lncRNA SNHG16对胶质瘤U251细胞增殖、侵袭和迁移的影响

目的 探讨沉默长链非编码RNA核内小RNA宿主基因16（SNHG16）对胶质瘤U251细胞增殖、侵袭和迁移的影响。方法 实时荧光定量PCR检测正常胶质细胞HEB、NHA和胶质瘤细胞A172、U251、U87、SHG-4中SNHG16的表达。用siRNA沉默U251细胞SNHG16的表达，分为NC-siRNA组和SNHG16-siRNA组，CCK-8法检测细胞增殖，Transwell实验检测细胞侵袭，划痕实验检测细胞迁移。结果 与正常胶质细胞HEB和NHA比较，胶质瘤细胞A172、U251、U87和SHG-4的SNHG16表达水平明显升高（P<0.05）。与NC-siRNA组比较，SNHG16-siRNA组细胞增殖能力、细胞侵袭能力和细胞迁移能力均明显降低（P<0.05）。结论 SNHG16在胶质瘤细胞中高表达，特异性沉默SNHG16基因可以抑制胶质瘤细胞的增殖、侵袭和迁移能力。     

Evi1基因对胶质瘤细胞株U87/U251增殖、侵袭的影响

目的探讨Evi1基因在不同级别胶质瘤组织中的表达,以及调控该基因对胶质瘤细胞株U87和U251细胞增殖、细胞周期和侵袭影响,为靶向癌基因治疗胶质瘤提供客观依据。方法首先通过实时定量PCR(qRT-PCR)分别检测人脑不同级别胶质瘤组织以及U87和U251细胞株中Evil基因的表达水平。用合成的小分子干扰RNA(small interfering RNA,siRNA)干扰下调U87和U251细胞Evi1基因表达作为干扰组,同时设空白和阴性对照组。qRT-PCR检测转染前后Evi1基因在U87和U251细胞中的表达和转染效率。CCK-8法检测实验组细胞的增殖能力,流式细胞仪分析Flow Cytometer(FCM)其细胞周期。Transwell以及划痕试验检测各组U87和U251的侵袭和迁移能力。以及用qRT-PCR分析下调Evi1基因后U87和U251细胞基质金属蛋白酶2(MMP2)的表达水平。结果 Evi1基因在胶质瘤组织中呈现高表达,且随着胶质瘤级别程度增加其表达水平亦上升,其中Ⅲ和Ⅳ级的胶质瘤组织以及U87、U251细胞Evi1基因表达明显高于非瘤脑组织(均P0.05)。通过siRNA下调Evi1基因后,胶质瘤细胞株U87和U251的增殖减缓(均P0.05),其侵袭和迁移能力明显下降(均P0.01),并能阻滞其细胞周期G1期(P0.05)。干扰组U87和U251细胞MMP2表达水平明显降低(均P0.01)。结论 Evi1基因在胶质瘤中呈高表达,干扰U87和U251细胞Evi1基因可抑制胶质瘤的增殖和侵袭。     

lncRNA SNHG7对胶质瘤细胞侵袭、迁移的影响

目的 探讨长链非编码RNA（lncRNA）小核仁RNA宿主基因7（SNHG7）与胶质瘤生存预后的关系,及其对胶质瘤细胞侵袭和迁移的影响。方法 选取2015年6月至2016年3月手术切除的胶质瘤组织80例（术后随访截止2020年4月）和50例颅脑损伤颅内减压术中切取的非肿瘤脑组织为对照,用RT-PCR法检测lncRNA SNHG7的表达水平。体外培养胶质瘤U87细胞,转染不同质粒敲低lncRNA SNHG7表达,用Transwell实验检测细胞侵袭和迁移能力;双荧光素酶报告基因实验检测lncRNA SNHG7对miR-4516的调控作用。结果 胶质瘤组织lncRNA SNHG7表达量明显高于对照组（P<0.05）;多因素Cox回归分析显示,lncRNA SNHG7高表达是胶质瘤病人不良生存预后的独立影响因素（P<0.05）;生存曲线分析显示,高表达组胶质瘤病人中位总生存期较低表达组明显缩短（P<0.05）。敲低lncRNA SNHG7表达,明显抑制U87细胞侵袭和迁移力（P<0.05）。双荧光素酶报告基因实验证实lncRNA SNHG7靶向上调miR-4516表达,上调miR-4516可逆转敲低lncRNA SNHG7表达对胶质瘤U87细胞侵袭和迁移能力的作用（P<0.05）。结论 胶质瘤组织lncRNA SNHG7呈高表达,与病人不良生存预后有关。lncRNA SNHG7通过靶向调控上调miR-4516表达促进胶质瘤细胞的侵袭和迁移。     

