Effects Of P16 On The Biological Behavior Of Cervical Cancer Cells Under Different Oxygen Conditions

Objective:To determine the optimal concentration and action time of CoCl₂drug to simulate hypoxia,and to detect the expression of hypoxia inducible factor-1α（HIF-1α）in cervical cancer tissues under the conditions of appropriate concentration and action time;The migration,invasion,proliferation,cloning ability,cell cycle and apoptosis of tumor cells were observed after p16 gene silencing under different oxygen conditions.Methods:1.The hypoxia model in vitro was established by using CoCl₂ reagent;2.Human cervical cancer He La cells were cultured in vitro,and different concentrations of CoCl₂（0,50,100,200,400,800μmol/L）were used to act on human cervical cancer He La After different time（6,12,24,48,72 hours）,the cell counting reagent（Cell Counting Kit-8,CCK8）method was used to detect the effect of hypoxia on the proliferation activity of human cervical cancer He La cells under the action of different concentrations and different times.Determine the appropriate concentration and action time of the drug;study the expression of HIF-1αprotein in cervical cancer He La cells by Western Blot;3.Use si RNA technology to induce p16 gene silencing in cervical cancer He La cells 4.Using Wound Healing assay,Transwell assay,CCK8 method and Colony Formation assay to detect cervical cancer He La cells after p16 gene silencing under different oxygen conditions The changes of migration,invasion,proliferation and cloning ability of cervical cancer He La cells were detected by PI（FITC Annexinv-PI）method after silencing of p16gene under different oxygen conditions.Results:1.The proliferation of cervical cancer He La cells was mostly inhibited by different concentrations of CoCl2 drugs.When the CoCl2concentration increased,the cell proliferation activity showed a downward trend,and the cell viability decreased in a time-and dose-dependent manner.With the increase of oxygen time,the cell proliferation viability first gradually increased and then decreased slowly.Compared with the control group,the cell viability of He La cells treated with CoCl2 decreased in a time-and dose-dependent manner.Considering the time and dose,400u M CoCl2 treated He La cells for 24 h and the cell viability decreased.difference.2.Western blotting results showed that compared with the control group,HIF-1αprotein expression was up-regulated in cervical cancer He La cells.3.Under different oxygen conditions,after the silencing of the p16 gene,the comparison between the normoxia and hypoxia groups was statistically significant（P<0.05）.Therefore,it can be concluded that the silencing of the p16 gene under hypoxic conditions is better than that under normoxia conditions.Increased migration rate of cervical cancer He La cells.4.Under different oxygen conditions,after the silencing of the p16 gene,the comparison between the normoxia and hypoxia groups was statistically significant（P<0.05）.Therefore,it can be concluded that the silencing of the p16 gene under hypoxic conditions is better than that under normoxia conditions.The cell invasive ability of cervical cancer He La cells was significantly improved.5.Under different oxygen conditions,after the p16 gene was silenced,the comparison between the normoxia and hypoxia groups was statistically significant（P<0.05）.Therefore,it can be concluded that the p16 gene silenced under hypoxic conditions was better than that under normoxic conditions.The cell proliferation rate of cervical cancer He La cells is increased.6.Under different oxygen conditions,after the silencing of the p16 gene,the comparison between the normoxia and hypoxia groups was statistically significant（P<0.05）.Therefore,it can be concluded that the silencing of the p16 gene under hypoxic conditions is better than that under normoxia conditions.the relative clone formation rate of cervical cancer He La cells was increased.7.Under different oxygen conditions,after the silencing of the p16 gene,the comparison between the normoxia and hypoxia groups was statistically significant（P<0.05）.Therefore,it can be concluded that the silencing of the p16 gene under hypoxic conditions is better than that under normoxic conditions.the cell proliferation index and S phase cell ratio of cervical cancer He La cells increased.8.Under different oxygen conditions,after the silencing of the p16 gene,the comparison between the normoxia and hypoxia groups was statistically significant（P<0.05）.Therefore,it can be concluded that the silencing of the p16 gene under hypoxic conditions is better than that under normoxic conditions.the apoptosis rate of cervical cancer He La cells decreased.Conclusion:1.The proliferation activity of cervical cancer He La cells is inhibited by hypoxia,and there is a big difference when the concentration of CoCl2 is 400μmol/L and treated for 24 hours;2.24 hours,and 400u M CoCl₂ concentration,HIF-1αThe protein is highly expressed in cervical cancer He La cells;3.After sip16 was transfected into He La cells under normoxic conditions to deplete its expression,the migration rate,invasion ability,proliferation rate and relative clone formation rate of cervical cancer cells were all higher than 4.Under hypoxic conditions,sip16 was transfected into cervical cancer He La cells to deplete the migration rate and invasion ability after expression.proliferation rate,relative clone formation rate were increased,and the cell proliferation index and the ratio of S phase cells in the cell cycle were increased,and the apoptosis rate showed a downward trend.Therefore,improving the hypoxic microenvironment in tumor cells and the therapeutic targeting of p16 gene may be a promising therapeutic strategy for cervical cancer.