The Effects Of Growth And Development In Sika Deer Antler Chondrocyte Cells By AR Small Interfering RNAs

The estradiol and testosterone levels in plasma of the stag decreased from peak at the same time, maintaining a lower level in the whole period. The low level of androgen is advantageous to the male deer. It determined androgen receptor transcription in red deer antler tissue. Androgen combining with AR forming complexes regulates the growth and development of deer antler, and AR is controlled by the receptor gene. It is bound to affect the structure and function of AR and composites, and ultimately leads to its biological effection changing, influencing the antler growth if AR gene mutation. Firstly, cloning the partial sequences of the chondrocytes AR gene, and then designing siRNAs according to AR target genes. The siRNA of AR gene imports the antler chondrocytes by liposome through siRNA interference technologies in this study, to regulate AR protein expression, and affect antler chondrocytes proliferation, cycle and cell apoptosis, and to study the action mechanism on development of antler chondrocyte cells. The results were listed as follow:(1) The partial AR gene sequence is 2346 bp, and sequences alignment indicates the sequence is for the sika deer AR c DNA, and open reading frame coding 781 amino acids in sika deer. The sequence had been submitted to Genbank, and registration number: KT599413.1.(2) Multiple sequences alignment results showed that AR gene and protein has highly homology in Cervus nippon, Bos taurus, Ovis aries, Capreolus. And Bos taurus, Ovis aries, Capreolus nucleotide homology were 98%; and Human nucleotide homology was 89%; and Bos taurus, Ovis aries amino acid homology were 98%; and Homo sapiens amino acid sequence homology was 87%.(3) The cells transfected with the siRNAs by Lipofectamine? RNAiMAX for 48 h, and RT-PCR analysis results show that the relative expression of AR mRNA of siRNA group is decreased significantly comparing with the control group(P<0.01); Western Blot results show that the expression of AR protein of siRNA group was significantly lower than the control group(P<0.05), indicating that the effect of siRNA interference is significant.(4) The results of cell apoptosis and hormone levels of testosterone and estradiol show that the difference was not significant comparing to the negative control group(P>0.05); and cell cycle results showed that the siRNA group arrested the cell cycle at S phase(P<0.01); the cell proliferation results indicated that siRNA-treated cells were able to significantly inhibit cell proliferation, and androgen can promote the growth of deer antler chondrocytes.The results show that AR plays an important role on the growth of Sika Deer antler chondrocytes. The relative expression of AR mRNA and AR protein is decreased significantly. AR siRNA inhibits cell proliferation by regulating the S phase of cell cycle of chondrocytes, and androgen promotes the growth and development of deer antler chondrocytes indirectly.