上调E-cadherin的表达而下调N-cadherin和Vimentin的表达.结论:柴胡皂苷D通过逆转EMT改变抑制人成骨肉瘤细胞MG-63的迁移和侵袭.     

miR-548d抑制骨肉瘤细胞的增殖和迁移

目的:探讨miR-548d对骨肉瘤细胞增殖和迁移的影响。方法:通过Real time PCR及Western blot检测miR-548d和KRAS在30例骨肉瘤组织以及293、MG63和U2OS细胞中的表达情况。对30例骨肉瘤组织中miR-548d和KRAS含量的相关性进行分析，随后通过报告基因实验印证miR-548d对KRAS的靶向作用。MG63细胞中分别过表达或沉默miR-548d后，通过Western blot实验分析KRAS的变化情况，MTT实验观察miR-548d对细胞增殖的影响，Transwell实验观察miR-548d对其迁移能力的影响。结果:骨肉瘤组织及细胞中miR-548d表达较低，KRAS表达较高。在骨肉瘤组织中miR-548d与KRAS的表达呈负相关。报告基因实验证明miR-548d可以直接打靶KRAS。Western blot指出miR-548d可以抑制KRAS的表达。最后MTT和Transwell实验指出miR-548d可以抑制MG63细胞的增殖与迁移。结论:miR-548d可以通过打靶KRAS抑制骨肉瘤细胞的增殖和迁移。     

miRNA-95在骨肉瘤组织中的表达及其对MG-63细胞增殖和侵袭的影响

目的：研究miRNA-95在骨肉瘤组织和细胞株中的表达情况及其对骨肉瘤MG-63细胞增殖、凋亡、细胞周期和侵袭 能力的影响。方法：以实时荧光定量PCR检测15例骨肉瘤组织及其癌旁组织（标本收集自2015年1月至2018年1月青岛市海 慈医疗集团外科手术的病例）和骨肉瘤细胞株（MG-63、 U2OS、143B和HOS）与正常人成骨细胞株hFOB1.19中的miRNA-95表 达。利用Lipofectamine 2000将miRNA-95 mimics和miRNA-95 inhibitors分别转染至人骨肉瘤MG-63细胞株中,并设置miRNANC对照组,CCK-8法检测各组细胞增殖活力变化,流式细胞术检测各组细胞周期和凋亡变化,Transwell方法检测各组细胞侵袭 能力变化,双荧光素酶活性实验检测并验证miRNA-95在MG-63细胞中的靶向基因。结果：miRNA-95在人骨肉瘤组织中的表 达水平显著高于癌旁组织（P<0.01）, 在MG-63、 U2OS、143B和HOS细胞中的表达水平显著高于hFOB1.19,且在MG-63细胞中表 达水平最高（P<0.01）。与miRNA-NC对照组相比,miRNA-95 mimics组中MG-63细胞的增殖活力显著上升、细胞凋亡率显著下 降而侵袭率显著上升（均P<0.01）、 而miRNA-95 inhibitors组中MG-63细胞增殖活力显著下降、细胞周期被阻滞、细胞凋亡率显著 上升而侵袭率显著下降（均P<0.01）;miRNA-95在骨肉瘤MG-63细胞中靶向上皮膜蛋白-1（epithelial membrane protein-1,EMP-1） 基因发挥作用。结论：miRNA-95在人骨肉瘤组织和细胞中均呈高表达,抑制骨肉瘤MG-63细胞中miRNA-95表达能够促进细 胞凋亡进而抑制细胞增殖、细胞周期及侵袭能力,该作用可能通过靶向EMP-1基因而发挥。     

Down-regulation of c-Met and Bcl2 by microRNA-206, activates apoptosis,and inhibits tumor cell proliferation,migration and colony formation

