| Background&ObjectiveHepatocellular carcinoma (HCC) is one of the most common malignant tumorsworldwide. As a tumor marker for liver cancer, alpha-fetoprotein (AFP) reflects the load oftumors, which makes it conducive to the diagnosis and treatment evaluation of liver cancer.However, AFP is not effective for the prognosis of liver cancer. Although it is reported thatthere is an increasing number of tumor markers, their clinical application values either arevery limited or need extensive verification. It still remains a key issue in the liver cancerresearch field to further identify diagnostic and prognostic molecular markers, and todevelop a means to accurately predict recurrence, metastasis and prognosis. As aheparin-binding factor, midkine (MK) is directly involved in tumor growth and metastasis.Given that MK is often highly expressed in most malignant tumors, it is expected that itwill become a new tumor marker. Epithilial-mesenchymal transition (EMT) is a process inwhich epithelial cells undergo remarkable morphological changes characterized byincreased cell mobility and activity. This study has aimed to develop a method to calculateserum MK concentrations and evaluate its value in HCC diagnosis. This method can beserved as a foundation for the measurement of serum heparin-binding molecule levels.Besides, this study has established the exprerimental evidence to prove that MK inducesEMT in HCC cells; it has further investigated the role of MK in promoting HCCdevelopment by inducing EMT.Method1. A heparin-enzyme linked immunosorbent assay (Heparin-ELISA) for determination ofMK was developed using heparin-antibody sandwich technique.(1)As a heparin-binding factor, serum MK levels were detected by heparin-antibodysandwich ELISA.(2) Mark the standard curve.(3) Analytical sensitivity test. (4) Recovery rate test.(5) Linear analysis.(6) Precision test.(7) Comparison with a commercial MK ELISA kit.2. Preliminary research about the detection of the total concentration of MK and PTN insera using a heparin-double antibody sandwich ELISA.3. Detection of serum MK levels in HCC patients.4. The in vitro study of EMT induced by MK, the process influencing the proliferation andinvasion of HCC cells(1) Cell proliferation assay.(2) Transwell assay.(3) Immunoflurescence.(4) Western blot analysis.5. Detection of the expressions of MK and the EMT markers and the numbers ofcirculating tumor cells (CTCs) in HCC specimens.(1) Detecting the expressions of MK in HCC tissues using the method ofimmunohistochemistry (IHC).(2) Detection the expressions of vimentin and E-cadherin in HCC tissues.(3) Detection CTCs in HCC patients.(4) Correlation analysis of MK expressions in HCC tissues with MK levels in HCCsera, E-cadherin expressions in HCC tisuues, the contents of CTCs in HCC blood, andclinical pathologic features of HCC.Results1. The method of heparin-antibody sandwich ELISA for MK assay has been developed,meeting the requirement of experiment basically.(1) The best working concentration of heparin biotin and mouse antihuman monoclonal midkine antibody was2μg/mL and0.75μg/mL, respectively.(2) The standard curve equation was y=1.2308x-0.0094, r=0.9973.(3) The assay had an analytical sensitivity of0.047ng/mL.(4) The range of recovery rate was90.6%~102.0%.(5) Satisfactory lnear results were obtained between0.096ng/mL and1.436ng/mL,and the linear equation was y=1.1387x+0.011, r=0.998.(6) Within-run and between-day coefficients of variation were6.3%~6.9%and9.0%~9.8%, respectively.(7) Compared with a commercial hMK ELISA kit, the results had no significantdifference (P=0.058). Result of this method correlated well with a commercial hMKELISA kit (r=0.9779).2. The result of two factor assay reflected that this method could detect the total quantity oftwo heparin-binding factors at one time.3. Serum MK levels of the healthy volunteers and HCC were (0.259±0.061) and(0.501±0.111) ng/mL, respectively, with a significant difference between HCC patientsand healthy volunteers (P<0.05). The postitive rate of MK in HCC was85.9%using theserum cut-off value of0.382ng/mL.4. The in vitro study of EMT induced by MK in HCC cells indicated that10nM MKsignificantly enhanced the proliferation and invasion of Hep3B cells, moreover, HCC cellsdisplayed an alterd mesenchymal morphology, vimentin expression was up-regulated whileE-cadherin was down-regulated.5. The results of clinical research of HCC specimens:(1) MK was overexpressed in tumor tissues compared with paratumor tissues fromHCC patients.(2) Serum MK levels correlated well with MK expressions in tumor tissues from HCC(P <0.01).(3) MK expressions in tumor tissues correlated well with intra-hepatic metastasis and TNM stage (P <0.05).(4) In tumor tissues, vimentin expression was up-regulated while E-cadherin wasdown-regulated. There was a negative correlation between MK expression and E-cadherinexpression in HCC tissues (P <0.05).(5) CTCs were detected in the blood samples from10of40(80%), with an average of12.6±13.2(mean±SD), correlating well with MK expression in HCC tissues.ConclusionThe heparin-antibody sandwich ELISA could meet the requirement basically. It canapply to the detection of total concentration of two heparin-binding factors. Afterstimulated by MK, the changes in the morphology and molecular character of HCC cellssuggested that MK could induce EMT, which could promote the proliferation and invasionof those cells. The positive rate of MK expression in HCC patients correlated well withintra-hepatic metastasis and TNM stage. There was a negative correlation between MKexpression and E-cadherin expression in HCC tissues. Therefore， we can make aconclusion that MK induces EMT and futher advances the intra-hepatic metastasis andblood dissenmination of HCC cells. |
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