Value of endonuclease domain containing 1 in progression of prostate cancer

AIM: To analyze the difference of endonuclease domain containing 1 (ENDOD1) expression between benign prostatic hyperplasia (BPH) tissues and prostate cancer (PCa) tissues and to investigate the effect of ENDOD1 on the biological function of human prostate cancer cells. METHODS: The BPH samples (n=20) and PCa samples (n=21) were processed and analyzed according to the instruction of immunohistochemical (IHC) staining. The mRNA and protein levels of ENDOD1 in the normal prostate epithelial cells and prostate cancer cells were evaluated by RT-qPCR and Western blot, respectively. The recombinant plasmids pCMV-N-Flag-ENDOD1 was constructed and was transfected into the human prostate cancer cells. The proliferation, apoptosis, migration and invasion abilities of the prostate cancer cells were evaluated by MTT assay, flow cytometry, Transwell migration and Matrigel invasion assays, respectively. RESULTS: The analysis of variance of the immunoreactivity score showed that PCa tissues with high Gleason score displayed significantly lower ENDOD1expression than that with low Gleason score and BPH (P<0.05). The expression of ENDOD1 at mRNA and protein levels in PC3 cells and DU145 cells was significantly lower than that in the LNCap cells (P<0.05). The proliferation of DU145 transfected with ENDOD1 was inhibited. The flow cytometry indicated that ENDOD1 over-expression in the DU145 cells resulted in a notable increase in G₀/G₁ phase arrest (P<0.05), but the apoptotic rates showed no statistical difference. The results of Transwell assay showed that migration and invasion abilities of the cells were also inhibited after transfection with over-expressing ENDOD1 plasmid (P<0.05). CONCLUSION: The expression of ENDOD1 significantly decreased in prostate cancer with high Gleaon score. Meanwhile, the ENDOD1 is specifically down-regulated in androgen independent prostate cancer (AIPC) cell lines. Over-expression of ENDOD1 remarkably inhibits the proliferation, migration and invasion abilities of AIPC.     

Relationship between phosphoglycerate kinase 1 and clinical features of prostate cancer and its role in prognosis

AIM: To study the expression and prognostic functions of phosphoglycerate kinase 1 (PGK1) in prostate cancer. METHODS: The prostatic samples were collected from the patients with prostate cancer and benign prostatic hyperplasia (BPH) in TCM-Integrated Hospital of Southern Medical University from Jan 2013 to Dec 2013. The protein expression of PGK1 in the prostate specimens was detected by immunohistochemical analysis and Western blot. Furthermore, the correlations of PGK1 expression with the clinicopathological features and prognosis of prostate cancer were also evaluate. RESULTS: The expression of PGK1 in the prostate specimens was significantly up-regulated compared with the BPH individuals. In addition, the expression of PGK1 was significantly correlated with the local infiltration, Gleason score, TNM grade, bone metastasis, and serum prostate-specific antigen (PSA) concentration. Finally, bone metastasis, serum PSA level and PGK1 expression were independent risk factors for prostate cancer illustrated by Cox analysis, and high expression of PGK1 was correlated with poor prognosis. CONCLUSION: PGK1 expression is an independent risk factor for prostate cancer, and it might act as a prognostic biomarker for prostate cancer.     

Changes of serum T-PSA,F-PSA and F/T ratio in patients with prostate cancer (PCa) and its clinical significance

AIM: To evaluate the changes of serum total prostate specific antigen (T-PSA), free prostate specific antigen (F-PSA) and the ratio of F-PSA to T-PSA (F/T) in patients with prostate cancer and its clinical significance. METHODS: The concentrations of T-PSA and F-PSA in serum were measured by micropartical enzyme immunoassay (MEIA) using AxSYM System, and the F/T ratio was calculated. RESULTS: Before operation, the concentrations of T-PSA and F-PSA in patients with PCa were much higher and F/T ratio was significantly lower than that in patients with benign prostate hyperplasia (BPH). T-PSA and F-PSA levels decreased, but F/T ratio increased after operation in PCa and BPH. F/T ratio in 83.5% PCa and 6.5% BPH was less than 0.16. To diagnosis PCa, the sensitivity of F/T ratio was 83.5%, and the specificity was 86.7%. CONCLUSION: Serum T-PSA, F-PSA and F/T ratio are important parameters for the early diagnosis of prostate cancer.     

