Decreased miR-30b-5p expression by DNMT1 methylation regulation involved in gastric cancer metastasis

miRNAs have emerged as crucial regulators in the regulation of development as well as human diseases, especially tumorigenesis. The aims of this study are to evaluate miR-30b-5p expression pattern and mechanism in gastric carcinogenesis due to which remains to be determined. Expression of miR-30b-5p was analyzed in 51 gastric cancer cases and 4 cell lines by qRT-PCR. The effect of DNA methylation on miR-30b-5p expression was assessed by MSP and BGS. In order to know whether DNMT1 increased miR-30b-5p promoter methylation, DNMT1 was depleted in cell lines AGS and BGC-823. The role of miR-30b-5p on cell migration was evaluated by wound healing assays. Decreased expression of miR-30b-5p was found in gastric cancer samples. In tumor, the expression level of miR-30b-5p was profound correlated with lymph node metastasis (P = 0.019). The level of miR-30b-5p may be restored by DNA demethylation and DNMT1 induced miR-30b-5p promoter methylation. In vitro functional assays implied that enforced miR-30b-5p expression affected cell migration, consistent with tissues analysis. Our findings uncovered that miR-30b-5p is significantly diminished in gastric cancer tissues, providing the first insight into the epigenetic mechanism of miR-30b-5p down-regulation, induced by DNMT1, and the role of miR-30b-5p in gastric cancer carcinogenesis. Overexpression of miR-30b-5p inhibited cell migration. Thus, miR-30b-5p may represent a potential therapeutic target for gastric cancer therapy.     

Aberrant hypermethylation of miR-9 genes in gastric cancer

Carcinogenesis of the stomach involves multiple steps including genetic mutation or epigenetic alteration of tumor suppressor genes or oncogenes. Recently, tumor suppressive miRNAs have been shown to be deregulated by aberrant hypermethylation during gastric cancer progression. In this study, we demonstrate that three independent genetic loci encoding for miR-9 (miR-9-1, miR-9-2 and miR-9-3) are simultaneously modified by DNA methylation in gastric cancer cells. Methylation-mediated silencing of these three miR-9 genes can be reactivated in gastric cancer cells through 5-Aza-dC treatment. Subsequent analysis of the expression levels of miR-9 showed that it was significantly down-regulated in gastric cancers compared with adjacent normal tissues (P value < 0.005). A similar tendency toward a tumor-specific DNA methylation pattern was shown for miR-9-1, miR-9-2 and miR-9-3 in 72 primary human gastric cancer specimens. Ectopic expression of miR-9 inhibited cell proliferation, migration and invasion, suggesting its tumor suppressive potential in gastric cancer progression.     

miR-125b靶向MCL1抑制胃癌 MGC-803细胞增殖

探讨mi R-125b对胃癌MGC-803细胞增殖的影响及机制,为阐明胃癌发病的分子机制提供实验依据.采用q RT-PCR和原位杂交,检测mi R-125b在正常胃黏膜(NGM)和胃癌(GAC)组织中的表达.将mi R-125b导入胃癌MGC-803细胞,观察mi R-125b高表达对MGC-803细胞增殖的影响.利用Targetscan 6.2软件及荧光素酶报告基因检测,分析mi R-125b对MCL1基因的靶向性作用.构建MCL1干扰载体,观察干扰MCL1基因表达对MGC-803细胞增殖的影响.结果发现,mi R-125b在胃癌组织中低表达,其表达与胃癌的分化程度及患者预后呈正相关,与TNM分期、淋巴结转移呈负相关(P0.01).mi R-125b高表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01);mi R-125b与MCL1基因的3′UTR(2 613~2 620)结合,抑制MCL1的m RNA及蛋白质表达(P0.01);沉默MCL1基因表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01).从而得出结论,mi R-125b在胃癌组织中低表达,其表达与胃癌组织分化程度、TNM分期、淋巴结转移及患者预后密切相关;mi R-125b靶向抑制MCL1基因表达,活化caspase-3信号通路,抑制MGC-803细胞增殖.     

