Effect of miR-4270 Suppression on Migration in Hepatocellular Carcinoma Cell Line (HepG2)

Background: Liver transplantation and surgical resection are two major strategies for treatment of hepatocellular carcinoma (HCC) patients. One approach to treating HCC is the suppression of metastasis to other tissues. Herein, we aimed to study the effect of miR-4270 inhibitor on migration of HepG2 cells as well as activity of matrix metalloproteinase (MMP) these cells in order to find a strategy to suppress metastasis in future. Methods: HepG2 cells were treated with 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 nM of miR-4270 inhibitor, and then the cell viability was measured by trypan blue staining. Afterwards, cell migration and MMP activity of HepG2 cells were assessed by wound healing assay and zymography, respectively. The MMP gene expression was determined by real-time reverse transcription polymerase chain reaction. Results: Results showed that miR-4270 inhibitor decreased the cell viability of HepG2 cells in a concentration-dependent manner. Also, inhibition of the miR-4270 reduced invasion, MMP activity, and expression of MMP genes in HepG2 cells, respectively. Conclusion: Our findings suggest that miR-4270 inhibitor decreases in vitro migration, which could help find a new approach for HCC therapy patients.

Ovarian expression of follicle stimulating hormone and activin receptors genes in a prenatally-androgenized rat model of polycystic ovary syndrome in adulthood.

BACKGROUND: The expression of genes involved in basic pathways, such as folliculogenesis and steroidogenesis may be affected following prenatal androgen exposure. Besides, exposure to androgens during prenatal life plays a central role in developing polycystic ovary syndrome (PCOS) in females in later life. In the present study, we aimed to examine the expression of the follicle stimulating hormone receptor (FSHR) and activin receptor (actR) genes in ovarian granulosa cells (GCs) of a prenatally-androgenized rat model of PCOS in adulthood. METHODS AND RESULTS: In the adult rat model of PCOS and their controls (n = 8 in each group), different phases of the estrous cycle were determined by vaginal smear. Total RNA was extracted from the ovarian GCs using the TRIzol protocol, a reverse transcription kit was used for complementary DNA (cDNA) synthesis, and the expression of FSHR and actR genes was measured by SYBR-Green Real-Time PCR. GraphPad Prism was used for statistical analysis of data, and the t-Student's test was used to compare the results between the two groups. PCOS rats had longer and irregular estrous cycles compared to controls. The expression of FSHR and actR genes were significantly decreased in the rat model of PCOS compared to control rats. In PCOS rats, genes expression ratios for FSHR and actR were 0.91 ± 0.11 times (P = 0.008) and 0.42 ± 0.13 times (P = 0.048) less than controls, respectively. CONCLUSION: Reduced expression of the FSHR and actR genes in ovarian GCs may be one of the mechanisms mediating PCOS-related disorders, especially abnormal ovarian folliculogenesis and ovulation dysfunction, following exposure to androgens during fetal life.

New evidence on tumor suppressor activity of PTEN and KLLN in papillary thyroid carcinoma.

This study aimed to address the hypothesis that the expression of PTEN and KLLN tumor suppressor genes could diminish in papillary thyroid cancer (PTC) compared to paired normal tissue (PNT) and multinodular goiter (MNG). PTEN and KLLN expressions were assessed at both mRNA and protein levels in 82 tissue samples, including 30 PTC, 30 PNT, and 26 MNG using SYBR-Green Real-Time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Bioinformatics studies were performed to evaluate the genomic location and the genes promoter region. The mRNA expression of PTEN and KLLN in PTC was significantly lower than PNT (PTEN, P = 0.0033; KLLN, P = 0.0005). A significant decrease in the mRNA level of KLLN was also observed in PTC than MNG (P = 0.0304). Decreased level of PTEN mRNA (odds ratio=0.391; P = 0.013) or KLLN mRNA (odds ratio=0.023; P = 0.025) was associated with an increased risk of PTC tumorigenesis. Areas under the ROC curve for PTEN and KLLN were 0.69 and 0.78, respectively. PTEN and KLLN protein expressions in PTC compared to PNT or MNG were not significantly different. The bioinformatics studies revealed the sequence near the promoter region is lowly conserved across species. Four GC boxes were found upstream of the PTEN transcription start site (TSS), and one TATA box and one GC box were found upstream of KLLN TSS. The results suggest PTEN and KLLN are the two tumor suppressor genes that decreasing or loss of both of them occurs in sporadic PTC tumorigenesis. It appears they could have a promising application in both diagnostic and therapeutic areas.

Overexpression of mir-129-1, miR-146b, mir-183, and mir-197 in follicular thyroid carcinoma and adenoma tissues.

