miR-9通过靶向E盒结合锌指蛋白2调控小细胞肺癌的恶性生物学行为及其可能的机制

目的：探讨 miR-9 通过靶向 E 盒结合锌指蛋白 2（zinc finger E-box binding homeobox 2，ZEB2）对小细胞肺癌（small cell lung cancer，SCLC）细胞生物学行为的调控作用，分析miR-9在SCLC中的作用及其工作机制。方法：采用qPCR、WB和免疫组化方法检测于2018年2月至2019年11月于河北医科大学第四医院肿瘤内科接受手术治疗的67例SCLC患者癌组织及癌旁组织中ZEB2的表达。采用TargetScan预测miR-9的潜在靶基因并通过双荧光素酶报告基因试验、qPCR和WB法进行验证。CCK-8法、流式细胞术和Transwell实验检测miR-9和ZEB2 过表达对 NCI-H446 的生物学行为影响，WB法检测对细胞中E-cadherin，N-cadherin和Vimentin蛋白表达的影响。利用miR-9过表达NCI-H446细胞构建SCLC裸鼠移植瘤模型，观察miR-9对裸鼠移植瘤生长的影响。结果：SCLC组织中ZEB2的mRNA和蛋白表达水平明显高于癌旁正常组织（P<0.01）。miR-9在ZEB2的3'' UTR上具有潜在的结合位点，与对照组相比，miR-9过表达组NCI-H446细胞中ZEB2的mRNA和蛋白表达水平明显降低（P<0.01），细胞增殖、迁移和侵袭能力均显著下降（P<0.05或P<0.01），促EMT蛋白表达减少，而同时过表达ZEB2能够逆转上述影响。体内实验中，miR-9过表达组移植瘤体积、重量均明显低于对照组（P<0.05或P<0.01）。miR-9组裸鼠肿瘤组织中和ZEB2蛋白的表达均较对照组明显降低（P<0.01）。结论：miR-9通过靶向调控ZEB2从而抑制SCLC细胞的生物学行为以及NCI-H446裸鼠移植瘤的生长。     

miR-21对视网膜母细胞瘤细胞增殖的影响及作用机制

目的　探讨MicroRNA-21（miR-21）对视网膜母细胞瘤(RB)细胞增殖的影响及作用机制。方法　采用Real-time PCR技术检测miR-21在正常视网膜组织和确诊RB组织中的表达情况；然后在转染的基础上运用MTT检查RB细胞存活率、流式细胞仪检测细胞凋亡率。Western blot法检测与细胞凋亡相关蛋白PDCD4、Bax、Bcl-2的表达。结果　与正常视网膜组织miR-21表达 （0.703±0.071）相比，RB组织miR-21为高表达（2.214±0.162），差异有统计学意义（P<0.01）。在Weri-Rb-1细胞中，与NC组(2.245±0.213)相比，miR-21抑制剂转染后明显降低了miR-21的表达水平，miR-21 inhibitor组为0.683±0.075,差异有统计学意义 （P<0.01）。两组细胞转染后24 h、48 h、72 h、96 h，MTT测定法检测细胞活力结果显示:两组24 h的A值比较，差异无统计学意义 （P>0.05），miR-21 inhibitor 组在 48 h、72 h、 96 h的A值均低于 NC 组，差异均有统计学意义 （均为P<0.01）。流式细胞术检测结果显示:NC组凋亡细胞在总细胞中百分比为（3.045±0.301）%和（4.832±0.493）%，miR-21 inhibitor组凋亡细胞在总细胞中百分比为（2.593±0.257）%和（40.167±4.014）%，miR-21 inhibitor组Weri-Rb-1细胞的凋亡率明显高于miR-21 NC组(P<0.01)。Western blot检测结果显示:NC组PDCD4表达（0.192±0.045）相比miR-21 inhibitor组（0.683±0.091）表达明显减少，NC组Bax的蛋白表达水平（0.143±0.036）相比miR-21 inhibitor组（1.192±0.054）也明显减少，差异均有统计学意义（P<0.01)，NC组Bcl-2蛋白表达（0.864±0.038）相比miR-21 inhibitor组（0.257±0.026）明显增多，差异有统计学意义（P<0.05)。结论　miR-21是RB的促癌基因，miR-21抑制剂可以通过降低miR-21表达抑制肿瘤细胞的增殖、促进细胞凋亡，这一过程与PDCD4、Bax、Bcl-2等凋亡相关蛋白有关。     

