胶质瘤中miR-383与其靶向circ_001634相互调控机制及作用研究

目的研究脑胶质瘤中circ_001634的表达以及与miR-383的相互调控关系及在胶质瘤发生发展中的作用。方法实时定量聚合酶链反应(Real-time PCR)检测脑胶质瘤组织中circ_001634的表达情况,及分别过表达circ_001634与miR-383后对相互的影响,非锚定依赖性生长实验检测转染circ_001634后胶质瘤U251细胞生长情况,Western blot检测miR-383下游基因CCND1蛋白表达情况。结果 circ_001634在胶质瘤中的表达显著升高;过表达circ_001634能抑制胶质瘤细胞生长,且能下调miR-383表达,并抑制CCND1表达;同时过表达miR-383,也能够抑制circ_001634表达。结论胶质瘤特异性circ_001634表达升高是miR-383表达下调和功能受抑制重要调控机制,circ_001634与miR-383的相互调控很可能是胶质瘤发生发展的重要原因。     

Circ_0000003通过调控miR-338-3p/IRS2轴调控甲状腺癌细胞增殖、转移和凋亡

目的探究circ0000003对甲状腺癌(TC)细胞增殖、转移和凋亡的影响及其机制。方法 CGTH W-3细胞分为转染组-1(转染空白质粒)、转染组-2(转染pcDNA-circ0000003)、转染组-3(共转染miR-338-3p mimics和pcDNA-circ0000003);TPC-1细胞分为转染组-4(转染control si-RNA)、转染组-5(转染si-circ0000003)、转染组-6(共转染miR-338-3p inhibitors和si-circ0000003)。用定量逆转录聚合酶链式反应(qRT-PCR)检测TC组织和细胞系中circ0000003表达。用蛋白质印迹法检测TC组织中胰岛素受体底物2(IRS2)及凋亡相关蛋白的表达,用CCK-8实验检测细胞增殖,用Transwell实验检测细胞迁移和侵袭能力,用双荧光素酶报告基因实验验证circ0000003与miR-338-3p之间的靶向关系。结果 Circ0000003在甲状腺癌组织(3.41±0.27)中的表达水平显著较癌旁组织(1.03±0.11)高(P<0.05)。转染组-1,转染组-2,转染组-4,转染组-5在96 h时吸光度分别为0.70±0.07,0.85±0.07,0.71±0.07和0.57±0.04;迁移细胞数目分别为(124.33±6.18),(171.00±12.19),(71.00±6.19)和(126.30±7.93)个;侵袭迁移细胞数目分别为(89.00±11.86),(126.33±7.93),(86.67±8.86)和(56.33±7.93)个。上述指标,转染组-2与转染组-1比较,转染组-5与转染组-4比较,差异均有统计学意义(均P<0.05)。结论 Circ0000003通过调节miR-338-3p/IRS2轴促进TC细胞的增殖、迁移和侵袭,并抑制细胞凋亡。     

Mid-arm muscle circumference as an indicator of osteoporosis in community-dwelling older men

BackgroundOsteoporosis is an underdiagnosed disease and is lack of convenient and cost-efficient screening tool. We undertook a study to determine whether mid-arm muscle circumference (MAMC) was associated with osteoporosis.DesignData were retrieved from the National Health and Nutrition Examination Survey (NHANES) III participants (aged 40–90 years). We divided the MAMC into tertile groups (T1, T2, and T3). Femoral neck bone density was analyzed because it was the reference skeletal site for defining osteoporosis in epidemiological studies. Participants with T- scores ≤ –2.5 were categorized as having osteoporosis. Multivariate logistic regression models were used to evaluate the associations between the MAMC tertiles and osteoporosis.ResultsAfter adjustment for multiple covariates, osteoporosis was significantly inversely associated with the MAMC tertiles in the male group (T2/T1: OR 0.47, 95 % CI 0.30–0.75 and T3/T1: OR 0.34, 95 % CI 0.18–0.64), whereas nonsignificant association was found in the female group (T2/T1: OR 0.92, 95 % CI 0.70–1.20 and T3/T1: OR 0.84, 95 % CI 0.47–1.53). Subgroup analyses (40–64 and ≥65 years old; BMI <25 and ≥25 kg/m²) revealed consistent results.ConclusionThe MAMC is an economical and practical tool that may assist in screening and early diagnosis of osteoporosis for the older men.     

