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ERRα Inhibition Of RNA Interference On The Expression Of Apoptosis Of Caski

Posted on:2019-01-16Degree:MasterType:Thesis
Country:ChinaCandidate:L LiFull Text:PDF
GTID:2394330566970516Subject:Obstetrics and gynecology
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OBJECTIVE: To use RNA interference technology as a means to determine whether ERRα-siRNA specifically affects the expression of ERRα in Caski cells and to determine the apoptotic rate of cervical cancer Caski cells by targeting ERRα gene knockdown impact.METHODS: Pre-experimental infections of cervical cancer Caski cells were performed using a cell-infection technique using a well-constructed,safe and effective ERRα-siRNA lentivirus,to verify that ERRα-siRNA is effective in cervical Caski cell infection.The optimal infection index(MOI)and optimal culture conditions for virus-infected Caski cells were determined;formal infection experiments were performed to construct stable Caski-ERRα-NC(blank lentiviral vector negative control)cell lines and Caski-ERRα-siRNA(KD group)cell lines were used to detect the expression of ERRα mRNA in cervical cancer Caski-ERRα-NC and Caski-ERRα-si RNA cell lines by RT-PCR.The best small-interfering RNA fragment for silence.Flow cytometry was used to determine the apoptosis rate of the best knockout cell line in Caski-ERRα-NC cell line and Caski-ERRα-si RNA cell line.The endogenous silence of ERRα was used to determine the decline of cervical Caski cells.What is the impact of the death rate?RESULTS: Pre-infection experiments demonstrated that the ERRα small interfering RNA used in this experiment could effectively infect cervical cancer Caski cells,and the infection effect was the best under the conditions of MOI=10,culture medium with normal medium and infection adjuvant,and the infected cells.Stable expression;From the results of the infection experiment,it was found that the KD1/2/3 group was significantly different from the NC group(KD1 vs.NC,P<0.05,KD2 vs.NC,P<0.05;KD3 vs.NC,P<0.05)demonstrated that all three ERRα siRNA fragments played an endogenous role in knocking out the ERRα gene in cervical cancer Caski cells,and that KD2 group had the best knockout effect on ERRα(62.4% gene knockout rate).).Flow cytometry was used to detect the apoptotic rate of Caski cells silenced by ERRα in KD2 group,Caski cells infected only by empty virus vector in NC group,and Caski cells treated without any treatment in CON group under the same culture conditions.Theresults showed that KD2 the group was significantly more significant than the NC group(p<0.001).CONCLUSION: The knock-out of ERRα gene in cervical cancer Caski cells results in an increase in the apoptosis rate of cervical Caski cells.In other words,the gene silencing of ERRα can increase the apoptosis of cervical cancer Caski cells,which proves that ERRα may be a new target for cervical cancer targeted therapy.It is of great significance for the future treatment of cervical cancer and its prognostic evaluation.
Keywords/Search Tags:Cervical cancer Caski cells, Estrogen receptor-related-receptor alpha, Gene knockout, RNA interference, Small interfering RNA, Apoptosis
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