miR-21靶向调节MARCKS基因影响人乳腺癌细胞多西紫杉醇敏感性的研究

目的:探讨miR-21对MARCKS基因表达的调控及其与乳腺癌MDA-MB-231细胞株多西紫杉醇耐药的关系。方法:通过基因芯片筛选到miR-21在亲代敏感细胞株(MDA-MB-231/S)与多西紫杉醇耐药细胞株(MDA-MB-231/Doc)中存在差异表达,应用实时荧光定量(RT-qPCR)方法验证此差异,同时分别检测MDA-MB-231/S细胞和MDA-MB-231/Doc细胞转染miR-21模拟物和抑制物后的表达变化;通过靶基因预测软件预测miR-21与MARCKS基因的关系;采用四甲基偶氮唑蓝(MTT)法、流式细胞术、RT-qPCR和蛋白质印迹法检测miR-21表达变化对三阴性乳腺癌细胞多西紫杉醇耐药性的影响,并探讨其与MARCKS基因表达的关系。结果:与MDA-MB-231/S相比,miR-21在MDA-MB-231/Doc中的表达水平明显升高(2.03±0.06)倍(P0.05)。与阴性对照组比,MDA-MB-231/S细胞转染miR-21 mimics后miR-21表达水平明显增高(2.26±0.07)倍(P0.05),而MARCKS基因在mRNA和蛋白水平上均明显下调。MDA-MB-231/Doc细胞转染miR-21 inhibitors后,miR-21表达水平是阴性对照组的(0.36±0.03)倍(P0.05),MARCKS基因的mRNA和蛋白水平高于其阴性对照组。转染miR-21 mimics后的MDA-MB-231/S细胞IC50显著高于其阴性对照组(P0.05),相反,与阴性对照组相比,MDA-MB-231/Doc细胞转染miR-21 inhibitors后,其IC50显著降低(P0.05)。MDA-MB-231/S转染miR-21 mimics后,miR-21表达量明显上调,MARCKS基因分别是阴性对照组的(3.564±0.336)倍和(2.019±0.268)倍(P0.05)。结论:miR-21可能通过靶向调节MARCKS基因增强乳腺癌细胞对多西紫杉醇的耐药性。     

LncRNA OIP5-AS1 regulates radioresistance by targeting DYRK1A through miR-369-3p in colorectal cancer cells

Object

This study aimed to investigate the role of lncRNA OIP5-AS1 in regulating radioresistance of colorectal cancer (CRC) cells.

The role of OAT2 (SLC22A7) in the cyclic nucleotide biokinetics of human erythrocytes

The present study was conducted to characterise the transporter(s) responsible for the uptake of cyclic nucleotides to human erythrocytes. Western blotting showed that hRBC expressed OAT2 (SLC22A7), but detection of OAT1 (SLC22A6), or OAT3 (SLC22A8) was not possible. Intact hRBC were employed to clarify the simultaneous cyclic nucleotide egression and uptake. Both these opposing processes were studied. The K_m‐values for high affinity efflux was 3.5 ± 0.1 and 39.4 ± 5.7 μM for cGMP and cAMP, respectively. The respective values for low affinity efflux were 212 ± 11 and 339 ± 42 μM. The uptake was characterised with apparently low affinity and similar K_m‐values for cGMP (2.2 mM) and cAMP (0.89 mM). Using an iterative approach in order to balance uptake with efflux, the predicted real K_m‐values for uptake were 100–200 μM for cGMP and 50–150 μM for cAMP. The established OAT2‐substrate indomethacin showed a competitive interaction with cyclic nucleotide uptake. Creatinine, also an OAT2 substrate, showed saturable uptake with a K_m of 854 ± 98 μM. Unexpectedly, co‐incubation with cyclic nucleotides showed an uncompetitive inhibition. The observed K_m‐values were 399 ± 44 and 259 ± 30 μM for creatinine, in the presence of cGMP and cAMP, respectively. Finally, the OAT1‐substrate para‐aminohippurate (PAH) showed some uptake (K_m‐value of 2.0 ± 0.4 mM) but did not interact with cyclic nucleotide or indomethacin transport.     

miR-34b调控肠道病毒71型在人横纹肌肉瘤细胞中的复制及机制

【背景】研究发现microRNAs(miRNAs)可以参与调控病毒在宿主细胞内感染和复制的过程。【目的】研究miR-34b对肠道病毒71型(Enterovirus71,EV71)在宿主细胞内的复制及其可能机制。【方法】在人横纹肌肉瘤(Rhabdomyosarcoma,RD)细胞中转染miR-34b mimics和Inhibitor,通过Western blot和Real-time PCR实验检验EV71病毒的复制和表达情况。随后利用双荧光素酶报告系统验证miR-34b与潜在靶点eIF4E的相互作用,并检测miR-34b对RD细胞中eIF4E mRNA表达水平的影响。【结果】miR-34b可以促进病毒在RD细胞中的复制和表达,而miR-34b抑制剂有抑制病毒复制的作用,细胞内miR-34b可以通过作用于靶基因eIF4E调控EV71在宿主细胞中的复制过程。【结论】揭示了miR-34b在EV71病毒复制过程中的调控作用及机制,研究EV71病毒与宿主miRNAs的相互作用机制为进一步阐明EV71病毒感染与复制机理奠定了基础。     

Bone marrow-derived mesenchymal stem cells repair severe acute pancreatitis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling

BackgroundSevere acute pancreatitis (SAP) is associated with high morbidity and mortality. Bone marrow mesenchymal stem cells (BMSCs) have shown obvious protective effect on SAP. However, little is known about the underlying mechanism. The objective of this study is to unravel the role and regulatory mechanism of miR-181a-5p in BMSCs-mediated pancreatic repair.MethodsBMSCs were isolated from Sprague-Dawley rats and characterized by flow cytometry and Oil Red O staining. Sodium taurocholate- and caerulein-induced models were used as SAP models in vivo and in vitro, respectively. Pancreatic injury were evaluated by H&E and histopathological analysis, as well as by measuring levels of amylase, lipase and cytokines. qRT-PCR and western blotting were performed to detect the level of miR-181a-5p and the protein levels of PTEN/Akt, respectively. ELISA was conducted to detect the levels of TNF-α, IL-1β, IL-6, angiopoietin, IL-4, IL-10 and TGF-β1. The apoptotic rate of AR42 J cells was quantitated by concurrent staining with Annexin-V-FITC and PI.ResultsBMSCs significantly attenuated pancreatic injury in SAP rats by reducing inflammatory infiltration and necrosis, and this effect was abolished by CXCR4 agonist AMD3100. ADM3100 exhibited more severe pancreatic injury and decreased miR-181a-5p levels in the pancreas and serum compared to SAP group. Overexpression of miR-181a-5p in BMSCs (BMSCs-miR-181a-5p) markedly potentiated the protective effect of BMSCs by reducing histological damage and levels of amylase and lipase. Moreover, BMSCs-miR-181a-5p dramatically reduced levels of angiopoietin, TNF-α, IL-1β and IL-6, but induced the levels of IL-4 and IL-10. In caerulein-treated AR42 J cells, co-culturing of BMSCs-miR-181a-5p alleviated caerulein-induced increase of amylase and lipase, and apoptosis via PTEN/Akt/TGF-β1 signaling.ConclusionBMSCs alleviate SAP and reduce inflammatory responses and apoptosis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling. Hence, BMSCs-miR-181a-5p could serve as potential therapeutic target for SAP.     

Regulatory effects of lncRNA ATB targeting miR-200c on proliferation and apoptosis of colorectal cancer cells

This investigation was intended to elucidate whether long noncoding RNA (lncRNA)-activated by transforming growth factor-β (ATB) interacting with miR-200c could mediate colorectal cancer (CRC) progression, offering potential strategies for diagnosing and treating CRC. Here totally 315 patients with CRC were recruited, and their CRC tissues and adjacent normal tissues were gathered. Concurrently, four colon cancer cell lines (ie, SW620, Lovo, HCT116, and SW480) and the human colon mucosal epithelial cell line (NCM460) were also purchased. Moreover, si-ATB, si-NC, miR-200c mimic, miR-200c inhibitor, and miR-NC were prepared for transfection into the CRC cells, and their effects on CRC cell lines were evaluated based on the conduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and flow cytometry assay. Eventually, the Luciferase reporter gene assay was carried out to judge if there existed a targeted relationship between ATB and miR-200c. The results of Cox regression analyses suggested that overexpressed lncRNA ATB, underexpressed miR-200c, poor tumor differentiation, lymph-vascular invasion, and perineural invasion were symbolic of shortened survival of the patients with CRC (all P < .05). Besides, transfection of pcDNA3.1-ATB and miR-200c inhibitor could boost the viability and proliferation of Lovo and SW620 cell lines (all P < .05). Meanwhile, the expressions of p53 and p21 were also reduced under treatments of pcDNA3.1-ATB and miR-200c inhibitor (P < .05). In addition, CDK2 seemed to reverse the contribution of miR-200c to intensifying viability and proliferation of Lovo and SW420 cell lines (P < .05). Furthermore, ATB might downregulate miR-200c expression by targeting it (P < .05), and CDK2 was subjected to dual regulation of both ATB and miR-200c (P < .05). In conclusion, the lncRNA ATB/miR-200c/CDK2 signaling was responsible for intensified proliferation and prohibited apoptosis of CRC cells, which might provide effective approaches for diagnosing and treating CRC.     

A single-nucleotide polymorphism in lnc-LAMC2-1:1 interferes with its interaction with miR-128 to alter the expression of deleted in colorectal cancer and its effect on the survival rate of subjects with ovarian cancer

This study aimed to identify the association between lnc-LAMC2-1:1 polymorphism rs2147578 and the recurrence of ovary cancer, as well as to study the underlying mechanism of rs2147578 in ovary cancer. Real-time polymerase chain reaction, Western blot analysis, immunohistochemistry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Logrank test, and Kaplan-Meier analysis were carried out to explore the role of rs2147578 in ovary cancer. No obvious difference was observed concerning all clinical characteristics among 90 patients genotyped as CC (N = 28), CG (N = 38), and GG (N = 24) in their rs2147578 polymorphism. In addition, the subjects carrying the CC genotype had longer recurrence-free survival time and showed a lower level of malignancy compared with those carrying CG and GG genotypes. Lnc-LAMC2-1:1 and miR-128 were lowly expressed in the CC group, while deleted in colorectal cancer (DCC) was highly expressed in the CC group. Furthermore, DCC was identified as a target gene of miR-128, and miR-128 mimics decreased the luciferase activity of cells cotransfected with wild-type DCC 3′-untranslated region. Lnc-LAMC2:1-1 directly targeted and affected miR-128 expression, and the G allele in lnc-LAMC2-1:1 rs2147578 upregulated miR-128 expression. Transfection with a miR-128 precursor evidently downregulated the expression of lnc-LAMC2-1:1, miR-128, and DCC expression, but did not affect the expression of ABCC5 and body mass index. Finally, miR-128 precursor promoted cell proliferation and inhibited cell apoptosis. Compared with lnc-LAMC2-1:1 rs2147578C allele, the G allele increases the risk of ovarian cancer by reducing the binding between lnc-LAMC2-1:1 and miR-128-3p, which in turn further decreases the expression of DCC and inhibits cell apoptosis.