MiR-205 determines the radioresistance of human nasopharyngeal carcinoma by directly targeting PTEN

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance severely reduces NPC radiocurability. Here, we have established a radio-resistant NPC cell line, CNE-2R, and investigate the role of miRNAs in radioresistance. The miRNAs microarray assay reveals that miRNAs are differentially expressed between CNE-2R and its parental cell line CNE-2. We find that miR-205 is elevated in CNE-2R. A target prediction algorithm suggests that miR-205 regulates expression of PTEN, a tumor-suppressor. Introducing miR-205 into CNE-2 cells suppresses PTEN protein expression, followed by activation of AKT, increased number of foci formation and reduction of cell apoptosis post-irradiation. On the other hand, knocking down miR-205 in CNE-2R cells compromises the inhibition of PTEN and increases cell apoptosis. Significantly, immunohistochemistry studies demonstrate that PTEN is downregulated at late stages of NPC, and that miR-205 is significantly elevated followed the radiotherapy. Our data conclude that miR-205 contributes to radioresistance of NPC by directly targeting PTEN. Both miR-205 and PTEN are potential predictive biomarkers for radiosensitivity of NPC and may serve as targets for achieve successful radiotherapy in NPC.Key words: nasopharyngeal carcinoma, PTEN, miRNA, miR-205, radioresistance     

miR-200a Regulates Epithelial-Mesenchymal to Stem-like Transition via ZEB2 and β-Catenin Signaling

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133⁺ side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.     

Circulating miR-17, miR-20a,miR-29c,and miR-223 Combined as Non-Invasive Biomarkers in Nasopharyngeal Carcinoma

Background

MicroRNAs have been considered as a kind of potential novel biomarker for cancer detection due to their remarkable stability in the blood and the characteristics of their expression profile in many diseases.

Methods

We performed microarray-based serum miRNA profiling on the serum of twenty nasopharyngeal carcinoma patients at diagnosis along with 20 non-cancerous individuals as controls. This was followed by a real-time quantitative Polymerase Chain Reaction (RT-qPCR) in a separate cohort of thirty patients with nasopharyngeal carcinoma and thirty age- matched non-cancerous volunteers. A model for diagnosis was established by a conversion of mathematical calculation formula which has been validated by analyzing 74 cases of patients with nasopharyngeal carcinoma and 57 cases of non-cancerous volunteers.

Results

The profiles showed that 39 and 17 miRNAs are exclusively expressed in the serum of non-cancerous volunteers and of patients with nasopharyngeal carcinoma respectively. 4 miRNAs including miR-17, miR-20a, miR-29c, and miR-223 were found to be expressed differentially in the serum of NPC compared with that of non-cancerous control. Based on this, a diagnosis equation with Ct difference method has been established to distinguish NPC cases and non-cancerous controls and validated with high sensitivity and specificity.

Conclusions

We demonstrate that the serum miRNA-based biomarker model become a novel tool for NPC detection. The circulating 4-miRNA-based method may provide a novel strategy for NPC diagnosis.     

MiR-205 determines the radioresistance of human nasopharyngeal carcinoma by directly targeting PTEN

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance severely reduces NPC radiocurability. Here, we have established a radio-resistant NPC cell line, CNE-2R, and investigate the role of miRNAs in radioresistance. The miRNAs microarray assay reveals that miRNAs are differentially expressed between CNE-2R and its parental cell line CNE-2. We find that miR-205 is elevated in CNE-2R. A target prediction algorithm suggests that miR?205 regulates expression of PTE N, a tumor-suppressor. Introducing miR-205 into CNE-2 cells suppresses PTE N protein expression, followed by activation of AKT, increased number of foci formation and reduction of cell apoptosis postirradiation. On the other hand, knocking down miR-205 in CNE-2R cells compromises the inhibition of PTE N and increases cell apoptosis. Significantly, immunohistochemistry studies demonstrate that PTE N is downregulated at late stages of NPC, and that miR-205 is significantly elevated followed the radiotherapy. Our data conclude that miR-205 contributes to radioresistance of NPC by directly targeting PTE N. Both miR-205 and PTE N are potential predictive biomarkers for radiosensitivity of NPC and may serve as targets for achieve successful radiotherapy in NPC.     

鼻咽癌组织miR-20b-5p、miR-325-3p表达水平与放疗敏感性和预后的关系研究

摘要 目的：探讨鼻咽癌组织微小核糖核酸（miR）-20b-5p、miR-325-3p表达水平与放射治疗敏感性和预后的关系。方法：选取2017年11月至2019年6月我院收治的84例确诊为鼻咽癌并拟进行放射治疗的患者设为鼻咽癌组,另选取同期收治的42例慢性鼻咽炎患者为对照组,比较鼻咽癌组织及鼻咽部炎症组织中miR-20b-5p、miR-325-3p表达水平,分析鼻咽癌组织中miR-20b-5p、miR-325-3p表达水平与鼻咽癌患者临床病理特征的关系。根据鼻咽癌患者放疗敏感性评估结果分为敏感组和抵抗组,比较两组miR-20b-5p、miR-325-3p表达水平。随访3年,Kaplan-Meier法及Cox回归分析法分析miR-20b-5p、miR-325-3p表达水平与鼻咽癌患者生存预后的关系。结果：鼻咽癌组miR-20b-5p、miR-325-3p表达水平均高于对照组（P＜0.05）。不同T分期、N分期、临床分期患者在miR-20b-5p、miR-325-3p高表达组与低表达组中的占比比较存在统计学差异（P＜0.05）。完成7~8周放疗后3个月评估患者放疗抵抗率36.90%,抵抗组miR-20b-5p、miR-325-3p表达水平均高于敏感组（P＜0.05）。miR-20b-5p高表达鼻咽癌患者的累积生存时间短于miR-20b-5p低表达患者（P＜0.05）;miR-325-3p高表达鼻咽癌患者的累积生存时间短于miR-325-3p低表达患者（P＜0.05）。单因素、多因素Cox回归分析显示,年龄＞60岁、T3/T4期、miR-20b-5p高表达、miR-325-3p高表达是鼻咽癌患者预后不良的独立危险因素（P＜0.05）。结论：鼻咽癌组织中miR-20b-5p、miR-325-3p均异常高表达,其表达水平与肿瘤浸润深度、淋巴结转移、临床分期及放疗敏感性有关,且miR-20b-5p、miR-325-3p高表达患者放疗后预后不良风险更大。     

Inhibition of miR-191 contributes to radiation-resistance of two lung cancer cell lines by altering autophagy activity

Background

Lung cancer is the leading cause of cancer-related morbidity and mortality all over the world. Surgery resection, radiotherapy, chemotherapy, immunotherapy and combined treatments have been discovered and well established for treatments. However, low survival rate of five years after clinical treatments mainly due to recurrence of stress-resistant cancer cells calls for better understanding and new ideas. Our project aimed to understand the forming process of stress resistant lung cancer cells after radiotherapy.

Methods

Two classic non-small cell lung cancer (NSCLC) cell lines A549 and H1299 initially were radiated with a ¹³⁷Cs gamma-ray source with doses ranging from 0 to 12 Gy to generate radiation-resistant cancer cells. 8 Gy of radiation was regard as a standard dosage since it provides effective killing as well as good amount of survivals. The expression levels of autophagy-related proteins including Beclin-1, LC3-II and p62 were studied and measured by both western blot and quantitative real-time polymerase chain reaction (real-time RT-PCR).

Results

Increased Beclin-1, LC3-II and decreased p62 have been observed in radiation-resistant cells indicating elevated autophagy level. Decreased miR-191 in radiation-resistant cells performed by Taqman qRT-PCR also has been seen. Two binding sites between Beclin-1 and miR-191 suggest potential association between.

Conclusions

It is reasonable to speculate that inhibition of miR-191 expression in lung cancer cells would contribute to the establishment of radiation-resistant cells via mediating cellular autophagy. Therefore, miR-191 is a potential target for therapy in treating radiation-resistant lung cancer.     

Relative expressions of miR-205-5p, miR-205-3p, and miR-21 in tissues and serum of non-small cell lung cancer patients

Objective was to assess and compare the relative expressions of miR-205-5p, miR-205-3p, and miR-21-3p in tissues and serum among non-small cell lung carcinoma (NSCLC) patients, benign pulmonary conditions patients, and healthy volunteers. Serum samples were obtained between October 2011 and September 2012 from 20 NSCLC patients undergoing surgical treatment, 20 patients diagnosed with a benign lung disease (pulmonary tuberculosis, pneumonia, chronic obstructive pulmonary disease, or interstitial pneumonia) (lesion group), and 20 healthy volunteers (control group). NSCLC patients provided cancer tissues and cancer-adjacent normal tissues during surgery (paired specimens). Quantitative RT-PCR was used to assess miR-205-5p, miR-205-3p, and miR-21-3p expressions in serum and tissue samples. The relative expressions of miR-205-5p and miR-205-3p were significantly higher in NSCLC tissues compared with cancer-adjacent paired specimens (both P < 0.001). In the serum, significantly higher miR-205-5p, miR-205-3p, and miR-21-3p relative expressions were observed in the NSCLC group compared with the two other groups (all P < 0.001). The relative expressions of miR-205-5p and miR-21-3p in NSCLC tissues and serum were significantly correlated (r = 0.553, P = 0.011; and r = ?0.541, P = 0.014, respectively), while no significant correlation was observed for miR-205-3P (P = 0.120). Expressions of miR-205-5p and miR-205-3P in squamous cell carcinoma specimens were significantly higher than in lung adenocarcinoma specimens (both P = 0.001). Similarly, higher serum miR-205-5p and miR-205-3p levels were observed in squamous cell carcinoma patients (P = 0.033 and P = 0.002, respectively). In this preliminary and novel study, miR-205-5p was more useful as a marker for NSCLC than miR-205-3p or miR-21, indicating a potential for future applications in NSCLC diagnosis and prognosis.     

miR-143 inhibits proliferation and metastasis of nasopharyngeal carcinoma cells via targeting FMNL1 based on clinical and radiologic findings

Mounting evidence has reported that microRNA-143 (miR-143) is involved in the development of multiple cancers. To investigate the underlying mechanisms of miR-143 regulating proliferation and metastasis in nasopharyngeal carcinoma (NPC) cells, we evaluated the levels of miR-143 and formin-like protein 1 (FMNL1) in NPC tissues. The results of qRT-PCR and Western blot analysis showed that the expression of miR-143 was decreased, while FMNL1 was increased in NPC tissues. The expression of miR-143 was significantly elevated in NPC cells compared with that of human nasopharyngeal epithelial cells. The results of MiRcode prediction, dual-luciferase reporter, and Western blot analysis assays indicated that miR-143 negatively regulated the expression of FMNL1 (r² = 0.4365P = 0.0001). Overexperssion of miR-143 or FMNL1 knockdown inhibited cell proliferation, migration, and invasion in NPC cells (P < 0.05). Ectopic expression of FMNL1 undermined the inhibition effect of miR-143 on proliferation, migration, and invasion in NPC cells. The findings of this study revealed that miR-143 functioned as a tumor suppressor and inhibited the NPC progression by targeting FMNL1.     

微小RNA-205 在甲状腺乳头状癌中的表达及临床意义

目的:观察微小RNA(microRNA,miRNA,miR)-205在甲状腺乳头状癌(PTC)中的表达并探讨其临床意义。方法:收集自2014年1月至2014年12月在我院甲状腺外科住院治疗的甲状腺乳头状癌患者的术后新鲜病理组织45例,其中男14例,女31例,年龄24-69岁,平均45.5岁。结节性甲状腺肿28例,癌旁正常甲状腺组织5例。提取各组织中的miRNA,应用实时荧光定量聚合酶链反应(RT-q PCR)方法检测miR-205的表达情况。结果:甲状腺乳头状癌miR-205的表达量较非肿瘤组织(结节性甲状腺肿、癌旁组织)明显下调[(1.06±1.76)vs(3.19±4.88),P=0.038]。伴淋巴结转移的PTC组织中miR-205表达量明显低于无淋巴结转移的PTC组织[(1.21±1.80)vs(9.59±1.60),P=0.003]。miR-205的相对表达与PTC患者性别、年龄及浸润与否均无显著相关性,而肿瘤直径呈显著相关性。结论:miR-205在PTC中的表达异常下调,可能与PTC的发生、侵袭和转移有关。     

Involvement of cholesterol in hepatitis B virus X protein-induced abnormal lipid metabolism of hepatoma cells via up-regulating miR-205-targeted ACSL4

