OBJECTIVETo explore the effects of lentivirus-mediated RNA interference silencing HMGA2 on proliferation and expressions of cyclin B2 and cyclin A2 in HL-60 cell line.

METHODSThe protein and mRNA expressions of HMGA2 in HL-60 cells transduced by recombinant lentivirus producing HMGA2 gene short hairpin (shRNA) were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis; The effects of the lentivirus on cell proliferation inhibiting rate, the ability of cell proliferation and cell cycle were analyzed by soft agar colony formation assay and FCM, respectively; The protein and mRNA expressions of cyclin B2 and cyclin A2 were also examined by Western-blot and RT-PCR.

RESULTSRecombinant lentivirus producing HMGA2 shRNA was successfully constructed, which was identified by PCR and sequencing; Stable HMGA2-deficient HL-60 cell line was established by puromycin, its mRNA and protein expression inhibition rates were (80.66 ± 7.98)% and (76.35 ± 12.72)%, respectively. Silencing of endogenous HMGA2 resulted in efficient inhibition of the cellular proliferative activity, low and flat of the cell growth curve and the lack of typical character of exponential growth. FCM revealed significant more cell cycle G(2)/M arrest \[(30.00 ± 5.78)%\] in HL-60 cell line transfected specific shRNA than control group \[(13.90 ± 4.07)%\] (P < 0.05). The cyclin B2 mRNA and protein expression inhibition rates in stable HMGA2-deficient HL-60 cell line were (67.55 ± 7.69)% and (51.77 ± 4.81)%, respectively, while the expression of cyclin A2 had no significant change compared with control group.

CONCLUSIONRNAi silencing of HMGA2 down-regulated cyclinB2, significantly inhibited the proliferation of HL-60 cells and induced the accumulation of HL-60 cells in the G(2)/M phase. Thus, HMGA2 may be an important target for anti-leukemia therapy.

Cell Proliferation ; Cyclin A2 ; genetics ; Cyclin B2 ; genetics ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HL-60 Cells ; HMGA2 Protein ; genetics ; Humans ; Lentivirus ; RNA Interference ; RNA, Small Interfering ; genetics

Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited tumor syndrome characterized by development of various combinations of tumors in multiple endocrine glands, including the pituitary, parathyroid or pancreas. MEN1 results from mutations in tumor suppressor gene Men1, which encodes nuclear protein menin. Menin has been shown to preferentially repress cell proliferation in endocrine tissues including pancreatic beta cells. Herein, the present study was to explore the potential mechanisms underlying menin in repressing cell proliferation in mice MEN1 insulinoma. In the Gene Set Enrichment Analysis (GSEA), Ccnb2 (encoding cyclin B2) was up-regulated in pancreatic islets of Men1-excised mice after 14-day tamoxifen-feeding. Immunofluorescence with antibody against cyclin B2 revealed that the expression of cyclin B2 was greatly increased in MEN1 insulinoma. In Men1(-/-) cells, Men1 ablation leaded to an increase in cyclin B2 expression. Immunofluorescent staining by phospho-H3S10 antibody revealed the increasing number of Men1(-/-) cells in mitosis. Cells were seeded at a density of 5 × 10(4), then counted on day 2, 4 and 6, and the cell growth curve revealed Men1 ablation increased the cell proliferation. In contrast, knockdown of cyclin B2 by shRNA diminished the number of cells in mitosis and reduced cell proliferation. Further, chromatin immunoprecipitation (ChIP) assay indicated that menin affected the histone modification of the promoter of Ccnb2 by reducing the level of histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 acetylation but not affecting the level of histone H3 lysine 9 tri-methylation (H3K9me3) or histone H3 lysine 27 tri-methylation (H3K27me3). Our results suggest that menin may inhibit MEN1 insulinoma by suppressing cyclin B2 expression via histone modification.
Animals ; Cell Proliferation ; Cyclin B2 ; genetics ; metabolism ; Histones ; metabolism ; Insulinoma ; metabolism ; pathology ; Mice ; Mice, Knockout ; Multiple Endocrine Neoplasia Type 1 ; genetics ; Mutation ; Pancreatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; genetics

AIMTo analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double-strand break repair.

METHODSTwo lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 (Ccna1); p53- and Ccna1; Atm-double knockout mice.

RESULTSThe block in spermatogenesis observed in Cyclin A1-/- (Ccna1-/-) testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccna1-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccna1-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1.

CONCLUSIONCcna1-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.

Animals ; Ataxia Telangiectasia Mutated Proteins ; Cell Cycle ; genetics ; physiology ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin A ; genetics ; metabolism ; Cyclin A1 ; Cyclin B ; Cyclin B2 ; DNA ; genetics ; DNA Repair ; genetics ; physiology ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Expression Regulation ; genetics ; physiology ; Male ; Mice ; Mice, Knockout ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Spermatogenesis ; genetics ; physiology ; Testis ; cytology ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism