Effect Of Downregulated APC-Cdh1 On The Cell Phase Of Hela Cells

ObjectiveIt is one of important mechanism that aberrant cell cycle control causes cell deregulated proliferation in malignant cancer. Skp2 is one of many factors controlling cell cycle, and is paid close attention to increasingly as a new cancer gene. Skp2 is also one important substrate of APC-Cdh1. APC-Cdh1 plays important role on regulating cell cycle, it is unclear whether downregulated APC-Cdh1 can cause apoptosis on proliferation cells and how to influence cell cycle. So Cdh1 small interfering RNA (siRNA) eukaryotic expression vector was constructed and its expression was evaluated in Hela cells. After APC-Cdh1 was downregulated, the expression of Skp2 was detected and effect was investigated on the Hela cell cycle.Methods1. Construction and identification of Cdh1 small interfering RNA eukaryotic vectorThe interfering sequence and control sequence for Cdh1 small hairpin RNA (shRNA) were designed based on pENTRTM /H1/TO vector and the oligonucleotides were synthesized. These oligonucleotides were annealed and ligated into linearized pENTRTM/H1/TO vector respectively. After confirmation by DNA sequencing, positive recombinant plasmids were transfected into Hela cells respectively by the liposome method. Forty-eight hours later, total cellular RNA and total protein were extracted ,then the expression of Cdh1 was analyzed by Real Time-PCR and western blot.2. Effect of downregulated APC-Cdh1 on the cell phase of Hela cellsSuccessfully constructed Cdh1 small interfering RNA eukaryotic vectors pENTR/shCdh1 and pENTR/shcontrol were transfected into Hela cells. After 24h, Zeocin 300 mg/L was added to screen them. After 10 d, successfully screened cells were cultivated amplifiedly. The cell cycle and the cellular apoptosis were examined by fluorescence activated cell sorter. Cell proliferation was evaluated by incorporating 5-bromodeoxyuridine(Brdu). The expression of Skp2 mRNA was detected by Real-Time PCR.Results1. After confirmation by DNA sequencing, we constructed Cdh1 siRNA eukaryotic expression vector and control vector successfully, named as pENTR/shCdh1 and pENTR/shcontrol respectively. Compared with the Hela cells transfected with pENTR/shcontrol or untransfected, the transfected pENTR/shCdh1 cells showed significant suppression in the expression of Cdh1(P<0.05).2. As compared with control group, the cell distribution of G0-G1 phase was significantly decreased while the cell distribution of S phase was significantly increased. There were no significant differences between the apoptosis rate of both groups. Cell proliferation and the expression of Skp2 mRNA in pENTR/shCdh1 transfected Hela cells were significantly increased compared with pENTR/shcontrol transfected Hela cells.ConclusionThe Cdh1 siRNA eukaryotic expression vector is successfully constructed and it can knockdown the expression of Cdh1 in Hela cells. Downregulated Cdh1 can increase the cell distribution of S phase ,but has no significant effect on apoptosis.