Hax-1对胶质瘤细胞增殖、迁移和侵袭的作用

目的 探讨造血干细胞特异性相关结合蛋白-1（Hax-1）对胶质瘤细胞增殖、迁移和侵袭的影响。方法 选择2018年9月至2019年6月手术切除并得到术后病理证实的胶质瘤组织35例和颅脑损伤内减压术切除的正常脑组织35例,采用qRT-PCR检测Hax-1 mRNA水平;同时检测胶质瘤细胞系（U87、A172、T98及U343）和人星形胶质细胞（NHAs）Hax-1 mRAN水平。使用Lipofectamine 2000法将Hax-1 siRNAs和阴性对照siRNA转染U87细胞构建Hax-1低表达细胞株,将Hax-1高表达质粒和PCDNA3.1空载质粒转染U343细胞构建Hax-1过表达细胞株,使用蛋白印迹法验证转染效果,采用CCK-8法检测细胞增殖,采用Transwell小室实验检测细胞迁移和侵袭,免疫印迹法检测NF-κB信号通路相关蛋白表达水平（NF-κB、CCND1、C-myc、MMP-2、MMP-9）。结果 胶质瘤组织Hax-1 mRNA水平明显高于正常脑组织（P<0.05）;胶质瘤细胞系（U87、A172、T98及U343）Hax-1 mRNA水平明显高于人星形胶质细胞（P<0.05）,其中U87细胞Hax-1 mRNA水平最高,U343细胞最低。U343细胞转染Hax-1过表达质粒后,Hax-1蛋白水平显著增高（P<0.05）,细胞增殖、侵袭、迁移能力均明显增强（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显增高（P<0.05）。U87细胞转染Hax-1 siRNA后Hax-1蛋白水平显著降低（P<0.05）,细胞增殖、侵袭、迁移能力均明显降低（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显降低（P<0.05）。结论 胶质瘤组织Hax-1呈高表达,明显促进肿瘤细胞增殖、侵袭和迁移,其机制可能与激活NF-κB信号通路有关。     

RNA干涉CD44基因抑制脑胶质瘤细胞U251的增殖与侵袭性研究

目的通过合成靶向分化抗原簇44(CD44)基因的siRNA片段,下调CD44在脑胶质瘤细胞U251中的表达,揭示其在胶质瘤增殖、迁移和侵袭中所起的作用。方法根据CD44基因序列设计并合成的siRNA片段转染U251细胞,利用荧光实时定量(qRT-PCR)检测细胞中CD44基因mRNA水平的变化;蛋白印迹实验(Western blot)检测CD44基因蛋白水平的变化;四甲基偶氮唑蓝染色(methyhhiazolyl tetrazolium,MTT)法检测U251细胞增殖;单细胞划痕愈合实验、Transwell小室侵袭实验检测U251细胞的迁移与侵袭能力变化。结果体外合成的CD44-siRNA能有效抑制U251细胞中CD44基因在mRNA水平与蛋白水平的表达(P0.05),并抑制细胞的增殖(P0.05)和迁移侵袭能力(P0.05)。结论 CD44-siRNA能够有效抑制U251脑胶质瘤细胞的增殖及其迁移侵袭力,为以CD44为靶点的肿瘤基因治疗奠定了基础。     

长链非编码RNA SNHG12在神经系统疾病中研究进展

小核仁RNA宿主基因12(small nucleolar RNA host gene 12,SNHG12),其表达水平的异常常改变体内细胞的增殖、自噬及凋亡,与肿瘤及某些特定疾病的发生发展明显相关,可作为这些疾病筛查与诊断的生物标记物。本文结合SNHG12的结构及功能,阐述SNHG12的表达水平上调或下调在脑胶质瘤细胞生长、缺血性脑卒中的病理生理的作用机制。     

