Study Of The Role And Mechanism Of GNL3L In The Progression Of Esophageal Squamous Cell Carcinoma

Background:Esophageal cancer(EC)ranked sixth in global cancer mortality,with a 5-year survival rate of less than 20%,and a median survival time of less than 1 year.EC falls into two types with different epidemiology and pathology: esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).Among them,ESCC is the most common type,accounting for approximately more than 90% of all EC.Despite the adoption of non-invasive screening and endoscopic techniques,which enable early detection and treatment measures application,nearly half of the patients are still diagnosed with metastasis or unresectable tumor.EC is insensitivity to chemotherapy,and the application of targeted drugs failed to improve the survival rate significantly.Therefore,it is particularly necessary to further elucidate the molecular mechanism of the development and progression of EC to provide not only new targets for drug development,but also new markers to predict the prognosis of patients,so as to better guide treatment and improve patient prognosis.GNL3L(guanine nucleotide-binding protein-like 3-like)is a new,evolutionarily conserved GTP binding nucleoprotein,which belongs to HSR1-MMR1 subfamily members of GTPase.In recent years,the role of GNL3L in cell proliferation and apoptosis has received increasing attention.The role of GNL3L in tumorigenesis and progression has been demonstrated in human gastric cancer,breast cancer,cervical cancer,hepatocellular cancer,colorectal cancer and glioma cells.For example,in colorectal cancer,miR-4454 overexpression suppressed GNL3L production and ameliorated drug resistance of cancer through NF-?B pathway.However,the role of GNL3L in ESCC cells and the associate signaling pathways have not been clearly investigated.Therefore,this study intended to investigate the biological functions of GNL3L in the proliferation,migration invasion and apoptosis of ESCC,further elucidate its mechanism of action,and provide new strategies and corresponding theoretical basis for targeted therapy and prognosis prediction of ESCC.Methods:(1)The expression and clinical value of GNL3L in ESCC.1)The expression of GNL3L in ESCC and normal esophageal tissues and its impact on the prognosis of patients were analyzed through the Gene Expression Profiling Interactive Analysis(GEPIA)database.2)Correlation analysis by Linked Omics further confirmed the clinical value of GNL3L;gene enrichment analysis was performed to predict possible signal pathways.(2)The biological function of GNL3L in the progression of ESCC.1)Using GNL3L siRNA,GNL3L overexpression plasmids and negative control NC to transfect Eca-109 and KYSE-150 ESCC cells,and established GNL3L-knockdown and GNL3L-overexpression cell lines.2)The successful establishment of expected cell lines was confirmed by Western Blot assay.The effects of knockdown and overexpression of GNL3L on the proliferation,migration,invasion and apoptosis of ESCC cells were detected by using CCK8 assay,clone formation assay,cell scratch assay,Transwell assay and flow cytometry.Western Blot was utilized to detect the expression levels of apoptosis-related proteins(Bcl-2,Bax and Caspase3-p17)in GNL3L knockdown cells.3)Establish the subcutaneous tumor model of human derived ESCC in nude mouse,and divided into NC group,GNL3L-KD group and GNL3L-OE group.The changes of tumor size and weight were recorded.(3)Explore the related signaling pathway of GNL3L in ESCC progression.1)Using Western Blot to detect related protein expression in RAS pathway.2)Cells were treated with antineoplaston A10,a natural inhibitor of RAS signaling pathway,and CCK8 assay was used to detect cell proliferation flow cytometry was used to detect cell apoptosis.(4)Statistical analysis.All in vitro experiments were repeated for three times separately.All data in this experiment were expressed as mean ± SD.Graph Pad Prism 7 was used for graphing and statistical analysis.A p value less than 0.05 was considered statistically significant.Results:(1)Differential expression of GNL3L in ESCC and its clinical values.GNL3L expression level in ESCC tissue was significantly higher,and patients with high GNL3L expression levels had a shorter survival time.GNL3L expression levels were related with residual tumor tissues races.Genes related to GNL3L were abundantly expressed in tumor apoptosis pathway and RAS signaling pathway.(2)GNL3L affects the proliferation,migration,invasion and apoptosis of ESCC.Knockdown GNL3L significantly inhibited the proliferation,migration and invasion of ESCC cells,promoted cell apoptosis;while overexpression of GNL3L possessed the opposite effects.And the apoptotic biological function was realized through Bcl-2/Bax axis and Caspase cascade reaction.In nude mice subcutaneous tumor model,the inhibition of tumor proliferation of GNL3L knockdown were further confirmed,so as to the pro-proliferation function of GNL3L overexpression.(3)GNL3L affects the biological behavior of ESCC through the RAS signaling pathway.Knockdown and overexpression of GNL3L affected the expression of RAS pathway protein,such as Ras and Raf.The GNL3L overexpressing cell line was treated with antineoplaston A10,and the cell proliferation and anti-apoptosis effect of GNL3L were inhibited.Conclusions:In this study,we showed that GNL3L expression was increased in ESCC and related to prognosis.The expression level of GNL3L affected the proliferation,migration and invasion of ESCC cells,and inhibited cell apoptosis.GNL3L might influence the expression of RAS pathway protein,and Antineoplaston A10,a natural RAS pathway inhibitor,inhibited the pro-proliferation and anti-apoptosis effects of GNL3L,we supposed that GNL3L realized its biological function through RAS signaling pathway.The pro-proliferation effects of GNL3L were further confirmed in the subcutaneous tumor model of human derived ESCC in nude mouse.