miR-182靶向NUMB调控结直肠癌细胞增殖、迁移和侵袭的机制研究

目的:研究miR-182对结直肠癌细胞HT-29增殖、迁移和侵袭的影响，并探讨其机制。方法:运用qRT-PCR检测人正常结肠上皮细胞HCoEpiC、结直肠癌细胞HT-29中miR-182、NUMB的表达；将anti-miR-con组（转染anti-miR-con）、anti-miR-182组（转染anti-miR-182）、pcDNA组（转染pcDNA）、pcDNA-NUMB组（转染pcDNA-NUMB）、anti-miR-182+si-con组（anti-miR-182和si-con共转染）、anti-miR-182+si-NUMB组（anti-miR-182和si-NUMB共转染）均用脂质体法转染HT-29细胞；Western blot检测各组细胞中NUMB的蛋白表达；Transwell检测各组细胞的迁移和侵袭；MTT法检测各组细胞的增殖；双荧光素酶报告基因检测实验检测各组细胞的荧光素酶活性。结果:与人正常结肠上皮细胞HCoEpiC相比，结直肠癌细胞HT-29中miR-182表达显著升高，NUMB表达显著降低（P<0.05）；抑制miR-182、过表达NUMB均可下调HT-29细胞的增殖、迁移、侵袭；NUMB是miR-182的靶标。敲减NUMB可逆转抑制miR-182对HT-29细胞增殖、迁移、侵袭的下调作用。结论:miR-182可促进结直肠癌细胞的增殖、迁移、侵袭，其机制可能与靶向NUMB有关，将可为miR-182靶向治疗结直肠癌提供依据。     

Molecular miR-19a in Acute Myocardial Infarction: Novel Potential Indicators of Prognosis and Early Diagnosis

Objective: Due to the increasing annual incidence rate of disability and mortality in patients with acute myocardial infarction (AMI), the need for an appropriate diagnostic tool has become a crucial urgent issue. An increase in biomarkers and protein levels in response to AMI can be used as a predictive biomarker with different sensitivities and specificities. This study aimed at investigating the role of miR-19a as a biomarker with acceptable sensitivity and specificity for early diagnosis of AMI. Methods: We studied 175 patients with AMI admitted within 12 h of symptom onset and 90 healthy subjects as control group. Patients were divided into two groups, including group I (normal vessels and no significant artery stenosis) and primary percutaneous coronary intervention (PCI) group II (patients with more than 50% stenosis in vessels and severe atherosclerosis) diagnosed by angiography. The expression level of miR-19a was evaluated by the real-time polymerase chain reaction and other serum chemistries were also analyzed. Results: The results demonstrated that circulating miR-19a levels were significantly increased in patient groups compared to the control group (2.88 ± 1.06 vs. 5.93 ± 1.28, P<0.0001). We also found that miR-19a levels were higher in group II (134.62-fold) than group I (15.42-fold). The upper levels of miR-19a were significantly correlated with the increased serum levels of CK-MB (ρ=0.29, P<0.0001), CTn I (ρ=0.4, P<0.0001) and creatinine (ρ=0.27, P<0.0001). In addition, Receiver Operating Characteristic (ROC) analysis revealed that circulating miR-19a had considerable diagnostic accuracy for the patients with normal vessel with an AUC of 0.930 (95% CI: 0.697-0.765) and for PCI patients with an AUC of 0.966 (95% CI: 0.748-0.784). Conclusion: Circulating miR-19a possibly has prognostic value to be used as a promising molecular target for early diagnosis and prognosis of AMI.     

