Expression Levels Of miR-222 Research Articles

The Association between Inflammatory Cytokines and miRNAs with Slow Coronary Flow Phenomenon.

Slow coronary flow (SCF) is a coronary artery disorder. Several inflammatory mediators have been reported to be associated with vascular homeostasis and endothelial dysfunction. The aim of this study was to investigate the association between cytokines and miRNAs in patients with SCF compared to the controls. In this regard, blood samples were acquired from 45 SCF patients and 45 age- and sex-matched healthy control subjects. Serum and peripheral blood mononuclear cells (PBMCs) were separated. Expression levels of miRNAs and cytokines in PBMCs were measured by real-time PCR. As a final point, serum levels of cytokines were quantified by ELISA. Expression levels of miR-1, miR-133, miR-208a, miR-206, miR-17, miR-29, miR-223, miR-326, and miR-155 as considerable indicators of inflammatory function significantly increased in SCF patients while the expression levels of miR-15a, miR-21, miR-25, miR-126, miR-17, miR-16 and miR-18a as considerable indicators of anti-inflammatory function significantly decreased in patients with SCF compared to the control group. Additionally, serum IL-1β, IL-8, and TNF-α concentrations were significantly higher in the SCF group than controls. However, no significant differences were observed in IL-10 production in SCF patients compared to the controls. This study provided the potential role of miRNAs as biomarkers for SCF diagnosis as well as suitable markers for monitoring coronary artery disease (CAD) development in these patients. More investigations are still necessary to unravel the detailed essential mechanisms of circulating miRNA levels in patients with heart failure and SCF.

Clinical value of serum microRNA-195 expression in invasive ductal carcinoma of the breast

BackgroundMicroRNAs (miR) are small molecules, with around 17–25 nucleotides in length. Dysregulation of miRNA expression can be used as a diagnostic biomarker of cancer because of their high stability in human serum. MiR-195 has been implicated as a diagnostic biomarker of breast cancer. Therefore, this study aimed to identify the expression levels of miR-195 and its diagnostic role in patients of breast cancer (BC). MethodsThe expression patterns of miR-195 in the serum was analysed from a total number of 100 female with invasive ductal carcinoma in addition to healthy control subjects. Isolation of total RNA was done, samples were measured using SYBR green-based real-time RT-PCR technology. ResultsCirculating levels of miR-195 were found to be significantly decreased in BC group compared to that of control group (p < 0.001), the serum miR-195 was associated with numerous clinicopathological considerations of breast cancer as serum miR-195 expression decreased significantly in the presence of nodal (p = 0.044) and distant metastasis (p = 0.049) in BC cases, moreover it showed significant negative correlation with tumour stage, grade and primary tumour extent (p < 0.001). Receiver operating characteristic (ROC) curve was conducted for discrimination between BC cases and control groups, miR-195 expression showed excellent AUCs (0.987), with the best cut off value of 0.996, sensitivity was 90%, specificity was 100%, PPV was 100%, NPV was 87% and accuracy was 94%. Regression analysis revealed that higher CA15-3 and lower miRNA195 were considered as independent predictors for BC diagnosis. ConclusionOur data showed that serum miR-195 expressions were down-regulated in breast cancer females; furthermore, this downregulation was associated with higher specificity in diagnosis BC cases and so it could be used as a non-invasive diagnostic marker in BC.

Overexpression of microRNA-96 is associated with poor prognosis and promotes proliferation, migration and invasion in cholangiocarcinoma cells via MTSS1.

MicroRNA-96 (miR-96) has been revealed serve an oncogenic role in various types of cancer. However, the role of miR-96 in cholangiocarcinoma (CCA) development and progression is yet to be elucidated. Thus, the aim of the present study was to investigate the role of miR-96 in CCA. The expression pattern of miR-96 in CCA tissues and cell lines was evaluated using reverse transcription-quantitative PCR analysis. Kaplan-Meier curves and Cox regression analyses were conducted to investigate the prognostic significance of miR-96 in CCA. Cell Counting Kit-8 and Transwell assays were performed to identify the functions of miR-96. The association between miR-96 and metastasis suppressor-1 (MTSS1) was verified using a dual-luciferase assay. The results demonstrated that miR-96 expression levels were increased in CCA tissues and cell lines compared with those in adjacent normal tissues and normal human intrahepatic biliary epithelial cell lines, respectively. High expression levels of miR-96 were significantly associated with lymph node metastasis, differentiation and TNM stage. In addition, upregulated expression of miR-96 was associated with a poorer prognosis and was predicted to be a prognostic factor in patients with CCA. Overexpression of miR-96 in vitro promoted CCA cell proliferation, migration and invasion. Additionally, MTSS1 was identified as a direct target of miR-96. The results of the present study indicated the clinical and biological importance of miR-96 as an oncogene in CCA. miR-96 may represent an independent prognostic biomarker and may promote CCA cell proliferation, migration and invasion by targeting MTSS1.

