miR-126 regulated breast cancer cell invasion by targeting ADAM9

Accumulating evidence has shown that microRNAs (miRNAs) deregulation is commonly observed in human malignancies and crucial to cancer metastasis. Herein, we demonstrated that miR-126 play a suppressor role in human breast cancer cells invasion through the direct repression of a disintegrin and metalloprotease 9 (ADAM9). MiR-126 expression was investigated in forty cases of breast cancer specimens by real-time PCR. Transwell assay was conducted to explore the effects of miR-126 on the invasion of human breast cancer cell lines. The impact of miR-126 overexpression on putative target ADAM9 was subsequently confirmed by Western blot analysis. Our results indicated that miR-126 expression was frequently down-regulated in breast cancer specimens compared with adjacent normal tissues (P<0.05). Overexpression of miR-126 significantly reduced (P<0.05) the protein levels of ADAM9, further suppressed (P<0.05) breast cancer cell invasion in vitro. Meanwhile, knockdown of ADAM9 by small interfering RNA (siRNA) also inhibited (P<0.05) breast cancer cell invasion. Thus, our study revealed that miR-126 may act as a tumor suppressor via inhibition of cell invasion by downregulating ADAM9 in breast cancer development.     

Role of miR-101 in pheochromocytoma patients with SDHD mutation

This study aimed to screen the potential diagnostic biomarkers for distinguishing the malignant pheochromocytoma (PCC) from benign PCC. A total of 59 patients with PCC (benign and malignant) were enrolled in this study. The expression level of miRNAs in patients with different kind PCCs (healthy control, benign, malignant, malignant with or without SDHD mutation, adrenal and extra-adrenal) was analyzed using the qRT-PCR analysis. Besides, the diagnosis accuracy of miRNA in PCC samples was analyzed using the ROC analysis. Moreover, level of miR-101 in serum was detected by qRT-PCR analysis and serum VEGF level in patients with PCC was detected using the ELISA kit. Compared with benign PCC, miR-101 level was higher in patients with malignant PCC (P < 0.05), while the level of miR-513-5p and miR-26b showed no difference between malignant PCC and benign PCC (P > 0.05). miR-101 expression was significantly increased in malignant tumor tissue with SDHD mutation (P < 0.05) and in extra-adrenal tissues (P < 0.05), respectively. Besides, AUCs for miR-101 in PCC samples was 0.79 and for which in PCC samples with non-SDHD mutation was 0.77. Besides, serum miR-101 in malignant PCC was high but showed no difference among groups (P > 0.05). Moreover, serum VEGF level in malignant tumors was significantly high compared with benign tumor, as well as that in malignant PCC with SDHD mutation (P < 0.05). Our study suggested that SDHD mutation may enhance the overexpression of miR-101 in malignant tumors and miR-101 may be a potential diagnostic biomarker for malignant PCC and benign PCC.     

Interaction Between Serum/Glucocorticoid-Regulated Kinase 1 and Interleukin-6 in Chronic Rhinosinusitis

PurposeSerum/glucocorticoid-regulated kinase 1 (SGK1) has recently emerged as a critical regulator of inflammatory diseases. In this study, we examined SGK1 expression and its possible pathogenic roles in chronic rhinosinusitis (CRS).MethodsImmunohistochemistry, western blotting, Bio-Plex assay, enzyme-linked immunosorbent assays, and quantitative real-time polymerase chain reaction were performed to assess protein and gene expression levels. The mRNA expression levels of SGK1 and interleukin-6 (IL-6) were extracted from a CRS database to perform correlation analysis. Stable cell lines with SGK1 overexpression (16HBE) and knockdown (A549) were constructed to investigate the interaction between SGK1 and IL-6 in vitro.ResultsSGK1 exhibited strong cytoplasmic and nuclear staining in the epithelial layers and the lamina propria of nasal polyps (NPs) and in the mucosal tissues of CRS without nasal polyps (CRSsNP). The mRNA and protein expression levels of SGK1 and IL-6 were significantly increased in NPs and CRSsNP tissues, compared to control tissues. SGK1 phosphorylation was significantly greater in NPs than in CRSsNP tissues (P < 0.01). The mRNA levels of SGK1 and IL-6 were significantly correlated (P < 0.001, r = 0.649). Exposure to IL-6 significantly increased SGK1 expression in cultured dispersed NP cells, 16HBE cells, and A549 cells. IL-6 expression was significantly down-regulated in SGK1-overexpressing 16HBE cells (P < 0.01) and significantly up-regulated in SGK1-knockdown A549 cells (P < 0.05). Administration of GSK650394, a SGK1 inhibitor, significantly increased IL-6 self-induced mRNA expression in cultured dispersed NP cells and 16HBE cells.ConclusionsThe interaction between SGK1 and IL-6 may play an anti-inflammatory role in IL-6-induced inflammation in the pathogenesis of CRS.     

