Plasma Extracellular Vesicle miRNAs Can Identify Lung Cancer,Current Smoking Status,and Stable COPD

Lung cancer remains the leading cause of cancer related mortality worldwide. We aimed to test whether a simple blood biomarker (extracellular vesicle miRNAs) can discriminate between cases with and without lung cancer. Methods: plasma extracellular vesicles (EVs) were isolated from four cohorts (n = 20 in each): healthy non-smokers, healthy smokers, lung cancer, and stable COPD participants. EV miRNA expression was evaluated using the miRCURY LNA miRNA Serum/Plasma assay for 179 specific targets. Significantly dysregulated miRNAs were assessed for discriminatory power using ROC curve analysis. Results: 15 miRNAs were differentially expressed between lung cancer and healthy non-smoking participants, with the greatest single miRNA being miR-205-5p (AUC 0.850), improving to AUC 0.993 in combination with miR-199a-5p. Moreover, 26 miRNAs were significantly dysregulated between lung cancer and healthy smoking participants, with the greatest single miRNA being miR-497-5p (AUC 0.873), improving to AUC 0.953 in combination with miR-22-5p; 14 miRNAs were significantly dysregulated between lung cancer and stable COPD participants, with the greatest single miRNA being miR-27a-3p (AUC 0.803), with two other miRNAs (miR-106b-3p and miR-361-5p) further improving discriminatory power (AUC 0.870). Conclusion: this case control study suggests miRNAs in EVs from plasma holds key biological information specific for lung cancer and warrants further prospective assessment.     

Atrial Fibrillation in Heart Failure Is Associated with High Levels of Circulating microRNA-199a-5p and 22–5p and a Defective Regulation of Intracellular Calcium and Cell-to-Cell Communication

MicroRNAs (miRNAs) participate in atrial remodeling and atrial fibrillation (AF) promotion. We determined the circulating miRNA profile in patients with AF and heart failure with reduced ejection fraction (HFrEF), and its potential role in promoting the arrhythmia. In plasma of 98 patients with HFrEF (49 with AF and 49 in sinus rhythm, SR), differential miRNA expression was determined by high-throughput microarray analysis followed by replication of selected candidates. Validated miRNAs were determined in human atrial samples, and potential arrhythmogenic mechanisms studied in HL-1 cells. Circulating miR-199a-5p and miR-22-5p were significantly increased in HFrEF patients with AF versus those with HFrEF in SR. Both miRNAs, but particularly miR-199a-5p, were increased in atrial samples of patients with AF. Overexpression of both miRNAs in HL-1 cells resulted in decreased protein levels of L-type Ca²⁺ channel, NCX and connexin-40, leading to lower basal intracellular Ca²⁺ levels, fewer inward currents, a moderate reduction in Ca²⁺ buffering post-caffeine exposure, and a deficient cell-to-cell communication. In conclusion, circulating miR-199a-5p and miR-22-5p are higher in HFrEF patients with AF, with similar findings in human atrial samples of AF patients. Cells exposed to both miRNAs exhibited altered Ca²⁺ handling and defective cell-to-cell communication, both findings being potential arrhythmogenic mechanisms.     

Association between Circulating MicroRNAs (miR-21-5p,miR-20a-5p,miR-29b-3p,miR-126-3p and miR-101-3p) and Chronic Allograft Dysfunction in Renal Transplant Recipients

Chronic allograft dysfunction (CAD) is a major condition affecting long-term kidney graft survival. Serum microRNA (miRNA) has been reported as a biomarker for various conditions of allograft injuries. The upregulation of miR-21 is the best-known miRNA change in graft tissue, urine and plasma. However, the correlation of plasma miR-21 with the severity of CAD remains unclear. In our study, 40 kidney transplantation recipients with late graft survival for more than 10 years were enrolled. The CAD group (n = 20) had either an eGFR between 15 to 60 mL/min or a biopsy-proved chronic allograft nephropathy or rejection. The control group (n = 20) had an eGFR ≥ 60 mL/min without proteinuria and hematuria for a consecutive 3 months before the study. We performed RNA sequencing to profile the miRNAs expression. There were six differentially expressed miRNAs in the CAD group. Among them, miR-21-5p and miR-101-3p were decreased, and miR-20a-5p was increased. We found that miR-21-5p, miR-20a-5p and miR-101-3p all participated in the TGF-beta pathway. We demonstrated that decreased miR-21-5p and miR-101-3p, and increased miR-20a-5p were the novel CAD-associated miRNAs in the TGF-beta pathway. These findings may pave the way for developing early prediction miRNAs biomarkers for CAD, and possibly developing therapeutic tools in the field of kidney transplantation.     

