| Background Head and neck squamous cell carcinoma(HNSCC) ranks sixth among cancers worldwide, includes tumors of the oral cavity, oropharynx, and larynx. Survival rates for HNSCC have remained unchanged throughout the last three decades, and half of all cases die within 5 years of diagnosis. The presence of lymph node metastasis affects more than 50 % of HNSCC patients and it is one of the most important prognostic indicators associated with poor long survival rates. At present, the most effective treatment is surgery. And only taking measures as soon as possible, tumor is likely to be removed completely. Only in this way can we prevent the tumor progression to ensure the patient's life and health. Micro RNAs(mi RNAs) are endogenous RNAs that play important generegulatory roles in animals via sequences pecific interactions with the 3?UTR of cognate m RNA targets, causing suppression of translation and m RNA decay. Subsets of mi RNAs have been identified as potential diagnostic and prognostic markers in malignant tumors. At present, the cure rate of Head and neck squamous celll carcinoma is still low. So it is necessary to find high sensitivity and specificity of mi RNA as markers of diagnosis or treatment of Head and neck squamous celll carcinoma.In this study, we detected the expression level of the mi R-645 in Head and neck squamous cancer tissue with different metastatic potential by the real-time quantitative PCR technique, and analyzed the correlation between the expression level and clinical pathological parameters of Head and neck cancer. We detected the expression level of mi R-645 in 2 cases of Head and neck squamous cancer cells using real-time quantitative PCR, and adopted the method of transient transfection to transfer the mi R-645 mimics or mi R-645 inhabitor into the Head and neck squamous cell carcinoma lines in order to obtain high or low expression cell model of the mi R-645. In order to understand the mi R-645 effects on cancer cell biological behavior, and to further clarify the role of mi R-645 in Head and neck squamous cell carcinoma occurrence and development. we explored the proliferation of tumor cells by CCK8 method, detected the invasion by Transwell essay, studied the tumor cell migration ability by wound healing assay, and then studied the tumor cell cloning ability by soft agar assay. In addition, we use the statistical analysis to explore the expression of mi R-645 and the relationship with IFIT2, which laid the foundation for further study of the molecular mechanism of the mi R-645 regulation.Part One The study of the expression level of the mi R-645 and the correlation with clinical pathological parameters in Head and Neck squamous cell carcinomaObjective To explore the expression level of the mi R-645 in the tissues of Head and Neck squamous cell carcinoma with different metastatic potential and the correlation with clinical pathological parameters in Head and Neck squamous cell carcinoma.Methods The expression level of the mi R-645 was detected by real-time quantitative PCR in tissues of Head and Neck squamous cell carcinoma, which contain 76 cases tissues with lymphatic metastasis ? 51 cases tissue without lymphatic metastasis. The correlation between the expression level of cancer tissues and clinical pathologicalparameters of Head and Neck squamous cell carcinoma were analyzed by independent-samples T tests and one-way ANOVA.Results 1.The expression level of the mi R-645 in tissues of Head and Neck squamous cell carcinoma different metastatic potential. The results of real-time quantitative PCR demonstrated that mi R-645 was significantly lower in metastatic cancer tissues than tissues without lymphatic potential(p<0.05). The expression level of the mi R-645 in two groups were 2.71±0.24?1.58±0.23, respectively. 2.We analylized the correlation between the expression level of cancer tissues and clinical pathological parameters of Head and Neck squamous cell carcinoma. Medians of the relative expression values and clinicopathological factors were presented in Table. Statistically significant associations between the mi R-645 expression levels and metastatic rates and pathological grade were observed. In the HNSCC tissues with severe histological signs(vascular emboli, perineural invasion, diffuse infiltration), the expression level of mi R-645 was also significantly higher than the expression level in the tissues with non-severe histological signs. However, there was no significant correlation between the expression level of mi R-645 and age, sex, tumor size, site, smoking history, alcohol history.Brief Summary 1.The expression level of mi R-645 was significantly higher than the expression level in the tissues with non-severe histological signs, suggesting that the up-regulated of the mi R-645 might promote the metastatic potential. 