Screening Of RNA-binding Proteins Associated With Prognosis And Analyzing The Biological Function Of BOP1 In Lung Adenocarcinoma

Objectives To screen the abnormally expressed RBPs associated with prognosis in lung adenocarcinoma(LUAD),and to explore the expression,clinical significance,potential biological function and specific regulatory mechanisms of the key RNA-binding protein BOP1 in LUAD.Methods(1)The transcriptome data and the corresponding clinical data of LUAD were downloaded from the Cancer Gene Atlas(TCGA)database by using R software packages.Differentially expressed RBPs were identified by using the DESeq2 package.Then,the gene co-expression network analysis,survival analysis,least absolute shrinkage and selection operator(LASSO)regression analysis were implemented to obtained core RBPs associated with LUAD prognosis.(2)The expression and prognostic value of BOP1 in 33 tumors were analyzed by using multi-omics data.A series of analyses were conducted to further explore the mutations and copy number alterations of BOP1 in LUAD.The relationship between BOP1 expression and clinical results of patients with LUAD were also assessed.(3)The expression of BOP1 in LUAD tissues and cells was verified by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction(RT-qPCR).(4)LUAD cell line A549 or PC-9 was stably transfected by lentivirus(LV-BOP1 or LV-pCDH-BOP1)to increase or knock down BOP1 expression.The clonogenic assay,wound healing test and transwell assay were used to evaluate the proliferation,migration and invasion abilities of all tumor cells.(5)The expression of JNK pathway-,EMT-and angiogenesis-related genes in A549 and PC-9 cells with BOP1 overexpression and knockdown were detected by western blotting and/or quantitative PCR.(6)A549 cells transfected with LV-pCDH-BOP1 or LV-pCDH vector were injected into nude mice to construct a subcutaneous xenotransplant tumor model.Immunohistochemical techniques were used to further verify the mechanism of BOP 1 promoting the growth of LUAD.Results(1)The differentially expressed RBPs between LUAD tumor tissue and adjacent non-tumor lung tissue were identified,including 124 highly expressed RBPs and 40 low expressed RBPs in tumor tissue.Among them,BOP 1,EXO1,GAPDH,IGF2BP1 and SMAD9 are key RBPs closely related to the overall survival of patients with LUAD.(2)The mRNA of BOP1 was abnormally expressed in 27 types of tumors including LUAD,and its expression level was correlated with the overall survival(OS)of patients with 14 types of cancer including LUAD.The expression of BOP1 protein was significantly higher in at least 6 tumors including LUAD than that in corresponding normal tissues.(3)The level of BOP1 in LUAD cells was higher than that in normal bronchial epithelial cells(P<0.05),and the expression of BOP 1 was significantly increased in LUAD compared with adjacent non-tumor lung tissue(P<0.05).The expression of BOP1 was positively related to the clinical stage and tumor size of patients with LUAD(P<0.05),and negatively related to the OS and disease-specific survival(DSS)of tumor patients(P<0.05).LUAD patients who with high BOP1 expression were more likely to develop lymph node metastasis.(4)The expression of BOP 1 in LUAD is closely related to a lot of biological pathways,such as tumorigenesis,DNA damage repair,immune regulation,epithelial-mesenchymal transition(EMT),RNA processing and cell cycle.(5)The proliferation,migration and invasion abilities of PC-9 and A549 cells with up-regulated BOP1 expression were significantly stronger than those of corresponding wild-type LUAD cells and tumor cells transfected with empty vector(P<0.05),while the proliferation,migration and invasion abilities of BOP1-silenced A549 and PC-9 cells were significantly reduced(P<0.05).(6)The expression levels of JNK and phosphorylated JNK(p-JNK)in BOP 1-overexpressing cells were significantly increased(P<0.05),the expressions of EMT-related marker proteins Snail and N-cadherin were significantly augmented,E-cadherin was obviously reduced(P<0.05),and the expressions of angiogenesis-related genes VEGFA and PCNA were significantly higher than those in the control group(P<0.05),while downregulation of BOP 1 showed that the expressions of JNK,p-JNK,Snail,N-cadherin,VEGFA and PCNA were significantly decreased(P<0.05),E-cadherin was obviously augmented(P<0.05).Compared with the control group,the promoting effect of BOP1 on the phosphorylation of JNK,as well as its downstream effector gene c-Jun,Snail,N-cadherin,PCNA and VEGFA was significantly weakened when treating with SP600125(a specific inhibitor of JNK)in BOP 1-overexpressing A549 and PC-9 cells(P<0.05),while the inhibitory effect on the expression of E-cadherin were also significantly weakened(P<0.05).(7)Compared with the control groups,the outcomes of the tumorigenesis research in nude mice indicated that the overexpression of BOP 1 could significantly promote the growth of the xenotransplanted tumor,while BOP1 silencing the growth rate of the xenotransplanted tumor was significantly slowed down(P<0.05).After treating with SP600125,the growth of LUAD xenografts showed an overall inhibiting trend,but the weight and volume of the xenografts in the BOP1 overexpression group were not significantly different from those in control groups(P>0.05),while the volume of xenografts in the BOP1 silencing group was significantly smaller than that in the control group(P<0.05),and no significant difference was found between the weight of the transplanted tumor and the control group(P>0.05).Moreover,compared with the empty vector group,the proportion of Ki67 positive cells in the transplanted tumor tissue of the BOP 1 overexpression group was significantly increased(P<0.05).(8)The results of microvessel density(MVD)counting of CD34-labeled LUAD vascular endothelial cells showed that the MVD in tumor tissues with high BOP1 expression was significantly higher than that in low BOP1 expression tissues(P<0.05),and the MVD was positively correlated with BOP1 expression in LUAD samples(r=0.443,P<0.01).Conclusions(1)The differentially expressed RBPs between LUAD tumor tissue and adjacent non-tumor lung tissue were identified.Among them,BOP1,EXO1,GAPDH,IGF2BP1 and SMAD9 are key RBPs closely related to the overall survival of patients with LUAD.(2)The expression of BOP1 was significantly increased in LUAD tissues and cells,was positively correlated with the tumor size and clinical stage,was negatively related to the OS and DSS,and patients with high BOP1 expression are more likely to develop lymph node metastasis.(3)Over-expression of BOP1 enhanced the proliferation,migration and invasion of LUAD A549 and PC-9 cells.(4)BOP1 regulate EMT and angiogenesis by activating the JNK/c-Jun signaling pathway to promotes the growth and progression of LUAD.