Tumor-suppressive microRNA silenced by tumor-specific DNA hypermethylation in cancer cells

MicroRNA (miRNA) genes, located in intergenic or intragenic non-coding regions of the genome, are transcribed and processed to small non-protein-coding RNA of approximately 22 nucleotides negatively regulating gene expression. Some miRNA have already been reported for their genetic alterations, aberrant expression and oncogenic or tumor-suppressive functions. After 2008, there has been a striking increase in the number of publications reporting tumor-suppressive miRNA (TS-miRNA) silenced epigenetically in various types of cancers, suggesting important clinical applications for miRNA-based molecular diagnosis and therapy for cancers. Here, we introduce a correlation of the gene silencing of TS-miRNA through CpG island hypermethylation with the genomic distances between intergenic and intragenic miRNA genes or protein-coding host genes and CpG islands located around these genes. Furthermore, we also discuss the potential of miRNA replacement therapy for cancers using double-stranded RNA mimicking TS-miRNA.     

CHD5, a tumor suppressor that is epigenetically silenced in lung cancer

Chromodomain helicase DNA binding protein 5 (CHD5) is a potent tumor suppressor that serves as a master regulator of a tumor-suppressive network. Examination of the role played by CHD5 in a wide range of human cancers is warranted. In this study, we focused on the epigenetic modification and tumor-suppressive role of CHD5 in lung cancer. We measured CHD5 mRNA and protein expression in lung cancer cells, lung cancer tissues, and their corresponding noncancerous lung tissues using real-time PCR and Western blot analysis. We then determined the methylation status of the CHD5 promoter in these samples using methylation-specific sequencing and analyzed CHD5 re-expression in lung cancer cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. Next, the lung cancer cell clones stably expressing EGFP-CHD5 protein or EGFP protein, respectively, were obtained and the effects of restored CHD5 expression on cell proliferation, colony formation, and tumorigenicity were assessed. CHD5 expression ranged from low to absent in the lung cancer cell lines and tissues examined; the CHD5 promoter was hyperethylated in these samples. Treatment with 5-aza-dC resulted in a localized decrease in methylation density and an increase in CHD5 expression. Clonogenicity and tumor growth were abrogated in A549 and H1299 cells upon restoration of CHD5 expression. A significant reduction in clonogenicity was observed; an average of 47.83 ± 4.6% reduction for A549-EGFP-CHD5 was observed compared to A549-EGFP, and an average of 56.39 ± 5.3% reduction for H1299-EGFP-CHD5 was observed compared to H1299-EGFP. A549-EGFP exhibited an average tumor size of 452.3 ± 36.5 mm(3), whereas A549-EGFP-CHD5 exhibited an average tumor size of only 57.7 ± 18.5 mm(3). Thus, our findings indicate that CHD5 is a potential tumor suppressor gene that is inactivated via an epigenetic mechanism in lung cancer.     

miR-152 functions as a tumor suppressor in colorectal cancer by targeting PIK3R3

Accumulating evidence showed that microRNA-152 (miR-152) was frequently downregulated, and functioned as tumor suppressor in many cancers, but little is known about its biological role and intrinsic regulatory mechanisms in colorectal cancer (CRC). Here, we explored the potential role of miR-152 in CRC and the possible molecular mechanisms. Our results proved that miR-152 expression was downregulated in CRC cell lines and tissue samples, and its expression was inversely correlated with advanced tumor-node-metastasis (TNM) stage (P?<?0.01) and lymph node metastasis (P?<?0.01). Function assays demonstrated that restoring the expression of miR-152 in CRC cells dramatically reduced the cell proliferation and cell migration and invasion and promoted apoptosis and caspase-3 activity in vitro, as well as suppressed tumor growth in vivo. Mechanistic investigations defined phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) as a direct and functional downstream target of miR-152. In addition, we also found that PIK3R3 expression was upregulated and was inversely correlated with miR-152 expression in clinical CRC tissues. Downregulation of PIK3R3 mimicked the tumor-suppressive effects of miR-152 overexpression in CRC cells. Taken together, these results elucidated the function of miR-152 in CRC progression and suggested that miR-152 might function as tumor suppressor in CRC by targeting PIK3R3.     

