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While a number of coding genes have explained the anticancer activity of ginsenoside Rh2, little is known about noncoding RNAs. This study was performed to elucidate the regulatory activity of long noncoding RNA (lncRNA) CFAP20DC-AS1, which is known to be downregulated by Rh2. MiR-3614-3p, which potentially binds CFAP20DC-AS1, was screened using the LncBase Predicted program, and the binding was verified by assaying the luciferase activity of a luciferase/lncRNA recombinant plasmid construct. The competitive endogenous RNA (ceRNA) relationship of the two RNAs was further validated by quantitative PCR after deregulation of each RNA using siRNA. The effect of miRNA and target genes on the MCF-7 cancer cell growth was determined by monitoring proliferation and apoptosis in the presence of Rh2 after deregulating the corresponding gene. The miRNA decreased the luciferase activity of the luciferase/CFAP20DC-AS1 fusion vector, confirming the binding. SiRNA-based deregulation of CFAP20DC-AS1 attenuated the expression of miR-3614-3p and vice versa. In contrast to CFAP20DC-AS1, miR-3614-3p was upregulated by Rh2, inhibiting proliferation but stimulating apoptosis of the MCF-7 cells. Target genes of miR-3614-3p, BBX and TNFAIP3, were downregulated by Rh2 and the miRNA but upregulated by the lncRNA. Rh2 inhibits CFAP20DC-AS1, which obscures the association of the lncRNA with miR-3614-3p, resulting in the suppression of oncogenic BBX and TNFAIP3. Taken together, the Rh2/CFAP20DC-AS1/miR-3614-3p/target gene axis contributes to the antiproliferation activity of Rh2 in cancer cells. [ABSTRACT FROM AUTHOR]

Ginsenoside Rh2, a major bioactive ingredient abundant in red ginseng, has an antiproliferative effect on various cancer cells. In this study, we report a novel long noncoding RNA, C3orf67-AS1, which was identified as being hypermethylated at a CpG site of the promoter by Rh2 in MCF-7 cancer cells. Rh2-induced hypermethylation was responsible for the lower gene expression; the expression was recovered following treatment with a methyltransferase inhibitor, 5-aza-2′-deoxycytidine. When C3orf67-AS1 was downregulated by a siRNA, the cell growth rate was decreased, demonstrating the RNA's oncogenic activity. Accordingly, breast cancer patients showed a lower methylation and higher expression level of C3orf67-AS1. Within 800 kb flanking C3orf67-AS1 on the chromosome, eight genes were found, and four genes including C3orf67 (the sense strand gene of C3orf67-AS1) were downregulated by Rh2. In particular, C3orf67 was downregulated when C3orf67-AS1 was suppressed by a siRNA; however, the expression of C3orf67-AS1 was not affected by C3orf67. Taken together, this study identifies a novel noncoding RNA, C3orf67-AS1, of which the expression could be suppressed by Rh2 via promoter methylation, thereby mediating the anti-proliferative effect of the ginsenoside. [ABSTRACT FROM AUTHOR]

Ginsenoside Rg3 is a key metabolite of ginseng and is known to inhibit cancer cell growth. However, the epigenetics of CpG methylation and its regulatory mechanism have yet to be determined. Genome-wide methylation analysis of MCF-7 breast cancer cells treated with Rg3 was performed to identify epigenetically regulated genes and pathways. The effect of Rg3 on apoptosis and cell proliferation was examined by a colony formation assay and a dye-based cell proliferation assay. The association between methylation and gene expression was monitored by RT-PCR and Western blot analysis. Genome-wide methylation analysis identified the "cell morphology"-related pathway as the top network. Rg3 induced late stage apoptosis but inhibited cell proliferation up to 60%. Hypermethylated TRMT1L, PSMC6 and NOX4 were downregulated by Rg3, while hypomethylated ST3GAL4, RNLS and KDM5A were upregulated. In accordance, downregulation of NOX4 by siRNA abrogated the cell growth effect of Rg3, while the effect was opposite for KDM5A. Notably, breast cancer patients with a higher expression of NOX4 and KDM5A showed poor and good prognosis of survival, respectively. In conclusion, Rg3 deregulated tumor-related genes through alteration of the epigenetic methylation level leading to growth inhibition of cancer cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer recurrence caused by tamoxifen resistance hampers chemotherapy in breast cancer patients. The reasons behind the resistance were investigated by screening epigenetically regulated genes through analysis of methylation data from tamoxifen-resistant MCF-7 cells. MMP1 locus was found to be hypomethylated at a promoter CpG site and its expression was upregulated in the cell line, which was verified by the drug-resistant tumor tissues from breast cancer patients (n = 28). Downregulating MMP1 using a short hairpin RNA inhibited the growth of resistant cells and increased sensitivity to tamoxifen in vitro as well as in a xenografted mouse model in vivo. This study suggests that MMP1 is potentially a target gene to control tamoxifen resistance in breast cancer. Background: Tamoxifen (tam) is widely used to treat estrogen-positive breast cancer. However, cancer recurrence after chemotherapy remains a major obstacle to achieve good patient prognoses. In this study, we aimed to identify genes responsible for epigenetic regulation of tam resistance in breast cancer. Methods: Methylation microarray data were analyzed to screen highly hypomethylated genes in tam resistant (tamR) breast cancer cells. Quantitative RT-PCR, Western blot analysis, and immunohistochemical staining were used to quantify expression levels of genes in cultured cells and cancer tissues. Effects of matrix metalloproteinase-1 (MMP1) expression on cancer cell growth and drug resistance were examined through colony formation assays and flow cytometry. Xenografted mice were generated to investigate the effects of MMP1 on drug resistance in vivo. Results: MMP1 was found to be hypomethylated and overexpressed in tamR MCF-7 (MCF-7/tamR) cells and in tamR breast cancer tissues. Methylation was found to be inversely associated with MMP1 expression level in breast cancer tissues, and patients with lower MMP1 expression exhibited a better prognosis for survival. Downregulating MMP1 using shRNA induced tam sensitivity in MCF-7/tamR cells along with increased apoptosis. The xenografted MCF-7/tamR cells that stably expressed short hairpin RNA (shRNA) against MMP1 exhibited retarded tumor growth compared to that in cells expressing the control shRNA, which was further suppressed by tam. Conclusions: MMP1 can be upregulated through promoter hypomethylation in tamR breast cancer, functioning as a resistance driver gene. MMP1 can be a potential target to suppress tamR to achieve better prognoses of breast cancer patients. [ABSTRACT FROM AUTHOR]

Background: MTO1 and MRPL41 are nuclear-encoded mitochondrial genes encoding a mitochondrial tRNA-modifying enzyme and a mitochondrial ribosomal protein, respectively. Although both genes have been known to have potential roles in cancer, little is known about their molecular regulatory mechanism, particularly from an epigenetic approach. In this study, we aimed to address their epigenetic regulation through the estrogen receptor (ER) in breast cancer.Methods: Digital differential display (DDD) was conducted to identify mammary gland-specific gene candidates including MTO1 and MRPL41. Promoter CpG methylation and expression in breast cancer cell lines and tissues were examined by methylation-specific PCR and real time RT-PCR. Effect of estradiol (E2), tamoxifen, and trichostatin A (TSA) on gene expression was examined in ER + and ER- breast cancer cell lines. Chromatin immunoprecipitation and luciferase reporter assay were performed to identify binding and influencing of the ER to the promoters.Results: Examination of both cancer tissues and cell lines revealed that the two genes showed an opposite expression pattern according to ER status; higher expression of MTO1 and MRPL41 in ER- and ER+ cancer types, respectively, and their expression levels were inversely correlated with promoter methylation. Tamoxifen, E2, and TSA upregulated MTO1 expression only in ER+ cells with no significant changes in ER- cells. However, these chemicals upregulated MRPL41 expression only in ER- cells without significant changes in ER+ cells, except for tamoxifen that induced downregulation. Chromatin immunoprecipitation and luciferase reporter assay identified binding and influencing of the ER to the promoters and the binding profiles were differentially regulated in ER+ and ER- cells.Conclusions: These results indicate that different epigenetic status including promoter methylation and different responses through the ER are involved in the differential expression of MTO1 and MRPL41 in breast cancer. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of the study was to evaluate the methylation status of the interleukin-10 (IL-10) gene in breast cancer tissues compared with normal and benign breast disease tissues. Between 2000 and 2001, we used paraffin-embedded specimens of 30 normal, 31 benign and 72 breast cancer tissues from the National Cancer Center, Korea. The methylation patterns of the IL-10 gene were evaluated using bisulfite DNA sequencing and the expression levels of IL-10 mRNA were evaluated using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). The methylation rates of the IL-10 gene were significantly lower in malignant tumors than in benign and normal tissues (normal; 63.3%, benign; 74.2%, cancer; 45.8%, p = 0.02). The methylation density rates of the IL-10 gene were also significantly lower in malignant tumors (normal; 59.68 ± 7.12%, benign; 48.89 ± 7.45%, cancer; 30.56 ± 4.18%, p = 0.001). Tissues with aberrant methylation of the IL-10 gene showed significantly lower rates of mRNA expression compared with unmethylated cases (12.5% vs. 68.0%, p = 0.012). The mRNA expression of tissues with unmethylated IL-10 was upregulated approximately ten thousand-fold compared to those with IL-10 methylation in the real-time RT-PCR experiment. IL-10 methylation demonstrated a significant association with lower expression of Ki-67 (9.36 ± 2.43 vs. 19.68 ± 3.42, p = 0.02). IL-10 methylation in cancer tissues is lower than that in normal and benign breast tissues, and DNA hypomethylation in the gene influences gene activation. Our data suggest that hypomethylation of the IL-10 gene can be involved in the process of breast carcinogenesis. [Copyright &y& Elsevier]

Ginsenoside Rg3 exerts antiproliferation activity on cancer cells by regulating diverse noncoding RNAs. However, little is known about the role of long noncoding RNAs (lncRNAs) or their relationship with competitive endogenous RNA (ceRNA) in Rg3-treated cancer cells. Here, a lncRNA (ATXN8OS) was found to be downregulated via Rg3-mediated promoter hypermethylation in MCF-7 breast cancer cells. SiRNA-induced downregulation of ATXN8OS decreased cell proliferation but increased apoptosis, suggesting that the noncoding RNA possessed proproliferation activity. An in silico search for potential ATXN8OS-targeting microRNAs (miRs) identified a promising candidate (miR-424-5p) based on its high binding score. As expected, miR-424-5p suppressed proliferation and stimulated apoptosis of the MCF-7 cells. The in silico miR-target-gene prediction identified 200 potential target genes of miR-424-5p, which were subsequently narrowed down to seven that underwent hypermethylation at their promoter by Rg3. Among them, three genes (EYA1, DACH1, and CHRM3) were previously known oncogenes and were proven to be oppositely regulated by ATXN8OS and miR-424-5p. When taken together, Rg3 downregulated ATXN8OS that inhibited the tumor-suppressive miR-424-5p, leading to the downregulation of the oncogenic target genes. [ABSTRACT FROM AUTHOR]

Abstract Cold atmospheric plasma (CAP) has gained attention for use in cancer treatment owing to its ability to preferentially induce cancer cell death; however, the involved molecular mechanism remains to be elucidated. Herein, an epigenetic effect of CAP on cancer cells was examined by performing a genome-wide ChIP-seq for H3K4me3 in MCF-7 breast cancer cell line. Consequently, 899 genes showed significantly changed methylation level at H3K4 with constructing "Cellular Compromise, DNA Replication, Recombination, Repair, and Cell Cycle" as the top network. Comparisons with expression array data revealed a coincidence between histone modification and gene expression for 18 genes, and the association was confirmed by ChIP-PCR and qRT-PCR for selected genes. The expression of the affected genes, such as HSCB and PRPS1, was recovered when a histone demethylase JARID1A was inhibited. Furthermore, JARID1A was induced by CAP via the reactive oxygen species signaling. The two genes are known as oncogenes and show a higher expression in breast cancer tissue, and this was supported by the decreased colony formation ability of MCF-7 cells when the cells were treated with siRNAs against each gene. Taken together, these data indicate that CAP inhibits cancer cell proliferation by modulating the methylation level of H3K4 corresponding to oncogenes. Graphical abstract fx1 Highlights • Cold atmospheric plasma induced genome-wide alteration of H3K4me3 in the MCF-7. • The H3K4me3 of HSCB and PRPS1 was increased by cold atmospheric plasma. • Inhibition of a histone demethylase (JARID1A) recovered expression of HSCB and PRPS1. • Cold atmospheric plasma inhibits cancer cell proliferation by modulating H3K4me3. [ABSTRACT FROM AUTHOR]

Cancer recurrence, which is frequently accompanied by chemotherapy, has been a challenge in cancer treatment. This study was carried out to examine the potential applications of the reactive oxygen species (ROS)-producing cold atmospheric plasma (CAP) to overcome the cancer cells’ drug resistance, which has been emerging as an alternative therapeutic tool for cancer. For this, we developed a tamoxifen (Tam)-resistant MCF-7 (MCF-7/TamR) breast cancer cell model and examined the effect of CAP on the recovery of Tam sensitivity at the cellular and molecular level. The ROS level was increased 1.9-fold in CAP-treated MCF-7/TamR cells compared to the non-treated cell. CAP was proven to restore sensitivity by up to 50% for MCF-7/TamR cells against Tam after CAP treatment. The comparison of genome-wide expression between the acquisition of Tam resistance and CAP treatment identified 20 genes that commonly showed significant expression changes. Notably, all the genes except two have been oppositely dysregulated in the two cellular statuses, and the majority of them are known to contribute to the acquisition of Tam resistance. The protein expression of selected genes, MX1 and HOXC6, was recovered to that of their parental cell by CAP. Furthermore, the dysregulation of MX1 and HOXC6 in MCF-7/TamR alleviated the drug sensitivity recovery effect of CAP. Taken together, CAP inhibited the growth of Tam-resistant MCF-7 cancer cells and reset it to the Tam-sensitive status by restoring the expression of drug resistance–related genes. These findings may lend credence to CAP as an alternative or complementary tool in the treatment or prevention of Tam-resistant cancer. [ABSTRACT FROM AUTHOR]