Decreased miR-124 contributes to the epithelial-mesenchymal transition phenotype formation of lung adenocarcinoma cells via targeting enhancer of zeste homolog 2

IntroductionMiR-124, a tumor suppressor, is involved in regulating various cellular processes. The purpose of this study was to investigate the possible function of miR-124 in LA (lung adenocarcinoma) cells.AimsMiR-124 expression levels in the 54 pairs of LA tissues (and corresponding non-tumor tissues) obtained at the Sixth People’s Hospital of Yancheng City and in LA cells were assessed by qRT-PCR. Colony formation assay, wound healing assay, transwell assays, attachment/detachment, western blotting and immunofluorescence assays were performed to assess the function of miR-124 on proliferation, migration and epithelial-to-mesenchymal (EMT) phenotypes in LA cells in vitro. Enhancer of zeste homolog 2 (EZH2) is identified as a target of miR-124 by bioinformatics analysis and luciferase reporter assays. Rescue assays were applied to verify the relationship between miR-124 and EZH2.ResultsMiR-124 was down-regulated in LA tissues (compared to adjacent non-tumor tissues), and was down-regulated in 3 out of 4 lung cancer cell lines compared to immortalized, non-tumorigenic bronchial epithelial cells. Forced expression of miR-124 significantly suppressed tumor cell proliferation, migration and inhibited the EMT process. On the contrary, deletion of miR-124 could obviously promote cell proliferation, migration and facilitate the formation of EMT phenotype. Bioinformatics analysis and luciferase reporter assays confirmed that EZH2 was a target gene of miR-124 and was negatively correlated with the level of miR-124 in cancer tissues.ConclusionOur current study suggested that miR-124 was a tumor suppressor in LA, and miR-124 was associated with LA cell EMT phenotype formation via targeting EZH2.     

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。     

miR-122 Inhibits Hepatocarcinoma Cell Progression by Targeting LMNB2

In the present study, we investigated the role of miR-122 in hepatocarcinoma progression and explored the mechanism. In hepatocarcinoma tissues and cells, we used qRT-PCR to validate the miR-122 expression level. Next, we used colony formation by crystal violet staining assay to compare cell proliferation ability, and we used scratch test or Transwell assay to compare cell migration or invasion ability. We then conducted bioinformatics or luciferase reporter gene assay to prove the regulation effect of miR-122 on lamin B2 (LMNB2), and the biological function of LMNB2 was analyzed. We used nude mouse tumorigenicity assay to test the inhibition effect of miR-122 ASO therapy against hepatocarcinoma. miR-122 was reduced in hepatocarcinoma tissues compared to the paracarcinoma tissues, which was relatively low or high in hepatocarcinoma cell line SMMC7721 or Hep3B, and overexpressed miR-122 inhibited proliferation, migration, and invasion in hepatocarcinoma cells. Additionally, some reports showed that LMNB2 was regulated by miR-122, which inhibited the expression of LMNB2. Moreover, LMNB2 functioned to promote cell proliferation, migration, and invasion. We could achieve the inhibition of hepatocarcinoma using miR-122 therapy through decreasing LMNB2 expression in vivo. Our data indicated that miR-122 could inhibit hepatocellular carcinoma cell progression by targeting LMNB2 and as a therapeutic target for hepatocarcinoma treatment.     

Knockdown of PVT1 inhibits IL-1β-induced injury in chondrocytes by regulating miR-27b-3p/TRAF3 axis

Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) has been identified to implicate in the progression of osteoarthritis (OA). However, the mechanism underlying PVT1 in OA development remains largely unknown. This study aimed to investigate the effect of PVT1 on interleukin-1 beta (IL-1β)-induced injury in chondrocytes and explore potential mechanism. The cartilage tissues from 25 OA patients and normal controls were collected. Human transformed chondrocytes C28/I2 were stimulated by IL-1β. The levels of PVT1, microRNA (miR)-27b-3p, and tumor necrosis factor receptor-associated factor 3 (TRAF3) were detected by quantitative real-time polymerase chain reaction or western blot. IL-1β-induced injury was investigated by cell viability, apoptosis, autophagy and inflammatory response using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometry, western blot and enzyme linked immunosorbent assay, respectively. The target association between miR-27b-3p and PVT1 or TRAF3 was explored by luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. We found that PVT1 expression was enhanced in OA patients and IL-1β-treated C28/I2 cells. Silence of PVT1 promoted cell viability and autophagy but suppressed apoptosis and inflammatory response in IL-1β-treated C28/I2 cells. miR-27b-3p was confirmed as a target of PVT1 and its deficiency reversed the suppressive effect of PVT1 knockdown on IL-1β-induced injury. TRAF3 was a target of miR-27b-3p and attenuated the effect of miR-27b-3p on IL-1β-induced injury in C28/I2 cells. Moreover, TRAF3 expression was positively regulated by PVT1 via sponging miR-27b-3p. Collectively, knockdown of PVT1 increased cell viability and autophagy but inhibited apoptosis and inflammatory response in chondrocytes treated by IL-1β via up-regulating miR-27b-3p and down-regulating TRAF3.     

miR-451a suppresses the development of breast cancer via targeted inhibition of CCND2

Extensive research has indicated that miRNAs are crucial for the occurrence and progression of cancers. miR-451a, involved in breast cancer (BC), is one of the miRNAs. This study focused on the mechanism by which miR-451a regulates BC. The levels of miR-451a in BC tissues and cell lines were examined using quantitative real-time polymerase chain reaction (qRT-PCR). Kaplan‒Meier analysis showed that this was intimately related to the patient's overall survival rate. Functional experiments revealed the negative effects of miR-451a on the abilities of BC cells to multiply (tested by Cell Counting Kit-8), migrate (tested by wound healing assay), and invade (tested by Transwell assay) and its positive effects on apoptosis (tested by flow cytometry). Western blotting indicated that the expression of tumor-related proteins was affected by miR-451a. Moreover, in vivo experiments suggested that tumor growth was clearly restrained by an miR-451a agonist in a xenograft tumor model. Bioinformatic analysis indicated that miR-451a directly targeted Cyclin D2 (CCND2), as demonstrated by the luciferase reporter assay. An opposite change in the level of CCND2 and miR-451a in BC was indicated by qRT-PCR, western blotting, and immunohistochemistry. Subsequently, functional experiments and western blotting analysis confirmed that CCND2 accelerated BC progression, which was regulated by miR-451a. Cumulatively, research on miR-451a may be valuable for BC treatment.     

益气逐瘀方对缺氧诱导乳鼠心肌细胞凋亡及miR-24/Bim通路的影响

目的研究益气逐瘀方(参元丹)对缺氧诱导乳鼠心肌细胞凋亡及miR-24/Bim通路的影响。方法分离并培养乳鼠心肌细胞,建立心肌细胞缺氧模型,Lipofectamine2000转染miR-24 mimic/inhibitor至心肌细胞,实验分为正常组、缺氧组、参元丹组、参元丹+miR-24 mimic组、参元丹+miR-24 inhibitor组,治疗组予参元丹含药血清干预。比色法检测心肌细胞培养基上清液LDH、CPK活性,MTT法检测心肌细胞存活率,Annexin V/PI双染流式细胞术检测心肌细胞凋亡,qRT-PCR检测心肌细胞miR-24、Bim mRNA表达。结果与低氧组相比,参元丹组、参元丹+miR-24 mimic组、参元丹+miR-24 inhibitor组均能显著降低细胞培养基上清液LDH、CPK活性(P<0.05),提高心肌细胞存活率(P<0.05),降低心肌细胞凋亡(P<0.05),升高心肌细胞miR-24 mRNA表达(P<0.05),降低Bim mRNA表达(P<0.05);治疗组之间比较,参元丹+miR-24 mimic组作用更显著(P<0.05)。结论益气逐瘀方参元丹能够减轻缺氧诱导乳鼠心肌细胞损伤及细胞凋亡,其作用机制可能与其上调miR-24表达,同时抑制其靶基因Bim mRNA表达有关。