Not hMSH2 but hMLH1 is frequently silenced by hypermethylation in endometrial cancer but rarely silenced in pancreatic cancer with microsatellite instability

Disruption of the DNA mismatch repair (MMR) system has been found to play an important role in sporadic human cancers of several organs such as colorectum, stomach, endometrium, and pancreas. In cancers of the former three organs, disruption of the MMR system is mainly caused by hypermethylation of the hMLH1 gene. We investigated the expression of the hMLH1 and hMSH2 proteins immunohistochemically in pancreatic and endometrial cancers with high frequency microsatellite instability (MSI-H). Loss of expression of hMLH1 was found in none of seven pancreatic cancer, whereas eight (57%) of 14 endometrial cancer showed loss of expression of hMLH1. On the other hand, one (14%) of seven pancreatic cancers and two (14%) of 14 endometrial cancers showed loss of hMSH2 expression. We further analyzed the methylation status at the promoter region of the hMLH1 and hMSH2 genes and found hypermethylation of hMLH1 at the promoter region in the great majority of endometrial cancers with loss of expression. However, no pancreatic cancer showed hypermethylation. We then further analyzed 22 pancreatic cancer cell lines and obtained similar results. These results suggested that MSI-H in pancreatic cancer is probably caused by different mechanisms from those of other sporadic cancers with MSI-H.     

Klotho is silenced through promoter hypermethylation in gastric cancer

As one of major epigenetic changes to inactivate tumor suppressor genes in human carcinogenesis, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes and predict the prognosis of cancer patients. In the present study, we found KL (klotho) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. KL expression was downregulated in primary gastric carcinoma tissues (n=22, p<0.05) and all of gastric cancer cells lines examined. Ectopic expression of KL inhibited the growth of gastric cancer cells partially through the induction of apoptosis, demonstrating a tumor suppressive role of KL in gastric cancer. Demethylation with 5-aza-2'-deoxycytidine (Aza) increased KL expression and KL promoter was hypermethylated in gastric cancer cell lines as well as some of primary gastric carcinoma tissues (47/99) but none of normal gastric tissues. Importantly, promoter methylation of KL was significantly associated with the poor outcome of gastric cancer patients (p=0.025, Log-rank test), highlighting the relevance of epigenetic inactivation of KL in gastric carcinogenesis. As a summary, we found that KL is a novel tumor suppressor gene epigenetically inactivated in gastric cancer and promoter methylation of KL could be used to predict the prognosis of gastric cancer patients.     

ADAM23, a possible tumor suppressor gene, is frequently silenced in gastric cancers by homozygous deletion or aberrant promoter hypermethylation

Array-based comparative genomic hybridization (CGH-array) has a powerful potential for high-throughput identification of genetic aberrations in cell genomes. We identified a homozygous loss of ADAM23 (2q33.3) in the course of a program to screen a panel of gastric cancer (GC) cell lines (1/32, 3.1%) for genomic copy-number aberrations using our custom-made CGH-array. Infrequent homozygous deletion of ADAM23 was also seen in primary gastric tumors (1/39, 2.6%). ADAM23 mRNA was expressed in normal stomach tissue, but not in the majority of GC cell lines without homozygous deletion of this gene. Expression of ADAM23 mRNA was restored to gene-silenced GC cells after treatment with 5-aza 2'-deoxycytidine. The methylation status of the ADAM23 CpG island, which showed promoter activity, correlated inversely with its expression. Methylation of this CpG island was observed both in GC cell lines and in primary GC tissues; in primary tumors with a hypermethylated CpG island, expression of ADAM23 was lower than in adjacent noncancerous tissues. Moreover, restoration of ADAM23 in GC cells reduced their numbers in colony-formation assays. These results suggest that genetic or epigenetic silencing by hypermethylation of the ADAM23 CpG-rich promoter region leads to loss of ADAM23 function, which may be a factor in gastric carcinogenesis.     