Hsa-miRNA-206 (miR-206), highly expressed in skeletal muscle, has recently been discovered to have anticancer properties in different tissues. However, the role of miR-206 on lung cancer is still ambiguous. In this study, we investigated the role of miR-206 on the development of lung cancer. The results indicated that miR-206 expression was suppressed in lung cancer tissues and very low levels were found in non-small cell lung cancer (NSCLS) cell liness. Transient transfection of miR-206 into cultured A549 and SK-MES-1 cells led to significant decrease in cell growth, migration, invasion and colony formation, and promoted cell apoptosis. Using bioinformatics, we identified putative miR-206 binding sites within the 3′-untranslated region (3′-UTR) of the human c-Met and Bcl2 mRNA. The expression of c-Met and Bcl2 proteins were shown to be down-regulated after treated with miR-206 by subsequent Western blot and qRT-PCR analysis. Conversely, up-regulation of c-Met and Bcl2 were confirmed in tissue samples of human lung cancer, with its level inversely correlated with miR-206 expression. In addition, miR-206 also decreased the gene expression of MMP-9, CCND1 and CCND2 while increased the gene expression of p57 (Kip2) in A549 and SK-MES-1 cells. Taken together, our results demonstrated that miR-206 suppressed c-Met and Bcl2 expression in NSCLS and could function as a potent tumor suppressor in c-Met/Bcl2-over expressing tumors. Inhibition of miR-206 function could contribute to aberrant cell proliferation, migration, invasion and apoptosis, leading to NSCLS development.     

miR-369靶向调节SOX4表达对骨肉瘤细胞增殖和凋亡的调控作用

目的:观察微小RNA（miRNA,miR）-369通过调节SOX4对骨肉瘤MG-63细胞增殖、凋亡的影响。方法:miR-369体外模拟物（mimics）转染,构建miR-369上调表达模型,以转染阴性对照链为对照组,CCK-8法和流式细胞技术分别检测细胞增殖率和凋亡率变化;Real-time PCR法检测SOX4 mRNA表达水平;双荧光素酶活检检测证实miR-369与SOX4相互作用关系;Western-blot法检测不同组间SOX4及Bcl-2家族相关蛋白表达水平变化。结果:miR-369 mimics转染后,MG-63细胞中miR-369表达水平较对照组明显提高(P=0.009);miR-369 mimic转染后24 h及48 h后细胞相对存活率均明显低于对照组;流式细胞仪结果显示mimic组细胞凋亡率较对照组明显提高（P=0.031）;Real-time PCR和Western blot实验分别证实miR-369 mimic转染后,SOX4在mRNA和蛋白水平表达均明显抑制;而Bcl-2家族相关蛋白表达水平发生明显变化。结论:miR-369表达上调可以抑制骨肉瘤细胞增殖并诱导凋亡,这些作用与靶向下调SOX4表达有关。     

miR-185通过靶向调控CDC42基因表达抑制骨肉瘤MG63细胞的增殖和迁移

目的：分析miR-185以及细胞分裂周期蛋白42(CDC42)在骨肉瘤组织和细胞中的表达情况,初步探究miR-185是否通过调控CDC42影响骨肉瘤MG63细胞的增殖与迁移。方法：选取2020年1月至2021年1月于衡水市第四人民医院经病理确诊为骨肉瘤的的28例患者的癌组织及癌旁组织,采用免疫组化法检测骨肉瘤组织中CDC42的表达,采用qPCR法检测骨肉瘤组织中miR-185的表达。双荧光素酶报告基因实验验证CDC42基因与miR-185间的靶向关系。根据转染物不同,将MG63细胞分为miR-185 mimic组、miR-NC组、miR-185 inhibitor组、NC-inhibitor组、CDC42组（转染CDC42过表达载体）及阴性对照（NC）组,采用划痕愈合实验、CCK-8法和流式细胞术分别检测miR-185和CDC42表达对MG63细胞迁移、增殖和周期的影响。构建骨肉瘤MG63细胞裸鼠移植瘤模型,采用免疫组化法、qPCR法和WB法检测过表达或敲降miR-185对移植瘤组织中Ki67与CDC42表达的影响。结果：与癌旁组织相比,骨肉瘤组织中miR-185表达明显降低,而CDC...