Anxa5Enhance Lymphatic Metastatic Potential Of Mouse Hepatocarcinoma Cell Line Hca-P

Background: Lymphatic metastasis is a major factor that influences the prognosis andsurvival of patients with malignant tumors. The study of lymphatic metastaticmechanism provides basis for their clinical diagnosis and treatment. Anxa5(AnnexinA5) is a member of Annexins. Accumulated studies showed that the deregulation ofAnxa5was closely associated with tumor progression, invasion, metastasis and drugresistance. However, the relationship of Anxa5with lymphatic metastasis is poorlystudied. Mouse hepatocarcinoma cell Hca-F with high metastatic potential (75%) andHca-P with low metastatic potential (25%), a pair of synogeneic hepatocarcinomaascites cell lines with similar genetic background, are ideal experimental subjects forlymph node metastasis (LNM) study. Previous studies from our group found that Anxa5was increased by216%in Hca-F compared to Hca-P cell at protein level, whichimplicates that Anxa5might potentially increase lymphatic metastasis of mousehepatocarcinoma.Objective:1. To detect the differential expression levels of Anxa5in Hca-P and Hca-Fcell lines.2. To construct pcDNA3.1-V5/HisB-Anxa5eukaryotic expression plasmidand obtain monoclonal Hca-P cell with stably up-regulated Anxa5.3. To observe thechanges in morphology and submicrostructure of Hca-P cells following Anxa5up-regulation.4. To investigate the effect of Anxa5up-regulation on the proliferation,migration, invasion, tumorigenicity and lymph node metastatic ability of Hca-P cell.The cuurent work aims to provide a new input for further studying the lymphatic metastatic mechanisms of tumors.Methods:1. Western blot was performed to detect the differential expression levels ofAnxa5in Hca-P and Hca-F cell lines.2. RT-PCR was performed to amplify thefull-length coding sequence of mouse Anxa5. The amplified Anxa5sequence wascloned into pcDNA3.1-V5/HisB vector, the constructed pcDNA3.1-V5/HisB-Anxa5plasmid was then transfected into Hca-P cells. The monoclonal Hca-P cell with stablyup-regulated Anxa5was obtained by G418screening and limited dilution. Hca-P cellline transfected with pcDNA3.1-V5/HisB empty plasmid was also obtained as thecontrol. Protein expression levels of Anxa5in corresponding Hca-P cells were detectedby Western blot.3. The changes in morphology and submicrostructure of correspondingHca-P cells with Anxa5up-regulation were observed by using inverted microscopy andtransmission electron microscopy, respectively.4. CCK-8assay was performed todetermine the influence of Anxa5up-regulation on Hca-P cell proliferation ability. Theinfluence of Anxa5up-regulation on the migriation and invasion abilities of Hca-P wasmeasured by Transwell chamber assay. The foot-pad subcutaneous injection method wasdone to study the effect of Anxa5up-regulation on Hca-P cell tumorigenicity and lymphnode metastasis for mice.Results:1. Anxa5was increased by155%in Hca-F compared to Hca-P cell at proteinlevel.2. The enzyme digestion and sequencing results showed that the cloning plasmidpMD19-T-Anxa5and eukaryotic expression plasmid pcDNA3.1-V5/HisB-Anxa5weresuccessfully constructed.3. Following Anxa5up-regulation, the size of Hca-P cell andthe number of rough intracellular endoplasmic reticula and mitochondria increased,mitochondria swelled, mitochondrial crista and matrix density decreased.4. Themonoclonal Anxa5-Hca-P-1, Anxa5-Hca-P-2and Anxa5-Hca-P-3cell lines with stableAnxa5over-expression were obtained. Compared with Control-Hca-P, the proteinexpression levels of Anxa5in Anxa5-Hca-P-1, Anxa5-Hca-P-2and Anxa5-Hca-P-3celllines increased by15.0%,20.3%and67.7%, respectively.5. Compared withControl-Hca-P, the proliferation rates of Anxa5-Hca-P-1and Anxa5-Hca-P-2cell linesincreased significantly, while, the proliferation rate of Anxa5-Hca-P-3decreased significantly.6. Compared with Control-Hca-P, the migration abilities of Anxa5-Hca-P-1, Anxa5-Hca-P-2and Anxa5-Hca-P-3cell lines increased by68.11%,60.15%and167.39%, respectively.7. Compared to Control-Hca-P, the invasion ability of Anxa5-Hca-P-1, Anxa5-Hca-P-2and Anxa5-Hca-P-3cell lines increased by47.18%,57.57%and100.03%, respectively.8. The expression level of Anxa5showed promote effect ontumor apparent malignancy, but no effect on tumor size.9. The degree of lymph nodemetastases of Anxa5-Hca-P-1and Anxa5-Hca-P-3cell ines were higher than that ofControl-Hca-P, and the degree of lymph node metastasis of Anxa5-Hca-P-3was higherthan that of Anxa5-Hca-P-1.Conclusion:1. Anxa5expressed in both Hca-P and Hca-F cells. The protein level ofAnxa5was increased by155%in Hca-F compared to Hca-P cell.2. Following Anxa5up-regulation, the size of Hca-P cell and the number of rough intracellular endoplasmicreticula and mitochondria increased, mitochondria swelled, mitochondrial crista andmatrix density decreased.3. Anxa5up-regulation increased the proliferation abilitiy ofHca-P cells, interestingly, its up-regulation to a certain degree showed inhibitory effecton Hca-P proliferation.4. Anxa5up-regulation resulted in significant enhancement inmigration ability of Hca-P cells, the more up-regulation of Anxa5, the moreenhancement in migration ability.5. Anxa5up-regulation resulted in significantenhancement in invasion ability of Hca-P cells, the more up-regulation of Anxa5, themore enhancement was observed for Hca-P invasion ability.6. The expression of Anxa5was relevant with tumor apparent malignant grade and had nothing to do with tumorsize.7. Anxa5up-regulation promoted LNM. The degree of LNM was positivelycorrelated with Anxa5expression level; Taken together, Anxa5plays a crucial role inmurine hepatocarcinoma cell lymphatic metastasis and has potential role for use as anovel biomaker for therapy of hepatocarcinoma.