Ras Association Domain Family 1A Research Articles

A noninvasive method for the detection of foetal DNA in early pregnancy based on differential methylation pattern of Ras association domain family member 1A

Background The detection of foetal DNA and extravillus trophoblasts (EVTs) in early pregnancy in cervical and uterine samples offers a potential pathway for non-invasive prenatal diagnostics. However, the challenge lies in effectively quantifying these samples. This study introduces a novel approach using the Ras association domain family 1 A (RASSF1A), which exhibits hypermethylation in foetal cells and hypomethylation in maternal cells. The differential methylation pattern of RASSF1A provides a unique biomarker for quantifying foetal cells in cervical and intrauterine samples. Methods This study was conducted between September 2022 and May 2023. In total, 23 samples (12 cervical cell samples and 11 intrauterine samples) were collected from women in the Sichuan Jinxin Women & Children Hospital, Jingxiu District, Chengdu, China. The cervical cell samples were collected via lavage and brush techniques, and the intrauterine cell samples were obtained via uterine lavage. These samples were collected as part of a broader effort to advance our understanding of foetal cell dynamics during early pregnancy. The sampling methods were chosen for their minimally invasive nature and their potential in capturing a representative cell population from the respective sites. After digestion of the cell samples using a methylation-sensitive restriction enzyme cocktail, a critical step to differentiate between maternal and foetal DNA, the quantitative polymerase chain reaction (qPCR) of RASSF1A and β-actin (ACTB) were employed to measure foetal DNA and cell concentrations. Immunofluorescence techniques targeting histocompatibility complex, class I G (HLA-G) and GATA binding protein 3 (GATA-3) were employed to detect EVTs in the cell samples and in decidual tissue, with the latter providing an additional layer of confirmation for the presence of foetal cells. Results The results showed no hypermethylated RASSF1A was detected in any of the cervical samples, irrespective of whether the samples were obtained by brush or lavage. However, an average of 17,236 ± 7490 foetal cells per sample were detected in the uterine lavage samples. Foetal cells accounted for approximately 0.14% ± 0.10% of the total cell population in these samples. The presence of EVTs in these samples was confirmed by their expression of both HLA-G and GATA-3. Conclusion The detection of foetal cells in uterine cavity samples based on hypermethylation of RASSF1A and quantification of foetal cells can be used to prenatal screening. GATA-3 can be used to label EVTs.

RASSF1A promotes radiosensitivity in nasopharyngeal carcinoma by promoting FoxO3a and inhibiting the Nrf2/TXNRD1 signaling pathway.

Radiotherapy is widely used as the first-line treatment for nasopharyngeal carcinoma (NPC). However, the resistance of some patients to treatment lowers its clinical effectiveness. Compared to typical epithelial cells, NPC markedly lowers the Ras-association domain family 1A (RASSF1A) protein expression. RASSF1A overexpression sensitizes NPC cells to radiotherapy. Mechanistically, RASSF1A promotes the expression of Forkhead box O3a (FoxO3a) in the nucleus and inhibits the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway via binding to the Kelch-like ECH-associated protein 1 (Keap1) promoter. Through elevating intracellular ROS levels, RASSF1A overexpression inhibits the expression of thioredoxin reductase 1 (TXNRD1), a crucial Nrf2 target gene, and increases NPC sensitivity to radiation. Immunohistochemical staining of NPC tissue sections revealed that the expression of RASSF1A is negatively correlated with that of TXNRD1. The traditional Chinese medicine component andrographolide (AGP), which induces RASSF1A expression, increased the sensitivity of NPC cells to radiotherapy in vitro and in vivo. Our findings implied that RASSF1A increases the sensitivity of NPC to radiation by increasing FoxO3a expression in the nucleus, inhibiting the Nrf2/TXNRD1 signaling pathway, and elevating intracellular ROS levels. AGP targets RASSF1A and may be a promising adjuvant sensitizer for enhancing radiosensitivity in NPC.

Ras Association Domain Family 1A Gene Promoter Methylation as a Biomarker for Chronic Viral Hepatitis C-related Hepatocellular Carcinoma.

