miR-124 Inhibits Growth and Invasion of Gastric Cancer by Targeting ROCK1

MicroRNAs (miRNAs) act as critical regulators of genes involved in many biological processes. Aberrant alteration of miRNAs have been found in many cancers, including gastric cancer (GC), but the molecular mechanisms are not well understood. Herein, we investigated the role of miR-124 in GC. We found that its expression was significantly reduced in both GC tissue samples and cell lines. Forced expression of miR-124 suppressed GC cell proliferation, migration, and invasion. Furthermore, the Rho-associated protein kinase (ROCK1) was identified as a direct target of miR-124 in GC cells. Finally, silencing of ROCK1 showed similar effects as miR-124 overexpression, while supplementation of ROCK1 remarkably restored the cell growth and invasion inhibited by miR-124. Together, our data demonstrate that miR-124 acts as a tumor suppressor bytargeting ROCK1, and posit miR-124 as a novel strategy for GC treatment.     

Cinobufacin Suppresses Cell Proliferation via miR-494 in BGC-823 Gastric Cancer Cells

Cinobufacin is used clinically to treat patients with many solid malignant tumors. However, the mechanismsunderlying action remain to be detailed. Our study focused on miRNAs involved in cinobufacin inhibition of GCcell proliferation. miRNA microarray analysis and real time PCR identified miR-494 as a significant cinobufacinassociatedmiRNA. In vivo, ectopic expression of miR-494 inhibited the proliferation and induced apoptosis ofBGC-823 cells on CCK-8 and flow cytometry analysis. Further study verified BAG-1 (anti-apoptosis gene) to beatarget of miR-494 by luciferase reporter assay and Western blotting. In summary, our study demonstrated thatcinobufacin may inhibit the proliferation and promote the apoptosis of BGC-823 cells. Cinobufacin-associatedmiR-494 may indirectly be involved in cell proliferation and apoptosis by targeting BAG-1, pointing to use as apotential molecular target of cinobufacin in gastric cancer therapy.     

非小细胞肺癌中miR-19a和miR-19b的表达及临床意义

目的:分析miR-19a、miR-19b在非小细胞肺癌中的表达及其与临床病理特征的关系。方法:非小细胞肺癌手术标本61例及其癌旁正常组织,提取总RNA,采用实时定量PCR方法检测miR-19a、miR-19b在非小细胞肺癌及其癌旁正常组织中的表达,并分析其与临床病理特征的关系。结果:miR-19a、miR-19b在非小细胞肺癌组织中的表达高于对应的癌旁正常肺组织,其表达与临床分期、病理类型及淋巴结有无转移相关（P<0.05）。而在不同年龄、性别和吸烟史患者间差异无统计学意义（P>0.05）。结论:miR-19a、miR-19b高表达与非小细胞肺癌的临床分期、病理分型及有无淋巴结转移密切相关,miR-19a、miR-19b有可能成为非小细胞肺癌的重要肿瘤标志物之一。     

miR-19a和miR-19b在骨肉瘤中的表达及其临床意义

目的：探讨miR-19a和miR-19b在骨肉瘤组织及配对瘤旁组织中的表达及其与临床病理特征的相关性。方法：收集32对骨肉瘤和配对的瘤旁组织,运用实时荧光定量PCR（qPCR）检测骨肉瘤组织及其瘤旁组织中miR-19a和miR-19b的表达,分析其与骨肉瘤临床病理特征的相关性及其临床意义。结果：qPCR结果显示骨肉瘤组织中miR-19a和miR-19b的平均表达量较瘤旁组织中均明显上调（P均 < 0.05）。二者的表达水平呈正相关（r=0.685,P=0.000）。miR-19a和miR-19b的表达与骨肉瘤病理分级之间存在正相关（r=0.478,P=0.027）;miR-19a的表达与临床分期呈正相关（r=0.365,P=0.031）且与预后相关。Cox回归多因素分析发现miR-19a可作为骨肉瘤患者预后的影响因子（P=0.037）。结论：miR-19a和miR-19b在骨肉瘤中表达上调,在骨肉瘤的发生发展中可能发挥重要的作用,其中miR-19a可能作为骨肉瘤独立的预后标志物。     

