gga-miR-140-3p抑制马立克病病毒转化细胞MSB1增殖、迁移和侵袭

以马立克病病毒(MDV)感染SPF白来航鸡,收集脾和肝来源的淋巴瘤,利用实时荧光定量PCR(qRTPCR)进行检测,结果显示gga-miR-140-3p在感染MDV的肿瘤化脾和肝淋巴瘤中低表达。为揭示该miRNA在马立克病(MD)肿瘤转化过程中的作用,以该miRNA为研究对象,MDV转化细胞系MDCC-MSB1为试验材料,分别转染miRNA激动剂或阴性对照,检测细胞增殖和迁移,并检测与细胞侵袭密切相关的基因MMP2和MMP9mRNA表达情况。结果显示,与阴性对照组相比,转染gga-miR-140-3p激动剂后,细胞增殖在24、36和48h后发生显著(P<0.05)抑制,在60和72h后发生极显著(P<0.01)抑制。转染激动剂后,细胞迁移数也显著(P<0.05)减少。转染激动剂后,MMP2在转染后24h转录显著下调(P<0.05),转染后48和72h转录极显著(P<0.01)下调,MMP9基因在转染后48h转录显著(P<0.05)下调。gga-miR-140-3p作为在MDV感染组织中异常表达的miRNA,能够影响MD肿瘤淋巴细胞的特征——增殖、迁移和侵袭,提示该miRNA可能参与MD肿瘤转化过程。

Dosage Effects of Fadrozole on Growth and Development of Sex-Reversed Genetic Female Chickens

Fadrozole, an aromatase inhibitor, can masculinize genetic female chickens and high-dose decreases the hatchability. Therefore, it is important to study the growth and development of sex-reversed females after hatch. Chick embryos from a population of CAU3 egg-type were treated with different concentrations of Fadrozole prior to the sexual differentiation at E3.0 （st18）. At hatch, the phenotypic sex and genetic sex were identified by vent sexing and genetic diagnosis with CHD1, respectively. Body weight and shank length of sex reversal were tested at 8 and 20 wk, respectively. Testicular development, oviduct and ovarian degeneration were observed and serum concentration of estradiol and testosterone were tested with radioimmunoassay （RIA） at 30 wk. The results showed that body weight and shank length of sex- reversed females were not significantly different between low-dose groups （0.1, 0.3, and 0.5 mg for F1, F2, and F3, respectively） and high-dose groups （1.0 and 1.3 mg for F4 and F5, respectively） （P〉0.05）. Left and right testes or ovotestes in F2, F3, F4, and F5 groups were heavier than that of in F1 group （P〈0.05）. While the gonad weight of treatment groups were less than that in male control （P〈0.05）, oviduct weight in F2, F3, F4, and F5 groups were significant differences compared with female control and F1 group （P〈0.05）. Egg number from onset of laying egg to 30 wk in F4 and F5 groups were less than in female control, F1 and F2 groups （P〈0.05）. Serum testosterone level in F5 group was significant higher compared with female control, F1, F2, F3, and F4 groups （P〈0.05）, but significant lower compared with male control （P〈0.05）. While concentration of serum estradiol in F5 group was significant lower compared with female control, F1, F2, and F4 groups （P〈0.05）. In conclusion, the concentration of Fadrozole do not affect postnatal growth of sex-reversed female chicken and the degree of sex-reversed females elevate with the increase of Fadrozole concentration at sex maturity.

Knockdown of the Meq gene in Marek’s disease tumor cell line MSB1 might induce cell apoptosis and inhibit cell proliferation and invasion

Marek’s disease(MD),a highly cell-associated and contagious disease of chickens caused by Marek’s disease virus(MDV)can result in neural lesions,immunosuppression and neoplasia in chicken.The Meq gene is an important oncogene in the MDV genome,and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines.An experiment was conducted to elucidate the role of Meq in MD tumor transformation.RNA interference technology was used to block its expression,and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1.A small interfering RNA with an interference efficiency of 70%(P<0.01)was transfected into MSB1 cells to knock down the expression of Meq gene.The cell proliferation,cycle and apoptosis were detected post-Meq knockdown.The results showed that MSB1 cell proliferation was downregulated remarkably at 48 h(P<0.01),60 h(P<0.05)and 72 h(P<0.01)post-Meq knockdown.The cell cycle was unaffected(P>0.05).B-cell lymphoma 2 gene(BCL2)was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway.The activity of caspase-6 was upregulated(P<0.05)significantly and BCL2 gene expression was downregulated(P<0.05)significantly post-Meq knockdown,suggesting cell apoptosis might be induced.MSB1 cell migration did not exhibit any obvious change(P>0.05)post-Meq knockdown,but the expression of two genes(matrix metalloproteinase 2(MMP2)and MMP9)that are correlated closely to cell invasion was downregulated(P<0.05)remarkably post-Meq knockdown.The Meq knockdown might affect the main features of tumorous cells,including proliferation,apoptosis,and invasion,suggesting that the Meq gene might play a crucial role in interfering with lymphomatous cell transformation.

Expression analysis for candidate genes associated with eggshell mechanical property

Damaged eggshells result in losses of eggs.The genetic mechanism of variable eggshell strength is still unclear.The current study was conducted to verify whether the eggshell calcification related genes,CALB1,SPP1,DMP4,BMP2 and SLIT2,were associated with eggshell mechanical property.For this purpose quantitative PCR（q-PCR） analysis was performed to detect gene expression between two groups of hens laying strong and weak eggs.The hens were selected from 360 White Leghorn layers at 60 wk to ensure that the strong and weak eggs differed significantly in breaking strength but not in eggshell thickness and weight.Using this special strong/weak eggshell model,we found that the expression of CALB1 in the uterus of strong shell group was about 3-fold higher（P〈0.05） than that in weak shell group.The DMP4 expression was significantly higher（2-fold,P〈0.05） in the uterus of weak shell group than that in strong shell group.However,no difference was observed for genes of SPP1,SLIT2 and BMP2 between these two groups.The current study provides a new insight to investigate the association of candidate genes with eggshell mechanical property.