siRNA-mediated silencing of c-kit in mouse primary spermatogonial cells induces cell cycle arrest.
Several genes/gene products are known to act in a concert to regulate the process of spermatogenesis. One such gene is c-kit, a transmembrane tyrosine kinase receptor which plays an indispensable role in the maturation and differentiation of spermatogonial germ cells (SGCs). In the present study, siRNA approach was used to assess the role of c-kit in survival and proliferation of murine primary SGCs. The effect of different concentrations of anti-c-kit siRNA-1 and siRNA-2 (0.15, 0.315, 0.625, 1.25, 2.50, 5, and 10 nM) on c-kit protein and mRNA expression at post-transfection time (0, 6, 12, 24, 48, and 72 hours) was assessed using an array of techniques such as flow cytometry, ELISA, Western blot, and RT-PCR. Transfection of cells with anti-c-kit siRNAs (0.15-10 nM) at various time points after (0-72 hours) showed significant knockdown c-kit mRNA and protein expression. MTT, Alamar blue assays, and RT-PCR were used to investigate the effects of c-kit silencing on survival, proliferation, distribution, and apoptosis of cells. Experiments were also conducted to determine the effects of c-kit knockdown on cell cycle distribution, DNA laddering, and apoptosis. The results indicated that the transfection with anti-c-kit siRNA induces DNA fragmentation and cell cycle arrest at G(2)/M phase leading to significant reduction in cell viability and proliferation. In addition, enhanced suppression of c-kit protein in P815 cells was observed after transfection as compared to ES-E14TG2alpha cells, suggesting early onset of c-kit protein repression in P815 cells leading to prolongation in cell doubling time. In conclusion, our data provide the first evidence of specific knockdown of c-kit expression in mouse primary SGCs, which emphasizes the critical role played by c-kit in germ cell survival, proliferation, and apoptosis.
Sikarwar
Arun P
AP
Immunology Laboratory, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Parel, Mumbai, India.
Reddy
K V R
KV
D000818:Animals; D002453:Cell Cycle / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes / Q000378:metabolism; D020868:Gene Silencing; D007202:Indicators and Reagents / Q000378:metabolism; D008297:Male; D034801:Mastocytoma / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D013093:Spermatogonia / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D019009:Proto-Oncogene Proteins c-kit; D016650:Fluorescein-5-isothiocyanate