ABSTRACT
【Objective】 To explore the relationship of fat mass and obesity-associated protein (FTO) with the m6A modification and expression level of DKK2 in the process of myocardial fibrosis. 【Methods】 Cardiac fibroblasts (CFs) were grouped as follows: Control group, AngⅡ-treated group, AngⅡ+EV group (transfected with empty vector and negative control siRNA and then treated with AngⅡ), AngⅡ+FTO-O group (transfected with FTO overexpression vector and then treated with AngⅡ), and AngⅡ+FTO-O+DKK2 siRNA group (treated with AngⅡ after co-transfection of FTO overexpression vector and DKK2 siRNA). Mice were divided into the following groups: Control group (sham operation group), AMI group (constructing acute myocardial infarction model), AMI+EV group (AMI mice were intraperitoneally injected with nanoparticles containing empty vector), and AMI+FTO-O group (AMI mice were intraperitoneally injected with nanoparticles containing FTO overexpression vector). Then, the expressions of FTO and DKK2 were detected by fluorescence quantitative PCR and Western blotting, the m6A modification level of DKK2 was detected by RNA binding protein immunoprecipitation, the cell viability was detected by CCK-8, the cardiac function of AMI mice was evaluated, and the cardiac pathological changes of mice were detected by HE and Masson staining. 【Results】 AngⅡ inhibited the expression of FTO, thereby enhancing the m6A modification level of DKK2 and downregulating the expression of DKK2 (P<0.05). AngⅡ promoted cell viability and enhanced the expressions of α-SMA, collagen Ⅰ and collagen Ⅲ (P<0.05). FTO overexpression significantly blocked the above-mentioned regulatory effects of AngⅡ (P<0.05), but DKK2 siRNA could antagonize the effect of FTO overexpression on AngⅡ. The expressions of FTO and DKK2 were downregulated in AMI mice, and the m6A modification level of DKK2 was increased (P<0.05). When FTO was overexpressed, the expressions of FTO and DKK2 in AMI mice were significantly restored, the m6A modification level of DKK2 and myocardial fibrosis were significantly reduced (P<0.05), and the cardiac pathological changes were significantly improved. 【Conclusion】 FTO can promote the expression of DKK2 by reducing the m6A modification level of DKK2, thereby inhibiting the progression of myocardial fibrosis. This indicates that FTO/DKK2 pathway is a key pathway in regulating myocardial fibrosis.
ABSTRACT
Objective The aims of this study were to investigate the expression of DKK2,a WNT signaling pathway regula-tor,in nephroblastoma cells and tissues of children,the effect on the proliferation of nephroblastoma SK-NEP-1 cells,and to explore its mechanism. Methods The relative expression of DKK2 in nephroblastoma cells and tissues was analyzed by qRT-PCR and im-munoblotting assays. Overexpressing DKK2 SK-NEP-1 cells were set as the experimental group( DKK2 group);the blank control plasmid group was set as a control group( Vector group),transfected with pcDNA3. 1 ( +) -Flag-DKK2 plasmid( Experimental group)and pcDNA3. 1( +) -Flag-Vector plasmid(Control group). The over-expression of DKK2 was confirmed in SK-NEP-1 cells by RT-PCR and immunoblotting. CCK-8 and cell cloning assays were used to determine the effect of DKK2 on cell prolifera-tion;flow cell cycle and apoptosis assays were used to confirm the effect on cell proliferation in overexpressed DKK2 cells. The xen-graft formation assay in nude mice was to verify the effect of DKK2 on proliferation in overexpressed DKK2 cells;the mechanism of DKK2 in inhibitory proliferation was analyzed by qRT-PCR,Western blotting and immunohistochemistry. Results Compared with normal renal epithelial tissues,DKK2 mRNA was down-regulated in children with nephroblastoma,and the difference was statistically significant(P<0. 001). Compared with the control group,transfected DKK2 cell viability was significantly inhibited after treatment for 24,48 and 72 h( P<0. 05),cell clone formation in the experimental group was significantly inhibited(31. 11% ± 2. 14% ) ( P<0. 05),the cell cycle in the experimental group was significantly arrested at the G1 phase(P<0. 001),and the apoptosis rate in the experimental group was significantly increased(P<0. 001). Compared with the control group,the tumor weight and volume in nude mice were significantly low in the experimental mice which were injected DKK2 overexpression cells(P<0. 001). Active-β-cate-nin and downstream genes were significantly inhibited in over-expressed DKK2 SK-NEP-1 cells. Conclusion DKK2 is down-regulated in human cutaneous nephroblastoma and participates in the mechanism of nephroblastoma by antagonizing Wnt/β-catenin signaling pathway.
ABSTRACT
Objective To study the expression and the function of DKK2 and to explore its potential mechanisms in breast cancer.Methods The expression of DKK2 was detected by RT-PCR in normal breast tissues and breast cancer cells.we have transfected DKK2 into breast cancer cell lines MDA-MB-231 and MCF-7.The cells before transfection were used as control group and marked with Vector.The cells after transfection were used as experimental group and marked with DKK2.Furthermore by qRT-PCR and Western-blot,the expression of DKK2,Notch signaling pathway and related factors were analyzed.We also detected the function of DKK2 by cloning assay,Transwell assay and proliferation assay.Results No expression of DKK2 was found in breast cancer cell lines MDA-MB-231 or MCF-7,with relatively high expression in normal breast tissue.The number of apoptotic cells was 2.57±1.18 before transfectionin in cell line MDA-MB-231,and 49.53±8.27 after transfection.The difference was statistically significant between the two groups (P＜0.005).The relative colony formation rate of MDA-MB-231 cells and MCF7 cells after transfection accounted for 20.44％ and 15.21％,respectively.The difference was statistically significant by t test.The number of apoptosis cells in MB231/DKK2 group was 49.53± 8.27 and that in MB231 / Vector group was 2.57±1.18.The difference was statistically significant (P＜0.005).The number of migrated cells in MB231/DKK2 group was 112.0±8.1 and that in MB231/Vector group was 178.0±12.0.The difference was statistically significant (P＜0.005).The mRNA expression of Notch 1 in group MB231/Vector was recorded as 1.The mRNA expression of JAG1 in MB231/DKK2 group was 0.2891.The difference was also statistically significant (P＜0.005).Conclusions Restored expression of DKK2 in silenced breast cells suppresses breast cancer cell proliferation and migration through repressing Notch signaling.DKK2-Notch signaling pathway may be its potential molecular mechanism to function in breast cancer.DKK2 may be one of the target genes for early diagnosis and treatment of breast cancer.
ABSTRACT
Objective: To investigate the expression level of dickkopf 2 (DKK2) gene in human bladder cancer cell lines and tissues, and the effects of DKK2 overexpression on proliferation and migration of bladder cancer cells, and to explore the possible mechanism. Methods: The relative expression levels of DKK2 mRNA and protein in human bladder cancer cell lines and tissues were detected by RT-PCR, real-time fluorescent quantitative PCR and Western blotting, respectively. The bladder cancer T24 cells with low expression of endogenous DKK2 gene were transfected with DKK2 overexpression plasmids, the expression levels of DKK2 mRNA and protein were measured by RT-PCR, real-time flu orescent quantitative PCR and Western blotting, respectively. Then the proliferation, colony formation, migration, invasion and cell cycle were detected by CCK-8, plate clone formation assay, scratch wound healing assay, Transwell assay and FCM assay, respectively. Furthermore, the expressions of proliferation-and migration-related molecules in T24 cells after DKK2 overexpression were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: The expressions of DKK2 mRNA and protein were downregulated in human bladder cancer 5637, T24, SW780, J82, HT-1 1 97 and HT-1 376 cells as compared with the normal bladder SV-HUC-1 cells (all P < 0.01), and those in human bladder cancer tissues were also downregulated as compared with the normal bladder tissues and paired para-cancerous tissues (all P < 0.001). After transfection with DKK2 overexpression plasmids, the expression levels of DKK2 mRNA and protein were increased, the proliferation, colony formation, migration and invasion of bladder cancer T24 cells were inhibited (all P < 0.05), and the cell cycle was arrested in G0/G1 phase (P < 0.001). Furthermore, the mRNA and protein expressions of p21 and E-cadherin were significantly upregulated (both P < 0.001), and the expressions of cyclin D1, vimentin and N-cadherin were downregulated in T24 cells with DKK2 overexpression (all P < 0.001). Conclusion: DKK2 gene is low-expressed in human bladder cancer cells and tissues, and it may be a potential tumor suppressor gene involved in the progression of human bladder cancer.
ABSTRACT
OBJECTIVES: The inhibitory effect of Dickkopf (Dkk)-1 on osteoblastic differentiation through blocking Wnt signaling has been well studied. However, the role of other members of the subfamily of Dkks remains unclear. We have examined the role of different Dkks on osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. METHODS: Osteoblastic differentiation was induced by treatment of Wnt-3a with Dkks or vehicle in C3H10T1/2 cells and alkaline phosphatase (ALP) activity was measured. Serum deprivation induced apoptosis was performed with pre-treatment of Dkks or vehicle in MC3T3-E1 cells and methyl thiazolyl tetrazolium (MTT) assay was done. RESULTS: Dkk-2 at low concentrations (5 and 20 nM) and Dkk-3, -4 at any concentrations (5 to 100 nM) significantly increased Wnt-3a-induced ALP activity, whereas Dkk-2 at high concentration (100 nM) significantly reduced. Treatment of Dkk-2, -3 and -4 at high concentration (100 nM) showed significant decreases of Wnt/beta-catenin transcriptional activity, whereas no effects were seen at low concentration (20 nM). In parallel experiments, treatment of Dkk-1 showed robust dose dependent inhibition not only in ALP activity but also in Wnt/beta-catenin transcriptional activity. Dkk-2, -3 and -4 increased serum deprivation-induced apoptosis in MC3T3-E1 mouse osteoblasts, while Dkk-1 had no effect. CONCLUSIONS: We found that unlike Dkk-1, Dkk-3 and -4 stimulated early osteoblastic differentiation at various concentrations regardless of their inhibitory effects on Wnt/beta-catenin transcriptional activity at high concentration. Dkk-2 had a biphasic effect where the lower doses significantly increased ALP activity while the high dose was inhibitory. Dkk-2, -3 and -4 stimulated osteoblast apoptosis whereas Dkk-1 had no effect.