| Objective:In our early studies, we have shown that proto-oncogenes c-myc and neul up-regulate during primordial follicle assembly and development using the microarray approach. In this experiment, we further investigated the role and the possible related-signaling pathway of proto-oncogenes c-myc and neul in the initiation of primordial follicle growth in rats by using RNA interference and microarray methods.Methods:Lentivirus-mediated c-myc and neul RNA interference vectors were constructed. RT-PCR was used to detect the mRNA levels of proto-oncogenes c-myc and neul. Western blot was used to measure the expression of c-Myc and Neul protein. The growth of primordial follicles was observed after knocking down the expression of proto-oncogenes c-myc and neul in the ovary. The gene expression levels were studied using Illumina rat RatRef-12 whole genome expression chips.Results:1. Knocking down proto-oncogenes c-myc and neul in the ovary, mRNA and protein levels of c-myc and neul were decreased significantly (both P<0.01).2. The numbers of the primordial follicles in the c-myc and neul knocked-down groups were observed a significant increase (P<0.01) and the numbers of the primary follicles and secondary follicles were observed a significant decrease (P<0.05), comparing with the blank control and negative control groups. The negative control group had no significant change (P>0.05), comparing with the blank control group.3. Analysis of the ovaries genes demonstrated that 2896 differentially expressed genes were identified between the 0 d and 8 d blank control groups during the normal initiation of primordial follicle growth. Among these genes, the expression of 1457 genes were up-regulated (i.e. Wnt4, Wnt5a, Notch4, Hey2, c-Myc, Neul) (Diffscore>+20) and 1439 down-regulated genes (Diffscore<-20). Compared with the 8 d blank control group, there were 33 up-regulated genes and 61 down-regulated genes, including 6 genes related to c-myc signal pathway (i.e. Ptch1, Stat1, IL-1βPtpn6, c-Myc, Rac2) in the lentivirus c-myc shRNA group; And there were 160 up-regulated genes and 191 down-regulated genes, including 2 genes related to neu1 signal pathway (i.e. Cln3, Neul) in the lentivirus neul shRNA group; Besides there were 187 and 83 differentially expressed genes in the lentivirus NC shRNA group and lentivirus pGCL-GFP group respectively, but none differecence in the expression of proto-oncogenes c-myc and neul were found.Conclusions:1. Proto-oncogenes c-myc and neul possibly played a positive role in the initiation of primordial follicle growth.2. After proto-oncogene c-myc in the ovary was knocked down, there was a significant change in the expression of genes related to c-myc signal pathway (i.e. Ptchl, Statl, IL-ip, Ptpn6, RacI) during the process of the initiation of primordial follicle growth.3. The Wnt and Notch signaling pathways including proto-oncogene c-myc might be involved in the initiation of primordial follicle growth.4. After proto-oncogene neul in the ovary was knocked down, there was a significant change in the expression of genes related to neul signal pathway (i.e. Cln3) during the process of the initiation of primordial follicle growth. |
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