AIM:To explore the relationship between DNA methyltransferase 1 (DNMT1) and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) and its biological significance in primary HCC.METHODS: We carried out an immu...AIM:To explore the relationship between DNA methyltransferase 1 (DNMT1) and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) and its biological significance in primary HCC.METHODS: We carried out an immunohistochemical examination of DNMT1 in both HCC and paired non-neoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC cell line SMMC-7721.RESULTS: DNMT1 protein expression was increased in HCCs compared to histologically normal non-neoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated signifi cantly with poor tumor differentiation (P=0.014). There were more cases with DNMT1 overexpression in HCC with HBV (42.85%) than in HCC without HBV (28.57%). However, no signif icant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen (PCNA), even in HCC cell lines where DNMT1 was stably decreased.CONCLUSION: The f indings implied that DNMT1 plays a key role in HBV-related hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.展开更多
Objective: To better understand the contribution of dysregulated DNA methyltransferase 1(DNMT1) expression to the progression and biology of clear cell renal cell carcinoma(ccRCC).Methods: We examined the differences ...Objective: To better understand the contribution of dysregulated DNA methyltransferase 1(DNMT1) expression to the progression and biology of clear cell renal cell carcinoma(ccRCC).Methods: We examined the differences in the expression of DNMT1 in 89 ccRCC and 22 normal tissue samples by immunohistochemistry. In addition, changes in cell viability, apoptosis, colony formation and invading ability of ccRCC cell lines(786-0 and Caki-1) were assessed after transfection with DNMT1 siRNA.Results: We found DNMT1 protein was significantly higher expressed in ccRCC than that of in no-tumor tissues(56.2% and 27.3%, respectively, P=0.018). The expression of DNMT1 was strongly associated with ccRCC tumor size, tumor pathology stage, histological grading, lymph node metastasis, vascular invasion, recurrence and prognosis. Moreover, knockdown of DNMT1 expression significantly inhibited ccRCC cell viability, induced apoptosis, decreased colony formation and invading ability.Conclusions: Expression of DNMT1 protein is increased in ccRCC tissues, and DNMT1 expression is associated with poor prognosis of patients. Experiments in vitro further showed DNMT1 played an essential role in proliferation and invasion of renal cancer cells. Moreover, targeting this enzyme could be a promising strategy for treating ccRCC, as evidenced by inhibited cell viability, increased apoptosis, decreased colony formation and invading ability.展开更多
This study was designed to clone cDNA of goat DNA methyltransferase 1(DNMT1) gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat...This study was designed to clone cDNA of goat DNA methyltransferase 1(DNMT1) gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer(NT) embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization(IVF) embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned(GenBank no.FJ617538).Furthermore,an effective interfering shRNA(pRNAD2) was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls(P < 0.05),reaching the similar rates to IVF embryos(P > 0.05).In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.展开更多
DNA methyltransferase 1(DNMT1)is a useful biomarker for lung cancer in early clinical diagnosis.A rapid magnetic chemiluminescence immunoassay(MCLIA)for DNMT1 in human serum has been developed.Horseradish peroxidase(H...DNA methyltransferase 1(DNMT1)is a useful biomarker for lung cancer in early clinical diagnosis.A rapid magnetic chemiluminescence immunoassay(MCLIA)for DNMT1 in human serum has been developed.Horseradish peroxidase(HRP)-second-Ab was used to labeled polyclonal antibodies of anti-DNMT1.DNMT1 in sample integrates with specific immunomagnetic beads and can constitute a supersandwiched immunoreaction.In magnetic field,nonspecific materials can be separated.After luminescent substrate luminol-H2O2-BIP was added,the relative light unit(RLU)of HRP was detected and was discovered to be directly proportional to the content of DNMT1 in sample.The correlative variables involved in the MCLIA value were optimized and the methodological evaluation was carried out.After optimization,in the range of0.5–128 ng/mL,the linear regression equation was y=0.5014 x+1.769(x was logCDNMT1,y was relative luminescence units(RLU)/RLU0),and the limit of detection was 0.01 ng/mL.The RSD of intra-and interassays were 15.8%–16.9%and 14.3%–18.1%,respectively.The recovery was from 70.0%to 106.2%.Furthermore,paralleled with purchasable enzyme-linked immunosorbent assay(ELISA)kits,MCLEIA had lower detection limit,wider linear range and shorter detection time.Therefore,the MCLEIA established in this study could be used for the sensitive detection of DNMT1 in serum sample.展开更多
基金Supported by National Natural Science Foundation of China,No.30470950
文摘AIM:To explore the relationship between DNA methyltransferase 1 (DNMT1) and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) and its biological significance in primary HCC.METHODS: We carried out an immunohistochemical examination of DNMT1 in both HCC and paired non-neoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC cell line SMMC-7721.RESULTS: DNMT1 protein expression was increased in HCCs compared to histologically normal non-neoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated signifi cantly with poor tumor differentiation (P=0.014). There were more cases with DNMT1 overexpression in HCC with HBV (42.85%) than in HCC without HBV (28.57%). However, no signif icant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen (PCNA), even in HCC cell lines where DNMT1 was stably decreased.CONCLUSION: The f indings implied that DNMT1 plays a key role in HBV-related hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.
