Stability Of miRNAs Research Articles

Digestion of Plant Dietary miRNAs Starts in the Mouth under the Protection of Coingested Food Components and Plant-Derived Exosome-like Nanoparticles.

The regulatory functions of plant miRNAs on mammalian bodies are controversial, mainly because stability of the miRNAs in the digestive tract, as the prerequisite for their cross-kingdom effects, has somehow been overlooked. Hence, as the first stage of food ingestion, stability of plant miRNAs in human saliva has been investigated. The results show that plant miRNAs are of considerable resistance against salivary digestion, as surviving miRNAs more than 20 fM are detected. The stability varies dramatically, which can be explained by the difference in tertiary structure, governing their affinities to RNase. Surprisingly, miRNAs of low initial concentrations can end up with high survival rates after digestion. Plant miRNAs can be loaded into exosome-like nanoparticles (ELNs) and microcapsules formed by food components, both of which protect the miRNAs from being degraded in human saliva. Overall, plant miRNAs can apply certain strategies to maintain constant concentrations, paving the way for their potential cross-kingdom effects.

Recent Progress in Nanomaterials Modified Electrochemical Biosensors for the Detection of MicroRNA.

MicroRNAs (miRNAs) are important non-coding, single-stranded RNAs possessing crucial regulating roles in human body. Therefore, miRNAs have received extensive attention from various disciplines as the aberrant expression of miRNAs are tightly related to different types of diseases. Furthermore, the exceptional stability of miRNAs has presented them as biomarker with high specificity and sensitivity. However, small size, high sequence similarity, low abundance of miRNAs impose difficulty in their detection. Hence, it is of utmost importance to develop accurate and sensitive method for miRNA biosensing. Electrochemical biosensors have been demonstrated as promising solution for miRNA detection as they are highly sensitive, facile, and low-cost with ease of miniaturization. The incorporation of nanomaterials to electrochemical biosensor offers excellent prospects for converting biological recognition events to electronic signal for the development of biosensing platform with desired sensing properties due to their unique properties. This review introduces the signal amplification strategies employed in miRNA electrochemical biosensor and presents the feasibility of different strategies. The recent advances in nanomaterial-based electrochemical biosensor for the detection of miRNA were also discussed and summarized based on different types of miRNAs, opening new approaches in biological analysis and early disease diagnosis. Lastly, the challenges and future prospects are discussed.

Non-Coding RNAs in Human Breast Milk: A Systematic Review.

Breast milk is the primary source of nutrition and hydration for the newborn infant but also plays an important role in the child’s first immune defense. Additionally, several breast milk factors have been implicated in immune-related health outcomes later in life, including immunoglobulins, cytokines, chemokines, growth factors and, more recently, non-coding RNA (ncRNA) species. In this systematic review, we provide a comprehensive summary of the current literature on endogenous ncRNAs found in human breast milk. Thirty (30) relevant studies were identified and, whilst the majority studies focused on microRNAs (miRNAs), there is evidence that breast milk contains high quantities of RNA which also include long-coding RNAs, circular RNAs, as well as other short RNAs and fragmented tRNA and rRNAs. Among studies investigating miRNAs, miR-148a-3p, miR-30a/d-5p, miR-22-3p, miR-146b-5p, miR-200a/c-3p, and the 5p end of the let-7 miRNAs were commonly reported among the top 10 miRNAs in the cell, lipid, and skim milk fractions of breast milk. Methodological difference and small sample sizes limit the possibility of conclusively identifying which maternal and infant characteristics affect the miRNA profile. The highly expressed miRNAs were generally reported to be similar across lactational stage, milk fraction, maternal and infant characteristics, or infant growth and health. All the same, individual studies identify potential differences in miRNA expression levels which should be confirmed by future studies. Stability, uptake, and physiological functions of miRNAs were also considered in several studies. Breast milk miRNAs are relatively resistant to a range of harsh conditions and uptake experiments suggest that extracellular vesicles containing miRNAs and circular RNAs can be taken up by intestinal epithelial cells. Although the evidence regarding the functional effect of breast milk miRNAs is limited, the predicted functions range from metabolic and biosynthetic processes to signaling pathways, cellular adhesion, communication, growth, and differentiation. Finally, this systematic review highlights some of the methodological challenges and knowledge gaps which can help direct future research in this field. In particular, it is important to further investigate the bioavailability of miRNAs in different milk fractions, and to characterize other ncRNAs which are largely unstudied.Systematic Review RegistrationPROSPERO https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=138989, identifier CRD42020138989.