Background One of the most prevalent aberrant epigenetic modifications found in hepatocellular carcinoma (HCC) is abnormal DNA methylation. Our study aimed to evaluate serum Ras association domain family 1A (RASSF1A) gene promoter methylation in patients with chronic viral hepatitis C (HCV)-associated liver cirrhosis with and without HCC as a potential new marker for the early detection of HCC. Methodology The 60 participants who participated in the trial were divided into the following three groups: 20 patients with newly diagnosed primary HCC on top of HCV-related liver cirrhosis, 20 patients with HCV-related liver cirrhosis, and 20 age- and sex-matched healthy individuals as a control group. All participants underwent methylation-specific polymerase chain reaction testing to detect the blood level of the RASSF1A gene's methylated promoter. Results Methylated RASSF1A was found in 30% of patients with liver cirrhosis caused by HCV and in 65% of patients with HCC, but not in any of the controls. It was discovered that the serum methylation RASSF1A had an accuracy of 82.50% and an area under the curve (AUC) of 0.825 for separating HCC patients from healthy controls. With an AUC of 0.675 and an accuracy of 67.50%, it was able to differentiate patients with HCC from those with HCV-related liver cirrhosis. Additionally, there was no statistically significant association between RASSF1A methylation status and HCC mass size (p = 0.449). Conclusions Serum RASSF1A promoter methylation status detection could be useful for detecting HCC early, especially in high-risk individuals such as those with HCV.

DNA methylation influences the CTCF-modulated transcription of RASSF1A in lung cancer cells.

Ras-association domain family 1A (RASSF1A) is one of the most methylated genes in lung cancer (LC). We investigate whether the high DNA methylation level of RASSF1A can relieve the resistance of RASSF1A to LC by inhibiting RASSF1A's transcription factor binding to RASSF1A. RASSF1A expression in tissues and cells was tested utilizing quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. RASSF1A expression and RASSF1A methylation level in LC cells exposed to 5-Aza-dc were assessed by qRT-PCR and quantitative methylation-specific PCR. The association between CTCF and RASSF1A was assessed using hTFtarget, ChIP, and luciferase reporter gene analysis. The effects of 5-Aza-dc, CTCF, and RASSF1A on cell biological behaviors and epithelial-mesenchymal transition (EMT)-related markers were assessed by cell function experiments and Western blot. Moreover, we constructed the xenograft tumor and pulmonary nodule metastasis models, and assessed tumor volume and weight. RASSF1A expression and pulmonary nodule metastasis were tested utilizing qRT-PCR, Western blot, and H&E staining. RASSF1A was under-expressed in LC tissues and cells. 5-Aza-dc enhanced RASSF1A level and weakened RASSF1A methylation level in LC cells. RASSF1A silencing neutralized 5-Aza-dc-mediated repressing effects on LC cell biological function and EMT. The loss of CTCF binding to RASSF1A in LC cells was associated with DNA methylation. The effect of 5-Aza-dc on RASSF1A level, LC cell malignant behaviors, and EMT-related factors were strengthened by CTCF upregulation. RASSF1A overexpression suppressed LC tumor growth and pulmonary nodule metastasis in vivo. DNAmethylation blocked the modulation of RASSF1A expression by CTCF and relieved the resistance of RASSF1A to LC.

Role of MST1 in the regulation of autophagy and mitophagy: implications for aging-related diseases.

As a key mechanism to maintain cellular homeostasis under stress conditions, autophagy/mitophagy is related to the occurrence of metabolic disorders, neurodegenerative diseases, cancer, and other aging-related diseases, but the relevant signal pathways regulating autophagy have not been clarified. Mammalian sterile 20-like kinase 1 (MST1) is a central regulatory protein of many metabolic pathways involved in the pathophysiological processes of aging and aging-related diseases and has become a critical integrator affecting autophagic signaling. Recent studies show that MST1 not only suppresses autophagy through directly phosphorylating Beclin-1 and/or inhibiting the protein expression of silent information regulator 1 (SIRT1) in the cytoplasm, but also inhibits BCL2/adenovirus E1B protein-interacting protein 3 (BNIP3)-, FUN14 domain containing 1 (FUNDC1)-, and Parkin (Parkinson protein 2)-mediated mitophagy by interacting with factors such as Ras association domain family 1A (RASSF1A). Indeed, a common pharmacological strategy for anti-aging is to induce autophagy/mitophagy through MST1 inhibition. This article reviews the role and mechanism of MST1 in regulating autophagy during aging, to provide evidence for the development of drugs targeting MST1.

