The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: miR-21 and promoter methylation.
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ABSTRACT:
SUBMITTER: Yazdani Y
PROVIDER: S-EPMC4717643 | biostudies-literature | 2016 Jan
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe oncogenic microRNAs (miRNAs) miR-21 and miR-31 negatively regulate tumor-suppressor genes. Their potential as serum biomarkers has not been determined in human colorectal cancer (CRC).MethodsTo determine whether miR-21 and miR-31 are secretory miRNAs, we screened expression in medium from 2 CRC cell lines, which was followed by serum analysis from 12 CRC patients and 12 control subjects. We validated expression of candidate miRNAs in serum samples from an independent cohort of 186 CRC patients, 60 postoperative patients, 43 advanced adenoma patients, and 53 control subjects. We analyzed miR-21 expression in 166 matched primary CRC tissues to determine whether serum miRNAs reflect expression in CRC. Patient survival analyses were performed by Kaplan-Meier analyses and Cox regression models. All statistical tests were two-sided.ResultsAlthough miR-21 was secreted from CRC cell lines and upregulated in serum of CRC patients, no statistically significant differences were observed in serum miR-31 expression between CRC patients and control subjects. In the validation cohort, miR-21 levels were statistically significantly elevated in preoperative serum from patients with adenomas (P < .001) and CRCs (P < .001). Importantly, miR-21 expression dropped in postoperative serum from patients who underwent curative surgery (P < .001). Serum miR-21 levels robustly distinguished adenoma (area under the curve [AUC] = 0.813; 95% confidence interval [CI] = 0.691 to 0.910) and CRC (AUC = 0.919; 95% CI = 0.867 to 0.958) patients from control subjects. High miR-21 expression in serum and tissue was statistically significantly associated with tumor size, distant metastasis, and poor survival. Moreover, serum miR-21 was an independent prognostic marker for CRC (hazard ratio = 4.12; 95% CI = 1.10 to 15.4; P = .03).ConclusionsSerum miR-21 is a promising biomarker for the early detection and prognosis of CRC.
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Project description:We previous found the expression level of PTEN was low in the chronic lymphocytic leukemia (CLL) patients. To assess the pathogenic contribution of the low expression of PTEN, we determined PTEN-regulating miRNA interference, PTEN promoter methylation and PTEN gene mutation condition in CLL. One hundred and fifty-four previously untreated CLL patients and 200 cases of healthy controls were sequenced in exons 5-9 of PTEN. None of single nucleotide polymorphism site or mutation was detected in the coding sequences of those exons. Methylation of PTEN promoter was found in one (1.33%) of the 75 patients with CLL, but none of the 25 age-matched control subjects. We found that PTEN was a potential target of miR-26a and miR-214, which had been confirmed following dual-luciferase reporter assays, reverse transcription polymerase chain reaction and Western blotting. High expression of miR-26a was associated with advanced Binet stage (P=0.012), p53 aberrations (P=0.014) and inferior time to first treatment (P=0.038), and high expression of miR-214 was only associated with p53 aberrations (P=0.041). Inhibition of miR-26a or miR-214 could induce more apoptosis in primary cultured CLL cells. These findings support miR-26a and miR-214 down-regulate expression of PTEN in CLL, but not PTEN mutation or promoter methylation.
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Project description:BackgroundPhosphatase and tensin homolog (PTEN) acts as a tumor suppressor gene. Inactivation of PTEN has been reported in various types of cancers. PTEN promoter methylation possibly underlies PTEN inactivation, which results in tumorigenesis. The aim of this study was to investigate whether PTEN promoter methylation contributes to PTEN inactivation in ameloblastoma and its associated protein expression.Material and methodsIn total, 20 fresh-frozen ameloblastoma samples were evaluated for PTEN promoter methylation using methylation-specific polymerase chain reaction (MS-PCR). A subset of 10 paraffin-embedded ameloblastoma samples was examined for PTEN expression through immunohistochemistry. Four primary cultured ameloblastoma cells were investigated for PTEN promoter methylation and PTEN transcriptional expression via reverse transcription PCR.ResultsPTEN promoter methylation was detected in 65% (13/20) of the ameloblastoma samples. Of 10 ameloblastoma samples, 4 exhibited reduced PTEN expression. Of 5 samples with methylated PTEN, 3 (60%) were associated with loss of PTEN expression. However, PTEN expression was detected in 4 (80%) of 5 samples with unmethylated PTEN. In addition, 3 (75%) of 4 primary ameloblastoma cell cultures exhibited an inverse correlation between PTEN promoter methylation and PTEN transcription level.ConclusionsPTEN promoter methylation is found in a number of ameloblastomas but not significantly correlated with loss of PTEN expression. Genetic or epigenetic mechanisms other than PTEN promoter methylation may contribute to PTEN inactivation in ameloblastoma tumor cells.
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Project description:Long noncoding RNAs (lncRNAs) regulate multiple biological effects in cancers. Recently, RNA methylation has been found to modify not only coding RNAs but also some noncoding RNAs. How RNA methylation affects lncRNAs to affect colorectal cancer (CRC) progression remains elusive. The expression of LINC01559 was explored through RNA sequencing, quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). The preliminary exploration of its function was performed using Western blotting (WB) and immunohistochemistry (IHC). Functional experiments in vitro and in vivo were conducted to explore the biological functions of LINC01559 in CRC. The LINC01559/miR-106-5p/PTEN axis was verified through fluorescence in situ hybridization (FISH), luciferase assays, and rescue experiments. RIP-sequencing, m6A RNA immunoprecipitation (MeRIP) assays and bioinformatic analysis were conducted to determine the upstream mechanism of LINC01559. The results showed that LINC01559 was downregulated in CRC compared with normal controls. Lower expression of LINC01559 in CRC patients predicted a poor prognosis. In addition, PTEN was found to be positively correlated with LINC01559, and miR-106b-5p could be the link between LINC01559 and PTEN. Then, silencing LINC01559 restored the malignant phenotype of CRC cells, while cotransfection of miR-106b-5p inhibitor neutralized this effect. Mechanistically, we found abundant m6A modification sites on LINC01559. Then, we uncovered these sites as potential targets of METTL3 through experiments in vivo. The results revealed a negative functional regulation of the LINC01559/miR-106b-5p/PTEN axis in CRC progression and explored a new mechanism of METTL3-mediated m6A modification on LINC01559. These results elucidate a novel potential therapeutic target for CRC treatment.
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Project description:New biomarkers are needed for the prognosis of advanced colorectal cancer, which remains incurable by conventional treatments. O6-methylguanine DNA methyltransferase (MGMT) methylation and protein expression have been related to colorectal cancer treatment failure and tumor progression. Moreover, the presence in these tumors of cancer stem cells, which are characterized by CD133 expression, has been associated with chemoresistance, radioresistance, metastasis, and local recurrence. The objective of this study was to determine the prognostic value of CD133 and MGMT and their possible interaction in colorectal cancer patients.MGMT and CD133 expression was analyzed by immunohistochemistry in 123 paraffin-embedded colorectal adenocarcinoma samples, obtaining the percentage staining and intensity. MGMT promoter methylation status was obtained by using bisulfite modification and methylation-specific PCR (MSP). These values were correlated with clinical data, including overall survival (OS), disease-free survival (DFS), tumor stage, and differentiation grade.Low MGMT expression intensity was significantly correlated with shorter OS and was a prognostic factor independently of treatment and histopathological variables. High percentage of CD133 expression was significantly correlated with shorter DFS but was not an independent factor. Patients with low-intensity MGMT expression and ?50% CD133 expression had the poorest DFS and OS outcomes.Our results support the hypothesis that MGMT expression may be an OS biomarker as useful as tumor stage or differentiation grade and that CD133 expression may be a predictive biomarker of DFS. Thus, MGMT and CD133 may both be useful for determining the prognosis of colorectal cancer patients and to identify those requiring more aggressive adjuvant therapies. Future studies will be necessary to determine its clinical utility.
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Project description:Background/aimAlthough some mutations of KRAS proto-oncogene, GTPase (KRAS) have been associated with the prognosis and therapeutic management of colorectal cancer (CRC), the epigenetic mechanisms (DNA methylation and microRNA expression) that regulate wild-type KRAS expression in patients with CRC are poorly known. The aim of this study was to establish whether there is a relationship between the expression of the wild-type KRAS gene, the methylation status of its distal promoter, and miR-143 and miR-18a-3p levels in samples of sporadic CRC.Patients and methodsA total of 51 cases of sporadic CRC with wild-type KRAS were analyzed. The expression levels of KRAS mRNA, miR-18a-3p, miR-143, and KRAS protein, as well as methylation in the distal promoter of the KRAS gene were evaluated.ResultsIn the analyzed cases, KRAS mRNA expression was detected in 51.1%; wild-type KRAS protein was found in the membrane in 31.4% and in the cytoplasm in 98% of cases. An inverse relationship of marginal significance was observed between miR-18a-3p and KRAS protein expression in the cytoplasm (odds ratio=0.14, 95% confidence interval=0.012-1.092; p=0.08). The methylation status of the distal promoter of KRAS at four CpG islands was analyzed in 30 cases (58.8%): partial methylation of the four CpG islands evaluated was observed in two cases (6.7%). In these cases, KRAS protein expression was not evidenced at the membrane level; miR-18a-3p expression was not detected either but high expression of miR-143 was observed.ConclusionNo association was found between the expression levels of KRAS mRNA, miR-18a-3p, miR-143 and methylation status. Methylation status was detected with low frequency, thus being the first report of methylation in wild-type KRAS.
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Project description:PurposeTo evaluate the effect of the overexpression of miR-21 on the properties of pterygium and examine whether miR-21 promotes the proliferation of pterygium cells through targeting the PTEN/AKT signaling pathway.MethodsInformation regarding patient gender, age, and pterygium severity was gathered. Expression of miR-21 was obtained through examination of excised pterygium tissues and superior conjunctiva tissues with real-time PCR. Human pterygium fibroblasts (HPFs) were obtained from pterygium surgery and subjected to primary culture. The HPF cell lines were divided into a negative control group, an miR-21 inhibitor group, and an miR-21 inhibitor + VO-Ohpic trihydrate group, and then the cell viability and apoptosis and the expression of PTEN and AKT were examined.ResultsFifty-eight subjects with unilateral primary pterygium were included. An increase in the miR-21 levels in pterygium tissue was evident compared with that in the paired normal conjunctival tissues (independent-samples t test, p<0.01). As the severity of the pterygium increased, the miR-21 levels increased (p=0.004, rs=0.373, Spearman's rank correlation coefficient). The miR-21 inhibitor suppressed the proliferation and induced apoptosis of HPF cells through increasing the PTEN expression, and further decreasing the expression of p-AKT, which could be reversed by the PTEN inhibitor VO-Ohpic trihydrate.ConclusionsAberrant miR-21 overexpression in the pterygium could target PTEN, which contributes to abnormal proliferation of the HPF cells through depressing the PTEN/AKT pathway. The results also suggested the potential of miR-21 and the PTEN/AKT pathway as a novel therapeutic strategy for pterygium.
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