G Protein Regulated Inducer Of Neurite Outgrowth 1 Is A Potential Marker For Non-small Cell Lung Cancer Prognosis

BackgroundLung cancer is still the leading cause of cancer death in both men and women around the world.A total of 2.2 million new lung cancer diagnoses are expected in 2020,accounting for 11.4 percent of the global cancer burden.Each year,the number of people diagnosed with lung cancer rises.The most frequent histological type of lung cancer is non-small cell lung cancer(NSCLC),which accounts for more than 85 percent of all lung malignancies.NSCLC doesn't really have early obvious symptoms.At the time of diagnosis,around 40-50 percent of NSCLC patients have locally advanced or metastatic illness and have lost the opportunity of radical surgery.There are a variety of treatment methods for NSCLC.Surgical treatment is the first choice for early NSCLC,while a combination of various methods is used for the advanced NSCLC patients.Chemotherapy remains a moderately successful treatment option for advanced NSCLC.Clinical proofs of concept for targeted therapy in patients with sensitive gene mutations have been generated.Immunotherapy,as an emerging force in the treatment of NSCLC,has also made breakthrough progress.However almost all patients develop resistance after a period of treatment,which will eventually lead to tumor recurrence or metastasis.The prognosis for recurrent disease and relapsing patients is very poor and it is even worse in those who relapse during the first 24 months.As a result,the 5-years survival rate of patients with advanced NSCLC is only between 4%to 17%.Therefore,there is an urgent need for novel therapeutic strategies to improve survival of patients with NSCLC.The occurrence and development of NSCLC are affected by many factors such as heredity,environment and are closely related to the structure and expression regulation of a large number of related genes in human body.Recently,new insights on molecular mechanism of NSCLC have lead to further search for tumor markers and viable therapeutic targets that can improve the amount of lung cancer early detection and treatment.G protein regulated inducer of neurite outgrowth 1(GPRIN1)is a protein encoding gene.The GPRIN1 protein consists of 856 amino acids and mainly localized on the cell membrane and cytoplasmic vesicles.GPRIN1 protein can specifically recognize the activated G protein which acts like a "molecular switch" in signal transduction between cell membrane receptor and effector protein.G protein play a substantial role in cell growth,proliferation,differentiation and the maintenance of cellular signal transduction.Changes in the structure and function of G protein can lead to abnormal cell signal transduction.Some of these aberrant signaling circuits may promote cancer cell proliferation and the spread of primary tumor lesions to other organs.Aberrant activity or expression of G protein have also been identified in several cancers,such as lung cancer,stomach cancer,breast cancer,colorectal cancer and so on.GPRIN1 gene mutations can lead to dysregulation of G protein activity,which is related to cancer initiation and progression.According to the TCGA gene bank's study of RNA sequencing data and clinical data,we found that GPRIN1 was differentially expressed in lung adenocarcinoma and normal lung tissue.The expression level of GPRIN1 was closely related to the clinicopathological characteristics and prognosis of lung adenocarcinoma patients.Therefore,it can be speculated that GPRINI plays a critical role in the pathogenesis of NSCLC.So far,no systematic studies have been conducted on the role and mechanism of GPRIN1 in NSCLC.As a result,elucidating the function and regulation mechanism of GPRIN1 in NSCLC may aid in the development of novel therapeutic opportunities for NSCLC prevention and treatment.ObjectiveIn this study,we examined the expression of GPRIN1 gene in NSCLC and analyzed its correlation with clinical characteristics,survival and prognosis of NSCLC patients.We also studied the effects of GPRIN1 gene on proliferation,clonal formation,migration,invasion and epithelial mesenchymal transformation(EMT)of NSCLC cells.We created an NSCLC cell xenograft model in nude mice to see if GPRIN1 has any effect on the formation of xenograft tumors.MethodsPart ?:1.Before this study,we obtained data related to GPRIN1 gene expression and clinical characteristics of patients with lung adenocarcinoma by using data array analysis of TCGA database.2.We selected 100 paraffin-embedded tissue samples of non-small cell lung cancer and 40 adjacent normal lung tissue samples to make tissue microarray.3.GPRIN1 expression was detected using immunohistochemistry in 100 NSCLC tumors and 40 neighboring normal lung tissues.4.We used statistical methods to analyze the difference of GPRIN1 expression in NSCLC tissues and adjacent normal lung tissues.5.We also analyzed the correlation between GPRIN1 expression and clinical characteristics of NSCLC patients,such as age,sex,smoking history,TNM stage and tissue differentiation.6.According to the follow-up data of NSCLC patients,we figured out the correlation between GPRIN1 expression and patient survival.Part ?:1.A549,NCI-H209,NCI-H460,Calu-1 human lung cancer cell lines and normal control cells(BEAS2B)were were cultured in vitro.2.qRT-PCR were conducted to detect GPRIN1 expression in lung cancer cells and normal control cells.3.Western-blot assay were conducted to detecte the expression of GPRIN1 protein in each group.4.Cell lines with knockdown/over-expression of GPRIN1 were constructed,and a negative control group was constructed.5.CCK8 was used to measure the effect of knockdown or over-expression of GPRIN1 on proliferation ability of NSCLC cells.6.Plate colony formation was used to dectect the effect of knockdown or over-expression of GPRIN1 on the clonogenic ability of NSCLC cells.7.The effect of GPRIN1 knockdown or overexpression on the migratory ability of NSCLC cells was investigated using the Transwell cell migration and invasion experiment.8.By using the Western-blot experiment,the effects of GPRIN1 on the expression of E-cadherin,N-cadherin,Vimentin,and Snail proteins in EMT were discovered.Part ?:1.The stable expression cell lines of GPRIN1 knockout group,GPRIN1 overexpression group and their corresponding control groups were constructed by lentivirus vector.2.To create a subcutaneous tumor model in nude mice,transfected A549 cells and Calu-1 cells were implanted subcutaneously.3.Every week,the tumor volume was determined by measuring and recording the long and short diameters of nude mice in each group with a vernier caliper.4.At the fifth week,and tumors were completely stripped and weighed after death of nude mice.5.Immunohistochemistry was used to detect the expression of Ki-67,E-cadherin,N-cadherin,and Snail proteins in subcutaneously implanted tumors of nude mice.ResultsPart ?:1.GPRIN 1 protein was mostly found in the cell membrane and cytoplasm of NSCLC cells,according to immunohistochemical staining study of GPRIN 1 protein expression in 100 NSCLC tissues.2.The high expression rate of GRPIN1 protein in 100 NSCLC tissues was 65%(65/100).In the normal group,the high expression rate was just 27.5 percent(11/40).These findings suggested that GPRIN 1 protein expression was higher in NSCLC than in normal lung tissues(P<0.05).3.The high expression of GPRIN 1 protein was correlated with the degree of tumor differentiation in NSCLC patients(P<0.05).The level of GPRIN1 expression in NSCLC patients had no significant relationship with their gender,age,smoking status,T,N,M,or TNM stage(P>0.05).The higher the level of GPRIN1 expression,the lower the degree of tumor tissue differentiation,according to these findings.4.Patients with high GPRIN 1 protein expression had a significantly shorter overall survival time than those with low GPRIN1 protein expression,suggesting that high GPRIN1 expression was associated with poor prognosis(P=0.004).5.GPRIN1 expression level,N,M,and TNM stage were all associated to OS in NSCLC patients,according to the Univariate Cox regression analysis(P<0.05).GPRIN1 expression level and TNM stage were found to be independent adverse prognostic variables in NSCLC patients in the Multivariate Cox regression analysis(P<0.05).Part ?:1.In lung cancer cell lines A549,NCI-H209,NCI-H460 and CALU-1,GPRIN1 mRNA expression was substantially higher than in normal lung epithelial cell lines BEAS2B(P<0.01).2.We used A549 cell line to construct a low GPRIN1 expression group and CALU-1 cell line to construct a high GPRIN1 expression group.At the same time,their negative control group was constructed.3.The qRT-PCR and Western-blot showed that the expression of GPRIN1 mRNA and protein in A549 cells reduced compared to the control group after the GPRIN1 gene was knocked down.After overexpression of GPRIN1 gene,the mRNA and protein expression of GPRIN1 increased in Calu-1 cells.The difference was statistically significant(P<0.01).4.The proliferation rate of A549 cells after GPRIN1 knocked down was considerably lower than the control group in a CCK8 cell proliferation experiment(P<0.01).The proliferation rate of CALU-1 cells which overexpressed GPRIN1 was significantly increased compared with the control group(P<0.01).5.The number of cell clones created in the knockdown GPRIN1 group was much higher than in the control group,according to a clone formation experiment(P<0.01).The number of cell clones formed in GPRIN1 overexpression group was lower than that in control group(P<0.01).6.The migration and invasion capabilities of A549 cells were considerably reduced after GPRIN1 was knocked down compared to the control group in the Transwell cell migration and invasion experiment(P<0.01).After overexpression of GPRIN1,the migration and invasion ability of CALU-1 cells were significantly enhanced compared and the difference was statistically significant(P<0.01).7.In A549 cells,knocking down GPRIN1 expression lowered the expression of N-cadherin,Vimentin,and Snail,while increasing the expression of E-cadherin in the Western-blot experiment(P<0.01).The opposite effect was observed in CALU-1 cells overexpressing GPRIN1(P<0.01).Part ?:1.Tumor growth was slower in the GPRIN1 deletion group than in the control group,whereas tumor growth was faster in the GPRIN1 overexpression group than in the control group(P<0.05).2.The weight of subcutaneous tumors in nude mice after GPRIN1 deletion was lower than in the control group,and the weight of subcutaneous tumors in nude mice after GPRIN1 overexpression was higher than in the control group(P<0.05).3.The expression level of Ki-67 protein in tumor tissue sections of nude mice reduced after GPRIN1 deletion compared to the control group,and the expression level of Ki-67 protein in tumor tissue sections increased after GPRIN1 overexpression compared to the control group(P<0.05).4.After GPRIN1 deletion,E-cadherin expression in nude mice tumor tissues was higher than in the control group,whereas N-cadherin and snail expression were significantly reduced.When GPRIN1 was overexpressed,E-cadherin expression reduced compared to the control group,whereas N-cadherin and snail protein expression increased compared to the control group.(P<0.05).ConclusionsPart ?:1.GPRIN1 protein expression was higher in NSCLC tissues than in neighboring normal lung tissues.2.GPRIN1 protein is mainly located in the cell membrane and cytoplasm of NSCLC cells3.The expression level of GPRIN1 protein was correlated with the degree of tissue differentiation,regional lymph node classification and clinical stage of NSCLC patients.4.GPRIN1 protein expression is an independent predictor of NSCLC prognosis.Part ?:1.GPRIN1 gene expression was higher in NSCLC cells than in normal lung epithelial cells.2.The high expression of GPRIN1 can promote the proliferation,clonal formation,migration and invasion of NSCLC cells.3.GPRIN1 can promote NSCLC invasion and migration through EMT by regulating the expression of-related factors.Part ?:1.Knockout of GPRIN1 can inhibit tumor growth in nude mice,and overexpression of GPRIN1 can promote tumor growth in nude mice.2.Knockout of GPRIN1 decreased the proliferation of NSCLC cells in vivo,and overexpression of GPRIN1 promoted the proliferation of NSCLC cells in vivo.3.Knockout of GPRIN1 can inhibit the EMT process of NSCLC cells in nude mice,and overexpression of GPRIN1 can promote EMT in tumor cells.