Primary Investigation Of The Tumor Suppressor Mechanism Of STGC3 Gene On Nasopharyngeal Carcinoma Cell Line CNE2

Objective: Previous investigation indicated that overexpression of STGC3 could inhibit the proliferation of the cultured CNE2 cell line. To examine the effect of STGC3 on tumorigenicity of CNE2 cell line and explore its mechanism in nude mice so as to further investigate the tumor suppressor role of STGC3.Methods: The Tet/pTRE-STGC3/CNE2 cell line was planted under the front leg skin of nude mice induced by Dox via intra-peritoneal injection. The mRNA and protein levels of STGC3 in transplanted tumor tissues were detected with RT-PCR, Western blotting and immunohistochemistry. The apoptosis ratio of the tumor cells was analyzed by flow cytometry. Bcl-2 and Bax proteins were examined by immunohistochemistry method. Immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, PDQuest software analysis, peptide mass fingerpringting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) and Mascot database searching were used to separate and identify differentially expressed proteins when STGC3 was highly expressed in the tumor tissue. Heat shock 70 kDa protein was detected in tumor tissues of Tet/pTRE/CNE2 and Tet/pTRE-STGC3/CNE2 in nude mice.Results: The results indicated that STGC3 was mainly expressed in nucleus. A high level of STGC3 expression could inhibit the tumorigenicity of the CNE2 cell line in nude mice. Tumor grew slowly, later and smaller. Cell apoptotic percentage increased, Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated in Tet/pTRE-STGC3/CNE2 cell line induced by Dox (P<0.01). Proteomic experiment indicated that Peptidyl-prolyl cis-trans isomerase A, Cofilin-1, Prohibitin and Guanine nucleotide-binding protein subunit beta 2-like 1 were up-regulated and Serine/threonine-protein kinase WNK1, Splicing factor arginine/serine-rich 1 and Heat shock 70kDa protein 8 isoform 2 variant were down-regulate when STGC3 was highly expressed. The proteins involved in cytoskeleton, cell apoptosis, cell immunity, cell proliferation and signal pathway. Heat shock 70 kDa proteins detected in the tumor tissues of Tet/pTRE /CNE2 and Tet/pTRE-STGC3/CNE2 in nude mice confirmed the proteomic experimental results, which indicated that proteomic results reflected the protein expression condition in tumor tissues fairly well.Conclusion:1 STGC3 could suppress the proliferation of the Tet/pTRE-STGC3/CNE2 cell line through raising the apoptosis ratio by up-regulated the Bax protein expression and down-regulated the Bcl-2 protein expression.2 Over-expression of STGC3 might make the tumorigenisity of CNE2 cell line in nude mice decrease via increasing the expression of Peptidyl-prolyl cis-trans isomerase A,Cofilin-1,prohibitin and Guanine nucleotide-binding protein subunit beta 2-like 1 and down-regulating Serine/threonine-protein kinase WNK1,Splicing factor arginine/serine-rich 1 and Heat shock 70kDa protein 8 isoform 2 variant expression. Objective: STGC3 over-expression could inhibit the proliferation of CNE2 cell line in vitro and vivo. Otherwise, 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. This paper was designed to investigate the effect of progesterone on Tet/pTRE-STGC3/CNE2 cell line and its related molecular mechanism.Methods: The proliferative capacity of the CNE2 cell line and Tet/pTRE-STGC3/CNE2 cell line in the culture medium with progesterone was evaluated by the means of the microculture tetrazolium assay (MTT). A series of methods, including immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, Coomassie brilliant blue staining, Imaging Master software analysis, peptide mass fingerpringting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Mascot database searching, were used to separate and identify differentially expressed proteins induced by progesterone on Tet/pTRE-STGC3/CNE2 cells.Results: (1) MTT assay showed that the proliferation of CNE2 cell line and Tet/pTRE-STGC3/CNE2 cell line induced by Dox were significantly inhibited by progesterone (10-9mol/L-10-5mol/L) in concentration-dependent manner. The inhibitive ratios were 0.8%-34.3% and 24.1%-50.9% respectively. The inhibitive ratio of progesterone on Tet/pTRE-STGC3/CNE2 cell line was more obvious than that on CNE2 cell line. (2) After 2-DE, there were 785±38, 756±41 spots in Tet/pTRE-STGC3/CNE2 cell line induced by Dox and treated with progesterone and PBS, respectively. 8 differential proteins were primaryly identified by MALDI-TOF-MS. Progesterone could increase the expression of Triosephosphate isomerase,Cofilin-1,Heterogeneous nuclear ribonucleoprotein C2, Peptidyl-prolyl cis-trans isomerase A and Hypothetical protein and down-regulate Heterogeneous nuclear ribonucleoprotein A2/B1 isoform 2 and Keratin 13 expression. The proteins involved in cytoskeleton, cell differentiation, cell immunity and cell metabolism.Conclusion:1 Progesterone could enhance the tumor suppressor effect of STGC3 on CNE2 cell proliferation.2 Progesterone could enhance the suppressive function of STGC3 via increasing the expression of Triosephosphate isomerase,Cofilin-1,Heterogeneous nuclear ribonucleoprotein C2, Peptidyl-prolyl cis-trans isomerase A and Hypothetical protein and down-regulating Heterogeneous nuclear ribonucleoprotein A2/B1 isoform 2 and Keratin 13 expression.