| Background & objective: Liver cancer is the sixth most common malignant tumor and the fourth leading cause of cancer-related deaths worldwide.Most of the liver cancers are hepatocellular carcinoma(HCC).Due to the lack of specific clinical symptoms in early HCC and the lack of simple and easy screening methods,many patients with HCC are have entered to the advanced stage at the time of diagnosis.Compared with early HCC,advanced HCC has poor prognosis with limited treatments.Hepatitis B virus(HBV)infection is the leading cause of HCC in Chinese population.For this reason,it is particularly necessary to screen HCC regularly and early in patients with chronic hepatitis B(CHB)or cirrhosis.Compared with serum,the urine is non-invasive,simple and easy to operate,and is an ideal biomarker detection method.This study aims to discover new diagnostic or screening biomarkers in the urine of patients with HBV-related HCC and assess its efficacy in diagnosing HCC.At present,the most widely used and most recognized diagnostic marker for HCC is alpha-fetoprotein(AFP).However,the biological function of AFP in HBV-infected HCC cells is still unclear.This study is intended to clarify it by further experiments.Methods: We collected urine specimens from a discovery cohort(n = 40)and a validation cohort(n = 140)for biomarker studies.The discovery cohort contains four groups: HCC(n=10),liver cirrhosis(LC,n=10),chronic Hepatitis B(CHB,n=10)and healthy controls(n=10);the validation cohort contains 3 groups: HCC(n = 75,of which 48 were newly diagnosed,27 were postoperative patients with HCC),LC(n = 51)and CHB(n = 14).Isobaric tags for relative and absolute quantification(iTRAQ)technique combined with high performance liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS)was used to identify urine differentially expressed proteins(DEPs)when compared HCC with non-HCC in the discovery cohort.Tissue-specific expression analysis(TSEA)was used to identify the histologic origin of the DEPs.The target proteins were identified based on fold change of DEPs and a series of bioinformatics methods(the PANTHER database for functional enrichment experiments,the STRING online database for constructing protein-protein interaction network).The presence or absence of the target proteins in the urine and the molecular weights were verified by Western blotting.The concentrations of the target proteins in urine were determined by enzyme linked immunosorbent assay(ELISA).To clarify the biological function of AFP in HBV-infected hepatoma cells,we transfected shRNA lentivirus targeting human AFP into HepG2.2.15 and HepGAD38 cells.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot were used to assess the knockdown efficiency of AFP.Cell proliferation was assessed by MTT,apoptosis was detected by flow cytometry,and cell invasion and migration were measured by Transwell assay.Statistical analysis are as follows: the comparison of target proteins levels were analyzed by the Mann-Whitney U test,the pearson correlation was used to determine the correlation between two consecutive variables,the recipient operational characteristics(ROC)curve was used to assess the efficacy of the target urine proteins in diagnosing HCC,the Delong's test was used to compared the ROC curves,the P value of less than 0.05 was considered statistically significant.Results: Through proteomics techniques and analysis,we identified a total of 96 urine DEPs of HCC,of which 74 were up-regulated,22 were down-regulated.These proteins are mainly secreted proteins,and the signaling pathway is mainly enriched in blood coagulation,CCKR signal pathway and plasminogen activation cascade.Through protein-protein interaction network analysis,we identified 15 key proteins: CP,FGA,AFP,CST3,TGOLN2,HSP90B1,VCAN,LTBP1,IGFBP7,ORM1,AMBP,FGB,ORM2,RBP4 and CAMP,these proteins are thought to play an important role in the development of HCC.In addition,compared with previous serum proteomics studies,a total of 6 proteins are shared in both serum and urine: ORM1,LBP,ANPEP,GSN,AFP and SERPINA3,by integrating the top 10 DEPs,we finally identified AFP and ?1 acid glycoprotein(ORM1)as our target proteins for subsequent studies.Western Blot experiments confirmed that AFP and ORM1 were present in the urine,and their corresponding band positions were 70 kd and 45 kd,respectively,which were consistent with the actual molecular weights of the proteins.By ELISA,we obtained the concentration of u-AFP and ù-ORM1 in 140 patients in the validation cohort.The levels of u-AFP in the HCC group [109.9(3,3308.9)pg/ml] was significantly higher than that in the LC group [3(3,3)pg/ml,p = 0.002] and the CHB group [3(3,3)pg/ml,p < 0.001].In addition,we found a strong correlation between u-AFP and serum AFP or AFP-L3.The Pearson correlation coefficient was 0.673(p < 0.0001)and 0.944(p < 0.0001),respectively,indicating that u-AFP may be derived from blood circulation.The levels of u-ORM1 in HCC group [1370.6(522.2,10909.2)ng/ml] was significantly higher than that in LC group [574.6(320,1056.4)ng/ml,p < 0.001],but no significant difference with the CHB group(p = 0.286).In order to evaluate the clinical application value of both urine proteins,we plotted the ROC curve.The area under the ROC curve(AUC)of u-AFP was 0.795(95%CI: 0.706-0.886).When the diagnostic threshold was set to 25.8 pg/ml,the sensitivity of u-AFP in diagnosing HCC was 63.5% and the specificity was 95.4%.Although u-ORM1 was less effective in diagnosing HCC,its AUC was 0.705(95% CI: 0.604-0.807),but when combined with u-AFP,the diagnostic efficiency was significantly improved,and the AUC reached 0.864(95% CI: 0.791-0.937),sensitivity was 80.9%,specificity was 85.5%.In addition,we performed qualitative diagnosis analysis on u-AFP and u-ORM1.u-AFP was deemed as positive when greater than 3pg/ml,and positive for u-ORM1 when greater than 1284.9ng/ml.The positive prediction value of u-AFP alone was 90.9%,and the diagnostic sensitivity of parallel combination of u-AFP and u-ORM1 was 85.1%.It is expected to be further developed into a diagnostic test strip.Cell functional experiments showed that knockdown of AFP significantly inhibited the proliferation,invasion and migration of HBV-infected cells.After AFP knockdown,HBV-DNA in cell supernatant decreased by about 50%,HBsAg showed no significant change and HBeAg increased by about 20%,indicating that AFP has a significant effect on HBV-DNA replication.AFP knockdown promotes the expression of IFN1 A and downstream genes,including OAS1,OAS2,ISG15 and RNASEL.This indicates that AFP may promote HBV-DNA replication through inhibiting IFN? signaling pathway,although this conclusion needs to be confirmed by more experiments.Conclusions: Urinary AFP is a potential biomarker for the diagnosis or screening of HCC and the diagnostic efficacy can be improved when combined with urine ORM1.These two proteins are hopefully to be developed as a diagnostic test strip.AFP can promote the proliferation,invasion and migration of HBV-infected HCC cells,and may promote HBV-DNA replication through inhibiting IFN? signaling pathway. |
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