miR-106a和miR-24-1在结肠癌中的表达及意义

 目的
检测结肠癌与癌旁组织miR-106a和miR-24-1的表达差异及其与c-myc的表达是否具有关联性。方法选临床病理诊断明确的结肠癌患者,手术后取癌组织标本提取RNA,RT-PCR检测原癌基因c-myc在结肠癌组织与癌旁组织中的表达。TaqMan荧光定量PCR检测结肠癌与癌旁组织miR-106a和miR-24-1的表达差异。结果c-myc表达阳性的癌组织表达miR-106a要明显高于癌旁组织,其差异具有统计学意义（P<0.05）;c-myc表达阴性的癌组织表达miR-106a要高于癌旁组织（P<0.05）,结肠癌组织与癌旁组织miR-24-1的表达无差异。结论结肠癌组织c-myc阳性组和c-myc阴性组表达miR-106a均高于癌旁组织,miR-106a有可能作为结肠癌的筛查目标之一;结肠癌组织c-myc阳性组和c-myc阴性组表达miR-24-1与癌旁组织无差异,还需更多研究证实其与结肠癌的关系。     

miR-214在胃癌患者外周血中的表达及其临床意义

目的探讨miR-214在胃癌中的表达及临床意义。方法采用实时荧光定量PCR法,检测40例胃癌患者癌组织及癌旁组织中miR-214的差异表达;同时检测血浆miR-214在56例胃癌患者及40例正常组织中的表达。结果miR-214在胃癌组织中的表达水平明显高于癌旁组织;与正常组织比较,胃癌患者中的血浆miR-214表达明显增高;血浆miR-214表达与患者性别、年龄无关,与疾病分期及生存期密切相关,差异具有统计学意义(P<0.05);Cox回归分析发现疾病分期、miR-214的表达可作为独立的预后因子(P<0.05)。结论循环miR-214表达与胃癌肿瘤分期和生存时间相关,检测循环miR-214可能作为评估胃癌患者临床预后的靶基因。     

微小RNA-133b在胃癌中的表达及意义

目的 研究miR-133b在胃癌细胞、正常胃黏膜上皮永生化细胞、胃癌组织及相应癌旁组织中的表达,探讨miRNA对胃癌发生发展的调控作用。方法 培养7种胃癌细胞株及正常胃黏膜上皮细胞,并收集56例胃癌患者癌组织及相应癌旁组织,提取细胞及组织中总的Small RNA,采用real-timePCR法检测miR-133b在胃癌细胞及组织中的表达,分析miR-133b的表达与细胞分化程度、临床病理特征的关系。结果 miR-133b在胃癌细胞及组织中均表达下调,差异具有统计学意义。miR-133b的低表达与细胞分化程度、TNM分期及有无淋巴结转移显著相关。结论 miR-133b在胃癌细胞及胃癌组织中的表达明显下调,可能作为抑癌基因参与胃癌的发生、发展。     

A novel SOCS5/miR-18/miR-25 axis promotes tumorigenesis in liver cancer

The role of miRNAs with tumor suppressive activity in liver cancer has been well studied. However, little is known about potential oncomiRs in HCC. In our study, we conducted a systematic evaluation of candidate oncomiRs and found that upregulation of miR-18a and miR-25 in HCC was associated with poor patient survival and promoted proliferation in HCC cell lines. These two miRNAs belong to the polycistronic paralogous miR-17-92 and miR-25-106b clusters respectively. Although the members of both clusters are often upregulated in HCC, the contribution of individual miRNAs in these clusters to HCC tumorigenesis is not fully understood. We validated SOCS5 as a bona fide target of both miRNAs, and established, for the first time, the tumor suppressive role of SOCS5 in liver cancer. We further investigated the mechanism by which SOCS5 contributes to tumorigenesis, demonstrated that this SOCS5/miR-18a/miR-25 axis regulates the tumor suppressor TSC1 and downstream mTOR signaling, and highlighted the potential therapeutic use of miR-18a and miR-25 inhibition in restoring SOCS5 levels in HCC.     

吡格列酮对胃癌细胞生长的影响及其机制

 目的研究过氧化物酶体激活物活化受体γ(PPARγ)在人胃癌MGC803中的表达及其配体吡格列酮(PGZ)对胃癌细胞生长的影响。方法用不同浓度的吡格列酮处理人胃癌MGC803细胞,采用RT-PCR法检测MGC803中PPARγ的表达变化;MTT法观察细胞增殖抑制作用;Hoechst33342/PI双荧光活细胞染色法和DNA凝胶电泳技术分析细胞凋亡。结果MGC803中存在PPARγ表达;PGZ能显著抑制MGC803生长(P<0.05),IC50值为9.01×10-6μmol/L,且作用呈剂量依赖性;高浓度PGZ能明显诱导凋亡产生;PGZ作用MGC803细胞后PPARγ表达呈剂量依赖性上调趋势(P<0.05)。结论吡格列酮依赖激活PPARγ能在体外抑制肿瘤细胞生长并诱导其凋亡,提示PPARγ可能是胃癌治疗的一个新的分子靶点。     