A Clinical Study on the Expression of miR-195 in Cardiac Carcinoma

Background: Recently, some studies have revealed that microRNAs (miRs; miRNAs) mainly regulate gene expression in the transcription of microRNAs. However, the role of microRNAs in cell development, differentiation, proliferation, and other physiological processes remains unclear. Objectives: The present study aimed to investigate the expression level of microRNA-195 in the pathological tissues of patients with cardiac carcinoma and its clinical effects. Methods: Patients with primary cardiac carcinoma were enrolled as the study subjects. The tumor and adjacent tissue samples for cancer pathology were obtained during the operation. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of microRNA-195 and its correlation with the clinicopathological features of cardiac carcinoma was analyzed. Results: A total of 64 patients were included in this study. Firstly, the expression rate of miR-195 in cardiac carcinoma tissues showed a significant decreasing trend (3.65 ± 0.42 vs. 2.05 ± 0.33) (P < 0.001) and the actual expression level of miR-195 in pathological tissues of cardiac carcinoma was negatively correlated with the malignant degree of pathological tissues (P = 0.028), invasion (P = 0.037), and lymph node metastasis of cardiac carcinoma (P = 0.023), but miR-195 was not correlated with age (P = 0.615) and gender (P = 0.465). Conclusions: The expression of microRNA-195 is closely correlated to the severity of cardiac carcinoma and the occurrence of lymph node metastasis.

Baicalin attenuates lipopolysaccharide-induced renal tubular epithelial cell injury by inhibiting the TXNIP/NLRP3 signalling pathway via increasing miR-223-3p expression.

Baicalin has been used in China to treat inflammation-related diseases, such as inflammation-induced acute kidney injury (AKI). However, the specific mechanism of baicalin remains unclear. To observe the protective effects of baicalin on lipopolysaccharide (LPS)-induced inflammatory injury of renal tubular epithelial cells (HK-2 cells) and to explore its protective mechanism. LPS (1 mg/L) was used to induce an HK-2 cell inflammatory injury model in vitro. The cells were divided into seven groups: the normal control group, LPS-induced group, LPS plus 5 μmol/L baicalin treatment group, LPS plus 15 μmol/L baicalin treatment group, LPS plus 25 μmol/L baicalin treatment group, LPS plus 50 μmol/L baicalin treatment group, and LPS plus 75 μmol/L baicalin treatment group. 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide (MTT) assay was employed for detecting the relative survival rate of HK-2 cells. Enzyme-linked immunosorbent assay was used for detecting the levels of inflammatory factors, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α). Moreover, the expression of inducible nitric oxide synthase (iNOS); cyclooxygenase-2 (COX-2); nuclear factor kB65 (NF-κB65); phosphorylated NF-κB inhibitory protein-α (p-IκB-α); NF-κB inhibitory protein (IκB); human thioredoxin interacting protein (TXNIP); and human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) were determined by Western blot analysis. The expression levels of NLRP3 and TXNIP mRNA and miR-223-3p were determined by RT-PCR. Results found that the relative survival rate of HK-2 cells treated with different baicalin concentrations was significantly increased (P<0.05) and the levels of the inflammatory factors IL-6, IL-1β, and TNF-α were significantly decreased (P<0.05) compared with those of the LPS-induced group. The expression levels of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 and the genes expressions of TXNIP and NLRP3 were significantly decreased in the cells (P<0.05), while the expression level of miR-223- 3p was significantly increased (P<0.05). These changes were induced in a dose-dependent manner. The results suggest that baicalin significantly inhibited the expression of inflammation-related proteins and alleviated LPS-induced inflammatory injury in HK-2 cells. The mechanism may be associated with the inhibition of activation of the TXNIP/NLRP3 inflammatory pathway, which might be mediated by increased expression of miR-223-3p. Thus, NLRP3 is a regulatory target of miR-223-3p.