The Role of microRNAs in Osteoporosis Diagnostics

AbstractMicroRNAs (miRNAs) are short (18–24 nucleotides) non-coding RNA sequences that regulate gene expression via binding of messenger RNA. It is estimated that miRNAs co-regulate the expression of more than 70% of all human genes, many of which fulfil important roles in bone metabolism and muscle function. In-vitro and in-vivo experiments have shown that the targeted loss of miRNAs in distinct bone cell types (osteoblasts and osteoclasts) results in altered bone mass and bone architecture. These results emphasize the biological relevance of miRNAs for bone health.MiRNAs are not only considered as novel bone biomarkers because of their biological importance to bone metabolism, but also on the basis of other favorable properties: 1) Secretion of miRNAs from cells enables “minimally invasive” detection in biological fluids such as serum. 2) High stability of miRNAs in serum enables the retrospective analysis of frozen blood specimens. 3) Quantification of miRNAs in the serum is based on the RT-PCR - a robust method that is considered as the gold standard for the analysis of nucleic acids in clinical diagnostics.With regard to osteoporosis, it has been shown that many of the known risk factors are characterized by distinct miRNA profiles in the affected tissues: i) age-related loss of bone mass, ii) sarcopenia, iii) changes in estrogen metabolism and related changes Loss of bone mass, and iv) diabetes. Therefore, numerous studies in recent years have dealt with the characterization of miRNAs in the serum of osteoporosis patients and healthy controls, and were able to identify recurring miRNA patterns that are characteristic of osteoporosis. These novel biomarkers have great potential for the diagnosis and prognosis of osteoporosis and its clinical outcomes.The aim of this article is to give a summary of the current state of knowledge on the research and application of miRNA biomarkers in osteoporosis.

Connection between miRNA Mediation and the Bioactive Effects of Broccoli (Brassica oleracea var. italica): Exogenous miRNA Resistance to Food Processing and GI Digestion.

Broccoli (Brassica oleracea var. italica) and its bioactive compounds are associated with beneficial health effects, which might be enabled, at least in part, through miRNA regulation, despite recent controversial studies suggesting that exogenous dietary miRNAs may reach host circulation and target cells to regulate gene expression. Here, a computational analysis was performed to explore the processes and pathways associated with genes targeted either by (1) host-expressed miRNAs (endogenous) modulated by the bioactive compounds in broccoli or (2) miRNAs derived from broccoli (exogenous). In addition, the stability of exogenous miRNAs from broccoli was assessed after broccoli was subjected to the usual processing methods and in vitro digestion-simulating gastrointestinal (GI) conditions. Overall, bioinformatic results show that the anticarcinogenic and cancer-preventive properties attributed to cruciferous vegetables might be mediated, at least in part, through miRNA-related mechanisms. Moreover, results show that broccoli-derived miRNAs can survive common food-processing conditions and GI digestion.

MRNA and microRNA stability validation of blood samples under different environmental conditions

RNA molecules, including mRNAs and microRNAs (miRNAs), have been used for forensic body fluid identification. Specific body fluids present unique mRNA expression patterns, while miRNAs identifying body fluids are mainly differentially expressed. miRNAs are thought to be more stable than mRNAs, although this lacks adequate supporting data. In this study, we addressed perceived concerns regarding the stability of miRNAs and mRNAs in blood samples. The samples used in this study involved three groups. First, environmentally-degraded blood stain samples were exposed to a range of environmental conditions over 1–360 days to degrade naturally. Second, simulated-degraded samples were prepared using RNase A or high temperature (80 °C). Furthermore, two authentic casework samples that were proven to be degraded from short tandem repeat (STR) profiles were analyzed. mRNAs and miRNAs present in the same blood samples were simultaneously detected through reverse transcriptase qPCR (RT-qPCR). Furthermore, mRNAs expression was determined by an mRNA multiplex PCR system. Our results showed that both mRNAs and miRNAs were stable in dry environments. The stability of miRNAs was relatively higher than that of mRNAs in humid environments or at high temperature. RNase A had the most serious impact on RNA stability, both mRNA profiles and miRNAs expression patterns were altered. The results of this study provide data and support to demonstrate that miRNAs represent more stable RNA molecules in body fluid identification compared to mRNAs.

Small RNA expression and miRNA modification dynamics in human oocytes and early embryos.

Small noncoding RNAs (sRNAs) play important roles during the oocyte-to-embryo transition (OET), when the maternal phenotype is reprogrammed and the embryo genome is gradually activated. The transcriptional program driving early human development has been studied with the focus mainly on protein-coding RNAs, and expression dynamics of sRNAs remain largely unexplored. We profiled sRNAs in human oocytes and early embryos using an RNA-sequencing (RNA-seq) method suitable for low inputs of material. We show that OET in humans is temporally coupled with the transition from predominant expression of oocyte short piRNAs (os-piRNAs) in oocytes, to activation of microRNA (miRNA) expression in cleavage stage embryos. Additionally, 3′ mono- and oligoadenylation of miRNAs is markedly increased in zygotes. We hypothesize that this may modulate the function or stability of maternal miRNAs, some of which are retained throughout the first cell divisions in embryos. This study is the first of its kind elucidating the dynamics of sRNA expression and miRNA modification along a continuous trajectory of early human development and provides a valuable data set for in-depth interpretative analyses.

Isolation of Cell-Free miRNA from Biological Fluids: Influencing Factors and Methods.

A vast wealth of recent research has seen attempts of using microRNA (miRNA) found in biological fluids in clinical research and medicine. One of the reasons behind this trend is the apparent their high stability of cell-free miRNA conferred by small size and packaging in supramolecular complexes. However, researchers in both basic and clinical settings often face the problem of selecting adequate methods to extract appropriate quality miRNA preparations for use in specific downstream analysis pipelines. This review outlines the variety of different methods of miRNA isolation from biofluids and examines the key determinants of their efficiency, including, but not limited to, the structural properties of miRNA and factors defining their stability in the extracellular environment.

Dysregulated plasma microRNAs as potential biomarkers of aging and Alzheimer´s disease

The constant aging of the population worldwide leads to an increased prevalence of age-related diseases like neurodegenerative disorders. Alzheimer´s disease (AD) as one of the most widespread form of neurodegeneration is defined by the gradual deterioration of memory and cognitive functions. The pathological changes emerge years before the onset of clinical symptoms. Small non-coding RNAs, known as microRNAs (miRNAs), act like post-transcriptional gene regulators that trigger translational inhibition or mRNA degradation. The biological role and stability of miRNAs in peripheral biofluids imply their candidate role as diagnostic and prognostic markers. Our study aimed to detect and validate candidate miRNA biomarkers related to AD neurodegeneration. Transcriptomic analysis was executed on AD patients compared to healthy age-matched controls to determine expression levels of miRNAs in plasma samples. The examination of samples identified a panel of dysregulated miRNAs differentiating the AD individuals from healthy controls. Our data suggest the diagnostic potential of microRNAs in AD and other neurodegenerative diseases.

MicroRNAs in childhood nephrotic syndrome.

The discovery of microRNAs (miRNAs) has opened up new avenues of research to understand the molecular basis of a number of diseases. Because of their conservative feature in evolution and important role in the physiological function, microRNAs could be treated as predictors for disease classification and clinical process based on the specific expression. The identification of novel miRNAs and their target genes can be considered as potential targets for novel drugs. Furthermore, currently, the circulatory and urinary exosomal miRNAs are gaining increasing attention as their expression profiles are often associated with specific diseases, and they exhibit great potential as noninvasive or minimally invasive biomarkers for the diagnosis of various diseases. The remarkable stability of these extracellular miRNAs circulating in the blood or excreted in the urine underscored their key importance as biomarkers of certain diseases. There is voluminous literature concerning the role of microRNAs in other diseases, such as cardiovascular diseases, diabetic nephropathy, and so forth. However, little is known about their diagnostic ability for the pediatric nephrotic syndrome (NS). The present review article highlights the recent advances in the role of miRNAs in the pathogenesis and molecular basis of NS with an aim to bring new insights into further research applications for the development of new therapeutic agents for NS.

Advances in reciprocal regulation mechanisms of microRNAs and target genes

<p indent="0mm">MicroRNAs (miRNAs), which are encoded by endogenous genes, are a class of single-stranded non-coding RNAs with approximately <sc>22 nt</sc> in length. They act on target genes by binding to Argonaute (AGO) family proteins to form miRNA-induced silencing complexes (miRISCs), thereby functioning at the transcriptional and post-transcriptional levels. With the in-depth study of miRNAs, it has been found that in addition to messenger RNAs (mRNAs), non-coding RNAs (ncRNAs), primary miRNAs (pri-miRNAs), and DNAs can also act as the target genes of miRNAs. By mRNA or ncRNA cleavage, DNA transcription silence or activation, pri-miRNA processing, and mRNA translation, miRNAs regulate almost all cell proliferation/differentiation, individual growth/development, and homeostasis. Since miRNAs are differentially expressed in various cell types and play a role in the whole development process, studies on miRNAs have focused on their origins, biogenesis, and mechanisms. However, the mechanisms of miRNA transformation, especially the rapid change of miRNAs under specific conditions, have not been well addressed. Recently, it has been discovered that target genes can regulate the biogenesis, degradation, and protection of miRNAs in a sequence-dependent manner, indicating that the regulation of miRNAs and target genes is not unidirectional, but reciprocal. The target genes modulate the biogenesis of miRNAs by mediating the processing, stability, and nuclear export of pri-miRNAs or precursor miRNAs (pre-miRNAs). The modulation depends on the binding of miRNAs to the complementary sequence on the target genes. However, no general binding pattern represents the modulation of target genes on miRNA biogenesis. Extensive complementarity between target genes and miRNAs is a crucial requirement for the target-directed miRNA degradation (TDMD). Central bulge with varied length is a mismatched region in the center of an extensive complementarity and is essential to trigger TDMD. The mechanisms of TDMD are controversial. It may be due to the exonuclease-induced miRNA degradation, which is dependent on tailing and trimming; or the ZSWIM8 ubiquitin ligase-triggered miRNA degradation, which is independent of tailing and trimming. Organisms increase miRNA stability through target genes and coordinate with TDMD to maintain miRNA homeostasis. Elevated accumulation of the passenger strand in AGO is found in the process of target genes promoting miRNA stability. However, it is unclear whether the process will trigger a transformation of the guide strand. Although the study on the regulation of miRNAs by target genes is still in infancy, it provides a novel perspective for the regulation of miRNAs. In this review, the latest progress in the regulation of miRNAs by target genes was outlined in detail, the conditions, mechanisms, and functions of their interaction were summarized, and future research directions on miRNAs transformation were proposed in order to provide a theoretical basis for further understanding the interaction between miRNAs and target genes<italic> in vivo</italic> and the development of miRNA-based therapeutic approaches.

Regulation of RNA stability at the 3' end.

RNA homeostasis is regulated by a multitude of cellular pathways. Although the addition of untemplated adenine residues to the 3' end of mRNAs has long been known to affect RNA stability, newly developed techniques for 3'-end sequencing of RNAs have revealed various unexpected RNA modifications. Among these, uridylation is most recognized for its role in mRNA decay but is also a key regulator of numerous RNA species, including miRNAs and tRNAs, with dual roles in both stability and maturation of miRNAs. Additionally, low levels of untemplated guanidine and cytidine residues have been observed as parts of more complex tailing patterns.

The persistence and stability of miRNA in bloodstained samples under different environmental conditions

miRNA markers have been an area of forensic interest to identify body fluid sources in recent years. In this study, reverse transcription and quantitative real time polymerase chain reaction (RT-qPCR) were performed to detect the existence of blood-specific miRNA markers in bloodstained samples under different environmental conditions, Blood samples from 6 individuals were deposited onto glass plates and exposed to different temperature, humidity, ultraviolet light intensity, and natural condition. When samples were stored to a series of estimated test times, total RNA was extracted and the Ct values of the target RNAs were detected, targets included two miRNA markers (hsa-miR-16-5p, hsa-miR-451a) and one reference gene (U6 snRNA). Analysis results showed that miR-451a represented strong stability and could be detected at all detection points. Meanwhile, each RNAs exhibited unique degradation characteristics, compared to U6, miRNAs showed stronger stability. Additionally, rain had an adverse effect on RNAs stability and accelerates its degradation rate.