Association of miR-193b Down-regulation and miR-196a up-Regulation with Clinicopathological Features and Prognosis in Gastric Cancer

Dysregulated expression of microRNAs (miRNAs) has been shown to be closely associated with tumordevelopment, progression, and carcinogenesis. However, their clinical implications for gastric cancer remainelusive. To investigate the hypothesis that genome-wide alternations of miRNAs differentiate gastric cancer tissuesfrom those matched adjacent non-tumor tissues (ANTTs), miRNA arrays were employed to examine miRNAexpression profiles for the 5-pair discovery stage, and the quantitative real-time polymerase chain reaction (qRTPCR)was applied to validate candidate miRNAs for 48-pair validation stage. Furthermore, the relationshipbetween altered miRNA and clinicopathological features and prognosis of gastric cancer was explored. Amonga total of 1,146 miRNAs analyzed, 16 miRNAs were found to be significantly different expressed in tissues fromgastric cancer compared to ANTTs (p<0.05). qRT-PCR further confirmed the variation in expression of miR-193band miR-196a in the validation stage. Down-expression of miR-193b was significantly correlated with Laurentype, differentiation, UICC stage, invasion, and metastasis of gastric cancer (p<0.05), while over-expression ofmiR-196a was significantly associated with poor differentiation (p=0.022). Moreover, binary logistic regressionanalysis demonstrated that the UICC stage was a significant risk factor for down-expression of miR-193b (adjustedOR=8.69; 95%CI=1.06-56.91; p=0.043). Additionally, Kaplan-Meier survival curves indicated that patientswith a high fold-change of down-regulated miR-193b had a significantly shorter survival time (n=19; mediansurvival=29 months) compared to patients with a low fold-change of down-regulated miR-193b (n=29; mediansurvival=54 months) (p=0.001). Overall survival time of patients with a low fold-change of up-regulated miR-196a (n=27; median survival=52 months) was significantly longer than that of patients with a high fold-changeof up-regulated miR-196a (n=21; median survival=46 months) (p=0.003). Hence, miR-193b and miR-196a maybe applied as novel and promising prognostic markers in gastric cancer.     

MiR-218-5p通过靶向抑制TDP1表达促进鱼藤酮诱导的胃癌细胞凋亡

目的 探讨过表达miR-218-5p和抑制TDP1的表达对鱼藤酮诱导损伤胃癌细胞凋亡的影响,阐明其可能的作用机制。方法 采用RT-PCR检测人正常胃黏膜上皮细胞和四种胃癌细胞中miR-218-5p及TDP1表达水平,并分析其相关性。双荧光素酶报告基因验证miR-218-5p对TDP1的靶向调控作用。采用1.0 μmol/L鱼藤酮诱导胃癌细胞损伤,流式细胞术检测细胞周期及凋亡率。Western blot检测细胞线粒体中TDP1水平及细胞Bax、Cyt-c蛋白的表达。结果 miR-218-5p在胃癌细胞中低表达（P<0.05）,TDP1高表达（P<0.01）,两者表达呈负相关（R²=0.9580, P=0.0212）。与对照组比较,损伤组SGC-7901细胞发生G1期阻滞,凋亡率升高（P<0.01）。与损伤组比较,miR-218-5pmimic组SGC-7901细胞G1期阻滞加剧,细胞凋亡率进一步升高（P<0.01）,Bax及Cyt-c表达上调（P<0.01）,而线粒体中TDP1蛋白水平降低（P<0.01）;TDP1过表达组细胞G1期阻滞得到缓解,凋亡率降低（P<0.01）,线粒体中TDP1蛋白水平升高（P<0.01）,Bax及Cyt-c表达降低（P<0.01）。结论 miR-218-5p可靶向抑制TDP1表达,诱导胃癌细胞凋亡,其作用机制可能与抑制线粒体DNA损伤修复及功能维持、激活线粒体内源性凋亡途径有关。     

miR-19a acts as an oncogenic microRNA and is up-regulated in bladder cancer

Background

The application of microRNAs (miRNAs) as potential biomarkers and therapy targets has been widely investigated in many kinds of cancers. The discovery of tumor associated miRNAs in serum of patients supported the use of plasma/serum miRNAs as noninvasive means of cancer detection. However, the aberrant expression of miRNAs in bladder cancer patients and their intensive roles and mechanisms in bladder cancer are poorly understood.