A panel of DNA methylation biomarkers for detection and improving diagnostic efficiency of lung cancer

Lung cancer remains the leading cause of cancer deaths worldwide. Although low-dose spiral computed tomography (LDCT) screening is used for the detection of lung cancer in a high-risk population, false-positive results of LDCT remain a clinical problem. Here, we developed a blood test of a novel panel of three established lung cancer methylation biomarkers for lung cancer detection. Short stature homeobox 2 gene (SHOX2), ras association domain family 1A gene (RASSF1A), and prostaglandin E receptor 4 gene (PTGER4) methylation was analyzed in a training cohort of 351 individuals (197 controls, 154 cases) and validated from an independent cohort of 149 subjects (89 controls, 60 cases). The novel panel biomarkers distinguished between malignant and benign lung disease at high sensitivity and specificity: 87.0% sensitivity [95% CI 80.2–91.5%], 98.0% specificity [95% CI 94.9–99.4%]. Sensitivity in adenocarcinoma, squamous cell carcinoma, small cell lung cancer, and other lung cancer was 89.0%, 87.5%, 85.7%, and 77.8%, respectively. Notably, cancer patients in stage I and II showed high diagnostic sensitivity at 82.5% and 90.5%, respectively. Moreover, the diagnostic efficiency did not show bias toward age, gender, smoking, and the presence of other (nonlung) cancers. The performance of the panel in the validation cohort confirmed the diagnostic value. These findings clearly showed that this panel of DNA methylation biomarkers was effective in detecting lung cancer noninvasively and may provide clinical utility in stand-alone or in combination with current imaging techniques to improve the diagnosis of lung cancer.

REX-1 Represses RASSF1a and Activates the MEK/ERK Pathway to Promote Tumorigenesis in Prostate Cancer.

Epigenetics play an important role in the pathogenesis of prostate cancer; it is urgent to investigate vital transcription factors in methylation regulation with the aim to develop novel treatment strategies targeting prostate cancer. As a member of the zinc finger protein family, REX-1 (reduced expression-1) is a transcription factor that has been reported to be closely linked to the development of several cancers. So far, the expression level and precise function of REX-1 in prostate cancer remain largely unknown. Here, we show that REX-1 was overexpressed in prostate cancer clinical tissues, and its expression level was closely correlated with patient prognosis. REX-1 affected prostate tumor growth in vivo by MEK/ERK phosphorylation. Mechanistic studies indicated that REX-1 recruited DNMT3b (DNA methyltransferase 3b), inhibited the transcription of RASSF1a (RAS association domain family 1a), and further modulated methylation of RASSF1a promoter. Intervention of the REX-1/DNMT3b/RASSF1a axis may shed light on the development of novel therapeutic approaches for prostate cancer treatment. IMPLICATIONS: REX1 overexpression recruits DNMT3b and downregulates RASSF1a by promoter methylation, suggesting that epigenetic intervention may contribute to prostate cancer treatment.

RAS-association domain family 1A regulates the abnormal cell proliferation in psoriasis via inhibition of Yes-associated protein.

Psoriasis is a chronic, inflammatory skin disease with a high incidence and recurrence; however, its exact pathogenesis and aetiology remain unclear. This study aimed to analyse the effect of the upstream negative regulator RAS‐association domain family 1A (RASSF1A) on Yes‐associated protein (YAP) in psoriasis. Skin lesions of 22 patients with psoriasis and 19 healthy controls were used. Human epidermal keratinocytes stimulated by M5 (IL‐1α, IL‐17, IL‐22, TNF‐α and oncostatin M) were used to establish a psoriatic cell model. BALB/c mice treated with topical imiquimod were used to establish a psoriatic mouse model. As the methylation level of RASSF1A increased, its expression in psoriatic patients and mice model decreased. Addition of the methylation inhibitor 5‐Aza‐CdR or RASSF1A‐overexpressing lentivirus vector increased RASSF1A and reduced YAP expression; meanwhile improved skin lesions, reduced cell proliferation, induced cell cycle arrest in the G0/G1 phase, increased apoptosis, reduced inflammatory cytokines and activities of ERK, STAT3 and NF‐κB signalling pathways. The results indicated that RASSF1A could play a role in the treatment of psoriasis by inhibiting YAP expression. Based on these findings, targeted drugs that can inhibit the methylation or increase the expression of RASSF1A may be useful for treating psoriasis.

