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Arthritis is a kind of autoimmune disease, which includes many circumstances that affect joints, the tissues surrounding the joints, and other connective tissues. Osteoarthritis (OA) and rheumatoid arthritis (RA) are the common arthritis seen in many populations. Researchers have made extensive studies on all types of arthritis, novel drugs are being developed by many laboratories, but yet no treatment option is available for these diseases and need new insight into the molecular pathways and pathophysiology of all types of arthritis. MicroRNAs (miRNAs), a class of non-coding RNAs, have shown to be played a plenty of roles in both a suppressive and a promoting role in disease pathogenesis and progression. Among the classes of microRNAs, miR-21 is a widespread miRNA commonly upregulated in many diseases and suggesting that it plays an important role in cell proliferation, apoptosis, and invasion. It is highly expressed in osteoclast precursors and the pro-osteoclastogenic nature of miR-21 makes it a promising candidate as a therapeutic target to treat bone-related disorders. Up to now, there are few papers that demonstrate the role of miR-21 in arthritis and related bone disorders and the number of studies related to different types of arthritis is sparse. Therefore, the main thrust of this paper is to provide an overview of the current clinical evidence and significance of miR-21 in arthritis and bone-related inflammation disorders. We summarize the important research findings surrounding the role of miR-21 and its involvement in the treatment of different types of arthritis.

Diabetes is one of the most significant health issues across the world. People identified with diabetes are more vulnerable to various infections and are at a greater risk of developing cardiovascular diseases. The plant-based food we consume often contains many sterol-based bioactive compounds. It is well documented that these compounds could effectively manage the processes of insulin metabolism and cholesterol regulation. Insulin resistance followed by hyperglycemia often results in oxidative stress level enhancement and increased reactive oxygen species production. At the molecular level, these changes induce apoptosis in pancreatic cells and hence lead to insulin insufficiency. Studies have proved that plant sterols can lower inflammatory and oxidative stress damage connected with DNA repair mechanisms. The effective forms of phyto compounds are polyphenols, terpenoids, and thiols abundant in vegetables, fruits, nuts, and seeds. The available conventional drug-based therapies for the prevention and management of diabetes are time-consuming, costly, and with life-threatening side effects. Thereby, the therapeutic management of diabetes with plant sterols available in our daily diet is highly welcome as there are no side effects. This review intends to offer an overview of the present scenario of the anti-diabetic compounds from food ingredients towards the therapeutic beneficial against diabetes.

Introduction: Hypertension (HTN) is one of the major public health complications throughout the world. Although progress has been made in HTN research, early diagnosis and treatment of HTN are yet to be flourished. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression in many diseases including HTN. According to our previous report, there is direct evidence showing that miR-510 is involved in HTN. Hypothesis: We assessed the hypothesis that to decipher the critical pathways of miR-510 and PTEN in regulation of PI3K/AKT Pathways, so it can be used as a therapeutic pathway as well as biomarker. Finally, we intend to study how anti-miR-510 reduces the HTN in the hypertensive induced rat model. Methodology: Proliferation, migration, invasion and apoptosis capacity of miR-510, Anti-miR-510, PTEN and eNOS were evaluated in HUVEC Cells. The expression pattern of miR-510, anti-miR-510 and eNOS, PTEN are analyzed by qPCR and western blot The relationship between miR-510, Anti-miR-510 and PTEN are investigated by luciferase assay Deoxycorticosterone acetate (DOCA) of 70mg/kg and 1% NaCl salt induced hypertension model (Sprague Dawley rats) was used in this study. According to the dose dependent study, anti-miR-510 was administered intravenously. Histological analyses are performed to examine the hypertrophy by using cardiac tissues fixed in 1% paraformaldehyde. Results and conclusion: Our results suggested that miR-510 can directly influence the PTEN expressions in HUVEC cells. Furthermore, the in vitro experiments clearly indicated that miR-510/PI3K/AKT/PTEN axis involved in HTN. Anti-miR-510 delivery can reduce the progression of HTN in the hypertensive induced rat model. Though miR-510 is involved in HTN, but its molecular mechanism is almost unknown. So, these analyses suggested that the correlation between miR-510, PTEN, anti-miR-510 are the critical step in gene expression and regulations. Thus, all these analyses could lead in identification of therapeutic target and non-invasive biomarker for HTN. Our in vivo experiments suggesting that anti-miR-510 may act as a novel therapeutic molecule for HTN treatment.

