Difference Analysis On The Tumorigenicity Of STGC3 Transfected CNE2 Cell Line In Female And Male Nude Mice

Objective: Our previous study indicated that there were a delay in tumor formation and a dramatic reduction in tumor size when CNE2 cell line overexpressed STGC3 were injected into nude mice and the tumor size in female nude mice was highly smaller than that in male. To identify the above results, the proliferation effect ofβ-estradiol on on CNE2 cell line over-expressied STGC3 was examined in vivo. The samples were enlarged and the tumorigenicity difference of the pcDNA3.1(+)/STGC3/CNE2 cell line in female and male nude mice were further investigated.Methods: The recombinant pcDNA3.1(+)/STGC3 plasmid was constructed and transfected into CNE2 cell line with lipofectamine 2000. CNE2 cell clones with stable STGC3 expression were obtained through G418 selection and identified by RT-PCR and Western blotting methods. The effect ofβ-Estradiol on growth rate of pcDNA3.1(+)/STGC3/CNE2 cell line was examined by cytometry. The pcDNA3.1(+)/STGC3/CNE2 cell line was inoculated subcutaneously in nude mice. Tumorigenicity diversity was analyzed in female and male nude mice. STGC3 expression was detected in tumor xenografts in nude mice by RT-PCR, immunohistochemistry and Western blotting methods. Cell morphologic changes were observed by microscope. Flow cytometry was used to analyze cell cycle.Results: ER protein was expressed in nucleus and cytoplasm. The results indicated that pcDNA3.1(+)/STGC3/CNE2 cell growth rate decreased after treated withβ-estradiol in vitro. Tumorigenicity test showed that there were significant differences in tumor size and weight between pcDNA3.1(+)/STGC3/CNE2 and control cases (P<0.05). Furthermore, tumor size and weight had significant differences between male and female nude mice in the STGC3 transfected cases and tumor growth velocity of pcDNA3.1(+)/STGC3/CNE2 in the female nude mice was the lowest in all cases (P<0.05). No significant difference was found between male and female nude mice in other control cases (P>0.05). There were more cells blocked in G0/G1 phase in tumor xenografts of pcDNA3.1(+)/STGC3/CNE2 cell line of the female nude mice case than those in other cases.Conclusion:1β-estradiol might enhance the effect of STGC3 to inhibit CNE2 cell proliferation in vivo.2 There was gender difference in STGC3 on inhibiting the tumorigenicity of CNE2 cell line in vitro. The tumor suppressor effect of STGC3 on CNE2 cell growth in female nude mice was stronger than that in male and the cells blocked in G0/G1 phase.Part 2 Difference analysis of the protein expression patern in tumor tissues of the STGC3-transfected CNE2 line in female and male nude miceObjective: The tumor suppressor effect of STGC3 on CNE2 cell growth in female nude mice was stronger than that in male and the cells blocked in G0/G1 phase. To further explore its mechanism, proteomic technologies was employed to screen and identify the differentially expressed proteins between tumor tissues of pcDNA3.1(+)/STGC3/CNE2 cell line in male and female nude mice.Methods: The total proteins of the tumor tissues of pcDNA3.1(+)/STGC3/CNE2 cell line in the male and female nude mice were separated by immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis. After a modified Neuhoff's colloidal coomassie blue G-250 staining, image scanning and PDQuest software analysis, the differentially expressed protein spots were incised from gels and digested by trypsin in gel. Then matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for peptides mass analysis. The acquired peptide mass fingerprints (PMFs) were used to search for matches in SWISS-PROT and NCBI databases with Mascot software. The functions of the differenyially expressed proteins were further analyzed according to bioinformatic resources. Heat shock 70 kDa protein was detected in the tumor tissue of pcDNA3.1(+)/STGC3/CNE2 cell line in female and male nude mice.Results: using 2-DE, well-resolved and reproducible 2-DE pattern of tumor tissues of pcDNA3.1(+)/STGC3/CNE2 in male and female nude mice were established. There were 807±18 spots matched in tumor tissues of pcDNA3.1(+)/STGC3/CNE2 in female nude mice. For tumor tissues of pcDNA3.1(+)/STGC3/CNE2 in male nude mice, the number of average spots of 3 gels was 843±32. The average match rate was 87.2%. The differentially up-regulated, clear and well spots were signed, then sected and degested. The fingerprints of the spots were established and researched in related database in Mascot software. 5 up-regulated spots and 4 down-regulated spots in tumor tissues of pcDNA3.1(+)/STGC3/CNE2 in female nude mice were identified. Fumarate hydratase, Thioredoxin peroxidase, IgE-dependent histamine-releasing factor, Serine/threonine-protein kinase WNK1 and Ribosomal protein P0 variant were up-regulated and Heat shock 70kDa protein, Cofilin-1, L-lactate dehydrogenase B chain and Guanine nucleotide-binding protein subunit beta 2-like 1 were down-regulated in tumor tissues of pcDNA3.1(+)/STGC3/CNE2 in female nude mice compared those in male nude mice. These proteins involved in cytoskeleton, cell metabolism, molecular chaperones, immunological regulation and signal pathways. Heat shock 70 kDa protein detected in the tumor tissue of pcDNA3.1(+)/STGC3/CNE2 cell line in female and male nude mice confirmed the proteomic experimental results, which indicated that the proteomic results reflected the protein expression condition in the tumor tissue of pcDNA3.1(+)/STGC3/CNE2 cell line in female and male nude mice.Conclusion:1 Differentially expressed proteins existed in the tumor tissues of pcDNA3.1(+)/STGC3/CNE2 in male and female nude mice.2 9 differentially expressed proteins were screened and identified. Fumarate hydratase, Probable thioredoxin peroxidase, IgE-dependent histamine-releasing factor, Serine/threonine-protein kinase WNK1 and Ribosomal protein P0 variant were up-regulated and Heat shock 70kDa protein, Cofilin-1, L-lactate dehydrogenase B chain and Guanine nucleotide-binding protein subunit beta 2-like 1 were down-regulated in tumor tissues of pcDNA3.1(+)/STGC3/CNE2 in female nude mice compared those in male nude mice.3 These proteins might enhance the tumor suppressor effect of STGC3 on CNE2 cell line.