凉血通瘀方干预高血压大鼠急性脑出血模型对脑组织中差异miRNA表达的影响＊

目的 研究凉血通瘀方对高血压大鼠急性脑出血模型脑组织miRNA表达的影响，对差异表达的miRNA靶基因进行分析，探索凉血通瘀方可能的药效机制。方法 将自发性高血压大鼠随机分成对照组（B）和实验组（C）。适应性饲养一周后，C组灌胃凉血通瘀方，B组灌胃等体积生理盐水，连续5天，每天1次。构建脑出血模型后收集脑组织，借助全转录组测序技术获得miRNA表达量，与miRBase数据库比对获取已知miRNA，使用miRDeep2预测新miRNA。差异分析软件为DESeq2，筛选阈值为|log₂FC| ≥1 并且P <0.05。对显著差异表达的miRNA进行靶基因预测，对靶基因进行GO功能、KEGG通路富集和PPI网络分析。结果 实验组和对照组对比，共发现21个显著差异表达的miRNA，上调有9个，下调有12个，共预测得到1243个有统计学意义的靶基因。GO富集分析发现，生物过程中突触囊泡分泌的调节、神经递质分泌的调节和神经递质运输的调节占前三位，神经元投射终点、全膜、质膜区域和细胞投射则是主要的细胞成分。分子功能分别为小GTPase绑定、底物特异性跨膜转运蛋白活性和离子跨膜转运体活性。通路分析结果显示，靶基因在癌证通路、pI3K-Akt信号通路、人类乳头瘤病毒感染、神经活性配体-受体相互作用和MAPK通路等分布广泛。采用STRING网站和Cytoscape软件，根据MCC算法筛选出ADRA2C、CASR、CCL28、CCR1、DRD2、GNAT3、GRM2、DYNC1LI1、GABBR1、GNAI1等核心靶基因。结论 凉血通瘀方对脑出血急性期鼠脑组织内miRNA的表达有重要影响；显著差异表达miRNAs可能通过靶向核心基因调控凉血通瘀方干预急性脑出血的病理过程及预后。     

miR-145调控肝癌腹水模型Th9细胞升高的机制研究

目的:探讨miR-145调控肝癌腹水模型Th9细胞升高的作用机制。方法:构建小鼠H22肝癌腹水模型。造模两周后处死小鼠并分离出脾脏组织，流式细胞术分析肝癌腹水组（MA组）与正常对照组（Control 组）小鼠脾脏Th9细胞表达水平。ELISA法检测脾脏IL-9表达水平。RT-PCR法检测脾脏miR-145表达水平。Western Blot法检测脾脏中PI3K/Akt/mTOR/P70S6K/HIF-1α相关蛋白表达水平。分选小鼠脾脏CD4+T细胞，随机分为miR-145 mimics组和NC组，分别应用miR-145-5P mimics、阴性对照寡核苷酸（negative control，NC）进行转染，RT-PCR检测各组miR-145、HIF-1α mRNA及IL-9 mRNA表达水平。结果:与Control 组相比，MA组Th9细胞及其细胞因子IL-9表达均升高（P<0.05），miR-145表达降低（P<0.05），p-PI3K、p-Akt、mTOR、p-mTOR、p-P70S6K、HIF-1α蛋白均升高（P<0.05）。与NC组相比，miR-145 mimics组miR-145显著升高，HIF-1α mRNA及IL-9 mRNA表达下降。结论:肝癌腹水中Th9细胞升高可能与miR-145下降导致的PI3K/Akt/mTOR/P70S6K/HIF-1α通路激活有关。     