2.The expression level of mi R-645 was significantly correlated with histologic grade?lymphatic metastatic and histologic signs of severity, suggesting that mi R-645 might play an important role in the occurrence and development of HNSCC, which may be a promising biomarker in the early diagnosis of HNSCC.Part Two The effects of the mi R-645 on biological behaviors in human HNSCC cell linesObjective Explore the effects of the mi R-645 on biological behaviors in human HNSCC cell lines through exogenous intervention measures to chang the expression of mi R-645.Method: Through real-time quantitative PCR detection the expression of mi R-645 in two HNSCC cell lines. through transient transfection mi R-645 mimics into HN4 cell to obtain the high expression cell mode mi R-645. And the experiment was divided into three group, such as: blank group?negetive group?transfection mi R-645 mimics group. through transient transfection mi R-645 inhibitor into HN12 cell to obtain the low expression cell mode mi R-645. And the experiment was divided into three group, such as: blank group?negetive group?transfection mi R-645 inhibitor group. The proliferation of tumor cells was explored by CCK8 and soft agar assay, the cell invasion was detected by transwell essay, and the tumor cell migration ability was studied by wound healing assay. In addition, we use the statistical analysis to explore the expression of mi R-645 and the relationship with IFIT2.Results 1. Through real-time quantitative PCR, the results demonstrate the expression of mi R-645 is higher in HN12 cells than HN4 cells. The results are 2.07±0.250? 0.97±0.21 respectively, and the difference was statistically significance(P=0.003). 2. Through real-time quantitative PCR, the results demonstrate expression of mi R-645 which transfected mi R-645 mimics is significantly higher than blank group and negative group in HN4 cells, and the difference was statistically significance(P<0.05); expression of mi R-645 which transfected mi R-645 inhabitor is significantly lower than blank group and negative group in HN12 cells, and the difference wasstatistically significance(P<0.05). 3. Cell proliferation rate of HN4 transfected mi R-645 mimics is significantly higher than the negative group in the fifth and seventh day, and the difference was statistically significance(P<0.05); Cell proliferation rate of HN12 transfected mi R-645 mimics is significantly lower than the negative group in the fifth and seventh day, and the difference was statistically significance(P<0.05). 4. Cell number breaking through the PVDF membrane of HN4 transfected mi R-645 mimics is significantly higher than the negative group, and the difference was statistically significance(P<0.05); Cell number breaking through the PVDF membrane of HN12 transfected mi R-645 inhabitor is significantly lower than the negative group, and the difference was statistically significance(P<0.05); 5. Cell migration ability of HN4 transfected mi R-645 mimics is significantly higher than negative group, and the difference was statistically significance(P<0.05); Cell migration ability of HN12 transfected mi R-645 inhabitor is significantly lower than the negative group, and the difference was statistically significance(P<0.05); 6. Cell clonality of HN4 transfected mi R-645 mimics is significantly higher than the negative group, and the difference was statistically significance(P<0.05); Cell migration ability of HN12 transfected mi R-645 inhabitor is significantly lower than the negative group, and the difference was statistically significance(P<0.05); 7. There is native correlation between mi R-645 and IFIT2. The correlation coefficient is 0.181, which has the statistical significance(P=0.001).Brief Summary 1. In vitro experiments, we successful implementation of the expression of mi R-645 rise or fall. In addition, increasing the expression of mi R-645 could promote the proliferation?invasion?migration ability and clone formation of the HNSCC cell lines. So, it may play of promote cancer. 2. IFIT2 may be the target gene of mi R-645, and mi R-645 may impose influence on cell biology function through regulating IFIT2 expression.Conclusion 1. mi R-645 expression level is raised in Head and Neck Squamous carcinoma tissue, and it is closely associated with the transfer and prognosis of the cancer. 2. The chang of mi R-645 can significantly affect the biological behavior of Head and Neck Squamous carcinoma cell, such as: proliferation?invasion?metastasis. 3. mi R-645 may directly regulate the expression of IFIT2, which may affect the biological behavior of Head and Neck Squamous carcinoma cell. |
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