Plasma circulating miR-126 in patients with coronary artery heart disease and its effect on vascular endothelial cells

AIM: To investigate the role of plasma circulating miR-126 and miR-16 in the patients with coronary artery heart disease and to explore the influence of miR-126 on vascular endothelial cells. METHODS: Plasma total RNA was isolated from 52 patients with stable coronary artery disease and 52 healthy volunteers. The circulating miR-126 and miR-16 in those people were detected using specific primers. Endothelial cell line EA.hy926 was transfected with a miR -126 inhibitor, and total RNA of the cells was isolated 30 h after transfection to detect the expression level of vascular endothelial growth factor (VEGF). RESULTS: The expression of plasma circulating miR-126 was significantly decreased in the patients with coronary artery heart disease compared with healthy controls (P<0.05). No significant difference of circulating miR-16 between the patients with coronary artery heart disease and healthy controls was observed (P>0.05). The expression of VEGF in the endothelial cell line EA.hy926 transfected with miR-126 inhibitor was 2.08 times higher than that in negative control cells 30 h after transfection (P<0.05). CONCLUSION: Plasma circulating miR-126 is significantly decreased in the patients with coronary artery heart disease. Plasma circulating miR-16 in the patients with coronary artery heart disease and in the healthy controls is stable. miR-126 negatively regulates the expression of VEGF in vascular endothelial cells.     

Detection of miR-122 expression in serum by Taqman probe real-time fluorescence quantitative PCR and its clinical significance

AIM: To detect the serum level of miR-122 expression by the technique of Taqman probe real-time fluorescence quantitative PCR for identifying its clinical significance. METHODS: The stem-loop RT primer, PCR primer and Taqman probe of miR-122 and U6 snRNA were designed. The expression of miR-122 in the serum samples of 27 cases of preoperative hepatocellular carcinoma (HCC), 15 cases of hepatitis B, 15 cases of hepatitis C, 15 cases of healthy control (HC), 11 cases of postoperative hepatocellular carcinoma (PHCC) and 10 cases of recurrence after postoperative hepatocellular carcinoma was detected by Taqman probe real-time fluorescence quantitative PCR. U6 snRNA was used as the internal control. RESULTS: The results showed that the method of Taqman probe real-time fluorescence quantitative PCR could detect the amplification signal of serum miR-122. The expression level of serum miR-122 in the patients with HCC, hepatitis B, hepatitis C and recurrence was higher than that in HC and the patients with PHCC. Meanwhile, the serum level of miR-122 in the patients with hepatitis C was higher than that in the patients with HCC, hepatitis B and recurrence. However, no difference of miR-122 expression level among HCC, hepatitis B and recurrent patients was observed. The miR-122 level was lower in PHCC patients than that in HCC and recurrent patients. In hepatitis B virus surface antigen (HBsAg) and/or hepatitis B virus e antigen (HBeAg) positive patients, the miR-122 level was higher than that in the negative ones. The miR-122 level in hepatitis C antibody (HCV-Ab) positive patients was raised compared with the negative ones. The serum level of alanine aminotransferase (ALT) was positively correlated with the serum level of miR-122 (r=0.34, P<0.05). The miR-122 expression level in the patients with serum AFP≥400 μg/L was higher than that in the patients with serum AFP<400 μg/L. CONCLUSION: The method of Taqman probe real-time fluorescence quantitative PCR can detect the serum level of miR-122 expression. Serum miR-122 might be used as a new biomarker of liver diseases, especially in the early diagnosis of primary hepatocellular carcinoma, the curative effect of surgical operation and the prognosis.     

