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Objective:To investigate the role of SUMO E3 ligase ZNF451 in DNA damage repair and explore the underlying mechanism in non-small cell lung cancer A549 cells and cervical cancer HeLa cells.Methods:A549 cells and HeLa cells were irradiated with γ-ray irradiation or treated with etoposide. Cell proliferation viability was detected by the cell counting kit-8 assay. Protein expression was detected by Western blot assay. DNA damage repair level was detected by DR-GFP plasmid system, and the spatial positioning was detected by immunofluorescence.Results:Etoposide decreased the expression level of ZNF451 in a dose- and time- dependent manner. After treatment with 30, 50, 80 μmol/L etoposide, the cell viability were reduced after the knockdown of ZNF451 in A549 and HeLa cells(A549: t = 27.62, 25.61, 5.32, P<0.01; HeLa: t = 30.77, 21.28, 4.18, P<0.01). Furthermore, ZNF451 was recruited at DNA damage sites. A co-localization and endogenous interaction were found between ZNF451 and γ-H2AX after the treatment of irradiation or etoposide. Moreover, the expression level of γ-H2AX was significantly increased after treatment with 30, 50, 80 μmol/L etoposide(A549: t = 6.12, 10.67, 4.68, P<0.01; HeLa: t = 7.94, 9.81, 15.12, P<0.01)and the repair efficiency of NHEJ was reduced in ZNF451 knockdown cells( t = 18.60, P<0.05). Finally, the immunofluorescence assay showed that ZNF451 was co-localizated with 53BP1 and MDC1 after irradiation or etoposide treatment. Conclusions:Knockdown of ZNF451 inhibits cell proliferation and increases the level of DNA damage in A549 and HeLa cells. ZNF451 was recruited to DNA damage sites after DSBs and participated in NHEJ repair by co-localizing with DNA damage repair factor 53BP1/MDC1.
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We report the first known ethnic Malay patient with laminin alpha-2 (merosin) deficiency (MDC1A), a subtype of congenital muscular dystrophy (CMD)as a result of novel LAMA2 gene mutations. The 21-month-old female presented with hypotonia at birth and gross motor delay of her distal lower limbs. Physical examination showed generalised hypotonia, hyporeflexia and myopathic facies but good cognitive functions. Serum creatine kinase was elevated and white matter changes were detected in the brain MRI. Muscle biopsy showed dystrophic changes with complete laminin α2 deficiency by immunohistochemistry. Mutation analysis of LAMA2 showed compound heterozygote at exon 21, c.2888delG(p.Gly963Alafs*111) and exon 34, c.4886dupC(p.Pro1629Profs*40) leading to premature stop codon for each of the frameshift mutations. Patient review at seven years of age showed satisfactory cognitive functions despite having contractures and weakness. Genetic testing of LAMA2 related muscular dystrophy facilitated the earlier diagnosis of MDC1A and genetic counselling for this family. MDC1A
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Objective To investigate the effects of inhibition of MDC1 protein expression on xenografted tumors in nude mice,and to observe the histopathological and cellular changes in nude mice.Methods Three pairs of effective and control short hairpin RNA targeting MDC1 mRNA were designed and cloned into the pSIH1-H1-copGFP vector.Real-time PCR and Western blot were used to determine the mRNA and protein expression of MDC1.After selection by copGFP reporter gene,cells were divided into negative transfection group (ECA109-N) and MDC1 transfection group (ECA109-M).The transfected cells were injected into nude mice.The mice were divided into ECA109 group,ECA109-N group,and ECA109-M group.Each group was divided into irradiation subgroup and non-irradiation subgroup.The changes in tumor size after irradiation were evaluated in each group.Western blot was used to measure the expression of CHK1,CHK2,and CHK2T68 in xenografted tumors.Flow cytometry was used to analyze the cell cycle distribution and apoptosis of tumor cells in nude mice.The variance analysis was used to compare the mean of multiple groups,and the SNK-q test was used in the two two groups.Results The pMDC1-shRNA plasmid was successfully constructed and used to transfect ECA109 cells.ECA109-M cells were obtained by stable transfection with the recombinant plasmid.All inoculated nude mice survived with visible xenografted tumors at the underside of the paw in about one week.There was no swelling and wound in inoculation sites.There was no significant difference in tumor size between different groups (P＞0.05).The tumor growth in the ECA109 group and the ECA109-N group significantly slowed down after irradiation with a dose of 15 Gy (P＜0.05).Compared with the other two groups,the ECA109-M group had a significant smaller tumor size,significantly slower relative tumor growth,and significantly higher growth inhibition (all P＜0.05).The q value of the ECA109-M group was 1.36.In the ECA109-M group,there were no significant changes in the protein expression of CHK1 and CHK2 after irradiation (P＞ 0.05);however,the phosphorylation of CHK2T68 protein was significantly reduced after irradiation (P＜0.05).There were no significant differences in cell cycle distribution or the proportion of apoptotic cells in tumor tissue between the three groups (P＞0.05).Conclusions Inhibition of MDC1 protein expression by RNA interference can effectively inhibit the growth of xenografted tumors after irradiation in the nude mice by increasing their radiosensitivity.
