miR-21-5p在急性有机磷农药中毒患者血清中的表达意义及与CK-MB、AMS水平的相关性分析

目的探究miR-21-5p在急性有机磷农药中毒患者血清中的表达意义及与肌酸磷化脢-同功脢MB(CK-MB)、血清淀粉酶(AMS)水平的相关性。方法选择2018年9月至2019年12月在我院救治的60例急性有机磷农药中毒患者作为中毒组,50例正常体检者作为对照组。入院后采取患者静脉血3 ml,qPCR检测急性有机磷农药中毒患者血清miR-21-5p表达水平,ELISA法检测急性有机磷农药中毒患者血清CK-MB、AMS水平。分析两组间血清miR-21-5p表达水平的差异及其与CK-MB、AMS水平的相关性。结果qRT-PCR结果显示,中毒组血清miR-21-5p相对表达量为1.48±0.36,明显高于对照组的1.05±0.31,差异有统计学意义(P<0.05)。ELISA结果显示,中毒组血清CK-MB和AMS相对表达量明显高于对照组的相对表达量,差异均有统计学意义(P<0.05)。血清miR-21-5p与CK-MB和AMS水平呈正相关性(P<0.05)。血清miR-21-5p的表达与患者是否有呼吸衰竭和胰腺炎有关(P<0.05)。结论miR-21-5p在急性有机磷农药中毒患者血清中的表达显著升高且与CK-MB、AMS水平呈正相关。     

血清miR-122和miR-155在丙戊酸钠诱导小鼠仔鼠肝损伤中的表达

目的 探讨血清miR-122和miR-155在丙戊酸钠（VPA）致小鼠仔鼠肝损伤过程中的动态表达及其与肝损 伤的关系。方法 4周龄昆明小鼠按随机数字表法分为对照组30只和实验组56只。实验组又分为7组,分别给予 VPA 375 mg·kg-1·d-1连续灌胃1、3、5、7、14、21和28 d;对照组又分为5组,分别于1、7、14、21和28 d 给予1 mL生理盐 水灌胃,留取血清及肝组织。HE染色观察肝组织病理改变;全自动生化分析仪测定血清丙氨酸转氨酶（ALT）和天冬 氨酸转氨酶（AST）;实时荧光定量聚合酶链式反应（qPCR）检测血清miR-122和miR-155表达水平。结果 肝组织 病理学结果显示,实验组肝细胞出现不同程度的空泡变性。3、5、7 d时肝细胞胞浆内可见少量炎性细胞,21 d时大 量肝细胞变性坏死,28 d时大量肝细胞再生。血清ALT 与AST均呈现下降-升高的趋势。实验组给药后miR-122 表达水平下调,7 d 时降至最低点（下降了 81%）,之后上调;而 miR-155 的表达趋势与 miR-122 相反,7 d 时达到最 高点,是对照组的2.88倍。miR-122与ALT和AST呈正相关（rs分别为0.965、0.900,均P＜0.05）,miR-155与AST呈 负相关（rs=-0.811,P＜0.05）。结论 miR-122和miR-155参与了VPA药物性肝损伤的发生,有望成为VPA药物性肝 损伤的早期预警指标。     

Cationic lipid nanoparticles for therapeutic delivery of siRNA and miRNA to murine liver tumor

miR-122, a liver-specific tumor suppressor microRNA, is frequently down-regulated in hepatocellular carcinoma (HCC). LNP-DP1, a cationic lipid nanoparticle formulation, was developed as a vehicle to restore deregulated gene expression in HCC cells by miR-122 delivery. LNP-DP1 consists of 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), egg phosphatidylcholine, cholesterol and cholesterol-polyethylene glycol. In vitro, LNP-DP1-mediated transfection of a miR-122 mimic to HCC cells down-regulated miR-122 target genes by > 95%. In vivo, siRNAs/miRNAs encapsulated in LNP-DP1 were preferentially taken up by hepatocytes and tumor cells in a mouse HCC model. The miR-122 mimic in LNP-DP1 was functional in HCC cells without causing systemic toxicity. To demonstrate its therapeutic potential, LNP-DP1 encapsulating miR-122 mimic was intratumorally injected and resulted in ~ 50% growth suppression of HCC xenografts within 30 days, which correlated well with suppression of target genes and impairment of angiogenesis. These data demonstrate the potential of LNP-DP1-mediated microRNA delivery as a novel strategy for HCC therapy.From the Clinical EditorIn this study, LNP-DP1 –a cationic lipid nanoparticle formulation –is reported as a vehicle to restore deregulated gene expression in hepatic carcinoma cells by siRNA and miRNA delivery using a mouse model. Further expansions to this study may enable transition to clinical trials of this system.     