Follicular thyroid carcinoma (FTC) is responsible for approximately 10% of thyroid malignancies. Since this type of malignancy indicates no capsular and vascular invasions, adenoma and follicular carcinoma of thyroid are not distinguishable. It has been proved that microRNAs, which regulate approximately 30% of coding proteins, have an association with follicular thyroid adenoma (FTA) and carcinoma of the thyroid. Therefore, the aim of this study was to assess the expression of some miRNAs for detecting the most appropriate miRNA as potential biomarker in the diagnosis of FTA and FTC patients. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was applied to determine the expression levels of miR-129-1, miR-146b,-183 and miR-197 in 48 cases (16 FTC, 16 FTA and 16 hyperplasia/multinodular goiter (MNG) cases). The significance of miRNA differential expression levels among groups were assessed using Multivariate test by Statistical Package for Science Software (SPSS v.20) and Graph Pad Prism v.8. Results indicated that all of the miRNAs had significant overexpression in FTC and FTA versus MNG cases, and also increased expression level in FTC in comparison with FTA, however it was not significant. The results of ROC curve analysis determined the significant overexpression and prognostic value of miR-129-1 in FTA cases and miR-146b in both FTA and FTC cases compared to MNG group. Although all of the earlier mentioned microRNAs were overexpressed in FTC and FTA cases, the ROC curve results demonstrated that miR-129-1 had agreeable AUC for FTA cases. Therefore, it seems that it's cut-off point could be helpful in distinguishing between FTA and multinodular goiter cases. On the other hand, although miR-146b has excellent diagnostic value in both FTA and FTC groups, it seems that this microRNA is unable to act as a specific biomarker to discriminate between FTA and FTC cases. This data need to be confirmed in a large cohort study and other biological samples such as plasma.

Validation of Reference Genes for Normalization of Relative qRT-PCR Studies in Papillary Thyroid Carcinoma.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including 'GUSB and HPRT1' or 'GUSB and HMBS' or 'GUSB and PGM1' were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.

Dietary inorganic nitrate attenuates hyperoxia-induced oxidative stress in obese type 2 diabetic male rats.

AIMS: Hyperoxia has beneficial metabolic effects in type 2 diabetes. However, hyperoxia exacerbates already existing oxidative stress in type 2 diabetes. Nitrate, a nitric oxide donor, is an effective new treatment in type 2 diabetes and also has antioxidant properties. The aim of this study was to determine whether nitrate administration can attenuate hyperoxia-induced oxidative stress in obese type 2 diabetic rats. MAIN METHODS: Fifty-six male Wistar rats (190-210 g) were divided into 8 groups: Controls (non-treated, nitrate-treated, O2-treated, and nitrate + O2-treated) and diabetes (non-treated, nitrate-treated, O2-treated, and nitrate + O2-treated). Diabetes was induced using high-fat diet and low-dose of streptozotocin (30 mg/kg). Rats in intervention groups, were exposed to 95% oxygen and consumed sodium nitrate (100 mg/L) in drinking water. Serum fasting glucose, oxidized (GSSG) and reduced (GSH) glutathiones, total oxidant status (TOS), catalase and superoxide dismutase (SOD) activities, and total antioxidant capacity (TAC) were measured after intervention. Oxidative stress index (OSI) was calculated as TOS/TAC ratio. KEY FINDINGS: Diabetic rats had increased oxidative stress and hyperoxia exacerbated it. In O2-diabetic rats, nitrate decreased GSSG (102.7 ±â€¯2.1 vs. 236.0 ±â€¯20.1 µM, P < 0.001), TOS (67.7 ±â€¯7.3 vs. 104 ±â€¯3.8 µM, P < 0.001), and OSI (0.44 ±â€¯0.04 vs. 0.91 ±â€¯0.07, P < 0.001) and increased catalase (2.8 ±â€¯0.13 vs. 1.8 ±â€¯0.21 KU/L, P = 0.014), SOD (53.4 ±â€¯1.5 vs. 38.4 ±â€¯1.2 U/mL, P < 0.001), GSH (43.7 ±â€¯1.4 vs. 17.8 ±â€¯0.5 mM, P = 0.003), TAC (152.5 ±â€¯1.9 vs. 116.7 ±â€¯5.0 mM, P < 0.001), and GSH/GSSG ratio (0.43 ±â€¯0.01 vs. 0.08 ±â€¯0.01, P = 0.005). Nitrate also potentiated effects of hyperoxia on decreasing fasting glucose. SIGNIFICANCE: Our results showed that dietary nitrate attenuates hyperoxia-induced oxidative stress in type 2 diabetic rats.

Altered gene expression of hydrogen sulfide-producing enzymes in the liver and muscles tissues of hyperthyroid rats.

Thyroid hormones have a role in the regulation of hydrogen sulfide (H2 S) biosynthesis. In this study, we determined the effects of hyperthyroidism on H2 S levels in various tissues and messenger RNA (mRNA) expression of cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in the liver and muscles of the rat. Sixteen male Wistar rats were divided into the hyperthyroid and the control groups. Hyperthyroidism was induced by adding l-thyroxine (12 mg/L) to drinking water for a period of 21 days. H2 S concentrations in serum, liver, aorta, heart, and soleus muscles, as well as mRNA expressions of CBS, CSE, and 3-MST in these tissues were measured at Day 21. Hyperthyroid rats had lower H2 S levels in the serum compared with controls (14.7 ± 1.4 vs. 25.7 ± 1.6 µmol/L, p < 0.001). Compared with controls, hyperthyroid rats had lower levels of H2 S in the aorta (89%), heart (80%), and soleus (103%) muscles, but higher levels in the liver (35%). Hyperthyroidism decreased the ratio of CBS/CSE mRNA expression in the liver and the CSE/CBS mRNA expression in the muscles by decreasing CBS levels in liver (34% cf. controls) and CSE levels in the aorta, heart, and soleus muscles (respectively, 51%, 7%, and 52% cf.). In addition, hyperthyroidism decreased the mRNA expression of 3-MST in the liver (51%) and aorta (33%), and increased it in the heart (300%) and soleus muscle (182%). In conclusion, hyperthyroidism increased H2 S levels in the liver and decreased it in muscles; these effects are at least in part due to increases and decreases in expression of CSE in the liver and muscles, respectively. These data indicate an association between thyroid hormone status and gene expression of the H2 S-producing enzymes in the rat.

Effects of long-term nitrate supplementation on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in male obese type 2 diabetic rats.

PURPOSE: Supplementation with inorganic nitrate to boost the nitrate-nitrite-nitric oxide (NO) pathway, may act as a potential therapeutic agent in diabetes. The aim of this study was to determine the effects of nitrate on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in obese type 2 diabetic rats. METHODS: Male Wistar rats were divided into 4 groups: Control, control + nitrate, diabetes, and diabetes + nitrate. Diabetes was induced using a high-fat diet and low-dose of streptozotocin. Sodium nitrate (100 mg/L in drinking water) was administered simultaneously for two months. Serum levels of fasting glucose, insulin, and lipid profiles were measured every 2-weeks. Glycated hemoglobin (HbA1c) was measured monthly. Serum thiobarbituric reactive substances (TBARS) level and catalase activity were measured before and after treatment. At the end of the study, glucose, pyruvate, and insulin tolerance tests were done. Glucose-stimulated insulin secretion (GSIS) and insulin content from isolated pancreatic islets were also assessed; mRNA expression of iNOS as well as mRNA expression and protein levels of GLUT4 in insulin-sensitive tissues, and serum IL-1ß were determined. RESULTS: Nitrate supplementation in diabetic rats significantly improved glucose tolerance, lipid profiles, and catalase activity as well as decreased gluconeogenesis, fasting glucose, insulin, and IL-1ß; although it had no significant effect on GSIS, islet insulin content, HbA1c, and serum TBARS. Compared to the controls, in diabetic rats, mRNA expression and protein levels of GLUT4 were significantly lower in the soleus muscle (54% and 34%, respectively) and epididymal adipose tissue (67% and 41%, respectively). In diabetic rats, nitrate administration increased GLUT4 mRNA expression and protein levels in both soleus muscle (215% and 17%, respectively) and epididymal adipose tissue (344% and 22%, respectively). In diabetic rats, nitrate significantly decreased elevated iNOS mRNA expression in both the soleus muscle and epididymal adipose tissue. CONCLUSION: Chronic nitrate supplementation in obese type 2 diabetic rats improved glucose tolerance, insulin resistance, and dyslipidemia; these favorable effects were associated with increased mRNA and protein expression of GLUT4 and decreased mRNA expression of iNOS in insulin-sensitive tissues, and with decreased gluconeogenesis, inflammation, and oxidative stress.

Transient Congenital Hypothyroidism Alters Gene Expression of Glucose Transporters and Impairs Glucose Sensing Apparatus in Young and Aged Offspring Rats.

BACKGROUND/AIMS: Transient congenital hypothyroidism (TCH) could disturb carbohydrate metabolism in adulthood. Aging is associated with increased risk of type 2 diabetes. This study aims to address effects of TCH on mRNA expressions of glucose transporters (GLUTs) and glucokinase (GcK) in islets and insulin target tissues of aged offspring rats. METHODS: The TCH group received water containing 0.025% 6-propyl-2-thiouracil during gestation. Offspring from control and TCH groups (n=6 in each group) were followed until month 19. Gene expressions of GLUTs and GcK were measured at months 3 and 19. RESULTS: Compared to controls, aged TCH rats had higher GLUT4 expression in heart (4.88 fold) and soleus (6.91 fold), while expression was lower in epididymal fat (12%). In TCH rats, GLUT2 and GcK expressions in islets were lower in young (12% and 10%, respectively) and higher in aged (10.85 and 8.42 fold, respectively) rats. In addition, liver GLUT2 and GcK expressions were higher in young (13.11 and 21.15 fold, respectively) and lower in aged rats (44% and 5%, respectively). CONCLUSION: Thyroid hormone deficiency during fetal period impaired glucose sensing apparatus and changed glucose transporter expression in insulin-sensitive tissues of aged offspring rats. These changes may contribute to impaired carbohydrate metabolism.