Expression and Clinical Significance of MicroRNA-195 in Patients with Diffuse Large B Cell Lymphoma

To investigate the expression and clinical significance of microRNA-195 in patients with diffuse large B cell lymphoma(DLBCL). Sixty patients with DLBCL were selected from nearly four years in our hospital, and at the same time 30 healthy people with physical examination of the same period and with the same age in our hospital were choosed as control group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the miR-195 expression of the patients and controls, the relationship between miR-195 expression and clinicopathological characteristics of DLBCL and survival time of patients was analyzed. The expression level of miR-195 in DLBCL patients was significantly lower than that in the controls (P<0.001). The expression level of miR-195 closely related with tumor diameter, IPI score and Ann Arbor stage of patients with DLBCL. Overall survival(OS) time of DLBCL patients with highly expressed miR-195 was significantly longer than that of patients with low expression (P<0.001). miR-195 expression decrease in DLBCL patients, and miR-195 closely relates with tumor characteristics of patients with DLBCL. DLBCL patients with higher expression of miR-195 show longer overall survival time.

Expression and Clinical Significance of MicroRNA-195 in Patients with Diffuse Large B Cell Lymphoma

To investigate the expression and clinical significance of microRNA-195 in patients with diffuse large B cell lymphoma(DLBCL). Sixty patients with DLBCL were selected from nearly four years in our hospital, and at the same time 30 healthy people with physical examination of the same period and with the same age in our hospital were choosed as control group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the miR-195 expression of the patients and controls, the relationship between miR-195 expression and clinicopathological characteristics of DLBCL and survival time of patients was analyzed. The expression level of miR-195 in DLBCL patients was significantly lower than that in the controls (P<0.001). The expression level of miR-195 closely related with tumor diameter, IPI score and Ann Arbor stage of patients with DLBCL. Overall survival(OS) time of DLBCL patients with highly expressed miR-195 was significantly longer than that of patients with low expression (P<0.001). miR-195 expression decrease in DLBCL patients, and miR-195 closely relates with tumor characteristics of patients with DLBCL. DLBCL patients with higher expression of miR-195 show longer overall survival time.

Role of ADAM9 and miR-126 in the development of abdominal aortic aneurysm

Background and aimsAbdominal aortic aneurysm (AAA) is a life-threatening vascular disease when aortic rupture occurs, especially for elders. There is an urgent need to understand the mechanisms of AAA formation and development at molecular level. Our previous study showed that disintegrin and metalloprotease 10 (ADAM10) played an important role in abdominal aortic aneurysm formation. In this study, we investigated the effects of another ADAM protein (ADMA9) in AAA formation. Method and resultsUsing AngII treated human aortic smooth muscle cells (HASMCs) and human aortic endothelial cells (hAoECs) as in vitro AAA model and murine AAA model, ADAM9 was overexpressed suggesting that ADAM9 may play important roles in AAA formation. Further investigation showed that ADAM9 induced inflammation leading to increased macrophage infiltration. ADAM9 was also found to induce cell apoptosis. AKT/NF-κB pathway was activated in murine AAA. Bioinformatic analysis showed that the 3’ UTR of ADMA9 was a potential target of miR-126. We investigated the potential of using miR-126 to modulate ADAM9 expression. The expression level of miR-126 was decreased and inversely correlated with the expression of ADAM9 in the in vitro AAA model. Further investigation showed that miR-126 negatively regulated gene expression of ADAM9 and suppressed the production of inflammatory cytokines. miR-126 was also found to improve cell survival and significantly reduce AAA formation in murine AAA. ConclusionsOur data revealed a link between ADAM9 and AAA formation, providing an approach to control AAA development using miR-126, possibly through modulation of the expression level of ADAM9.

MiR-195 regulates CD40 to maintain Th17/Treg balance in rats with non-alcoholic fatty liver disease