circRNA_001569通过miR-145/HBXIP轴影响乳腺癌细胞的恶性生物学行为

目的：探讨 circRNA_001569 通过 miR-145/HBXIP 轴在乳腺癌细胞增殖、侵袭、迁移中发挥的作用。方法:收集2016年1月至2019年1月期间衡水市人民医院收治的30例乳腺癌患者的癌组织和癌旁组织。qPCR检测circRNA_001569在乳腺癌组织、癌旁组织以及细胞系中的表达。生物信息学工具预测miR-145的靶基因，RNA免疫沉淀（RNA immunoprecipitation，RIP）和双荧光素酶报告基因实验检测 miR-145 或靶基因之间的相互作用 ；向乳 腺 癌 MDA-MB-231 和 MCF-7细胞中转染si-circRNA_001569、miR-145 mimics或miR-145 inhibitor，建立基因过表达或沉默的细胞模型，qPCR和Western blotting分别检测转染对相关基因和蛋白表达的影响，CCK-8法、Transwell实验检测转染对细胞增殖、侵袭和迁移的影响。结果:在乳腺癌组织和乳腺癌细胞中，circRNA_001569 和 HBXIP 均呈高表达、miR-145 呈低表达。RIP 分析和双荧光素酶实验证实了 miR-145 与circRNA_001569和HBXIP之间的靶向关系；circRNA_001569或HBXIP过表达促进MDA-MB-231和MCF-7细胞的增殖、侵袭和迁移（均 P<0.01），而 miR-145 过表达起相反的作用（均 P<0.01）。结论:circRNA_001569 可能通过下调 miR-145 的表达、上调HBXIP的表达从而促进乳腺癌细胞的增殖、侵袭和迁移。     

Plasma miRNA-based signatures in CRC screening programs

Colorectal cancer (CRC) screening programs help diagnose cancer precursors and early cancers and help reduce CRC mortality. However, currently recommended tests, the fecal immunochemical test (FIT) and colonoscopy, have low uptake. There is therefore a pressing need for screening strategies that are minimally invasive and consequently more acceptable to patients, most likely blood based, to increase early CRC identification. MicroRNAs (miRNAs) released from cancer cells are detectable in plasma in a remarkably stable form, making them ideal cancer biomarkers. Using plasma samples from FIT-positive (FIT+) subjects in an Italian CRC screening program, we aimed to identify plasma circulating miRNAs that detect early CRC. miRNAs were initially investigated by quantitative real-time PCR in plasma from 60 FIT+ subjects undergoing colonoscopy at Fondazione IRCCS Istituto Nazionale dei Tumori, then tested on an internal validation cohort (IVC, 201 cases) and finally in a large multicenter prospective series (external validation cohort [EVC], 1121 cases). For each endoscopic lesion (low-grade adenoma [LgA], high-grade adenoma [HgA], cancer lesion [CL]), specific signatures were identified in the IVC and confirmed on the EVC. A two-miRNA-based signature for CL and six-miRNA signatures for LgA and HgA were selected. In a multivariate analysis including sex and age at blood collection, the areas under the receiver operating characteristic curve (95% confidence interval) of the signatures were 0.644 (0.607–0.682), 0.670 (0.626–0.714) and 0.682 (0.580–0.785) for LgA, HgA and CL, respectively. A miRNA-based test could be introduced into the FIT+ workflow of CRC screening programs so as to schedule colonoscopies only for subjects likely to benefit most.     

MicroRNA‐204‐5p: A novel candidate urinary biomarker of Xp11.2 translocation renal cell carcinoma

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early‐stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC‐TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)‐204‐5p levels in urinary exosomes of 40‐week‐old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA‐204‐5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR‐204‐5p‐containing exosomes. Notably, we also observed increased miR‐204‐5p levels in urinary exosomes in 20‐week‐old renal PRCC‐TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40‐week‐old Tg mice, suggesting that miR‐204‐5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR‐204‐5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC‐TFE3 fusion gene. These findings suggest that miR‐204‐5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC.     

miRNA141表达的调控对人前列腺癌DU145细胞恶性生物行为的影响

目的：研究microRNA-141（miR-141）表达的调控对人前列腺癌细胞系DU145细胞增殖、周期、凋亡、侵袭和迁移等恶性生物学行为的影响及其作用机制。方法：利用脂质体Lipofectamine2000将miR-141 mimics（miR-141 up组）和miR-141 inhibi‐tiors（miR-141 down 组）分别转染至人前列腺癌DU145细胞中，同时设置未转染组（Control组）和miRNA无义序列转染组（NC组）。qPCR检测转染前后各组DU145细胞中miR-141表达变化，MTT方法检测各组DU145细胞的增殖活力和对顺铂（DDP）作用敏感性变化，流式细胞术检测各组DU145细胞周期和顺铂作用下凋亡率变化，Transwell方法检测各组细胞侵袭和迁移能力的变化，Western blotting检测各组细胞中VEGF、EGFR蛋白表达量变化。结果：与Control组和NC组相比，miR-141 down组DU145细胞中miRNA-141表达水平下降为（0.18±0.08），其细胞增殖活力显著下降而对DDP敏感性显著上升、细胞周期被阻滞于G0+G1期而细胞凋亡率显著上升至（46.67±5.86）%、细胞侵袭率和迁移率分别显著下降至（44.34±8.32）%和（57.73±6.19）%、VEGF和EGFR相对表达量分别下降至（0.47±0.06）和（0.36±0.06）（上述各指标分别P<0.05或P<0.01）。miR-141 up组DU145细胞中miR‐NA-141表达水平上升为（4.23±0.53），其细胞增殖活力显著上升而对DDP敏感性显著下降、细胞周期提前进入S和G2期而细胞凋亡率显著下降至（18.77±4.24）%、细胞侵袭率和迁移率分别显著上升至（89.94±6.34）%和（94.44±5.84）%、VEGF和EGFR相对表达量分别上升至（0.89±0.07）和（0.73±0.06）（上述各指标分别P<0.05或P<0.01）。结论：miR-141在前列腺癌DU145细胞中发挥促癌因子作用，其下调表达能够显著抑制DU145细胞增殖活力、周期、侵袭与迁移，而促进对DDP的敏感性和细胞凋亡，其机制可能与其抑制VEGF和EGFR蛋白表达有关。     

