| BackgroundCa19-9 is one of the most widely used tumor markers in the diagnosis and treatment of pancreatic diseases.Accurate CA19-9 baseline value is of great significance for guiding differentiation of benign and malignant pancreatic diseases,tevaluation of resectability of pancreatic cancer,evaluation of treatment response and prognosis prediction.However,it has been observed in clinical practice that the level of CA19-9 in patients with pancreatic disease complicated with obstructive jaundice is significantly increased,so that the measured value of CA19-9 cannot truly reflect the true level of CA19-9 secreted by tumor,thus affecting the diagnosis of disease and clinical decision-making in patients with pancreatic cancer.Therefore,it is urgent to develop the correction formulas of CA19-9 in patients with benign and malignant obstructive jaundice to guide clinical practice and avoid improper clinical decision-making.ObjectiveExplore influencing factors of CA19-9 measured value.Derive correction formulas of CA19-9 real value before the biliary decompression in order to help surgeons make proper clinical decision for obstructive jaundice patients.MethodsObstructive jaundice patients who underwent biliary decompression between January 2014 and January 2019 in Peking Union Medical College Hospital,with specific clinical or pathological diagnosis as well as CA19-9 value measured within one month before and after biliary decompression,were brought into our study.Demographic data,imaging data,serological test results,pathological results,operation data,diagnosis and other data were collected according to the electronic medical record system.Mann-whitney U test was used to compare the differences between groups.Spearman rank sum test was used to evaluate the correlation between CA19-9 and other indicators.Binary linear regression was used to explore the correction formulas of CA19-9.ResultsOur study brought into 121 patients(102 malignant patients and 19 benign patients).CA19-9 value(p=0.048),TBil value(p=0.048)and neutrophil percentage(p=0.013)in malignant group were significantly higher than in benign group,and in contrary,RBC count(p=0.047)in malignant group was significantly lower than that in benign group.In malignant patients,CA19-9 value has no definite correction with TBil value,but was strongly correlated with AST(p<0.0001),GGT(p=0.029)and neutrophil percentage(p=0.013).CA19-9 value of 24.5%malignant patients did not decline significantly or even increased after biliary decompression,while CA19-9 value of 89.5%benign patients decline significantly.There was no significant difference in the average decline rate of TBil in benign and malignant patients,and the average decline rate of CA19-9 value in malignant patients was significantly lower than that in benign patients(p=0.014).By screening independent variables with the best imitative effect,the correction formula for predicting the true CA19-9 value in malignant patients before biliary decompression was:CA19-9true=0.63 ×CA19-9measured-20.3;the correction formula for predicting the true CA19-9 value in benign patients before biliary decompression was:CA19-9true=0.085 ×CA19-9measured+60.4.In regard to differential diagnosis of benign and malignant diseases,the diagnostic efficacy of CA19-9 value after biliary decompression(AUC=0.699)and CA19-9 predicted true value obtained by correction formula(AUC=0.695)was significantly higher than that of CA19-9 value before biliary decompression(AUC=0.598).ConclusionsBenign and malignant patients with obstructive jaundice have significant differences in indicators such as CA19-9 in baseline.Average TBil decline rates after biliary decompression in two groups were similar,but average CA19-9 decline rate in malignant patients was significantly lower than that in begine patients.We successfully obtained the correction formulas for predicting CA19-9 true value using CA19-9 measured value before biliary decompression as independent variable.With help of our correction formulas,patients with benign and malignant obstructive jaundice can be better differentiated.Our correction formulas also have a good application prospect in evaluation for resectablility,treatment response and prognosis prediction of pancreatic cancer.BackgroundPancreatic cancer has insidious onset,high malignancy and poor prognosis.Chemotherapy is an important part of the treatment for pancreatic cancer,in which gemcitabine(GEM)based combined chemotherapy is one of the first-line chemotherapy regimen for pancreatic cancer.However,resistance for GEM has always been a bottleneck problem leading to recurrence and death of pancreatic cancer patients,and the current combined chemotherapy regimen or combined targeted therapy regimen with GEM cannot solve the problem of GEM resistance.Therefore,it is of great significance to explore the mechanism of GEM resistance from a new molecular biological perspective to enhance the efficacy of GEM sensitization and