The Role And Mechanism Of SGOL1 In The Development Of Non-small Cell Lung Cancer

Background and Objective: Lung cancer is the tumor with the highest mortality rate in the world,which is largely due to the absence of obvious early symptoms.At present,the main screening method is low-dose spiral CT screening,but it has its limitations,such as certain radiation damage,high cost,higher requirements for imaging doctors,false positive and false negative,etc.There is still no ideal method for early molecular diagnosis of lung cancer.Therefore,the study of the potential molecular mechanism of the occurrence and development of lung cancer is helpful to develop new diagnostic and predictive biomarkers and effective treatment strategies.Shugoshin-1(SGOL1)is one of the human homologues of yeast Shugoshin,which is located in the centromere region,which can prevent the precocious division of the centromere cohesion complex and maintain the stability of the chromosome.Genetic instability caused by human chromosome abnormalities may lead to tumorigenesis.In recent years,the role of SGOL1 in tumors has attracted much attention,and its role in tumors is controversial.Previous studies have shown that the low expression of SGOL1 in colorectal cancer is related to the poor prognosis of colorectal cancer,while the high expression of SGOL1 in liver cancer is related to the poor prognosis of liver cancer.At present,the role of SGOL1 in lung cancer is not clear.In this study,lentiviral vector was used to regulate the expression of SGOL1 in non-small cell lung cancer cell lines,and then to study the effect of SGOL1 on the biological behavior of tumor cells and its possible molecular mechanism.Methods(1)The expression of SGOL1 in non-small cell lung cancer tissues and its relationship with clinicopathological features were analyzed by bioinformatics.(2)The m RNA expression level of SGOL1 in normal lung epithelial cells and several non-small cell lung cancer cells were detected by q RT-PCR assays.Western Blot assays were used to detect the protein expression of SGOL1 in normal lung epithelial cells and sevaral non-small cell lung cancer cells.(3)Through the silence SGOL1 expression of SH-RNA lentiviral vector(SH-SGOL1)and lentiviral blank vector(SH-NC),two kinds of lung cancer cell lines A549 and NCI-H2405 in logarithmic phase were transfected in vitro.A549 cells stable strain and NCI-H2405 cells stable strain in silencing SGOL1 group and control group were successfully constructed,and the transfection effects were verified by q RT-PCR tests and Western Blot experiments.(4)CCK8 assays and clone formation assays were conducted to detect the effect of SGOL1 on the proliferation abilities of lung cancer cells in vitro using the A549 cells stable strain and the NCI-H2405 cells stable strain of the silenced SGOL1 group and the control group.The effects of SGOL1 on the metastasis and invasion of lung cancer cells in vitro were detected by scratch healing assays and Transwell assays.(5)The expression level of PKM2 in the TCGA database,was positively correlated with the expression level of SGOL1.Western Blott assays were used to detect the expression of PKM2 in A549 cell stable strain and NCI-H2405 cell stable strain in silenced SGOL1 group and control group.(6)The lentiviral vectors expressing PKM2(PKM2-plasmid)were transfected into A549 cells stable strain and NCI-H2405 cells stable strain cultured in logarithmic phase and treated with silenced SGOL1.A549 cells stable strain and NCI-H2405 cells stable strain overexpressing PKM2 were successfully constructed in silenced SGOL1 group,and the transfection effects were verified by q RT-PCR tests and Western Blot experiments.(7)The A549 cells stable strain and NCI-H2405 cells stable strain of silenced SGOL1 control group,silenced SGOL1 group and silenced PKM2 group were used to detect the changes of proliferation ability of lung cancer cells in vitro by CCK8 assays and clone formation assays,and the changes of metastatic and invasive abilities of lung cancer cells in different groups were detected by scratch healing tests and Transwell tests in vitro.Results(1)GEO database showed that the expression levels of SGOL1 m RNA in nonsmall cell lung cancer tissues were significantly increased,and the differences were statistically significant(****P<0.0001).Through the detection of normal lung epithelial cells and several non-small cell lung cancer cells by q RT-PCR assays,it was found that the m RNA levels of SGOL1 in lung cancer cells were significantly higher than that of normal lung epithelial cells,and the same results were obtained by using Western Blot assays to detect the protein levels of cell lines.The correlation between SGOL1 and the over survival times of lung cancer patients were analyzed by GEO database(GSE37745).It was found that the over survival times of non-small cell lung cancer patients with high expression of SGOL1 were significantly lower than that of patients with low expression of SGOL1,and the differences were statistically significant(P=0.0036).(2)The expressions of SGOL1 in A549 and NCI-H2405 cells were silenced by lentivirus-mediated SH-RNA,and the silencing effects were confirmed by Western Blot assays.The abilities of cell proliferation were detected by CCK8 assays,the results showed that the abilities of cells proliferation in the group of silenced SGOL1expression(SH-RNA)were significantly lower than that in the control group(SH-NC),and the abilities of cells clone formation were detected by clone formation assays,the results showed that the abilities of cells clone formation in the group of silenced SGOL1expressions(SH-RNA)were significantly lower than that in the control group(SH-NC),P<0.05.The cells migration abilities were detected by scratch tests,the results showed that the cells migration abilities of the group with silenced SGOL1 expressions(SHRNA)were significantly lower than that of the control group(SH-NC),and the invasive abilities of cells were detected by Transwell assays,the results showed that the invasive abilities of cells in the group of silenced SGOL1 expressions(SH-RNA)were significantly lower than that of the control group(SH-NC),P<0.05.(3)A positive correlation was identified between expression levels of PKM2 and SGOL1 in the TCGA database(r=0.38,P=0).Western Blot results showed that knockdown of SGOL1 in A549 and NCI-H2405 cells down-regulated PKM2 as well.In addition,co-interventions of SGOL1 and PKM2 were performed in A549 and NCIH2405 cells to clarify the interaction mechanism between them.The results from CCK8 assays,clone formation assays,scratch tests,Transwell assays showed that overexpression of PKM2 in lung cancer cells could partially reverse the regulatory effects of SGOL1 knockdown on proliferation,migration and invasion(P<0.05).Conclusions(1)SGOL1 were highly expressed in non-small cell lung cancer tissues and cells,and were associated with poor clinical prognosis.(2)Silenced SGOL1 expression can inhibit the proliferation,migration and invasion of non-small cell lung cancer cells.(3)Overexpression of PKM2 can partially reverse the regulatory effects of SGOL1 knockdown on proliferation,migration and invasion.