Effect And Mechanism Of STING On Proliferation,migration And Invasion Of Cervical Cancer

Background Cervical cancer is one of the most common gynecological cancers.There are about 570,000 new cases and 310,000 deaths worldwide each year.About 95% of cervical cancer cases are caused by persistent infection with the high-risk human papillomavirus(HPV).Cervical cancer remains a health crisis for women,despite the availability of vaccines to prevent the high-risk type of HPV.Therefore,it is necessary to develop a new direction for prevention and treatment of cervical cancer.Stimulator of interferon genes(STING)is a transmembrane protein,which is an important molecule in cytoplasmic DNA sensing pathway.More and more evidence suggests that the STING pathway,in addition to protecting the host against multiple pathogenic attacks,also plays a crucial role in cancer.In colorectal cancer,ovarian cancer and melanoma,STING signals associated with anti-tumor T cell initiation and type I interferon are severely suppressed to evade immune surveillance.The low expression of STING in gastric cancer tissues is related to tumor size,tumor invasion depth,lymphatic metastasis,clinicopathological stage and survival rate.Knocking out STING can promote proliferation,clone formation,migration and invasion of gastric cancer cells.In addition,a number of studies have shown that STING agonists can play an anti-tumor role as single drug or combined surgical treatment,radiotherapy,chemotherapy,and immunotherapy,making up for the deficiency of traditional therapy.Objective At present,there are few studies related to the occurrence and development of STING in cervical cancer,and the specific role of STING in cervical cancer is still unclear.In order to study the relationship between STING and the occurrence and development of cervical cancer,the expression of STING in human keratinocytes,cervical epithelial immortalized cells,and four cervical cancer cell lines(He La,Si Ha,C33 A,and Ca Ski)was detected in this study.The effects of STING on the proliferation,migration,invasion and apoptosis of cervical cancer cells were investigated by constructing stable transfected cervical cancer cell lines with silent STING and overexpression of STING.In addition,this study further explored the possible mechanism by which STING affects the proliferation,migration and invasion ability of cervical cancer cells.In vivo experiments were carried out to verify the results of in vitro experiments by constructing a subcutaneous xenograft tumor model in nude mice,providing a new strategy for reducing the recurrence or metastasis of cervical cancer patients and a new approach for gene targeted therapy of cervical cancer.Methods Part 1: Effects of STING on proliferation,migration,invasion and apoptosis of cervical cancer in vitro 1.Expression of STING m RNA and protein in human keratinocytes,cervical epithelial immortalized cells and four cervical cancer cell lines(He La,Si Ha,C33 A and Ca Ski)cultured in vitro were detected by q RT-PCR and western blot.2.Designed and constructed sh RNA lentiviral vector targeting STING gene,and transfected into He La and Si Ha cell lines;Overexpressing lentiviral vector of STING gene was designed and constructed,and transfected into C33 a cell line.Stable transfected cell lines overexpressing and silencing STING genes were constructed and identified respectively.3.The effects of STING on the proliferation,migration and invasion ability of cervical cancer cells were investigated by CCK8 experiment,plate cloning experiment,cell scratch experiment and Transwell chamber experiment.Part 2: Exploration of the signal pathway regulating biological behavior of cervical cancer cells by STING 1.After down-regulating the expression of STING,the expression level of total ?-catenin protein,activated ?-catenin protein and nucleus ?-catenin protein were detected by western blot,and the expression level of downstream target genes(cyclin D1,c-Myc,MMP9)in the wnt pathway were detected.2.After up-regulating the expression of STING,the expression level of total ?-catenin protein,activated non-phosphorylated ?-catenin protein and nucleus ?-catenin protein and downstream target genes(cyclin D1,c-Myc,MMP9)in the Wnt pathway were detected by western blot.3.Immunoprecipitation assay was used to detect whether STING was bound to ?-catenin;4.When wnt/?-catenin pathway inhibitor was combined with down-regulating the expression of STING,the expression level of total ?-catenin protein,activated ?-catenin protein and nucleus ?-catenin protein and downstream target genes(cyclin D1,c-Myc,MMP9)in the wnt pathway were detected by western blot.5.When wnt/?-catenin pathway inhibitor was combined with down-regulating the expression of STING,using CCK8 experiment,plate cloning experiment and Transwell chamber experiment to explore whether the effects of down-regulating STING expression on the proliferation,migration and invasion ability of cervical cancer cells could be reversed by ?