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The Effect of miR-1468 on Growth Hormone in Pituitary Cells of Yanbian Yellow Cattle

The Effect of miR-1468 on Growth Hormone in Pituitary Cells of Yanbian Yellow Cattle

Tai-hua Jin1, An-gang Lou2,Jiu-xiu Ji1, Cheng-dou Cui2, Long-zheng Yu2 andLi-zeng Guan1*

1College of Agriculture and Forestry Science, Linyi University, Linyi 276005, China
2 Agriculture College, Yanbian University, Yanji 133000, China
 
Tai-hua Jin and An-gang Lou contributed equally to this work.

*      Corresponding author: guanlizeng@163.com

Fig. 1.

Expression level of miR-1468 in pituitary cells of Yanbian yellow cattle. (A) The expression level of miR-1468 in pituitary cells transfected with the mimics (miR-1468-mi group) and mimics reference substance (NC group) of miR-1468. Compared with NC group, the column marked by ** showed significant difference (P<0.01); (B) The expression level of miR-1468 in pituitary cells transfected with the inhibitor (miR-1468-in group) and inhibitor reference substance (iNC group) of miR-1468. U6 was used as an internal reference.

Fig. 2.

Effect of miR-1468 on GH mRNA transcription level in pituitary cells of Yanbian yellow cattle. (A) The relative transcription level of GH mRNA in pituitary cells transfected with miR-1468 minics. Mimics (miR-1468-mi group) and mimics reference substance (NC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with NC group, the column marking** showed extremely significant difference (P<0.01); (B) The relative transcription level of GH mRNA in pituitary cells transfected with miR-1468 inhibitor. Inhibitor (miR-1468-in group) and inhibitor reference substance (iNC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with iNC group, the column marking* showed significant difference (P<0.05). β-actin was used as an internal reference.

Fig. 4.

Prediction of target relations for miR-1468.

Fig. 5.

Luciferase activity determination. (A) The changes of luciferase activity after PirGLO- CREB1-3’UTR normal plasmid co-transfecting with miR-1468 mimics (miR-1468-mi group) and mimics reference substance (NC group) for 48 h. Compared with NC group, the column marking** showed extremely significant difference (P<0.01); (B) The changes of luciferase activity after PirGLO-CREB1-3’UTR mutant plasmid plasmid co-transfecting with miR-1468 mimics (miR-1468-mi group) and mimics reference substance (NC group) for 48 h. Compared with NC group, the column without marking**or* showed no significant difference (P>0.05).

Fig. 6.

Effect of miR-1468 on transcription level of CREB1 mRNA in pituitary cells of Yanbian yellow cattle. (A) The relative transcription level of CREB1 mRNA in pituitary cells transfected with miR-1468 minics. Mimics (miR-1468-mi group) and mimics reference substance (NC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with NC group, the column marking** showed extremely significant difference (P<0.01); (B) The relative transcription level of CREB1 mRNA in pituitary cells transfected with miR-1468 inhibitor. Inhibitor (miR-1468-in group) and inhibitor reference substance (iNC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with iNC group, the column marking** showed extremely significant difference (P<0.01). β-actin was used as an internal reference.

Fig. 7.

Effect of miR-1468 on CREB1 protein expression level in pituitary cells of Yanbian yellow cattle. (A) The relative expression level of CREB1 protein in pituitary cells transfected with miR-1468 minics. Mimics (miR-1468-mi group) and mimics reference substance (NC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with NC group, the column marking** showed extremely significant difference (P<0.01); (B) The relative expression level of CREB1 protein in pituitary cells transfected with miR-1468 inhibitor. Inhibitor (miR-1468-in group) and inhibitor reference substance (iNC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with iNC group, the column marking* showed significant difference (P<0.05). β-actin was used as an internal reference. Electrophoresis of the CREB1 and β-actin gene fragment was from two separate gels.

Fig. 3.

Effect of miR-1468 on GH protein expression level in pituitary cells of Yanbian yellow cattle. (A) The relative expression level of GH protein in pituitary cells transfected with miR-1468 minics. Mimics (miR-1468-mi group) and mimics reference substance (NC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with NC group, the column marking** showed extremely significant difference (P<0.01); (B) The relative expression level of GH protein in pituitary cells transfected with miR-1468 inhibitor. Inhibitor (miR-1468-in group) and inhibitor reference substance (iNC group) of miR-1468 were transfected into pituitary cells of Yanbian yellow cattle, with three replicates in each group. Compared with iNC group, the column marking** showed significant difference (P<0.01). β-actin was used as an internal reference. Electrophoresis of the GH and β-actin gene fragment was from two separate gels.

Pakistan Journal of Zoology

August

Vol. 53, Iss. 4, Pages 1201-1601

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