DNMT3B Expression Might Contribute to Abnormal Methylation of RASSF1A in Lager Colorectal Adenomatous Polyps.

Background It is pretty well known that DNA methyltransferases (DNMTs) are actively involved in abnormal cell growth. The goal of the current study is to explore the correlation between DNMT expression and colorectal adenomatous polyps (CAPs). Method Twenty pairs of CAP samples with a diameter ≥ 10 mm and corresponding normal colorectal mucosa (NCM) tissues from patients were used in the present study. The expression levels and activity of DNA methyltransferases (DNMTs) were measured in the CAP tissues. The global methylation and the promoter methylation level of 3 kinds of tumour suppressor gene were detected. Results mRNA and protein levels of DNMT3B were found to be elevated in the CAP tissues compared with the control tissue. Additionally, the methylation of long interspersed nuclear elements-1 (LINE-1/L1) was decreased in the CAP tissue. Furthermore, methylation of the promoter of a tumour suppressor gene Ras association domain family 1A (RASSF1A) was increased in the CAP tissues, while the mRNA levels of RASSF1A were decreased. Conclusions These results suggest that the overexpression of DNMT3B may contribute to a role in the genesis of CAPs through the hypomethylation of chromosomes in the whole cell and promoter hypermethylation of RASSF1A.

MiR-602 Mediates the RASSF1A/JNK Pathway, Thereby Promoting Postoperative Recurrence in Nude Mice with Liver Cancer.

PurposeAt present, there are few studies on the mechanisms underlying postoperative recurrence of liver cancer, and the mechanism of action of miR-602 in postoperative recurrence of liver tumors is not clear. Our goals were to investigate the effects of miR-602 on the expression of the Ras-associated domain family 1A (RASSF1A) gene and the regulation of primary and recurrent hepatic tumors to clarify the molecular mechanisms of miR-602 in postoperative hepatocellular carcinoma.MethodsWe constructed a mouse liver orthotopic tumor model and a mouse liver recurrent tumor model. We measured the expression levels of the RASSF1A gene and then analyzed the effects of miR-602 on the regulation of RASSF1A. We transiently transfected the miR-602 gene into cells that stably overexpressed RASSF1A and examined relevant indicators to elucidate the mechanisms by which miR-602 regulates the RASSF1A/c-Jun N-terminal kinase (JNK) pathway in recurrence and dormancy in liver cancer.ResultsRASSF1A expression was inversely related to that of JNK, activating transcription factor 2 (ATF-2), and c-Jun in SMMC7721 cells stably transfected with the RASSF1A gene and in recurrent mouse tumor tissues. After transient transfection of cells with miR-602 mimic or miR-602 inhibitor, the expression of miR-602 was inversely related to that of RASSF1A.ConclusionMiR-602 might inhibit the JNK signaling pathway by inhibiting the expression of RASSF1A, thereby promoting recurrence of liver cancer after surgery. The low expression levels of miR-602 in liver cancer tissues were closely related to postoperative recurrence; they could be used as a marker to judge the prognosis of patients with liver cancer.

Tricistronic expression of MOAP-1, Bax and RASSF1A in cancer cells enhances chemo-sensitization that requires BH3L domain of MOAP-1.

