miR-182-5p在膀胱癌中的表达及其机制

  目的  探究miR-182-5p及FOXO3在膀胱癌中的表达意义及其参与膀胱癌可能的机制。  方法  收集2017年6月至2019年6月64例于郑州人民医院行手术切除膀胱癌患者的组织标本和临床资料,采用RT-qPCR检测膀胱癌组织及膀胱癌细胞中的miR-182-5p和FOXO3表达,按照其表达水平分为miR-182-5p高表达组和miR-182-5p低表达组、FOXO3高表达组和FOXO3低表达组,采用Western blot实验检测FOXO3蛋白的表达。采用Kaplan-Meier生存曲线分析miR-182-5p和FOXO3与膀胱癌患者预后的相关性。采用双荧光素酶报告实验分析miR-182-5p与FOXO3的靶向关系,并采用实时荧光定量PCR（RT-qPCR）和Western blot检测转染miR-182-5p抑制剂对FOXO3表达的影响;分别采用CCK-8、Transwell实验和流式细胞术检测转染miR-182-5p抑制剂对T24细胞增殖、侵袭和凋亡的影响。  结果  miR-182-5p在膀胱癌组织中的表达量为2.27±0.66,显著高于癌旁组织的1.04±0.36,两者比较差异具有统计学意义（P<0.001）,与肿瘤大小、TNM分期、淋巴结转移有关（P<0.05）;FOXO3在膀胱癌组织中低表达,与肿瘤大小、分化程度、TNM分期及淋巴结转移有关（P<0.05）;miR-182-5p高表达组、FOXO3低表达组的患者的生存率显著低于miR-182-5p低表达组、FOXO3高表达组（P=0.038,P=0.004）。T24细胞中miR-182-5p高表达（P<0.05）。抑制miR-182-5p上调FOXO3表达,抑制miR-182-5p表达能抑制T24细胞的增殖和侵袭,促进细胞凋亡（P<0.05）。  结论  miR-182-5p在膀胱癌组织中高表达,而FOXO3低表达。miR-182-5p促进膀胱癌细胞的增殖和侵袭,抑制其凋亡,可能与负调控FOXO3有关。      

乳腺癌患者组织中miR-6861-5p的表达及临床意义

目的:研究乳腺癌患者组织中微小RNA-6861-5p（miR-6861-5p）的表达水平及临床意义。方法:完全随机选取本院2013年7月至2017年7月住院患者乳腺癌组织139例、良性病变组织101例及癌旁组织79例,实时定量PCR检测miR-6861-5p表达水平并分析乳腺癌组织miR-6861-5p的表达与临床病理特征的相关性。以43例完成随访患者治疗前miR-6861-5p表达量的中位数,将其分为miR-6861-5p高表达组（24例）和低表达组（19例）,Log-rank单因素分析比较两组患者的生存期差异。结果:乳腺癌组织中miR-6861-5p的RNA表达水平（9.242±3.188）显著高于良性病变组织（7.224±3.249）（P<0.01）及癌旁组织（7.221±2.594）（P<0.01）,其表达水平与肿瘤分期、淋巴结转移情况及激素受体表达相关（P<0.05）;miR-6861-5p在Luminal B-like型的表达量（10.910±2.386）显著高于TNBC亚型（7.605±2.565）（P<0.001）;miR-6861-5p高表达组的3年生存率低于miR-6861-5p低表达组（P=0.023）。结论:乳腺癌组织中miR-6861-5p的表达水平显著增高,是乳腺癌疾病进展、淋巴结转移及分子分型的潜在生物标志物。     