Antinociceptive Effects of Amiloride and Benzamil in Neuropathic Pain Model Rats

Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in neuropathic pain has not been investigated. In this study, we investigated the effect of the intrathecal amiloride and benzamil in neuropathic pain model, and also examined the role of ASICs on modulation of neuropathic pain. Neuropathic pain was induced by L4-5 spinal nerve ligation in male Sprague-Dawley rats weighing 100-120 g, and intrathecal catheterization was performed for drug administration. The effects of amiloride and benzamil were measured by the paw-withdrawal threshold to a mechanical stimulus using the up and down method. The expression of ASICs in the spinal cord dorsal horn was also analyzed by RT-PCR. Intrathecal amiloride and benzamil significantly increased the paw withdrawal threshold in spinal nerve-ligated rats (87%±12% and 76%±14%, P=0.007 and 0.012 vs vehicle, respectively). Spinal nerve ligation increased the expression of ASIC3 in the spinal cord dorsal horn (P=0.01), and this increase was inhibited by both amiloride and benzamil (P<0.001 in both). In conclusion, intrathecal amiloride and benzamil display antinociceptive effects in the rat spinal nerve ligation model suggesting they may present an alternative pharmacological tool in the management of neuropathic pain at the spinal level.     

Down-regulation of nectin-4 inhibits apoptosis in systemic lupus erythematous (SLE) through targeting Bcl-2/Bax pathway

Purpose: The purpose of this study was to investigate the potential role of nectin-4 in systemic lupus erythematous (SLE) cell apoptosis during the disease development and its potential mechanism. Methods: Human peripheral blood mononuclear cells (PBMCs) were obtained for the isolation of monocytes and T lymphocytes. siRNA-nectin-4 plasma was constructed for the transfection into T cells using Lipofectamine 2000 reagent. siRNA with no nectin-4 sequence was transfected into T cells for the control group. mRNA expression of nectin-4 in cells was analyzed using RT-PCR method. Effect of netin-4 expression on T cell apoptosis was analyzed with Annexin V-FITC cell apoptosis kit. Moreover, effects of nectin-4 expression on cell apoptotic-related proteins expressions were detected using western blotting analysis. Results: Nectin-4 was significantly overexpressed in cells from SLE group compared with healthy control (HC) group (P<0.05). When T cells were transfected with sinectin-4, nectin-4 slicing increased cell apoptosis in HC group but significantly decreased apoptosis in SLE group (P<0.05). Nectin-4 slicing significantly decreased CD40L and CD17 expressions in SLE (P<0.05), but performed no effect on CD11a expression. Moreover, nectin-4 down-regulation could significantly decrease Bcl-2, Bcl-XL, and caspase-6 expressions but increase Bax level in SLE group. Conclusion: The data presented in this study suggested that nectin-4 may be a therapeutic target for SLE through affecting the cell apoptosis.     

Expression of miR-32 in human non-small cell lung cancer and its correlation with tumor progression and patient survival

Introduction: miR-32 has recently been found to be implicated in many critical processes in various types of human cancer. However, its clinical significance in human non-small cell lung cancer (NSCLC) has not yet been elucidated. In the present study, we investigated the expression of miR-32 in NSCLC and analyzed its association with clinical features and prognosis of NSCLC patients. Methods: Quantitative real-time PCR (qRT-PCR) was used to measure expression level of miR-32 in lung cancer cell lines, normal bronchial epithelial cells, 90 pairs of tumor samples and adjacent non-tumor tissues. To determine its prognostic value, overall survival was evaluated using the Kaplan-Meier method. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis. Results: The expression of miR-32 was significantly decreased in lung cancer cell lines and NSCLC tissues compared with normal bronchial epithelial cells and adjacent non-tumor tissues (P < 0.05). This reduction of miR-32 was associated with tumor stage and lymph node metastasis (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with low miR-32 expression had shorter overall survival time than those with high miR-32 expression (P < 0.05). Univariate analysis revealed statistically significant correlations between overall survival and miR-32 level, tumor stage and lymph node metastasis (P < 0.05). Furthermore, miR-32 levels, tumor stage and lymph node metastasis were independently associated with overall survival (P < 0.05). Conclusions: Our results provided the first evidence that down-regulation of miR-32 was correlated with NSCLC progression, and miR-32 might be a potential molecular biomarker for predicting the prognosis of patients.     