RNAi干扰脑胶质瘤CEP55表达对胶质瘤细胞LN229功能影响的相关性研究

目的探讨中心体相关蛋白55(CEP55)在胶质瘤组织及正常脑组织中的表达差异,以及通过RNAi技术敲低CEP55表达对胶质瘤细胞系LN229功能的影响。方法收集南京医科大学附属脑科医院25例胶质瘤手术标本及10例正常脑组织标本,采用实时荧光定量PCR(qRT-PCR)及Western blot检测35例样本中CEP55的表达水平。通过siRNA敲低LN229细胞系中CEP55的表达后,采用细胞计数、划痕及Transwell实验检测其对细胞增殖、迁移及侵袭功能的影响。结果 CEP55在脑胶质瘤组织中的表达量明显高于正常脑组织。干扰CEP55的表达后,胶质瘤细胞LN229的功能明显降低。结论 CEP55在胶质瘤中高表达,并参与影响了胶质瘤细胞系LN229的增殖、迁移、侵袭功能。CEP55今后可能成为胶质瘤治疗的新靶点。     

LncRNA HNF1A-AS1在胶质瘤中的表达及功能研究

目的 研究长链非编码RNA (long non-coding RNA,LncRNA) HNF1A-AS1在胶质瘤中的表达及其对胶质瘤细胞增殖、迁移、侵袭及凋亡的影响。方法 运用实时定量PCR技术检测HNF1A-AS1在胶质瘤组织与正常脑组织以及胶质瘤细胞与正常胶质细胞之间的表达差异。利用特异小干扰RNA (si RNA)构建低表达的胶质瘤细胞系A172和U251。通过CCK-8实验、EDU实验、Transwell、流式实验研究胶质瘤细胞增殖、迁移、侵袭、细胞凋亡情况,Western blot研究与细胞迁移、侵袭及凋亡相关蛋白的表达变化。结果 LncRNA HNF1A-AS1在胶质瘤组织和胶质瘤细胞系中高表达。抑制HNF1A-AS1表达后,胶质瘤细胞增殖、侵袭、迁移能力降低,细胞凋亡率增加。Western Blot结果显示:抑制HNF1A-AS1表达后,一些与细胞迁移、侵袭、凋亡相关蛋白的表达水平明显改变。结论 LncRNA HNF1A-AS1在胶质瘤中高表达,抑制HNF1A-AS1表达能降低胶质瘤细胞的增殖、迁移、侵袭能力,同时增加胶质瘤细胞的凋亡比率。因此,HNF1A-AS1可以作为脑胶质瘤的潜在治疗靶点。     

RNA结合蛋白RBM38表达对胶质瘤细胞增殖、迁移、侵袭能力的影响

目的探讨RNA结合蛋白RBM38(RNA结合基序蛋白38,RNA-binding motif protein 38)在胶质瘤组织和细胞中的表达水平及其对胶质瘤细胞增殖、迁移、侵袭和凋亡的调节作用。方法采用实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)检测胶质瘤组织和瘤旁正常组织的RBM38表达水平。采用q RT-PCR和Western blot检测胶质瘤细胞U87、U251和正常星形胶质细胞HA1800的RBM38表达水平。用免疫组化染色检测59例胶质瘤组织的RBM38表达水平,并分析其与临床病理资料的关系。将靶向RBM38的特异性小干扰RNA运用脂质体介导转染法转染至胶质瘤细胞U87和U251。应用CCK-8(cholecystokinin-8)增殖实验、划痕实验、Transwell小室实验、流式细胞仪测凋亡细胞,检测RBM38对于胶质瘤细胞增殖、迁移、侵袭和凋亡能力的影响。采用q RT-PCR检测抑制RBM38表达后肿瘤细胞P53基因的表达水平。结果相较于瘤旁的脑组织,胶质瘤组织RBM38的表达量明显升高(P 0. 05)。胶质瘤细胞中的RBM38表达水平明显高于正常胶质细胞(均P 0. 001)。免疫组化结果显示,与低级别胶质瘤相比,高级别胶质瘤的RBM38表达水平更高(P 0. 05)。抑制RBM38的表达后,胶质瘤细胞U87和U251的增殖速率明显下降、迁移距离变短、能透过生物膜的细胞数目明显下降(均P 0. 05)。下调了RBM38的表达后胶质瘤细胞的凋亡比率升高(P 0. 05)。被抑制了RBM38表达的胶质瘤细胞P53基因的表达水平升高(P 0. 05)。结论 RBM38在胶质瘤组织及细胞系中呈高表达,RBM38的高表达与胶质瘤的临床分级密切相关。下调RBM38的表达后能抑制胶质瘤细胞的增殖、迁移、侵袭能力,促进细胞的凋亡,并使P53基因的表达水平增高;可能为临床诊断和治疗胶质瘤提供新的靶点。     

