Function And Mechanism Of GBP1 In The Development And Progression Of Cervical Cancer

Objective:As a new biomarker,GBP1 plays an important role in many physiological processes,such as anti-infection,differentiation and migration of endothelial cells,so it is a multifunctional protein involved in many biological processes.At present,the role of GBP1 in tumor pathogenesis,drug resistance and immunotherapy has become the new hotspot.GBP1 plays different roles in different cancers.In this study,the potential biological function of GBP1 in cervical cancer was explored by bioinformatics combined with some experiments in vitro and in vivo,in order to lay the foundation for GBP1 as a new target for diagnosis and treatment of cervical cancer.Methods:1)The cervical cancer data in TCGA,HPA and other data were analyzed by various R-packages and online tools,and the cervical cancer tissue chips of clinical samples were stained by multiple immunofluorescence histochemistry,in order to analyze the expression of GBP1in clinical samples,its relationship with tumor infiltrating T lymphocytes and PD-1/PD-L1immune checkpoint molecules,Co X survival analysis was used to explore the relationship between GBP1 and prognosis of cervical cancer.2)The expression of GBP1 in Caski cell was inhibited in vitro by RNA interference,GBP1 was overexpressed by lentivirus overexpression test in Caski cell,the growth changes of Caski cells before and after transfection were detected by CCK8 test,the invasion ability changes were detected by Transwell chamber method,and the apoptosis changes was detected by flow cytometry.Nude mouse tumorigenesis experiment was used to construct a transplanted tumor model of cervical cancer and detect the growth of the tumor in vivo.3)RNA transcriptome sequencing was performed on Caski cell lines with over-expression and suppressed expression of GBP1.After data quality control,the differentially expressed genes were pathway enrichment analysis,then the data of alternative splicing events are mined and the alternative splicing genes are enriched and analyzed.Enhanced RNA immunoprecipitation technique was used to detect the binding of GBP1 to RNA in Caski cell line.Immunocoprecipitation combined with mass spectrometry tandem technique was used to detect GBP1-specific interacting proteins in GBP1 stably transformed cell lines,and these proteins were enriched and analyzed to find alternative splicing factors bound to GBP1.By analyzing the RNA sequencing data of He La cell line after HNRNPK overexpression(GSE148385 data set)and the e CLIP sequencing data of HNRNPK protein in K562 cell line and Hep G2 cell line(Encode data set),combined with the RNA sequencing data of GBP1 inhibited expression and overexpression in Caski cell line,the target gene of GBP1regulating alternative splicing through HNRNPK was explored.Results:1)TCGA online database analysis showed that GBP1-m RNA was widely expressed in 33 common malignant tumors,and cervical cancer ranked the top three.In cervical cancer,GBP1 has a significant positive correlation with genes such as GBP4,CXCL10,TAP1,STAT1 and GBP1P1,and a significant negative correlation with genes such as SNORC,VPS37D,SPINK1,HES6 and CDHR3.The expression of GBP1 was significantly correlated with19 immunosuppressant molecules,35 immunostimulant molecules and 26 chemokines.Through six algorithms,it was found that GBP1 had a strong positive correlation with most immune infiltrating cells.The expression of GBP1 in CESC was significantly correlated with long overall survival rate,disease-specific survival rate and progression-free survival rate,and the hazard ratios were 0.61,0.50 and 0.49,respectively.Immunohistochemical results in HPA database showed that GBP1 protein could be detected in both cervical adenocarcinoma and cervical squamous cell carcinoma,mainly expressed in cell membrane and cytoplasm.Analysis of single cell sequencing data showed that GBP1 was expressed in all kinds of common cell types in cervical cancer tissue microenvironment.Based on the analysis of Cell Miner online database,it was found that the sensitivity of Cediran IB,Blu-667,JNJ-42756493 and Pazopanib were related to the expression of GBP1.In 104 clinical samples of cervical cancer,the percentage of CK+GBP1+cells was positively correlated with the percentage of CK+PD-L1+cells(r_s=0.844,P<0.01).The strong positive expression of GBP1 in cancer cells was a prognostic risk factor for cervical cancer,and the HR values of univariate and multivariate analysis were 3.736 and 2.541,respectively.2)Compared with si-NC transfection group,the inhibition rate of GBP1 m RNA and protein in si-GBP1 transfection group reached 86.4%,and 75.0%;the proliferation ability of si-GBP1 transfection group at 48 hours and 72 hours was 87.1%and 82.5%respectively,which indicated that the expression of GBP1 was inhibited and the cell proliferation was inhibited;the invasive ability of si-GBP1 transfection group decreased to 30.0%of that of si-NC transfection group,indicating that inhibiting GBP1 expression inhibited cell invasion;the percentage of apoptotic cells in the si-GBP1 transfection group increased from 16.51%to 24.40%,indicating that the inhibition of GBP1 expression promoted the apoptosis of Caski cells.In the experiment of GBP1 overexpression,compared with OE-NC transfection group,the m RNA expression of GBP1 in OE-GBP1 transfection group increased to 6.9 times;the proliferation ability of OE-GBP1 transfection group was 1.08 times,1.29 times and 1.20times at 24 hours,48 hours and 72 hours,respectively,indicating that overexpression of GBP1 promoted cell proliferation;the invasion ability of OE-GBP1 transfection group increased to 2.65 times,which indicated that overexpression of GBP1 promoted the invasion of Caski cells.Compared with OE-NC transfection group,the proportion of apoptotic cells in OE-GBP1 transfection group did not change.In the subcutaneous tumorigenesis test of nude mice,compared with the negative control group,the final tumor volume and weight of GBP1 overexpression group increased significantly(P<0.01).It shows that GBP1 overexpression could promote tumor growth in animals.3)The RNA transcriptome sequencing of Caski cell line after overexpression and inhibition of GBP1confirmed that the differentially expressed genes of GBP1 were closely related to apoptosis,viral carcinogenesis and other pathways.At the same time,it was found that the expression of GBP1 was related to a large number of RNA alternative splicing events,suggesting that GBP1 may play a carcinogenic role by directly or indirectly affecting the alternative splicing of target genes in cervical cancer.However,the i RIP-seq test proved that GBP1 is not an RNA binding protein,indicating that it can not be used as an alternative splicing factor to cause cancer by alternative splicing.Immunocoprecipitation combined with mass spectrometry tandem technology found that the classical alternative splicing factors such as DHX15,HNRNPK,PRPF6,S ART1,SRSF2,SRSF7,U2AF1 and U2AF2 were specific interacting proteins of GBP1.Through the analysis of sequencing data and online data,combined with existing reports,GBP1-HNRNPK-MIR4435-2HG and GBP1-HNRNPK-CD44 are two valuable regulatory pathways for carcinogenesis of GBP1 in cervical cancer.Conclusion:GBP1 is widely expressed in cervical cancer,and is associated with poor prognosis,so it is a potential cancer-promoting molecule.Although GBP1 is related to a large number of alternative splicing events,it is not an alternative splicing factor.It indirectly regulates the alternative splicing of target genes including CD44 and MIR4435-2HG by combining with a series of classical alternative splicing factors such as HNRNPK,and finally produces a cancer-promoting effect.