| Part I The expression and clinical significance of HMGA2 in nasopharyngeal carcinomaObjective:To compare the differences of High-mobility group protein 2 (HMGA2) expression in nasopharyngeal carcinoma tissue and non-tumoral inflammatory nasopharynx tissues, and to analysis the relationship between the HMGA2 levels and clinicopathological characteristics and prognosis.Methods:1. By real-time quantitative PCR to detect HMGA2 mRNA expression of NPC (n=72) and non-tumoral inflammatory nasopharynx (n=20) paraffin specimens and analyze its relationship with the clinical pathologic characteristics.2. Using immunohistochemistry to detect HMGA2 expression of NPC (n=124) and and non-tumoral inflammatory nasopharynx (n=20) paraffin specimens, and analyzes its relationship with clinical pathological factors.3. The 3 years disease-free survival differences between the low HMGA2 mRNA expression and high HMGA2 mRNA expression group were analyzed by Kaplan-Meier survival curve.4. The differences of group 3 year survival rate in low HMGA2 protein expression group and high HMGA2 protein expression group were estimated by Log-rank test. And the pronositc value of HMGA2 was estimated by the Cox regression model.Results:1. The HMGA2 mRNA relative expression level in NPC tissue (median 0.294) was significantly higher than the non-tumoral inflammatory nasopharynx (median 0.045) (P< 0.05). Moreover, HMGA2 mRNA expression in NPC tissue posivively correlated with two years and metastasis of nasopharyngeal carcinoma patients factors (P< 0.01).2. HMGA2 high protein expression (59/124, positive rate:47.58%), non-tumoral inflammatory nasopharynx has no HMGA2 expression(positive rate:0).The HMGA2 expression in NPC was significantly higher than that of inflammatory nasopharynx (x2=16.12, P< 0.01). HMGA2 protein expression in NPC posivively correlated with N staging, TNM stage,2 years related recurrence and metastasis(P< 0.01).3. Disease-free survival rate in 3 years of HMGA2 high mRNA expression group was significantly lower than low HMGA2 mRNA expression group (HR,3.52; 95% CI:1.34 7.79; P=0.01).4. The 3 year survival rate in all NPC was 78.8%(95% CI:0.708-0.852),3 year survival rate of low and high HMGA2 protein expression group was 90.8%(95% CI: 0.806-0.957) and 66.1%(95% CI:0.525-0.766) respectively, the difference was statistically significant (P<0.001). Univariate analysis showed that T stage, N stage, TNM staging and HMGA2 were predictors of poor prognosis. Multivariate analysis showed that only N stage (HR:7.892,95%CI:2.731-22.807, P<0.001) and HMGA2 ((HR:2.683,95%CI:1.185-6.077, P=0.018)) were independent predictors of poor prognosis.Conclusion:These results demonstrated that HMGA2, an independent prognostic factor, may promote NPC occurrence and progression, and HMGA2 may be considered a potential therapeutic target in NPC.Part Ⅱ The expression of HMGA2 contributes to nasopharyngeal carcinoma cells malignant biological behaviorsObjective:The aim of this study was to investigate the effect of HMGA2 in regulating proliferation, migration and invasion in NPC in vitro.Methods:1. The expression of HMGA2 mRNA and protein were estimated by qRT-PCR and Western blot in human immortalized nasopharyngeal epithelium NP-69 cell line and human nasopharyngeal carcinoma cell line CNE-1, CNE-2,5-8F,6-10B, C666-1 and HNE-1.2. Selected relatively high express HMGA2 cells, CNE-1 and CNE-2, transient transfection si-HMGA2. The viability and metastatic ability were analyzed by Cell Counting Kit-8 (CCK8), colony formation assay, and transwell assay. The changes of cell cycle were evaluated by flow cytometry.Results:1. HMGA2 mRNA expression in each NPC cell lines was significantly higher than that of NP-69 (P< 0.01). Also, HMGA2 protein high expression in NPC cells, little expression in NP-69, the results were statistically significant (P< 0.01).2. In addition, following knockdown of HMGA2 by si-HMGA2, the growth rate was markedly inhibited in CNE-1 and CNE-2 cells on 2th day,3th day,4th day (P<0.001). Transwell showed that the cells migrated to the lower chamber is 41.22 ±4.45 and 45.34 ±6.11 in the si-HMGA2 CNE-1 and si-HMGA2 CNE-2 group, and the cells migrated to the lower chamber were 141.34±5.22 and 157.36±6.41, the differences were statistically significant (P< 0.001). Invasive experiments showed that the cells of si-HMGA2 group migrated