Stroke is an important neurological disease in which blood flow to the brain is interrupted and it is becoming an increasing non-communicable disease in developing countries. Current treatment options for stroke is modifying lifestyle practice, diabetes treatment, drugs, and other factors management, but yet no cure is available in sight for the disease, despite it requires new insight into the molecular and therapeutic targets. In general, MicroRNAs (miRNAs) are small noncoding RNAs considered as of greater biological importance and controls molecular signaling pathways in diabetic pathogenesis. Among the reported MiRNAs, MIR-21 is considered to be an important MiRNA, which is frequently elevated in many types of types of strokes, suggesting that it plays an important role in cell proliferation, and apoptosis. Until now, there is no research paper that signifying the role of miR-21 in all types of strokes and the number of studies on the different category of strokes is limited, so in this paper, we are highlighting the recent investigations related to the significance of miR-21 in different types of strokes based on the up-to-date reports. It was found that MiR-21 was found to be normally up and down regulated in all types of strokes, however; we summarize the important research findings related to the role of miR-21 in different types of strokes.

MicroRNAs are a novel group of universally present non-coding RNA that has been implicated in the wide-ranging biological process and are critical in the manifestation of diverse disease including diabetes mellitus (DM). Although there are so many numbers of micro RNAs involved in DM none of them effective for the suppression of Type I DM (T1DM), hence there is an urgent need for identifying exact microRNAs that may serve as a candidate biomarker for T1DM. Moreover, we know that expressed sequence tags (ESTs) are random single pass sequences reads which displays the condition/tissue-specific transcribes of an organism. In the present study, we have applied bioinformatics tools to identify hsa-mir-7110-5p from diabetes mellitus using EST resources. Our studies revealed that hsa-mi-7110 was confirmed in the T1DM specific EST sequence among the predicted miRNA secondary structure. It has been known that miRNAs are known to be an important factor involved in the initiation and progression of T1DM. Based on the above information, we also deciphered the molecular mechanism and the role of miR-7110 from T1DM patients and found that miR-7110 was a novel prognostic, diagnostic and therapeutic target for T1DM.

Myeloid leukemia (ML) is heterogeneous cancer classified by abnormal growth of myeloid cells due to genetic aberrations and mutations. It is generally categorized by clonal disorders of hematopoietic stem cells and differentiation. The molecular mechanism behind the myeloid malignancies is not yet known, but recent sequencing analysis reveals all the mutated factors. As we know that there is currently no compromise on therapy for such types of malignancies and at the present painful process like chemotherapy and radiation therapy are not effective for the treatment of ML, so there is an urgent need to develop a non-invasive biomarker for different types of ML. MicroRNAs (MiRNAs) is a small non-coding RNAs that have been involved in a wide range of biological function and it is the main cause of the manifestation of many diseases. Among the reported MiRNAs, MIR-21 is considered to be an important MiRNA, which is frequently elevated in many types of types of cancer, suggesting that it plays an important role in cancer progressions. So far, there is no paper that signifies the role of miR-21 in all types of ML and the number of studies on the different category of ML is sparse. Therefore, the main thrust of this paper is to provide an overview of the current clinical evidence and significance of miR-21 in ML. It was found that MiR-21 was found to be normally upregulated in all types of ML, however, we summarize the important research findings surrounding the role of miR-21 in different types of ML.

