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.     

Targeting miR-21 with AS-miR-21 suppresses aggressive growth of human tongue squamous cell carcinoma in vivo

MicroRNAs (miRNAs) are involved in many human malignant tumors. Notably, miR-21 was identified to contribute to tumorigenicity. To investigate the repressive effect of targeting miR-21 with AS-miR-21 on proliferation of tongue squamous cell carcinoma (TSCC). We established the Tca8113-luc cell line with stable luciferase expression using pGL6-luciferase (pGL6-luc) plasmid transfection. TSCC xenograft models were characterized by high tumorigenicity rate and stable growth. Intratumor injection of Oligofectamine™-mediated AS-miR-21 significantly inhibited TSCC growth. The suppression of malignant phenotype was also accompanied by decreased photon signals, rare necrosis foci, smaller nucleuses, weakly stained nucleuses, atypia reversal and tumor angiogenesis reduction. Additionally, miR-21 expression was markedly decreased in TSCC xenografts and the apoptotic index was increased. Intratumor injection of AS-miR-21 into TSCC xenografts could reduce expression of miR-21, promote apoptosis of TSCC cells and inhibit TSCC proliferation.     

Radiotherapy suppressed tumor-specific recruitment of regulator T cells via up-regulating microR-545 in Lewis lung carcinoma cells

Objective: Radiotherapy is an important treatment for cancer. The main irradiated action is thought to be the irreversible damage to tumor cell DNA, but recent studies showed that high dose radiotherapy related to the tumor immune response. This study was designed to determine the relationship between Lewis lung tumor radiosensitivity and CD4+CD25+ regulatory T cells (Tregs) infiltration and elucidate the underlying mechanisms in vitro. Methods: With tumor transplantation method to establish mice Lewis lung tumor mice model, to observe the inhibition rate of radiotherapy to tumor growth. Proliferation profiles of CD4+CD25+ Tregs and CD4+ T cells were assessed by flow cytometry. MiR-545 and CCL-22 mRNA were determined by Quantitative Real-Time PCR. CCL-22 protein was determined by western blot assay. Results: Radiotherapy caused a time-dependent inhibition of tumor growth as well as a decrease in the percentage of tumor-infiltrating CD4+CD25+ Tregs of CD4+ T cells compared with no treatment group. And the miR-545 was significantly upregulated and CCL-22 was significantly down-regulated in irradiated tumor and Lewis lung cancer cells. In Lewis lung cancer cell transfection experiments, mimic or inhibitor for miR-545 negatively regulated CCL-22 expression when cells treated or treated without irradiation. Silenced miR-545 promotes CD4+CD25+ Treg proliferation. Additionally, silenced miR-545 reversed radiosensitivity of Lewis lung cancer. Conclusion: Radiotherapy suppressed specific recruitment of regulator CD4+CD25+ Treg cells in Lewis lung carcinoma via up-regulating microR-545.     

Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts

Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1 and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.     

Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts

Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.     

MiR-191 inhibits TNF-α induced apoptosis of ovarian endometriosis and endometrioid carcinoma cells by targeting DAPK1

Emerging evidence showed that miRNA dysregulation is involved in the development of endometriosis and may contribute to pathological process of endometriosis associated ovarian cancer (EAOC). miR-191 is one of the most differentially expressed miRNAs in pairwise comparisons among healthy controls, endometriosis, and EAOC patients. However, its regulative network in endometriosis and EAOC are still not clear. This study explored the role of miR-191 in TNF-α induced cell death in ovarian endometriosis and endometrioid carcinoma cells. Based on tissues samples collected from healthy controls, endometriosis, and EAOC patients, this study verified significantly higher expression of miR-191 in endometriosis and endometrioid cancer. Interestingly, we also observed inverse expression trend between miR-191 and DAPK1, a positive mediator of programmed cell death. By conducting luciferase assay, we confirmed miR-191 can directly target DAPK1 and regulate its expression. Functionally, we also found DAPK1 can promote TNF-α induced cell death. DAPK1 knockdown in endometriosis CRL-7566 cells can weaken its response to TNF-α induced cell death, while its overexpression in endometrioid cancer cells CRL-11731 enhanced the response. These functions of DAPK1 can be directly modulated by miR-191. Therefore, the miR-191-DAPK1 axis may play an important role modulating the response of ovarian endometriosis and endometrioid carcinoma cells to death-inducers and might contribute malignant transformation of endometriosis.     