PurposeThis study aims to explore the relationship between miR-195 and CD40 and its effect on Th17/Treg balance in rats with non-alcoholic fatty liver disease (NAFLD). MethodsWe established rat models of NAFLD and made seven groups, Normal group (without modeling), Model group (model rats), NC group (model rats injected with negative control vector), miR-195 OE group (model rats injected with miR-195 mimic), anti-miR-195 group (model rats injected with miR-195 inhibitor), Si-CD40 group (model rats injected with CD40 silencing vector), and anti-miR-195+Si-CD40 group (model rats injected with miR-195 inhibitor and CD40 silencing vector). Dual-luciferase reporter gene assay verified the targeting relationship between miR-195 and CD40. The mRNA and protein expression levels of miR-195, CD40 as well as Th17/Treg associated cytokines in the liver tissues were detected. The pathological changes of liver tissues were detected, and the liver lesion scoring was carried out. The liver coefficient was calculated. The levels of liver function related indices, and Th17/Treg associated cytokines and inflammatory factors in serum were determined. The proportions of Th17/Treg cells in serum were determined by flow cytometry. ResultsCompared with Normal group, miR-195 expression level in liver tissues of rats in other six groups was significantly reduced (all P < 0.05); the serum levels of AST, ALT, GGT, IL-17, TNF-α, IL-23, IL-6, IL-8, TC, TG, HDL, and LDL, and the Th17/Treg ratio, as well as the mRNA and protein expression levels of CD40, RORyt, IL-17, TNF-α, IL-23, and IL-8 in liver tissues were significantly increased (all P < 0.05); while the mRNA and protein expression levels of Foxp3, and IL-10 level were significantly reduced (all P < 0.05). Compared with Model group, the above parameters showed an opposite trend in miR-195 OE group and Si-CD40 group were significantly reduced (all P < 0.05). Moreover, anti-miR-195 group could aggravate the imbalance of Th17/Treg cells in rats with NAFLD and promote inflammatory response. Compared with anti-miR-195 group, the combined treatment in anti-miR-195+Si-CD40 group can partially avoid the imbalance of Th17/Treg cells, and inhibit inflammatory response. ConclusionOverexpression of miR-195 can reduce the Th17/Treg ratio to maintain Th17/Treg balance by inhibiting CD40 expression in rats with NAFLD.

Therapeutic effect of Resveratrol in the treatment of osteoarthritis via the MALAT1/miR-9/NF-κB signaling pathway.

The aim of the current study was to explore the role of Resveratrol (Res) in osteoarthritis (OA) and its underlying mechanism. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to determine the relative expression levels of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), microRNA-9 (miR-9), nuclear factor kappa B subunit 1 (NF-κB1), interleukin (IL)-6, matrix metallopeptidase 13 (MMP-13) and caspase-3 in vitro and in the in vivo model of OA, as well as examining the effect of Res on MALAT1, miR-9 and NF-κB1, IL-6, MMP-13 and caspase-3 expression levels. Immunohistochemical analysis was performed to examine NF-κB1 and MMP-13 protein levels in the in vivo model of OA. Dual-luciferase reporter assays were used to confirm the regulatory relationship between miR-9 and MALAT1 and NF-κB1, as well as examining the effect of Res on the transcriptional activation of MALAT1 promoter. Furthermore, the effect of Res on cell proliferation in vitro was examined by MTT assay. The relative mRNA expression levels of MALAT1 and NF-κB1 were significantly increased, while miR-9 expression was significantly decreased in the OA group compared with the sham group. Treatment with Res partially reversed the effects of OA on MALAT1, NF-κB1 and miR-9 expression. Similarly, the relative protein expression levels of NF-κB1, IL-6, MMP-13 and caspase-3 were significantly increased in the OA group compared with the sham group; however, treatment with Res partially reversed the effects of OA on the protein expression levels of NF-κB1, IL-6, MMP-13 and caspase-3. MALAT1 and NF-κB1 were identified as potential target genes of miR-9, and dual-luciferase assays were used to examine the effect of miR-9 on the luciferase activity of 3′UTR MALAT1 and NF-κB1. Treatment with Res suppressed the transcriptional activation of the MALAT1 promoter, thereby inhibiting MALAT1 expression. Additionally, the relative expression level of miR-9 significantly increased following treatment with Res in a dose-dependent manner, while the relative protein expression levels of NF-κB1, IL-6, MMP-13 and caspase-3 significantly decreased following treatment with Res compared with the control. Furthermore, treatment with Res significantly increased the growth rate of chondrocytes in a dose-dependent manner compared with the control. Taken together, these results suggest that direct targeting of the MALAT1/miR-9/NF-κB1/IL-6, MMP-13/caspase-3 axis may be a novel therapeutic strategy for the treatment of OA.

A study of serum miRNA-122 in hepatitis C and associated hepatocellular carcinoma

