The Detection And Significance Of Associated Proteins With Lymphatic Metastasis Of Mouse Hepatocarcinoma On Cytology, Histology And Serology

Hepatocellular carcinoma (HCC) is a kind of high malignant and high mortalitydigestive system tumor. Lymphatic metastasis is an important clinical determinant forthe prognosis of HCC, therefore it is essential to investigate the molecular mechanismsof lymphatic metastasis and seek potentially therapeutic target. Impressive progressionhas been made in diagnosis and treatment of HCC. However，biomarkers that indicatethe risk of invasion and metastatic potential of HCC and can be widely used in clinicalsettings are not currently available. Discovering the protein markers associated withtumor metastasis of HCC and exploring the molecular mechanism have been the focusof the translational study for HCC.Hca-F (>70%) and its syngeneic cell line Hca-P (<30%) are mousehepatocarcinoma ascites cell lines with different lymphatic metastatic potential, haveproved to be an ideal model for explore HCC involved to lymphatic metastasis. In ourprevious study, we applied many molecular biology techniques to find differentiallyexpressed genes and proteins for lymphatic metastasis between the two cell lines.Membrane associated proteins A7(Anxa7), Gelsolin (Gsn) and(Galectin-3，Gal-3)showed high expression in the Hca-F cells. Lymphangiogenesis is the premiseof lymphatic metastasis, the role and correlation between the expression of VEGF-C and lymph node metastasis have been proved in animal experiment and clinical trialThe relationships between Annexin A7and VEGF-C in HCC are poorlyunderstood. To the best of our knowledge, no one has reported on the proteinco-expression of Annexin A7and VEGF-C in HCC.Serum and tissue are optimal sources for discovery and analysis of cancerbiomarker. Serum AnnexinA7have not been detected to date, while serum Gelsolin、Galectin-3、ALG-2and SODD are also unclear in HCC.The current work was firstly to identify potential proteins from serum related toHCC invasive progression using mouse hepatocarcinoma cell lines with differentmetastasis potentials by experimental animal studies validation. Such characteristicchanges may serve as indicators of real-time monitoring for development of the disease,they are important for diagnosis and therapy.PartⅠ: The influence on VEGF-C expression after tumor cellsregulated by Annexin A7in vitroObjective: To detect VEGF-C expression after Hca-P cells stably transfected withAnnexin A7in vitro.Methods: RNA and proteins were extract from Hca-P, PAnxa7-Up, PAnxa7-DownandPAnxa7-NCcells, the relative mRNA and proteins levels of VEGF-C were determined byReal-time qRT-PCR and Western blot analysis, respectively. ELISA was performed todetect VEGF-C expression in cell supernatant.Results:1: The gene expression of Annexin A7and VEGF-C in PAnxa7-Downcellswere respectively0.51folds and1.86folds compared to PAnxa7-NCcells, while they wererespectively1.96folds and0.46folds in PAnxa7-Upcells compared to PAnxa7-NCcells.2:The protein expression of Annexin A7was down-regulated42%, VEGF-C wasup-regulated70%; the expression of Annexin A7was up-regulated32%, VEGF-C wasdown-regulated55%.3: VEGF-C increased obviously in PAnxa7-Downcell supernatant; while decreased obviously in PAnxa7-Upcell supernatant.Conclusion: Expression and secretion of VEGF-C increased after Annexin A7were down-regulated; expression and secretion of VEGF-C reduced after Annexin A7were up-regulated.PartⅡ:Dynamic changes of VEGF-C after tumor cells regulated byAnnexin A7in vivoObjective: To explore the dynamic changes of Annexin A7and VEGF-C inprimary tumors and in serum in vivo.Methods: Hca-F, Hca-P, PAnxa7-Downand PAnxa7-Upwere injected into the footpads of615mice and made a model of lymphatic metastasis. Once a week until8week afterinoculation, tumors derived from Hca-F, Hca-P, PAnxa7-Downand PAnxa7-Upgrew.Lymphatic metastasis rates of each group were detected by HE stain from2week to8week. The dynamic changes of Annexin A7and VEGF-C proteins in primary tumorwere detected by western blot from2week to8week. The dynamic changes of AnnexinA7and VEGF-C proteins in serum were detected by ELISA every week.Results:1. Lymphatic metastasis appeared at the third week in Hca-F and PAnxa7-Down;Lymphatic metastasis appeared at the fourth week in Hca-P; Lymphatic metastasisappeared at the fifth week in PAnxa7-Up.2. The protein expression trends of Annexin A7showed gradual decrease, whileVEGF-C showed gradual increase in four groups; we found negative correlationsconfirmed between Annexin A7expression and lymph node metastasis potential indifferent tumor groups except PAnxa7-Down, while a positive correlations confirmedbetween VEGF-C expression and lymph node metastasis potential in different tumorgroups except PAnxa7-Down.3.The serum Annexin A7/VEGF-C level were