Diagnostic value of serum miR-21 for diabetic nephropathy

AIM:To evaluate the diagnostic value of serum miR-21 as a molecular biomarker in the patients with diabetic nephropathy (DN). METHODS:Twenty-five patients with DN, 50 patients with type II diabetes ［divided into DMA group and DMB group base on urinary microalbumin (UmAlb), 25 in each group］, 25 patients with diabetic nephropathy-induced uremia (UM) and 25 normal controls (NC) were included in the study. Total RNA was extracted from the serum samples for determining the miR-21 level using real-time PCR. The relationship between miR-21 level and clinical parameters of the patients with diabetic nephropathy was analyzed. RESULTS:The relative expression level of serum miR-21 was significantly lower in DN group than that in DMA group, DMB group and NC group, and was significantly higher than that in UM group (all P<001). The serum miR-21 level showed significant negative correlation with cystatin C, UmAlb, UmAlb/urinary creatinine in DN patients (P<0.01,P<0.05,P<0.05). For discriminating the DN patients from NC, DMA, DMB, DMA＋NC, DMB＋NC and DMA＋DMB, the areas under the receiver operating characteristic curve (AUC-ROC) for serum miR-21 were 0848 (95% CI: 0737~0959), 0896 (95% CI: 0812~0980), 0782 (95% CI: 0641~0922), 0838 (95% CI: 0743~0933), 0796 (95% CI: 0675~0917) and 0808 (95% CI: 0704~0911), and the sensitivity and specificity were 800% and 720%, 720% and 880%, 720% and 840%, 760% and 770%, 760% and 820%, 700% and 860%, respectively. For discriminating the DN patients from DMA＋DMB＋NC, the AUC-ROC was 0845 (95% CI: 0752~0939), and the sensitivity and specificity were 760% and 773%, respectively. CONCLUSION: Serum miR-21 can serve as a molecular diagnostic marker for diagnosis of diabetic nephropathy.     

Effect of abnormal expression of PDK4 on glycolysis and proliferation in prostate cancer cells

AIM To investigate the expression of pyruvate dehydrogenase kinase 4 （PDK4） in prostate cancer tissue and its effect on glycolysis and growth of prostate cancer cells. METHODS Immunohistochemistry was used to compare the expression differences of PDK4 protein in benign prostatic hyperplasia （BPH） and prostate cancer tissues. The expression levels of PDK4 in normal prostatic epithelial cells （RWPE-1） and different prostate cancer cell lines （PC3, LNCaP, DU145 and C4-2） were detected by RT-qPCR and Western blot. Recombinant plasmid carrying PDK4-shRNA was constructed, and the expression of PDK4 in prostate cancer PC3 cells was down-regulated by transfection with PDK4-shRNA. The changes in glycolysis level of PC3 cells before and after transfection were determined by cell glycolysis kit, and the effects of PDK4 on the viability and cell cycle distribution of PC3 cells were detected by CCK-8 assay and flow cytometry. RESULTS In prostate cancer tissues, the expression level of PDK4 protein was significantly higher than that in BPH tissues （P<0.05）, and the analysis of immunohistochemical score showed that prostate cancer tissues with high Gleason score displayed significantly higher PDK4 expression than those with low Gleason score （P<0.05）. Compared with normal prostatic epithelial cells, RT-qPCR and Western blot results indicated that the expression level of PDK4 was also significantly increased in prostate cancer cell lines （P<0.05）. In addition, CCK-8 assay results showed that the viability of prostate cancer PC3 cells was significantly decreased after knockdown of PDK4 expression （P<0.05）. The results of flow cytometry demonstrated that knockdown of PDK4 expression in PC3 cells resulted in a notable increase in G₀/G₁ phase arrest （P<0.05）. CONCLUSION PDK4 is highly expressed in prostate cancer tissues and cell lines, and significantly increases in prostate cancer with high Gleason score. In addition, down-regulation of PDK4 expression significantly inhibits glycolysis and growth of prostate cancer cells, resulting in cell cycle arrest at G₀/G₁ phase.     

Effect of abnormal expression of miR-141 on malignant biological behaviors of human hepatocarcinoma cells

AIM: To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells. METHODS: The RNA from SMMC-7721 cells and HL-7702 cells was extracted. SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine. After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. The changes of migration ability were investigated by Transwell invasion assay. RESULTS: The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells transfected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P < 0.05). The percentages of G₁ phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migration ability was inhibited (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P < 0.05). When miR-141 was down-regulated, the percentages of G₁ phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P < 0.05). CONCLUSION: miR-141 is down-regulated in human hepatocarcinoma cell line. Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells. miR-141 may function as a tumor suppressor gene during HCC development.     

Baicalin inhibits high glucose-induced apoptosis of mouse glomerular mesangial cells via miR-141/Sirt1 signaling pathway

AIM:To investigate the therapeutic mechanism of baicalin for diabetic nephropathy involving microRNA-141 (miR-141)/silent information regulator 1 (Sirt1) signaling pathway. METHODS:Mouse glomerular mesan-gial cell line SV40-MES-13 was treated with high glucose (HG, 25 mmol/L glucose) to establish diabetic nephropathy cell model. Baicalin at 100 μmol/L was used to treat glomerular mesangial cells. qPCR and Western blot were performed to determine the expression levels of miR-141 and Sirt1. The regulatory relationship between miR-141 and Sirt1 was detected by dual-luciferase assay. The apoptosis of glomerular mesangial cells was analyzed by flow cytometry. RESULTS:Compared with control group, the cells treated with HG showed increased levels of miR-141 and apoptosis, and Sirt1 expression was decreased (P<0.01). Baicalin and miR-141 inhibitor suppressed the HG-induced effect on the levels of miR-141, Sirt1 and apoptosis. Knockdown of Sirt1 expression reversed the effect of miR-141 inhibitor on the levels of miR-141, Sirt1 and apoptosis. Over-expression of miR-141 reversed the effect of baicalin on the glomerular mesangial cells treated with HG. Up-regulation of Sirt1 abolished the effect of miR-141 over-expression on the glomerular mesangial cells. CONCLUSION:Baicalin inhibits the apoptosis of mouse glomerular mesangial cells via miR-141/Sirt1 signaling pathway, thus attenuating diabetic nephropathy.     

Association between plasma level of microRNA-21 and pulmonary hypertension in heart failure patients with preserved ejection fraction

AIM: To investigate the correlation between microRNA-21 (miR-21) and pulmonary hypertension(PH) in the patients with heart failure with preserved ejection fraction (HFpEF). METHODS: From January 2014 to February 2016, 102 HFpEF patients presenting to Jiangxi Provincial People's Hospital were enrolled in this study. The patients were divided into PH-HFPEF group (n=36, PASP ≥ 50 mmHg) and HFpEF group (n=40, PASP<50 mmHg). Another 36 age-and sex-matched subjects served as healthy controls. The plasma level of miR-21 and its correlations with clinic data were examined, and multivariate logistic regression analysis was performed to identify the independent predictors of PH-HFpEF. Receiver operating characteristic (ROC) curve was analyzed, and the area under the curve (AUC) was calculated. The sensitivity and specificity for PH-HFPEF diagnosis were also determined. RESULTS: Age, plasma endothelin-1 (ET-1), interleukin-6 (IL-6), brain natriuretic peptide (BNP) and left atrial diameter (LAD) were significantly higher in PH-HFPEF group than those in HFPEF group (P<0.05). The level of miR-21 was significantly increased in PH-HFPEF group compared with HFpEF group and healthy control group. In addition, the miR-21 level was correlated with PASP (r=0.267, P=0.000), plasma IL-6 (r=0.302, P=0.013) and left ventricular mass index (LVMI) (r=0.515, P=0.036). Plasma IL-6 was positively correlated with plasma ET-1 (r=0.622, P=0.002). PASP was positively correlated with plasma IL-6 (r=0.36, P=0.023), ET-1 (r=0.76, P=0.004), BNP (r=0.43,P=0.031), and LAD (r=0.39, P=0.044). ROC curve showed that the AUC of miR-21 was 0.80. Multivariate logistic regression analysis showed that miR-21, LAD and BNP were the independent predictors of PH-HFpEF. CONCLUSION: Increased plasma miR-21 level is positively correlated to PH-HFpEF, and is an independent predictor of PH-HFpEF, suggesting that plasma miR-21 might be a useful new biomarker for predicting PH in HFpEF patients.     

Molecular mechanism of miR-29a inhibiting malignant phenotypes in prostate cancer cells

AIM:To investigate the biological functions of microRNA-29a (miR-29a) in prostate cancer and the molecular mechanism of miR-29a over-expression inhibiting malignant phenotypes of prostate cancer cells. METHODS:The levels of miR-29a expression in the prostate cancer tissues and cells were detected and analyzed using gene microarray and bioinformatics. The expression levels of miR-29a and lysine (K)-specific demethylase 4B (KDM4B) mRNA in prostate cancer tissues, paracarcinomatous tissues, 4 prostate cancer cell lines (PC3, DU145, LNCaP and ArCaP) and normal prostate epithelial cell line (RWPE-1) were measured by real-time PCR. PC3, DU145, LNCaP and ArCaP cells were transfected with pGenesil-1-miR-29a plasmid using transient transfection. The cell viability, colony formation rate and apoptotic rate were analyzed by MTT assay, colony formation assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The protein expression of KDM4B was determined by Western blot. RESULTS:The results of gene microarray and bioinformatic analysis indicated that differential expression of miR-29a was found in the prostate cancer tissues and the paracarcinomatous tissues. The levels of miR-29a in the prostate cancer tissues and prostate cancer cells were significantly decreased, while the mRNA levels of KDM4B were notably increased compared with the paracarcinomatous tissues and RWPE-1 cells, respectively (P<0.05). Compared with negative control (pGenesil-1) group, the cell viability and colony formation rate were significantly decreased, the apoptotic rate was significantly increased, and the protein expression of KDM4B was notably inhibited in the prostate cancer cells with miR-29a over-expression (P<0.05). The cell viability was significantly enhanced, and the apoptosis was significantly inhibited in the prostate cancer cells with KDM4B over-expression (P<0.05). CONCLUSION:Low expression of miR-29a was found in the prostate cancer tissues and cells. miR-29a over-expression inhibits the growth of prostate cancer cells and induces apoptosis. The mechanism may be associated with inhibiting the protein expression of KDM4B.     

Analyzing clinical characteristic of miR-196b in human colorectal cancer using TCGA and effect of miR-196b on 5-FU resistance of CRC cell line

AIM: To investigate the clinical characteristics of microRNA (miR)-196b in colorectal cancer (CRC) and to study its biological function in 5-fluorouracil (5-FU) resistance. METHODS: miRNA sequence dataset and the corresponding clinical data of CRC patients were downloaded from The Cancer Genome Atlas (TCGA). Expression level and clinical characteristics of miR-196b in CRC patients were analyzed using SPSS 17.0. CRC cell line overexpres-sing miR-196b was established using transient transfection method. MTS test was used to evaluate the effect of miR-196b overexpression on 5-FU resistance. RESULTS: miR-196b expression was associated with lymph node metastasis and TNM stage (P<0.05), but not related with age and sex. Lymph node metastasis and distant metastasis were independent prognostic factors for rectal patients (P<0.05). The expression level of miR-196b was not associated with survival condition of rectal patients. The viability of the cells overexpressing miR-196b treated with different concentrations of 5-FU was significantly higher than that in control group (P<0.05). CONCLUSION: miR-196b may be a potential biomarker of TNM stage and lymph node metastasis in CRC. miR-196b increases the 5-FU resistance of CRC cells.     

Changes of miR-155-5p in serum of cervical cancer patients and effects of miR-155-5p on cervical cancer cells

AIM:To detect the serum level of miR-155-5p in the patients with different cervical diseases, and to analyze its effects on the proliferation, cell cycle and apoptosis of cervical cancer cells. METHODS:SYBR Green I real-time quantitative PCR was used to detect the level of miR-155-5p in the serum of the patients with different cervical diseases. miR-155-5p mimic or inhibitor was used to increase or decrease the expression of miR-155-5p in cervical cancer cells. The proliferation, cell cycle and apoptosis were measured by CCK-8 assay and flow cytometry. RESULTS:The serum level of miR-155-5p in cervical cancer group was higher than that in cervicitis group and healthy group. No statistical difference of the serum miR-155-5p level between cervical intraepithelial neoplasia group and cervical cancer group was observed. Compared with blank group, liposome group and negative control group, the proportion of S-phase cells increased and apoptotic cells decreased in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p mimic. The proportion of G2/M-phase cells increased significantly in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p inhibitor. CONCLUSION: Compared with healthy controls, the serum level of miR-155-5p in the cervical cancer patients increases, and may act as a novel tumor molecular marker for diagnosis of cervical cancer. miR-155-5p has no significant effect on the proliferation, cell cycle and apoptosis of HeLa cell. miR-155-5p may promote SiHa cells to enter S phase and inhibit the apoptosis of SiHa cells.     

Elevated levels of serum IL-6, ICAM-1 and P-selectin in stable survivors with liver transplantation

AIM: To study the profile of serum IL-6， ICAM-1 and P-selectin in stable survivors with clinical liver transplantation (LTx). METHODS: Flow cytometric analysis was used to determine the phenotype of T cell subsets in peripheral blood mononuclear cells （PBMCs） from stable survivors with liver transplantation (n=22), and healthy volunteers (n=12). Serum levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF-α) and interleukin (IL-6), intercellular adhesion molecules (ICAM-1) and P-selectin in stable survivors with liver transplantation and healthy volunteers were assessed by enzyme-linked immunoabsordent assay (ELISA). Recently performed 6 cases of liver transplantation were also dynamically observed in this study. RESULTS: Percentage of CD4+ T cells， CD8+ T cells and CD3+ T cells, as well as ratio of CD4 to CD8 were no difference between two groups (P>0.05). However, a significant higher percentage of CD3+CD25+ T cells was found in stable liver transplantation group as compared to healthy group (P<0.05). Significantly increased concentrations of IL-6, ICAM-1 and P-selectin were found in stable liver transplantation group as compared to healthy group (P<0.05). A high TNF-α level was detected in stable liver transplantation group while no significant difference was found as compared to healthy volunteers group (P>0.05). There was not found no regular change of serum cytokines (IL-6, TNF-α) and adhesion molecules (ICAM-1, P-selectin) in 6 liver transplanted patients during post-operation from day 1 to day 30, indicating that was associated with the different status of patients before or after transplantation. CONCLUSIONS: Our data suggesting that increased levels of ICAM-1 and P-selectin, appears to participate in the processing of immunoregulation to transplanted livers, whereas elevated concentrations of IL-6 appear to be involved in the repair of the injury induced by TNF-α in allo-transplanted livers.     

Alteration of serum IL-6, IL-8, TNF-α and sICAM-1 in patients with pre-menstruation recurrent aphthous ulceration

AIM:To investigate the serum levels of interleeukin-6(IL-6), interleukin-8(IL-8), tumor necrosis factor-alpha(TNF-α) and soluble intercellular adhesion molecule-1 (sICAM-1)in female patients with pre-menstruation recurrent aphthous ulceration(RAU).METHODS:Serum levels of IL-6, IL-8, TNF-α and sICAM-1 in 21 pre-menstruation RAU patients were examined using ELISA technique, and compared to 10 healthy individuals and 22 the female RAU patients unrelated to menstrual cycle.RESULTS:The serum levels of IL-6, IL-8, TNF-α in patients with pre-menstruation RAU were not only significantly higher than that in the normal control group(P＜0.01), but also higher than that in the RAU patients without pre-menstruous recurrence (P＜0.01).The level of serum TNF-α in the RAU patients without pre-menstruous recurrence was slightly higher than that in the normal control group(P＜0.05).The level of sICAM-1 had not changed.CONCLUSION:The serum levels of IL-6, IL-8 and TNF-α increase in pre-menstruation RAU patients, which might play a role in local lesion of RAU.     

Expression of microRNA-1284 in gastric cancer and underlying mechanism

AIM: To evaluate the correlation between microRNA-1284 (miR-1284) and gastric cancer, and to investigate the underlying mechanism. METHODS: The expression of miR-1284 was examined by real-time PCR in 63 gastric cancer (GC) tissue samples and 63 non-malignant adjacent tissue samples. The correlation between miR-1284 and the clinicopathological feature of GC was analyzed. Lentiviral vector containing miR-1284 was constructed and transfected into GC SGC-7901 cells. After transfection, the expression of miR-1284 was examined by real-time PCR. The cell activity was evaluated by CCK-8 assay. The cell cycle and apoptosis were determined by flow cytometry. The ability of cell migration was measured by wound-healing assay. The potential target gene of miR-1284 was predicted by online bioinformatic softwares. The expression of JAG1 mRNA was examined by real-time PCR. The protein levels of JAG1, Notch1 and NF-κB were analyzed by Western blotting. RESULTS: Compared with non-malignant adjacent tissue samples, the results of real-time PCR showed significant downregulation of miR-1284 in 42 GC tissue samples (P<0.05). The expression level of miR-1284 was not significantly associated with age and gender of the patients, tumor size, TNM staging and lymph node metastases (P>0.05), but significantly associated with histologic grading (P<0.05). Compared with LV-NC-GFP group and control group, after transfection of miR-1284 in LV-miR-1284 group, the expression of miR-1284 was significantly increased (P<0.05), the percentages of apoptotic cells and the cells in G₀/G₁ phase were significantly increased (P<0.05), the cells activity and ability of migration were significantly decreased (P<0.05), and the expression of JAG1, Notch1 and NF-κB was significantly decreased (P<0.05). CONCLUSION: The inhibitory effect of miR-1284 on gastric cancer may be associated with the regulation of its targeting gene JAG1.     

Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.     

Effect of microRNA-7 knockdown on ConA-induced acute liver injury in mice

AIM: To study the effect of microRNA-7 (miR-7) knockdown (KD) on concanavalin A (ConA)-induced acute liver injury (ALI) in mice.METHODS: Wild type (WT) mice and miR-7KD mice were received ConA (30 mg/kg) to induced acute liver injury model by intraperitoneal injection, and the morphological changes, liver weight and weight index were measured 48 h later. The pathological changes of the liver tissues were observed by HE staining. The levels of serum alanine aminotransferase (ALT), IL-4 and IFN-γ were detected by ELISA. The proportional changes of CD4⁺ T cells and the relative levels of IL-4 and IFN-γ were analyzed by flow cytometry.RESULTS: The color of the liver tissue became lighter, and the weight and weight index were changed significantly in miR-7KD mice compared with control group (P<0.05). HE staining showed that the inflammatory cell infiltration was increased in the liver of miR-7KD mice. Moreover, the level of serum ALT was significantly increased (P<0.05). The serum level of IFN-γ elevated significantly (P<0.01), while the IL-4 levels decreased significantly (P<0.01) in the serum of miR-7KD mice. Furthermore, the proportion of CD4⁺ T cells and relative IFN-γ cells increased obviously (P<0.01).CONCLUSION: miR-7 knockdown promotes the pathogenesis of the ConA-induced acute liver injury in mice.