cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

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.

Aberrant DNA methyltransferase 1 expression in clear cell renal cell carcinoma development and progression

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 ecRCC 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 DNMTI 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.

Suppressing Effects of Down-regulating DNMT1 and DNMT3b Expression on the Growth of Human Cholangiocarcinoma Cell Line

Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members （DNMT1 and DNMT3b） in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.

Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1

Objective:To explore whether casticin(CAS)suppresses stemness in cancer stem-like cells(CSLCs)obtained from human cervical cancer(CCSLCs)and the underlying mechanism.Methods:Spheres from He La and Ca Ski cells were used as CCSLCs.DNA methyltransferase 1(DNMT1)activity and m RNA levels,self-renewal capability(Nanog and Sox2),and cancer stem cell markers(CD133 and CD44)were detected by a colorimetric DNMT activity/inhibition assay kit,quantitative real-time reverse transcription-polymerase chain reaction,sphere and colony formation assays,and immunoblot,respectively.Knockdown and overexpression of DNMT1 by transfection with sh RNA and c DNA,respectively,were performed to explore the mechanism for action of CAS(0,10,30,and 100 nmol/L).Results:DNMT1 activity was increased in CCSLCs compared with He La and Ca Ski cells(P<0.05).In addition,He La-derived CCSLCs transfected with DNMT1 sh RNA showed reduced sphere and colony formation abilities,and lower CD133,CD44,Nanog and Sox2 protein expressions(P<0.05).Conversely,overexpression of DNMT1 in He La cells exhibited the oppositive effects.Furthermore,CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in He La-derived CCSLCs(P<0.05).Interestingly,DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness.As expected,DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in He La cells.Conclusion:CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation,suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.

Epigenetic marks are modulated by gender and time of the day in the hippocampi of adolescent rats:a preliminary study

Although the involvement of gender in epigenetic machinery in peripheral tissues during the neonatal period has been suggested, the gender-related epigenetic profile of brain areas during the adolescent period is rarely exploited. Furthermore, the influence of time of day on hippocampal acetylation marks has been demonstrated in young adult and aged rats; however, there are no studies reporting epigenetic changes in the adolescent period. Therefore, this study aimed to investigate the effects of gender on hippocampal DNA methyltransferase 1 content and histone deacetylase（HDAC） activity of adolescent rats at different time points, specifically early morning and afternoon. Both epigenetic markers increased significantly in the hippocampi of female rats compared to the male group, an indicator of reduced transcriptional activity. In addition, HDAC activity during the early morning was higher compared to afternoon groups in both male and female rats, while DNA methyltransferase 1 content was not altered by the time of day. Our findings demonstrate that hippocampal DNA methylation and histone acetylation status can be influenced by gender during the adolescent period, while the time of the day impacts HDAC activity.

Effect on proliferation and apoptosis of T24 cell lines via silencing DNMT1 with RNA interference

Expression of DNA methyltransferase 1(DNMT1),which plays an important role on aberrantly methylated CpG in the promoter regions of tumor sup-pressor genes(TSGs),is higher in bladder cancer cells than in normal bladder cells.Therefore,its overexpression is closely related to tumor formation.In this study,the eukaryotic vector pshRNA-DNMT1 was constructed and transfected into T24 cells.Levels of DNMT1 mRNA and protein were detected by reverse transcription-polymerase chain reaction(RT-PCR)and western blot.Relative to the blank control at the 24th,48th and 72nd hour after transfection of pshRNA-DNMT1,the inhibitory rates of DNMT1 mRNA levels in T24 cells were 28.44%,52.48%,70.91%,respectively.Those of DNMT1 proteins were 24.27%,57.79%,and 77.74%,respectively.Proliferation and apoptosis were assayed by MTT and flow cytometry with Annexin-V-FITC/PI staining.The growth inhibition rates of pshRNA-DNMT1 at the 24th,48th and 72nd hour after transfection of pshRNA-DNMT1 were(4.34¡0.76)%,(9.87¡1.54)%and(13.78¡1.93)%,respectively.There were statistically significant differ-ences between pshRNA-DNMT1 and the control blank at each time points(P,0.01);24,48 and 72 hours after T24 cells were transfected by pshRNA-DNMT1,the apoptosis rates of pshRNA-DNMT1 were(3.87¡0.81)%,(8.69¡1.23)%and(11.46¡1.24)%,respectively(P,0.01 vs blank control).Based on this case,our conclu-sion is that the recombinant plasmid pshRNA-DNMT1 can silence the expression of gene DNMT1 mRNA and protein effectively,and to some extent,it also can inhibit the proliferation of bladder cancer cell and promote the cellular apoptosis.

Protein phosphatase 6 (Pp6) is crucial for regulatory T cell function and stability in autoimmunity

Regulatory T(T_(reg))cells constitute a dynamic population that is critical in autoimmunity.T_(reg) cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities.However,recent studies demonstrated that certain inflammatory conditions induce T_(reg) cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells.Therefore,the identification of novel targets crucial to both T_(reg) cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity.In this study,we found that conditional Pp6 knockout(cKO)in T_(reg) cells led to spontaneous autoinflammation,immune cell activation,and diminished levels of FoxP3 in CD4^(+)T cells in mice.Loss of Pp6 in T_(reg) cells exacerbated two classical mouse models of T_(reg)-related autoinflammation.Mechanistically,Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308,leading to impaired FoxP3 expression in T_(reg) cells.In summary,our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling,thus maintaining T_(reg) cell stability and preventing autoimmune diseases.

Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter

The retinoblastoma gene product(pRb)is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation.We found that twelve weeks after transfection of the exogenous active(ΔB/X andΔр34)or inactive(ΔS/N)forms of RB into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous RB,despite being G-418 resistant.However,the consequences of the transient production of exogenous RB had different effects on the cell fate.TheΔB/X andΔр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation(AD).On the contrary,theΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2.Additionally,the PPARγ2 promoter in undifferentiatedΔS/N cells was hypermethylated,but all except−60 position CpG became mostly demethylated after cells exposure to AD.We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis,production of active exogenous RBs results in an AD-promoting epigenetic state.These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.