miR-21通过下调PTEN增强人脑胶质瘤细胞对卡莫司汀耐药

目的:探讨微小RNA-21(miR-21)在增强人脑胶质瘤细胞对卡莫司汀(BCNU)耐药中的作用及其可能的作用机制。方法: 用jetPRIME将miR-21mimics及阴性对照序列转入人脑胶质瘤细胞SWOZ2,用实时荧光定量PCR法检测BCNU敏感株SWOZ2与BCNU耐药株SWOZ2-BCNU、SWOZ2-miR-21mimics细胞与对照组细胞中miR-21表达差异,用CCK-8检测这两对细胞对BCNU敏感度的差异,用Westernblotting检测这两对细胞中第10号染色体同源缺失性磷酸酶及张力蛋白(PTEN)、磷酸化蛋白激酶B(p-Akt)和P-糖蛋白(P-gp)表达的差异。结果:SWOZ2-BCNU细胞miR-21的表达水平明显高于SWOZ2细胞,转染组SWOZ2-miR-21mimics细胞中miR-21的表达量明显高于对照组;SWOZ2-BCNU细胞BCNU的半数抑制浓度(IC₅₀)明显高于SWOZ2细胞,转染组细胞BCNU的IC₅₀明显高于对照组;与SWOZ2细胞相比,SWOZ2-BCNU细胞中PTEN蛋白的表达明显降低,p-Akt和P-gp蛋白的表达都明显升高;转染后,与对照组相比,转染组细胞中PTEN蛋白的表达明显降低,而p-Akt和P-gp蛋白的表达皆明显升高。结论:miR-21可能通过下调PTEN蛋白表达,增强人脑胶质瘤细胞对BCNU耐药。     

The use of cationic MPEG-PCL-g-PEI micelles for co-delivery of Msurvivin T34A gene and doxorubicin

In our previous study, a series of triblock copolymers based on MPEG-PCL-g-PEI were successfully synthesized, and the physicochemical properties of their self-assembled micelles were also investigated. Here, a further evaluation of these micelles was carried out, including in vitro drug release behavior, body distribution as well as blood compatibility. The developed MPEG-PCL-g-PEI micelles was labeled with ⁹⁹Tc for tracing the body distribution of micelles after i.v. injection, and the results showed that the MPEG-PCL-g-PEI micelles mainly concentrated in the tumor tissue. Meanwhile, the anti-tumor activity on both B16F10 subcutaneous tumor model and lung metastasis model was tested and the results indicated that DOX-loaded micelles could significantly inhibit tumor growth as compared with free doxorubicin, which was accompanied by significantly increased apoptosis of tumor cells. By introduction of gene Msurvivin T34A in combination with chemotherapies in the treatment of lung metastasis tumor, it could greatly reduce systemic toxicity as well as improved the anti-tumor efficiency. These results demonstrated that it is possible to use cationic MPEG-PCL-g-PEI micelles for effectively co-delivering functional gene and chemotherapeutic agent, and thus improving anti-tumor effect and systemic toxicity.     

Multifunctional nanocarrier mediated co-delivery of doxorubicin and siRNA for synergistic enhancement of glioma apoptosis in rat

As the most fatal malignancy in brain, glioma cannot be effectively treated with the conventional chemotherapy and thus techniques which may improve the chemotherapeutic effect are of great importance in clinical glioma treatment. Based on the folate-targeted multifunctional nanocarrier developed in our lab, effective co-delivery of DOX and siRNA into rat C6 glioma cells over-expressing folate receptors was achieved. Although cell apoptosis was initiated even at low DOX doses such as 0.5 μg/mL in the DOX-alone treatment mediated by the folate-targeted nanocarrier, anti-apoptotic response in C6 cells was activated as well, as revealed by molecular biological investigations. Delivery of BCL-2 siRNA using the folate-targeted nanocarrier can effectively suppress the anti-apoptotic response and sensitized C6 cells to DOX treatment both in vitro and in vivo. In particular, animal studies using the in situ rat C6 glioma model showed that the folate-targeted co-delivery of BCL-2 siRNA and DOX caused not only an obvious down-regulation of the anti-apoptotic BCL-2 gene but also a remarkable up-regulation of the pro-apoptotic Bax gene, resulting in the significantly elevated level of caspase-3 activation and remarkable cell apoptosis in tumor tissues. Our results strongly demonstrated the synergistic effect of siRNA and DOX in inducing glioma C6 cell apoptosis, upon which an excellent therapeutic effect was achieved using the folate-targeted co-delivery strategy as indicated by the effective tumor growth inhibition and prolonged rat survival time in the animal test.     

