Regulatory Mechanism Of STAT3 Mediated Autophagy In Cervical Cancer Stem Cells

BackgroundCervical cancer is a common gynecological tumor,and the incidence rate is increasing year by year and younger.It is also a disease that seriously endangers women' s health,and the mortality rate ranks second in female malignant tumors.At present,although drug and gynecological surgery have achieved certain effects on tumors,the general prognosis of malignant tumors in the advanced stage has not changed.In recent years,although scholars at home and abroad have made in-depth research on cervical cancer,the specific pathogenesis of cervical cancer has not yet been fully clarified.Therefore,it is urgent to further explore the specific pathogenesis of cervical cancer in clinical practice,and provide new ideas for improving the treatment and prognosis of cervical cancer,especially advanced and recurrent cervical cancer.Some studies have found that signal transducers and activators of transcription(STAT3)is an important regulatory gene,which is closely related to cervical cancer recurrence,lymphatic metastasis and prognosis of patients,and is important for cervical cancer cells.The regulation can cause abnormalities in the regulation of autophagy and affect the progression of the tumor.At the same time,a large number of studies have shown that autophagy is closely related to the proliferation,differentiation and metastasis of cervical cancer cells.Objective1.To study the relationship between STAT3 and proliferation,apoptosis and autophagy of cervical cancer HeLa cells,and to lay a foundation for the subsequent study of whether STAT3-mediated autophagy plays a similar regulatory role in cervical cancer stem cells.2.The role of STAT3 gene in the autophagy of cervical cancer stem cells and its possible molecular mechanisms were studied using CRISPR/Cas9 gene editing technology.Methods1.To study the relationship between STAT3 and proliferation,apoptosis and autophagy of cervical cancer HeLa cells1)Transiently transfected cervical cancer cells to regulate STAT3 protein expression,'construct STAT3 group with different protein expression;2)Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)assay to detect STAT3 mRNA expression in each group level;3)CCK-8 assay,flow cytometry to detect the proliferation and apoptosis of different STAT3 protein expression groups;4)Observing apoptotic bodies and autophagosomes in each group by electron microscopy;5)The expressions of apoptosis-related proteins Bcl-2,Caspase-3 and autophagy-related genes Beclinl and LC3-? in cervical cancer cells were detected by western blot analysis in STAT3 high expression group,control group and low expression group.2.Extraction and identification of cervical cancer stem cells from cervical cancer Hela and SiHa cells1)Separation and culture of cervical cancer stem cells by serum-free suspension culture of tumor stem cells;2)Induction of cervical cancer stem cell differentiation into adherent cells by adding 20%serum;3)Detection of cell surface molecular marker CD133 by flow cytometry CD44 expression identifies cervical cancer stem cells;4)CCK-8 assay detects the proliferation of cervical cancer stem cells;5)Cell plate cloning assay detects the cloning rate of cervical cancer stem cells;6)Flow cytometry detects apoptosis rate of cervical cancer stem cells.3.Regulatory effect of STAT3-mediated autophagy on human cervical cancer stem cells1)CRISPR/Cas9 gene editing technology to construct STAT3 mutant monoclonal cancer cervical cancer Hela cervical cancer stem cell stable transfectant;2)autophagy fluorescence(MDC staining)detection of cervical cancer stem cell stable transgenic autophagy;3)qPCR method The mRNA expressions of autophagy-related proteins Beclinl,LC3B,P62 and Bcl-2 in the stable cells of cervical cancer stem cells were detected.Results1.STAT3 can promote the proliferation of cervical cancer HeLa cells,inhibit its apoptosis and autophagy1)Successfully constructed three groups of HeLa cervical cancer cells with different expression levels of STAT3 protein,qRT-PCR confirmed that the transfection effect was satisfactory;2)CCK-8 experiment,flow cytometry to detect cell proliferation and withering of different STAT3 protein expression groups The ability to die suggests that STAT3 can significantly increase the proliferation of cervical cancer HeLa cells,and STAT3 can also significantly inhibit the apoptosis of cervical cancer HeLa cells;3)Electron microscopic observation of apoptotic bodies and autophagosomes in each group showed Up-regulation of STAT3 expression can inhibit the apoptosis and autophagy of cervical cancer cells;4)Western blot detection of Bcl-2,Caspase-3,Beclin1 and LC3-? mRNA expression in each group of cells indicates that STAT3 promotes HeLa cell proliferation it may be associated with decreased expression of Caspase-3,Beclinl and LC3-? genes.2.Successfully isolated and identified cervical cancer stem cells from cervical cancer Hela and SiHa cells1)Separation and culture of cervical cancer stem cells by serum-free suspension culture of tumor stem cells;2)Induction of cervical cancer stem cell differentiation into adherent cells by adding 20%serum;3)Detection of cell surface molecular marker CD133 by flow cytometry And the expression of CD44 identified cervical cancer stem cells;4)CCK-8 assay to detect the proliferation of cervical cancer stem cells showed that cervical cancer stem cells have higher proliferative capacity;5)Cell plate cloning experiments to detect the cloning rate of cervical cancer stem cells it shows that cervical cancer stem cells have higher cloning rate than parental cells;6)Flow cytometry detection of apoptosis rate of cervical cancer stem cells shows that the apoptosis rate of cervical cancer stem cells is decreased compared with the parental cells.3.Regulatory effect of STAT3-mediated autophagy on human cervical cancer stem cells1)Successfully constructed three STAT3 mutant monoclonal Hela cervical cancer stem cell stable transfectants by CRISPR/Cas9 gene editing technology,which were high expression group,high expression control group,low expression group and low expression control group;2)Phagocytosis(MDC staining)was used to detect autophagy in cervical cancer stem cells,indicating that STAT3 plays an important role in the regulation of autophagy in cervical cancer stem cells.Inhibition of STAT3 expression can significantly promote autophagy in cervical cancer stem cells;3)qPCR method was used to detect the mRNA expression of Beclinl,LC3B,P62 and Bcl-2 in the stable cells of cervical cancer stem cells.The results showed that STAT3 promoted the proliferation of cervical cancer stem cells and increased the expression of Bcl-2 protein.STAT3 inhibits the autophagy of cervical cancer stem cells,which may be related to the decrease of Beclinl and LC3B and the expression of P62 protein.At the same time,STAT3 is involved in the autophagy regulation of human cervical cancer stem cells,and the molecular mechanism of autophagy regulation may be suggested.The STAT3/Bcl-2/Beclin-1 axis acts.Conclusions1.STAT3 mediates the development of HeLa cells in cervical cancer,and STAT3 as a target provides a new way to reverse cervical cancer HeLa cells.2.Cell populations with stable cervical cancer stem cell properties can be isolated from Hela cell lines by using tumor stem cell serum-free suspension culture method.3.STAT3 may be a target gene for autophagy activation of cervical cancer stem cells.4.The STAT3/Bcl-2/Beclin-1 axis participates in and plays a role in the development of STAT3-mediated autophagy in human cervical cancer stem cells.