Involvement of methylation of group of miRNA genes in regulation of expression of <Emphasis Type="Italic">RAR-beta2</Emphasis> and <Emphasis Type="Italic">NKIRAS1</Emphasis> target genes in lung cancer

To date, there are more than 2000 known human miRNAs, each of which may be involved in the regulation of hundreds of protein-coding target genes. In turn, the methylation of CpG islands affects the miRNA gene expression. Our aim was to evaluate the role of methylation in the regulation of miRNA gene expression and, consequently, in the regulation of the expression of target genes in primary lung tumors. Using a common collection of samples of non-small-cell lung cancer, we have performed a comprehensive study, including an analysis of the methylation status and level of expression of some miRNA genes and their potential target genes on chromosome 3, i.e., RAR-beta2 and NKIRAS1. The increased frequency of methylation in lung tumors compared to histologically normal tissue was revealed for miR-9-1 and miR-34b/c genes with significant statistics (P < 0.05 by Fisher’s exact test) and for miR-9-3 and miR-193a was marginally significant (P < 0.1). A significant correlation was revealed between the changes in methylation and level of expression of miR-9-1 gene (P ≈ 5 × 10⁻¹² by Spearman) in lung tumors, which suggests the role of methylation in the regulation of expression of these miRNA genes. Furthermore, a statistically significant negative correlation (P ≈ 3 × 10⁻¹² to 5 × 10⁻¹³ by Spearman) was found between changes in the levels of expression of miR-9-1 and miR-17 and RAR-beta2 target genes, as well as between the changes in the level of expression of miR-17 and NKIRAS1 that were not previously analyzed. The inverse relationship between the levels of expression of miRNA genes and their target genes is consistent with the known mechanism of the suppression of the expression of protein-coding genes under the action of miRNA. For the first time, significant correlations (P ≈ 3 × 10⁻¹⁰ to 4 × 10⁻¹³ by Spearman) were shown between changes in the methylation status of miRNA genes (miR-9-1, miR-9-3, miR-34b/c, miR-193a) and the level of expression of the RAR-beta2 target gene and changes in the methylation status of miR-34b/c, and miR-193a and the level of expression of the NKIRAS1 target gene in the primary lung tumors, which suggests the possibility of indirect effects of the methylation of miRNA genes on the level of expression of target genes.     

Circulating microRNA-133, microRNA-17 and microRNA-25 in serum and its potential diagnostic value in gastric cancer

Gastric cancer is one of the most common malignancies in the world and is considered as the most lethal gastrointestinal cancer. microRNAs (miRNAs) can be very important in detecting a disease at an early stage. The aim of this study was to investigate the microRNA-17 (miR-17), miR-25, and miR-133b in the serum of gastric cancer subjects. Serum samples were obtained from 120 gastric cancers and 102 healthy subjects. We evaluated expression levels of miR-17, miR-25 and miR-133b by quantitative real-time polymerase chain reaction. Our results showed that in the patients with gastric cancer, the expression level of miR-17 and miR-25 were significantly increased compared with the control group (P < 0.5), while the expression level of miR-133b was significantly decreased in patient groups compared with control cases (P < 0.5). It seems that expression of miRNAs in Iranian patients with gastric cancer is similar to other patients in other populations. These findings suggested that miR-17, miR-25 and miR-133b could be introduced as potential diagnostic candidates for the detection in gastric cancer patients in the early stage.     

The Value of MiR-383, an Intronic MiRNA,as a Diagnostic and Prognostic Biomarker in Intestinal-Type Gastric Cancer

MicroRNAs, a class of gene expression regulatory non-coding RNAs, participate in the pathogenic mechanisms of gastric cancer which is one of the life-treating cancers. Due to its aberrant expression in some types of human cancer, miR-383 has the value of being investigated in relation to cancer treatment and diagnosis. MiR-383 is placed in intron of SGCZ, a protein-coding gene, which is subject to dysregulation in various diseases. The purpose of the current study was to investigate the contribution of miR-383 to intestinal-type gastric adenocarcinoma tumorigenesis. The expression level of miR-383 was investigated by qRT-PCR in pairs of tumorous and adjacent tumor-free tissues of 40 patients with gastric cancer during endoscopy. Also, the susceptibility of miR-383 as a tumor marker and the relationship between its aberrant expression and clinicopathological features were determined. qRT-PCR data showed that miR-383 was dysregulated during gastric tumorigenesis. MiR-383 was dramatically downregulated up to sevenfold in intestinal-type gastric adenocarcinoma compared with adjacent tumor-free tissues (P < 0.001). Misregulation of miR-383 did not reveal a significant correlation with clinical characteristics. The ROC area of 80% with 76% sensitivity and 84% specificity was determined by P < 0.001. The current study demonstrated downregulation of miR-383 in intestinal-type gastric adenocarcinoma. Downregulation of miR-383 might be used as a potential tumor marker for the diagnosis of gastric cancer or could be a potential target for gene therapy.     

A miR-570 binding site polymorphism in the B7-H1 gene is associated with the risk of gastric adenocarcinoma

Single nucleotide polymorphisms (SNPs) in putative microRNA (miRNA) target sites (miRSNPs) could affect the binding of miRNA with the target and contribute to the susceptibility of human cancers. However, the role of miRSNPs in gastric cancer susceptibility remains largely unknown. Since the over-expression of B7-H1 protein has been reported to be closely related to disease progression of gastric cancer, we investigated the possible role of miRSNPs at the 3′-untranslated region (3′-UTR) of B7-H1 in the risk of developing gastric cancer. In this association study on 205 gastric adenocarcinoma patients and 393 non-cancer controls, we found that the genotype distribution of a common C>G polymorphism (rs4143815) was significantly different between the cases and controls (P = 1.32 × 10^?8). Compared with CC homozygotes, GG homozygotes and G allele carriers showed 3.73-fold (P = 2.98 × 10^?8) and 1.85-fold (P = 0.002) increased risk of gastric adenocarcinoma, respectively. Stratified analyses indicated that variant genotypes had a strong association with the clinic-pathological features of gastric cancer including differentiation grade, depth of tumor infiltration, and tumor node metastasis (TNM) stage (P < 0.001). Luciferase reporter assay indicated that this SNP might be responsible for aberrant B7-H1 protein expression in gastric cancer by disrupting the interaction between miR-570 and B7-H1 mRNA. These results are consistent with our hypothesis and indicate that genetic polymorphisms influencing B7-H1 expression modify cancer susceptibility.     

Aberrant hypermethylation of miR-9 genes in gastric cancer

Carcinogenesis of the stomach involves multiple steps including genetic mutation or epigenetic alteration of tumor suppressor genes or oncogenes. Recently, tumor suppressive miRNAs have been shown to be deregulated by aberrant hypermethylation during gastric cancer progression. In this study, we demonstrate that three independent genetic loci encoding for miR-9 (miR-9-1, miR-9-2 and miR-9-3) are simultaneously modified by DNA methylation in gastric cancer cells. Methylation-mediated silencing of these three miR-9 genes can be reactivated in gastric cancer cells through 5-Aza-dC treatment. Subsequent analysis of the expression levels of miR-9 showed that it was significantly down-regulated in gastric cancers compared with adjacent normal tissues (P value < 0.005). A similar tendency toward a tumor-specific DNA methylation pattern was shown for miR-9-1, miR-9-2 and miR-9-3 in 72 primary human gastric cancer specimens. Ectopic expression of miR-9 inhibited cell proliferation, migration and invasion, suggesting its tumor suppressive potential in gastric cancer progression.     

Epigenetic silencing of miR-203 in Kazakh patients with esophageal squamous cell carcinoma by MassARRAY spectrometry

Dysregulation of miR-203 by promoter methylation is associated with the development of various cancers. We aimed to explore the underlying link between promoter methylation and miR-203 expression in Kazakh esophageal squamous cell carcinoma (ESCC). MassARRAY® System spectrometry was used to quantitatively analyze the DNA methylation of 32 CpG sites within miR-203 in 99 Kazakh ESCC and 46 normal esophageal tissues (NETs) with similar population characteristics. We conducted real-time PCR to detect miR-203 expression levels and evaluated their association with methylation. Eleven CpG units within miR-203 promoter were frequently hypermethylated in ESCC compared with NETs (P < 0.05). The hypermethylation of several CpG units positively correlated with age, lower esophagus, constrictive type of ESCC, and moderately differentiated ESCC. Given the involvement of human papillomavirus (HPV) in etiology of ESCC was confirmed from our previous reports, herein we found that CpG units within miR-203 in HPV16-positive ESCC are more heavily methylated. Furthermore, miR-203 expression showed a nearly 4.5-fold decrease in ESCC than NETs (0.206 ± 0.336 vs. 0.908 ± 1.424, P < 0.001) and was significantly associated with lymph node metastasis (P = 0.012). The expression of miR-203 with 11 completely hypermethylated CpG units was approximately 6.5-fold lower than that with at least 1 unmethylated CpG unit (P < 0.001) and especially the CpG_15.16 and CpG_31.32 with higher methylation levels in ESCC tissues exhibited lower expression levels of miR-203, which indicated a reverse association between miR-203 methylation and expression. Hypermethylated miR-203 is a potential biomarker and targeted delivery of miR-203 could therefore serve as a preventive or therapeutic strategy for Kazakh ESCC.     

