The role of CDK1 siRNA interference in cell cycle and cell apoptosis

In the present report,cyclin-dependent kinase1(CDK1)siRNA was transfected into cells to silence the CDK1 gene expression and study its role in the cell cycle and cell apoptosis.The siRNA targeting CDK1 gene was chemically synthesized and transfected into Hela cells by lipofectamine 2000.The expression levels of CDK1 gene and protein were examined by real-time quantitative polymerase chain reaction(PCR)and Western blot,respectively.The cell cycle was analyzed by using DNA content analysis byflow cytometry.Cell apoptosis was detected by the Annexin V/PI method.The morphological changes of transfected cells were examined under the microscopy by Wright-Giemsa stain.CDK1 gene was successfully silenced by its siRNA,and the CDK1 protein expression level was decreased significantly,especially from 48th h to 60th h after transfection.The DNA content analysis showed that transfection of CDK1 siRNA led to cells accumulating in G2/M phase.There was no significant difference in the apoptotic rate between transfected cells and the control cells after transfection of CDK1 siRNA for 48 or 60 h.More double nucleus or multinucleus cells could be seen under the microscopy among the transfected cells.The decreased CDK1 expression by siRNA silencing gave rise to cell cycle arrest in G2/M phase but did not induce apoptosis.

HIF-1α siRNA Leads to Apoptosis of Pancreatic Cancer Cells under Hypoxic Conditions

OBJECTIVE To explore the role （HIF-1α） in the proliferation and cells under hypoxic conditions. of hypoxic inducible factor-1α apoptosis of pancreatic cancer METHODS A cassette encoding small interference RNA （siRNA） targeting HIF-1α mediated by recombinant adeno-associated virus （rAAV） was constructed, giving rAAV-siHIE rAAV-siHIF or rAAV- hrGFP was transfected into exponentially growing MiaPaCa2 cells under hypoxic conditions. Then, the expression of HIF-1α mRNA and protein, the proliferation and apoptosis of MiaPaCa2 cells were examined, using real-time PCR, Western Blot, MTT and TUNEL, respectively. RESULTS Under hypoxic conditions, rAAV-siHIF inhibited the expression of HIF-1α mRNA and protein in MiaPaCa2 cells. At the same time, rAAV-siHIF decreased MiaPaCa2 cell proliferation and induced apoptosis. However, rAAV-hrGFP had no effect on the expression of HIF-1α as well as the proliferation and apoptosis of MiaPaCa2 cells under hypoxic conditions. CONCLUSION Under hypoxic conditions, HIF-1α plays a key role in the proliferation of MiaPaCa2 cells, and inhibition of HIF- 1α expression can lead to MiaPaCa2 cell apoptosis.

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies,transfection of exogenous nucleic acids including plasmid DNA and small interference RNA(siRNA)are generally performed.However,the cellular processing and the fate of these nucleic acids remain elusive.By investigating the cellular behavior of transfected nucleic acids using confocal imaging,here we show that when siRNA was cotransfected into cultured cells with other nucleic acids,including single-stranded RNA oligonucleotides,single and double-stranded DNA oligonucleotides,as well as long double-stranded plasmid DNA,they all aggregate in the same cytoplasmic granules.Interestingly,the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity,suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event.Our results argue against the claim that the siRNAaggregating granules are the functional site of RNA interference(RNAi).Taken together,our studies suggest that,independent of their types or forms,extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules“transfection granules”.