miR-224和 miR-378e在大肠癌组织中的表达及其临床意义

【摘要】 目的 探讨miR-224 和 miR-378e 在原位大肠癌癌组织和癌旁正常黏膜组织中的表达差异， 并分析其临床意义。 方法 miRNA 表达谱芯片技术检测大肠癌癌组织和癌旁组织标本中表达差异的miRNA， 运用荧光实时定量聚合酶链反应（real-time PCR）验证其结果，并分析其与临床病理资料之间的关系。 结果 miRNA 表达芯片分析发现， 与正常黏膜组织比较， miR-224在大肠癌组织中表达显著上调， miR-378e在大肠癌组织中表达显著下调， 并被 real-time PCR 所证实。 miR-224 在不同组织学类型癌组织中表达差异有统计学意义， miR-378e 在不同浸润深度癌组织中表达差异有统计学意义， miR-224 表达与组织学类型有关， miR-378e 表达与大肠癌浸润深度有关。 结论miR-224具有潜在的癌基因作用， miR-378e具有潜在的抑癌基因作用， miR-224和 miR-378e可作为     

MicroRNA-378-3p/5p represses proliferation and induces apoptosis of oral squamous carcinoma cells via targeting KLK4

Oral squamous cell carcinoma (OSCC) is one of the most common types of head and neck neoplasm. Down-regulation of hsa-microRNA-378 (miR-378) has been proved in OSCC tissues, suggesting that miR-378 might play crucial roles in the progression of OSCC. The present study aimed to evaluate the effect of miR-378-3p/5p on the proliferation and apoptosis of OSCC in vitro and in vivo. According to the results, lentivirus-mediated overexpression of miR-378 lowered the colony formation efficiency, blocked cell cycle progression, and decreased the percentage of Ki-67 positive cells, whereas knockdown of miR-378-3p/5p led to the opposite results. Furthermore, the apoptosis of OSCC cells was induced by the overexpression of miR-378 as evidenced by decreasing Bcl-2/Bax ratio, increasing cleaved caspase-9, cleaved caspase-3, and cleaved PARP levels, and promoting the release of cytochrome c into the cytoplasm. However, the above results were reversed by miR-378-3p/5p silencing. In addition, the overexpression of miR-378 inhibited the activation of PI3K/AKT signalling pathway. Conversely, miR-378-3p/5p knockdown resulted in the inactivation of PI3K/AKT signalling pathway. Mechanically, we validated that miR-378-3p/5p could target kallikrein-related peptidase 4 (KLK4), and enforced overexpression of KLK4 counteracted miR-378 overexpression-induced apoptosis. Finally, tumourigenesis in nude mice was suppressed by the overexpression of miR-378, which was promoted by miR-378-3p/5p silencing. Taken together, these results suggest that miR-378 may be a potential target in the diagnoses and treatment of OSCC.     

血浆中micro RNA-122表达量与肝癌手术前后肝损伤的相关性

目的研究血浆中micro RNA-122（miR-122）的表达量与肝癌手术前后肝损伤的相关性。方法利用荧光定量检测30名健康人与30例肝癌患者的术前miR-122及丙氨酸氨基转移酶（ALT）的表达量,并进行比较,且检测30例肝癌患者的术前,术后第1、3、7天的血浆中miR-122及ALT,探讨miR-122与ALT的相关性。结果术前肝癌患者血浆中miR-122以及ALT的表达量均显著高于健康人群（P〈0.05）。患者术前,术后第1、3、7天血浆中的miR-122均与ALT呈正相关。结论血浆miR-122与肝切除术肝功能损伤相关,有望成为肝癌肝切除术前后肝功能损伤的检测指标。     

核苷类药物与干扰素治疗时乙型病毒性肝炎患者血清miRNA的表达

目的检测核苷类药物治疗与干扰素治疗时乙型病毒性肝炎患者血清中micro RNA(miRNA)表达的变化,并通过统计分析研究其临床意义。方法选取50例患者,分别在用药治疗前、治疗中与治疗后检测血清中6种miRNAs(miR-122,miR-223,miR-26,miR-602,let-7a和let-7f)的表达差异。结果 miR-602,let-7a,let-7f的表达量随治疗时间延长而下降,且与耐药有显著关联。结论乙型病毒性肝炎患者血清中miR-602,let-7a,let-7f表达量的检测具有潜在的临床价值。     

Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release

Aim： Paeonol （2＇-hydroxy-4＇-methoxyacetophenone） from Cortex moutan root is a potential therapeutic agent for atherosclerosis. This study sought to investigate the mechanisms underlying anti-inflammatory effects of paeonol in rat vascular endothelial cells （VECs） in vitro.
Methods： VECs were isolated from rat thoracic aortas. The cells were pretreated with paeonol for 24 h, and then stimulated with ox-LDL for another 24 h. The expression of microRNA-21 （miR-21） and PTEN in VECs was analyzed using qRT-PCR. The expression of PTEN protein was detected by Western blotting. TNF-α release by VECs was measured by ELISA.

Results： Ox-LDL treatment inhibited VEC growth in dose- and time-dependent manners （the value of IC50 was about 20 mg/L at 24 h）. Furthermore, ox-LDL （20 mg/L） significantly increased miR-21 expression and inhibited the expression of PTEN, one of downstream target genes of miR-21 in VECs. In addition, ox-LDL （20 mg/L） significantly increased the release of TNF-α from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose- and time-dependent manners. Moreover, paeonol （120 μmol/L） prevented ox-LDL-induced increases in miR-21 expression and TNF-α release, and ox-LDL-induced inhibition in PTEN expression. A dual-luciferase reporter assay showed that miR-21 bound directly to PTEN＇s 3＇-UTR, thus inhibiting PTEN expression. In ox-LDL treated VECs, transfection with a miR-21 mimic significantly increased miR-21 expression and inhibited PTEN expression, and attenuated the protective effects of paeonol pretreatment, whereas transfection with an miR-21 inhibitor significantly decreased miR-21 expression and increased PTEN expression, thus enhanced the protective effects of paeonol pretreatment.

Conclusion： miR-21 is an important target of paeonol for its protective effects against ox-LDL-induced VEC injury, which may play critical roles in development of atherosclerosis.     

MiR-122 increases sensitivity of drug-resistant BEL-7402/5-FU cells to 5-fluorouracil via down-regulation of bcl-2 family proteins

To investigate the changes in drug sensitivity of miR-122 transfected BEL-7402/5-FU cells. MiR-122 and negative miRNA expression vectors were constructed and stably transfected into BEL-7402/5-FU cells. Real-time RT-PCR was used to detect the level of miR-122, Bcl-XL, Bcl-2 and P53 mRNA. Western Blotting was used to detect Bcl-2, Bcl-XL and P53 protein expression. Drug sensitivity of the cells to 5-fluorouracil (5-FU) was analyzed with MTT and flow cytometry. Compared with negative miRNA transfectants or untreated cells, mRNA and protein expression level of Bcl-2, Bcl-XL in stable miR-122 transfectants were decreased. Accordingly, P53 protein expression showed a significant up-regulation; MTT results showed that after incubation with 5-FU, miR-122 transfectants had higher cell inhibitory rates than negative miRNA or untreated cells; flow cytometry results demonstrated that apoptosis rate increased in miR-122 transfected cells, compared with negative miRNA or untreated cells. After addition of 5-FU (10 and 100 micromol/I), miR-122 transfected cells showed higher apoptosis rate than negative miRNA or untreated cells. MiR-122 can specifically down-regulate the expression of Bcl-2 and Bcl-XL, and increase P53 activity in BEL-7402/5-FU cells, which increased cells spontaneous apoptosis and sensitize cells to 5-FU. Therefore, MiR-122 can be used as a potential therapy agent against human hepatoblastoma.     