hTERT methylation and expression in gastric cancer

Gastric cancer is the second most prevalent cause of cancer death worldwide. DNA methylation is a common event in gastric carcinogenesis. hTERT seems to be the rate-limiting determinant of telomerase activation, which is responsible for stability and life span. hTERT hypermethylation has been associated with telomerase expression. In the present study, we investigated the promoter methylation status and hTERT protein expression in gastric cancer and normal mucosa samples. One hundred and nine gastric cancer and 53 normal mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analysed using peroxidase in 55 gastric cancer and 18 normal gastric mucosa samples. This is the first study evaluating hTERT methylation status in gastric carcinogenesis. We did not observe hTERT protein expression in normal gastric mucosa. Moreover, hTERT expression was observed in 80% of tumours and was associated with gastric cancer (p?<?0.0001). Partial methylation was the most frequent pattern in gastric samples, even in normal mucosa. The frequency of specimens presenting hypermethylation was significantly higher in tumours than in normal mucosa samples (p?=?0.0002), although the presence of hypermethylated promoter was not associated with a higher frequency of hTERT expression. A low correlation between hTERT protein expression and methylation was verified in gastric cancer samples. There was a clear difference in the frequency of hTERT expression and methylation within tumoral and non-tumoral tissues. Methylation status and telomerase expression may be useful for the diagnosis of gastric cancer and may have an impact on the anti-telomerase strategy for cancer therapy.     

The effect of decitabine on the expression and methylation of the PPP1CA,BTG2, and PTEN in association with changes in miR-125b,miR-17, and miR-181b in NALM6 cell line

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent pediatric cancer. DNA methylation and changes in the microRNAs (miRNAs) expression are known to be important causes of B-ALL. Decitabine as a DNA methyltransferase inhibitor agent is able to induce hypomethylation in several tumor suppressor genes. Much evidence has proven BTG2, PPP1CA, and PTEN act as tumor suppressor genes in many malignancies. In this case control study, the messenger RNA (mRNA) expression of PPP1CA, BTG2, and PTEN genes using quantitative real-time polymerase chain reaction (rRT-PCR) in Nalm6 cell line and five patients suffer from ALL with mean age 5.6 years were determined in compare with seven normal healthy donors age and sex matched. qRT-PCR analysis revealed that the expression levels of PPP1CA, BTG2, and PTEN genes were significantly decreased in Nalm6 ([FC] = 0.46, [FC] = 0.046, [FC] = 0.54) and according to the Methylation-specific PCR (MSP) analysis, these genes were hypermethylated in Nalm6. In next step, the effects of decitabine treatment on the methylation and expression of these genes in association with changes in miR-125b, miR-17, and miR-181b expression levels were evaluated in optimal concentration 2.5 µM of decitabine. Our data showed that decitabine is able to restore the expression levels of aforementioned genes and downregulate expression levels of oncomiRs; including miR-125b, miR-17, and miR-181b in Nalm6 cell line. Therefore, it seems that decitabine can be used as a potential drug for the first line treatment of patients with B-ALL, but further in vivo investigation is necessary.     