血清miR-21在糖尿病肾病中的诊断价值

 目的：评价血清miR-21对糖尿病肾病的诊断价值,探讨糖尿病肾病诊断的分子标志物。方法：对25例糖尿病肾病患者、25例II型糖尿病患者A［尿微量白蛋白（UmAlb）＜1429 mg/L］、25例II型糖尿病患者B（UmAlb＞1429 mg/L）、25例糖尿病肾病引发的尿毒症患者及25例正常对照组的血清标本进行总RNA提取,并进行miR-21的实时荧光定量PCR检测,分析血清miR-21相对表达量与相关临床指标的关系,评估血清miR-21对糖尿病肾病的诊断效能。结果：miR-21在糖尿病肾病患者血清中的相对表达量显著低于正常对照组、糖尿病A组及B组,同时显著高于尿毒症组(均P＜001),糖尿病肾病患者血清miR-21水平与胱抑素C、UmAlb、UmAlb/尿肌酐呈显著负相关（P<001,P<005,P<005)。经logistic回归及ROC曲线分析,血清miR-21在糖尿病肾病患者与正常对照组、糖尿病A组、糖尿病B组、糖尿病A组＋正常对照组、糖尿病B组＋正常对照组和糖尿病A组＋糖尿病B组中诊断糖尿病肾病的曲线下面积（AUC）为0848(95%CI：0737~0959)、0896(95%CI：0812~0980)、0782(95%CI：0641~0922)、0838(95%CI：0743~0933)、0796(95%CI：0675~0917)和0808(95%CI：0704~0911),敏感性和特异性分别为800%和720%、720%和880%、720%和840%、760%和770%、760%和820%、700%和860%;在4组人群中诊断糖尿病肾病的AUC为0845(95%CI：0752~0939),敏感性和特异性分别为760%和773%。结论：血清miR-21可作为糖尿病肾病的分子诊断标志物。     

Multifunctional QD-based co-delivery of siRNA and doxorubicin to HeLa cells for reversal of multidrug resistance and real-time tracking

Co-delivery of siRNA and chemotherapeutic agents has been developed to combat multidrug resistance in cancer therapy. Recently, we developed a series of quantum dots (QDs) functionalized by β-cyclodextrin (β-CD) coupled to amino acids, some of which can be used to facilitate the delivery of siRNA. In this study, two CdSe/ZnSe QDs modified with β-CD coupled to L-Arg or L-His were used to simultaneously deliver doxorubicin (Dox) and siRNA targeting the MDR1 gene to reverse the multidrug resistance of HeLa cells. In this co-delivery system, Dox was firstly encapsulated into the hydrophobic cavities of β-CD, resulting in bypass of P-glycoprotein (P-gp)-mediated drug efflux. After complex formation of the mdr1 siRNA with Dox-loaded QDs via electrostatic interaction, significant down-regulation of mdr1 mRNA levels and P-gp expression was achieved as shown by RT-PCR and Western blotting experiments, respectively. The number of apoptotic HeLa cells after treatment with the complexes substantially exceeded the number of apoptotic cells induced by free Dox only. The intrinsic fluorescence of the QDs provided an approach to track the system by laser confocal microscopy. These multifunctional QDs are promising vehicles for the co-delivery of nucleic acids and chemotherapeutics and for real-time tracking of treatment.     

miR-21 inhibitor suppresses proliferation and migration of nasopharyngeal carcinoma cells through down-regulation of BCL2 expression

This study is to investigate the expression of miR-21 in nasopharyngeal carcinoma (NPC) cells, and the effect of miR-21 in the biological behavior and expression of B-cell lymphoma 2 (BCL2) in NPC cells. Paired NPC and adjacent non-tumor tissues were obtained from 53 patients who underwent primary surgical resection of NPC tissues. Luciferase reporter assay was performed to test whether BCL2 is a direct target of miR-21. Methylthiazolyl blue tetrazolium assay and colony assay were used to evaluate the effect of miR-21 on NPC cell proliferation. Transwell and wound-healing assays were carried out to test the effect of low expression of miR-21 on cancer cell migration and invasion. QRT-PCR and Western blotting were used to measure the levels of mRNA and protein expression, respectively. Tumor tissues showed a positive correlation between the levels of miR-21 and BCL2 protein expression. Cells transfected with miR-21 inhibitor healed slower compared the control (P < 0.05). In addition, cell migration was notably inhibited by the down-regulation of miR-21 in vitro (P < 0.05). The reduction in miR-21 expression showed a remarkable effect on the biological behavior of NPC cell clone formation (P < 0.05). Low expression of miR-21 by transfection with miRNA expression plasmid led to a decrease in BCL2 expression, which was accompanied by reduced migration and proliferation of the cancer cells. Our results demonstrated that miR-21 inhibitor down-regulated BCL2 expression level, suggesting that BCL2 might be a target gene for the initiation and development of NPC cells.     