Circulating Small EVs miRNAs as Predictors of Pathological Response to Neo-Adjuvant Therapy in Breast Cancer Patients

Neo-adjuvant therapy (NAT) is increasingly used in the clinic for the treatment of breast cancer (BC). Pathological response to NAT has been associated with improved patients’ survival; however, the current techniques employed for assessing the tumor response have significant limitations. Small EVs (sEVs)-encapsulated miRNAs have emerged as promising new biomarkers for diagnosis and prediction. Therefore, our study aims to explore the predictive value of these miRNAs for the pathological response to NAT in BC. By employing bioinformatic tools, we selected a set of miRNAs and evaluated their expression in plasma sEVs and BC biopsies. Twelve miRNAs were identified in sEVs, of which, miR-21-5p, 221-3p, 146a-5p and 26a-5p were significantly associated with the Miller–Payne (MP) pathological response to NAT. Moreover, miR-21-5p, 146a-5p, 26a-5p and miR-24-3p were independent as predictors of MP response to NAT. However, the expression of these miRNAs showed no correlation between sEVs and tissue samples, indicating that the mechanisms of miRNA sorting into sEVs still needs to be elucidated. Functional analysis of miRNA target genes and drug interactions revealed that candidate miRNAs and their targets, can be regulated by different NAT regimens. This evidence supports their role in governing the patients’ therapy response and highlights their potential use as prediction biomarkers.     

Identification of miR-20a-5p as Robust Normalizer for Urine microRNA Studies in Renal Cell Carcinoma and a Profile of Dysregulated microRNAs

Renal cell carcinoma (RCC) is the third most frequent urinary malignancy and one of the most lethal. Current diagnostic and follow-up techniques are harmful and unspecific in low-grade tumors. Novel minimally invasive markers such as urine microRNAs (miRNAs) are under study. However, discrepancies arise among studies in part due to lack of consent regarding normalization. We aimed to identify the best miRNA normalizer for RCC studies performed in urine samples together with a miRNA profile with diagnostic value and another for follow-up. We evaluated the performance of 120 candidate miRNAs in the urine of 16 RCC patients and 16 healthy controls by RT-qPCR followed by a stability analysis with RefFinder. In this screening stage, miR-20a-5p arose as the most stably expressed miRNA in RCC and controls, with a good expression level. Its stability was validated in an independent cohort of 51 RCC patients and 32 controls. Using miR-20a-5p as normalizer, we adjusted and validated a diagnostic model for RCC with three miRNAs (miR-200a-3p, miR-34a-5p and miR-365a-3p) (AUC = 0.65; Confidence Interval 95% [0.51, 0.79], p = 0.043). let-7d-5p and miR-205-5p were also upregulated in patients compared to controls. Comparing RCC samples before surgery and fourteen weeks after, we identified let-7d-5p, miR-152-3p, miR-30c-5p, miR-362-3p and miR-30e-3p as potential follow-up profile for RCC. We identified validated targets of most miRNAs in the renal cell carcinoma pathway. This is the first study that identifies a robust normalizer for urine RCC miRNA studies, miR-20a-5p, which may allow the comparison of future studies among laboratories. Once confirmed in a larger independent cohort, the miRNAs profiles identified may improve the non-invasive diagnosis and follow-up of RCC.     

