Effects Of STGC3 Gene Down-expression On NP69 Cells And Its Molecular Mechanisms

[Objectives] To explore the roles of STGC3 gene in carcinogenesis of nasopharyngeal carcinoma and its molecular mechanisms, effects of knocked-down STGC3 by sh RNA were investigated on cell growth and proliferation, malignant abilities of migration, invasion and tumorigenicity of immortalized nasopharyngeal epithelial NP69 cell line in vitro and in vivo, and the differentially expressed proteins were analysed with i TRAQ coupled with mass spectrometry in NP69 cell line with stable low expression of STGC3 gene.After verification of some differentially expressed proteins, the expression and function of them were detected in nasopharyngeal carcinoma cell lines and tissues.[Methods] sh RNA targeting STGC was designed to establish expression plasmid which was confirmed by double enzyme digestion and DNA sequencing, and then transfected into NP69 cell line and selected by G418 to establish a stable cell line with low expression of STGC3. After that, the cell cycle, cell growth and proliferation were tested by flow cytometry and MTT, respectively.The changes in abilities of colongenic, invasion, migration and tumorigenicity were measured by cell colony formation assay, transwell experiments and inoculation in nude mice, respectively. Subsequently, the total proteins were extracted and quantified from the NP69 cells with low expression of STGC3 and the control cells respectively and information of abundance about protein expression were obtained by i TRAQ coupled with mass spectrometry after proteolysis, and then the differentially expressed proteins were screeninged by searching related database and the interactional networks among proteins were constructed by bioinformatics IPA( Ingenuity Pathways Analysis).Then the important protein molecules with different abundance were verified by RT-PCR and Western Blotting in p RNAi-U6.1/sh RNA/STGC3/NP69 cells, and their expression in nasopharyngeal carcinoma cell lines and tissues were detected. After intervention of function of the differentially-expressed proteins, effects on cell growth, proliferation, invasion and migration ability of nasopharyngeal carcinoma CNE1, CNE2 cells and NP69 cells with low expression of STGC3 were observed, and the expression of protein molecules in signal pathways related to differentially-expressed proteins were detected at the same time.[Results] The recombinant vector of p RNAi-U6.1/sh RNA/STGC3 targeting to STGC3 was successfully constructed and verified by bacteria liquid PCR, double enzyme digestion and DNA sequencing. The expression of STGC3 m RNA in NP69 cell line was significantly decreased and verified by RT-PCR after transfection with this recombinant vector, and this cell line with STGC3 low expression was named p RNAi-U6.1/sh RNA/STGC3/NP69, and the cell line transfected with p RNAi-U6.1/scramble plasmid was accordingly named p RNAi-U6.1/scramble/NP69.According to the results from MTT assay cell growth curve showed that proliferation of p RNAi-U6.1/sh RNA/STGC3 /NP69 cells was faster than that of NP69 and p RNAi-U6.1/scramble /NP69 cells on the 4th day.Numbers and volume of cell colonies of p RNAi-U6.1/sh RNA/STGC3/NP69 cell line were much more than that of the controls showed by colongenic experiment(p < 0.05). Numbers of p RNAi-U6.1/ sh RNA/STGC3/NP69 cells in(G2+S) phase were notably higher than that of control group which was showed by FCM analysis, indicating that low expression of STGC3 gene can promote cell proliferation and improve colongenic ability of NP69 cells by affecting cell cycle progression.Numbers of migrated p RNAi-U6.1/ sh RNA/ STGC3/NP69 cells were much more than that of p RNAi-U6.1/ scramble/NP69 and NP69 cell line which were showed by Transwell with package of matrigel or not,suggesting that lower expression of STGC3 gene in NP69 cell line significantly enhanced its capability of cell migration and invasion.However, during the period of 8 weeks the xenografted tumor did not form after subcutaneous inoculation with these three kinds of cell lines in nude mice, respectively.After successfully transfection with p RNAi-U6.1/sh RNA/STGC3, a total of 83 kinds of proteins with different expression were showed by i TRAQ isotope labeling and mass spectrometry. Of them 45 kinds of proteins such as m TOR, TPM2, Cofilin-1, ANXA3, Musashi-2, Kindlin-2were up-regulated and 38 were down-regulated including Beclin-1,TSC-22, AMOTL2 and so on.The function of these 83 kinds of protein molecules was involved in protein synthesis, cell growth and proliferation,cytoskeleton, signal transduction and autophagy, and which formed the complex network with interaction among them.The enhanced expression of m TOR and reduced Beclin-1 were validated through RT-PCR, Western Blotting and immunohistochemical detection in p RNAi-U6.1/sh RNA/STGC3/NP69 cell line. The expression of m TOR and Beclin-1 were also detected in nasopharyngeal carcinoma tissues, CNE1 and CNE2 cell lines in which there were enhanced m TOR and reduced Beclin-1.The positive expression rate of m TOR protein in nasopharyngeal carcinoma tissues of patients with clinical Ⅲ, Ⅳstage and with lymph node metastasis were higher than that in patients with clinical Ⅱstage and without lymph node metastasis,respectively. The expression of Beclin-1 protein was negatively correlated with m TOR in nasopharyngeal carcinoma tissue.After that, effects of rapamycin with different concentrations(0, 1, 2,4 and 8umol/L) on cell growth, proliferation, invasion and migration of CNE1, CNE2 and p RNAi-U6.1/sh RNA/STGC3/NP69 cell lines were observed. The results showed that rapamycin can significantly inhibit cell growth, proliferation, invasion and migration of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines corresponding to the concentration gradient and time of rapamycin, but there were no obvious inhibitory effects on CNE1 cells after rapamycin treatment with different concentration and time. After treatment with rapamycin for 24 h at the same concentration, the inhibition rate on cell growth and proliferation of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines were markedly lower than that at 48 h and 72 h, but there was no statistical difference between the inhibition rate at 48 h and 72 h. After treatment with 8umol/L rapamycin for 48 h, migratory and invasive abilities of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines were significantly suppressed.The results of Western blot showed that the expression of P-p70S6 K and cyclin D1 in these two kinds of cell lines were reduced, but there were no obvious changes at expression of m TOR, and p70S6 K protein after treatment with 8μmol/L rapamycin for 48 h. These results suggested that rapamycin can inhibit cell proliferation, migratory and invasive abilities of p RNAi-U6.1/sh RNA/STGC3/NP69 and CNE2 cell lines through suppressing expression of P-p70s6 k and Cyclin D1, but there were no obvious effects on their expression and abilities of CNE1 cell line.[Conclusions]1) Knock-Down STGC3 by recombinant vector p RNAi-U6.1/sh RNA/STGC3 can lead to different expression of 83 kinds of proteins including enhanced m TOR and reduced Beclin-1, and promote growth and proliferation, enhance abilities of migration and invasion, and result in partly malignant transformation of NP69 cell line.2) Partly malignant transformation of NP69 cell line result from Knock-Down STGC3 maybe related to m TOR signal pathway.