Tumor-related microRNAs Research Articles

Edodes Cultured Extract Regulates Immune Stress During Puberty and Modulates MicroRNAs Involved in Mammary Gland Development and Breast Cancer Suppression.

Immune stressors, such as lipopolysaccharides (LPS), profoundly affect microbiota balance, leading to gut dysbiosis. This imbalance disrupts the metabolic phenotype and structural integrity of the gut, increasing intestinal permeability. During puberty, a critical surge in estrogen levels is crucial for mammary gland development. However, inflammation originating from the gut in this period may interfere with this development, potentially heightening breast cancer risk later. The long-term effects of pubertal inflammation on mammary development and breast cancer risk are underexplored. Such episodes can dysregulate cytokine levels and microRNA expression, altering mammary cell gene expression, and predisposing them to tumorigenesis. This study hypothesizes that prebiotics, specifically Lentinula edodes Cultured Extract (AHCC), can counteract LPS's adverse effects. Using BALB/c mice, an acute LPS dose was administered at puberty, and breast cancer predisposition was assessed at 13 weeks. Cytokine and tumor-related microRNA levels, tumor development, and cancer stem cells were explored through immunoassays and qRT-PCR. Results show that LPS induces lasting effects on cytokine and microRNA expression in mammary glands and tumors. AHCC modulates cytokine expression, including IL-1β, IL-17A/F, and IL-23, and mitigates LPS-induced IL-6 in mammary glands. It also regulates microRNA expression linked to tumor progression and suppression, particularly counteracting the upregulation of oncogenic miR-21, miR-92, and miR-155. Although AHCC slightly alters some tumor-suppressive microRNAs, these changes are modest, highlighting a complex regulatory role that warrants further study. These findings underscore the potential of dietary interventions like AHCC to mitigate pubertal LPS-induced inflammation on mammary gland development and tumor formation, suggesting a preventive strategy against breast cancer.

Phase Separation of DNA-Encoded Artificial Cells Boosts Signal Amplification for Biosensing.

Life-like hierarchical architecture shows great potential for advancing intelligent biosensing, but modular expansion of its sensitivity and functionality remains a challenge. Drawing inspiration from intracellular liquid-liquid phase separation, we discovered that a DNA-encoded artificial cell with a liquid core (LAC) can enhance peroxidase-like activity of Hemin and its DNA G-quadruplex aptamer complex (DGAH) without substrate-selectivity, unlike its gelled core (GAC) counterpart. The LAC is easily engineered as an ultrasensitive biosensing system, benefiting from DNA's high programmability and unique signal amplification capability mediated by liquid-liquid phase separation. As proof of concept, its versatility was successfully demonstrated by coupling with two molecular recognition elements to monitor tumor-related microRNA and profile cancer cell phenotypes. This scalable design philosophy offers new insights into the design of next generation of artificial cells-based biosensors.

A biologically stable, self-catalytic DNAzyme machine encapsulated by metal-phenolic nanoshells for multiple microRNA imaging

DNAzyme machines play critical roles in the fields of cell imaging, disease diagnosis, and cancer therapy. However, the applications of DNAzyme machines are limited by the nucleases-induced degradation, non-specific binding of proteins, and insufficient provision of cofactors. Herein, protected DNAzyme machines with different cofactor designs (referred to as ProDs) were nanoengineered by the construction of multifunctional metal-phenolic nanoshells to deactivate the interferential proteins, including nucleases and non-specific binding proteins. Moreover, the nanoshells not only facilitate the cellular internalization of ProDs but provide specific metal ions acting as cofactors of the designed DNAzymes. Cellular imaging results demonstrated that ProDs could effectively and simultaneously monitor multiple tumor-related microRNAs in living cells. This facile and rapid strategy that encapsulates DNAzyme machines into the protective metal-phenolic nanoshells is anticipated to extend to a wide range of functional nucleic acids-based biomedical applications.

Cruciate DNA probes for amplified multiplexed imaging of microRNAs in living cells.

The real-time imaging of low-abundance tumor-related microRNAs (miRNAs) in living cells holds great potential for early clinical diagnosis of cancers. However, the relatively low detection sensitivity and possible false-positive signals of a probe in complex cellular matrices remain critical challenges for accurate RNA detection. Herein, we developed a novel aptamer-functionalized cruciate DNA probe that enabled amplified multiple miRNA imaging in living cells via catalytic hairpin assembly (CHA). The cross-shaped design of the cruciate DNA probe improved the stability against nucleases and acted as a modular scaffold for CHA circuits for efficient delivery into tumor cells. The cruciate DNA probe allowed self-assembly through thermal annealing and displayed excellent performance for sensitive miRNA detection in vitro. The cruciate DNA probe could be internalized into nucleolin-overexpressed cells specifically via cell-targeting of the AS1411 aptamer, achieving amplified fluorescence imaging and quantitative evaluation of the expression of miRNAs in living cells. Through the simultaneous detection of intracellular multiple miRNAs, the developed cruciate DNA probe could provide more accurate information and reduce the chances of false positive signals for cancer diagnosis. This approach offers a new opportunity for promoting the development of miRNA-related biomedical research and tumor diagnostic applications.

Self-propelled quantum dots-based spherical nucleic amplifier for ratiometric imaging of intracellular microRNAs

Real-time in-situ visualization and sensitive detection of thimbleful tumor-related microRNAs (miRNAs) in living cells is still unmet. Especially, poor amplification efficiency and resistance against complex intracellular environment hinder amplifiers’ widespread applications in vivo . Herein, we report enzyme-free self-propelled quantum dots-based spherical nucleic acids (QDs-based SNAs, also named TSD-QDs) as a ratiometric amplifier for stable, efficient, and sensitive detection and imaging of miRNAs in biological samples and living cells. The ratiometric amplifier is proved possesses outstanding stability in human serum and cells, as well as prominent recognition capability for miRNAs. More importantly, the ingeniously designed tail sequence of fuel strand (FS) could lead to high operating efficiency and excellent detection performance of TSD-QDs for non-enzyme detection in vitro (in the range of 10 fM-150 nM with a limit of detection (LOD) of 5.8 fM). Furthermore, the spherical nucleic acids (SNAs) structure can endow TSD-QDs with favorable photostability and high resistance against to the nuclease, thus benefit its applications in intracellular miRNA-21 monitoring via fluorescence imaging. With these advantages, the proposed TSD-QDs based amplifier enables accurate and effective monitoring of the miRNAs expression levels in living cells (A549, normal QSG-7701, Hela and MCF-7 cells), and poses great potential in medical diagnostics and biomedical applications.

MicroRNA-621 functions as a metastasis suppressor in colorectal cancer by directly targeting LEF1 and suppressing Wnt/β-catenin signaling

AimsColorectal liver metastasis (CRLM) is the leading death-causing among colorectal cancer (CRC) patients. Recently, a novel tumor-related microRNA, miR-621, has been identified as a tumor suppressor in diverse tumor types, but its role in CRLM remains unclear and requires further investigation. Main methodsTo elucidate novel regulators of CRLM progression, we used a well-established CRLM animal model. After serially transplanting human colon carcinoma cell lines Caco-2 into the liver, we obtained liver metastatic variants that exhibited a strong ability for invasion and metastasis. High-throughput sequencing was conducted on these newly established cell lines. After comparison and prediction between the two cell lines: parental Caco-2 (hereafter referred to as F0) and F3, miR-621 was identified as a candidate regulator for lymphoid enhancer-binding factor 1 (LEF1) expression. Further validation was achieved with dual-luciferase reporter assay. Key findingsThe gain- and loss-of-function validation showed that miR-621 inhibits cell viability, cell cycle progression, colony formation, and proliferation in vitro. Meanwhile, miR-621 could reverse EMT malignant phenotype. LEF1, an important downstream mediator of activated Wnt/β-catenin signaling pathway, was validated as the direct functional target of miR-621. miR-621 interacts directly with the LEF1 3′-UTR and post-transcriptionally suppresses LEF1 expression. Moreover, LEF1 overexpression reversed the effect of miR-621. LEF1 silencing counteracted miR-621 down-regulation-induced effects. Further in vivo experiments revealed that miR-621 over-expression suppressed CRLM, but LEF1 abrogated the inhibitory effect of miR-621. SignificanceMiR-621 is a vital tumor suppressor in CRC and could be a promising anti-cancer therapeutic target.

Sensitive detection of microRNAs using polyadenine-mediated fluorescent spherical nucleic acids and a microfluidic electrokinetic signal amplification chip

The identification of tumor-related microRNAs (miRNAs) exhibits excellent promise for the early diagnosis of cancer and other bioanalytical applications. Therefore, we developed a sensitive and efficient biosensor using polyadenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) for miRNA analysis based on strand displacement reactions on gold nanoparticle (AuNP) surfaces and electrokinetic signal amplification (ESA) on a microfluidic chip. In this FSNA, polyA-DNA biosensor was anchored on AuNP surfaces via intrinsic affinity between adenine and Au. The upright conformational polyA-DNA recognition block hybridized with 6-carboxyfluorescein-labeled reporter-DNA, resulting in fluorescence quenching of FSNA probes induced by AuNP-based resonance energy transfer. Reporter DNA was replaced in the presence of target miRNA, leading to the recovery of reporter-DNA fluorescence. Subsequently, reporter-DNAs were accumulated and detected in the front of with Nafion membrane in the microchannel by ESA. Our method showed high selectivity and sensitivity with a limit of detection of 1.3 pM. This method could also be used to detect miRNA-21 in human serum and urine samples, with recoveries of 104.0%–113.3% and 104.9%–108.0%, respectively. Furthermore, we constructed a chip with three parallel channels for the simultaneous detection of multiple tumor-related miRNAs (miRNA-21, miRNA-141, and miRNA-375), which increased the detection efficiency. Our universal method can be applied to other DNA/RNA analyses by altering recognition sequences.

Simultaneous Imaging of Dual microRNAs in Cancer Cells through Catalytic Hairpin Assembly on a DNA Tetrahedron.

Accurate detection and imaging of tumor-related microRNA (miRNA) in living cells hold great promise for early cancer diagnosis and prognosis. One of the challenges is to develop methods that enable the identification of multiple miRNAs simultaneously to further improve the detection accuracy. Herein, a simultaneous detection and imaging method of two miRNAs was established by using a programmable designed DNA tetrahedron nanostructure (DTN) probe that includes a nucleolin aptamer (AS1411), two miRNA capture strands, and two pairs of metastable catalytic hairpins at different vertexes. The DTN probe exhibited enhanced tumor cell recognition ability, excellent stability and biocompatibility, and fast miRNA recognition and reaction kinetics. It was found that the DTN probe could specifically enter tumor cells, in which the capture strand could hybridize with miRNAs and initiate the catalytic hairpin assembly (CHA) only when the overexpressed miR-21 and miR-155 existed simultaneously, resulting in a distinct fluorescence resonance energy transfer signal and demonstrating the feasibility of this method for tumor diagnosis.

Poly-adenine-mediated spherical nucleic acid probes for live cell fluorescence imaging of tumor-related microRNAs.

Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells. polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells. The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

A designer DNA tetrahedron-based molecular beacon for tumor-related microRNA fluorescence imaging in living cells.

The accurate and effective imaging of tumor-related miRNA in living cells has been playing an increasingly important role in cancer imaging. However, due to the low miRNA content and complex intracellular microenvironment, the current imaging methods of miRNAs in living cells still have some limitations. In this work, we developed a designer nanoprobe of tetrahedral DNA framework (TDF) combined with MB (termed TDFM nanoprobe) for the efficient fluorescence imaging of tumor-related miRNA-214 in living cells. In cell-free experiments, we demonstrated that the TDFM nanoprobe has sensitive detection and good specificity by fluorescence measurements. Before the TDFM nanoprobe was used for intracellular miRNA-214 fluorescence imaging, we confirmed its intracellular stability and negligible cytotoxicity by a standard MTT assay. In intracellular imaging experiments, we observed the strong fluorescence signal exhibited by the cells incubated with the TDFM nanoprobe using confocal fluorescence microscopy, which indicated that the TDFM nanoprobe was suitable for detecting and imaging tumor-related miRNA-214 in living cells. Furthermore, under the optimal incubation conditions, we employed the TDFM nanoprobe to study differences in the expression levels of tumor-related miRNA-214 in human breast cancer cells (MCF-7) and human umbilical vein endothelial cells (HUVEC). The TDFM nanoprobe we designed shows great potential to be applied in the development of DNA nanodevices, providing an improved strategy for the fluorescence imaging of miRNAs in living cells.

Prognostic Role of FGFR3 Expression Status and Tumor-Related MicroRNAs Level in Association with PD-L1 Expression in Primary Luminal Non-Muscular Invasive Bladder Carcinoma.

Background: bladder cancer is one of the most common urinary tract malignancies. Establishment of robust predictors of disease progression and outcome is important for personalizing treatment of non-muscular invasive bladder carcinoma (NMIBC). In this study we evaluated association of PD-L1 expression with other prognostic biomarkers, such as expression of miRNA-145 and miRNA-200a, FGFR3 gene expression, and mutation status in tissue specimens of the luminal subtype of newly diagnosed high and low grade NMIBC. Methods: twenty patients with primary luminal NMIBC were enrolled in the study. Tumor grade and risk level were determined in accordance with European Organization for Research and Treatment of Cancer (EORTC) guidelines and World Health Organization (WHO) classification. Neoplasm molecular subtype and PD-L1 expression level were assessed by immunohistochemistry. We used real-time PCR to evaluate the expression of microRNAs and FGFR3. We detected FGFR3 hotspot mutations in codons 248 and 249 by Sanger sequencing. Results: high grade primary luminal NMIBC showed comparatively higher expression of PD-L1 and microRNA-145 than a low grade tumor, whereas the latter had a higher FGFR3 expression and hotspot mutation rate. The tumor grade (HR = 571.72 [11.03–2.96] p = 0.002), PD-L1 expression (HR = 2.33 [0.92–1.92] p = 0.012), and FGFR3 expression (HR = 0.08 [0.17–0.42] p = 0.003) were associated with relapse-free survival. Conclusions: tumor grade in association with PD-L1 and FGFR3 expression can be considered as a complex predictor for primary luminal NMIBC progression.

MicroRNA-183-5p contributes to malignant progression through targeting PDCD4 in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains one of the most common malignant tumors worldwide. The present study aimed to investigate the biological role of microRNA-183-5p (miR-183-5p), a novel tumor-related microRNA (miRNA), in HCC and illuminate the possible molecular mechanisms. The expression patterns of miR-183-5p in clinical samples were characterized using qPCR analysis. Kaplan–Meier survival curve was applied to evaluate the correlation between miR-183-5p expression and overall survival of HCC patients. Effects of miR-183-5p knockdown on HCC cell proliferation, apoptosis, migration and invasion capabilities were determined via Cell Counting Kit-8 (CCK8) assays, flow cytometry, scratch wound healing assays and Transwell invasion assays, respectively. Mouse neoplasm transplantation models were established to assess the effects of miR-183-5p knockdown on tumor growth in vivo. Bioinformatics analysis, dual-luciferase reporter assays and rescue assays were performed for mechanistic researches. Results showed that miR-183-5p was highly expressed in tumorous tissues compared with adjacent normal tissues. Elevated miR-183-5p expression correlated with shorter overall survival of HCC patients. Moreover, miR-183-5p knockdown significantly suppressed proliferation, survival, migration and invasion of HCC cells compared with negative control treatment. Consistently, miR-183-5p knockdown restrained tumor growth in vivo. Furthermore, programmed cell death factor 4 (PDCD4) was identified as a direct target of miR-183-5p. Additionally, PDCD4 down-regulation was observed to abrogate the inhibitory effects of miR-183-5p knockdown on malignant phenotypes of HCC cells. Collectively, our data suggest that miR-183-5p may exert an oncogenic role in HCC through directly targeting PDCD4. The current study may offer some new insights into understanding the role of miR-183-5p in HCC.

