Interplay of miR-542, miR-126, miR-143 and miR-26b with PI3K-Akt is a Diagnostic Signal and Putative Regulatory Target in HPV-Positive Cervical Cancer.

Human papillomavirus accounts for 99.7% of all cervical cancer cases worldwide. The viral oncoproteins alter normal cell signaling and gene expression, resulting in loss of cell cycle control and cancer development. Also, microRNAs (miRNAs) have been reported to play a critical role in cervical carcinogenesis. Especially these are not only appropriate targets for therapeutic intervention in cervical cancer but also early diagnostic signals. The given study tries to improve the sparse knowledge on miRNAs and their role in this physiological context. Deregulated miRNAs were identified by analyzing the raw data of the well-founded GSE20592 dataset including 16 tumor/normal pairs of human cervical tissue samples. The dataset was quantified by a conservative strategy based on HTSeq and Salmon, followed by target prediction via TargetScan and miRDB. The comprehensive pathway analysis of all factors was performed using DAVID. The theoretical results were subject of a stringent experimental validation in a well-characterized clinical cohort of 30 tumor/normal pairs of cervical samples. The top 31 miRNAs and their 140 primary target genes were closely intertwined with the PI3K-Akt signaling pathway. MiR-21-3p and miR-1-3p showed a prominent regulatory role while miR-542, miR-126, miR-143, and miR-26b are directly targeting both PI3K and AKT. This study provides insights into the regulation of PI3K-Akt signaling as an important inducer of cervical cancer and identified miR-542, miR-126, miR-143, and miR-26b as promising inhibitors of the PI3K-Akt action.

Characterization of the first microRNA in human CDH1 that affects cell cycle and apoptosis and indicates breast cancers progression.

The E-cadherin protein (Cadherin 1, gene: CDH1), a master regulator of the human epithelial homeostasis, contributes to the epithelial-mesenchymal transition (EMT) which confers cell migratory features to the cells. The EMT is central to many pathophysiological changes in cancer. Therefore, a better understanding of this regulatory scenario is beneficial for therapeutic regiments. The CDH1 gene is approximately 100 kbp long and consists of 16 exons with a relatively large second intron. Since none microRNA (miRNA) has been identified in CDH1 up to now we screened the CDH1 gene for promising miRNA hairpin structures in silico. Out of the 27 hairpin structures we identified, one stable RNA fold with a promising sequence motive was selected for experimental verification. The exogenous validation of the hairpin sequence was performed by transfection of HEK293T cells and the mature miRNA sequences could be verified by quantitative polymerase chain reaction. The endogenous expression of the mature miRNA provisionally named CDH1-i2-miR-1 could be confirmed in two normal (HEK293T, HUVEK) and five cancer cell lines (MCF7, MDA-MB-231, SW480, HT-29, A549). The functional characterization by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a suppression of HEK293T cell proliferation. A flow cytometry-based approach showed the ability of CDH1-i2-miR-1 to arrest transfected cells on a G2/M state while annexin staining exemplified an apoptotic effect. BAX and PTEN expression levels were affected following the overexpression with the new miRNA. The in vivo expression level was assessed in 35 breast tumor tissues and their paired nonmalignant marginal part. A fourfold downregulation in the tumor specimens compared to their marginal controls could be observed. It can be concluded that the sequence of the hub gene CDH1 harbors at least one miRNA but eventually even more relevant for the pathophysiology of breast cancer.

miR-21-5p, miR-141-3p, and miR-205-5p levels in urine-promising biomarkers for the identification of prostate and bladder cancer.

BACKGROUND: Early detection of cancers improves patients' survival and decreases the treatment cost. Unfortunately, the current methods for diagnosis of bladder and prostate cancers, two most common urothelial malignancies, suffer from a low sensitivity and specificity. MicroRNAs, as a group of endogenously produced non-coding RNAs, regulate gene expression and their expression is observed to be altered in many cancers and cancer progression phenomena. The remarkable stability of microRNAs in biofluids and their unique expression pattern in different pathological conditions make them an appealing, noninvasive diagnostic method in cancer diagnosis. Our objective is to identify microRNAs as biomarkers in urine samples of bladder and prostate cancers to improve the existing diagnostic methods in this field. MATERIALS AND METHODS: In this study, urine samples from 110 men with either bladder (n = 45) or prostate (n = 23) cancer, benign prostatic hyperplasia (n = 22) and healthy controls (n = 20) were collected. qPCR was used to evaluate the expression level of miR-21-5p, miR-141-3p, and miR-205-5p in these samples. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. RESULTS: The analysis of the data revealed that miR-21-5p, miR-141-3p, and miR-205-5p are differentially expressed in urine of bladder and prostate cancer patients. All these three microRNAs were upregulated in these samples and they were also able to differentiate benign prostatic hyperplasia from malignant cases. The statistical analyses revealed a good specificity for each individual microRNA. CONCLUSION: The results show that these three urine-based microRNAs might be a good choice to implement a specific and non-invasive diagnostic tool for bladder and prostate cancer. The expression pattern of all three microRNAs was particularly useful to distinguish benign and invasive tumors in prostate cases. From the patients' perspective the improvement of the diagnostic situation is awaited eagerly.