血清miR-6861-5p检测在乳腺癌临床诊治中的价值探讨

  目的  研究miR-6861-5p在乳腺癌患者血清中的表达,并探讨miR-6861-5p表达在乳腺癌患者临床诊治中的价值。  方法  通过实时荧光定量PCR法（RT-qPCR）检测2012年1月至2015年6月山东省德州市第二人民医院112例乳腺癌患者、37例乳腺良性病变和53例健康女性血清miR-6861-5p相对表达量,分析血清miR-6861-5p表达与乳腺癌患者临床病理和术后复发之间的关系,并探讨其表达对乳腺癌的临床诊断价值。  结果  乳腺癌患者血清miR-6861-5p相对表达量为（7.99±1.63）,显著高于良性病变患者（6.45±1.06）（P＜0.05）和健康研究对象（6.43±1.28）（P＜0.05）;血清miR-6861-5p表达与乳腺癌患者淋巴结转移、肿瘤组织ER、PR和HER-2表达、TNM分期、分子分型以及组织学分期显著相关（均P＜0.05）;肿瘤切除后乳腺癌患者血清miR-6861-5p表达量显著降低;治疗前血清miR-6861-5p诊断乳腺癌的敏感度和特异度分别为69.6%和77.8%,且与乳腺癌患者术后复发率有关。  结论  miR-6861-5p在乳腺癌患者血清中表达上调,是术前乳腺癌诊断、肿瘤分期、癌细胞转移和术后监测乳腺癌复发的潜在生物标志物。      

miR-26b-3p对乳腺癌细胞生物学行为的影响及作用机制研究

背景与目的：miRNA是一类长度为21~23个核苷酸的单链非编码RNA分子，其作用机制主要为靶向于mRNA的3’非翻译区（3’ untranslated region，3’UTR）从而抑制其靶基因的表达。miRNA在肿瘤的发生、发展过程中发挥着关键作用，探讨miR-26b-3p对乳腺癌生物学行为的影响及作用机制。方法：通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测miR-26b-3p在三种乳腺癌细胞系MCF-7、MDA-MB-231和MDA-MB-453中的表达，选取miR-26b-3p表达水平最低的乳腺癌细胞转染miR-26b-3p mimics后，采用细胞计数试剂盒（cell counting kit-8，CCK-8）法检测细胞的增殖，采用transwell迁移和侵袭实验检测细胞迁移和侵袭能力，通过小动物活体成像及裸鼠移植瘤模型检测miR-26b-3p对乳腺癌细胞裸鼠移植瘤生长和转移的影响，采用双荧光素酶报告基因分析检测miR-26b-3p与锌指E盒结合同源盒基因1（zinc finger E-box binding homeobox 1，ZEB1）的相互作用，采用RTFQ-PCR和蛋白质印迹法（Western blot）检测ZEB1的表达。结果：乳腺癌细胞系MDA-MB-453中miR-26b-3p表达最低，在MDA-MB-453细胞中转染miR-26b-3p mimics后，miR-26b-3p的表达水平显著升高（P<0.05），细胞的增殖能力显著降低（P<0.05），细胞的迁移（P<0.001）和侵袭能力（P<0.01）显著降低。过表达miR-26b-3p可抑制裸鼠体内乳腺癌移植瘤的生长和转移。miR-26b-3p可与ZEB1的3’UTR结合，抑制ZEB1的表达。结论：miR-26b-3p可靶向于ZEB1，抑制乳腺癌细胞的增殖、迁移和侵袭，抑制乳腺癌的生长和转移。     

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA–target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA–mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.     

成骨肉瘤细胞SOSP-9607中miRNA的克隆与验证

背景与目的:microRNA(miRNA)是一类非编码蛋白,并参与转录后调节的单链小分子RNA(约20～25个碱基),对基因表达具有重要调控作用,与肿瘤的发生存在着密切的关系.本研究拟克隆成骨肉瘤细胞系SOSP-9607中miRNA,并对部分功能性基因进行验证.方法:以SOSP-9607细胞作为实验对象,分离提取细胞内小片段RNA(≤200 nt),多聚腺苷酸化和5'连接子连接后,通过RT-PCR进行反转录和扩增,克隆到pCR 4-TOPO载体上得到大约109 bp DNA片段,测序后经过生物信息学分析确定miRNA的表达情况.根据当前miRNA的研究现状,在克隆到的miRNA中选取部分功能性miRNA和新发现miRNA,合成相应探针后与从SOSP-9607细胞、HeLa细胞和成骨肉瘤组织中分离出的小片段RNA进行Northern印迹验证.结果:克隆测序后得到182个克隆体,经生物信息学分析发现有47个miRNA(25种),其中含有23种已知的miRNA和2种Nature杂志上预测的miRNA(miR-165和miR-166).选取的三条实体瘤相关的miRNA(miR-21、miR-20a、miR-17-5p)以及两条预测的miRNA进行Northern印迹验证,miR-21、miR-20a、miR-17-5p在SOSP-9607细胞和成骨肉瘤组织中均表达,预测的miR-165和miR-166在SOSP-9607细胞和成骨肉瘤组织中亦均表达,但miR-166在HeLa细胞中没有表达.结论:克隆了SOSP-9607细胞中的miRNA,并验证了部分功能性miRNA的表达,提示miRNA与肿瘤发生可能有密切的关系.     

Plasma Circulating Mirnas Profiling for Identification of Potential Breast Cancer Early Detection Biomarkers

Objective: This study aimed to characterize the miRNA expression profiles from plasma samples of our local breast cancer patients in comparison to healthy control by using miRNA PCR Array. Methods: In this study, plasma miRNA profiles from eight early-stage breast cancer patients and nine age-matched (± 2 years) healthy controls were characterized by miRNA array-based approach, followed by differential gene expression analysis, Independent T-test and construction of Receiver Operating Characteristic (ROC) curve to determine the capability of the assays to discriminate between breast cancer and the healthy control. Results: Based on the 372-miRNAs microarray profiling, a set of 40 differential miRNAs was extracted regarding to the fold change value at 2 and above. We further sub grouped 40 miRNAs of breast cancer patients that were significantly expressed at 2-fold change and higher. In this set, we discovered that 24 miRNAs were significantly upregulated and 16 miRNAs were significantly downregulated in breast cancer patients, as compared to the miRNA expression of healthy subjects. ROC curve analysis revealed that seven miRNAs (miR-125b-5p, miR-142-3p, miR-145-5p, miR-193a-5p, miR-27b-3p, miR-22-5p and miR-423-5p) had area under curve (AUC) value > 0.7 (AUC p-value < 0.05). Overlapping findings from differential gene expression analysis, ROC analysis, and Independent T-Test resulted in three miRNAs (miR-27b-3p, miR-22-5p, miR-145-5p). Cohen’s effect size for these three miRNAs was large with d value are more than 0.95. Conclusion: miR-27b-3p, miR-22-5p, miR-145-5p could be potential biomarkers to distinguish breast cancer patients from healthy controls. A validation study for these three miRNAs in an external set of samples is ongoing.     

miR-221在胃癌中的表达及临床诊断意义

目的:研究微小RNA-221（microRNA-221,miR-221）在胃癌中的表达及应用于胃癌临床诊断的潜在可能。方法:收集原发性胃癌患者及健康对照组血清标本各30例;miRNA 表达谱分析确定miR-221的差异化表达;利用多元线性回归模型分析miR-221与胃癌临床病理因素(性别、年龄、肿瘤大小、TNM分期、肿瘤部位、淋巴结转移及组织类型等)之间的关系。利用受试者工作特征(ROC)曲线评估miR-221诊断胃癌的敏感性及特异性;通过约登指数(Youden index)确定其最佳诊断截断值(cut-off value)。结果:miR-221在胃癌标本中的表达水平（4.52±1.78）显著高于健康对照组（2.38±1.37）,差异有显著统计学意义（P＜0.01）。miR-221的表达与胃癌患者性别、年龄、肿瘤大小、肿瘤部位及组织类型等因素无明显相关性;而与TNM分期和淋巴结转移有显著相关性。miR-221诊断胃癌的截断值为4.214,诊断敏感性为76.3%,特异性为87.6%。结论:miR-221在胃癌中存在显著异常高表达,在诊断胃癌方面具有潜在临床应用价值。     