抑癌因子miR-10a抑制Tiam1表达对胃癌细胞凋亡和迁移的影响

目的:探讨miR-10a抑制Tiam1表达对胃癌细胞凋亡和侵袭的影响。方法:获取胃上皮组织细胞及胃癌组织细胞,利用q PCR及Western blot实验检测两种细胞中mi R-10a表达与Tiam1的m RNA及蛋白表达水平,同时检测胃癌细胞S746T及正常胃粘膜细胞RGM-1和NGEC中mi R-10a表达与Tiam1蛋白表达水平。通过将mi R-10a mimic和mi R-10a inhibitor转染HS746T细胞,利用流式细胞术检测HS746T的细胞周期和细胞凋亡,TranswellTM实验检测HS746T细胞的侵袭能力,qPCR及Western blot实验检测凋亡相关蛋白caspase3、caspase9和Bax以及周期相关蛋白P21表达水平;荧光素酶活性分析实验检测Tiam1是mi R-10a的作用靶点。已构建的Tiam1高表达的Tiam1-pcDNA3.1质粒和敲除Tiam1基因的PX458质粒分别转染HS746T细胞,通过流式细胞术及TranswellTM实验检测HS746T细胞的凋亡及侵袭能力。结果:与胃上皮组织细胞相比,早期胃癌临床组织细胞中mi R-10a表达降低,Tiam1的m RNA及蛋白表达升高;mi R-10a的表达与早期胃癌患者的肿瘤转移密切相关,与年龄、性别和肿瘤分期无关;与正常胃粘膜细胞RGM-1和NGEC相比,胃癌细胞HS746T中的mi R-10a表达降低,而Tiam1蛋白表达升高;mi R-10a可抑制HS746T细胞侵袭,促进细胞凋亡,使其停滞于G0/G1期;mi R-10a靶向作用于Tiam1基因的3'非翻译区(3'UTR),减少Tiam1的蛋白表达;Tiam1可抑制HS746T细胞凋亡,促进HS746T细胞侵袭。结论:mi R-10a靶向作用于Tiam1基因的3'UTR,抑制HS746T细胞的增殖及侵袭,促进HS746T细胞凋亡。     

MiR-19a/b modulate the metastasis of gastric cancer cells by targeting the tumour suppressor MXD1

The microRNAs 19a and 19b, hereafter collectively referred to as miR-19a/b, were recognised to be the most important miRNAs in the oncomiRs—miR-17-92 cluster. However, the exact roles of miR-19a/b in cancers have not been elucidated. In the present study, miR-19a/b was found to be over-expressed in gastric cancer tissues and significantly associated with the patients'' metastasis of gastric cancer. Using gain or loss-of-function in in vitro and in vivo experiments, a pro-metastatic function of miR-19a/b was observed in gastric cancer. Furthermore, reporter gene assay and western blot showed that MXD1 is a direct target of miR-19a/b. Functional assays showed that not only MXD1 had an opposite effect to miR-19a/b in the regulation of gastric cancer cells, but also overexpression of MXD1 reduced both miR-19a/b and c-Myc levels, indicating a potential positive feedback loop among miR-19a/b, MXD1 and c-Myc. In conclusion, miR-17-92 cluster members miR-19a/b facilitated gastric cancer cell migration, invasion and metastasis through targeting the antagonist of c-Myc -- MXD1, implicating a novel mechanism for the malignant phenotypes of gastric cancer.     

microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.     

Long non-coding RNA ZFAS1 regulates the malignant progression of gastric cancer via the microRNA-200b-3p/Wnt1 axis

Gastric cancer is a common malignant tumor. Studies from our laboratory or others have shown that long non-coding RNA (lncRNA) zinc finger antisense (ZFAS)1 often acts as an oncogene. However, the molecular underpinnings of how ZFAS1 regulates gastric cancer remain to be elucidated. Results showed that ZFAS1 expression was upregulated, and microRNA-200b-3p (miR-200b) expression was downregulated in gastric cancer tissues. MiR-200b overexpression suppressed the proliferation, cell cycle process, and Wnt/β-catenin signaling of gastric cancer cells. Subsequently, we identified miR-200b is a target of ZFAS1 and Wnt1 is a target of miR-200b. Furthermore, promotion of cancer malignant progression and activation of Wnt/β-catenin signaling induced by ZFAS1 was counteracted by increasing miR-200b expression. In vivo, ZFAS1 knockdown suppressed the tumorigenesis with the upregulated miR-200b and the inactive Wnt/β-catenin signaling. Summarily, we demonstrated a critical role of miR-200b in gastric cancer, and ZFAS1 can promote malignant progression through regulating miR-200b mediated Wnt/β-catenin signaling.