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1.
Theriogenology ; 161: 65-73, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33296745

RESUMO

This study aims to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in bovine cumulus cells in order to reveal by which mechanisms ASH1L regulates epigenetic modification and apoptosis in cumulus cells. The location of ASH1L and the methylation pattern of H3K36 were detected using immunofluorescence staining in cumulus cells. Quantitative PCR (qPCR) and western blotting were used to screen for effective siRNA targeting the ASH1L gene. Also, the mRNA expression levels of apoptosis-related genes and polycomb inhibitory complex genes were estimated by qPCR after knocking down the ASH1L gene in bovine cumulus cells. Cell proliferation and apoptosis were measured with the CCK-8 method and Annexin V-FITC by flow cytometry, respectively. The results of immunofluorescence analysis showed that ASH1L is located in the nucleus of bovine cumulus cells and is distributed in a dotted pattern. ASH1L knockdown in cumulus cells induced a decrease in the levels of H3K36me1/2/3 methylation (P < 0.05). Additionally, ASH1L knockdown inhibited cell proliferation, increased the apoptosis rate, and upregulated the expression of apoptosis genes CASPASE-3, BAX and BAX/BCL-2 ratio (P < 0.05). Meanwhile, the mRNA expression levels of EZH2 and SUZ12, two subunits of PRC2 protein, were increased in cells with ASH1L knockdown (P < 0.05). Therefore, the expression of ASH1L methyltransferase and its function in on the apoptosis of bovine cumulus cells were first studied. The mechanism by which ASH1L regulates the histone methylation and apoptosis in cumulus cells was also revealed.


Assuntos
Apoptose , Células do Cúmulo , Animais , Bovinos , Proliferação de Células , Células do Cúmulo/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Metilação
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 27(1): 33-5, 2011 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-21208561

RESUMO

AIM: To construct a cell line which stably expresses human chemokine receptor 6 (CCR6). METHODS: The human CCR6 cDNA and plasmid G were co-transfected into HEK 293 cells and the clones stably expressing CCR6 were picked out. The expression of CCR6 in HEK293 cells was detected by RT-PCR, Western blotting, immunofluorescence test, calcium mobilization and in vitro chemotaxis assay. RESULTS: The transfected HEK293 cells could stably express functional human CCR6. CONCLUSION: Successfully establish a cell line which stably express human CCR6 and lays the foundation for its biological function's study and specific antagonist screening.


Assuntos
Receptores CCR6/biossíntese , Receptores CCR6/genética , Western Blotting/métodos , Linhagem Celular Transformada , Clonagem Molecular , DNA Complementar/genética , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Plasmídeos/genética , Receptores CCR6/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
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