BACKGROUND: The discovery of a cluster of short non-coding RNAs called microRNAs (miRNAs) has become an important event in molecular biology. One of its representatives, miR-122 plays a large role in regulating the expression of genes involved in carbohydrate, lipid metabolism, and iron metabolism in the body. In experimental studies it was shown that in addition to regulatory functions, miR-122 is involved in the pathogenesis of hepatitis C, providing the life cycle of the virus in the cell. The shift of emphasis in the study of miR-122 from basic research into clinical medicine seems to be a promising area of personalized medicine.&#x0D; AIMS: to determine the clinical significance of miR-122 in acute and chronic hepatitis C and associated hepatocellular carcinoma.&#x0D; MATERIALS AND METHODS: A total of 407 people were examined, including 17 patients with acute hepatitis C (AHC), 158 patients with chronic hepatitis C (CHC) and 62 patients with HCC associated with hepatitis C. Comparison groups consisted of 84 healthy individuals and 62 patients with clinically pronounced cirrhosis of a non-infectious etiology. In each cohort, the relative miR-122 level was determined in the blood of patients. The analysis was performed in PCR using the Qubit microRNA Assay Kit -100 for the quantitative determination of microRNAs (Thermo Fisher Scientific, USA). Relative miR-122 expression values were calculated by the formula 2 -CT using U6 snRNA as a reference RNA.&#x0D; RESULTS: The highest miR-122 level in serum was found in patients with AHC at the height of the icteric period. The level of miR-122 showed a direct correlation with the activity of hepatic transaminases in patients with AHC (r = 0.72) and CHC (r = 0.44). An analysis of miR-122 level relative to the degree of liver fibrosis in patients with chronic hepatitis C showed that, as liver fibrosis progresses, the level of miR-122 expression decreases. The decrease in miR-122 expression in patients with severe fibrosis was universal and did not depend on the etiology of the disease. The development of HCC in the presence of chronic hepatitis C was accompanied by a decrease in the level of miR-122 by 10 times on average compared to patients with chronic hepatitis C.&#x0D; CONCLUSIONS: The determination of the expression level of miR-122 in serum can be used in laboratory monitoring of the management of patients with HC as an indicator of the severity of liver damage in AHC and the rate of formation of liver fibrosis in CHC. Evaluation of possibility of using miR-122 as a predictor of the development of HCC in the outcome of HC requires additional studies of the specificity and sensitivity of the test and comparison of the obtained data with the results of using generally accepted protein tumor markers.

Expression of MiR-335 and its target metalloproteinase genes: clinical significance in breast cancer

Background Early diagnosis of breast cancer decreases mortality rate; therefore, novel diagnostic methods are urgently required. In this study, authors aimed to investigate the role of serum-derived miR-335 in breast cancer, and the expression of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) and evaluating their feasibility and clinical utility as biomarkers for the early detection of breast cancer. Materials and methods Blood samples were collected from a total of 210 individuals who were enrolled in this study. The participants were divided into newly diagnosed breast cancer patients (n = 115), patients with benign breast lesions (n =55) and healthy individuals as control group (n =40). The expression profile of miR-335, MMP2 and MMP9 were determined using quantitative polymerase chain reaction (qPCR). Results MiR 335 expression level was down-regulated in primary breast cancer group as compared to benign breast group and healthy individuals with 98% and 94.9% sensitivity and specificity, respectively. MMP2 and MMP9 showed significantly higher expression levels in breast cancer group as compared to both benign and healthy group and reporting 92.7% and 93% sensitivity, respectively. The relations between investigated markers and pathologic types, staging, grading, and lymph node involvement were significant with these factors. Expression level of miR-335 was decreased with increased MMP2 and MMP9 at significant level. Conclusion MiR-335, MMP2, and MMP9 can be used as diagnostic markers in breast cancer.

Inhibition of miR-217 Protects Against Myocardial Ischemia–Reperfusion Injury Through Inactivating NF-κB and MAPK Pathways

Recent studies have demonstrated that miRNAs play a vital role in regulating myocardial ischemia/reperfusion injury (MIRI). MiR-217 has been proven to be implicated in cardiac diseases such as chronic heart failure and cardiac myxoma. However, the role of miR-217 in MIRI is not clear. A mouse MIRI model was established and the myocardial infarct size was evaluated by TTC staining. The expression level of miR-217 in I/R group was determined by real-time polymerase chain reaction. Subsequently, MIRI mice and H9C2 cells were administrated with miR-217 inhibitor in vivo and in vitro, respectively. The levels of TNF-α and IL-6 were measured by commercially available ELISA kits. Blood and cell samples were collected for the measurement of lactate dehydrogenase (LDH) level and caspase-3 activity. Cell viability was assessed with the CCK-8 assay. We then explored the detailed molecular mechanisms by TargetScan 7.1 database and further studies were performed to prove the prediction by dual-luciferase reporter assay. Larger stainless infarct areas were observed in the MIRI group, accompanied by inceased serum LDH activity, indicating the mouse MIRI model was successfully established. MiR-217 was up-regulated in MIRI mice and hypoxia/reoxygenation-treated H9C2 cells. MiR-217 knockdown alleviated the MIRI in MIRI mouse model, and also attenuated the myocardial hypoxia/reoxygenation injury in H9C2 cells. Moreover, dual specificity protein phosphatase 14 (DUSP14) was proved to be a target of miR-217. Besides, further study indicated that inhibition of miR-217 protected against MIRI through inactivating NF-κB and MAPK pathways via targeting DUSP14. MiR-217 inhibition protected against MIRI through inactivating NF-κB and MAPK pathways by targeting DUSP14. This study may provide valuable diagnostic and factors and therapeutic agents for MIRI.