Overexpression of miR-221 inhibits proliferation and promotes apoptosis of human astrocytoma cells

microRNAs (miRNAs) play tumor-promoting roles in a variety of tumors. This study investigated the expression of miRNA-211 (miR-221) in human astrocytoma, and its effect on proliferation and apoptosis of human astrocytoma cells in vitro. miR-221 expression was detected in 10 astrocytoma tissues and 4 adjacent tissues by real-time quantitative PCR (qRT-PCR). miR-221 expression in situ was significantly higher in astrocytoma tissues than in adjacent tissues (P<0.05). To determine whether the upregulation of miR-221 could be associated with tumor development or progression, a synthetic miR-221 mimic was transiently transfected into U251 astrocytoma cells in vitro. qRT-PCR confirmed that the mimic significantly increased the expression of miR-221 in these cells. An MTT colorimetric assay indicated that proliferation was significantly higher in U251 cells transfected with miR-221 mimic than in scramble-transfected control cells (P<0.05). Further analysis of miR-221 transfected cells by flow cytometry revealed an altered cell cycle progression, with more cells in S and G1 phase, as well as an inhibition of apoptosis (P<0.05). These findings indicate that the upregulation of miR-221 in astrocytoma tissues may be associated with development or progression of these tumors. Thus, miR-221 should be explored as a potential molecular marker for the diagnosis and treatment of astrocytoma.     

Down-regulation of microRNA-124 is correlated with tumor metastasis and poor prognosis in patients with lung cancer

Introduction: MicroRNA-124 (miR-124) has been proven dysregulated in several human malignancies and correlated with tumor progression. However, its expression and clinical significance in non-small cell lung cancer (NSCLC) is still unclear. Thus, the aim of this study was to investigate the clinical significance of miR-124 expression in NSCLC. Methods: Expression levels of miR-124 in 92 pairs of NSCLC and adjacent non-tumor tissues were detected by quantitative real-time PCR (qRT-PCR). In order to determine its prognostic value, overall survival (OS) and disease-free survival (DFS) were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. Results: miR-124 expression level was significantly lower in NSCLC tissues compared with adjacent non-tumor tissues (P < 0.05). The 5-year OS of low miR-124 expression group was significantly shorter than that of high miR-124 expression group (P < 0.05). Moreover, the 5-year DFS of low miR-124 expression group was also significantly shorter than that of high miR-124 expression group (P < 0.05). In a multivariate Cox model, we found that miR-124 expression was an independent prognostic factor for both 5-year OS and 5-year DFS in NSCLC (P < 0.05). Conclusions: Our results offer the convincing evidence that miR-124 may play key roles in the progression of lung cancer and that the down-regulated expression of miR-124 may be independently associated with shorter OS and DFS of patients, suggesting that miR-124 might be a potential marker for further risk stratification in the treatment of lung cancer.     

穴位电针对慢性神经病理性疼痛大鼠脊髓前列腺酸性磷酸酶表达的影响

目的 观察电针对神经病理性痛大鼠脊髓前列腺酸性磷酸酶（PAP）的表达,及腺苷含量及大鼠痛阈变化的影响,探讨电针镇痛机制。方法 21只雄性SD大鼠采用慢性坐骨神经压缩模型(CCI),取“足三里”及“太冲”穴。设假手术组（sham组）、模型组(CCI组）和电针治疗组（EA组）。采用行为学检测大鼠术前、术后第7天和电针治疗后大鼠机械痛敏值和热敏痛阈值变化,采用Real-time PCR技术检测脊髓PAP mRNA的表达,高效液相色谱法检测脊髓腺苷含量。结果 相比CCI组大鼠,电针组大鼠机械痛敏值和热敏痛阈值明显提高（P<0.05, P<0.05）。电针组大鼠脊髓PAP mRNA表达和腺苷含量比CCI组大鼠显著增加（P<0.05）。与CCI组大鼠相比,电针组大鼠脊髓腺苷含量增加（P<0.01）。结论 电针可提高神经病理性痛大鼠机械痛阈值和热敏痛阈值,其机制可能是通过增加脊髓PAP mRNA的表达和腺苷含量发挥作用。     