Circulating miRNAs as Biomarkers for Mitochondrial Neuro-Gastrointestinal Encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disease for which there are currently no validated outcome measures for assessing therapeutic intervention efficacy. The aim of this study was to identify a plasma and/or serum microRNA (miRNA) biomarker panel for MNGIE. Sixty-five patients and 65 age and sex matched healthy controls were recruited and assigned to one of four study phases: (i) discovery for sample size determination; (ii) candidate screening; (iii) candidate validation; and (iv) verifying the performance of the validated miRNA panel in four patients treated with erythrocyte-encapsulated thymidine phosphorylase (EE-TP), an enzyme replacement under development for MNGIE. Quantitative PCR (qPCR) was used to profile miRNAs in serum and/or plasma samples collected for the discovery, validation and performance phases, and next generation sequencing (NGS) analysis was applied to serum samples assigned to the candidate screening phase. Forty-one differentially expressed candidate miRNAs were identified in the sera of patients (p < 0.05, log₂ fold change > 1). The validation cohort revealed that of those, 27 miRNAs were upregulated in plasma and three miRNAs were upregulated in sera (p < 0.05). Through binary logistic regression analyses, five plasma miRNAs (miR-192-5p, miR-193a-5p, miR-194-5p, miR-215-5p and miR-34a-5p) and three serum miRNAs (miR-192-5p, miR-194-5p and miR-34a-5p) were shown to robustly distinguish MNGIE from healthy controls. Reduced longitudinal miRNA expression of miR-34a-5p was observed in all four patients treated with EE-TP and coincided with biochemical and clinical improvements. We recommend the inclusion of the plasma exploratory miRNA biomarker panel in future clinical trials of investigational therapies for MNGIE; it may have prognostic value for assessing clinical status.     

MiR-10a in Pancreatic Juice as a Biomarker for Invasive Intraductal Papillary Mucinous Neoplasm by miRNA Sequencing

New biomarkers are needed to further stratify the risk of malignancy in intraductal papillary mucinous neoplasm (IPMN). Although microRNAs (miRNAs) are expected to be stable biomarkers, they can vary owing to a lack of definite internal controls. To identify universal biomarkers for invasive IPMN, we performed miRNA sequencing using tumor-normal paired samples. A total of 19 resected tissues and 13 pancreatic juice samples from 32 IPMN patients were analyzed for miRNA expression by next-generation sequencing with a two-step normalization of miRNA sequence data. The miRNAs involved in IPMN associated with invasive carcinoma were identified from this tissue analysis and further verified with the pancreatic juice samples. From the tumor-normal paired tissue analysis of the expression levels of 2792 miRNAs, 20 upregulated and 17 downregulated miRNAs were identified. In IPMN associated with invasive carcinoma (INV), miR-10a-5p and miR-221-3p were upregulated and miR-148a-3p was downregulated when compared with noninvasive IPMN. When these findings were further validated with pancreatic juice samples, miR-10a-5p was found to be elevated in INV (p = 0.002). Therefore, three differentially expressed miRNAs were identified in tissues with INV, and the expression of miR-10a-5p was also elevated in pancreatic juice samples with INV. MiR-10a-5p is a promising additional biomarker for invasive IPMN.     

Exosomal microRNAs miR-30d-5p and miR-126a-5p Are Associated with Heart Failure with Preserved Ejection Fraction in STZ-Induced Type 1 Diabetic Rats

Exosomal microRNAs (EXO-miRNAs) are promising non-invasive diagnostic biomarkers for cardiovascular disease. Heart failure with preserved ejection fraction (HFpEF) is a poorly understood cardiovascular complication of diabetes mellitus (DM). Little is known about whether EXO-miRNAs can be used as biomarkers for HFpEF in DM. We aimed to investigate the relationship between EXO-miRNAs and HFpEF in STZ-induced diabetic rats. We prepared STZ-induced diabetic rats exhibiting a type 1 DM phenotype with low body weight, hyperglycemia, hyperlipidemia and hypoinsulinemia. Histological sections confirmed atrophy and fibrosis of the heart, with collagen accumulation representing diabetic cardiomyopathy. Significant decreases in end-diastolic volume, stroke volume, stroke work, end-systolic elastance and cardiac output indicated impaired cardiac contractility, as well as mRNA conversion of two isoforms of myosin heavy chain (α-MHC and β-MHC) and increased atrial natriuretic factor (ANF) mRNA indicating heart failure, were consistent with the features of HFpEF. In diabetic HFpEF rats, we examined a selected panel of 12 circulating miRNAs associated with HF (miR-1-3p, miR-21-5p, miR-29a-5p, miR-30d-5p, miR-34a-5p, miR-126a-5p, miR-143-3p, miR-145-5p, miR-195-5p, miR-206-3p, miR-320-3p and miR-378-3p). Although they were all expressed at significantly lower levels in the heart compared to non-diabetic controls, only six miRNAs (miR-21-5p, miR-30d-5p, miR-126a-5p, miR-206-3p, miR-320-3p and miR-378-3p) were also reduced in exosomal content, while one miRNA (miR-34a-5p) was upregulated. Similarly, although all miRNAs were correlated with reduced cardiac output as a measure of cardiovascular performance, only three miRNAs (miR-30d-5p, miR-126a-5p and miR-378-3p) were correlated in exosomal content. We found that miR-30d-5p and miR-126a-5p remained consistently correlated with significant reductions in exosomal expression, cardiac expression and cardiac output. Our findings support their release from the heart and association with diabetic HFpEF. We propose that these two EXO-miRNAs may be important for the development of diagnostic tools for diabetic HFpEF.     

Analysis of the Applicability of microRNAs in Peripheral Blood Leukocytes as Biomarkers of Sensitivity and Exposure to Fractionated Radiotherapy towards Breast Cancer

Biomarkers for predicting individual response to radiation and for dose verification are needed to improve radiotherapy. A biomarker should optimally show signal fidelity, meaning that its level is stable and proportional to the absorbed dose. miRNA levels in human blood serum were suggested as promising biomarkers. The aim of the present investigation was to test the miRNA biomarker in leukocytes of breast cancer patients undergoing external beam radiotherapy. Leukocytes were isolated from blood samples collected prior to exposure (control); on the day when a total dose of 2 Gy, 10 Gy, or 20 Gy was reached; and one month after therapy ended (46–50 Gy in total). RNA sequencing was performed and univariate analysis was used to analyse the effect of the radiation dose on the expression of single miRNAs. To check if combinations of miRNAs can predict absorbed dose, a multinomial logistic regression model was built using a training set from eight patients (representing 40 samples) and a validation set with samples from the remaining eight patients (15 samples). Finally, Broadside, an explorative interaction mining tool, was used to extract sets of interacting miRNAs. The most prominently increased miRNA was miR-744-5p, followed by miR-4461, miR-34a-5p, miR-6513-5p, miR-1246, and miR-454-3p. Decreased miRNAs were miR-3065-3p, miR-103a-2-5p, miR-30b-3p, and miR-5690. Generally, most miRNAs showed a relatively strong inter-individual variability and different temporal patterns over the course of radiotherapy. In conclusion, miR-744-5p shows promise as a stable miRNA marker, but most tested miRNAs displayed individual signal variability which, at least in this setting, may exclude them as sensitive biomarkers of radiation response.     

Identification of miR-199a-5p,miR-214-3p and miR-99b-5p as Fibrosis-Specific Extracellular Biomarkers and Promoters of HSC Activation

Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) resulting in the formation of fibrous scars. In the clinic, liver biopsies are the standard diagnostic method despite the potential for clinical complications. miRNAs are single-stranded, non-coding RNAs that can be detected in tissues, body fluids and cultured cells. The regulation of many miRNAs has been linked to tissue damage, including liver fibrosis in patients, resulting in aberrant miRNA expression/release. Experimental evidence also suggests that miRNAs are regulated in a similar manner in vitro and could thus serve as translational in vitro–in vivo biomarkers. In this work, we set out to identify and characterize biomarkers for liver fibrosis that could be used in vitro and clinically for research and diagnostic purposes. We focused on miRNAs released from hepatic 3D cultures exposed to methotrexate (MTX), which causes fibrosis, and acetaminophen (APAP), an acute hepatotoxicant with no clinically relevant association to liver fibrosis. Using a 3D in vitro model, we corroborated compound-specific responses as we show MTX induced a fibrotic response, and APAP did not. Performing miRNA-seq of cell culture supernatants, we identified potential miRNA biomarkers (miR-199a-5p, miR-214-3p, niRNA-125a-5p and miR-99b-5p) that were associated with a fibrotic phenotype and not with hepatocellular damage alone. Moreover, transfection of HSC with miR-199a-5p led to decreased expression of caveolin-1 and increased α-SMA expression, suggesting its role in HSC activation. In conclusion, we propose that extracellular miR-214-3p, miR-99b-5p, miR-125a-5p and specifically miR-199a-5p could contribute towards a panel of miRNAs for identifying liver fibrosis and that miR-199a-5p, miR-214-3p and miR-99b-5p are promoters of HSC activation.     