MicroRNA-217 suppressed epithelial-to-mesenchymal transition through targeting PTPN14 in gastric cancer.

Background: Gastric cancer (GC) is the one of most common malignancies and its mechanism of metastasis remains unclear. The study was designed to investigate the effects of microRNA-217 on epithelial-to-mesenchymal transition. Methods: The expression levels of miR-217 in GC were assayed by real-time qPCR. Metastasis and invasion of cancer cell were assayed by transwell chamber. Double luciferase reporter gene was used to verify the target regulatory relationship between microRNA-217 and tyrosine–protein phosphatase non-receptor type 14 (PTPN14) on gastric cell lines. Epithelial-to-mesenchymal transition (EMT) markers were assayed by Western blot. Results: We found that miR-217 had a low level expression in gastric tumor tissues of 40 patients with GC, and a lower expression in the gastric tumor tissues of the patients with GC metastasis. Moreover, miR-217 markedly suppressed the metastasis and invasion of gastric cancer cell line in vitro. Furthermore, miR-217 inhibited the expression of PTPN14 by directly targeting its 3′UTR. Moreover, the down-regulation of PTPN14 reduced the metastasis and invasion, whereas up-regulation of PTPN14 led to the enhanced metastases and invasion of gastric cells. miR-217 induced the down-regulation of PTPN14 and inhibited the EMT in gastric cancer cells. Conclusion: miR-217 inhibited the EMT through directly targeting to the 3′UTR of PTPN14.

The role of miRNAs targeting K-ras and APC genes in colorectal cancer

The occurrence of abnormal expression patterns in different types of cancer suggests that micro RNAs (miRNAs) may play an important role in tumorigenesis. The aim of this study was to examine the expression levels of miRNAs known to be associated with the regulation of the expression levels of the APC and K-ras, which are important in the development of colorectal cancer (CRC). The expression levels of miR-27, miR-663, miR-217, miR-181d, APC and K-ras in the serum, tumor and adjacent tumor-free (healthy) tissues of the patients and serum of the healthy controls were investigated with qRT-PCR. Expression levels of miR-217, mR-181d, miR-663, miR-27 and K-ras were found to be higher in CRC tissues than in adjacent tumor-free tissues of the patients. In patient serum samples, miR-663 levels were statistically more elevated than in controls. In patient tumor tissues, miR-217, miR-181d and miR-27 expressions were found to be higher. Increased miR-181d and miR-217 expression levels are associated with increased K-ras expression in the tumor tissues, and the expression of K-ras, which takes part as an oncogene in the CRC development, might be regulated by these miRNAs (Tab. 4, Ref. 33).

MiR-126 on mice with coronary artery disease by targeting S1PR2.

To screen the differentially expressed micro ribonucleic acids (miRNAs) in the serum of coronary atherosclerosis patients, and to investigate their possible mechanisms of action. The differentially expressed serum miRNAs were screened from 3 coronary artery disease (CAD) patients and 3 healthy controls using miRNA expression profiles, which were verified using low-throughput quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) assay. 60 apolipoprotein E (ApoE)-/- mice were divided into model group, agomir-126 group, agomir-control (con) group, and antagomir-126 group using a random number table. They were fed with high-fat diets (21% fat and 0.15% cholesterol) ad libitum for 15 weeks to establish the mouse model of CAD. Then, hematoxylin and eosin (HE) staining was applied to detect the impact of miR-126 expression level on the tissue morphology in the thoracic aortic region. The influences of miR-126 expression level on the secretion levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined via enzyme-linked immunosorbent assay (ELISA). Western blotting assay was performed to examine the effects of miR-126 expression level on the expression levels of nuclear factor-kappa B (NF-κB) and vascular cell adhesion molecule-1 (VACM-1) in the tissues of the thoracic aortic region of the mice. The correlation between miR-126 expression level and sphingosine-1-phosphate receptor 2 (S1PR2) in the serum of CAD patients and animal models was analyzed by the Pearson correlation coefficient method. The targets of miR-126 were predicted using the bioinformatics method, and the direct targets were verified through investigations. Western blotting assay and ELISA were adopted to detect the impacts of miR-126 expression level on the expression and secretion levels of TNF-α, IL-1β, and IL-10 in S1P + oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Lentivirus-small hairpin RNA (shRNA) was utilized to knock down the expression level of S1RP2 to determine whether miR-126 affected the increase in the inflammation level in S1P + ox-LDL-induced HUVECs by targeting S1RP2. Compared with those in control group, 4 miRNAs (miR-126, miR-206, miR-4297, and miR-3646) in the serum of CAD patients exhibited the most significant expression differences, which increased by 6.72, 7.11, 13.57, and 21.22 times, respectively. The verification results of low-throughput RT-qPCR assay indicated that there were remarkable changes in the expression levels of the 4 selected miRNAs with differential expressions in comparison with those in control group, displaying statistically significant differences (p<0.01). The results of HE staining manifested that the coronary atherosclerotic plaques were reduced markedly in agomir-126 group, while notably more coronary atherosclerotic plaques were formed in the thoracic aortic region in antagomir-126 group. Meanwhile, the elevated expression level of miR-126 evidently lowered the expressions of serum TNF-α and IL-1β, but significantly increased the expression of IL-10 in the mouse model of CAD. According to the analysis results of the Pearson correlation coefficient method, the miR-126 expression level was negatively correlated with S1PR2 expression level in the serum of both CAD patients and animal models (r=-0.6123, r=-5.37). It was shown in bioinformatics prediction and luciferase reporter gene assay that miR-126 negatively regulated the S1PR2 expression by targeting the 3' untranslated region (UTR) of S1PR2 messenger RNA (mRNA). In the in vitro inflammation model, the increased expression level of miR-126 could relieve the inflammation in cells induced by S1P + ox-LDL. Based on the results of both Western blotting assay and ELISA, the differences in the expression and secretion levels of TNF-α, IL-1β, and IL-10, as well as the expression levels of signaling molecules of the NF-κB signaling pathway, in the cells were not statistically significant among miR-126 mimic treatment group, sh-S1PR2 group, and miR-126 mimic + sh-S1PR2 group, indicating that miR-126 affects the inflammation level in HUVECs by targeting S1PR2. MiR-126 represses the progression of coronary atherosclerosis in the mice by binding to S1PR2. The results of this research may propose a new mechanism of miR-126 in exerting its therapeutic effects and possess potential value for the treatment of CAD in the future.

The effect of Maras powder and smoking on the microRNA deregulation of oral mucosa.

Objective This study aimed to investigate the effects of Maras powder (a type of smokeless tobacco obtained from Nicotiana rustica Linn and mixed with the ashes of wood, especially from oak, walnut or grapevine) on the microRNA (miRNA) deregulation of oral mucosa, and it compares these effects with those of smoking.Methodology Oral mucosal samples were collected from 74 patients, consisting of 16 nonusers, 26 smokers, and 32 Maras powder users. Genes associated with oral cancer were selected and 90 microRNAs targeting these genes were identified. MicroRNA were isolated and purified using the microRNA isolation kit. MicroRNA were expressed using Fluidigm RT-PCR.Results A positive correlation between the duration of Maras powder use with miR-31 expression levels, and a negative correlation between the Maras powder chewing time and miR-372 expression levels was found. In addition, there is a negative correlation between the amount of Maras powder consumed and expression levels of miR-375, miR-378a, miR-145, and miR-10b; moreover, another negative correlation is observed between the number of cigarettes consumed and the expression levels of miR-23a, miR-23b, miR-203a, miR-200b, and miR-375. However, miR-200b and miR-92a levels were downregulated significantly more in Maras powder users when compared with smokers and nonusers (p<0.05).Conclusion The results show both chewing Maras powder and smoking have an effect on deregulation of miR-200b and miR-92a expressions. This leads to the belief that assessing the expression of these two miRNAs is a promising noninvasive method of analysis, especially in mutagen exposures. Finally, large-scale and high-throughput studies may help to identify an extensive miRNA expression profile associated with tobacco use and improve the understanding of oral malignancies.

NUP210 and MicroRNA-22 Modulate Fas to Elicit HeLa Cell Cycle Arrest.