Methods

Taqman probe stem-loop real-time PCR was used to accurately measure the levels of miR-19a in bladder cancer cell lines, 100 pairs of bladder cancer tissues and the adjacent non-neoplastic tissues and also the plasma collected from bladder cancer patients and normal controls. miR-19a mimics and inhibitors were transfected into bladder cancer cells to investigate its role on regulating cell proliferation which was measured by CCK-8 and colony formation assay. The target of miR-19a was identified by western blot and whether its regulatory role depends on its target was improved by a rescue experiment with miR-19a mimic and PTEN expression plasmid.

Results

miR-19a was significantly up-regulated in bladder cancer tissues and high-level of miR-19a was correlative with more aggressive phenotypes of bladder cancer. Meanwhile, gain or loss of function of miR-19a demonstrated that miR-19a can promote cell growth of bladder cancer cells and the further mechanism studies indicated that its oncogenic role was dependent on targeting PTEN. Furthermore, investigation of miR-19a expression in the plasma of bladder cancer patients showed that miR-19a was also increased in plasma of bladder cancer patients which strongly supported miR-19a could be developed as potential diagnostic marker of bladder cancer.

Conclusions

Our data indicated that miR-19a might act as an oncogenic microRNA in bladder cancer and was significantly up-regulated in bladder cancer carcinogenesis. The oncogenic role of miR19a in bladder cancer was dependent on targeting PTEN.     

F-box protein FBXO31 is down-regulated in gastric cancer and negatively regulated by miR-17 and miR-20a

FBXO31, a subunit of the SCF ubiquitin ligase, played a crucial role in neuronal development, DNA damage response and tumorigenesis. Here, we investigated the expression and prognosis value of FBXO31 in human primary gastric cancer (GC) samples. Meanwhile, the biological role and the regulation mechanism of FBXO31 were evaluated. We found that FBXO31 mRNA and protein was decreased dramatically in the GC tissue compared with the adjacent non-cancerous tissues. FBXO31 expression was significantly associated with tumor size, tumor infiltration, clinical grade and patients'' prognosis. FBXO31 overexpression significantly decreased colony formation and induced a G₁-phase arrest and inhibited the expression of CyclinD1 protein in GC cells. Further evidence was obtained from knockdown of FBXO31. Ectopic expression of FBXO31 dramatically inhibited xenograft tumor growth in nude mice. miR-20a and miR-17 mimics inhibited, whereas the inhibitor of miR-20a and miR-17 increased, the expression of FBXO31, respectively. miR-20a and miR-17 directly bind to the 3''-UTR of FBXO31. The level of miR-20a and miR-17 in GC tissue was significantly higher than that in surrounding normal mucosa. Moreover, a highly significant negative correlation between miR-20a (miR-17) and FBXO31 was observed in these GC samples. Therefore, effective therapy targeting the miR-20a (miR-17)-FBXO31-CyclinD1 pathway may help control GC progression.     

Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

The TSLC1 (tumor suppressor in lung cancer–1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non-cancerous gastric tissues, by bisulfite-SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO-III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non-cancerous gastric tissues, suggesting that this hypermethylation is a cancer-specific alteration. KATO-III and ECC10 cells retained two alleles of TSLC1 , both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi-allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers.     

miR-449a Suppresses Tumor Growth,Migration, and Invasion in Non-Small Cell Lung Cancer by Targeting a HMGB1-Mediated NF-kB Signaling Pathway

MicroRNAs (miRNAs) have been reported to be involved in many human cancers and tumor progression. The dysregulation of miR-449a is found in many types of malignancies and is associated with tumor growth, migration, and invasion. However, its expression and function in non-small cell lung cancer (NSCLC) still remains unclear. In our study, miR-449a was found to be downregulated in both NSCLC tissues and cell lines, and low miR-449a expression was obviously associated with tumor differentiation, TMN stage, and poor overall survival (OS). Moreover, we demonstrated that miR-449a could inhibit tumor proliferation, migration, and invasion in NSCLC. We also confirmed that HMGB1 was a direct target gene of miR-449a in NSCLC with dual-luciferase reporter assay, and upregulation of HMGB1 could reverse the miR-449a-induced suppression of growth, migration, and invasion in NSCLC cells. Last, we found that miR-449a suppressed tumor initiation and development through the NF- B signaling pathway. These results indicate that miR-449a functions as a tumor suppressor in NSCLC by targeting the HMGB1-mediated NF- B signaling pathway in NSCLC.     