Measuring Perceived Rehabilitation Needs of Caregivers of People with Schizophrenia in Mainland China

This paper reports the development and validation of the Wuxi version of the Rehabilitation Needs Questionnaire for Caregivers of People with Schizophrenia (PRNQ-C-WX) based on the original Hong Kong version (PRNQ-C-HK). PRNQ-C-WX was validated by exploratory factor analysis (EFA) using a convenience sample consisting of 200 caregivers of people with schizophrenia. EFA yielded an eight-factor solution accounting for 63.8 % of the total variance which resulted in a 50-item PRNQ-C-WX. The questionnaire has excellent internal consistencies. Its factor structure is similar to the Hong Kong version. Some suggestions for policy, service and research development in mental health in mainland China are made.     

Measuring Perceived Rehabilitation Needs of People with Schizophrenia in Mainland China

This paper reports the development and validation of the Wuxi version of the Rehabilitation Needs Questionnaire for People with Schizophrenia (PRNQ-S-WX) based on the original Hong Kong version. PRNQ-S-WX was validated by exploratory factor analysis (EFA) using a convenience sample of 250 people with schizophrenia. EFA yielded a 17-factor solution accounting for 81.3 % of the total variance which resulted in a 75-item PRNQ-S-WX. The questionnaire has sound internal consistencies. Its factor structure is similar to the Hong Kong version. Some suggestions for policy, service and research development in mental health in mainland China are made.     

New insights into cellular prion protein (PrP) functions: The “ying and yang” of a relevant protein

The conversion of cellular prion protein (PrP^c), a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrP^sc) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrP^c is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. Recent studies have confirmed its participation in basic physiological processes such as cell proliferation and the regulation of cellular homeostasis. Other studies indicate that PrP^c interacts with several molecules to activate signaling cascades with a high number of cellular effects. To determine PrP^c functions, transgenic mouse models have been generated in the last decade. In particular, mice lacking specific domains of the PrP^c protein have revealed the contribution of these domains to neurodegenerative processes. A dual role of PrP^c has been shown, since most authors report protective roles for this protein while others describe pro-apoptotic functions. In this review, we summarize new findings on PrP^c functions, especially those related to neural degeneration and cell signaling.     

Immunostaining for proliferating cell nuclear antigen: its role in determination of proliferation in routinely processed human brain tumor specimens

Alcohol- and formalin-fixed, paraffin-embedded samples of 71 brain tumors (35 gliomas, 22 metastatic carcinomas, 8 meningiomas and 6 other tumors) were investigated by immunocytochemistry with three different monoclonal antibodies against proliferating cell nuclear antigen (PCNA)/cyclin (19A2; 19F4; PC10). PC10 was found to work best; it is applicable to both alcohol- and formalin-fixed tumor samples. PCNA labeling indices (LIs) were compared in the same tumors with LIs obtained by Ki-67 immunostaining of frozen sections and by in vitro incubation with bromodeoxyuridine (BrdUrd); in the latter preparations, BrdUrd LIs could be compared with PCNA LIs in the very same areas of serial sections. In gliomas, PCNA LIs were 0.7–80.2% (mean 31.7%), in metastases 0–76.0% (mean 47.8%), and in meningiomas 0–53.0% (mean 19.3%). In general, PCNA LIs were highly significantly correlated with Ki-67 LIs (P=0.0002) and BrdUrd LIs (P=0.0001). However, when tumor subgroups are considered, only gliomas show a significant correlation with Ki-67 and BrdUrd LIs. Despite this statistical correlation, PCNA expression was out of proportion to proliferation indices as determined by both other methods in almost one third of all brain tumors. Immunocytochemistry for PCNA produces a broad spectrum of staining intensity of labeled nuclei, whose number is dependent upon the sensitivity of the immunocytochemical technique used. Thus, inter-oberserver and inter-laboratory variabilities in PCNA LI determination may occur. Overlapping of PCNA LIs between tumor subgroups of varying malignancy further limits the informational value for the individual case. In some classic meningiomas, high PCNA scores do not reflect the proliferative activity of the tumor, as Ki-67 and BrdUrd LIs are very low in these cases. We conclude that PCNA immunolabeling is of limited value in the individual tumor, mainly due to overexpression in many tumors, and at present cannot be recommended to replace Ki-67 and/or BrdUrd labeling methods for routine determination of proliferative activity in human tumor specimens.Supported in part by a grant from the Oncology Commission, Medical Faculty, University of Vienna     