miR-34c-3p与增殖细胞核抗原在三阴性乳腺癌组织和血清中的表达及意义

目的:检测miR-34c-3p与增殖细胞核抗原（PCNA）在三阴性乳腺癌(TNBC)组织和血清中的表达，分析二者的相关性，探讨其在TNBC发病中的意义。方法:选择2017年12月至2019年12月于空军军医大学第一附属医院就诊的72例TNBC患者作为实验组(A组)，以同期就诊的60例乳腺纤维腺瘤或乳腺腺病患者为对照组(B组)。采用Real-time RT-PCR 检测乳腺组织标本中miR-34c-3p mRNA及PCNA mRNA的表达；免疫组化检测两组乳腺组织中PCNA蛋白表达；ELISA法检测血清中PCNA的表达。并进一步分析癌组织中miR-34c-3p mRNA的表达与癌组织中PCNA mRNA及血清PCNA水平的相关性。结果:A、B组miR-34c-3p mRNA的相对表达量分别为0.884±0.159及1.255±0.131，A组明显低于B组，差异有统计学意义(P＜0.001)。A、B组PCNA mRNA的相对表达量分别为1.544±0.215及 1.230±0.204，A组表达高于B组，差异有统计学意义(P＜0.001)。免疫组化检测PCNA蛋白表达，A组阳性例数为50例，阴性例数为22例；B组阳性例数为21例，阴性例数为39例；A组阳性率明显高于B组，差异有统计学意义（P＜0.001）。两组患者血清中PCNA的表达，A组［(383.194±110.832） pg/ml］明显高于B组［(257.867±101.697） pg/ml］，差异有统计学意义(P＜0.001)。将A组TNBC患者肿瘤组织miR-34c-3p mRNA表达结果分别与PCNA mRNA表达结果及其血清中PCNA表达结果进行Pearson相关分析，结果显示:miR-34c-3p mRNA与PCNA mRNA及血清PCNA均呈明显负相关关系( P＜0.001、P=0.024<0.05)。结论:TNBC组织中miR-34c-3p表达下调，PCNA在癌组织及血清中均表达上调，二者在TNBC患者组织和血清中呈明显负相关关系。miR-34c-3p与 PCNA可能在TNBC的发病中起着重要的作用。     

MicroRNA-30a inhibits cell migration and invasion by downregulating vimentin expression and is a potential prognostic marker in breast cancer

Tumor recurrence and metastasis result in an unfavorable prognosis for cancer patients. Recent studies have suggested that specific microRNAs (miRNAs) may play important roles in the development of cancer cells. However, prognostic markers and the outcome prediction of the miRNA signature in breast cancer patients have not been comprehensively assessed. The aim of this study was to identify miRNA biomarkers relating to clinicopathological features and outcome of breast cancer. A miRNA microarray analysis was performed on breast tumors of different lymph node metastasis status and with different progression signatures, indicated by overexpression of cyclin D1 and β-catenin genes, to identify miRNAs showing a significant difference in expression. The functional interaction between the candidate miRNA, miR-30a, and the target gene, Vim, which codes for vimentin, a protein involved in epithelial-mesenchymal transition, was examined using the luciferase reporter assay, western blotting, and migration and invasion assays. The association between the decreased miR-30a levels and breast cancer progression was examined in a survival analysis. miR-30a negatively regulated vimentin expression by binding to the 3'-untranslated region of Vim. Overexpression of miR-30a suppressed the migration and invasiveness phenotypes of breast cancer cell lines. Moreover, reduced tumor expression of miR-30a in breast cancer patients was associated with an unfavorable outcome, including late tumor stage, lymph node metastasis, and worse progression (mortality and recurrence) (p < 0.05). In conclusion, these findings suggest a role for miR-30a in inhibiting breast tumor invasiveness and metastasis. The finding that miR-30a downmodulates vimentin expression might provide a therapeutic target for the treatment of breast cancer.     

Circulating miR-195 as a Therapeutic Biomarker in Turkish Breast Cancer Patients

Background: Dysregulation of miRNA expression may be used as a biomarker for specific tumours because it may contribute to development of cancer. Circulating miRNA profiles have been highlighted for their potential as predictive markers in heterogeneous diseases such as breast cancer. In the literature, there is evidence that miR-195 levels are differentially expressed pre- and post-operative periods in breast cancer patients. At the same time, miRNA expression levels may vary because of ethnic origins. This study aimed to determine expression levels and potential roles of miR-195 in Turkish breast cancer patients. Materials and Methods: The expression patterns of miR-195 were initially examined in breast cancer tissues (luminal A and B type) (n96). Subsequently, blood samples were prospectively collected from preoperative and postoperative Turkish breast cancer patients and disease free controls. Total RNA was isolated, and the expression level of miR-195 was quantified by real-time PCR. Results: We found that miR-195 level was altered in Turkish breast cancer patients, with down-regulation evident in breast cancer tissues compared to normal adjacent specimens. Furthermore, circulating levels of miR- 195 was significantly decreased in post-operative blood samples compared with pre-operative levels (p0.01 and <0.05). However, miR-195 was significantly increased in pre-operative blood samples of the luminal B type (p 0.04 and <0.05). Conclusions: This study represents the first report of a miR-195 expression profile in Turkish breast cancer patients. Our data suggests that miR-195 levels might be a clinically useful biomarker in the earliest stage of Turkish breast cancer patients.     

Goseki Grade and Tumour Location Influence Survival of Patients with Gastric Cancer

Background: Owing to the variability of histopathological features and biological behaviour in gastriccarcinoma, a great number of categorisation methods such as classical histopathologic grading, Laurenclassification, the TNM staging system and the newly presented Goseki grading method are used by pathologistsand other scientists. In our study, we aimed to investigate whether Goseki grade and tumour location havean effects on survival of gastric cancer cases. Materials and Methods: Eighty-four patients with gastricadenocarcinoma were covered in the investigation. The importance of Goseki grading system and tumour locationwere analysed in addition to the TNM staging and other conventional prognostic parameters. Results: Themedian survival time in our patients was 35 months (minimum: 5, maximum: 116). According to our findings,there was no relation between survival and tumour size (p=0.192) or classical histological type (p=0.270). Incontrast, the Goseki grade and tumour location significantly correlated with survival (p=0.007 and p<0.001,respectively). Additionally, tumours of the intestinal type had a longer median survival time (60.0 months) thandiffuse tumours (24.0 months). Conclusions: In addition to the TNM staging system, tumour location and theGoseki grading system may be used as significant prognostic parameters in patients with gastric cancer.     