Different stability of miRNAs and endogenous control genes in archival specimens of papillary thyroid carcinoma

BackgroundThe most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. However, there are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). Therefore, in this study we evaluated the impact of chosen EC and archival formalin-fixed, paraffin-embedded (FFPE) PTC tissue age on estimated target miRNA expression.MethodsRQ-PCR was used to determine expression levels of five miRNAs (miR-146b, miR-222, miR-21, miR-221 and miR-181b) and three different endogenous controls (RNU48, let-7a, miR-16), which were used to normalize the data. In total, 400 FFPE PTC tissues were analyzed that have been stored from 1 to 15 years.ResultsThe stability of commonly used ECs RNU48 and let-7a significantly differs from the stability of target miRNA in archival FFPE PTC tissues. Moreover, these differences have a great impact on miRNA expression results when FFPE tissue samples have been stored for a different period of time.ConclusionsIt is important to select an ECs not only stable in the tissue of interest but also with similar stability to target miRNA, especially when working with samples of different age.

Effect of vitrification on biogenesis pathway and expression of development-related microRNAs in preimplantation mouse embryos

Vitrification of embryos has been known as the most efficient cryopreservation method in assisted reproductive technology clinics. Vitrification of preimplantation embryo might be associated with altered gene expression profile and biochemical changes of vitrified embryos. Stringent regulation of gene expression in early embryonic stages is very critical for normal development. In the present study, we investigated the effect of vitrification on the canonical miRNA biogenesis pathway, and also the expression of developmental related miRNAs, in 8-cell and blastocyst mouse embryos. Although the expression pattern of the miRNA biogenesis pathway genes differed between 8-cell and blastocyst mouse embryos, vitrification did not affect the expression level of these genes in preimplantation embryos. The expression levels of miR-21 and let-7a were significantly decreased in vitrified 8-cell embryos and fresh blastocysts when compared with fresh 8-cell embryos. The expression of Stat3 was significantly reduced in blastocysts after vitrification. The alteration in the expression pattern of miRNAs, due to their mode of action, can affect broad downstream key developmental signaling pathways. Therefore, the blastocyst stage is the preferred point for embryo vitrification as they are less susceptible to cryo-damages regarding the stability of miRNAs related to the developmental and implantation competence of embryo.

Impact of processing method on donated human breast milk microRNA content.

Pasteurization of donated human milk preserves it for storage and makes it safe for feeding, but at the expense of its composition, nutritional values and functions. Here, we aimed to investigate the impact of Holder Pasteurization (HoP) and High Pressure Processing (HPP) methods on miRNA in human milk and to evaluate impact of these changes on miRNA functions. Milk samples obtained from women in 50th day of lactation (n = 3) were subjected either to HoP, HPP or remained unpasteurized as a control. Subsequently, miRNA was isolated from whole material and exosomal fraction and sequenced with Illumina NextSeq 500. Sequencing data were processed, read counts were mapped to miRNA and analyzed both quantitatively with DESeq2 and functionally with DIANA mirPath v.3. While HPP caused statistically insignificant decrease in number of miRNA reads compared to unprocessed material, HoP led to 82-fold decrease in whole material (p = 0.0288) and 302-fold decrease in exosomes (p = 0.0021) not leaving enough reads for further analysis. Changes in composition of miRNA fraction before and after HPP indicated uneven stability of individual miRNAs under high pressure conditions, with miR-30d-5p identified as relatively stable and miR-29 family as sensitive to HPP. Interestingly, about 2/3 of unprocessed milk miRNA content consists of only 10 distinct miRNAs with miR-148a-3p at the top. Functional analysis of most abundant human milk miRNAs showed their involvement in signaling pathways, cell communication, proliferation and metabolism that are obviously important in rapidly growing infants. Functions of miRNAs which suffered the greatest depletion during HPP were similar to roles of the majority of unprocessed human milk's miRNA, which indicates that those functions may be weakened although not completely lost. Our findings indicate that HPP is less detrimental to human milk miRNAs than HoP and should be considered in further research on recommended processing procedures for human milk banks.

Prediction of miRNA targets by learning from interaction sequences.