Breast cancer is more common among urban females. It has been estimated that in India there will be 155,000 new cases of breast cancer and 76,000 deaths due to breast cancer in 2015. Many factors play a key role in the progression of breast cancer but there is no evidence that proves or cures its progressions. Therefore, it is very important to take necessary early steps to screen, diagnose and treat the breast cancer as early as possible to save millions of lives. The present review high lights the significance of so many factors responsible for the progression of this disease, which includes pathological causes, roles of various molecular and biochemical markers, genetic mutations, role of micro RNA, gene expression analysis, cancer stem cells, apoptotic markers, prognosis, treatment and also emerging treatment modalities along with the existing ones like vaccines.

Mitochondrial dysfunction arises from an inadequate number of mitochondria, an inability to provide necessary substrates to mitochondria, or a dysfunction in their electron transport and a denosine triphosphate synthesis machinery. Occurrences of mitochondrial dysfunction are due to genetic or environmental changes in the mitochondria or in the nuclear DNA that codes mitochondrial components. Currently, drug options are available, yet no treatment exists in sight of this disease and needs a new insight into molecular and signaling pathways for this disease. microRNAs (miRNAs) are small, endogenous, and noncoding RNAs function as a master regulator of gene expression. The evolution of miRNAs in the past two decades emerged as a key regulator of gene expression that controls physiological pathological cellular differentiation processes, and metabolic homeostasis such as development and cancer. It has been known that miRNAs are a potential biomarker in both communicable and noncommunicable diseases. But, in the case of mitochondrial dysfunction in miRNAs, the number of studies and investigations are comparatively less than those on other diseases and dysfunctions. In this review, we have elaborated the roles of miRNAs in the mitochondrial diseases and dysfunctions.

Osteosarcoma (OS) is considered to be a malignant bone tumour that mainly affects the long bones, but it is also involved in other bones of the body. Currently, surgery and chemotherapy have achieved some response to patients with OS, but they are not increasing the survival rate as well as treatment options. Researchers made lot of drug options for OS, but yet, no treatment is existing in sight for the disease and needs a new insight into the molecular and signaling pathways for the disease. Now, it is necessary to develop a novel and alternative strategy for the prognosis, diagnosis and treatment options for OS. MicroRNAs (miRNAs) are a small non-coding RNA, and their size ranges from 18 to 22 nt in length. In the nucleus, miRNAs originate and transcribe into primary transcripts and later cleaved to produce stem loop-structured precursor nucleotides. microRNA 21 (miR-21) is considered to be a trivial marker for many diseases and has been upregulated in many cancers. Moreover, it plays a main role in proliferation, migration, invasion and apoptosis. miR-21 and its associated pathways are very important and play a critical role in the pathogenesis of OS and are considered to be a biomarker and a therapeutic target for OS. To our knowledge, there is no paper that demonstrates the responsibility and the role of miR-21 in OS and the number of studies related to miR-21 in OS is spare. Therefore, the main aim of this paper is to give an outline of the recent clinical investigation and importance of miR-21 in OS. It has been suggested that the up- and downregulation of miRNAs plays a crucial role in the pathogenesis and progression of OS. Normally, miR-21 was found to be upregulated in OS; however, we summarize the clinical relevance and the recent research findings associated with miR-21 in OS.

This letter is regarding an article which is recently published in Hepatology by Zhao Huanget al1 . Interestingly, the authors clearly described the role of long non coding RNA (lncRNA) H19 in the progression of hepatocellular carcinoma (HCC) via PPP1CA-induced inactivation of the p38MAPK pathway and Sponging miR-200b-3p. However, we have certain points which are not discussed in the paper.