基金supported by grants from National Natural Science Foundation of China (No. 30873097)
文摘Objective: To better understand the contribution of dysregulated DNA methyltransferase 1(DNMT1) expression to the progression and biology of clear cell renal cell carcinoma(ccRCC).Methods: We examined the differences in the expression of DNMT1 in 89 ccRCC and 22 normal tissue samples by immunohistochemistry. In addition, changes in cell viability, apoptosis, colony formation and invading ability of ccRCC cell lines(786-0 and Caki-1) were assessed after transfection with DNMT1 siRNA.Results: We found DNMT1 protein was significantly higher expressed in ccRCC than that of in no-tumor tissues(56.2% and 27.3%, respectively, P=0.018). The expression of DNMT1 was strongly associated with ccRCC tumor size, tumor pathology stage, histological grading, lymph node metastasis, vascular invasion, recurrence and prognosis. Moreover, knockdown of DNMT1 expression significantly inhibited ccRCC cell viability, induced apoptosis, decreased colony formation and invading ability.Conclusions: Expression of DNMT1 protein is increased in ccRCC tissues, and DNMT1 expression is associated with poor prognosis of patients. Experiments in vitro further showed DNMT1 played an essential role in proliferation and invasion of renal cancer cells. Moreover, targeting this enzyme could be a promising strategy for treating ccRCC, as evidenced by inhibited cell viability, increased apoptosis, decreased colony formation and invading ability.
基金funded by the Key Scientific and Technological Special Program of China (2008ZX08007004)
文摘This study was designed to clone cDNA of goat DNA methyltransferase 1(DNMT1) gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer(NT) embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization(IVF) embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned(GenBank no.FJ617538).Furthermore,an effective interfering shRNA(pRNAD2) was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls(P < 0.05),reaching the similar rates to IVF embryos(P > 0.05).In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.
基金supported by the National Natural Science Foundation of China(Nos.81402721,81573203,21605131)Science and Technology Department of Henan Province(No.22170004)
文摘DNA methyltransferase 1(DNMT1)is a useful biomarker for lung cancer in early clinical diagnosis.A rapid magnetic chemiluminescence immunoassay(MCLIA)for DNMT1 in human serum has been developed.Horseradish peroxidase(HRP)-second-Ab was used to labeled polyclonal antibodies of anti-DNMT1.DNMT1 in sample integrates with specific immunomagnetic beads and can constitute a supersandwiched immunoreaction.In magnetic field,nonspecific materials can be separated.After luminescent substrate luminol-H2O2-BIP was added,the relative light unit(RLU)of HRP was detected and was discovered to be directly proportional to the content of DNMT1 in sample.The correlative variables involved in the MCLIA value were optimized and the methodological evaluation was carried out.After optimization,in the range of0.5–128 ng/mL,the linear regression equation was y=0.5014 x+1.769(x was logCDNMT1,y was relative luminescence units(RLU)/RLU0),and the limit of detection was 0.01 ng/mL.The RSD of intra-and interassays were 15.8%–16.9%and 14.3%–18.1%,respectively.The recovery was from 70.0%to 106.2%.Furthermore,paralleled with purchasable enzyme-linked immunosorbent assay(ELISA)kits,MCLEIA had lower detection limit,wider linear range and shorter detection time.Therefore,the MCLEIA established in this study could be used for the sensitive detection of DNMT1 in serum sample.