Although important for apoptosis, the signaling pathway involving MOAP-1(Modulator of Apoptosis 1), RASSF1A (RAS association domain family 1A), and Bax (Bcl-2 associated X protein) is likely to be dysfunctional in many types of human cancers due to mechanisms associated with gene mutation and DNA hyper-methylation. The purpose of the present study was to assess the potential impact of generating physiologically relevant signaling pathway mediated by MOAP-1, Bax, and RASSF1A (MBR) in cancer cells and chemo-drug resistant cancer cells. The tricistronic expression construct that encodes MOAP-1, Bax, and RASSF1A (MBR) or its mutant, MOAP-1∆BH3L, Bax and RASSF1A (MBRX) was expressed from an IRES (Internal Ribosome Entry Site)-based tricistronic expression vector in human breast cancer cells, including MCF-7, MCF-7-CR (cisplatin resistant) and triple negative breast cancer cells, BMET05, for functional characterization through in vitro and in vivo models. Transient expression of MBR potently promoted dose-dependent apoptotic signaling and chemo-sensitization in the cancer cells, as evidenced by loss of cell viability, nuclei condensation and Annexin-V positive staining while stable expression of MBR in MCF-7 cells significantly reduced the number of MBR stable clone by 86% and the stable clone exhibited robust chemo-drug sensitivity. In contrast, MBRX stable clone exhibited chemo-drug resistance while transiently over-expressed MOAP-1ΔBH3L inhibited the apoptotic activity of MBR. Moreover, the spheroids derived from the MBR stable clone displayed enhanced chemo-sensitivity and apoptotic activity. In mouse xenograft model, the tumors derived from MBR stable clone showed relatively high level of tumor growth retardation associated with the increase in apoptotic activity, leading to the decreases in both tumor weight and volume. Expression of MBR in cancer cells induces apoptotic cell death with enhanced chemo-sensitization requiring the BH3L domain of MOAP-1. In animal model, the expression of MBR significantly reduces the growth of tumors, suggesting that MBR is a potent apoptotic sensitizer with potential therapeutic benefits for cancer treatment.

Aberrant SOCS3 Promoter Methylation as a Noninvasive Diagnostic Biomarker for Locally Advanced Prostate Cancer.

Objective:The aim of this study was to investigate the promoter methylation status of Rasassociated domain family 1A (RASSF1A), O-6-methylguanine-DNA methyltransferase (MGMT), Phosphatase with tensin homology (PTEN) and Suppressor of cytokine signaling 3 (SOCS3) tumor suppressor genes and evaluate the clinical utility of these genes as noninvasive, blood-based epigenetic biomarkers for the diagnosis of Prostate Cancer (PCa).Method:A total of 41 consecutive patients and 10 healthy control groups were enrolled in the study. Pyrosequencing was performed to analyze the methylation levels of the promoter regions of the four tumor suppressor genes in patients compared to healthy controls.Results:The promoter methylation levels of RASSF1A, MGMT, PTEN and SOCS3 did not differ between the patient and control groups. However, SOCS3 promoter methylation level was significantly higher for patients having locally advanced PCa compared to those having localizedPCa (p<0.05).ConclusionOur results indicated that SOCS3 could be a useful, noninvasive blood-based epigenetic biomarker for the diagnosis of locally advanced PCa.

DNA Methylation in Human Breast Cancer Cell Lines Adapted to High Nitric Oxide.

Nitric oxide (NO) exposure has been suggested to cause alterations in DNA methylation in breast cancer. We investigated the effect of NO on DNA methylation of promoters in cell lines of breast cancer. The methylation status of the promoters of breast cancer 1 (BRCA1), deleted in colon cancer (DCC), Ras-association domain family 1A (RASSF1A), O6-methylguanine-DNA methyltransferase (MGMT), and secreted frizzled related protein 1 (SFRP1) were analyzed in the parental and high nitric oxide-adapted cell lines of breast cancer using Illumina MiSequencing. Methylation of RASSF1A promoter in BT-20-HNO (74.7%) was significantly higher than that in BT-20 cells (72%) (p<0.05), whereas in MCF-7-HNO cells, methylation of MGMT promoter was found to have significantly decreased as compared to its parental cell line (45.1% versus 50.1%; p<0.0001). Promoter methylation of SFRP and DCC was elevated in T-47D-HNO relative to its parent cell line (p<0.05). Similarly to the double-edged effects of NO on tumorigenesis, its epigenetic effects through DNA methylation are diverse and contradictory in breast cancer.