Irrespective of positive developments of cancer treatment, the mortality due to various cancers remains high and the mechanisms of cancer initiation and the development also remains mysterious. As we know that microRNAs are considered to be a short noncoding RNA molecules consisting of 21–25 nucleotides (nt) in length and they silence their target genes by inhibiting mRNA translation or degrading the mRNA molecules by binding to their 3′-untranslated (UTR) region and play a very important role in cancer biology. Recent evidences indicate that miR-21 is over expressed in cancer stem cells and plays a vital role in cell proliferation, apoptosis, and invasion. Even though an increased expression level of miR-21 has been observed in cancer stem cells, studies related to the role of miR-21 in cancer stem cells are limited. The main aim of this mini review is to explain the potency of miR-21 in various cancer stem cells (CSCs) and as a new target for therapeutic interventions of cancer progression.

Over the years, considerable progress has been achieved in the management of androgen sensitive prostate cancer. However, inadequacies in the early diagnosis and treatment of prostate cancer remain, resulting in poor treatment and ineffective control of metastatic progression. MicroRNAs (miRNAs) are well known regulatory factor for the cellular and developmental processes and it has been revealed that many miRNAs contribute the initiation and progression of various cancers including prostate cancer. In the present study we have analyzed the expression and methylation status of miR-510 in normal prostate cell line (PNT1A and HPrEC), primary prostate cancer cell line (LNCaP and VCaP), advanced prostate cancer cell lines (DU145 and MDA PCa 2b) as well as in human prostate cancer biopsies and benign prostatic hyperplasia (BPH) samples. Our studies revealed that miR-510 was up-regulated in metastatic form of prostate cancer cell lines (DU145 and MDA PCa 2b) and in human prostate cancer biopsies but no significant changes were noticed in miR-510 expression in other cell lines (LNCaP, VCaP and PNT1A) as well as in BPH samples. At the same time, miR-510 promoter region was found to be methylated in LNCaP and PNT1A cell lines, but in case of DU145, PC-3 cell lines and prostate cancer biopsies, the miR-510 promoter region was found to be unmethylated. Thus the miR-510 may serve as a novel candidate for the diagnosis and as a new therapeutic target for prostate cancer treatment.

Genetic, environmental and demographic factors contribute to the development of essential hypertension. Genetic polymorphism of Rennin-angiotensin-aldosterone system (RAAS) has been extensively studied to determine the genetic susceptibility to hypertension. The insertion/deletion (I/D) angiotensin converting enzyme (ACE) polymorphism has been established as a cardiovascular risk factor in some population, but its association with essential hypertension is controversial. This study sought to determine the association of I/D polymorphism of the ACE gene in south Indian essential hypertensive subjects. A total of 208 clinically diagnosed essential hypertensive patients without any associated diseases and 220 healthy control subjects were included in this study. Distribution and allelic frequency of Insertion (I) and Deletion (D) polymorphism at the 287 base pair Alu repeat sequence in the intron 16 of ACE gene were analyzed. The distribution of II, ID, DD genotypes of ACE gene was 28.3%, 32.6% and 38.9% respectively in essential hypertensive patients and to 53.6%, 26.3% and 20% in controls. The allele frequency for D allele is 0.58 in essential hypertension as compared to 0.34 of control subjects. The genotype and allele frequency of ACE gene polymorphism is significantly differed in patients when compared to controls. In conclusion, the I/D polymorphism of ACE gene is associated with Indian essential hypertension.

Axl expression is deregulated in several cancer types, predicts poor overall patient survival and is linked to resistance to drug therapy. Here, we evaluated a library of natural compounds for inhibitors of Axl and identified dihydroartemisinin, the active principle of the anti-malarial drug artemisinin, as an Axl-inhibitor in prostate cancer. Dihydroartemisinin blocks Axl expression leading to apoptosis, decrease in cell proliferation, migration, and tumor development of prostate cancer cells. Dihydroartemisinin treatment synergizes with docetaxel, a standard of care in metastatic prostate cancer increasing overall survival of mice with human xenografts. Dihydroartemisinin control of miR-34a and miR-7 expression leads to inhibition of Axl expression in a process at least partially dependent on regulation of chromatin via methylation of histone H3 lysine 27 residues by Jumonji, AT-rich interaction domain containing 2 (JARID2), and the enhancer of zeste homolog 2. Our discovery of a previously unidentified miR-34a/miR-7/JARID2 pathway controlling dihydroartemisinin effects on Axl expression and inhibition of cancer cell proliferation, migration, invasion, and tumor formation provides new molecular mechanistic insights into dihydroartemisinin anticancer effect on prostate cancer with potential therapeutic implications.