Quantitative Prediction of Steatosis in Patients with Non-Alcoholic Fatty Liver by Means of Hepatic MicroRNAs Present in Serum and Correlating with Hepatic Fat

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease worldwide, but a reliable non-invasive method to quantify liver steatosis in primary healthcare is not available. Circulating microRNAs have been proposed as biomarkers of severe/advanced NAFLD (steatohepatitis and fibrosis). However, the use of circulating miRNAs to quantitatively assess the % of liver fat in suspected NAFLD patients has not been investigated. We performed global miRNA sequencing in two sets of samples: human livers from organ donors (n = 20), and human sera from biopsy-proven NAFLD patients (n = 23), both with a wide range of steatosis quantified in their liver biopsies. Partial least squares (PLS) regression combined with recursive feature elimination (RFE) was used to select miRNAs associated with steatosis. Moreover, regression models with only 2 or 3 miRNAs, with high biological relevance, were built. Comprehensive microRNA sequencing of liver and serum samples resulted in two sets of abundantly expressed miRNAs (418 in liver and 351 in serum). Pearson correlation analyses indicated that 18% of miRNAs in liver and 14.5% in serum were significantly associated with the amount of liver fat. PLS-RFE models demonstrated that 50 was the number of miRNAs providing the lowest error in both liver and serum models predicting steatosis. Comparison of the two miRNA subsets showed 19 coincident miRNAs that were ranked according to biological significance (guide/passenger strand, relative abundance in liver and serum, number of predicted lipid metabolism target genes, correlation significance, etc.). Among them, miR-10a-5p, miR-98-5p, miR-19a-3p, miR-30e-5p, miR-32-5p and miR-145-5p showed the highest biological relevance. PLS regression models with serum levels of 2–3 of these miRNAs predicted the % of liver fat with errors <5%.     

Adventitial Tertiary Lymphoid Organs as Potential Source of MicroRNA Biomarkers for Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology.     

Plasma miR-9-3p and miR-136-3p as Potential Novel Diagnostic Biomarkers for Experimental and Human Mild Traumatic Brain Injury

Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p < 0.01 for all), respectively, distinguishing rats with mTBI from naïve rats with 100% sensitivity and specificity. DdPCR further identified a subpopulation of mTBI patients with plasma miR-9-3p (n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at <2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population.     

Proteomic and miRNA Profiles of Exosomes Derived from Myometrial Tissue in Laboring Women

Myometrial contraction is essential for successful delivery. Recent studies have highlighted the vital roles of tissue-derived exosomes in disease diagnostic, prognostic, and therapeutic applications; however, the characteristics of uterine myometrium-derived exosomes are unclear. Here, we successfully isolated exosomes from myometrial tissues, human myometrial smooth muscle cells (HMSMCs), and human umbilical vein endothelial cells (HUVECs), then performed quantitative liquid chromatography-tandem mass spectrometry and miRNA sequencing to investigate the cargo of the exosomes. Fifty-two proteins and five miRNAs were differentially expressed (DE) in term non-labor and term labor myometrium-derived exosomes. Among them, seven proteins (SERPINE1, THBS1, MGAT1, VIM, FGB, FGG, and VWF) were differentially expressed both in the myometrial exosomes and tissues, three miRNAs (miR-363-3p, miR-203a-3p, and miR-205-5p) target 13 DE genes. The top three miRNA derived from HMSMCs (miR-125b-1-3p, miR-337-5p, and miR-503-5p) and HUVECs (miR-663a, miR-4463, and miR-3622a-5p) were identified. Two proteins, GJA1 and SLC39A14, exist in female blood exosomes and are highly expressed in HMSMCs exosomes, are also upregulated in the laboring myometrium, which verified increased in laboring blood samples, might be novel potential biomarkers for myometrial activation. The proteomic and miRNA profile of exosomes derived from laboring myometrium revealed some molecules in the exosomes that affect the intercellular communication and the function of the myometrium.     

miRNA Expression Is Increased in Serum from Patients with Semantic Variant Primary Progressive Aphasia

Primary progressive aphasia (PPA) damages the parts of the brain that control speech and language. There are three clinical PPA variants: nonfluent/agrammatic (nfvPPA), logopenic (lvPPA) and semantic (svPPA). The pathophysiology underlying PPA variants is not fully understood, including the role of micro (mi)RNAs which were previously shown to play a role in several neurodegenerative diseases. Using a two-step analysis (array and validation through real-time PCR), we investigated the miRNA expression pattern in serum from 54 PPA patients and 18 controls. In the svPPA cohort, we observed a generalized upregulation of miRNAs with miR-106b-5p and miR-133a-3p reaching statistical significance (miR-106b-5p: 2.69 ± 0.89 mean ± SD vs. 1.18 ± 0.28, p < 0.0001; miR-133a-3p: 2.09 ± 0.10 vs. 0.74 ± 0.11 mean ± SD, p = 0.0002). Conversely, in lvPPA, the majority of miRNAs were downregulated. GO enrichment and KEGG pathway analyses revealed that target genes of both miRNAs are involved in pathways potentially relevant for the pathogenesis of neurodegenerative diseases. This is the first study that investigates the expression profile of circulating miRNAs in PPA variant patients. We identified a specific miRNA expression profile in svPPA that could differentiate this pathological condition from other PPA variants. Nevertheless, these preliminary results need to be confirmed in a larger independent cohort.     

miR-22-3p and miR-30e-5p Are Associated with Prognosis in Cervical Squamous Cell Carcinoma

Alteration in expression of miRNAs can cause various malignant changes and the metastatic process. Our aim was to identify the miRNAs involved in cervical squamous cell carcinoma (SqCC) and metastasis, and to test their utility as indicators of metastasis and survival. Using microarray technology, we performed miRNA expression profiling on primary cervical SqCC tissue (n = 6) compared with normal control (NC) tissue and compared SqCC that had (SqC-M; n = 3) and had not (SqC-NM; n = 3) metastasized. Four miRNAs were selected for validation by qRT-PCR on 29 SqC-NM and 27 SqC-M samples, and nine metastatic lesions (ML-SqC), from a total of 56 patients. Correlation of miRNA expression and clinicopathological parameters was analyzed to evaluate the clinical impact of candidate miRNAs. We found 40 miRNAs differentially altered in cervical SqCC tissue: 21 miRNAs were upregulated and 19 were downregulated (≥2-fold, p < 0.05). Eight were differentially altered in SqC-M compared with SqC-NM samples: four were upregulated (miR-494, miR-92a-3p, miR-205-5p, and miR-221-3p), and four were downregulated (miR-574-3p, miR-4769-3p, miR-1281, and miR-1825) (≥1.5-fold, p < 0.05). MiR-22-3p might be a metastamiR, which was gradually further downregulated in SqC-NM > SqC-M > ML-SqC. Downregulation of miR-30e-5p significantly correlated with high stage, lymph node metastasis, and low survival rate, suggesting an independent poor prognostic factor.     

Gender Differences in microRNA Expressions as Related to Long-Term Graft Function in Kidney Transplant Patients

In recent studies, much has been discussed about biomarkers used in the evaluation of the transplanted graft function. However, there remains a lack of research regarding the long-term effects of microRNAs (miRNAs) on the different genders for kidney transplant (KTx) patients. In this study, we aim to assess the functions of miRNAs on long term outcomes of KTx patients by extracting differently expressed miRNAs between patients of normal graft function and graft dysfunction, while further analyzing their impact on the different genders. We analyzed the data of 40 patients who had received KTx for a period of more than ten years and included data regarding renal function, immuno-related markers and plasma miRNAs. Data were classified by gender for further studies. Twelve out of 17 females and 8 out of 23 males had undergone graft dysfunction. Renal function analysis showed significantly worse outcomes in the female patients. There were five differently expressed miRNAs between the female control group and female dysfunction group: miR-128-3p, miR-21-5p, miR-150-5p, miR-92a-3p and miR-15a-5p, and five between the male control group and male dysfunction group: miR-23a-3p, miR-126-3p, miR-142-3p, miR-223-3p and miR-26a-5p. Gender differences exist in incidences of kidney graft dysfunction, with male patients displaying better preservation in graft functions. Overall, these differently expressed miRNAs either enhance or suppress host immune responses. They can be predictive markers for graft survival and can also be important factors that lead to worse long term kidney graft function in females when compared to males.