When Hippo meets actin in the nucleus

ABSTRACTNuclear actin is exported from the nucleus via the Exportin-6 (XPO6)/RAN GTPase complex. We recently identified that RASSF1A and the hippo pathway kinase Mammalian STE20-like protein kinase 2 (MST2) play a pivotal role in nucleocytoplasmic shuttling of actin by regulating the association of XPO6 with RAN GTPase. Importantly, loss of Ras association domain family 1A (RASSF1A) and hippo signaling in cancer cells highlights a key mechanism by which nuclear actin promotes tumorigenesis.

A RASSF1A-HIF1\u03b1 loop drives Warburg effect in cancer and pulmonary hypertension

Hypoxia signaling plays a major role in non-malignant and malignant hyperproliferative diseases. Pulmonary hypertension (PH), a hypoxia-driven vascular disease, is characterized by a glycolytic switch similar to the Warburg effect in cancer. Ras association domain family 1A (RASSF1A) is a scaffold protein that acts as a tumour suppressor. Here we show that hypoxia promotes stabilization of RASSF1A through NOX-1- and protein kinase C- dependent phosphorylation. In parallel, hypoxia inducible factor-1 α (HIF-1α) activates RASSF1A transcription via HIF-binding sites in the RASSF1A promoter region. Vice versa, RASSF1A binds to HIF-1α, blocks its prolyl-hydroxylation and proteasomal degradation, and thus enhances the activation of the glycolytic switch. We find that this mechanism operates in experimental hypoxia-induced PH, which is blocked in RASSF1A knockout mice, in human primary PH vascular cells, and in a subset of human lung cancer cells. We conclude that RASSF1A-HIF-1α forms a feedforward loop driving hypoxia signaling in PH and cancer.

Abstract 2997: Epigenomic signatures of acquired platinum resistance in high grade serous ovarian cancer

Abstract Epithelial ovarian cancer (OC) is the most lethal gynecologic malignancy. The majority of advanced stage patients develop uniformly fatal resistance to standard platinum-based treatments. Epigenetic changes, particularly DNA methylation aberrations have been implicated in acquired resistance to platinum in OC. The goal of the current study was to identify methylation changes associated with the development of acquired resistance to platinum-based chemotherapy. We hypothesized that OC tumors harboring an aberrant DNA methylome could be reversed by DNMTi and re-sensitized to platinum treatment. To achieve our objective, we generated and compared tumor DNA methylation profiles from patients with recurrent platinum-resistant OC treated on a clinical trial with a DNA methyl transferase inhibitor, guadecitabine, before (n=65) and after (n=19) treatment (NCT01696032), and patients with primary (platinum-naïve) OC (n=20). Human ovarian surface epithelial cells (HOSE) were used as controls (n=5). We examined methylation levels across the genome using the Infinium Human Methylation 450 Bead Chip (Illumina). By comparing the methylome of platinum naive and platinum resistant ovarian tumors, normalized to HOSE, we identified 444 CpG island-containing gene promoters that became hypermethylated in resistant tumors. Of those, 45 gene promoters were hypermethylated at an FDR&amp;lt;0.05 and 19 genes at an FDR&amp;lt;0.05 and Δβ&amp;gt;0.1. Among those significantly hypermethylated genes, acquired promoter CpG island methylation of Ras Association Domain Family 1A (RASSF1A; a tumor suppressor gene in multiple cancer types) was observed. Of the 444 hypermethylated promoters in tumor resistant samples, 93 CpG islands became hypomethylated after guadecitabine treatment, suggesting that these promoters may be associated with reversal of resistance. Of those were the IFNA8 promoter, a gene known to be associated with anti-tumor effects in ovarian and other solid malignancies, and DEFB124, which is involved in the innate immune response. Pathways associated with immune reactivation were highly enriched by guadecitabine treatment, including IL-12 signaling and production of macrophages and MAPK and apoptosis signaling. Together, these results support that aberrant DNA methylation affecting gene networks associate with immune response plays a role in platinum resistant development and could be a therapeutic target. Citation Format: Fang Fang, Horacio Cardenas, Guanglong Jiang, Susan M. Perkins, Chi Zhang, Harold Keer, Yunlong Liu, Daniela Matei, Kenneth Nephew. Epigenomic signatures of acquired platinum resistance in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2997.

Study of the promoter methylation status of RASSF1A gene in oral squamous cell carcinoma

Oral cancer is one of the major global health problems. Methylation is one of the epigenetic modifications that play an important role in the transcriptional inactivation of tumor suppressor genes in human cancer. Aim: The present study was directed to assess the Ras-association domain family 1A (RASSF1 A) gene promoter methylation status in oral squamous cell carcinoma (OSCC) and its correlation to patient’s clinicopathological data. Material and methods: Formalin-fixed, paraffin-embedded tissue blocks of fifty patients with histopathologically confirmed OSCC in addition to 10 normal oral mucosa blocks were included in this study. Hematoxylin and Eosin (H&E) stain was used to confirm the diagnosis of cases as 16 well differentiated, 19 moderately differentiated and 15 poorly differentiated OSCC. Sections of each block were deparaffinized and prepared by DNA extraction for detection of RASSF1A gene methylation status by methylation-specific polymerase chain reaction (MSP) technique. Results: RASSF1A promoter methylation was not detected in normal cases, but it was found in 30% (15/50) of OSCC cases. The increase of methylation in OSCC was significant (p-value=0.045). The increase of methylation in poorly differentiated OSCC (6/15, 40%) was more than that.0 in moderately differentiated OSCC (7/19 36.8%) but both were significante in relation to normal mucosa (p-values= 0.021, 0.027 respectively). Well-differentiated OSCC was the lowest in methylation (2/16, 12.5%), while its increase than normal was not significant (p-value= 0.244). There was direct correlation between methylation and the lymph nodes metastasis. Conclusion: RASSF1A promoter methylation significantly associated with OSCC, the decrease of cell differentiation in histopathological stages and lymph node metastasis. Hence, RASSF1A promoter methylation could be used as a tumor marker in addition to histopathological stage and lymph nodes metastasis predictor in OSCC.

The effects of up-regulation of microRNA-148a on the expression of anti-oncogene preproenkephalin, p16 and ras association domain family 1A in pancreatic carcinoma AsPC-1 cells

Objective To investigate the effect of up-regulation of microRNA (miRNA, miR)-148a on the methylation and protein expression of anti-oncogene preproenkephalin (ppENK), p16, ras association domain family 1A (RASSF1A) and the proliferation of pancreatic cancer AsPC-1 cells. Methods MiR-148a mimics were transfected into AsPC-1 cells mediated by Lipofectamine 2000. AsPC-1 cells were devided into three groups, blank group (transfected Lipofectamine), mimincs-NC group (transfected negative mimics) and mimincs-148a group (transfected miR-148a mimics). At 72 h after transfection, the expression of miR-148a was analyzed by real-time quantitative polymerase chain reaction (Real-time PCR). The CpG island methylation status of anti-oncogene ppENK, p16, RASSF1A were measured by methylation specific polymerease chain reaction (MSP). The expression of DNA methy transferas 1 (DNMT1), ppENK, p16 and RASSF1A protein were detected with Western blotting. The cell proliferation activity was examined by methyl thiazol tetrazolium (MTT) assay. Results The expression of miR-148a in mimincs-148a group was significantly higher than those in blank group and mimincs-NC group (4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P=0.002), while the expression of DNMT1 protein in mimincs-148a group was significantly lower than those in blank group and mimincs-NC group (1.00±0.00 vs. 1.04±0.19 vs. 0.27±0.15, P=0.001). ppENK, p16 and RASSF1A genes were positive methylation in blank group and mimincs-NC group, while ppENK and p16 genes were negative methylation, and RASSF1A gene was partial methylation in mimincs-148a group. The expression of ppENK, p16 and RASSF1A protein in mimics-148a group were significantly higher than those in blank group and mimics-NC group (ppENK: 2.06±0.32 vs. 1.00±0.00 vs. 1.01±0.21, P=0.002; p16: 2.02±0.12 vs. 1.00±0.00 vs. 0.96±0.12, P=0.000; RASSF1A: 1.95±0.37 vs. 1.00±0.00 vs. 1.09±0.12, P=0.002). The cell proliferation activities in mimincs-148a group were significantly slower than those in in blank group and mimincs-NC group (P=0.026). Conclusion Up-regulation of miR-148a could lead to demethylation of ppENK, p16 and RASSF1A gene promoter CpG island and up-regulate its protein expression, and inhibit cell proliferation in pancreatic cancer AsPC-1 cells. The mechanism may be related to the inhibition of DNMT1 expression induced by miR-148a. Key words: Pancreatic cancer; MicroRNA-148a; DNA methy transferas 1; Anti-oncogene; Methylation; Cell proliferation

The relationship between RASSF1A promoter methylation and thyroid carcinoma: A meta-analysis of 14 articles and a bioinformatics of 2 databases (PRISMA).

DNA promoter methylation can suppresses gene expression and shows an important role in the biological functions of Ras association domain family 1A (RASSF1A). Many studies have performed to elucidate the role of RASSF1A promoter methylation in thyroid carcinoma, while the results were conflicting and heterogeneous. Here, we analyzed the data of databases to determine the relationship between RASSF1A promoter methylation and thyroid carcinoma. We used the data from 14 cancer-normal studies and Gene Expression Omnibus (GEO) database to analyze RASSF1A promoter methylation in thyroid carcinoma susceptibility. The data from the Cancer Genome Atlas project (TCGA) database was used to analyze the relationship between RASSF1A promoter methylation and thyroid carcinoma susceptibility, clinical characteristics, prognosis. Odds ratios were estimated for thyroid carcinoma susceptibility and hazard ratios were estimated for thyroid carcinoma prognosis. The heterogeneity between studies of meta-analysis was explored using H, I values, and meta-regression. We adopted quality criteria to classify the studies of meta-analysis. Subgroup analyses were done for thyroid carcinoma susceptibility according to ethnicity, methods, and primers. Result of meta-analysis indicated that RASSF1A promoter methylation is associated with higher susceptibility to thyroid carcinoma with small heterogeneity. Similarly, the result from GEO database also showed that a significant association between RASSF1A gene promoter methylation and thyroid carcinoma susceptibility. For the results of TCGA database, we found that RASSF1A promoter methylation is associated with susceptibility and poor disease-free survival (DFS) of thyroid carcinoma. In addition, we also found a close association between RASSF1A promoter methylation and patient tumor stage and age, but not in patients of different genders. The methylation status of RASSF1A promoter is strongly associated with thyroid carcinoma susceptibility and DFS. The RASSF1A promoter methylation test can be applied in the clinical diagnosis of thyroid carcinoma.

Impact of Natural Compounds on DNA Methylation Levels of the Tumor Suppressor Gene RASSF1A in Cancer.

Epigenetic inactivation of tumor suppressor genes (TSG) is a fundamental event in the pathogenesis of human cancer. This silencing is accomplished by aberrant chromatin modifications including DNA hypermethylation of the gene promoter. One of the most frequently hypermethylated TSG in human cancer is the Ras Association Domain Family 1A (RASSF1A) gene. Aberrant methylation of RASSF1A has been reported in melanoma, sarcoma and carcinoma of different tissues. RASSF1A hypermethylation has been correlated with tumor progression and poor prognosis. Reactivation of epigenetically silenced TSG has been suggested as a therapy in cancer treatment. In particular, natural compounds isolated from herbal extracts have been tested for their capacity to induce RASSF1A in cancer cells, through demethylation. Here, we review the treatment of cancer cells with natural supplements (e.g., methyl donors, vitamins and polyphenols) that have been utilized to revert or prevent the epigenetic silencing of RASSF1A. Moreover, we specify pathways that were involved in RASSF1A reactivation. Several of these compounds (e.g., reseveratol and curcumin) act by inhibiting the activity or expression of DNA methyltransferases and reactive RASSF1A in cancer. Thus natural compounds could serve as important agents in tumor prevention or cancer therapy. However, the exact epigenetic reactivation mechanism is still under investigation.