Role of miR-27a,miR-181a and miR-20b in gastric cancer hypoxia-induced chemoresistance

Despite the search for new therapeutic strategies for gastric cancer (GC), there is much evidence of progression due to resistance to chemotherapy. Multidrug resistance (MDR) is the ability of cancer cells to survive after exposure to chemotherapeutic agents. The involvement of miRNAs in the development of MDR has been well described but miRNAs able to modulate the sensitivity to chemotherapy by regulating hypoxia signaling pathways have not yet been fully addressed in GC. Our aim was to analyze miR-20b, miR-27a and miR-181a expression with respect to (epirubicin/oxaliplatin/capecitabine (EOX)) chemotherapy regimen in a set of GC patients, in order to investigate whether miRNAs deregulation may influence GC MDR also via hypoxia signaling modulation. Cancer biopsy were obtained from 21 untreated HER2 negative advanced GC patients, retrospectively analyzed. All patients received a first-line chemotherapy (EOX) regimen. MirWalk database was used to identify miR-27a, miR-181a and miR-20b target genes. The expression of miRNAs and of HIPK2, HIF1A and MDR1 genes were detected by real-time PCR. HIPK2 localization was assessed by immunohistochemistry. Our data showed the down-regulation of miR-20b, miR-27a, miR-181a concomitantly to higher levels of MDR1, HIF1A and HIPK2 genes in GC patients with a progressive disease respect to those with a disease control rate. Moreover, immunohistochemistry assay highlighted a higher cytoplasmic HIPK2 staining, suggesting a different role for it. We showed that aberrant expression of miR-20b, miR27a and miR-181a was associated with chemotherapeutic response in GC through HIF1A, MDR1 and HIPK2 genes modulation, suggesting a possible novel therapeutic strategy.     

奥沙利铂通过调控miRNA-7-5p/RAF-1促进胃癌SGC-7901细胞凋亡的研究

目的探讨奥沙利铂如何调控MAPK通路,抑制胃癌细胞的增殖。方法 NCBI检索文献,利用TargetScan、StarBase和miRBase数据库,进行GO分析与KEGG通路富集,找到相关miRNAs,预测靶基因。应用Real-time PCR、MTT、Hoechst33258、流式细胞术、细胞划痕实验、Western blot等方法分析人胃癌SGC-7901细胞的增殖、细胞周期、侵袭及蛋白表达情况。结果胃癌细胞中miR-7-5p显著低表达,RAF1与miR-7-5p存在互靶关系。miR-7-5p mimics与奥沙利铂均可促进SGC-7901细胞的凋亡,提高G1期细胞百分率(P<0.05),降低侵袭、迁移速度。caspase3、caspase9蛋白表达升高,Bcl-2/Bax比值降低(P<0.05)。结论过表达mi R-7-5p与奥沙利铂均可促进胃癌SGC-7901细胞的凋亡,提示奥沙利铂可能通过上调mi RNA-7-5p促进SGC-7901细胞的凋亡,降低侵袭、迁移速度。     

Aberrant Expression of miR-20a and miR-203 in Cervical Cancer

MicroRNAs (miRNAs) are small, non-coding RNAs that are critical regulators of various diseases. MicroRNA-20a (miR-20a) and microRNA-203 (miR-203) have previously shown significant alteration in a range of cancers.In this study, the expression levels of miR-20a and miR-203 in 100 cervical cancer tissues were detected byqRT–PCR and compared to patient matched-nontumor cervical tissues. Correlations between expression leveland clinicopathologic characteristics of cervical cancer were also analyzed. Finally, we studied the effect of miR-20a and miR-203 on cell proliferation in cervical cancer cell lines by MTT. We found that the expression levelof miR-20a (P<0.001) was significantly higher in cervical cancer patients than in healthy controls, while thatof miR-203 (P<0.001) was lower. Aberrant expression of miR-20a was correlated with lymph node metastasis(LNM), histological grade and tumor diameter, but down-regulated miR-203 was correlated with LNM only.Furthermore, we found that over-expression of miR-203 decreased cell proliferation, while reduction of miR-20a also prevented tumor progression. Our results support the involvement of miR-20a and miR-203 in cervicaltumorigenesis. We propose that miRNAs might be used as therapeutic agents for cervical cancer.