reverse drug resistance,so as to improve the overall survival of pancreatic cancer patients.RNA binding protein(RBP)is a kind of important proteins that regulate transportation,splicing,stability and translation of RNA.Abnormal expression of RBP often leads to a series of abnormal accumulation or degradation of downstream RNA resulting in various diseases.For example,the RNA-binding protein HuR is highly expressed in pancreatic cancer,which can promote proliferation,invasion and metastasis of pancreatic cancer cells and the tolerance to hypoxia and hypoglycemia by regulating the metabolism of downstream RNAs,like WEE1,PIM1,IDH1 and other mRNAs.However,there is a lack of systematic study on whether RBP plays a role in GEM resistance of pancreatic cancer,which RBP plays a significant role in GEM resistance of pancreatic cancer,and what specific role RBP plays in GEM resistance of pancreatic cancer.Therefore,it is of great significance to explore RBPs and their specific molecular mechanisms that play an important role in GEM resistance of pancreatic cancer for further understanding and solving GEM resistance of pancreatic cancer.ObjectiveScreen RBP related to GEM resistance of pancreatic cancer and its correlation with clinicopathological indicators.Clarify the effect of RNA-binding protein PUM2 on the malignant biological behaviors of pancreatic cancer,such as proliferation,migration and GEM resistance.Explore the specific mechanism of PUM2 regulating GEM resistance of pancreatic cancer.MethodsRBPs closely related to GEM resistance of pancreatic cancer were screened based on transcriptome sequencing,siRNA library proliferation and GEM resistance test results.Relationship between expression level of PUM2 and clinicopathological indicators was evaluated by immunohistochemical staining of tissue chip.SRB proliferation assay,GEM drug resistance assay and transwell cell migration assay were used to detect the effects of PUM2 on the malignant biological behaviors of pancreatic cancer cells in vitro by knocking down or overexpressing PUM2.The effects in vivo were explored by mice subcutaneous xenograft model.Furthermore,RIP-seq and RNA-seq were combined to explore the downstream mRNAs regulated by PUM2 in pancreatic cancer cells,and the regulation of PUM2 on downstream mRNAs was verified by qRT-PCR,Western Blot,RIP-qPCR,actinomycin D RNA stability assay,dual luciferase gene reporter assay and rescue experiments.Finally,combined with RIP-seq,RNA-seq and JSAPAR database,transcription factors with mutual regulation relationship with PUM2 were screened,and the regulatory relationship between the transcriptor factor and PUM2 was verified by qRT-PCR,Western Blot,RIP-qPCR,ChIPqPCR and f rescue experiments.ResultsBased on transcriptome sequencing results of GEM resistant pancreatic cancer cell line and the parent cell line,several RBPS were highly expressed in GEM-resistant pancreatic cancer cell line.We screened out the RNA-binding protein PUM2,which is most related to gemcitabine resistance of pancreatic cancer,through siRNA library and GEM resistance assay.Immunohistochemical staining of pancreatic cancer tissue chip suggested that high expression of PUM2 was an independent risk factor for poor prognosis of pancreatic cancer patients.In vitro functional experiments showed that PUM2 could promote proliferation,migration and resistance to gemcitabine of pancreatic cancer cells.In vivo experiments showed that knockdown of PUM2 inhibited the growth of subcutaneous transplanted tumor in mice and increased sensitivity to gemcitabine.Further,RNA-seq and RIP-seq were combined to explore the regulation role of PUM2 on downstream RNAs that promote GEM resistance in pancreatic cancer.We found that PUM2 up-regulated mRNA stability of several genes(ITGA3,ADAM 17,ASAP1,etc.)in the focal adhesion pathway.ITGA3 was confirmed to be the most significant downstream mRNA of PUM2 regulating gemcitabine resistance in pancreatic cancer by rescue experiments in vitro,and PUM2 could stabilize ITGA3 mRNA by binding to PUM binding element(PBE)in the 3’UTR region of ITGA3 mRNA.Finally,we found the mutual regulation relationship between transcription factor EGR1 and PUM2,that is PUM2 binding to 3’UTR region of EGR1 mRNA,and EGR1 binding to promoter region of PUM2 gene,resulting in a cascade effect that amplifying the role of PUM2 in pancreatic cancer drug resistance.ConclusionsRNA-binding protein PUM2 is closely related to the prognosis of pancreatic cancer patients.It promotes GEM resistance of pancreatic cancer by regulating mRNA stability of ITGA3 and other genes in focal adhesion pathway,and it has positive feedback regulation with transcription factor EGR1.The discovery of EGR1/PUM2/ITGA3 axis provides a solid experimental basis for the screening of chemotherapy regiments for pancreatic cancer patients in the future and the exploration of combination regimens reversing GEM resistance. |
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