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Objective To apply RNA interference technique for reducing the expression of MDC1 gene in esophageal carcinoma cell line ECA109, observe the changes in cell cycle and radiosensitivity after radiation, and discuss related mechanisms. Methods Three pairs of effective interference sequences and negative control sequences were synthesized for MDC1 mRNA sequence, and a recombinant plasmid was constructed with the vector pSIH1?H1?copGFP. RT?PCR and Western blot were used to determine the expression levels of MDC1 mRNA and protein. Colony?forming assay was applied to measure radiosensitivity, flow cytometry to determine cell cycle, Western blot to determine the expression of CHK1 and CHK2 proteins, and laser scanning confocal microscope to observe the number of MDC1 blotches inside the nucleus. One?way analysis of variance was used to analyze the differences between groups. Results The pSIH1?H1?copGFP plasmid was constructed successfully and ECA109 cells were infected to obtain ECA109M cells with stable transfection. The expression levels of MDC1 mRNA and protein in ECA109M cells were lower than those in ECA109N and ECA109 cells ( P= 0. 032 and 0. 041, respectively ) . After 5?Gy radiation, ECA109M cells had a lower proportion of G2+M cells than ECA109N and ECA109 cells ( P=0. 026) . After 5?Gy radiation, ECA109, ECA109N, and ECA109M cells had similar expression levels of CHK1 and CHK2 proteins ( P= 0. 345 and 0. 451, respectively ) , and ECA109M cells had a lower expression level of CHK2 T68 protein than ECA109 and ECA109N cells ( P=0. 012) . ECA109 cells had a D0 value of 3. 06 Gy and an SF2 value of 0. 91;the D0 values for ECA109N and ECA109M cells were 2. 90 Gy and 1. 88 Gy, respectively, and the SF2 values for them were 0. 89 and 0. 84, respectively ( P=0. 021 and 0. 037, respectively ) . Conclusions RNA interference can reduce the expression levels of MDC1 protein and cell cycle?related proteins, release cell cycle arrest, and enhance radiosensitivity in esophageal carcinoma ECA109 cells.
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AIM:To discover the effect of MCPH1 on the DNA damage induced by ionizing radiation in esoph-ageal cancer cells.METHODS:ECA109 cancer cells were radiated at dose of 8 Gy.The nuclear foci of relevant factors were detected 1 h after irradiation in the ECA109 cells after silence of MDC1 gene.A cell line was established that was sta-ble low expression of MCPH1.The nuclear foci induced by ionizing radiation after silence of MCPH1 were determined.RE-SULTS:The MCPH1 gene silenced ECA109 cell line was successfully constructed.A strong relationship between MDC1, MCPH1 andγ-H2AX was observed 1 h after 8 Gy irradiation.Silence of MDC1 did not affect the nuclear foci formation ofγ-H2AX and MCPH1.The nuclear foci of MDC1 but notγ-H2AX significantly reduced after silencing of MCPH1.CON-CLUSION:MCPH1 is located in the downstream of H2AX and upstream formation of MDC1, and regulates the nuclear fo-ci formation of MDC1 during DNA damage response.
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The congenital muscular dystrophies (CMDs) are a group of genetically and clinically heterogeneous hereditary myopathies with preferentially autosomal recessive inheritance, that are characterized by congenital hypotonia, delayed motor development and early onset of progressive muscle weakness associated with dystrophic pattern on muscle biopsy. The clinical course is broadly variable and can comprise the involvement of the brain and eyes. From 1994, a great development in the knowledge of the molecular basis has occurred and the classification of CMDs has to be continuously up dated. In the last number of this journal, we presented the main clinical and diagnostic data concerning the different subtypes of CMD. In this second part of the review, we analyse the main reports from the literature concerning the pathogenesis and the therapeutic perspectives of the most common subtypes of CMD: MDC1A with merosin deficiency, collagen VI related CMDs (Ullrich and Bethlem), CMDs with abnormal glycosylation of alpha-dystroglycan (Fukuyama CMD, Muscle-eye-brain disease, Walker Warburg syndrome, MDC1C, MDC1D), and rigid spine syndrome, another much rare subtype of CMDs not related with the dystrophin/glycoproteins/extracellular matrix complex.
As distrofias musculares congênitas (DMCs) são miopatias hereditárias geralmente, porém não exclusivamente, de herança autossômica recessiva, que apresentam grande heterogeneidade genética e clínica. São caracterizadas por hipotonia muscular congênita, atraso do desenvolvimento motor e fraqueza muscular de início precoce associada a padrão distrófico na biópsia muscular. O quadro clínico, de gravidade variável, pode também incluir anormalidades oculares e do sistema nervoso central. A partir de 1994, os conhecimentos sobre genética e biologia molecular das DMCs progrediram rapidamente, sendo a classificação continuamente atualizada. Os aspectos clínicos e diagnósticos dos principais subtipos de DMC foram apresentados no número anterior deste periódico, como primeira parte desta revisão. Nesta segunda parte apresentaremos os principais mecanismos patogênicos e as perspectivas terapêuticas dos subtipos mais comuns de DMC: DMC tipo 1A com deficiência de merosina, DMCs relacionadas com alterações do colágeno VI (Ullrich e Bethlem), e DMCs com anormalidades de glicosilação da alfa-distroglicana (DMC Fukuyama, DMC "Muscle-eye-brain" ou MEB, síndrome de Walker Warburg, DMC tipo 1C, DMC tipo 1D). A DMC com espinha rígida, mais rara e não relacionada com alterações do complexo distrofina-glicoproteínas associadas-matriz extracelular também será abordada quanto aos mesmos aspectos patogênicos e terapêuticos.
Sujets)
Humains , Dystrophies musculaires/congénital , Dystrophies musculaires/thérapieRésumé
The congenital muscular dystrophies (CMDs) are a group of genetically and clinically heterogeneous hereditary myopathies with preferentially autosomal recessive inheritance, that are characterized by congenital hypotonia, delayed motor development and early onset of progressive muscle weakness associated with dystrophic pattern on muscle biopsy. The clinical course is broadly variable and can comprise the involvement of the brain and eyes. From 1994, a great development in the knowledge of the molecular basis has occurred and the classification of CMDs has to be continuously up dated. We initially present the main clinical and diagnostic data concerning the CMDs related to changes in the complex dystrophin-associated glycoproteins-extracellular matrix: CMD with merosin deficiency (CMD1A), collagen VI related CMDs (Ullrich CMD and Bethlem myopathy), CMDs with abnormal glycosylation of alpha-dystroglycan (Fukuyama CMD, Muscle-eye-brain disease, Walker-Warburg syndrome, CMD1C, CMD1D), and the much rarer CMD with integrin deficiency. Finally, we present other forms of CMDs not related with the dystrophin/glycoproteins/extracellular matrix complex (rigid spine syndrome, CMD1B, CMD with lamin A/C deficiency), and some apparently specific clinical forms not yet associated with a known molecular mechanism. The second part of this review concerning the pathogenesis and therapeutic perspectives of the different subtypes of CMD will be described in a next number.
As distrofias musculares congênitas (DMCs) são miopatias hereditárias geralmente, porém não exclusivamente, de herança autossômica recessiva, que apresentam grande heterogeneidade genética e clínica. São caracterizadas por hipotonia muscular congênita, atraso do desenvolvimento motor e fraqueza muscular de início precoce associada a padrão distrófico na biópsia muscular. O quadro clínico, de gravidade variável, pode também incluir anormalidades oculares e do sistema nervoso central. A partir de 1994, os conhecimentos sobre genética e biologia molecular das DMCs progrediram rapidamente, sendo a classificação continuamente atualizada. Nesta revisão apresentaremos os principais aspectos clínicos e diagnósticos dos subtipos mais comuns de DMC associados com alterações do complexo distrofina-glicoproteínas associadas-matriz extracelular que são DMC com deficiência de merosina (DMC tipo 1A), DMCs relacionadas com alterações do colágeno VI (DMC tipo Ullrich e miopatia de Bethlem), DMCs com anormalidades de gliocosilação da alfa-distroglicana (DMC Fukuyama, DMC "Muscle-eye-brain" ou MEB, síndrome de Walker-Warburg, DMC tipo 1C, DMC tipo 1D), além da raríssima DMC com deficiência de integrina. Outras formas mais raras de DMC, não relacionadas com o complexo distrofina-glicoproteínas associadas-matriz extracelular também serão apresentadas (DMC com espinha rígida, DMC tipo 1B, DMC com deficiência de lamina A/C) e, finalmente, algumas formas clínicas com fenótipo aparentemente específico que ainda não estão associadas com um defeito molecular definido. A patogenia e as perspectivas terapêuticas dos principais subtipos de DMC serão apresentados em um próximo número, na segunda parte desta revisão.