Roles of the miR-139-5p/CCT5 axis in hepatocellular carcinoma: a bioinformatic analysis

Background: MiRNAs are pivotal regulators involved in proliferation, apoptosis, invasion, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, drug resistance and autophagy in hepatocellular carcinoma (HCC). The aim of this study was to investigate the influence of miR-139-5p and its target genes on the outcomes of HCC.Methods: Survival analysis of miR-139-5p in HCC was conducted in Kaplan-Meier plotter. Target genes of miR-139-5p were identified in TargetScan, miRTarBase and starBase. Gene Expression Omnibus (GEO) series were used for the validation of miR-139-5p target genes. Cox proportional regression model was also established.Results: In Kaplan-Meier plotter, 163 HCC patients were included. MiR-139-5p downregulation was significantly associated with unfavorable overall survival (OS) and disease-free survival (DFS) in HCC patients (all P < 0.001). MiR-139-5p was significantly downregulated in HCC tumors and human hepatoma cell lines (all P < 0.05). As a target gene of miR-139-5p, CCT5 was overexpressed in HCC tumor tissues and peripheral blood mononuclear cells (all P < 0.05). A negative correlation between CCT5 and miR-139-5p was found in TCGA dataset. CCT5 overexpression was significantly associated with worse OS in HCC patients (P < 0.001), which was validated in the {"type":"entrez-geo","attrs":{"text":"GSE14520","term_id":"14520"}}GSE14520 dataset (P = 0.017). CCT5 mRNA was significantly overexpressed in HCC patients with alpha-fetoprotein (AFP) > 300 ng/ml, BCLC staging B-C, TNM staging III and main tumor size > 5 cm (all P < 0.05). According to the Cox regression model of CCT5-interacting genes, HCC patients with high risk had poor OS compared to those with low risk in the TCGA dataset (P < 0.001), with the 1-year, 3-year, and 5-year ROC curves of an area under the curve (AUC) equal to 0.704, 0.662, and 0.631, respectively.Conclusions: MiR-139-5p suppresses HCC tumor aggression and conversely correlated with CCT5. The miR-139-5p/CCT5 axis might perform crucial functions in the development of HCC.     

MiR-139-3p通过靶向下调TRPV1缓解大鼠神经病理性疼痛

目的:研究miR-139-3p在大鼠神经病理性疼痛发生发展中的作用及其作用机制。方法:将大鼠随机分为假手术组(sham)、坐骨神经慢性压迫损伤模型组(chronic constriction injury group,CCI)、2 mg/kg miR-139-3p模拟物(mimic)鞘内注射组、4 mg/kg miR-139-3p mimic鞘内注射组。术后的第14 d,Real-time PCR法检测大鼠背根神经节(DRG)中miR-139-3p及TRPV1的表达。于注射前及注射后的第1、3、7、14 d,利用von Frey检测大鼠机械性痛阈及热辐射法检测大鼠热痛阈值。Western Blot法检测DRG中TRPV1的表达;ELISA法检测脊髓L4-L5组织中TNF-α及IL-1β的含量;双荧光素酶报告基因法检测miR-139-3p与TRPV1的靶向关系。结果:(1)与sham组相比较,miR-139-3p在CCI大鼠DRG中的表达显著下降,而TRPV1的表达显著上升(P0.05)。(2)与sham组相比较,CCI组中大鼠机械刺激缩足反射阈值及热刺激缩足反射潜伏期显著下降(P0.05);而与CCI组相比较,鞘内注射miR-139-3p mimic可显著促进大鼠机械刺激缩足反射阈值及热刺激缩足反射潜伏期的上升,抑制TRPV1的表达(P0.05)。(3)与sham组相比较,CCI组大鼠TNF-α及IL-1β含量显著上升(P0.05);而与CCI组相比较,鞘内注射miR-139-3p mimic可显著抑制TNF-α及IL-1β的产生(P0.05)。(4)miR-139-3p mimic转染可显著降低荧光素酶报告基因的荧光强度。结论:miR-139-3p通过靶向下调TRPV1的表达缓解大鼠神经病理性疼痛。     

Inhibitory effects of intrathecal p38β antisense oligonucleotide on bone cancer pain in rats

Objective: To evaluate the effects of intrathecal administration p38β antisense oligonucleotide on the development of bone cancer pain rats. Methods: Forty female SD rats weighing 180~220 g were randomly divided into 4 groups (n = 10 each): Group A (control group): intra-tibial injection of 3 μl Hank’s solution; group B (model group): intra-tibial injection of 3 μl MADB-106 mammary gland carcinoma cells of rats (4.8 × 10³/μl); group C (p38β-SODN 20 μg); group D (p38β-ASODN 20 μg). The model procedures in group C and D were same to those in the group B. From the 14^th day after operation, p38β-SODN 20 μg and p38β-ASODN 20 μg were respectively intrathecally administrated in group C and D once daily for 6 days whereas normal saline was for group A and B. Mechanical withdrawal threshold and radiant heat threshold of rat hind paws were measured before operation and every other day until 22 d of post-operation. The lumbar 4-6 spinal cord was removed on the 22^nd day. The expression of spinal p38β protein was determined by Western blot. Results: No significant differences in mechanical withdrawal threshold and radiant heat threshold were found at all time points in control group. During the first 6 days after operation there were obvious differences in radiant heat stimulus between control group between the other groups (P < 0.05); During 14-22 days after operation, mechanical pain threshold and radiant heat threshold between p38β-SODN group and Model group were significantly changed compared with that in control group (P < 0.05). However, the differences were not remarkable between control group and p38β-ASODN group (P > 0.05). The expression of p38β protein in lumbar spinal cord was significantly higher between p38β-SODN group and Model group than that in control group (P < 0.05). There was no significant difference in p38β protein expression between p38β-ASODN group and control group (P > 0.05). Conclusions: Hyperalgesia induced by bone cancer can be inhibited by intrathecal administration of p38β antisense oligonucleotide, which is achieved by reducing expression of p38β protein.     

MiR-19a targets suppressor of cytokine signaling 1 to modulate the progression of neuropathic pain

Purpose: we aimed to investigate whether miR-19a is associated with neuropathic pain and elucidate the underlying regulatory mechanism. Methods: We established a neuropathic pain model of bilateral chronic constriction injury (bCCI). Then bCCI rats were injected with mo-miR-19a, siR-SOCS1 or blank expression vector through a microinjection syringe via an intrathecal catheter on 3 day before surgery and after surgery. Behavioral tests, such as mechanical allodynia, thermal hyperalgesia and acetone induced cold allodynia, were performed to evaluate the pain threshold. Besides, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the expression of miR-19a and western blotting was carried out to measure the expression of SOCS1. Results: miR-19a expression levels were markedly increased in neuropathic pain models. Moreover, miR-19a significantly attenuated mechanical allodynia and thermal hyperalgesia, and similar results were obtained after knockdown of SOCS1 expression. However, miR-19a markedly increased the times that the rats appeared a sign of cold allodynia, and knockdown of SOCS1 expression had similar effects. Besides, the results of bioinformatics analysis and western blotting analysis were all confirmed that SOCS1 was a direct target of miR-19a in neuropathic pain models. Conclusions: Our finding indicate that SOCS1 is a direct target of miR-19a in neuropathic pain rats and miR-19a may play a critical role in regulating of neuropathic pain via targeting SOCS1.     

The Expression of MicroRNA-155 in Plasma and Tissue Is Matched in Human Laryngeal Squamous Cell Carcinoma

PurposeTumor-associated microRNAs have been detected in cancer, though whether plasma microRNA-155 (miR-155) could be a potential biomarker for laryngeal squamous cell carcinoma (LSCC) prognosis is unclear. We aimed to determine how miR-155 can be used to predict the clinical characteristics of patients with LSCC and correctly diagnose them.ResultsA total of 280 LSCC patients and 560 age- and sex-matched controls were included in the study. The miR-155 level was more up-regulated in LSCC tissue than in the non-tumor tissues (13.6±2.4 vs. 3.1±0.80, p<0.001). Additionally, a significantly higher miR-155 level in plasma samples from LSCC patients than in those of the controls (8.9±1.25 vs. 1.8±0.8, p<0.001) was reported. Tissue miR-155 showed an area under the curve (AUC) of 0.933, with a sensitivity of 82.6% and a specificity of 89.2%. The AUC for plasma miR-155 was 0.757, with a sensitivity of 58.4% and a specificity of 69.5%. When early LSCC in TNM I stage was considered, tissue miR-155 showed an area under the curve of 0.804, with a sensitivity of 85.2% and a specificity of 87.3%.ConclusionThe expression of tissue and plasma miR-155 were significantly up-regulated in patients with LSCC. Our work will serve as a basis for further investigation, preferably large-scale validation in clinical trials.