Impact Of Calcium Binding S100A8 And S100A9 Protein On Nasopharyngeal Carcinoma Cell Invasion And Migration

This paper contains two parts:(A) The impact of exogenous S100A8 and S100A9 protein on the invasion and migration of nasopharyngeal carcinoma cell; (B) The construction and expression of S100A8 and S100A9 eukaryotic co-expression plasmid.Part 1 Impact of Exogenous S100A8 and A100A9 Protein on Nasopharyngeal Carcinoma Cell Invasion and MigrationPurpose:To explore the impact of exogenous S100A8 and S100A9 protein on the proliferation, migration and invasion abilities of nasopharyngeal carcinoma cell as well as the MMPs expression changes in the process, thus providing theoretical foundation for relevant mechanisms researches about nasopharyngeal carcinoma cell invasion and migration by S100A8 and A100A9 protein.Method:(DCCK8 method is applied to measure the impact of different concentrations of S100A8 and S100A9 protein on the proliferation ability of CNE1 and CNE2 cell. ②The medium with 1μg/ml S100A8 and S100A9 protein compound is taken as the experiment group, and the medium without S100A8 and S100A9 protein compound is taken as the control group. Transwell chamber migration and invasion experiments are conducted respectively to detect the invasion and migration situations of the nasopharyngeal carcinoma cell. ③ The culture cell with 1μg/ml S100A8 and S100A9 protein compound in the medium is taken as the experiment group, and that without S100A8 and S100A9 protein compound in the medium is taken as the control group. After the cell culture, use the real time-PCR to detect the expression of the invasion and migration marker MMPs gene.Results:① As is shown by the CCK8 experiment results,0-5μg/ml S100A8/A9 protein can promote the cell proliferation of CNE1 and CNE2. When the concentration is higher than 30μg/ml, it shows the inhibition effect; ②Results of the Transwell cell migration experiment show the quantity of cell passing through the polycarbonate membrane in the experiment group is larger than that in the control group, and that the migration ability of cell in the experiment group is stronger than that in the control group, the difference of which is with statistical significance (P<0.05);③Results of the Transwell cell invasion experiment show the quantity of cell passing through the polycarbonate membrane in the experiment group is larger than that in the control group, and that the invasion ability of cell in the experiment group is stronger than that in the control group, the difference of which is with statistical significance (P<0.05);④According to the results of the MMPs gene test by real-time PCR, in contrast to the control group, the MMP2 and MMP7 gene expression of CNE1 cell in the experiment group has been up-regulated, the difference of which has the statistical significance (P<0.05). Its MMP9 and MMP12 gene expression has been down-regulated, the difference of which has the statistical significance (P<0.05); As for the CNE2 cell, its MMP1, MMP8, MMP9 and MMP12 gene expression has been down-regulated, the difference of which has the statistical significance (P<0.05).Conclusions:Low-concentration (<10μg/ml) S100A8/A9 protein can promote CNE1 and CNE2 cell proliferation, while the concentration exceeding 30μg/ml will inhibit the cell growth. Under the circumstance that the S100A8/A9 protein concentration is 1μg/ml, it may promote the CNE1 and CNE2 cell invasion and migration. the control group, the difference of which is with statistical significance (P<0.05);④According to the results of the MMPs gene test by real-time PCR, in contrast to the control group, the MMP2 and MMP7 gene expression of CNE1 cell in the experiment group has been up-regulated, the difference of which has the statistical significance (P<0.05). Its MMP9 and MMP12 gene expression has been down-regulated, the difference of which has the statistical significance (P<0.05); As for the CNE2 cell, its MMP1, MMP8, MMP9 and MMP12 gene expression has been down-regulated, the difference of which has the statistical significance (P<0.05).Conclusions:Low-concentration (<10μg/ml) S100A8/A9 protein can promote CNE1 and CNE2 cell proliferation, while the concentration exceeding 30μg/ml will inhibit the cell growth. Under the circumstance that the S100A8/A9 protein concentration is 1μg/ml, it may promote the CNE1 and CNE2 cell invasion and migration.Part 2 Construction of co-expression plasmid pBudCE4.1-S100A8-S100A9 and its expression in vitroPurpose:The purpose of this paper is to clone S100A8 and S100A9 gene from NPC cells along with the construction of expression plasmid and its expression in NPC cells, thus laying a foundation for research on the relationship between endogenous S100A8 and S100A9 protein and nasopharyngeal carcinoma.Method:The target gene S100A8 and S100A9 fragment of PCR amplification is inserted into the carrier pBudCE4.1 to construct the expression plasmid S100A8-S100A9-pBudCE4.1 which will be transfected to CNE1 via double enzyme digestion, PCR and sequencing identification and then be detected by real-time PCR and Western Blot.Results:The results of PCR, double enzyme digestion and nucleotide sequencing show that there is no mutation in S100A8 and S100A9 amino acid sequence (S100A8 has 1 single base mutation) and the expression plasmid S100A8-S100A9-pBudCE4.1 is correctly constructed. Additionally, the results of RT-PCR and Western Blot indicate that SI00A8 and S100A9 gene can transcribe and express correctly in CNE1.Conclusions:S100A8 and S100A9 gene can be successfully cloned with the construction of the expression plasmid S100A8-S100A9-pBudCE4.1 which suggests stable expression in NPC cells.