Peperomin E reactivates silenced tumor suppressor genes in lung cancer cells by inhibition of DNA methyltransferase

Advanced lung cancer has poor prognosis owing to its low sensitivity to current chemotherapy agents. Therefore, discovery of new therapeutic agents is urgently needed. In this study, we investigated the antitumor effects of peperomin E, a secolignan isolated from Peperomia dindygulensis, a frequently used Chinese folk medicine for lung cancer treatment. The results indicate that peperomin E has antiproliferative effects, promoting apoptosis and cell cycle arrest in non‐small‐cell lung cancer (NSCLC) cell lines in a dose‐dependent manner, while showing lower toxicity against normal human lung epidermal cells. Peperomin E inhibited tumor growth in A549 xenograft BALB/c nude mice without significant secondary adverse effects, indicating that it may be safely used to treat NSCLC. Furthermore, the mechanisms underlying the anticancer effects of peperomin E have been investigated. Using an in silico target fishing method, we observed that peperomin E directly interacts with the active domain of DNA methyltransferase 1 (DNMT1), potentially affecting its genome methylation activity. Subsequent experiments verified that peperomin E decreased DNMT1 activity and expression, thereby decreasing global methylation and reactivating the epigenetically silenced tumor suppressor genes including RASSF1A, APC, RUNX3, and p16INK4, which in turn activates their mediated pro‐apoptotic and cell cycle regulatory signaling pathways in lung cancer cells. The observations herein report for the first time that peperomin E is a potential chemotherapeutic agent for NSCLC. The anticancer effects of peperomin E may be partly attributable to its ability to demethylate and reactivate methylation‐silenced tumor suppressor genes through direct inhibition of the activity and expression of DNMT1.     

miR-23b as a potential tumor suppressor and its regulation by DNA methylation in cervical cancer

Background

The aberrant expression of miR-23b is involved in the development and progression of cancer. The aim of this study was to evaluate the potential role of methylation in the silencing of miR-23b in cervical cancer cell lines and to determine its expression in stages of malignant progression and in cervical cancer tissues HPV16-positive.

Methods

The methylation of the miR-23b promoter was determined in HeLa, SiHa, CaSki and C33A cells using a Human Cancer miRNA EpiTectMethyl II Signature PCR Array®. The cells were treated with 5-Aza-2′-deoxycytidine, and the expression of miR-23b, uPa, c-Met and Zeb1 was determined by qRT-PCR. miR-92a and GAPDH were used as controls. The expression of miR-23b was determined in cervical scrapes and biopsies of women without squamous intraepithelial lesions, with precursor lesions and with cervical cancer, all were HPV16-positive. The Fisher exact and Mann–Whitney tests were used to compare the differences of the expression of miR-23b, uPa, c-Met and Zeb1 among cell groups, and the difference among patients, respectively. The association between the expression of miR-23b and cervical cancer was determined by logistic regression with a confidence level of 95 %. A value of p?<?0.05 was considered statistically significant.

Results

In C33A, HeLa and CaSki cells, methylation was associated with decreased expression of miR-23b. After treatment with 5-Aza-CdR, the expression of miR-23b increased in all cell lines and the expression of c-Met decreased in HeLa cells, while uPa and Zeb1 decreased in C33A and CaSki cells. In SiHa cells the expression of uPa, c-Met and Zeb1 increased. The expression of miR-23b decreased in relation to the increase in the severity of the lesion and was significantly lower in cervical cancer. In women with premalignant lesions HPV16-positive, decreased levels of miR-23b increased the risk of cervical cancer (OR?=?36, 95 % CI?=?6.7-192.6, p?<?0.05).

Conclusions

The results suggest that the expression of miR-23b is regulated by the methylation of its promoter and is possible that this microRNA influence the expression of uPa, c-Met and Zeb1 in cervical cancer cells lines. In women with premalignant lesions and cervical cancer infected with HPV16, the expression level of miR-23b agree with a tumor suppressor gene.

     

The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma

Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.     

Cytoglobin is upregulated by tumour hypoxia and silenced by promoter hypermethylation in head and neck cancer

Background:

Cytoglobin (Cygb) was first described in 2002 as an intracellular globin of unknown function. We have previously shown the downregulation of cytoglobin as a key event in a familial cancer syndrome of the upper aerodigestive tract.

Methods:

Cytoglobin expression and promoter methylation were investigated in sporadic head and neck squamous cell carcinoma (HNSCC) using a cross-section of clinical samples. Additionally, the putative mechanisms of Cygb expression in cancer were explored by subjecting HNSCC cell lines to hypoxic culture conditions and 5-aza-2-deoxycitidine treatment.

Results:

In clinically derived HNSCC samples, CYGB mRNA expression showed a striking correlation with tumour hypoxia (measured by HIF1A mRNA expression P=0.013) and consistent associations with histopathological measures of tumour aggression. CYGB expression also showed a marked negative correlation with promoter methylation (P=0.018). In the HNSCC cell lines cultured under hypoxic conditions, a trend of increasing expression of both CYGB and HIF1A with progressive hypoxia was observed. Treatment with 5-aza-2-deoxycitidine dramatically increased CYGB expression in those cell lines with greater baseline promoter methylation.

Conclusion

We conclude that the CYGB gene is regulated by both promoter methylation and tumour hypoxia in HNSCC and that increased expression of this gene correlates with clincopathological measures of a tumour''s biological aggression.     

miR-488 acts as a tumor suppressor gene in gastric cancer

MicroRNAs (miRNAs) are small, non-coding RNAs that modulate development, cell proliferation, and apoptosis. The deregulated expression of microRNAs is found in carcinogenesis including gastric cancer (GC). In this study, we showed that the expression levels of miR-488 were downregulated in GC tissues compared to in non-tumor tissues. In addition, the expression of miR-488 was also lower in GC cell lines in contrast with the gastric epithelial cell line (GES). In addition, the expression level of miR-488 was negatively correlated with the TNM stage in GC patients, and lower miR-488 expression was found in tumors with advanced TNM stage. The ectopic expression of miR-488 suppressed the GC cell proliferation, cell cycle, colony information, and migration. PAX6 was identified as a direct target gene of miR-488 in HGC-27. Moreover, we found that the expression level of PAX6 was upregulated in the GC tissues compared with the non-tumor tissues. The PAX6 expression level was correlated with the cancer TNM stage, and higher PAX6 expression was found in tumors with advanced TNM stage. Furthermore, there was an inverse correlation between PAX6 and miR-488 expression levels in GC tissues. Therefore, these studies demonstrated that miR-488 might act as a tumor suppressor miRNA in the development of GC.     

Down-regulation of miR-212 expression by DNA hypermethylation in human gastric cancer cells

There has been few report discussing the expression and function of miR-212 in gastric cancer (GC). The aim of this pilot study was to investigate the expression of miR-212 in both gastric cancer tissues and gastric cancer cells and further explores the possible reasons for this change and the impact on the development of gastric cancer. qRT-PCR was used to detect the expression of miR-212 in primary GC tissues, adjacent normal tissues, gastric cancer cell lines BGC-823, SGC-7901, MKN-45, and normal gastric mucosa cell line GES. The expression of miR-212 was evaluated before and after treatment with methylation inhibitor-5-Aza-2'-deoxycitidine (5-Aza-dC), finally anti-miRNA and dual luciferase reporter assay were used to prove that MYC is a target gene of miR-212. The results showed that a significant reduction of miR-212 expression in GC tissues was observed compared to that in normal tissues (P = 0.002). At the same time, miR-212 expression level in normal gastric mucosa cell line GES was higher than that of in gastric cancer cell lines BGC-823, SGC-7901, and MKN-45 (P = 0.015, 0.008, 0.044, respectively). Computer sequence analysis showed the hypermethylation of CpG islands(CPI) in the promoter regions of miR-212 led to the lower expression of miR-212 in gastric cell strains (BGC-823 and SGC-7901). MiR-212 expression was significantly recovered after treatment with methylation inhibitor 5-Aza-dC (P = 0.016, 0.000, 0.015, respectively). Then, the results of AMOs transfection and dual luciferase reporter assay showed that Myc is a target of miR-212, which will be helpful to verify the function of miR-212 in carcinogenesis. The conclusion could be deduced from the study that decreased expression of miR-212 may be due to hypermethylation of CPI in gastric cancer cells, and miR-212 might act on the progression of gastric cancer through the potential target gene Myc.