MicroRNAs (miRNAs) are involved in a diverse variety of biological processes through regulating the expression of target genes in the post-transcriptional level. So, it is of great importance to discover the targets of miRNAs in biological research. But, due to the short length of miRNAs and limited sequence complementarity to their gene targets in animals, it is challenging to develop algorithms to predict the targets of miRNA accurately. Here we developed a new miRNA target prediction algorithm using a multilayer convolutional neural network. Our model learned automatically the interaction patterns of the experiment-validated miRNA:target-site chimeras from the raw sequence, avoiding hand-craft selection of features by domain experts. The performance on test dataset is inspiring, indicating great generalization ability of our model. Moreover, considering the stability of miRNA:target-site duplexes, our method also showed good performance to predict the target transcripts of miRNAs.

Stability and absorption mechanism of typical plant miRNAs in an in vitro gastrointestinal environment: basis for their cross-kingdom nutritional effects

Plant miRNAs, a group of 19–24 nt noncoding RNAs from plant foods, were recently found to have immunomodulatory and nutritional effects on mammalian and human bodies. However, how the miRNAs survive gastrointestinal (GI) environment and how the stable miRNAs are absorbed, which serve the basis for their biological functions, were not unraveled. Here, we investigated the stabilities of six typical plant miRNAs in simulated gastric and intestinal environments, and the absorption mechanisms by Caco-2 cells. The results showed that the miRNAs can survive the environment with certain concentrations. The mixture of food ingredients enhanced the stabilities of the plant miRNAs in the gastric conditions, while 2′-O-methyl modification protects the miRNAs in intestinal juice. The stabilities of the miRNAs vary significantly in the environment and are related to their secondary structures. The stable plant miRNAs can be absorbed by Caco-2 cells via clathrin- and caveolin-mediated endocytosis. Uptake of the miRNAs was sequence dependent, facilitated by NACh and TLR9, two typical receptors on cell membrane. The results suggest that some of plant miRNAs are stable in the mimic GI environment and can be absorbed by Caco-2 cells, underlying the potential of their cross-kingdom regulation effects.

Study of the preanalytical variables affecting the measurement of clinically relevant free-circulating microRNAs: focus on sample matrix, platelet depletion, and storage conditions.

IntroductionCirculating microRNAs (miRNAs) are emerging as potential biomarkers. However, the lack of preanalytical and analytical standardization limits their use. The aim of this study was to determine the expression of different miRNAs in plasma according to different collection and storage conditions.Materials and methodsVenous blood from 10 volunteers was collected in tubes spray-coated with dipotassium salt of ethylendiaminetetraacetic acid, either with (plasma-preparation tube, PPT) or without (K2EDTA) gel separator. Platelet-poor plasma (PPP) was also obtained from K2EDTA plasma. After storage under different conditions, miRNA-enriched total RNA was isolated from plasma and reverse transcribed. A panel of 179 miRNAs was assayed by quantitative polymerase chain reaction and the results were analysed by GenEx software. Detectability and stability of miRNAs were determined.ResultsThe number of undetected miRNAs was: 18, 24, and 22 in PPT; 83, 43, and 20 in K2EDTA; and 76, 106, and 104 in PPP samples, for plasma immediately frozen at - 80°C and plasma stored for 24h at room temperature or 4°C, respectively. Circulating miRNA expression in PPT samples was not affected by storage delay or temperature, while the percentage of up- and down-regulated miRNA in K2EDTA and PPP samples ranged from 2%, and 1% to 7%, and 5%, respectively.ConclusionsSample matrix, temperature and delay in storage strongly influence the expression level of plasma miRNAs. Our results indicate PPT tubes as the most suitable matrix to improve total miRNA detectability and stability, independently of temperature.

Laboratory Examination in Hepatocelullar Carcinoma

Hepatocellular carcinoma (HCC) is a malignant tumor originating from hepatocyte cells including primary malignant tumors of the epithelial liver. Hepatocellular carcinoma is the fifth most malignant in the world and the third largest cause of death of all malignancies. Diagnosis of HCC is obtained from the patient's history, examination of the patient, by imaging (ultrasonography, MRI or CT scan) and an increase in serum tumor biomarkers of alpha feto protein (AFP) (&gt; 400 ng mL). Early diagnosis is important to increase the patient's life expectancy and only 30-40% of patients are diagnosed at an early stage. Biomarkers in HCC not only help in diagnosing but also predict prognosis or recurrence and in choosing therapy. The stability of miRNA in blood circulation makes miRNA an ideal candidate for use in the diagnosis and treatment of cancer.