The Effect of Co-Administration of Death Camas (Zigadenus spp.) and Low Larkspur (Delphinium spp.) in Cattle

In many rangeland settings, there is more than one potential poisonous plant. Two poisonous plants that are often found growing simultaneously in the same location in North American rangelands are death camas (Zigadenus spp.) and low larkspur (Delphinium spp.). The objective of this study was to determine if co-administration of death camas would exacerbate the toxicity of low larkspur in cattle. Cattle dosed with 2.0 g of death camas/kg BW showed slight frothing and lethargy, whereas cattle dosed with both death camas and low larkspur showed increased clinical signs of poisoning. Although qualitative differences in clinical signs of intoxication in cattle co-treated with death camas and low larkspur were observed, there were not any significant quantitative differences in heart rate or exercise-induced muscle fatigue. Co-treatment with death camas and low larkspur did not affect the serum zygacine kinetics, however, there was a difference in the larkspur alkaloid kinetics in the co-exposure group. Overall, the results from this study suggest that co-exposure to death camas and low larkspur is not significantly more toxic to cattle than exposure to the plants individually. The results from this study increase our knowledge and understanding regarding the acute toxicity of death camas and low larkspur in cattle.     

药物肝损伤的潜在生物标志物循环miR-122的研究进展

药物诱导的肝损伤是药物研发失败或退市的主要原因之一,而传统的肝损伤评价指标因为种种缺陷如缺乏特异性或灵敏性而不能为药物的肝毒性评价提供早期、及时和可靠的信号。循环微RNA-122(miR-122)因其在肝脏特异性表达、以及其高度的稳定性和灵敏性成为目前肝毒性评价指标的研究热点。本综述主要从特异性、稳定性和灵敏性3方面系统地阐述循环miR-122成为肝毒性生物标志物的应用潜力,并对下一步的研究工作进行探讨。     

Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes

In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with ‘general metabolism’. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation.     

Polychlorinated biphenyl exposure alters the expression profile of microRNAs associated with vascular diseases

Exposure to persistent organic pollutants, including polychlorinated biphenyls (PCBs) is correlated with multiple vascular complications including endothelial cell dysfunction and atherosclerosis. PCB-induced activation of the vasculature subsequently leads to oxidative stress and induction of pro-inflammatory cytokines and adhesion proteins. Gene expression of these cytokines/proteins is known to be regulated by small, endogenous oligonucleotides known as microRNAs that interact with messenger RNA. MicroRNAs are an acknowledged component of the epigenome, but the role of environmentally-driven epigenetic changes such as toxicant-induced changes in microRNA profiles is currently understudied. The objective of this study was to determine the effects of PCB exposure on microRNA expression profile in primary human endothelial cells using the commercial PCB mixture Aroclor 1260. Samples were analyzed using Affymetrix GeneChip® miRNA 4.0 arrays for high throughput detection and selected microRNA gene expression was validated (RT-PCR). Microarray analysis identified 557 out of 6658 microRNAs that were changed with PCB exposure (p < 0.05). In-silico analysis using MetaCore database identified 21 of these microRNAs to be associated with vascular diseases. Further validation showed that Aroclor 1260 increased miR-21, miR-31, miR-126, miR-221 and miR-222 expression levels. Upregulated miR-21 has been reported in cardiac injury while miR-126 and miR-31 modulate inflammation. Our results demonstrated evidence of altered microRNA expression with PCB exposure, thus providing novel insights into mechanisms of PCB toxicity.     

Foamy macrophages in the liver of cattle fed Brachiaria brizantha hay

Liver and lymph nodes injuries characterized by clusters of foamy macrophages, some of them containing birefringent crystals, were observed in cattle fed on Brachiaria brizantha hay. The cattle were from an experimental group poisoned with Senecio brasiliensis known to cause hepatic fibrosis and hepatocyte megalocytosis. One of the animals developed photosensitivity but the exact cause wasn't determined since both plants were fed. The foamy macrophages were present from the 30th d of feeding. Early appearance of these lesions may be particular to the animal specie used or due to the presence of both toxic plants.     

Animals as Sentinels for Human Lead Exposure: A Case Report

Because human and nonhuman animals often share the same environment, there is potential concurrent exposure to toxicants. As a result, domestic animals can be used as sentinels for exposure of people to these agents. Here we present a case illustrating exposure of both humans and domestic animals to lead contamination in their environments. This case study occurred at a farm where cattle deaths were determined to have been caused by lead poisoning based on elevated postmortem tissue lead concentrations. Elevated blood lead concentrations were detected in the remaining cattle, a dog, a cat, and a pregnant woman (37.3 µg/dL) living on the farm. The range of blood lead concentrations in the domestic animals was 8.42 (cat) to 85.41 µg/dL (calf), although clinical signs of lead poisoning were not apparent in these animals. Further testing revealed the most likely source for lead exposure to be paint in the barn and home. Household dogs and cats have been used as sentinels for lead poisoning in humans, but cattle may also act as a sentinel species for environmental lead contamination.     

miR-21 regulates N-methyl-N-nitro-N′-nitrosoguanidine-induced gastric tumorigenesis by targeting FASLG and BTG2

MicroRNAs (miRNAs) are recently discovered regulators of gene expression and are important in the regulation of many cellular events. Evidence collected to date shows that miRNAs are altered after exposure to environmental toxicants. However, the role that miR-21 plays in the gastric tumorigenesis induced by environmental carcinogens remains largely unknown. The aim of this study was to characterize the regulatory role of miR-21 in the carcinogenic processes following exposure to the N-nitroso carcinogen N-methyl-N-nitro-N′-nitrosoguanidine (MNNG). We found a progressive dose- and time-dependent increase in miR-21 expression following treatment with MNNG. Dysregulated miR-21 affected both cell growth in GES-1 cells and the gastric tumorigenesis induced with MNNG. These data demonstrate the involvement of miR-21 in the malignant transformation and tumorigenesis activated by MNNG. We also established that the Fas ligand (FASLG) and B-cell translocation gene 2 (BTG2), regulated by miR-21, contribute to the transformation induced by MNNG in GES-1 cells. This is the first study to show that miR-21 is involved in chemical carcinogenesis in vivo and in vitro. The regulation by miR-21 of the gastric carcinogenesis induced by MNNG highlights the functional roles of miRNAs in chemical carcinogenesis, and offers a new explanation of the mechanisms underlying chemical carcinogenesis.     

Regulation of autophagy by miR-30d impacts sensitivity of anaplastic thyroid carcinoma to cisplatin

miR-30d has been observed to be significantly down-regulated in human anaplastic thyroid carcinoma (ATC), and is believed to be an important event in thyroid cell transformation. In this study, we found that miR-30d has a critical role in modulating sensitivity of ATC cells to cisplatin, a commonly used chemotherapeutic drug for treatment of this neoplasm. Using a mimic of miR-30d, we demonstrated that miR-30d could negatively regulate the expression of beclin 1, a key autophagy gene, leading to suppression of the cisplatin-activated autophagic response that protects ATC cells from apoptosis. A reporter gene assay demonstrated that the binding sequences of miR-30d in the beclin 1-3′ UTR was the region required for the inhibition of beclin 1 expression by this miRNA. We further showed that inhibition of the beclin 1-mediated autophagy by the miR-30d mimic sensitized ATC cells to cisplatin both in vitro (cell culture) and in vivo (animal xenograft model). These results suggest that dysregulation of miR-30d in ATC cells is responsible for the insensitivity to cisplatin by promoting autophagic survival. Thus, miR-30d may be exploited as a potential target for therapeutic intervention in the treatment of ATC.     

Genome-wide miRNA expression profiling of human lymphoblastoid cell lines identifies tentative SSRI antidepressant response biomarkers

Aim: Over 30% of patients with major depression do not respond well to first-line treatment with selective serotonin reuptake inhibitors (SSRIs). Using genome-wide expression profiling of human lymphoblastoid cell lines (LCLs) CHL1 was identified as a tentative SSRI sensitivity biomarker. This study reports on miRNAs implicated in SSRI sensitivity of LCLs. Methods: Eighty LCLs were screened from healthy adult female individuals for growth inhibition by paroxetine. Eight LCLs exhibiting high or low sensitivities to paroxetine were chosen for genome-wide expression profiling with miRNA microarrays. Results: The miRNA miR-151-3p had 6.7-fold higher basal expression in paroxetine-sensitive LCLs. This corresponds with lower expression of CHL1, a target of miR-151-3p. The additional miRNAs miR-212, miR-132, miR-30b*, let-7b and let-7c also differed by >1.5-fold (p < 0.05) between the two LCL groups. Conclusion: The potential value of these miRNAs as tentative SSRI response biomarkers awaits validation with lymphocyte samples of major depression patients. Original submitted 28 March 2012; Revision submitted 21 May 2012.