MiR-361-5p exerts tumor-suppressing functions in gastric carcinoma by targeting syndecan-binding protein.

MiR-361-5p, a tumor-related microRNA, has been reported to be implicated in the tumorigenesis and progression of diverse types of human malignancies; however, its role in gastric carcinoma remains unclear. This study aimed to explore the biological role of miR-361-5p in gastric carcinoma and clarify the potential mechanisms involved. In the present study, miR-361-5p was found to be significantly downregulated in both gastric carcinoma tissues and cell lines. Functional studies demonstrated that enhanced expression of miR-361-5p suppressed gastric carcinoma cell proliferation in vitro, inhibited tumor growth in vivo, and induced gastric carcinoma cell apoptosis. Moreover, the tumor-suppressing effects of miR-361-5p in gastric carcinoma were abrogated by the miR-361-5p inhibitor treatment. Notably, syndecan-binding protein was downregulated by miR-361-5p via direct binding to its 3' untranslated region in gastric carcinoma cells. Furthermore, syndecan-binding protein expression was discovered to be markedly upregulated and inversely correlated with miR-361-5p expression in gastric carcinoma tissues. Mechanistic studies revealed that restoring the expression of syndecan-binding protein alleviated miR-361-5p-induced inhibitory effects on proliferation of gastric carcinoma cells. Taken together, these findings suggest that miR-361-5p functions as a tumor suppressor in gastric carcinoma by directly targeting syndecan-binding protein and that miR-361-5p might be a novel therapeutic target for gastric carcinoma.

Single-excited double-emission CdTe@CdS quantum dots for use in a fluorometric hybridization assay for multiple tumor-related microRNAs.

A method is described for the simultaneous determination of hepatocellular carcinoma-associated microRNA-122 and microRNA-199a/b-3p. This probe consists of two kinds of nanomaterials. The first comprises CdTe@CdS core-shell quantum dots which, on excitation at 375nm give two emissions, with peak wavelengths at 543 (g-QDs) and at 627nm (r-QDs). The second comprises gold nanoparticles acting as a quencher. In the absence of the target, g-QD-N1 and r-QD-N2 are stable due to the fluorescence stability. With the addition of microRNA-122 and microRNA-199a/b-3p, g-QD-N1 and r-QD-N2 are conjugated to the surface of AuNP-S1/S2 through base complementary pairing. As a result, fluorescence resonance energy transfer (FRET) occurs, resulting in a decrease at 550nm and 635nm respectively, which can realize the simultaneous detection of two different microRNAs. Detection is achieved within 50min. The detection limits (3σ/k) are 0.2nM for microRNA-122 and 0.5nM for microRNA-199a/b-3p. The clinical applicability of the assay was demonstrated by detecting microRNAs in human serum and different cell lysates. Graphical abstractSchematic for the simultaneous determination of microRNA-122 and microRNA-199a/b-3p by FRET.

Downregulation of Transmembrane protein 40 by miR-138-5p Suppresses Cell Proliferation and Mobility in Clear Cell Renal Cell Carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) represents approximately 70% of RCC,as the most frequent histological subtype of RCC. MiR-138-5p, a tumor-related microRNA (miRNA), has been reported to be implicated in the diverse types of human malignancies, but its role in ccRCCremains unclearObjective: The study was designed to investigate the functional behaviors and regulatory mechanisms of miR-138-5p in ccRCCMaterials and Methods: Quantitative real-time PCR and western blotting analyses were performed to determine the expression of miR-138-5p and TMEM40 in ccRCC tissues. Pearson’s correlation coefficient was utilized to evaluate the correlation between miR-138-5p and TMEM40 expression. The function of miR-138-5p and TMEM40 in the cell proliferation, migration and invasion of ccRCC cells (786-O and ACHN) was assessed by CCK-8, colony formation, wound healing and transwell assay, respectively. A luciferase reporter assay was performed to confirm the direct binding of miR-138-5p to the target gene TMEM40Results: We found the expression of miR-138-5p was significantly down-regulated, while TMEM40 was remarkably up-regulated in ccRCC tissues. TMEM40 expression was discovered to be inversely correlated with miR-138-5p expression in ccRCC tissues. Functional studies demonstrated that miR-138-5p overexpression or TMEM40 knockdown significantly suppressed ccRCC cell proliferation, migration and invasion in vitro. Notably, we experimentally confirmed that miR-138-5p directly recognizes the 3’-UTR of the TMEM40 transcript and down-regulated its expression in ccRCC cells.Conclusions: Taken together, our findings provide the first clues regarding the role of miR-138-5p as a tumor suppressor in ccRCC by directly targeting of TMEM40