Susceptibility of human foetal brain tissue to cool- and freeze-storage

Human second trimester foetal brain tissue was stored for a period of 1–6 weeks under various conditions in an attempt to evaluate factors influencing its susceptibility (cell loss) and survivability. Post-storage viability of mesencephalon, striatum, cerebellum and occipital cortex was assessed by a protocol combining vital staining with cell density counts so that tissue viability and cell loss could be evaluated simultaneously; tissue survivability was evaluated by cell culture. A significant amount of cell loss occurred after 24 h storage at room temperature, after one week at 4°C and by two weeks at −20°C in all structures; storage at −196°C resulted in 17–21% cell loss at the end of a 6 week period. At −20°C the cryoprotective effect of 20% FCS was equivalent to that of 15% FCS + 7% DMSO combined, suggesting potential use of serum in replacement of chemical additives. The procedure for removal of DMSO was critical to cell viability and survivability: single step dilution led to 27–39% greater cell loss than slow, multi-step dilutions. In comparison to fresh, non-stored tissue, immunocytochemical characterization of in vitro propagated stored tissue revealed no changes in the populations of major constituent cell types including neurones, dopaminergic neurones, glial and fibroblast cells. These results provide information on possible conditions under which transplant tissue can be satisfactorily stored depending on the prevailing requirements.     

Interactions of living astrocytes in vitro: Evidence of the development of contact spacing

We have studied the behaviour of living, process-bearing astrocytes in vitro, observing groups of cells at daily intervals for up to 7 days. Each cell initially formed two processes, appearing bipolar in shape, and with further time in culture, grew additional processes and appeared stellate. As their processes grew, the interactions between astrocytes underwent characteristic changes. While bipolar, the cells appeared to avoid making contact, lying parallel to each other. As they became stellate, the astrocytes made extensive contact with neighbours, gradually forming extended, contacting networks in which their somas were regularly spaced (as previously described). The interactions which led to the establishing of such arrays were also evident. If two cells were initially close or adjacent, they extended short processes to contact each other; then, as their processes grew, their somas moved apart, until they were separated by 60-120 μm. If two cells were initially well separated, each directed processes towards the other until contact was made, often with striking precision, and their somas then moved together, until they were separated by 60-120 μm. These behaviours of contact, separation, and approach caused astrocytes to form clusters, within which their somas appeared regularly spaced, and may represent the interactions which occur among astrocytes during normal development to produce the regularly spaced arrays of astrocytes described in earlier studies of intact central nervous tissue. © 1994 Wiley-Liss, Inc.     

Use ofCr cell labelling to distinguish between release of radiolabelled amino acids from primary astrocyte cultures being due to efflux or cell damage

Continuous perfusion methods are widely used to monitor release of substances, particularly transmitters, from brain cell cultures growing as monolayers. However, if stimuli used to produce release also cause loss or lysis of cells, the appearance of label in the perfusate due to such effects will be indistinguishable from release. Using a perfusion method we have studied release of preloaded, radiolabelled amino acids from primary astrocyte cultures due to a variety of stimuli; hypotonic or high K⁺ media, activation of β-receptors or swelling-induced release due to isosmotic ethanol. In this study primary astrocyte cultures were simultaneously labelled with Na₂⁵¹CrO₄ and allowed to take up radiolabelledd-aspartate or taurine. It was found that while all of the above methods caused release of radiolabelled amino acids none caused release of⁵¹Cr into the perfusion fluid. In contrast, perfusion with 0.05% (v/v) Triton X-100 did lead to release of⁵¹Cr. Thus a variety of means of inducing swelling or shape changes in astrocytes causes true release of radiolabelled amino acids and simultaneously monitoring⁵¹Cr release seems a convenient means of distinguishing such release from cell loss or lysis.     

Neurofibromatosis tumor and skin cells in culture

Summary Structural proteins of cultured neurofibromatosis (NF) tumor and skin cells were studied with reference to control skin fibroblasts. In polyacrylamide gel electrophoresis (PAGE)/fluorography the banding patterns of the cell lysates were markedly similar. NF tumor cells, however, produced a 60 kD band with a stronger and a 48 kD band with a lighter protein staining and metabolic labeling intensity. Furthermore, skin cells were also characterized by a 26 kD protein and the tumor cells by a 22 kD protein with high metabolic labeling intensity. Neuraminidase/galactose oxidase/NaB³H₄-labeled NF skin and control skin cells possessed a 220 kD protein that was less intensively labeled in the tumor cells. The banding pattern of the skin cells was also characterized by a protein with slightly lower molecular weight (86 kD) than that of the tumor cell lysates (90 kD). In all cell lines studied indirect immunofluorescence stainings revealed bright arrays of vimentin type intermediary filaments but no desmin, cytokeratin, glial fibrillary acidic protein (GFAP), or neurofilament proteins. NF skin and control skin cells possessed well developed actin-containing bundles of microfilaments, while those of the tumor cells lacked a typical stress-fiber organization. The general morphology of the tumor cell cultures was also irregular. Transmission electron microscopy revealed no basic differences in the structure of intermediary filaments or microfilaments. The present data provide basic knowledge of neurofibromatosis skin and tumor cells and demonstrate that cultured cells originating from neurofibromas are defective in both their intracellular and extracellular organization.Financially supported by grants (to JP) from the Research and Scienctific Foundation of Orion Oy Ltd. and by institutional grants from the Turku University Foundation and the Sigrid Jusélius Foundation.     

In situ analysis of cell kinetics in human brain tumors

Summary A newly developed in vitro labeling method with bromodeoxyuridine (BrdU) identifies S phase cells in situ in freshly obtained surgical tissue of human brain tumors which is subsequently fixed and embedded in paraffin for BrdU immunovisualization. For the first time, the BrdU labeling index (LI) is successfully compared here with the LI obtained by immunostaining of frozen sections of the same tumors with monoclonal antibody Ki-67 which identifies all proliferating cells, i.e., the growth fraction. LIs were counted in at least five different areas with high density of labeled cells; at least 1,000 cells were counted. In 13 metastatic tumors, Ki-67 LI was 8.3%–62.6%, and BrdU LI was 5.1%–28.0%. In 18 gliomas, Ki-67 LI was 1.4%–19.3%, and BrdU LI was 0.2%–11.6%. In 7 meningiomas, Ki-67 LI was 0.3%–3.0%, and BrdU LI was 0%–2.0%. Statistical comparison of Ki-67 and BrdU LIs by linear regression analysis revealed a highly significant correlation: BrdU LI=0.99+0.34 Ki-67 LI (r=0.92,P<0.001). A significant heterogeneity of proliferation patterns may occur within one sample from area to area, as well as between different samples of the same tumor, especially in gliomas; thus, some subjective influence on LIs by arbitrary sampling and selection could occur in quantitative evaluation of in situ cell kinetics of human brain tumors. This study indicates that our in vitro BrdU-labeling method allows the in situ identification of S phase cells in excellently preserved fixed tumor tissue which is well suited for further histological examination. This method compares favorably with Ki-67 labeling of frozen sections and might emerge as a powerful new tool for the routine study of cell proliferation in surgical specimens of human brain tumors.Supported by funds from the Commission for Cancer Research of the Medical Faculty of the University of Vienna