PurposeCervical cancer is one of the most fatal diseases among women in under-developed countries. To improve cervical cancer treatment, discovery of new targets is needed. In this study, we investigated the expression of NUP210, miR-22, and Fas in cervical cancer tissues and their functions in cell cycle regulation.Materials and MethodsWe detected and compared the expression levels of NUP210, miR-22, and Fas in cervical cancer tissues with paired normal tissues using immunohistochemistry, Western blot, and real-time quantitative polymerase chain reaction. NUP210 was knocked down in HeLa cells via lentivirus, followed by cell cycle and proliferation analysis. Using a luciferase reporter assay, we explored the link between miR-22 and NUP210. We overexpressed miR-22 in HeLa cells and analyzed cell cycle and proliferation function. We then overexpressed miR-22 in NUP210 knockdown cells to explore the connection between Fas and miR-22-NUP210 signaling.ResultsWe found that NUP210 was overexpressed in cervical cancer patients. Knocking down NUP210 restored cell apoptosis and proliferation. We confirmed miR-22 as a regulator of NUP210 and verified that miR-22 was inhibited in cervical cancer development. We also found that restoring miR-22 expression could induce cell apoptosis. Finally, we found that miR-22-regulated expression of NUP210 could alter Fas expression and, in turn, elicit cell cycle arrest and proliferation.ConclusionmiR-22 in cervical cancer is downregulated, resulting in NUP210 overexpression and inhibition of Fas-induced cell apoptosis.

HPV16 E6/E7 promote the translocation and glucose uptake of GLUT1 by PI3K/AKT pathway via relieving miR-451 inhibitory effect on CAB39 in lung cancer cells.

Background:HPV16 E6/E7 proteins are the main oncogenes and only long-term persistent infection causes lung cancer. Our previous studies have shown that HPV16 E6/E7 protein up-regulates the expression of GLUT1 in lung cancer cells. However, whether E6 and E7 protein can promote the glucose uptake of GLUT1 and its molecular mechanism are unclear.Methods:The regulatory relationships of E6 or E7, miR-451, CAB39, PI3K/AKT, and GLUT1 were detected by double directional genetic manipulations in lung cancer cell lines. Immunofluorescence and flow cytometry were used to detect the effect of CAB39 on promoting the translocation to the plasma membrane of GLUT1. Flow cytometry and confocal microscopy were performed to detect the glucose uptake levels of GLUT1.Results:The overexpression both E6 and E7 proteins significantly down-regulated the expression level of miR-451, and the loss of miR-451 further up-regulated the expression of its target gene CAB39 at both protein and mRNA levels. Subsequently, CAB39 up-regulated the expression of GLUT1 at both protein and mRNA levels. Our results demonstrated that HPV16 E6/E7 up-regulated the expression and activation of GLUT1 through the HPV–miR-451–CAB39–GLUT1 axis. More interestingly, we found that CAB39 prompted GLUT1 translocation to the plasma membrane and glucose uptake, and this promotion depended on the PI3K/AKT pathway.Conclusion:Our findings provide new evidence to support the critical roles of miR-451 and CAB39 in the pathogenesis of HPV-related lung cancer.

MiR-132 Over Expression in Osteoarthritis may Regulate the Proliferation and Apoptosis of Fibroblast-like Synoviocytes via Targeting Sirtuin1

This present investigation aimed to explore the role and clinical significance of miR-132 in the pathogenesis of osteoarthritis. Twenty osteoarthritis patients and 20 healthy volunteers were enrolled in the present study. The synovial tissues and plasma of the patients and controls were collected and RT-qPCR was performed to determine the expression level of miR-132. In addition the osteoarthritis patient-derived fibroblast-like synoviocytes were isolated, cultured, transfected with miR-132 inhibitors and the proliferation and apoptosis of the cells was determined using MTT assay as well flowcytometry. Finally, the relationship between miR-132 and sirtuin1 in osteoarthritis was evaluated. miR-132 expression was significantly increased in both synovial tissues and plasma of osteoarthritis patients and results of receiver operating characteristic curve showed that both synovial and plasma miR-132 could serve as potential diagnostic biomarker for osteoarthritis with high sensitivity and specificity. Moreover, miR-132 inhibitors significantly inhibited the proliferation and promoted apoptosis of osteoarthritis patients-derived fibroblast-like synoviocytes. Finally, the expression of miR-132 and sirtuin1 was negatively correlated in osteoarthritis and sirtuin1 was confirmed to be a direct target of miR-132. miR-132 was up-regulated in osteoarthritis and miR-132 might promote the development of osteoarthritis through regulating the proliferation and apoptosis of osteoarthritis patients-derived fibroblast-like synoviocytes via targeting sirtuin1.