-catenin pathway inhibitor.Part 3: In vivo study of the effect of silent STING on the proliferation of cervical cancer cells 1.The He La control cell line and sh STING-He La cell line were used to construct subcutaneous transplantation tumor model in nude mice,and the influence of silencing STING transplantation tumor on the body weight of nude mice was explored;2.To verify the effect of down-regulating STING expression on the proliferation ability of cervical cancer cells in vivo by measuring the volume of transplanted tumor and tumor growth curve in nude mice;3.Explore the effect of down-regulating STING expression on transplanted tumor in nude mice by HE staining;4.Verify whether the He La cells with silent STING remain silent STING after tumor formation in vivo by immunohistochemical staining;5.To explore the effect of silencing STING on the proliferation of transplanted tumor by immunohistochemical staining with Ki67.Results: Part 1: 1.q RT-PCR and western blot showed that there were differences in the expression levels of STING in human keratinocytes,cervical epithelial immortalized cells and four cervical cancer cell lines.Compared with normal cells,the expression of STING in the HPV-negative cervical cancer cell line C33 A were significantly decreased,the expression of STING in HPV-positive cervical cancer cell line(He La?Si Ha?Ca Ski)were significantly increased.2.The sh RNA targeting STING was successfully synthesized and the sh RNA fragment with the highest interference efficiency was screened out.The levels of STING in sh STING-He La and sh STING-Si Ha stable cell lines were statistically significantly decreased.Over-expressing lentiviral vector of STING was successfully constructed.The levels of STING in C33A-STING stable cell lines were statistically significantly decreased.3.In vitro proliferation rate,clonal formation ability,migration and invasion ability of sh STING-He La and sh STING-Si Ha cells were increased.After overexpressing of STING gene,the proliferation rate,clone formation ability,migration and invasion ability of C33 a cells were significantly decreased.Part 2: 1.Western blot showed that the expression level of total ?-catenin protein,activated ?-catenin protein and nucleus ?-catenin protein were significantly increased after silencing STING,and the expression level of downstream target genes(cyclin D1,c-myc,MMP9)in the wnt/?-catenin pathway were also increased.2.After up-regulating the expression of STING,the expression level of total ?- catenin protein,activated ?-catenin protein and nucleus ?-catenin protein were significantly increased after silencing STING,and the expression level of downstream target genes(cyclin D1,c-myc,MMP9)in the wnt/?-catenin pathway were also significantly decreased.3.Immunoprecipitation assay showed that endogenous STING could bind ?-catenin;4.When wnt/?-catenin pathway inhibitor was combined with down-regulating the expression of STING,the expression level of total ?-catenin protein,activated nonphosphorylated ?-catenin protein,nucleus ?-catenin protein and the expression level of downstream target genes(cyclin D1,c-Myc,MMP9)in wnt/?-catenin pathway were significantly lower than that in the group with knockdown of STING alone.5.When wnt/?-catenin pathway inhibitor was combined with down-regulating the expression of STING,the proliferation rate,clonal formation ability,migration and invasion ability of cells were significantly lower than in the knockdown group of STING alone.Part 3: 1.After the successful construction of subcutaneous xenograft tumor model in nude mice,the body weight of nude mice in the experimental group was lower than that in the control group,but the trend was not statistically significant.2.Tumor volume and growth rate in sh STING-He La group were significantly higher than those in the control group.3.HE staining showed that the tumor lesions formed in vector-He La group were closely arranged,with dark nuclei and oval cell morphology.Tumor lesions formed in sh STING-He La group showed disordered cell arrangement and abundant vascular tissue.4.Immunohistochemical staining showed that the expression level of STING was lower than that of the control group after tumor-forming of sh STING-He La cell lines constructed in vitro.5.Immunohistochemical staining showed that the expression level of Ki67 insh STING-He La group was significantly higher than that in vector He La group.Conclusions 1.Different types of cervical cancer cells have different expression levels of STING,but the cell biological effects are the same.STING can inhibit the proliferation,migration and invasion of cervical cancer cells,and play a role of tumor suppressor gene in the occurrence and development of cervical cancer.2.STING down-regulates the expression levels of total ?-catenin protein,activated ?-catenin protein,nuclear ?-catenin protein and downstream target genes(cyclin D1,cMyc,MMP9)in the wnt/?-catenin signaling pathway of cervical cancer.3.ICG001,an inhibitor of Wnt/?-catenin pathway,can significantly reverse the up-regulation of Wnt/?-catenin pathway related proteins in cervical cancer cells induced by silencing STING.4.ICG001,an inhibitor of Wnt/?-catenin pathway,can significantly reverse the up-regulation of proliferation,migration and invasion of cervical cancer cells induced by silencing STING.5.STING inhibits the occurrence and development of cervical cancer by inhibiting the Wnt/?-catenin signaling pathway.6.Silencing STING can promote the proliferation of cervical cancer cells in vivo.