CircPAN3/miR-221/PTEN axis and apoptosis in myocardial Infarction: Quercetin's regulatory effects.

The circular RNA/microRNA/mRNA axis is a new layer of non-coding RNA(ncRNA)-based regulatory gene expression networks upstream of numerous cell signaling pathways. Circular RNAPAN3 (circPAN3) is involved in autophagy, fibrosis and apoptosis which are responsible for the reduction incardiac functional capacityfollowingmyocardial infarction(MI). However, the molecular mechanism of circPAN3 association with apoptosis is unknown. In addition, the relationship between quercetin as a cardioprotective factor in MI and circular RNA-dependent regulatory pathways has not yet been elucidated. MI was induced in Wistar rats using the left anterior descending artery (LAD) ligation method. One day after surgery, quercetin (30 mg/kg) was injected intraperitoneal (IP) every other day for two weeks. The expression of circPAN3 was increased in the MI group (P < 0.05). The increase in circPAN3 was accompanied by a decrease in miR-221 (P < 0.0001), an increase in PTEN (P < 0.0001), and cleaved caspase 3 (P < 0.001). Quercetin effectively reduced the expression of circPAN3 (P < 0.05), PTEN (P < 0.0001), and cleaved caspase 3 (P < 0.001), and increased the expression of miR-221 (P < 0.0001) and the ratio of p-AKT to p-PI3K (P < 0.001). The circPAN3/miR-221/PTEN pathway is an ncRNA-dependent apoptotic pathway in MI cardiac tissue. Quercetin effectively modulated this pathway, resulting in a reduction of cardiac tissue death and improvement in cardiac function after MI. This suggests that the circPAN3/miR-221 axis plays a role in apoptosis in MI, and quercetin can act as a protective candidate by modulating this pathway.

A novel PIK3CA hotspot mutation in Isfahanian breast cancer patients.

Somatic mutations of phosphatidylinositol 3-kinase (PI3K) catalytic subunit (PIK3CA) play an important role in tumorigenesis. Using PCR-SSCP, followed by sequencing, we examined the PIK3CA gene over the previously identified mutational hotspots in a panel of 50 breast cancer and 5 normal breast tissue, as well as 50 normal blood samples isolated from a population of Iranian patients. In the present study, the frequency of PIK3CA mutation was 14%. However, the previously identified hotspot mutations in exons 20 were completely absent in these breast cancer samples. Instead, we found a new hotspot mutation (3 of 50 samples) in exon 20.

Secondary toxic effect of graphene oxide and graphene quantum dots alters the expression of miR-21 and miR-29a in human cell lines.

For in vitro studies, non-toxic doses of nanomaterials are routinely selected by quantification of live cells after exposing to different concentrations of nanoparticles but considering only morphological changes or viability of cells is not sufficient to conclude that these nanomaterials are non-cytotoxic. Here we investigated if secondary toxicity is active in the cells exposed to non-toxic doses of graphene oxide (GO) and graphene quantum dots (GQDs). Non-cytotoxic dose of 15 µg mL-1 of GO (100 nm) and GQDs (50 nm) was selected according to MTT and Hoechst 33342/PI double staining assays. In order to investigate the secondary toxicity, the expression of miR-21, miR-29a and three genes at both mRNA and protein level were evaluated in MCF-7, HUVEC, KMBC/71 cells 4 and 24 h post exposure. Mitochondrial membrane potential (MMP) was assessed by Rhodamine 123 staining. According to our results, there was no significant decrease in viability of cells after exposure to the non-cytotoxic dose of GO and GQDs, but we observed significant alterations in the expression level of miR-21, miR-29a, Bax, Bcl2 and PTEN genes after treatment in all three cells. In addition to molecular changes, we observed alteration in mitochondrial activity at cellular level. However, we also observed that GO influenced the basal level of genes and MMP more compare to GQDs. Considering that all these genes are involved in breast tumor development and metastasis, the observed changes in miRNA expression and protein synthesis may alter cell fate and susceptibility and cause deviation in the desired outcome of GO and GQDs application in medical research.

