Targeting The STAT3-Bcl2 Pathway Promotes Apoptosis And Autophagy In Cervical Cancer Cells

Background:As the most common gynecological malignancy in the world,cervical cancer ranks third only to breast cancer and colorectal cancer in the incidence of malignant diseases among female patients.At present,in terms of the selection of treatmentmethods for cervical cancer,surgical focus resection can be used to eradicate or delay the development of the disease in patients with early detection,and the conventional treatment for advanced cervical cancer is concurrent chemoradiotherapy.however,the efficacy of chemoradiotherapy for most patients with advanced and middle stage islimited,and the recurrence and metastasis of patients with advanced and middle stage cervical cancer are still unresolved,so new methods are needed to overcome this problem.Signal transduction and transcriptional activation factor 3(signal transducers and activators of transcription,STAT3)exist in organism is the main role of gene transcription regulation,involved in cell growth,differentiation,proliferation and apoptosis,and a series of pathophysiological process of.It has also been found to be highly expressed in a variety of tumor tissues and cells,which is significantly related to the occurrence,development,recurrence,metastasis and prognosis of the tumor,and plays an important role in the relevant regulation.At the same time,there is a large amount of evidence that abnormal regulation of relevant oncogenes or tumor suppressor genes leads to abnormal apoptosis and autophagy of tumor cells,which leads to uncontrolled proliferation,differentiation and metastasis of cervical cancer cells,leading to tumor progression.Therefore,we intend to explore the role of STAT3 gene in the development of apoptosis and autophagy of cervical cancer cells and possible molecular mechanisms through CRISPR/Cas9 gene editing technology,so as to control the metastasis and recurrence of cervical cancer and provide a new idea for improving the survival rate of patients with advanced cervical cancer or recurrent metastatic cervical cancer.Methods:1.The expression of STAT3 gene,apoptosis-related gene bcl-2 and autophagy-related gene Beclinl in paraffin samples of cervical cancer tissues was detected by using tumor oncomine public database chip for bioinformatics analysis and immunohistochemistry.2.A monoclonal cervical cancer Siha cell strain with STAT3 mutation was constructed by CRISPR/Cas9 gene editing technology,and the effects of STAT3 knockout on the biological characteristics of cervical cancer Siha cells were observed by CCK8 method,cell clone formation experiment,transwell experiment and subcutaneous tumor implantation experiment in nude mice.3.With Western blot experiments in every expression of STAT3 and qPCR method stable group of cervical cancer cells turn plant autophagy related genes in Beclinl,Bcl-2 apoptosis related gene,P62,LC3-? protein and mRNA level changes.4.The differences in the number of apoptotic bodies and autophagosomes in cells of different STAT3 expression groups were detected by electron microscopy;The changes of autophagic flow in different expression groups were detected by chloroquine treatment.The interaction between STAT3 and Bcl2 was detected by confocal immunoassay.The differences of autophagosomes and autophagosomes in different treatment groups were observed by confocal microscopy with DAP/DALGreen staining.Result:1.Oncomine,a public database of cancer,was retrieved and the analysis results showed that mRNA expression levels of STAT3 gene,apoptosis-related gene bcl-2 and autophagy related gene Beclinl mRNA in cervical cancer tissues were significantly increased compared with adjacent normal tissues.2.Stably constructed monoclonal Siha cervical cancer cells with STAT3 mutation by CRISPR/Cas9 gene editing technology:low expression group(KO group),low expression control group(Vetor);Compared with the control group,Siha cells of cervical cancer were significantly inhibited in vitro proliferation,migration,clonal formation and tumorigenicity after STAT3 gene knockout.3.Western blot experiments in every expression of STAT3 and qPCR method stable group of cervical cancer cells turn plant autophagy related gene Beclinl,apoptosis related gene in the Bcl-2,P62,LC3-? protein and mRNA level changes,the results compared with control group,low STAT3 expression group of Beclin 1 in cervical cancer cells and LC3-the amount of protein and mRNA expression ? increases,while the Bcl-2 and P62 protein and mRNA expression levels drop.4.Compared with the control group,the number of apoptotic bodies and autophagosomes in the STAT3 knockout group increased,and the detection level of autophagy flow increased.Fluorescence co-localization experiment showed that STAT3 and Bcl2 had co-localization in space.Using confocal microscopy,autophagosomes and autophagosomes increased in the STAT3 knockout group compared with the control group.Conclusion:Stat3 is highly expressed in cervical cancer tissue,and after targeting the expression of stat3,it is a signal the expression level of the anti-apoptotic bcl2 was decreased,and the function of "protective autophagy" was activated,and the biological characteristics of the proliferation,migration,invasion and tumor formation of cervical cancer cells were suppressed,while using the stationary strain cells to verify that stat3 different of tumor cells were affected by apoptosis and autophagy activity in the body,which was performed to promote apoptosis and autophagy.In addition,we showed that stat3 and bcl2 have a common effect,suggesting that the development of the stat3 mediated cervical cancer may play a role in regulating the autophagum by the stat3-bcl2 pathway,so the molecular therapy of the target stat3 gene may improve the current clinical treatment of cervical cancer.