ABSTRACT
Objective To investigate the effect of fluoride exposure on expression of miRNA (miR)-200c and its target in human osteoblast Saos-2 cells.Methods Saos-2 cells were cultured in DMEM/F-12 medium and treated with fluoride (sodium fluoride,NaF).There were two groups including:control group (0 mg/L) and fluoride group (4 mg/L).Cells were harvested after 48 hours of culture with fluoride.The expression of miR-200c,the mRNA of alkaline phosphatase (ALP),osteocalcin (BGP),the target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and dual-specific phosphatase 1 (DUSP1) of miR-200c was detected by qRT-PCR.The protein expression of PTEN and DUSP1 was detected by Western blotting.Results The expressions of ALP,BGP mRNA and miR-200c in Saos-2 cells in the fluoride group (23.60 ± 1.87,9.41 ± 0.94,8.61 ± 0.26) were higher than those in the control group (1.00 ± 0.11,1.00 ± 0.07,1.00 ± 0.12).The differences were statistically significant (t =-24.084,-18.388,-8.687,P ＜ 0.05).The mRNA expressions of PTEN and DUSP1 in the fluoride group (0.63 ± 0.02,0.38 ± 0.02) were lower than those in the control group (1.02 ± 0.24,1.02 ± 0.24).The differences were statistically significant (t =3.327,5.454,P ＜ 0.05).The protein expressions of PTEN and DUSP1 in Saos-2 cells in the fluoride group (1.19 ± 0.10,0.83 ± 0.07) were lower than those in the control group (1.81 ± 0.14,1.44 ± 0.25).The differences were statistically significant (t =6.250,4.171,P ＜ 0.05).Conclusion Exposure to fluorine may increase the expression of miR-200c in Saos-2 cells,and fluorine may act on PTEN and DUSP1 through miR-200c,downregulates the mRNA and protein expression levels of PTEN and DUSP1.
ABSTRACT
Objective To investigate the regulation mechanism of silencing the DUSP1 gene on the release of proinflammatory cytokines in mice with acute pancreatitis(AP). Methods Two DUSP1 - siRNA and one scramble siRNA sequences were designed,and the sequence with higher silence efficiency was selected. Mice models with AP were established,and KM mice were divided into 6 groups:control group,AP group,AP + PD98059 group,AP + scram-ble group,AP + siRNA group and AP + PD98059 + siRNA group. Expressions of proinflammatory cytokines tumor necro-sis factor - α(TNF - α),interleukin(IL)- 1β and IL - 6 in serum were detected by using enzyme linked immunosor-bent assay(ELISA)after 12 h,24 h,48 h of modeling. Serum amylase levels were detected. The mRNA expression levels of DUSP1,TNF - α,IL - 1β and IL - 6 in pancreatic tissues were detected by using quantitative real time poly-merase chain reaction (qPCR). The protein expression levels of DUSP1,extracellular regulated protein kinases(ERK), c - Jun N - terminal kinase(JNK),p38,p - ERK,p - JNK and p - p38 in pancreatic tissues were detected by using Western blot. Results Compared with the control group,the other 5 groups displayed the increased expressions of TNF - α,IL - 1β,IL - 6 and amylase in serum,and expressions of DUSP1,TNF - α,IL - 1β,IL - 6,p - ERK,p -JNK,p - p38 in tissues,and there was a statistical significance (all P < 0. 05). Compared with the AP group,the AP +PD98059 + siRNA group showed the decreased DUSP1 expression in tissues,and there was a statistical significance (all P < 0. 05);the AP + PD98059 group had decreased expressions of TNF - α,IL - 1β,IL - 6 and amylase in serum,and expressions of TNF - α,IL - 1β,IL - 6,p - ERK,p - JNK,p - p38 in tissues,and there were statistical significances (all P < 0. 05);while the opposite results were observed in the AP + siRNA group with DUSP1 expression decreased. Conclusions The results support that silencing the DUSP1 gene promotes the release of proinflammatory cytokines through activating the mitogen - activated protein kinase signaling pathway in mice with AP.
ABSTRACT
The mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) belongs to the MAPK cascades which are central to cell proliferation and apoptosis. The carcinogenic role of MKP-1 has been reported in many types of cancer but it has rarely been investigated in breast cancer. The present study was designed to evaluate the MKP-1 mRNA expression and its possible regulation by methylation of MKP-1 promoter in the model of several breast cancer cell lines and tissues as well as controls. Our data demonstrate MKP-1 mRNA expression significantly decreased in five breast cancer cell lines compared to breast controls (P < 0.01). Using the methylation-specific PCR (MSP) analysis, the unmethylated reaction (U) is dominant in both normal cell lines and benign breast tumors (100% vs. 86.2%), whereas the methylated reaction (M) is dominant in both breast cancer cell lines and invasive breast tumors (100% vs. 57.2%). In terms of methylation ratio (M/M+U), methylation level in MKP-1 promoter is significantly higher in the invasive breast tumor tissues (n = 152) than in benign breast tumor tissues (n = 29) (P < 0.0001). Assessing the methylation ratio of the promoter of MKP-1 gene to diagnose the breast malignancy (invasive vs. benign), the area under the receiver-operating characteristic (ROC) curve was 0.809 (95% CI: 0.711-0.906, P < 0.001). The best performance for this prediction has a sensitivity of 76.32% and a specificity of 82.76% at the cutoff value of 0.38. Taken together, we firstly demonstrated that the promoter methylation of MKP-1 gene is a potential breast cancer biomarker for breast malignancy.