Quantification of circulating miR-517c-3p and miR-210-3p levels in preeclampsia.

MicroRNAs (miRNAs, miRs) are small regulatory non-coding RNAs that regulate gene expression by incomplete complementary attachment to the 3'UTR, 5'UTR, ORF and promoter regions of target mRNAs. We compared plasma levels of miR-210-3p and miR-517c-3p as cell-free microRNAs (cfmiRNAs) in preeclamptic (n = 20) and healthy women (n = 20). These miRs are responsible for cell growth and proliferation, placental hypoxia, immune response and apoptosis. We found higher expression levels of miR-210 and miR-517c in preeclamptic cases (+3.34 and +2.27 fold change, respectively). This is the first study that evaluates the plasma levels of miR-517c in preeclamptic cases by real time PCR (RT-PCR) technique. This study can lead to new opportunities for research about the roles of miRNAs in preeclampsia etiology or new biomarkers.

Statins: Complex outcomes but increasingly helpful treatment options for patients.

Statins are long known class of medicines and the most frequently prescribed drugs in cardiovascular pharmacotherapy, widely ordered not only in patients suffering from dyslipidemia, but also in patients with coronary artery disease, acute coronary syndromes, diabetes mellitus, stroke, hypertension, and chronic kidney disease, with or without coexisting dyslipidemia. However, several clinical trials have shown, that the advantages of statins goes beyond their reduction of the cholesterol level. Some crucial isoprenoid mediators which are highly essential for the activation of different intracellular/signaling proteins, that play important roles in multiple cellular mechanisms, are regulated by statins in addition to the inhibition of cholesterol biosynthesis. Moreover, anti-inflammatory intermediates and cytokines such as c-reactive protein, IL1, IL6, IL8, TNFA are affected downstream targets. Still, these numerous effects of statins such as anti-inflammatory effects, antioxidant effects, anti-proliferative, apoptotic, cell cycle regulatory and immunomodulatory effects, are primarily seen in conjunction with the inhibition of the HMG-CoA reductase. Other direct targets are missing. Beyond the classical application of statins, they were also tested to treat cancer with promising prospects, but still on a level of an adjuvant therapy option. Nevertheless the growing number of cancer studies and the increasing number of molecular players in affected pathways illustrates, that statins might be helpful in cancer therapeutics, despite the major part of the biological reaction network, which is regulated by statins, remains sketchy. It seems, that the statins still have some potential to improve established therapeutic procedures.

MicroRNA expression in serum samples of sulfur mustard veterans as a diagnostic gateway to improve care.

Sulfur mustard is a vesicant chemical warfare agent, which has been used during Iraq-Iran-war. Many veterans and civilians still suffer from long-term complications of sulfur mustard exposure, especially in their lung. Although the lung lesions of these patients are similar to Chronic Obstructive Pulmonary Disease (COPD), there are some differences due to different etiology and clinical care. Less is known on the molecular mechanism of sulfur mustard patients and specific treatment options. microRNAs are master regulators of many biological pathways and proofed to be stable surrogate markers in body fluids. Based on that microRNA expression for serum samples of sulfur mustard patients were examined, to establish specific microRNA patterns as a basis for diagnostic use and insight into affected molecular pathways. Patients were categorized based on their long-term complications into three groups and microRNA serum levels were measured. The differentially regulated microRNAs and their corresponding gene targets were identified. Cell cycle arrest, ageing and TGF-beta signaling pathways showed up to be the most deregulated pathways. The candidate microRNA miR-143-3p could be validated on all individual patients. In a ROC analysis miR-143-3p turned out to be a suitable diagnostic biomarker in the mild and severe categories of patients. Further microRNAs which might own a link to the biology of the sulfur mustard patients are miR-365a-3p, miR-200a-3p, miR-663a. miR-148a-3p, which showed up only in a validation study, might be linked to the airway complications of the sulfur mustard patients. All the other candidate microRNAs do not directly link to COPD phenotype or lung complications. In summary the microRNA screening study characterizes several molecular differences in-between the clinical categories of the sulfur mustard exposure groups and established some useful microRNA biomarkers. qPCR raw data is available via the Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110797.

Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of Sulfur Mustard-Exposed Veterans.

OBJECTIVE: In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims. MATERIALS AND METHODS: In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene. RESULTS: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. CONCLUSION: We demonstrate that non-miRNA reference genes have the least stabil- ity in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers.