| Background:Pancreatic adenocarcinoma(PAAD)is a very malignant tumor with a five-year survival rate of less than 10%.Because it lacks specific early clinical symptoms and clinical markers,many patients have missed the timing of surgery at presentation.There is extensive metabolic reprogramming in pancreatic cancer cells,which is closely related to the occurrence and development of pancreatic cancer.Rab10 is a protein of the RAS superfamily,and its high expression is associated with tumor proliferation,metastasis,and invasion,and leads to poor prognosis of malignant tumors.However,neither the role of Rab10 in PAAD nor related mechanisms have been revealed.Methods:In this study,we first used bioinformatics methods to obtain data from the TCGA database and the GTEx database to analyze the expression of Rab10 in PAAD.Then combined with the clinical follow-up data and transcriptome sequencing data of 179 PAAD patients in the TCGA database,the correlation between Rab10 and the survival time of PAAD patients was further analyzed to determine its potential clinical value.Then,the clinical value of Rab10 in PAAD was verified by collecting clinical specimens from PAAD patients collected from Northern Jiangsu People’s Hospital and the clinical data and follow-up data of the corresponding patients.RT-q PCR and Western blot techniques were used to detect m RNA and protein levels in cells,respectively.PAAD cell model with low expression of Rab10 was constructed by transient transfection technique,and cell phenotype experiments were performed: CCK-8 assay,cell scratch assay,Transwell migration assay,and aerobic glycolysis function of the cells was detected using glucose uptake assay,lactate production assay,pyruvate content assay,ATP content assay,OCR assay,and ECAR assay.By consulting the relevant literature as well as calling the database data again for analysis,we speculated that Rab10 may be involved in regulating GLUT1 expression and spatial distribution.The effect of Rab10 on the spatial distribution of GLUT1 in PAAD was observed by immunofluorescence.Stable low-expressing sh Rab10 cell lines were obtained using stable transfer techniques and screened,and the expression levels of associated GLUTs were identified using RT-q PCR and Western blot.GLUT1 was overexpressed in PAAD cells stably expressing low Rab10,and RTq PCR and Western blot were used to determine the expression of Rab10 and GLUT1 at the m RNA and protein levels and phenotypic experiments and aerobic glycolysis related tests were performed.Finally,we performed a subcutaneous tumor formation assay in nude mice to verify the effect of Rab10 on PAAD proliferation in vivo.Result:1.Data analysis of the TCGA database and the GTEx database suggests that Rab10 is highly expressed in PAAD tissues.Rab10 protein expression was assessed by immunohistochemical staining in 86 PAAD tissue samples and 55 adjacent noncancerous tissue samples collected from Northern Jiangsu People’s Hospital,of which70.9%(61/86)of PAAD samples showed high Rab10 expression,while this proportion was only 36.4%(20/55)in adjacent non-cancerous tissues,and the χ2 test suggested that Rab10 was differentially expressed in PAAD tissues and adjacent non-cancerous tissues.2.The clinical data and follow-up results of 86 PAAD specimens collected in this study were analyzed.The results showed that the expression of Rab10 was related to the T stage(T1 vs.T2-4,P<0.05)and Tumor grade(well differentiated or moderately differentiated vs.poorly differentiated or undifferentiated,P<0.05)of PAAD patients,but there was no significant difference with other clinical characteristics.Analysis of the clinical data of PAAD patients in the TCGA database revealed that patients with high Rab10 expression had shorter overall survival than those with low Rab10 expression(P<0.01).Kaplan-Meier survival curves were performed using log-rank tests using follow-up data and Rab10 expression in 86 PAAD patients in this study to validate these results.Cox univariate and multivariate results indicated that Rab10 expression(P<0.05),M stage(P<0.05)and pathological differentiation(P<0.01)were independent prognostic indicators.3.The results of RT-q PCR showed that Rab10 was highly expressed in PAAD cells(P<0.01).In the CCK-8 assay,the cell proliferation ability of the si Rab10 group was significantly lower than that of the control group(P<0.001);in the scratch assay and transwell migration assay,the cell migration ability of the si Rab10 group was significantly lower than that of the control group(P<0.001).In addition,PAAD cells knocked down Rab10 were also found to have significantly lower glucose uptake capacity(P<0.001),lactate production(P<0.001),pyruvate content(P<0.01),ATP content(P<0.001),OCR(P<0.001),and ECAR(P<0.001)than controls.The results of RT-q PCR and Western blot showed that while Rab10 expression was down-regulated,the expression of GLUT1 was also down-regulated(P<0.001),while overexpression of GLUT1 on this basis did not up-regulate Rab10 expression(P<0.001).In the following phenotypic experiments,PAAD cells overexpressing GLUT1 all showed stronger proliferation and migration ability than PAAD cells knocked down Rab10 only(both P<0.001).The results of aerobic glycolysis related experiments showed that aerobic glycolysis related indicators were significantly up-regulated in the sh Rab10+oe GLUT1 group compared with the sh Rab10 group(all P<0.001).4.In the mouse tumor formation experiment,the tumors in the low Rab10 expression group were significantly smaller than those in the control group(P<0.001).Conclusion:1.Rab10 expression in PAAD tissues is up-regulated compared with adjacent noncancerous tissues,and Rab10 expression is related to T stage and pathological differentiation of PAAD patients.2.Up-regulation of Rab10 predicts a poor prognosis in PAAD.3.Rab10 promotes PAAD proliferation and migration in vitro and promotes aerobic glycolysis.4.Rab10 up-regulates the expression of GLUT1.5.Rab10 promotes PAAD cell proliferation and migration via GLUT1 and promotes aerobic glycolysis. |
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