| Objective1.To identify the predictive effect of HOXA10 on survival and recurrence of patients after curative resection with pancreatic cancer,and establish clinical prediction models.2.To identify the effect of HOXA10 on the malignant biological behavior of pancreatic cancer,and explore the related mechanism.Methods1.Based on the The Cancer Genome Atlas(TCGA)database,we analyze the expression of HOXA gene family(HOXAs)in cancer and normal tissues of patients after curative resection with pancreatic cancer,and identify the relationship between the expression of HOXAs and overall survival(OS),and disease-free survival(DFS).Then,we select HOXAs whose expression is significantly different between cancer and normal tissues,or which are significantly related to prognosis for further study.2.In the study,123 patients after curative resection with pancreatic ductal adenocarcinoma(PDAC)in our hospital were enrolled in the training group from January2015 to August 2017,and 71 patients from our hospital and another hospital of our university were enrolled in the validation group from May 2013 to August 2017.The clinical data of the two groups were retrospectively analyzed.In the training group,the patients were divided into high expression group and normal or low expression group according to the expression of HOXA10.Univariate analysis and multivariate Cox regression analysis were used to determine the relationship between HOXA10 and survival,and recurrence after pancreatic surgery.Then creat clinical prediction models based on the result of Cox regression.In addition,calculate the C-index and AUC values,and draw the ROC curves and calibration curves.Finally,verify the models in the validation group.3.Construct the HOXA10 knockdown and overexpression cell lines of PDAC.Then calrify the effects of HOXA10 on cell phenotypes such as proliferation,migration,invasion,metastasis and apoptosis of PDAC,through MTT,clone formation,scratch healing assay,Transwell assay and cell cycle analysis.4.Construct the nude mouse subcutaneous xenograft tumor model with HOXA10 overexpression and control group,to clarify the effect of HOXA10 on the proliferation and metastasis of xenograft tumor in vivo.5.According to the sequencing data of the TCGA database and the high-throughput sequencing results of the eukaryotic transcriptome of the HOXA10 stable transfected cell line in this experiment,the cellular pathways affected by the abnormal expression of the HOXA10 were analyzed,and then verified by Western blot.Results1.Based on the analysis of TCGA database,the expression of HOXAs in cancer tissues was higher than that in the normal tissues,among which the expressions of HOXA3,HOXA4,HOXA5 and HOXA10 were significantly higher in cancer tissues(P < 0.05).The OS of the patients in the high expression group of HOXA10,HOXA11 and HOXA13 was significantly worse(P < 0.05).In addition,the DFS of the low expression groups of HOXA9 and HOXA10 was significantly higher than that of the high expression groups.2.Select HOXA3,HOXA4,HOXA5,HOXA10,HOXA11 and HOXA13 to further verify the expression in our center.Real-time PCR was used to quantitatively determine the expression of HOXAs in 123 pairs of cancer and adjacent tissues in our center.The results showed that HOXA3,HOXA4 and HOXA5 were lowly expressed,HOXA13 was normally expressed,and HOXA10 and HOXA11 were highly expressed in cancer tissues.The high expression of HOXA10 group had worse OS(P = 0.008)and recurrence-free survival(RFS)(P = 0.024).According to follow-up data,univariate and multivariate Cox regression analysis of OS and RFS were performed,and the risk factors for poor OS were ECOG score1(HR: 1.594,95% CI: 1.052-2.415,P = 0.028),increased levels of CA19-9(HR: 1.004,95%CI: 1.000-1.012,P = 0.035),high HOXA10 level(HR: 1.839,95% CI: 1.214-2.789,P =0.004),and increased tumor size(HR: 1.076,95% CI: 1.067-1.197,P = 0.048),intraoperative blood transfusion(HR: 1.504,95% CI: 1.212-1.862,P = 0.001),macrovascular invasion(HR: 1.763,95% CI: 1.157-2,687,P = 0.008).Risk factors for poorer RFS were ECOG score 1(HR: 1.712,95% CI: 1.097-2.670,P = 0.018),increased CA19-9 level(HR: 1.003,95% CI: 1.000-1.011,P = 0.025),high HOXA10 level(HR: 1.524,95% CI: 1.024-2.225,P = 0.037)and macrovascular invasion(HR: 1.179,95% CI: 1.053-1.320,P = 0.004).According to the multivariate analysis results,two clinical prediction models were established,and the C-index of the prediction model for postoperative OS and RFS were 0.726 and 0.642.Finally,the clinical prediction models were well applicable in the validation group.3.The phenotypic experiments suggested that the up-regulation of HOXA10 promoted the proliferation,invasion and metastasis of pancreatic cancer cells,and inhibits cell apoptosis.4.Nude mice subcutaneously transplanted tumor experiments showed that compared with the normal group,the HOXA10 overexpression group had larger tumor volume and faster tumor growth rate.5.HOXA10 is related to multiple pathways of tumorigenesis and development.This study shows that overexpression of HOXA10 can activate cell proliferation-related pathways and Akt/m TOR pathway to promote tumor proliferation,invasion and metastasis,and inhibit cell apoptosis.Conclusion1.The high expression of HOXA10 can be used as an independent risk factor for the survival and recurrence of pancreatic cancer patients after surgery.2.The clinical prediction models of survival and recurrence of patients after curative surgery with PDAC established in this study perform well.3.The up-regulation of HOXA10 can promote tumor proliferation,invasion and metastasis,inhibit cell apoptosis by activating cell proliferation-related pathways and Akt/m TOR pathway.The HOXA10 gene may serve as a potential target for pancreatic cancer therapy. |
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