miR-200、 miR -155及血管新生因子与复发性流产的相关性分析

目的 研究miR-200、miR-155及血管新生因子与原因不明复发性流产(unexplained recurrent spontaneousabortion,URSA)的相关性分析。 方法 选择2015年3月—2018年1月在青岛市妇女儿童医院妇产科就诊的URSA患者作为URSA组、要求终止妊娠的正常早孕患者作为对照组,检测绒毛组织中微小RNA(microRNA, miR)miR-200、miR-155、血管内皮生长因子(vascular endothelial growth factor,VEGF)、可溶性FMS样酪氨酸激酶1(soluble FMS like tyrosine kinase-1,sFlt-1)的表达量及血清中VEGF、sFlt-1的含量,对miR-200、miR-155靶向结合VEGF、sFlt-1进行生物信息学分析。 结果 URSA组的绒毛组织中miR-200(1.78±0.32 vs. 0.91±0.15)、sFlt-1(1.87±0.35 vs. 1.06±0.21)的相对表达量及血清中sFlt-1的含量[(12.39±2.31)ng/ml vs. (6.51±0.95)ng/ml]均高于对照组,差异有统计学意义(均P<0.05)。绒毛组织中miR-155相对表达量(0.60±0.10 vs. 0.93±0.16)、VEGF mRNA相对表达量(0.59±0.09 vs. 1.02±0.16)及蛋白表达量(0.62±0.07 vs. 1.04±0.18)、血清中VEGF的含量[(601.25±94.39)ng/ml vs. (935.12±132.47)ng/ml]低于对照组,差异有统计学意义(均P<0.05);URSA组患者绒毛组织中miR-200的表达量与血清中VEGF的含量、绒毛组织中VEGF的表达量均呈负相关,绒毛组织中miR-155的表达量与血清中sFlt-1的含量和绒毛组织中sFlt-1的表达量均呈负相关;miR-200、miR-155分别靶向结合VEGF、sFlt-1基因的3’UTR。 结论 miR-200表达增多、miR-155表达减少与URSA发生有关,miR-200靶向VEGF、miR-155靶向sFlt-1是介导该过程的可能机制。     

奥沙利铂通过调控miRNA-7-5p/RAF-1促进胃癌SGC-7901细胞凋亡的研究

目的探讨奥沙利铂如何调控MAPK通路,抑制胃癌细胞的增殖。方法NCBI检索文献,利用TargetScan、StarBase和miRBase数据库,进行GO分析与KEGG通路富集,找到相关miRNAs,预测靶基因。应用Real-time PCR、MTT、Hoechst33258、流式细胞术、细胞划痕实验、Western blot等方法分析人胃癌SGC-7901细胞的增殖、细胞周期、侵袭及蛋白表达情况。结果胃癌细胞中miR-7-5p显著低表达,RAF1与miR-7-5p存在互靶关系。miR-7-5p mimics与奥沙利铂均可促进SGC-7901细胞的凋亡,提高G1期细胞百分率(P<0.05),降低侵袭、迁移速度。caspase3、caspase9蛋白表达升高,Bcl-2/Bax比值降低(P<0.05)。结论过表达miR-7-5p与奥沙利铂均可促进胃癌SGC-7901细胞的凋亡,提示奥沙利铂可能通过上调miRNA-7-5p促进SGC-7901细胞的凋亡,降低侵袭、迁移速度。     

Extracellular vesicles transmitted miR-31-5p promotes sorafenib resistance by targeting MLH1 in renal cell carcinoma