Hypertension (HTN) is one of the most common emerging disease in developing countries. It alters endothelial cell structure and function, resulting in several diseases, such as cardiovascular disease, peripheral vasculopathy, cerebrovascular disease and nephropathy. Although much progress has been made in researching HTN in recent years, early diagnosis and treatment of HTN are not yet satisfactory, and progression control/treatment is still poor. MicroRNAs are well-known regulators of the physiological and developmental processes of HTN. Our results revealed that miR-510 was upregulated in blood samples from HTN patients, whereas no significant differences were observed in the control samples. Methylation analyses corroborated the miR-510 upregulation in patient samples. These results suggested that miR-510 can be used as a novel biomarker for diagnosis and as a new therapeutic target for HTN.

Background MicroRNAs (miRNAs) are small, highly conserved non-coding RNA molecules involved in the RNA silencing and post-transcriptional regulation of gene expression. miRNAs are well conserved in both plants and animals, and are thought to be a vital and evolutionarily ancient component of gene regulation and also act as oncogenes or tumor suppressors. It is known that Express Sequence Tags (EST) are a short sub-sequence of cDNA sequence, which contain information of condition or tissue specific transcripts (coding and non-coding) of an organism. Methods In the present study, we have applied the bioinformatics tools to identify miRNA from breast cancer using EST resource. Through bioinformatics approach, the presence of an EST encoding hsa-miR-17- 3p of breast cancer was identified. Results Further studies reveal that hsa-miR-17 is confirmed in the breast cancer specific EST sequence among the predicted miRNAs secondary structure. Moreover, miR-17-3p could be responsible for a tumor suppression, which plays a major role in human breast cancer. Conclusion Further studies are required to investigate the molecular mechanisms behind miR-17-3p involves in the suppression of breast cancer cells. Interestingly, our target analysis suggesting that all the targets involved in multiple signaling pathways in different cell regulations moreover, we need to have more number of in vitro and in vivo studies that prove miR-17-3p as candidate microRNA for breast cancer cells.

Hypertension (HTN) is a chronic medical condition that commonly affects the aging population worldwide. The prevalence of HTN is increasing in developing countries and is one of the leading causes of death in older individuals. HTN results from a complex interplay of genetic and environmental factors. Besides, HTN can result in various other health complications such as stroke and chronic kidney diseases, if not treated. Although various studies have explained the underlying mechanisms in the pathogenesis of HTN, limited information is available on their biomarkers. MicroRNAs (miRNAs) are RNA molecules that have been recognized as key regulators for HTN. miR-21 is a common microRNA that is has been reported to be significantly upregulated in HTN individuals. Hence, miR-21 can be a potential therapeutic target for HTN. The number of studies related to miR-21 on hypertension is limited. Therefore, the main thrust of this paper is to provide an overview of the current clinical evidence and significance of miR-21 in HTN.

Background MicroRNAs (miRNAs) are non-coding RNAs known to control a broad range of biological functions such as cellular proliferation, differentiation and programmed cell death. Recent reports showed that miRNAs can act as oncogenes or tumor suppressors, thereby, playing an important role in cancer initiation and progression. Moreover, we know that Expressed sequence tags (ESTs) are random single pass sequence reads, which displays the condition/tissue specific transcripts (coding and non-coding) of an organism. Methods In the present study, we have applied the bioinformatics approach to identify miRNA from prostate cancer using EST resource and its expressions were analyzed by quantitative reverse transcription PCR (qRT-PCR). Results Analysis of transcriptomics resource from the LNCaP cells revealed the presence of an EST encoding hsa-miR-3654. Presence of the premature candidate of miR-3654, demonstrates its expression in LNCaP cells. We further indentified that the expression level (Fold Induction) of miR-3654 in LNCaP was higher than the normal and androgen insensitive prostate cancer cell lines (PNT1A, PC-3). Conclusion we have identified the miR-3654 involved in prostate cancer progression using computational approach and hypothesized that the down regulation of miR-3654 could be responsible for a solid tumor to get cancer stem-like cell phenotype. Further studies are required to investigate the molecular mechanisms behind the STAT3 mediated miR-3654 repression and the associated metastasis.

A series of multifunctional codrugs (1-6) were synthesized to overcome the pro-oxidant effect associated with L-dopa (LD) therapy. Target compounds release LD and dopamine (DA) in human plasma after enzymatic hydrolysis, displaying an antioxidant effect superior to that of N-acetylcysteine (NAC). After intracerebroventricular injection of codrug 4, the levels of DA in the striatum were higher than those in LD-treated groups, indicating that this compound has a longer half-life in brain than LD.

Diabetes mellitus (DM) is a disease related to metabolic disorder and becoming an epidemic in developing countries. Current treatments for DM include modified lifestyle practice, obesity treatment, oral hypoglycemic agents, and insulin sensitizers, yet no cure is available in sight for the disease, despite it requires new insight into the molecular and pathophysiology. MicroRNAs (miRNAs) are small noncoding RNAs considered as of greater biological importance and controls molecular signaling pathways in diabetic pathogenesis. The microRNA 21 is an important microRNA frequently upregulated in all types of diseases suggesting that it plays an important role in cell proliferation, and apoptosis. The present mini review highlights the recent investigations related to the importance of miR-21 in different types of DM based on the up-to-date reports.

Despite promising developments of treatment, the mortality due to gastric cancer remains high and the mechanisms of gastric cancer initiation and the development also remains elusive. It has been reported that patients with positive serologic tests for H. pylori have a higher risk of the development of gastric cancer. microRNAs (miRNAs) are short non-coding RNA molecules consisting of 21-25 nucleotides (nt) in length. The miRNAs silence their cognate target genes by inhibiting mRNA translation or degrading the mRNA molecules by binding to their 3'-untranslated (UTR) regions and plays a very important role in cancer biology. Recent evidences indicate that miR-21 is overexpressed in tumour tissue, including gastric cancer and plays a vital role in tumour cell proliferation, apoptosis, invasion and angiogenesis. Elevated levels of miR-21 is associated with downregulation of tumour suppressor genes, such as programmed cell death 4 (PDCD4), tissue inhibitor of metalloproteinase 3, phosphatase and tensin homolog (PTEN), tropomyosin 1, ras homolog gene family member B, and maspin. Silencing of miR-21 through the use of a miR-21 inhibitor affected cancer cell viability, induced cell cycle arrest and increased chemosensitivity to anticancer agents indicating that miR-21 functions as an oncogene. Although an increased expression level of miR-21 has been observed in gastric cancer, studies related to the role of miR-21 in gastric cancer progression is very limited. The main thrust of this mini review is to explain the potency of miR-21 as a prognostic and/or diagnostic biomarker and as a new target for clinical therapeutic for interventions of gastric cancer progression.

The discovery of microRNAs (miRNAs) is one of the most significant breakthroughs in recent decades. miRNAs are a class of small, endogenous, non-coding RNAs (19–24 nt) that negatively regulate the expression of target genes by binding to their 3′ untranslated region and are involved in various biological processes. Three main approaches that are globally used for the detection of miRNA include direct cloning, forward genetics and computational approaches using bioinformatics.1 The bioinformatic approach uses the available transcriptomic and genomic data for mining miRNAs using a homology search with the existing miRNAs. In the current study, a computational approach was employed to identify potent miRNAs involved in the inflammatory response of the liver. The rat model of lipopolysaccharide (LPS)-induced liver inflammation was used, and the expression of the candidate miRNAs in hepatocytes was quantified by real-time polymerase chain reaction (qRT-PCR). Inflammation is the first step toward combating antigens and toward tissue recovery, and in some instances, it proceeds to a chronic state associated with debilitating autoimmune diseases, such as multiple sclerosis or even cancer. Chronic inflammation of liver tissues may lead to fibrosis, steatosis and cirrhosis, and may end with cancer. The response of liver tissue to chronic injury is stimulated by pro-inflammatory factors, such as bacterial endotoxin or interleukin-1.2 There is an increasing paradigm that miRNAs regulate different biological processes in various cell types within the liver, including liver pathology and cancer.3 The expression of MiR-517a, miR-892a and miR-106a* in bile was increased in patients with Ischemic type biliary lesions, a biliary complication encountered after liver transplantation.4 Although several miRNAs have been reported, miR-155, miR-146a and miR-125b are considered potent miRNAs involved in inflammation-mediated hepatocyte damage at multiple levels by targeting a pro-inflammatory cytokine production cascade where Toll-like receptor and intracellular signaling pathways determine pro- and anti-inflammatory cytokines.5 An increasing body of evidence has clearly shown that a differential expression of miRNAs is associated with liver diseases, such as hepatitis C and hepatitis B, metabolic disorders and drug abuse.6 Different approaches have been used to identify novel miRNAs that are involved in liver disorders. In our study, we used a computational approach that enabled us to identify two inflammatory responsive liver miRNAs from 89 liver EST sequences: rno-miR-24 and rno-miR-466i-5p. In the current study, the MiRBase database was used to identify miRNAs and their conserved homologues from liver ESTs under inflammatory conditions. In brief, the EST sequences (89, February 2013) of hepatic transcripts preferentially expressed during acute inflammation were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/nuccore) by searching ‘liver inflammation', and the inflammatory-responsive miRNAs were predicted as described by Thirugnanasambantham et al.1 The careful analysis of 89 liver EST sequences revealed 10 non-protein-encoding ESTs homologous to the miRNA sequences. A secondary structural analysis of the pre-miRNA-related sequence from the 10 non-coding ESTs revealed the presence of 2 miRNA encoding transcripts in the liver inflammatory EST sequences (Figure 1a and b). The source of the sequences, length of the precursor sequences and their minimum folding free energies and A+U content for the predicated miRNAs are shown in Figure 1c. Among the predicted miRNAs from the analyzed liver ESTs, rno-miR-24 was observed in the indirect strand and rno-miR-466i-5p was observed in the direct strand. rno-miR-466i-5p and rno-miR-24 had a minimum free energy of −29.74 and −48.1 kcal/mol, respectively, with an average of −38.92 kcal/mol. While considering the A+U percentage in the predicted miRNAs, it was approximately 50% and 47.4% in rno-miR-466i-5p and rno-miR-24, respectively. Figure 1 Inflammatory responsive miRNA identified using a computational approach and its stem-loop RT-PCR expression analysis in rat hepatocytes with LPS induction. (a) Hairpin structure of predicted Rattus norvegicus miRNA rno-miR-24; (b) hairpin structure of ... To further confirm the expression of the identified miRNAs in response to inflammation in liver cells, the hepatocytes were isolated from rat livers by the collagenase perfusion method,7 and inflammation was induced with LPS (1 µg/ml; Sigma-Aldrich, St Louis, MO, USA). Total RNA was extracted for the quantitative determination of COX-2, iNOS and the housekeeping gene GAPDH using real-time PCR analysis from stimulated and unstimulated cells at 4, 8, 12, 24, 36 and 48 h after LPS challenge. The forward and reverse primers used for inflammatory marker gene expressions were as follows: iNOS forward: 5′-ACAACAGGAACCTACCAGCTCA-3′, reverse: 5′-GATGTTGTAGCGCTGTGTGTCA-3; COX-2 forward: 5′-TGTATGCTACCATCTGGCTTCGG-3′, reverse: 5′-GTTTGGAAC AGTCGCTCGTCATC-3′, and GAPDH forward: 5′- GTATTGG GCGCCTGGTCACC-3′, reverse: 5′-CGCTCCTGGAAGATGGTGATGG-3′). The cycling condition was 35 cycles of 94 °C for 30 s, 60 °C for 45 s and 72 °C for 1 min followed by a final extension step at 72 °C for 7 min. Reverse transcription and quantitative expression analyses of rno-miR-24, rno-miR-466i-5p, and the endogenous control U6 were performed using real-time PCR as described previously.8 The primer sequences used for stem-loop RT-PCR were as follow: rno-miR-24 stem loop RT primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTTCTG-3′ forward primer 5′-GCGGCGGTGGCTCAGTACAGC-3′ rno-miR-466i-5p stem loop RT primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACACA-3′ forward primer 5′-GCGGCGGTGTGTGTGTGTGTG-3′ Universal reverse primer 5′-GTGCAGGGTCCGAGGT-3′ U6 RT primer 5′-AACGCTTCACGAATTTGCGTG-3′ U6 forward primer 5′-GCTCGCTTCGGCAGCACA-3′ U6 reverse primer 5′-GAGGTATTCGCACCAGAGGA-3′. The relative expression of target genes was stated as a fold ratio using the (ΔΔCT check) 2−ΔΔCT method. The expression patterns of the genes encoding inflammatory markers (Figure 1d) and identified miRNAs (Figure 1e) were analyzed in control and LPS-stimulated hepatocytes using real-time PCR. The expression of mRNA encoding COX-2 and iNOS were upregulated in proportion with LPS stimulation up to 24 h. We observed that the inflammatory markers COX-2 and iNOS reached the maximum level at 24 h (6.33- and 5.09-fold, respectively) and started to decline from 36 h (4.14- and 4.94-expression fold, respectively) after LPS treatment. However, the expression of both miRNAs was detected from 12 h after LPS treatment, in parallel with inflammation progression, as denoted by the expression of inflammatory markers. The maximum expression of both miR-24 and miR-466i-5p was reached 72 h (5.38 and 4.48 expression fold, respectively) after LPS treatment. In in vitro studies, increases in mRNA expression of COX-2 and iNOS levels upon LPS stimulation of hepatocytes were observed in this study. A similar expression pattern of iNOS was noticed from earlier studies in liver slices, where the expression was observed after 5 h of incubation with LPS and increased until 24 h.9 We found that the expression levels of both miRNAs were upregulated with LPS treatment. A similar result was observed with miR-155, a regulator of inflammation.5 Simultaneously, as denoted by the expression of inflammatory markers (COX-2 and iNOS), the inflammation was suppressed with the increase in expression of rno-miR-24 and rno-miR-466i-5p. Song et al.10 reported the upregulation of miR-466i in SST-treated chronic liver disease. In conclusion, our data identify two new miRNAs, miR-24 and miR-466i, which are upregulated in hepatocytes in response to inflammatory stimuli. This upregulation was reciprocally related with the inflammatory markers COX-2 and iNOS, suggesting that they are target genes in liver inflammation. The anti-inflammatory miRNAs identified with a computational approach are known to be involved in the regulation of inflammatory reactions. Furthermore, the analysis of inflammatory markers revealed that the level of inflammation was reduced with an increase in the expression level of the identified miRNA. Therefore, data from this study may open intriguing possibilities for the therapeutic value of the identified miRNAs as anti-inflammatory factors for liver injury.