circ_ 0061140 靶向 miR-6838-5p 促进非小细胞肺癌细胞的增殖、 阻滞细胞周期、 诱导细胞凋亡

目的 通过实验研究确认环状 RNA (circRNA) circ_ 0061140 是否能靶向 miR-6838-5p 调控非小 细胞肺癌细胞的增殖、 细胞周期和凋亡。 方法 采用逆转录-定量聚合酶链反应 (RT-qPCR) 检测 circ_ 0061140 和 miR-6838-5p 在非小细胞肺癌细胞中的表达情况。 将非小细胞肺癌 NCI-H1299 细胞分为 si-circ_ 0061140 组 (转染 si-circ_ 0061140; 低表达 circ_ 0061140)、 si-NC 组 (转染 si-NC; 阴性对照)、 NC 组 (未 转染; 空白对照)、 miR-6838-5p 组 (转染 miR-6838-5p 模拟物; 高表达 miR-6838-5p)、 miR-NC 组 (转染 miR-NC; 高表达 miR-6838-5p 的阴性对照)、 si-circ_ 0061140 + anti-miR-NC 组 (共转染 si-circ_ 0061140 与 anti-miR-NC)、 si-circ_ 0061140 + anti-miR-6838-5p 组 (共转染 si-circ_ 0061140 与 anti-miR-6838-5p)。 采用 Western 印迹检测细胞周期蛋白 D1 ( cyclinD1)、 活化的含半胱氨酸的天冬氨酸蛋白水解酶-3 ( cleaved caspase-3) 蛋白表达, MTT 法检测细胞增殖能力, 流式细胞仪检测细胞周期和凋亡情况。 双荧光素酶活性 检测 circ_ 0061140 和 miR-6838-5p 的靶向结合。 结果 非小细胞肺癌组织、 细胞 NCI-H1299、 NCI-H2170、 NCI-H1975 中的 circ_ 0061140 表达量均比癌旁组织或正常肺上皮细胞 BEAS-2B 高, miR-6838-5p 表达量比 癌旁组织或 BEAS-2B 细胞低 (P均 < 0. 05)。 si-circ_ 0061140 组或 miR-6838-5p 组 NCI-H1299 细胞的 CyclinD1 蛋白表达量、 增殖能力、 S 期细胞比例比 NC 组减少, Cleaved-caspase-3 蛋白表达量、 G0 / G1期细胞比 例、 凋亡率比 si-NC 组或 miR-NC 组增加 (P均 < 0. 05)。 circ_ 0061140 靶向调控 miR-6838-5p 的表达。 共转 染 si-circ_ 0061140、 anti-miR-6838-5p 可减弱转染 si-circ_ 0061140 对细胞生物学行为的作用。 结论 低表达 circ_ 0061140 通过靶向非小细胞肺癌细胞的 miR-6838-5p, 抑制细胞增殖、 阻滞 G0 / G1期细胞周期, 并加速 凋亡。     

Ginsenoside RG1-induced mesenchymal stem cells alleviate diabetic cardiomyopathy through secreting exosomal circNOTCH1 to promote macrophage M2 polarization

Diabetic cardiomyopathy (DCM) is a cardiac complication resulting from long-term uncontrolled diabetes, characterized by myocardial fibrosis and abnormal cardiac function. This study aimed at investigating the potential of ginsenoside RG1 (RG1)-induced mesenchymal stem cells (MSCs) in alleviating DCM. A DCM mouse model was constructed, and the effects of RG1-induced MSCs on myocardial function and fibrosis in diabetic mice were evaluated. RG1-induced MSCs were cocultured with high glucose-treated fibroblasts for subsequent functional and mechanism assays. It was discovered that RG1-induced MSCs secrete exosomes that induce macrophage M2 polarization. Mechanistically, exosomes derived from RG1-induced MSCs transferred circNOTCH1 into macrophages, activating the NOTCH signaling pathway. A competing endogenous RNA (ceRNA) regulatory axis consisting of circNOTCH1, miR-495-3p, and NOTCH1 was found to contribute to DCM alleviation.. This study unveiled that exosomal circNOTCH1 secreted by RG1-induced MSCs can alleviate DCM by activating the NOTCH signaling pathway to induce macrophage M2 polarization. This finding may contribute to the development of new therapeutic approaches for DCM.     

Emerging role of competing endogenous RNA and associated noncoding RNAs in thyroid cancer

Cancer of the thyroid is the most common endocrine malignancy. While treatment options are limited for individuals with medullary or anaplastic thyroid cancer, understanding the underlying mechanisms is vital to developing a successful thyroid cancer treatment strategy due to the tumor’s multistep carcinogenesis. Non-coding RNAs (ncRNAs) have been associated with thyroid cancer progression in several recent studies; however, the role of regulatory interactions among different types of ncRNAs in thyroid cancer remains unclear. Recently, competing endogenous RNA (ceRNA) has been discovered as a mechanism demonstrating regulatory interactions among non-coding RNAs, including pseudogenes, long non-coding RNAs (lnRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs). It has been concluded from the literature that numerous ceRNA networks are deregulated during the development, invasion, and metastasis of thyroid cancer, as well as in epithelial-mesenchymal transition (EMT) and drug resistance. Further understanding of these deregulations is important to develop diagnostic procedures for early detection of thyroid cancer and promising therapeutic options for effective treatment. The purpose of this review is to highlight the emerging roles of some newly found ceRNA members in thyroid cancer and outline the current body of knowledge regarding ceRNA, lncRNA, pseudogenes, and miRNAs.     

Hsa_circ_0041268 promotes NSCLC progress by sponging miR‐214‐5p/ROCK1

Circular RNAs hold significant regulatory functions during various tumors. However, the exact hsa_circ_0041268 roles in non‐small cell lung cancer (NSCLC) along with regulatory mechanism are unknown. In this study, RT‐qPCR was used to perceive hsa_circ_0041268 expressions in NSCLC cell lines. Our team constructed small interfering RNA for hsa_circ_0041268. NSCLC cell proliferation, migration, and tumorigenesis in nude mice were assayed to confirm hsa_circ_0041268 activities in NSCLC cells. We then used bioinformatics and luciferase reporter analyses to characterize the hsa_circ_0041268 downstream targets. The result shows that the expressions of hsa_circ_0041268 incremented in NSCLC cell lines and hsa_circ_0041268 downregulation decreased cell proliferation and migration. ROCK1 and miR‐214‐5p were hsa_circ_0041268 downstream targets. miR‐214‐5p downregulation or ROCK1 overexpression restored migration and proliferation abilities after hsa_circ_0041268 silencing. ROCK1 overexpression renovated migration and proliferation abilities after miR‐214‐5p overexpression. In vivo investigations confirmed that hsa_circ_0041268 downregulation inhibited tumor formation and metastasis in nude mice xenografts. Together, results demonstrated that hsa_circ_0041268 acted as tumor promoter through novel hsa_circ_0041268/miR‐214‐5p/ROCK1 axis, which highlighted its potential as NSCLC therapeutic agent.     

Circular RNA hsa_circ_0000285 regulates the microRNA‐599/G‐protein subunit gamma 12 (miR‐599/GNG12) axis to promote glioma progression

ObjectiveGlioma is the most common, rapidly progressing, lethal brain tumor. However, underlying mechanisms behind its abnormal progression remain largely unknown. This study aimed to investigate mechanism of action and effects of the hsa_circ_0000285 on glioma progression.MethodsRT‐qPCR was utilized to study RNA expression in glioma tissues and cell lines. The effects of hsa_circ_0000285 on glioma progression were studied by measuring cell proliferation and migration, apoptosis, tumor volume and weight in both glioma cells and xenograft glioma mice. The features of hsa_circ_0000285 were identified using chromatin fractionation and RNase digestion. Its mechanism of action was analyzed using bioinformatics, RNA‐binding protein immunoprecipitation, and luciferase reporter assay.ResultsWe found glioma tissues and cell lines were overexpressing hsa_circ_0000285. While hsa_circ_0000285 promoted cell proliferation and migration, it inhibited apoptosis in vitro. It also increased tumor volume and weight in vivo. Using bioinformatic analysis and verification experiments for studying its mechanisms, we confirmed that hsa_circ_0000285 sponged miR‐599, which negatively regulated GNG12 by binding to its mRNA.ConclusionHsa_circ_0000285 is overexpressed in the glioma and promotes its progression by directly regulating the miR‐599/GNG12 axis. This novel mechanism, therefore, shows that the hsa_circ_0000285/miR‐599/GNG12 axis may be a promising therapeutic target for glioma treatment.     

沉默circ9119调控miR-126抑制肝癌细胞的增殖、侵袭和自噬

目的:探讨环状RNA-9119（circular-RNA-9119,circ9119）在肝癌（hepatocellular carcinoma,HCC）中的作用及其作用机制。方法:qRT-PCR检测收集的组织样本和购买的细胞系中circ9119、miR-126的表达,并对细胞进行筛选。借助荧光原位杂交（FISH）实验、RNA结合蛋白免疫共沉淀（RIP）实验与双荧光素酶报告实验对circ9119和miR-126的关系进行明确。凋亡相关因子和自噬标记物借助qRT-PCR、Western blot测定。CCK8、Transwell、流式细胞术分别测定HCC细胞的增殖、侵袭和凋亡。裸鼠成瘤实验观测肿瘤生长状况。结果:HCC组织和细胞中,circ9119的表达上调、miR-126的表达下调（均P＜0.05）。circ9119能够作为miR-126的ceRNA对其进行调控。沉默circ9119有利于抑制HCC细胞的增殖、侵袭和自噬,促进细胞的凋亡,且能够抑制小鼠体内肿瘤的生长（均P＜0.05）。而过表达circ9119则变化与之相反。上调miR-126的表达与沉默circ9119中细胞变化趋势一致。上调miR-126的作用能够被过表达circ9119所逆转。结论:circ9119作为促癌因子负调控miR-126在HCC中发挥作用。     

Efficient and economic HPLC performance qualification

Analytical instrument qualification (AIQ) is a prerequisite for any analytical method validation and thus must be considered as a vital basis of analytical data integrity and quality in pharmaceutical analysis. There is a well-established system of qualification phases—Design Qualification, Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ). As HPLC systems are “off the shelf” equipment, Design Qualification may be disregarded here. IQ establishes that the instrument is received as designed and that it is properly installed. OQ is carried out modularly with the intention to ensure that the specific modules of the system and the whole system are operating according to the defined specifications. PQ as the last step of the initial qualification is supposed to ensure continued satisfactory performance of an instrument under actual running conditions over the anticipated working range during daily use. However, PQ is not a one time exercise, but is currently repeated regularly independently from routine use of the analytical system using standard reference test condition. But this approach, which is time consuming and expensive only provides a snapshot of system performance. As HPLC procedures generally require a system suitability test (SST) prior and/or after test, it might be far more reasonable and robust to use these SST data for a continuous PQ. The work presented here demonstrates that, under certain circumstances, satisfactory instrument performance assessment can be derived from system suitability tests and performance data from daily use as well. A generally accepted qualification list, consisting of only twelve critical parameters, was compiled in a first step. Some parameters such as injector or thermostatting accuracy were considered redundant while others were successfully incorporated in the proposed holistic approach. System suitability test data as well as OQ/PQ data were provided from different sources and evaluated. The promising results confirmed our concept of ongoing/continuous PQ as a major improvement in AIQ. This approach will not only help to reduce time and effort in the daily laboratory routine without losing data quality, but also avoid the critical re-evaluation of numerous analytical tests once a routine PQ fails.