(A) RT-PCR showed mRNA expression of ANXA2 in the NOK, HSC-3, SCC-4 and CAL27 cells
(A) RT-PCR showed mRNA expression of ANXA2 in the NOK, HSC-3, SCC-4 and CAL27 cells. migration and invasion of OSCC cells. ( 95%) /th th align=”remaining” rowspan=”1″ colspan=”1″ em P (-)-Nicotine ditartrate /em /th /thead Age?57471.2150.2701 ?57431.305(0.806?~?2.114)0.280GenderMale470.1020.7491Female441.081(0.666?~?1.752)0.753Tumor size? ?3.3471.4200.2331?3.3431.335(0.822?~?2.167)0.246TNM stageI?+?II498.1560.0041III?+?IV392.015(1.226?~?3.311)0.006Degree of differentiationLow405.6860.05810.069Moderate371.402 (0.760?~?2.588)0.279High520.711 (0.381?~?1.324)0.282Lymph node metastasisNo479.9980.0021Ysera372.310(1.345?~?3.967)0.002Invasion depth? ?5?mm464.1830.0411?5?mm401.811(1.008?~?3.254)0.047Tumor locationTongue430.1040.95010.951Gingiva461.074 (0.613?~?1.881)0.804Others481.103 (0.575?~?2.116)0.768ANXA2 expressionNegative5413.6760.0001Positive393.022(1.622?~?5.631)0.000 Open in a separate window Table 3 Cox multivariate analysis in individuals with OSSC. thead th align=”remaining” rowspan=”1″ colspan=”1″ Variables /th th align=”remaining” rowspan=”1″ colspan=”1″ em B /em /th th align=”remaining” rowspan=”1″ colspan=”1″ em SE /em /th th align=”remaining” rowspan=”1″ colspan=”1″ em Wald /em /th th align=”remaining” rowspan=”1″ colspan=”1″ em Sig /em /th th align=”remaining” rowspan=”1″ colspan=”1″ em HR ( 95%CI ) /em /th /thead Lymph node metastasis0.8230.2828.4890.0042.277 (1.309?~?3.960)ANXA2 expression1.1100.32311.8120.0013.034(1.611?~?5.712) Open in a separate window Manifestation of ANXA2 in OSCC cells The results of PCR and GRS european blot analysis showed the manifestation of ANXA2 in OSCC cell lines HSC-3, SCC-4 and CAL27 were all higher than the normal NOK cells (Fig.?3), consistent with the results in tissues. Additionally, the manifestation of ANXA2 in HSC-3 and SCC-4 cells were higher than that in CAL27 cell, so HSC-3 and SCC-4 cells were selected for the further experiments. Open in a separate windows Number 3 The mRNA and protein manifestation levels of ANXA2 in NOK, HSC-3, SCC-4 and CAL27. (A) RT-PCR showed mRNA manifestation of ANXA2 in the NOK, HSC-3, SCC-4 and CAL27 cells. (B) Western blot showed the protein manifestation of ANXA2 in the NOK, HSC-3, SCC-4 and CAL27 cells. (C) Assessment of the protein manifestation of ANXA2 in the NOK, HSC-3, SCC-4 and CAL27 cells. Data were offered as the mean??SD of at least three repeated experiments. * em P /em ? ?0.05, compared with NOK cells; # em P /em ? ?0.05, compared with HSC-3 and SCC-4 cells. Silencing of ANXA2 suppressed the proliferation of OSCC cells As demonstrated in Fig.?4, the manifestation level of ANXA2 mRNA and protein were significantly down-regulated in HSC-3 and SCC-4 cells after transfected with ANXA2-siRNA. Then CCK-8 assay was carried out to analyze the effect of ANXA2 within the proliferation ability of HSC-3 and SCC-4 cells (Fig.?5). The results showed the proliferation activity in ANXA2-siRNA group was significantly lower than that in the NC and blank organizations at 36?h and 48?h both in HSC-3 and SCC-4 cells ( em P /em ? ?0.05). Open in a separate window Number 4 Manifestation of ANXA2 after transfection with siRNA. (A) RT-PCR showed mRNA manifestation of ANXA2 after transfection with siRNA in HSC-3 and SCC-4 cells. (B) Western blot showed the protein manifestation of ANXA2 after transfection with siRNA in HSC-3 and SCC-4 cells. (C) Relative manifestation of ANXA2 after transfection with siRNA in HSC-3 and SCC-4 cells. Data were offered as the mean??SD of at least three repeated experiments. * em P /em ? ?0.05, compared with blank group; # em P /em ? ?0.05, compared with NC group. Open in a separate window Number (-)-Nicotine ditartrate 5 Effect of silencing ANXA2 manifestation on cell proliferation ability recognized by CCK-8. (A) Cell proliferation ability of HSC-3 cells; (B) Cell proliferation ability of SCC-4 cells. Data were offered as the mean??SD of at least three repeated experiments. * em P /em ? ?0.05, compared with blank group; # em P /em ? ?0.05, compared with NC group. Silencing of ANXA2 suppressed the migration ability of OSCC cells Wound healing assay was used to identify the effect of ANAX2-siRNA on migration ability of HSC-3 and SCC-4 cells. As demonstrated in Fig.?6, the migration rate of HSC-3 cells in the blank, NC and ANXA2-siRNA organizations were (52??4)%, (51??5)%, and (32??5)% at 24?h, (88??4)%, (86??5)%, and (62??4)% at 48?h, respectively. The migration rate of SCC-4 cells in the blank, NC and ANXA2-siRNA organizations were (50??8)% , (48??6)%, and (35??5)% at 24?h, (90??5)%, (89??4)%, and (63??5)% at 48?h, respectively. The results showed that ANXA2-siRNA significantly suppressed the migration of HSC-3 and SCC-4 cells ( em P /em ? ?0.05). Open in a separate window Number 6 Effect of silencing ANXA2 manifestation on migration ability of HSC-3 and SCC-4 cells recognized by wound healing assay. Silencing of ANXA2 suppressed the invasion ability of OSCC cells The invasion capabilities of HSC-3 and SCC-4 (-)-Nicotine ditartrate cells were analyzed by Transwell assay. As demonstrated in Fig.?7, the invasion cell figures were significantly reduced the ANXA2-siRNA group compared with the NC and blank organizations, indicating that.