Hepatitis B virus X protein (HBx) plays crucial roles in the development of hepatocellular carcinoma (HCC). The abnormal lipid metabolism is involved in the hepatocarcinogenesis. We previously reported that HBx suppressed miR-205 in hepatoma cells. In this study, we supposed that HBx-decreased miR-205 might contribute to the abnormal lipid metabolism according to the bioinformatics analysis. Interestingly, we showed that the expression levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) were negatively associated with those of miR-205 in clinical HCC tissues. Then, we validated that miR-205 was able to inhibit the expression of ACSL4 at the levels of mRNA and protein through targeting its 3′UTR. Strikingly, we found that HBx was able to increase the levels of cellular cholesterol, a metabolite of ACSL4, in hepatoma cells, which could be blocked by miR-205 (or Triacsin C, an inhibitor of ACSL4). However, anti-miR-205 could increase the levels of cholesterol in the cells. Moreover, we demonstrated that the levels of cholesterol were increased in the liver of HBx transgenic mice in a time course manner. Functionally, oil red O staining revealed that HBx promoted lipogenesis in HepG2 cells, which could be abolished by miR-205 (or Triacsin C). However, anti-miR-205 was able to accelerate lipogenesis in the cells. Interestingly, the treatment with Triacsin C could remarkably block the role of anti-miR-205 in the event. Thus, we conclude that miR-205 is able to target ACSL4 mRNA. The HBx-depressed miR-205 is responsible for the abnormal lipid metabolism through accumulating cholesterol in hepatoma cells.     

Mir-378a-5p抑制鼻咽癌细胞系CNE-1 细胞增殖以及肿瘤生长的初步研究*

目的： MiR-378a-5p是一种被认为在多种肿瘤发生过程中具有抑制肿瘤生长的微小RNA。然而miR-378a-5p在鼻咽癌中的 作用尚未见报道。因此,本文旨在通过临床样本的miRNA 表达谱分析以及细胞学实验从而揭示miR-378a-5p在鼻咽癌肿瘤发生过程中的作用。方法与结果：我们通过生物信息学的方法获取了鼻咽癌临床样本中miR-378a-5p的表达信息并通过与正常组织的 对比发现miR-378a-5p在鼻咽癌肿瘤组织中表达水平显著降低（P<0.01）。其次,我们发现高表达miR-378a-5p的鼻咽癌CNE-1 细 胞增殖速度显著较对照组降低（约40%~50%）。克隆形成实验证实了瞬时转染miR-378a-5p的鼻咽癌CNE-1 细胞的克隆形成数 量显著减弱。我们通过将稳定表达miR-378a-5p的CNE-1 细胞注射到裸鼠体内形成移植瘤并记录肿瘤生长曲线,结果显示 miR-378a-5p高表达组的裸鼠移植瘤体积明显较对照组小约50%,肿瘤重量显著降低(对照组0.33 g,处理组0.15 g)。结论：本研究通过对临床样本的分析以及在细胞和动物水平的实验验证揭示了miR-378a-5p具有抑制鼻咽癌肿瘤细胞增殖和肿瘤生长的作用。     

U6 is not a suitable endogenous control for the quantification of circulating microRNAs

Recently, microRNAs have been detected in serum and plasma, and circulating microRNA (miRNA) profiles have now been associated with many diseases such as cancers and heart disease, as well as altered physiological states. Because of their stability and disease resistance, circulation miRNAs appear to be an ideal material for biomarkers of diseases and physiological states in blood. However, the lack of a suitable internal reference gene (internal reference miRNA) has hampered research and application of circulating miRNAs. Currently, U6 and miR-16 are the most common endogenous controls in the research of miRNAs in tissues and cells. We performed microarray-based serum miRNA profiling on the serum of 20 nasopharyngeal carcinoma patients and 20 controls to detect the expressions of U6 and miRNAs. Profiling was followed by real-time quantitative Polymerase Chain Reaction (qPCR) in 80 patients (20 each with gastric cancer, nasopharyngeal carcinoma, colorectal cancer, and breast cancer) and 30 non-cancerous controls. qPCR was also performed to detect miRNAs in serum with repeated freezing and thawing. The results of microarray showed that with the exception of U6, Ct values of miR-16, miR-24, miR-142-3p, miR-19b and miR-192 in serum samples of nasopharyngeal carcinoma were greater than control samples. The results of 110 cases showed large fluctuations in U6 expression. The difference between the greatest and the least levels of expression was 3.29 for delta Ct values, and 1.23 for miR-16. The expressions of U6, miR-16 and miR-24 in serum subjected to different freeze–thaw cycles showed that U6 expression gradually decreased after 1, 2, and 4 cycles of freezing and thawing, while the expression of miR-16 and miR-24 remained relatively stable. Collectively, our results suggested that U6 is unsuitable as an internal reference gene in the research of circulating miRNAs.     

MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

Background

MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Methods

TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44^high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.

Results

MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44^high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.

Conclusions

MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.     

MiR-506 Suppresses Tumor Proliferation and Invasion by Targeting FOXQ1 in Nasopharyngeal Carcinoma

MiRNAs are small noncoding RNAs that play important roles in various biological processes including tumorigenesis. However, little is known about the expression and function of miR-506 in nasopharyngeal carcinoma (NPC). In this study, we showed that miR-506 was downregulated in nasopharyngeal carcinoma (NPC) cell lines and tissues. Ectopic expression of miR-506 dramatically suppressed cell proliferation, colony formation and invasion. Moreover, we identified the Forkhead box Q1 (FOXQ1) gene as a novel direct target of miR-506. MiR-506 exerts its tumor suppressor function through inhibition of the FOXQ1, which was involved in tumor metastasis and proliferation in various cancers. Furthermore, the expression of FOXQ1 is up-regulated in NPC cell lines and tissues. Taken together, our results indicate that miR-506 functions as a tumor suppressor miRNA in NPC and that its suppressive effects are mediated chiefly by repressing FOXQ1 expression.     

miR-106a-5p调控 PTEN对鼻咽癌细胞SUNE2增殖和迁移的抑制作用研究

目的研究miR-106a-5p对鼻咽癌细胞SUNE2增殖和迁移的影响。 方法将体外培养的鼻咽癌细胞SUNE2分成对照组（细胞未做任何处理）、Anti-NC组（转染阴性对照抑制剂）、Anti-miR-106a-5p组（转染miR-106a-5p抑制剂）、后期实验另设Anti-miR-106a-5p-inhibitor+si-NC组（转染miR-106a-5p抑制剂、siRNA阴性对照）、Anti-miR-106a-5p-inhibitor+si-PTEN组（转染miR-106a-5p抑制剂、PTEN siRNA）,采用Realtime PCR测定miR-106a-5p表达,MTT法检测增殖,Transwell小室检测细胞侵袭和迁移能力变化,用Western blot方法测定vimentin、E-cadherin蛋白表达。在线靶基因预测软件预测miR-106a-5p的靶基因可能为PTEN,双荧光素酶报告系统鉴定miR-106a-5p和PTEN的靶向关系。两组间比较用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与正常鼻咽上皮细胞NP69比较,鼻咽癌细胞CNE-2、HK1、SUNE2中miR-106a-5p水平（1.00±0.11比1.84±0.13、2.19±0.14、2.87±0.25）升高,差异具有统计学意义（P < 0.05）。与对照组、Anti-NC组比较,Anti-miR-106a-5p组鼻咽癌细胞miR-106a-5p水平（1.00±0.10、0.99±0.12比0.76±0.08）降低,OD值（0.56±0.05、0.57±0.04比0.32±0.02）,细胞侵袭数[（128.47±11.65）个、（129.84±10.93）个比（85.12±6.75）个],迁移数[（182.51±14.81）个、（180.68±17.64）个比（122.01±11.62）个],vimentin蛋白表达水平（0.84±0.09、0.82±0.07比0.30±0.05）降低,E-cadherin蛋白表达水平（0.29±0.04、0.28±0.05比0.76±0.08）升高,差异具有统计学意义（P均< 0.05）。与Anti-miR-106a-inhibitor+si-NC组比较,Anti-miR-106a-inhibitor+si-PTEN组细胞OD值（0.33±0.03比0.52±0.05）、侵袭数[（84.16±5.91）个比（105.79±8.63）个]、迁移数[（118.42±10.25）个比（164.28±12.05）个]、vimentin蛋白表达水平（0.34±0.06比0.68±0.05）均升高,E-cadherin蛋白表达水平（0.72±0.06比0.29±0.05）降低,差异具有统计学意义（P均< 0.05）。 结论miR-106a-5p可通过靶向调控PTEN抑制鼻咽癌细胞SUNE2增殖和迁移潜能。