found to be statistically higher than control; serum Annexin A7concentrations had a transient elevation before lymph nodemetastasis occurred in four groups; serum Annexin A7/VEGF-C concentrationgradually increased paralleling the lymph node metastatic potential increased in fourgroups; a positive correlation confirmed between serum Annexin A7level and lymphnode metastasis potential/serum VEGF-C level in four groups; a direct correlationconfirmed between serum VEGF-C level and lymph node metastasis potential in fourgroups.Conclusion:1. The protein expression trends of Annexin A7and VEGF-C showed crosscurrentin primary tumor.2. The increase of serum VEGF-C level can predict the occurrence of tumor.VEGF-C alone may be as histology and serological biomarkers of mousehepatocarcinoma lymphatic metastasis.3. The increase of serum Annexin A7level can predict the occurrence of tumor.Serum Annexin A7may be alone or combined VEGF-C as serological biomarkers ofmouse hepatocarcinoma lymphatic metastasis.Part Ⅲ: The influence on Gelsolin, Galectin-3, ALG-2and SODDexpression after tumor cells regulated by Annexin A7in vitroObjective: To detect the expression of Gelsolin, Galectin-3, ALG-2and SODDafter Hca-P cells stably transfected with Annexin A7in vitro.Methods: RNA and proteins were extract from Hca-P, PAnxa7-Up, PAnxa7-DownandPAnxa7-NCcells, the relative mRNA and proteins levels of Gelsolin, Galectin-3, ALG-2and SODD were determined by Real-time qRT-PCR and Western blot analysis,respectively.Results: The gene expression of Gelsolin, Galectin-3, ALG-2and SODD inPAnxa7-Upcells were respectively1.51folds,1.83folds,1.62folds and1.91foldscompared to PAnxa7-NCcells, while the protein expression of Gelsolin, Galectin-3, ALG-2 and SODD in PAnxa7-Upcells increased respectively53%,81%,38%and51%comparedto PAnxa7-NCcells.Conclusion: The expressions of Gelsolin, Galectin-3, ALG-2and SODD increasedafter Annexin A7was up-regulated.Part Ⅳ: Dynamic changes of Gelsolin, Galectin-3, ALG-2and SODDafter tumor cells regulated by Annexin A7in vivoObjective: To detect the dynamic changes of Gelsolin, Galectin-3, and ALG-2andSODD protein in primary tumors and in serum in vivo.Methods: Hca-F, Hca-P, PAnxa7-Downand PAnxa7-Upwere injected into the footpads of615mice and made a model of lymphatic metastasis. The dynamic changes of Gelsolin,Galectin-3, ALG-2and SODD proteins in primary tumor were detected by western blotfrom2week to8week. The dynamic changes of Gelsolin, Galectin-3, ALG-2andSODD proteins in serum were detected by ELISA every week.Results:1. Expression of Gelsolin and Galectin-3were both gradual increases in PAnxa7-Up,they were both gradual decrease in PAnxa7-Down; expression trends of ALG-2werefluctuant; expression of SODD appeared both in Hca-P and PAnxa7-Downtumor tissuesfrom4week, in Hca-F tumor tissues from3week, while in PAnxa7-Uptumor tissues from2week.2. We found a positive correlation confirmed between Gelsolin and Galectin-3andlymph node metastasis potential only in PAnxa7-Up; we found a positive correlationconfirmed between SODD and lymph node metastasis potential only in Hca-F andHca-P.3. The Gelsolin and Galectin-3levels in serum were found to be statistically higherthan control; the serum ALG-2and SODD were neither detected in four groups; serumGelsolin, Galectin-3, ALG-2and SODD concentration gradually increased paralleling the lymph node metastatic potential increased; The time of serum SODD initiallydetected were the same as the time of lymph node metastasis occurred in different tumorgroups except PAnxa7-Up.4. We found a positive correlation confirmed between serum Gelsolin andGalectin-3and lymph node metastasis potential/serum Annexin A7level only inPAnxa7-Up.5. We found a positive correlation confirmed between serum ALG-2and lymphnode metastasis potential/serum Annexin A7level/serum VEGF-C level in fourgroups.6. We found a positive correlation confirmed between serum SODD and lymphnode metastasis potential/serum Annexin A7level/serum VEGF-C level only inPAnxa7-Downand PAnxa7-Up.Conclusion:1. The increase of serum Gelsolin and Galectin-3levels can predict the occurrenceof tumor. They may be combined Annexin A7as histology and serological biomarkersof mouse hepatocarcinoma lymphatic metastasis.2. The increase of serum ALG-2level can predict the occurrence of tumor. SerumALG-2may be alone or combined Annexin A7or VEGF-C as serological biomarkers ofmouse hepatocarcinoma lymphatic metastasis. ALG-2may be alone as histologicalbiomarkers of mouse hepatocarcinoma lymphatic metastasis.3. The increase of serum ODD level can predict the occurrence and metastasis oftumor. Serum SODD may be alone or combined Annexin A7or VEGF-C as serologicalbiomarkers of mouse hepatocarcinoma lymphatic metastasis. SODD may be alone ashistological biomarkers of mouse hepatocarcinoma lymphatic metastasis.