Augmented miR-10b expression associated with depressed expression of its target gene KLF4 involved in gastric carcinoma

Previous studies have revealed several targets of miR-10b, such as syndecan-1, HOXD10, TBX5, and E-cadherin. In this study, we aimed to assess whether Krüppel-like factor 4 (KLF4) is a target gene of miR-10b in gastric cancer (GC). Targeting of KLF4 by miR-10b was confirmed by dual-luciferase reporter assays. The expression levels of miR-10b and KLF4 mRNA in 5 different gastric cancer cell lines and 65 pairs of gastric cancer tissues were detected by Real-time PCR. In addition, KLF4 protein in gastric cancer cell lines and 30 GC tissues was measured by western blotting and immunochemistry, respectively. KLF4 is a direct target gene of miR-10b in GC, and its expression is reduced by miR-10b at both mRNA and protein levels. In addition, the expression level of miR-10b was tendentiously upregulated in GC tissues while the expression levels of KLF4 mRNA and protein were decreased in gastric cancer tissues compared with normal adjacent tissue. There was a dramatically inverse correlation between the expression levels of miR-10b and KLF4 mRNA in GC (r = -0.339, P = 0.006). These findings indicate that miR-10b was upregulated in GC and may have a key role in GC pathogenesis and development through the downregulation of its target gene KLF4.     

HPV-16 E6 promotes cell growth of esophageal cancer via downregulation of miR-125b and activation of Wnt/β-catenin signaling pathway

High-risk human papillomavirus (HPV) is a possible cause of esophageal cancer. However, the molecular pathogenesis of HPV-infected esophageal cancer remains unclear. The expression levels of some microRNAs including miR-125b have been negatively correlated with HPV infection, and miR-125b downregulation is associated with tumorigenesis. In addition, Wnt/β-catenin signaling pathway has been suggested to play an important role in esophageal cancer (EC). We examined miR-125b and Wnt/β-catenin signaling pathway in HPV-16 E6 promoted tumor progression in EC. HPV-16 E6 transfection decreased markedly the expression levels of miR-125b and promoted the colony formation in the Eca 109 and Kyse 150 cell lines, and restoration of miR-125b expression level antagonized the increased colony formation in HPV-16 E6 transfected cell lines. We also demonstrated that overexpression of E6 upregulated the Wnt/β-catenin signaling activity via modulating the multiple regulators including TLE1, GSK3β, and sFRP4. Overexpression of miR-125b restored the expression levels of these proteins. Expression of miR-125b was lower in HPV-16 E6 positive esophageal cancer tissues, and was negatively correlated with E6 mRNA levels. Our results indicate that HPV-16 E6 promotes tumorigenesis in EC via down-regulation of miR-125b, and this underlying mechanism may be involved in the activation of the Wnt/β-catenin signaling pathway.     

miR-200a和miR-155在狼疮性肾炎患者肾组织中的表达及其作用机制探讨

目的：探讨miR-200a及miR-155在狼疮性肾炎(LN)患者肾组织中的表达及其在LN发病机制中的作用。方法通过实时荧光定量聚合酶链反应测定30例LN患者和15例健康对照组肾组织中miR-200a及miR-155的表达水平。结果 LN患者肾组织中miR-155的表达水平高于健康对照组,miR-200a的表达水平低于健康对照组,差异均具有统计学意义(P<0.01)。LN患者肾组织中miR-200a的表达水平与狼疮评分呈负相关(r=-0.391,P=0.032),miR-200a的表达水平与尿蛋白呈负相关(r=-0.395,P=0.034)。miR-155与狼疮评分呈正相关(r=0.593,P=0.001),miR-155表达水平与尿蛋白呈正相关(r=0.375,P=0.045),miR-155的表达水平与eGFR呈负相关（r=-0.59,P=0.001）。miR-200a的表达水平与eGFR之间无明显相关性(r=0.22,P=0.242)。结论肾组织中miR-155和miR-200a在狼疮性肾炎患者与健康对照组之间的表达水平有差异,且它们的表达水平与蛋白尿、肾功能及狼疮评分均相关。提示LN患者组织中miR-200a与miR-155的表达与LN患者肾功能恶化和狼疮疾病活动程度密切相关,可作为预测LN进展的重要标志物。     

miR-212 在乳腺浸润性导管癌 组织中的表达及效应分析

目的：分析miR-212在乳腺浸润性导管癌组织中的表达及与乳腺癌临床病理特征之间的相关性,并分析其效应。方法采用茎环RT- qPCR方法检测38例乳腺癌及癌旁正常乳腺组织标本中miR-212的表达,统计分析其表达水平与乳腺癌常用临床病理指标间的关系。不同浓度miR-212 inhibitor转染MCF7乳腺癌细胞,通过MTT法分析细胞活性,通过Tran- swel 实验检测细胞的侵袭能力。结果与癌旁正常乳腺组织比较,乳腺癌组织中miR-212的表达明显升高（P＜0.05）;分析miR-212表达水平与乳腺癌常用临床病理指标间的关系发现,miR-212表达水平与乳腺癌细胞与淋巴结转移、TNM分期相关及增殖指数（ki67）相关（P＜0.05）,与月经状况、肿块大小、雌激素和孕激素受体状态、Her-2表达未见显著相关性（P＞0.05）。细胞干预实验显示,与空白对照组、阴性对照组比较,miR-212 inhibitor可明显抑制MCF7细胞增殖活性（P＜0.05）和细胞迁移（P＜0.01）。结论乳腺癌组织高表达的miR-212在乳腺癌发展过程中发挥重要作用,有望成为乳腺癌防治的一个新靶点。