上/下调miR-21对结肠癌细胞的生物学作用及对西妥昔单抗药物敏感性的影响*

 目的： 探讨miR-21在结肠癌细胞中的生物学功能及对EGFR单抗西妥昔敏感性的影响。方法: 通过慢病毒载体的构建及包装生成上调/下调miR-21的慢病毒LV-miR-21和LV-anti-miR-21并感染人结肠癌RKO细胞,采用qRT-PCR、MTT、非锚定依赖性细胞生长、流式细胞术、CCK-8等技术检测上调/下调miR-21后细胞的miR-21表达水平、细胞增殖、克隆形成能力、细胞凋亡能力及对西妥昔单抗药物敏感性的变化。结果: LV-miR-21与LV-anti-miR-21慢病毒的滴度分别为3.0×1012 TU/L和2.0×1012 TU/L,将病毒颗粒分别感染人结肠癌RKO细胞,观察绿色荧光,感染效率在80%以上。LV-miR-21组的miR-21表达水平、细胞增殖能力和克隆形成能力均高于LV-anti-miR-21组,差异有统计学意义（P＜0.05）。而LV-anti-miR-21组的细胞早期凋亡率及西妥昔单抗对细胞的抑制率均高于LV-miR-21组（P＜0.05）。结论: miR-21可促进结肠肿瘤细胞的增殖。下调miR-21可以增加结肠癌细胞对靶向治疗药物西妥昔单抗的敏感性。     

Hyaluronic acid-chitosan nanoparticles for co-delivery of MiR-34a and doxorubicin in therapy against triple negative breast cancer

Metastatic relapse, development of drug resistance in cancer cells and adverse side effects of chemotherapeutic agents are the major obstacles for effective chemotherapy against triple-negative breast cancer. To address these problems, miR-34a, a potent endogenous tumor suppressive molecule in breast cancer, was co-encapsulated with doxorubicin (DOX) into hyaluronic acid (HA)-chitosan (CS) nanoparticles (NPs) and simultaneously delivered into breast cancer cells for improved therapeutic effects of drug. DOX-miR-34a co-loaded HA-CS NPs were successfully prepared through ionotropic gelation method in water. In vitro and in vivo experiments showed that miR-34a and DOX can be efficiently encapsulated into HA-CS NPs and delivered into tumor cells or tumor tissues and enhance anti-tumor effects of DOX by suppressing the expression of non-pump resistance and anti-apoptosis proto-oncogene Bcl-2. In addition, intracellular restoration of miR-34a inhibited breast cancer cell migration via targeting Notch-1 signaling. The obtained data suggest that co-delivery of DOX and miR-34a could achieve synergistic effects on tumor suppression and nanosystem-based co-delivery of tumor suppressive miRNAs and chemotherapeutic agents may be a promising combined therapeutic strategy for enhanced anti-tumor therapy.     

miR-21 and miR-146a: The microRNAs of inflammaging and age-related diseases

The first paper on “inflammaging” published in 2001 paved the way for a unifying theory on how and why aging turns out to be the main risk factor for the development of the most common age-related diseases (ARDs). The most exciting challenge on this topic was explaining how systemic inflammation steeps up with age and why it shows different rates among individuals of the same chronological age. The “epigenetic revolution” in the past twenty years conveyed that the assessment of the individual genetic make-up is not enough to depict the trajectories of age-related inflammation. Accordingly, others and we have been focusing on the role of non-coding RNA, i.e. microRNAs (miRNAs), in inflammaging. The results obtained in the latest 10 years underpinned the key role of a miRNA subset that we have called inflammamiRs, owing to their ability to master (NF-κB)-driven inflammatory pathways. In this review, we will focus on two inflammamiRs, i.e. miR-21−5p and miR-146a-5p, which target a variety of molecules belonging to the NF-κB/NLRP3 pathways. The interplay between miR-146a-5p and IL-6 in the context of aging and ARDs will also be highlighted. We will also provide the most relevant evidence suggesting that circulating inflammamiRs, along with IL-6, can measure the degree of inflammaging.     

pH 敏感的 PEG-PAsp（DIP）负载 miR-21-inhibitor 纳米聚合物囊泡的制备及传输在前列腺癌 PC-3细胞中的效应研究

目的：制备以生物纳米材料 pH 敏感嵌段共聚物聚乙二醇-聚天冬氨酸二异丙基氨基乙酯[PEG-PAsp (DIP)]传输 miR-21-inhibitor 聚合物囊泡,初步探讨 pH 敏感聚合物囊泡对前列腺癌细胞的毒性作用及传输效应。方法①通过静电作用,在室温条件下制备 PEG-PAsp (DIP)传输 miR-21-inhibitor 聚合物囊泡;②通过凝胶阻滞实验检测不同浓度下纳米聚合物囊泡(NPs)的复合情况;③通过 CCK-8实验检测不同浓度下 pH 敏感 NPs 对前列腺癌细胞 PC-3的毒性作用;④通过激光共聚焦实验,观察 PEG-PAsp (DIP)/ miR-21-inhibitor 纳米聚合物囊泡进入 PC-3细胞的传输效应。结果实验结果表明： N/ P 为20 PEG-PAsp (DIP)与 miR-21-inhibitor 已完全复合,粒径较小具有一定的正电荷,且对 PC-3细胞毒性较小;通过激光共聚焦实验,成功构建了纳米聚合物囊泡传输进入 PC-3前列腺癌细胞方法。结论成功制备了纳米聚合物囊泡,并初步探讨了纳米聚合物囊泡的表征、体外 PC-3细胞毒性试验与纳米聚合物囊泡结合 miRNA传输于 PC-3细胞的激光共聚焦实验,为下一步阐明反义 miR-21寡核苷酸诱导前列腺癌细胞凋亡的作用和分子机制打好基础。     

糖尿病大鼠肾组织中miR-21和SnoN表达的变化

目的探讨糖尿病(DM)大鼠肾组织中微小核糖核酸-21(miR-21)和核转录共抑制因子(SnoN)在肾纤维化过程中的表达变化及其可能机制。方法用链脲佐菌素复制DM大鼠模型,并设对照组(NC),每组n=8。10周后处死大鼠,观察肾组织形态变化;免疫组织化学染色、Western blot及RT-q PCR检测miR-21、SnoN、转化生长因子-β1(TGF-β1)、Smad3、p-Smad3(Ser423/425)、E钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、胶原蛋白Ⅰ(collagenⅠ)和胶原蛋白Ⅲ(collagenⅢ)的表达。结果与对照组相比,DM组肾组织p-Smad3(Ser423/425)、TGF-β1和α-SMA蛋白表达增加(P0.05),SnoN、E-cadherin蛋白表达减少(P0.05),但SnoN mRNA和miR-21表达明显上调(P0.05),并伴有collagenⅠ、collagenⅢ和FN在间质沉积增多。结论 TGF-β1可能上调miR-21表达,抑制SnoN翻译水平的表达,促进DN的纤维化病变。     

Effect of poly(dimethylsiloxane)-block-poly(oligo (ethylene glycol) methacrylate) amphiphilic block copolymers on dermal fibroblast viability and proliferation

Abstract

In this work, well-defined poly(dimethylsiloxane)-b-poly(oligo (ethylene glycol) methacrylate) (PDMS-b-POEGMA) amphiphilic block copolymers were synthesized and their effect on human dermal fibroblast were investigated. Anionic ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP) were used to synthesis the block copolymers. The molecular weight of synthesized copolymers ranged from 1000 to 2300?Da by changing the number of both PDMS and POEGMA units. It was found that the copolymer having low molecular weight decreased the fibroblast viability and proliferation by inducing apoptosis. It was proved by flow cytometry and TUNEL assay that human dermal fibroblast experienced apoptosis after exposure to synthesized amphiphilic copolymers. The results of this work suggest the use of PDMS-b-POEGMA amphiphilic copolymers with low molecular weight for hypertrophic scars remediation.     

Clinical Significance of miR-21-5p in Predicting Occurrence and Progression of Uremic Vascular Calcification in Patients with End-Stage Renal Disease

PurposeVascular calcification (VC) is a common complication of end-stage renal disease (ESRD). This study aimed to examine changes in the expression of miR-21-5p in ESRD patients with VC and to explore its clinical value in predicting the occurrence and progression of uremic VC.Materials and Methods120 ESRD patients were divided into patients without VC group (n=38) and patients with VC group (n=82). All patients were followed up for 2 years to evaluate VC progression. qRT-PCR was used to detect serum miR-21-5p levels. Receiver operating characteristic curves were constructed to assess diagnostic value. Kaplan-Meier and log-rank methods were utilized to calculate associations between VC progression and risk factors.ResultsSerum miR-21-5p levels were significantly higher in ESRD patients with VC than in those without VC and increased progressively with increasing disease severity. Serum miR-21-5p levels were able to distinguish patients with VC from those without VC, with an area under the curve value of 0.883, a sensitivity of 81.7%, and a specificity of 84.2%. After 2 years of follow-up, miR-21-5p expression had increased in patients with worse VC severity, compared with those with stable VC severity. Patients with high miR-21-5p levels were more likely to develop more severe VC, indicating an association between miR-21-5p and VC progression (log-rank p=0.002). Multivariable Cox regression analysis suggested that serum miR-21-5p is an independent predictive factor of VC progression in ESRD patients (hazard ratio=2.064, 95% confidence interval=1.225–3.478, p=0.006).ConclusionmiR-21-5p is overexpressed in the serum of ESRD patients with VC. Our results suggest that overexpression of miR-21-5p is closely associated with VC progression.     

LncRNA UCA1 sponges miR-204-5p to promote migration,invasion and epithelial-mesenchymal transition of glioma cells via upregulation of ZEB1

Long non-coding RNA urothelial carcinoma associated 1 (lncRNA UCA1) promotes cancer progression and enhances chemoresistance through miR-204-5p in a few cancers. However, no studies have investigated whether UCA1 regulates glioma metastasis through miR-204-5p and its target. In the present study, cell migration, invasion and epithelial-mesenchymal transition (EMT) were evaluated in glioma cells overexpressing UCA1. The relationships among UCA1, miR-204-5p and ZEB1 were examined by real-time PCR, western blotting and dual-luciferase reporter assays. The effect of UCA1 knockdown on xenograft tumor growth was investigated. The levels of miR-204-5p, fibronectin, COL5?A1 and ZEB1 in tumor tissues were also determined. The results showed that UCA1 overexpression promoted cell migration, invasion and EMT. UCA1 interacted with miR-204-5p and decreased its level. ZEB1 was identified as a direct target of miR-204-5p and miR-204-5p negatively regulated ZEB1 expression. Moreover, UCA1 sponged miR-204-5p and partially rescued the inhibitory effect of miR-204-5p on ZEB1. In our in vivo studies, UCA1 knockdown reduced tumor volume and tumor weight. In addition, the levels of fibronectin, COL5?A1 and ZEB1 were decreased, while miR-204-5p level was increased. The present study provides the first evidence that UCA1 promotes glioma metastasis through the miR-204-5p/ZEB1 axis, contributing to the understanding of the pathogenesis of glioma.     

Hepatitis C virus core protein expression leads to biphasic regulation of the p21 cdk inhibitor and modulation of hepatocyte cell cycle

Hepatitis C virus (HCV) Core protein is implicated in viral pathogenesis by the modulation of hepatocyte gene expression and function. To determine the effect of Core protein on the cell-cycle control of hepatocytes, a HepG2 cell line containing a Flag-tagged Core under the control of an inducible promoter was generated. Initial Core protein expression included the presence of unprocessed (191 aa) and processed (173 aa) forms of the Core proteins with the processed form becoming dominant later. Expression of the 191 aa form of Core protein corresponded to an increase in the expression of the p21, a decrease in cdk2-dependent kinase activity, and a decrease in the percentage of cells in S-phase along with an accumulation of cells in the G(0)/G(1) phase of the cell cycle. As the processed form accumulated, the p21 levels started to decline, suggesting that Core protein regulates p21 expression in a biphasic manner. These findings implicate Core protein in potentially modulating hepatocyte cell cycle differentially in the early stages of infection through biphasic regulation of p21 cdk kinase inhibitor.     

A single nucleotide substitution at the rib2 locus of the yeast mitochondrial gene for 21S rRNA confers resistance to erythromycin and cold-sensitive ribosome assembly

Summary We have studied a mutation (cs23) in the mitochondrial gene for 21SrRNA that affects the peptidyl transferase center of the ribosome and conditionally blocks the assembly of the 54S ribosomal subunit. Strains carrying this mutation are resistant to erythromycin and cold-sensitive for growth on nonfermentable carbon sources (Singh et al. 1978) Mitochondria isolated from mutant cells grown on glucose at 20°C, the nonpermissive temperature, were depleted of the 54S subunit and instead contained a novel 45S ribosomal particle. After mutant cells were shifted from 20°C to 32°C, 54S subunits were assembled, apparently from the 45S particles and pre-existing ribosomal proteins. DNA sequencing revealed that the mutant phenotype is a consequence of a C to A transversion at position 3993 of the 21SrRNA gene. Previously, C to U and C to G mutations have been identified at the same position in the 21S rRNA sequence. This position corresponds to C-2611 in the E. coli 23S RNA, a nucleotide that appears to be conserved in the large rRNA of all erythromycin-sensitive ribosomes.