| Cervical cancer is one of the most common malignant tumors in women,among which squamous cell carcinoma accounts for about 85%,adenocarcinoma accounts for about 10-15%,and squamous cell carcinoma accounts for 3%.At present,the main risk factor of cervical cancer is the continuous infection of high-risk human papillomavirus.However,the mechanism of tumor progression is complex,and the molecular mechanism of cervical cancer is still not fully understood.Autophagy is an evolutionally conserved lysosomal dependent metabolic pathway for degrading abnormal proteins,damaged organelles and exogenous microorganisms in cells,and it is also a catabolic process of cell recycling.Small RNA(miRNA,miRNA)was a kind of small molecular endogenous non-coding RNA,gene expression can be regulated at the post-transcriptional level by it.It can be involved in the process of tumorigenesis and development through the mechanism of locating tumor-associated genome,regulating epigenetics,processing related genes and their proteins.MiRNA targets and binds autophagy related genes to act on autophagy pathways,thereby regulating the process of autophagy and participating in the pathogenesis of a variety of malignant tumors,such as gynecological malignant tumors,but also other tumors:lung cancer,liver cancer,breast cancer and so on.Most of the studies on mir-1246 and tumors believe that it is an oncogene MiRNA,which can promote the occurrence and development of tumors.Our previous study found that mir-1246 can promote the growth,invasion and migration of SiHa cells of cervical squamous cell carcinoma and has the role of proto-oncogene,which can accelerate the development process of cervical squamous cell carcinoma.However,the relevant mechanism of mir-acting on the tumor development process is not determined by a single target or pathway.Whether mir-1246 is related to the regulation of autophagy in the process of promoting the growth and invasion of SiHa cells is not clear.In this study,we screened the target gene GAS1 related to mir-1246 and autophagy,and verified its binding to specific targets.By verifying the autophagy effect of mir-1246 and GAS1 on SiHa cells,as well as the influence on the growth and invasion ability of cells,it was confirmed that the regulation of autophagy by mir-1246 may be one of the molecular mechanisms for the occurrence and development of cervical squamous cell carcinoma.In addition,we also analyzed the expression of GAS1 in cervical squamous cell carcinoma,as well as its relationship with clinicopathological factors and prognosis.This study provides new train of thought for miR-1246 control the molecular mechanism of autophagy in cervical cancer,also provide new theoretical basis for targeted treatment options based on miR-1246 and GAS1.Part I mir-1246 regulates the autophagy of SiHa cells in cervical cancer and verified the its target gene GAS1Objective:To confirm mir-1246 regulates the autophagy of SiHa cells in cervical cancer,as well as its target gene GAS1.Methods:Using western blot to detect the autophagy key protein LC3 Ⅱ,P62 expression in miR-1246 mimic,miR-1246 inhibitor,negative control group(NC),and Blank control group(Blank).The number of autophagosomes in the four groups was compared by counting the number of GFP-LC3 light points under a fluorescence microscope.The target genes related to autophagy were predicted in the biological information database,Pubmed and KEGG databases to select the target genes.Western blot was used to detect the effect of mir-1246 on the protein expression of the target gene,double luciferase method was used to verify the specific binding targets of the target gene,and the recovery experiment experiment was used to verified that GAS1 was a functional target gene of mir-1246.Results:Compared with the NC group,LC3Ⅱ expression was decreased in mir-1246-inhibitor group(P=0.016)and increased in mir-1246-mimic group(P<0.001).On the contrary,The expression of P62 was up-regulated in mir-1246-inhibitor group(P<0.001)and down-regulated in mir-1246-mimic group(P=0.002).The expression of GFP-LC3 fusion protein in cells was observed under fluorescence microscope,Compared with the NC group,the number of GFP was decreased in the mir-1246-inhibitor group(P=0.008)and increased in the mir-1246-mimic group(P=0.006).According to the prediction in the biological information database and the query results in Pubmed and KEGG,GAS 1 was selected as the target gene.The western blot results showed that mir-1246 could down-regulate the expression of GAS 1,the double luciferase method confirmed that there were binding targets of mir-1246 on GAS1,and the recovery experiment verified that overexpression of GAS1 could reverse the enhanced autophagy effect induced by upregulation of mir-1246.Conclusion:GAS1 is an autophagy related target gene of mir-1246.By binding to the site on GAS1,mir-1246 down-regulates the expression of GAS1 and enhances the autophagy of SiHa cells.Part Ⅱ GAS1 regulating the autophagy and the influence on the proliferation,migration and invasion ability of cervical cancer SiHa cells Objective:To verify the regulating effect of GAS1 on the autophagy and the influence on proliferation,migration and invasion of of cervical cancer SiHa cells.Methods:Western blot was to use to detected the autophagy key protein LC3,P62 expression in GAS1 over-expression group(LV-GAS1),negative control group(NC)and blank control group(Blank),each group autophagy key protein LC3 Ⅱ,P62 expression;the changes of autophagy flow in each group were monitored by confocal microscopy;the autophagosomes or autophagic vesicles in each group were observed by transmission electron microscopy.CCK8 proliferation experiment,scratch experiment,Transwell migration and invasion experiment were used to verify the influence of GAS1 on the proliferation,migration and invasion ability of SiHa cells.The xenograft tumor model of nude mice was established,and the growth curves of xenograft tumor in each group were analyzed and compared.Results:Compared with the NC group,the expression of P62 was up-regulated in lv-gas1 group(P=0.016),while the expression of LC3II was down-regulated in LV-GAS1 group(P=0.010).After the addition of chloroquine,the protein amount of LC3 II in the NC+chloroquine group was significantly higher than that in the NC group(P<0.0001),while that in the LV-GAS1+chloroquine group was also slightly higher than that in the lv-gasl group(P=0.002),however,the increased level was significantly lower than that in the NC+chloroquine group.Compared with NC group,autophagy flow monitoring showed the autophagy flow in LV-GAS1 group was significantly reduced(P=0.009);transmission electron microscopy showed the number of autophagosomes in LV-GAS1 group was significantly decrease(P=0.020).The cell function experiment showed that the cell growth of LV-GAS 1 group was relatively slow compared with the NC group(P<0.001).The scratch healing rate decreased significantly at 24h and 48H(P<0.001,P<0.001).The number of transmembrane transfection was significantly decreased in the transwell experiment(P<0.001,P<0.001).In the tumor transplantation experiment of nude mice,the LV-GAS 1 group showed slower tumor growth(P<0.001)and smaller tumor volume than the NC group(P<0.001).Conclusion:GAS1 inhibited the autophagy of SiHa cells and the ability of cell proliferation,migration and invasion.Part Ⅲ The expression and clinical significance of GAS1 in cervical squamous cell carcinomaObjective:To analyze the expression of GAS1 and its relationship with clinicopathological factors and prognosis in cervical squamous cell carcinoma.Methods:Seventy-two cases of cervical squamous cell carcinoma tissue specimen(Ⅰa-Ⅱb)and 40 cases of normal cervical tissue specimen were selected in Department of gynecology,the Affiliated Tumor Hospital,Guangxi Medical University from July 2013 to June 2016.Real-time fluorescence quantitative PCR and immunohistochemical method were used to detect the difference of GAS1 expression between cervical squamous cell carcinoma and normal cervical tissue,and to analyze the correlation between GAS1 expression and clinicopathological factors,as well as its relationship withthe prognosis of patients.Results:The expression of GAS1 mRNA in cervical squamous cell carcinoma was significantly lower than that in normal cervical tissue,P<0.001.The high expression rate of GAS1 protein in cervical squamous cell carcinoma was significantly lower than that in normal cervical epithelium,P<0.001.The expression of GAS1 were correlated with tumor size(P=0.038),cervical interstitial infiltration depth(P<0.001),lymph-vascular space involvement(LVSI)(P=0.026),lymph node(LN)metastasis(P=0.010)and parametrial infiltration(P=0.015).The patients with low GAS1 expression have shorter disease free survival(DFS)and overall survival(OS)than those with high GAS1 expression(P=0.029;P=0.046).The results showed that the patients in GAS1-and LN+group had the lowest DFS and OS,followed by GAS 1+and LN+/GAS1-and LN-group,and GAS1-and LN-group had the highest DFS and OS(P=0.004,P=0.018);GAS1 and LVSI+group had the lowest DFS,followed by GAS1+and LVSI+/GAS 1-and LVSI-group,while GAS 1+and LVSI-group had the highest DFS(P=0.039).There was no statistical difference in OS among the groups(P=0.103).Conclusion:The expression of GAS1 in cervical squamous cell carcinoma is lower than that in normal cervical tissue,and it is correlated with some clinicopathological factors.Patients with low expression of GAS1 have relatively poor prognosis than patients with high expression of GAS1.GAS1 combined with lymph nodes and LVSI can enhance the evaluation of the prognosis. |
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