| Esophageal cancer(EC)is one of the most prevalent digestive tract malignancies in the world.In China,esophageal squamous cell carcinoma(ESCC)is the predominant histological type of EC,and Henan province contributes to the high incidence and mortality of EC.Because of the insidious onset of EC,with no dramatic symptoms to attract enough attention,most patients are normally diagnosed and treated at the middle or advanced stage.In recent years,a large number of studies have shown that autoantibodies against tumor-associated antigens(TAAs),in other words,tumor-associated autoantibodies(TAAbs),as well as microRNAs(miRNAs),could be stably existed in the blood circulation of tumor patients,with different expression profiles in different kinds of cancers,and thus serve as novel non-invasive tumor biomarkers to diagnose ESCC.PurposeSeven TAAbs(autoantibodies against HCCR-1,p53,p62,C-myc,NICD,hnRNP A2/B1 and MDM2,namely,anti-HCCR-1,anti-p53,anti-p62,anti-C-myc,anti-NICD,anti-hnRNP A2/B1 and anti-MDM2)and two mi RNAs(miR-21 and miR-25)were determined to calculate their expression levels in the plasma of ESCC patients and normal individuals,in order to evaluate the diagnostic values of those two kinds of tumor biomarker in the detection of ESCC,and thus to provide the theory basis for the realization of early discovery,early diagnosis and early treatment in ESCC.Methods1.The expression and purification of HCCR-1 recombinant proteins: The pre-constructed HCCR-1 recombinant expression plasmids(pET-30a(+)-HCCR-1)were transformed into the Escherichia coli BL21(DE3)competent cells to induce and express the HCCR-1 recombination protein.Then the protein was purified using nickel ion affinity chromatograph and determined using Bradford method.2.Plasma from 125 newly diagnosed hospitalized ESCC patients and 125 healthy individuals were collected.Those healthy controls were from a physical examination in the same period,and were gender-and age-matched with ESCC patients.3.The plasma expression levels of seven TAAbs were determined using indirect enzyme-linked immunosorbent assay(ELISA).4.The plasma expression levels of miR-21 and miR-25 were determined using real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR).5.The t test was used to compare the expression levels of TAAbs or mi RNAs between ESCC patients and healthy controls;the correlations between the concentrations of plasma TAAbs or miRNAs and clinicopathological features were evaluated by Fisher's exact probability test or ?2 test;the t test and one-way ANOVA were used to compare the expression differences of TAAbs or miRNAs in the plasma of ESCC patients between two groups or among three groups,respectively.The diagnostic values of single biomarker and multiple biomarkers in the detection of ESCC were evaluated,according to the evaluation method of diagnostic test.Besides,receiver operating characteristic(ROC)curves were conducted to calculate areas under the ROC curves(AUCs).Results1.HCCR-1 recombinant protein was successfully expressed and purified.Its concentration was 0.28 mg/mL.2.According to the results of ELISA of the seven selected TAAbs,except anti-NICD and-hnRNP A2/B1,the expression levels of the other five TAAbs(anti-HCCR-1,anti-p53,anti-p62,anti-C-myc and anti-MDM2)in ESCC group were significantly higher than those in the normal healthy controls(P < 0.05).3.The sensitivity of single TAAb to diagnose ESCC was from 7.2% to 23.2%,while the specificity ranged from 93.6% to 97.6%;the AUC ranged from 0.522 to0.728,which indicated a low diagnostic value.When tested those significant TAAbs in parallel,with anti-MDM2 as the starting point,which had the highest sensitivity,the sensitivity would increase from 23.2% to 60.8% in the diagnosis of ESCC,and AUC would elevate to 0.805.4.The expression levels of TAAbs in the plasma of ESCC patients were found tohave no significant difference in gender,age,and TNM stage(including T,N,and M stage)(all with P > 0.05).5.According to the results of qRT-PCR,miR-21 was over-expressed in ESCC patients compared with controls(P < 0.001),while no statistical difference was found about the expression levels of miR-25 between ESCC patients and controls(P > 0.05);the sensitivity and specificity of mi R-21 was 74.4% and 77.6%,respectively,and the ROC analysis showed the AUC of miR-21 was 0.804 when distinguishing ESCC patients from normal controls.6.The plasma expression levels of miR-21 and miR-25 were found to have no significant difference in gender,age,and M stage(all with P > 0.05),while were closely related to TNM stage,especially T stage(all with P < 0.01).The expression levels of miR-21 and miR-25 in ESCC patients in stage 0-I were dramatically higher than those in stage II(P < 0.001 and P = 0.043,respectively)and stage III-IV(P <0.001 and P = 0.017,respectively).Similarly,the expression levels of miR-21 and miR-25 in ESCC patients with Tis-T1 tumors were significantly higher than those with T2(P = 0.003 and P = 0.037,respectively)and T3 tumors(P < 0.001 and P =0.004,respectively).With regard to N stage,it was not related to miR-21(P > 0.05)but had some relation with miR-25(P < 0.001).7.The ROC analyses of miR-21 and miR-25 showed higher AUCs to diagnose early ESCC: the AUC was 0.857 for miR-21 and 0.627 for miR-25,when comparing ESCC patients in stage 0-I to all controls;their corresponding AUCs were 0.842 and0.656 when comparing ESCC patients with Tis-T1 tumors to all controls.8.The panel of five TAAbs and miR-21 hold the highest AUC(0.869)in the four combinations of miR-21 and TAAb(s),and the sensitivity and specificity was 76.8%and 84.8%,respectively.The AUC of the panel was 0.889 when distinguishing ESCC patients in stage 0-I while 0.882 when distinguishing Tis-T1 patients to all controls.Conclusions1.The five TAAbs(anti-HCCR-1,anti-p53,anti-p62,anti-C-myc and anti-MDM2)and miR-21 were the promising non-invasive biomarkers to diagnose ESCC.2.All TAAbs in the plasma of ESCC patients were found to have no relationwith gender,age,and TNM stage;both miR-21 and mi R-25 were found to have no relation with gender and age,while were related to TNM stage.3.Plasma miR-21 and miR-25 might be used to the early diagnosis of ESCC.4.The panel of five TAAbs and miR-21 had higher diagnostic value to diagnose ESCC,especially early ESCC. |
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