A Pilot Study Of Effect Of Silencing Nek2on He Biological Behaviors Of Skov3in Varian Cancer And The Relatrd Mechanism

Part1The effect of silencing Nek2on the Biological behaviors ofSKOV3of ovarian cancerObjective To design effective Nek2-siRNA transfected into SKOV3cellsof ovarian cancer, then, to observe the expression of Nek2and the effectof it on the multiplication,apoptosis,transference,invasion and cell cycleof SKOV3cells.Methods1. The expression of Nek2in the ovarian epithelium cancer was examinedby immunohistochemisthry.2. Three pairs of siRNA were designed by synthesized chemically suitableto the sequence of Nek2gene, and then be transfected into the SKOV3cells of ovarian cancer for forty-eight hours. The expression levels of Nek2mRNA and protein were examined by Real-time RT-PCR and Westernblotting after transfection, so as to screen the most effective siRNA. 3. Transfect Nek2-siRNA into SKOV3cells, the ability of multiplication ofSKOV3cells was examined by MTT; the change of apoptosis and cellcycle of SKOV3cells were examined by flow cytometry technology; theability of transference and invasion were examined by trans-well.Results1. The expression of Nek2in ovarian epithelium cancer was heightenedobviously.2. After transfection within Nek2-siRNA, the expression at both mRNAand protein levels of Nek2were down-regulated in SKOV3cells comparedwith the control(P＜0.05).Among them, the second group(sequence ofNek2-2) down-regulated most obviously.3. The ability of multiplication of the groups transfected by Nek2-siRNAfor48/72hours were lower than it of the blank control and negative controlgroups. The inhibition rates are32.1%,43.8%,(P＜0.05).4. Transfected by Nek2-siRNA for48hours,the apoptosis rate of the blankcontrol grpoup, negative control group and transfected group is(5.00±0.09)%,(5.53±0.24)%和(10.98±0.16)%,(P＜0.05)。 Theproportion of the cells in G2/M cell cycle of those groups is13.72%,12.27%和1.56%.Compared with the control, the numbers of the cells in G2/M cell cycle of transfected group are reduced obviously.(P＜0.05)5. Transfected by Nek2-siRNA for48hours,transference test showed thenumbers of the cells tans-well of the blank control grpoup,negative controlgroup and transfected group are(351.3±14.2),(360.7±11.1),(95.7±6.2).Compared with the control, the numbers of the transfected group are arereduced obviously.(P＜0.05). The invasion test showed the numbers ofthe cells tans-well of the blank control grpoup,negative control groupand transfected group are (340±17.3),(329.3±13.6),(27.3±5.1).Compared with the control, the numbers of the transfected group are arereduced obviously.(P＜0.05).Conclusion1. The expression of Nek2in ovarian epithelium cancer was high. And theexpression can be inhibited effectively by siRNA.2. The SKOV3cells of ovarian cancer transfected by Nek2-siRNA, andthen, the ability of multiplication was reduced; the apoptosis wasincreased; the ability of transference and invasion was reduced; the cellswere inhibited to G2/M cell cycle. All those induced that the cells could notgo through the mitotic phase and into the next cell cycle smoothly.3. Nek2can be used as the important target point of the inhibin to the metastasis and recrudescent of the ovarian cancer. Part2A pilot study of the related mechanism of the effect of silencingNek2on the biological behaviors of SKOV3in ovarian cancerObjective To investigate the mechanism of the effect of the multiplication,apoptosis,transference,invasion and cell cycle of SKOV3cells transfectedby Nek2-siRNA.Methods1. Transfected by Nek2-siRNA for48hours,the expressions of theapoptosis related factors-caspase-3,Bad and Bcl-2were examined byWestern blotting.2. Transfected by Nek2-siRNA for48hours,the expressions of thetransference and invasion related factors-MMP-2,MMP-9and TIMP-1were examined by Western blotting.3. Transfected by Nek2-siRNA for48hours,the expressions of the cellcycle related factors-cyclinB1,CDK1and P27were examined by Western 4. Transfected by Nek2-siRNA for48hours,the phosphorylation level ofthe ERK1/2was examined by Western blotting.Results1. The expressions of Caspase-3,Bad in SKOV3cells were increased; theexpression of Bcl-2was reduced after silencing Nek2(P＜0.05).2. The expressions of MMP-2,MMP-9in SKOV3cells were reduced; theexpression of TIMP-1was increased after silencing Nek2(P＜0.05).3. The expressions of cyclinB1,CDK1in SKOV3cells were reduced; theexpression of P27was increased after silencing Nek2(P＜0.05).4. The phosphorylation level of the ERK1/2in SKOV3cells wasreduced after silencing Nek2(P＜0.05).Conclusion1. Nek2inhibited can up-regulate the expressions of Caspase-3,Bad inSKOV3cells and down-regulate the expressions of Bcl-2.2. Nek2inhibited can up-regulate the expressions of TIMP-1in SKOV3cells and down-regulate the expressions of MMP-2and MMP-9. So that,the transference and invasion of ovarian cancer were inhibited. 3. Nek2inhibited can up-regulate the expressions of P27in SKOV3cellsand down-regulate the expressions of cyclinB1and CDK1. So that, theSKOV3cells going into G2/M phase was stopped.It induced that themitotic phase could not be started-up, and the cells could not go into thenext cell cycle, finally, the multiplication of the SKOV3cells of ovariancancer was inhibited.4. Nek2regulates the the biological behaviors of SKOV3of ovarian cancervia ERK signal pathway.