Sorafenib provides survival benefits in patients with advanced renal cell carcinoma (RCC), but its use is hampered by acquired drug resistance. It is important to fully clarify the molecular mechanisms of sorafenib resistance, which can help to avoid, delay or reverse drug resistance. Extracellular vesicles (EVs) can mediate intercellular communication by delivering effector molecules between cells. Here, we studied whether EVs are involved in sorafenib resistance of RCC and its possible molecular mechanisms. Using differential centrifugation, EVs were isolated from established sorafenib-resistant RCC cells (786-0 and ACHN), and EVs derived from sorafenib-resistant cells were uptaken by sensitive parental RCC cells and thus promoted drug resistance. Elevated exogenous miR-31-5p within EVs effectively downregulated MutL homolog 1 (MLH1) expression and thus promoted sorafenib resistance in vitro. Mice experiments also confirmed that miR-31-5p could mediate drug sensitivity in vivo. In addition, low expression of MLH1 was observed in sorafenib-resistant RCC cells and upregulation of MLH1 expression restored the sensitivity of resistant cell lines to sorafenib. Finally, miR-31-5p level in circulating EVs of RCC patients with progressive disease (PD) during sorafenib therapy was higher when compared to that in the pretherapy status. In conclusion, EVs shuttled miR-31-5p can transfer resistance information from sorafenib-resistant cells to sensitive cells by directly targeting MLH1, and thus magnify the drug resistance information to the whole tumor. Furthermore, miR-31-5p and MLH1 could be promising predictive biomarkers and therapeutic targets to prevent sorafenib resistance.     

A liquid-scintillation-based primary standardization of Pb

A new radioactivity solution standard of ²¹⁰Pb has been developed and will be disseminated by the National Institute of Standards and Technology (NIST) as standard reference material (SRM) 4337. This new ²¹⁰Pb solution standard is contained in a 5 mL flame-sealed borosilicate glass ampoule, consists of (5.133±0.002) g of a nominal 1 mol L⁻¹ nitric acid solution, has a density of (1.028±0.002) g mL⁻¹ at 20 °C, has carrier ion concentrations of about 11 μg Pb²⁺ and 21 μg Bi³⁺ per gram of solution, and is certified to contain a massic activity (9.037±0.22) kBq g⁻¹ as of the reference time 1200 EST, 15 June 2006. All of the uncertainties cited above correspond to standard uncertainties multiplied by a coverage factor k=2. The standardization for the ²¹⁰Pb content of the solution was based on 4πβ liquid scintillation (LS) measurements using CIEMAT/NIST ³H-standard efficiency tracing (CNET). Confirmatory determinations were also performed by high-resolution HPGe γ-ray spectrometry, by 2π spectrometry with a Si surface barrier detector of separated ²¹⁰Po, and by 4πβ(LS)–γ(NaI) anticoincidence counting.     

SNHG6靶向miR-101/TGFBR1促进AMI小鼠左心室心肌纤维化

目的探讨SNHG6对急性心肌梗死(AMI)小鼠左心室心肌的影响。 方法将30只雄性C57/BL6小鼠构建成AMI小鼠后随机均分为AMI组、AMI+SNHG6组、AMI+miR-101-3p组、AMI+SNHG6+miR-101-3p组、AMI+miR-101-3p+TGFBR1组,另设正常小鼠6只为假手术组。qRT-PCR检测AMI小鼠SNHG6、miR-101-3p表达水平。心脏彩超检测各组小鼠左室射血分数(LVEF);马松和天狼星红染色法以及免疫组化分析各组小鼠左心室心肌纤维化变化。将H9C2细胞株分为阴性对照组(转染空质粒)、SNHG6组(转染质粒SNHG6)、miR-101-3p组(转染质粒miR-101-3p)。Western blotting检测各组TGFBR1蛋白表达;采用双荧光素酶报告基因法预测并验证SNHG6/miR-101-3p/TGFBR1荧光素酶活性及调控机制。 结果AMI小鼠较假手术组SNHG6表达显著增加,miR-101-3p降低(P＜0.05)。与AMI组比较,AMI+SNHG6组小鼠LVEF降低,心肌纤维化程度加重(P＜0.05);AMI+miR-101-3p组LVEF升高,心肌纤维化程度减轻(P＜0.05)。AMI+SNHG6+miR-101-3p组较AMI+SNHG6组LVEF升高、心肌纤维化程度减轻(P＜0.05),而AMI+miR-101-3p+TGFBR1组较AMI+miR-101-3p组LVEF降低、心肌纤维化程度加重(P＜0.05)。双荧光素酶报告基因法验证显示,miR-101-3p组SNHG6、TGFBR1野生型质粒的荧光素酶活性较阴性对照组明显降低(P＜0.05)。 结论SNHG6抑制miR-101-3p上调TGFBR1加重AMI小鼠左心室心肌纤维化。