through Matrigel were 34.03±4.56 and 41.48±5.31 in CNE-1 and CNE-2, the cells of control group mitrated through the Matrigel were 113.83±4.18 and 135.33±4.36, two group were statistically significant (P<0.001). Clone formation rate was reduced by 45%,55% respectively(P< 0.001) in CNE-1 and CNE-2 si-HMGA2 group. By flow cytometry, G1 phase cells increased (P<0.001), and G2/Mphase cells proportion reduced (P<0.001) in si-HMGA2 group.Conclusion:HMGA2 might take part in some malignant biological behaviors including proliferation, tumor growth, migration, invasion and cell cycle.Part III The expression of HMGA2 contributes to nasopharyngeal carcinoma cells malignant biological behaviorsObjective:To assess the relationship between the expression of HMGA2 and epithelial-mesenchymal trasition markers and study the mechanism of HMGA2 promotes EMT.Methods:1. The expression of E-cadherin, Vimentin in 124 cases of NPC paraffin sample were evaluated by immunohistochemistry. And analyze the realationship between E-cadherin, Vimentin expression and clinical pathologic characteristics and prognosis.2. The relationship between HMGA2, E-cadherin and Vimentin were evaluated by Spearman rank correlation analysis.3. Following the silencing of HMGA2 in CNE-1 and CNE-2, the expression of E-cadherin, Vimentin and Snail were investgated by Western blot and cell immunofluorescence.4. Following the silencing of HMGA2, the key proteins expression of TGF-β/Smad pathway were analyzed by Western blot.Results:1. HMGA2 expression (showed in Part I), E-cadherin positive particles mainly located in cell membrane, Vimentin positive particles located in cytoplasm and/or part of the cell membrane. E-cadherin protein high expression(15/20, positive rate:75%) in 20 cases of chronic inflammation nasopharyngeal tissues, E-cadherin protein high expression(54/124, positive rate:43.55%) in 124 cases of NPC. And the results showed that E-cadherin expression in NPC lower than that of inflammatory tissues (x2=6.826, P<0.001); Vimentin express little in inflammatory tissues (positive rate:0), of high expression in NPC tissue (78/124positive rate:62.9%), means that Vimentin positive expression rate in NPC is higher than inflammatory tissue (x2= 27.449, P< 0.001).2. E-cadherin expression in NPC posivively correlated with N stage, TNM stage,2 years related recurrence and metastasis (P<0.01), Vimentin expression in NPC posivively correlated with N stage, TNM stage,2 years related recurrence and metastasis (P<0.01). In the low expression of E-cadherin group,3 year overall survival rates were 70%(95% CI:0.578-0.792), while in the high expression of E-cadherin group,3 year overall survival was 88.8%(95% CI:0.768-0.948). In the low expression of Vimentin group,3 year overall survival were 89.1%(95% CI: 0.757-0.953), in the high expression of Vimentin group 3 year overall survival rates were 71.8%(95% CI:0.604-0.804) (P<0.01).3. E-cadherin negatively correlated to the expression of Vimentin ((r=0.605, P< 0.001). The expression level of HMGA2 and Vimentin were positively correlated (r= 0.431, P<0.001), HMGA2 negatively correlated with the expression level E-cadherin (r=0.413, P< 0.001).4. By Western blot, in cell line CNE-1 and CNE-2, E-cadherin expression is relatively lowerer than the control, Vimentin expression is relatively higher than the control. after inhibiting HMGA2, E-cadherin expression up-regulated, and the Vimentin expression and Snail expression down-regulated. NPC cells Compared with control group, the inhibiting HMGA2 green fluorescent tags E-cadherin (located in cell membrane) expression high-rgulated, and red fluorescent tags Vimentin group (located in cytoplasm and cell membrane) expression down-rgulated.5. The TGF-βRⅡ, Smad3 and p-Smad3 expression were assessed by western blot, and inhibiting HMGA2, and found the key proteins of TGF-βRⅡ/Smad pathway were significantly down-regulated.Conclusion: HMGA2 expression and EMT epithelial markers E-cadherin negatively correlated, and mesenchymal markers Vimentin were positively correlated HMGA2 expression. By inhibiing HMGA2 can lead to E-cadherin increases, Vimentin reduced, and may be involved in NPC EMT process through TGF-β/Smad signaling pathways which affect the proliferation of NPC, metastasis and other malignant biological behavior. |
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