miR-4319通过靶向调控USP2抑制乳腺癌细胞侵袭

目的探讨miR-4319与泛素特异性蛋白酶2(USP2)表达的相关性以及miR-4319靶向USP2通过核转录因子κB(NF-κB)信号通路对乳腺癌细胞侵袭的影响。方法实时荧光定量PCR(qRT-PCR)检测miR-4319在正常乳腺癌上皮细胞(MCF10A)、低侵袭性乳腺癌细胞(MCF7)和高侵袭性乳腺癌细胞(MDA-MB-231)中的表达量。将MCF10A、MCF7和MDA-MB-231细胞分为6组进行转染:(1)MDA-MB-231/NC组,瞬时转染插入一段乱码序列(scramble 1),即作为miR-4319过表达对照质粒;(2)MDA-MB-231/miR-4319组,瞬时转染插入目的片段miR-4319质粒过表达miR-4319,即miR-4319过表达;(3)MDA-MB-231/miR-4319+Con组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达对照质粒,即miR-4319过表达以及USP2过表达对照;(4)MDA-MB-231/miR-4319+USP2组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达质粒,即miR-4319过表达和USP2过表达;(5)MDA-MB-231/miR-4319inhibitor组,瞬时转染miR-4319的反义序列抑制miR-4319的表达,即抑制miR-4319的表达;(6)MDA-MB-231/miR-4319inhibitor NC组,瞬时转染插入一段乱码序列(scramble 2),即作为抑制miR-4319组的对照组。qRT-PCR检测miR-4319在MDA-MB-231细胞中的转染效率。荧光素酶实验检测miR-4319与USP2 mRNA是否存在结合位点。qRT-PCR检测miR-4319在乳腺癌细胞中过表达后的USP2 mRNA表达水平。蛋白质印迹法检测过表达miR-4319或抑制miR-4319表达后的USP2蛋白表达水平。Transwell侵袭实验检测过表达miR-4319或USP2后MDA-MB-231细胞侵袭能力。双荧光素酶实验检测miR-4319对NF-κB信号通路活性的影响以及过表达USP2后miR-4319对NF-κB信号通路活性的影响。结果 qRT-PCR结果显示,miR-4319相对表达量在细胞MDA-MB-231(t=14.860,P<0.001)和MCF7(t=12.770,P<0.001)中分别为0.330±0.075和0.570±0.082,均低于细胞MCF10A(1.012±0.051);miR-4319相对表达量MDA-MB-231/miR-4319组(3.980±0.083)高于MDA-MB-231/NC组(1.009±0.058),差异有统计学意义,t=102.90,P<0.001。荧光素酶实验结果显示,pGL3-USP2 3’-UTR-WT报告载体与miR-4319质粒共转染后的荧光素酶活性下降,差异有统计学意义,t=15.740,P<0.001。qRT-PCR结果显示,USP2 mRNA相对表达量MDA-MB-231/NC组(1.013±0.058)和MDA-MB-231/miR-4319组(0.988±0.062)基本没有变化,差异无统计学意义,t=1.201,P=0.296。而蛋白质印迹法结果显示,USP2蛋白相对表达量miR-4319组(0.371±0.083)低于miR-4319/NC组(1.003±0.064),miR-4319/inhibitor组(1.982±0.093)高于inhibitor/NC组(1.104±0.072),USP2蛋白相对表达量的组间比较差异有统计学意义,F=212.4,P<0.001。Transwell侵袭实验结果显示,穿过Matrigel细胞数MDA-MB-231/miR-4319组(58.337±5.972)低于MDA-MB-231/NC组(192.371±5.476),差异有统计学意义,t=29.720,P<0.001;穿过Matrigel细胞数MDA-MB-231/miR-4319+USP2组(167.197±8.292)高于MDA-MB-231/miR-4319+Con组(63.283±10.397),差异有统计学意义,t=14.150,P=0.001。双荧光素酶结果显示,miR-4319/NF-κB-luc组荧光素酶活性(0.324±0.06)低于NC/pRL-TK组(1.024±0.080)、NC/NF-κB-luc组(2.023±0.110)和miR-4319/pRL-TK组(1.109±0.050),组间比较差异有统计学意义,F=237.1,P<0.001;miR-4319+USP2/NF-κB-luc组荧光素酶活性(2.032±0.12)高于miR-4319+USP2/pRL-TK组(1.094±0.100)、miR-4319+Con/pRL-TK组(1.063±0.080)和miR-4319+Con/NF-κB-luc组(0.334±0.050),组间比较差异有统计学意义,F=164.2,P<0.001。结论 miR-4319在乳腺癌细胞中低表达,miR-4319靶向结合USP2,过表达USP2逆转了miR-4319对乳腺癌细胞侵袭能力的抑制作用和NF-κB转录活性的抑制作用。miR-4319通过靶向结合USP2抑制NF-κB信号通路,从而抑制乳腺癌细胞的侵袭。