circRNA在肝癌中的研究进展

circRNA是一种新的内源性RNA，广泛存在于真核细胞中，参与转录或转录后基因表达调控，大部分属非编码RNA，是近几年RNA领域中的研究热点。circRNA主要通过拮抗miRNA来发挥“海绵作用”进而调控靶基因表达，与多种疾病有着密切联系，特别是在肿瘤中发挥重要作用。近年研究发现，circRNA对肝细胞肝癌（hepatocellular carcinoma，HCC）的发生、发展及转移具有重要调控作用。该文就circRNA与肝细胞肝癌关系的研究进展作一阐述。     

MicroRNA-145增强非小细胞肺癌细胞对吉非替尼敏感性及其机制探讨

目的:探讨微小RNA-145（microRNA-145，miR-145）对非小细胞肺癌细胞系吉非替尼耐药的作用及其可能的潜在机制。方法:实时荧光定量PCR（Q-PCR）方法检测正常细胞及非小细胞肺癌细胞中miR-145的表达差异；不同时间5 μmol/L吉非替尼干预肺癌细胞SPC-A-1和A549后，Q-PCR检测miR-145表达变化；miR-145 mimics和miR-145 inhibitors分别转染SPC-A-1和A549肺癌细胞后，CCK8检测细胞活力变化;双荧光素酶报告系统检测miR-145与ADAM19的靶向结合；miR-145 mimics转染SPC-A-1和A549肺癌细胞，Western blot检测ADAM19蛋白表达；Western blot检测5 μmol/L吉非替尼干预后，SPC-A-1和A549肺癌细胞中ADAM19蛋白的表达；miR-145 mimics转染SPC-A-1和A549肺癌细胞，再用5 μmol/L吉非替尼干预，Western blot检测ADAM19蛋白的表达；采用siRNA抑制ADAM19表达，再用5 μmol/L吉非替尼干预，CCK8检测细胞活力；采用BALB/C雌性裸鼠皮下接种肿瘤细胞的方法，观察上述效应。结果:Q-PCR结果显示，与正常肺上皮细胞系BEAS-2B相比较，肺癌细胞SPC-A-1和A549中miR-145表达明显降低；5 μmol/L吉非替尼干预SPC-A-1和A549细胞后，miR-145表达显著上调；转染miR-145 mimics后,CCK8结果显示两种细胞细胞活力下降；双荧光素酶报告系统结果显示，miR-145靶向结合ADAM19基因的3'-UTR区；Western blot结果显示，5 μmol/L吉非替尼诱导SPC-A-1和 A549细胞后，两种细胞中ADAM19蛋白表达均降低；miR-145 mimics转染SPC-A-1和A549细胞，之后给予5 μmol/L吉非替尼治疗，ADAM19蛋白表达下调；siRNA抑制SPC-A-1和A549细胞中ADAM19表达，再给予5 μmol/L吉非替尼治疗，CCK8结果显示，两种细胞的细胞活力显著降低；过表达BALB/C雌性裸鼠的miR-145后，显著抑制皮下肿瘤生长，并且增强吉非替尼的抗肿瘤效果。结论:miR-145显著增强肺癌细胞SPC-A-1和A549对吉非替尼的敏感性，其机制可能是通过靶向结合AMDM19基因的3'-UTR区域，从而抑制其表达。miR-145有可能成为治疗非小细胞肺癌吉非替尼耐药的有效靶点。