Multiplexed detection of micro-RNAs based on microfluidic multi-color fluorescence droplets.

In this work, simple, rapid, and low-cost multiplexed detection of tumor-related micro-RNAs (miRNAs) was achieved based on multi-color fluorescence on a microfluidic droplet chip, which simplified the complexity of light path to a half. A four-T-junction structure was fabricated to form uniform nano-volume droplet arrays with customized contents. Multi-color quantum dots (QDs) used as the fluorescence labels were encapsulated into droplets to develop the multi-path fluorescence detection module. We designed an integrated multiplex fluorescence resonance energy transfer system assisted by multiple QDs (four colors) and one quencher to detect four tumor-related miRNAs (miRNA-20a, miRNA-21, miRNA-155, and miRNA-221). The qualitative analysis of miRNAs was realized by the color identification of QDs, while the quantitative detection of miRNAs was achieved based on the linear relationship between the quenching efficiency of QDs and the concentration of miRNAs. The practicability of the multiplex detection device was further confirmed by detecting four tumor-related miRNAs in real human serum samples. The detection limits of four miRNAs ranged from 35 to 39pmol/L was achieved without any target amplification. And the linear range was from 0.1nmol/L to 1μmol/L using 10nL detection volume (one droplet) under the detection speed of 320 droplets per minute. The multiple detection system for miRNAs is simple, fast, and low-cost and will be a powerful platform for clinical diagnostic analysis. Graphical abstract.

A tetrahedral DNA nanoflare for fluorometric determination of nucleic acids and imaging of microRNA using toehold strands.

The authors describe a tetrahedral DNA nanostructure loaded with SYBR Green (SG-TDN) for fluorometric determination of nucleic acids. After intercalating into the TDN, fluorescence of SG is enhanced by 260-fold (exc 480nm, em 524nm), and the resulting SG-TDN nanoflare displays >7-fold stronger fluorescence than that of FAM-labeled TDN. The SG-TDNs were coupled to magnetic microparticles and polydopamine nanoparticles to construct multi-functional nanoprobes through sequence hybridization using a toehold strand. The method was applied to detect a stretch of microRNA sequence (20bp) in buffer and in undiluted serum with excellent selectivity, over awide linear range and with alow limit of detection (0.2nM). The probe was also applied for visualization of tumor-related microRNA in living cells via fluorescence imaging. Graphical abstract Schematic representation of tetrahedron-based DNA nanoflare for fluorometricnucleic acid determination in undiluted blood serum and living cells.

Target-assisted self-cleavage DNAzyme probes for multicolor simultaneous imaging of tumor-related microRNAs with signal amplification.

In this work, we have developed a novel and highly selective signal amplification strategy based on target-assisted self-cleavage DNAzyme probes for multicolor and simultaneous imaging of miRNA-222 and miRNA-223 in living cells. The fluctuation of miRNA-222 and miRNA-223 expression levels could also be monitored.

Platelet microparticles: small payloads with profound effects on tumor growth.

It has long been known that platelets facilitate metastasis formation (1). In cancer, platelets are known to contain tumor related microRNA and serve as a biomarker for cancer (2-4). Cancer related platelet profiles also associate with consensus molecular subtypes of colorectal cancer (5).

Comparative analysis of methods to determine DNA methylation levels of a tumor-related microRNA gene

Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation—bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight—by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R2=0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R2=0.864 and R2=0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison.