ABSTRACT
To explore the effect and mechanisms of demethylation drug zebularine on esophageal cancer cells apoptosis, ECA109 cells and KYSE170 cells were treated with zebularine at different concentrations (25, 50, 100, 200, and 400 μmol·L-1). The cell viability was measured by CCK-8. Flow cytometry was used to detect the cell apoptosis rate, Western blot was performed to determine the expression of apoptosis protein (Bcl-2, Bax, cleaved-caspase-3, and cleaved-PARP) and Wnt signal pathway molecules (β-catenin, cyclin D1, and c-Myc), real-time quantitative PCR was used to detect the expression level of negative regulatory genes of Wnt signaling pathway, methylation specific PCR (MSP) was used to detect the methylation status of secreted frizzled related protein 2 (SFRP2) and dickkopf 3 (Dkk3) genes. After knockdown of SFRP2 and Dkk3, the effect of zebularine on apoptosis was detected. The studies showed that zebularine could inhibit the activity of ECA109 and KYSE170 cells in a dose-dependent and time-dependent manner; zebularine could induce cell apoptosis, down-regulate the expression of Bcl-2 protein, up-regulate the expression of Bax, cleaved-caspase-3, and cleaved-PARP protein, and inhibit the expression of β-catenin, cyclin D1, and c-Myc protein (P < 0.05); the mRNA expression levels of Dkk3 and SFRP2 were significantly up-regulated by zebularine, while the methylation levels of SFRP2 and Dkk3 promoters were decreased; knockdown of SFRP2 and Dkk3 could reduce the apoptosis induced by zebularine. In summary, zebularine could reduce the methylation level of SFRP2 and Dkk3 gene promoter, promote the expression of SFRP2 and Dkk3 gene, and then induce the apoptosis of esophageal cancer cells by inhibiting Wnt/β-catenin signaling pathway.
ABSTRACT
Objective To explore the value of methylation of SDC2 and SFRP2 genes promoter in fecal DNA for colorectal cancer ( CRC) screening. Methods All stool samples were enrolled from Changhai Hospital of Naval Medical University, the Tenth People' s Hospital of Tongji University and the Seventh Medical Center of Chinese People's Liberation Army General Hospital. A total of 500 stool samples collected from March 2018 to December 2018 were allocated to CRC group ( 132 CRCs ) , adenoma group ( 38 advanced adenomas), healthy group (152 healthy individuals), interferential group (178 cases of benign colorectal disease or other non-colorectal tumors) and negative group (330 cases composed of healthy group and interferential group ) . The promoter methylation of fecal SDC2 and SFRP2 genes was detected by methylation-specific PCR (MSP) and compared with single gene methylation and the fecal immunochemical tests ( FIT) to evaluate its sensitivity and specificity. Results The stool sample analysis showed that the sensitivity of combined detection of SDC2 and SFRP2 in CRC group was 97. 73％ ( 129/132 ) , which was significantly higher than those of the single gene SDC2 test [ 70. 45％ ( 93/132) , P=0. 000] , single SFRP2 test [81. 82％ (108/132), P=0. 000] and FIT [69. 70％ (92/132), P=0. 000]. In adenoma group, the sensitivity of combined detection of SDC2 and SFRP2 was 57. 89％ (22/38), which was significantly higher than those of the single gene SDC2 test [ 15. 79％ ( 6/38 ) , P= 0. 000 ] and FIT [ 21. 05％ ( 8/38 ) , P=0. 021] , with no significant difference compared with that of SFRP2 test [ 47. 37％ ( 18/38) , P=0. 358] . In healthy group, the specificity of combined detection of SDC2 and SFRP2 was 98. 68％ (150/152), with no significant difference compared with those of single gene SDC2 test [ 100. 00％( 152/152) , P=0. 156] , single SFRP2 test [98. 68％ (150/152), P=1. 000] or FIT [95. 39％ (145/152), P=0. 091]. Specificities of combined detection of two genes in interferential and negative groups were 90. 45％ ( 161/178) and 94. 24％( 311/330) , which were significantly higher than 73. 03％( 130/178, P=0. 000) and 83. 33％( 275/330, P=0. 000) of FIT, respectively. Conclusion The combined detection test of methylation of SDC2 and SFRP2 is superior to single gene test, whose sensitivity of CRC and aggressive adenoma and specificity of distinguishing benign and malignant lesions are higher than FIT, which has potential application value.
ABSTRACT
Objective:SFRP2 gene is a member of the SFRPs family.The gene is located on chromosome 4q31.3 with 3 exons and 2 introns and first exons have higher density near the island of CpG.Many studies showed that the methylation level of SFRP2 gene and colon cancer,esophageal cancer,gastric cancer and other tumor occurrence relating to,development and prog nosis.This study aims to study the clinical characteristics of CpG SFRP2 promoter island hypermethylation in colorectal cancer and whether there is a certain correlation.Methods:by matrix assisted laser desorption ionization time of flight mass spectrometry for detecting specific CpG island methylation.Methylation status of SFRP2 promoter by Sequenom EpiTYPER was detected in 20 cases of normal tissue of colorectal cancer and tumor tissues.Results:Our study using multiple linear regression analysis in tumor tissue of SFRP2 methylation at promoter Ⅰ and Ⅱ,found that SFRP2 promoter methylation and clinical features of.SFRP2_01_CpG_5 significantly correlated (P=0.018),SFRP2_02_CpG_5 (P=0.018) associated with the location of the tumor,SFRP2_02_CpG_6,7,8,9(P=0.039) and the number of lymph node metastasis of.SFRP2_01_CpG_1.2(P=0.043),SFRP2_02_CpG_16 (P=0.044) correlated with tumor size.Conclusion:we from epigenetic aspects of its promoter CpG methylation level and colorectal cancer clinical and pathological features,found a correlation between clinical and pathological features of colorectal cancer and CpG methylation in its promoter,suggesting that the SFRP2 promoter may be at this stage of colorectal cancer and future biological genetics the progress of the potential surface markers.
ABSTRACT
Objective To understand the expression change of SFRP2 in human cervical cancer tissue and to investigate the effect of SFRP2 on cervical cancer cell proliferation.Methods The expression of SFRP2 in cervical cancer tissue was detected by using Western blot and qRT-PCR;the SFRP overexpressed human cervical cancer line was constructed by using lentivirus,the effect of SFRP2 on the proliferation of human cervical cancer cell line was analyzed by CCK-8 and plate cloning.The effect of SFRP2 on the expression of WNT pathway related proteins and genes in human cervical cancer cell was detected by Western Bolt and qRTPCR.Results Compared with paracancerous tissue,SFRP2 was lowly expressed in human cervical cancer tissue;overexpressed SFRP2 cervical cancer cell proliferation was inhibited;SFRP2 inhibiting cellular proliferation was occurred via WNT signal pathway.Conclusion The role of SFRP2 as a candidate gene for cervical cancer remains to be deeply studied.
ABSTRACT
Aim To investigate the alteration of Wnt/β-catenin signaling and sirtuins 1 in type 2 diabetic rats’ aorta and clarify its role in the development of di-abetes aortic disease. Methods The type 2 diabetes rat model was established by injection of streptozocin after five-week of high fat diet. The rats were randomly divided into control group, DM model group of 2 weeks, 4 weeks, 8 weeks and 12 weeks. Fasting blood glucose ( FBG ) , total cholesterol ( TC ) , triglyceride ( TG) , high density lipoprotein-cholesterol( HDL-C) , low density lipoprotein- cholesterol ( LDL-C ) and fast-ing insulin( FINS) levels were tested. HE staining was used to observe the pathological changes of aortal struc-tures. The alteration of Wnt2, β-catenin, TCF4, SIRT1 and sFRP2 in aortawas determined by Western blot and RT-PCR. Results Compared with control group, TC, TG, LDL-C levels of type 2 diabetic rats were significantly increased, HDL-C levels were signif-icantly reduced( P0. 05). But the expression of TCF4 and SIRT1 was enhanced continuously in DM compared with control group while sFRP2 decreased in the duration of DM development. Conclusions Wnt/β-catenin signa-ling pathway was activated in diabetic aortal injury by regulation of SIRT1 via sFRP2 . Further researches on its mechanism of actionin DM aorta injury may find a new therapeutic target for the disease.
ABSTRACT
BACKGROUND: Hair follicles undergo cycles of repeated growth and regression. The Wnt pathway plays an important role in the regeneration and differentiation of hair follicles. Sfrp2, a Wnt inhibitor, is involved in the developmental and disease processes of various cells and tissues by modulating the Wnt pathway. OBJECTIVE: The aim of this study was to understand the role of Sfrp2 in hair follicles through investigation of the Sfrp2 expression pattern in the skin and its effect on keratinocytes. METHODS: We investigated Sfrp2 mRNA expression and the expression of the wnt target genes, Ccnd1 and C-myc, at various mouse hair follicle developmental stages using Real-time polymerase chain reaction. We also investigated the effect of SFRP2 on the proliferation and differentiation of mouse keratinocyte cells by adding SFRP2 protein or overexpressing Sfrp2 using an in vitro culture system. RESULTS: Sfrp2 expression peaked in the catagen phase and remained high until telogen, and then declined at the beginning of the next anagen. An inverse relationship to Sfrp2 expression was found for the expression of the Wnt target genes, C-myc and Ccnd1. In addition, we also observed inhibited proliferation of mouse keratinocytes in the presence of SFRP2. CONCLUSION: These results suggest that Sfrp2 may play a role in the catagen phase by inhibiting the proliferation of keratinocyte and functioning as a Wnt inhibitor in keratinocytes.
Subject(s)
Animals , Mice , Genes, myc , Hair Follicle , Hair , Keratinocytes , Real-Time Polymerase Chain Reaction , Regeneration , RNA, Messenger , Skin , Wnt Signaling PathwayABSTRACT
Objective To investigate the promoter methylation status of SFRP1 and SFRP2 gene in gastric cardia adenocarcinoma (GCA). Methods Methylation specific PCR (MSP) method was used to examine the methylation status of the 5' CpG island of SFRP1 and SFRP2 gene in tumors and corresponding normal tissues. Results Methylation frequencies of SFRP1 and SFRP2 gene in tumor specimens were 87.2 % (82/94) and 83 %(78/94), which was significantly higher than that in corresponding normal tissues (14.9 % and 55.3 %, respectively) (P <0.001). Methylation frequencies of SFRP1 in lymph node metastasis group (96.4 %) was significantly higher than that in no lymph node metastasis group (73.7 %). Methylation frequencies of SFRP1 and SFRP2 gene in poor differentiation group were all higher than that in moderate and poor-moderate differentiation groups, but both of them did not show significant difference(P >0.05). 63 cases of GCA showed both of SFRP1 and SFRP2 gene simultaneous methylation, which including 36 cases of lymph node metastasis group, 27 cases of no lymph node metastasis group. Simultaneous methylation frequencies of SFRP1 and SFRP2 gene in lymph node metastasis group was higher than that in no lymph node metastasis group, poor differentiation group was higher than that in moderate and poor-moderate differentiation groups, but both of them did not show significant difference (P >0.05). Conclusion Promoter methylation of SFRP1 and SFRP2 might be related with oncogenesis of GCA and hypermethylation of SFRP1 gene might be related with the malignant behavior of GCA.