| Background and ObjectiveOral squamous cell carcinoma is the most common tumor that arises in the head and neck. To date, the major clinical therapy methods are still operation, supplemented with radiotherapy and chemotherapy. In recent decades, although the treatment technology is gradually improving, the patient's five-year survival rate is still less than 50%. With the deepening of molecular biology, cancer gene therapy research has become a new hot spot for cancer treatment. Therefore, studies of the molecular biology of the tumor are key issues to get insight into the mechanisms underlying the pathogenesis of oralsquamous cell carcinoma and, in turn, leading to more effective therapeutics finall y.The growth arrest-and DNA damage-indueible (gadd) 45 gene family, comprising Gadd45α/Gadd45a, Gadd45β/Gadd45b/myd118, and Gadd45γ/Gadd45g/cr6, is widely expressed in mammalian cells responding to stress stimuli.Gadd45a was the first stress-inducible gene found to be activated by the p53 tumour suppressor. Subsequently, Gadd45a was also shown to be a target of the BRCA1 (Breast Cancer Associated Protein 1). Gadd45a is a p53-regulated growth arrest and DN A-damage-inducible gene that is also regulated in a p53-independent manner. Gadd45a has been demonstrated to link to many important cellular processes such as DNA repair, chromatin accessibility, cell cycle checkpoints and genome stability. Whether Gadd45a plays a direct role in the progression and prognosis of oral squamous cell carcinoma remains unclear.In present study, we elucidate the significance of Gadd45a in the progression, treatment and prognosis of oral squamous cell carcinoma from three main points:1. The expression state and clinical significance of Gadd45a in human oral squamous cell carcinoma;2. To investigate the effect of Gadd45a-siRN A on cell growth and invasion of Tca8113 cells;3. To investigate the effect of Gadd45a-siRNA on radiosensitivity of Tca8113 cells.Materials and methods1. The expression status and clinical significance of Gadd45a in human oral squamous cell carcinoma1.1. The study included 106 patients with OSCC who underwent primary surgical resection at Qilu Hospital of Shandong University between 2003 and 2009. Sixty specimens of adjacent oral mucosa tissue were collected as control samples. The clini copathological information, including sex, age, tumor stage, and histological grade, was obtained from the clinical records.1.2. The Gadd45a protein expression in 106 cases of OSCC and 60 cases normal oral mucosa tissue were detected by immunohistochemistry.1.3. The relationship between the Gadd45a expression and the pathological characteristics was analyzed by SPSS statistical software. Chi-square test was used to analyse the data. Differences were considered significant at p< 0.05.2. The effect of Gadd45 a-siRN A on cell growth and invasion of Tca8113 cells2.1. The Gadd45a protein expression in Tca8113 cells was analyzed by immunofluorescence techniques.2.2. Examining the interference rate of siRNAs targeting Gadd45a: short interfering ribonucleic acid (siRNA) targeting Gadd45a or an irrelevant mRNA was chemically synthesized. Tca8113 cells were divided into three groups for transfection:lipofectamineTM 2000 only (mock), nonsense si-RNA and Gadd45a-siRNA. Mock and nonsense si-RNA were regarded as control groups. The constructed siRNAs were transfected into Tca8113 cells. Gadd45a expression was determined by real time quantitive RT-PCR and Western Blot techniques.2.3. The effect of Gadd45a gene silencing on proliferation of Tca8113 was tested by MTT after silencing gene of Gadd45a. The growth curve was draw to assess Tca8113 cell proliferation activity.2.4. The effect of Gadd45a gene silencing on cell cycle distribution of Tca8113 was analysed with flow cytometry at 24h after silencing Gadd45a gene. 2.5. The migration ability of Tca8113 after silencing Gadd45a gene was tested by Transwell chamber assays.3. The effect of G add45a-siRN A on rad iosensitivity of Tca8113 cells3.1. Irradiated with 0,2Gy,4Gy,8Gy, 10Gy ionizing radiation (IR), the survival fraction of Tca8113 cells were ascertained by MTT assays. The apoptosis and cell cycle of Tca8113 cells were detected by flow cytometry.3.2. Tca8113 cells were cultured in six-well plates until 70% confluence and then exposed to OGy,4Gy, lOGy IR respectively. The effect of IR on Gadd45a expression in human Tca8113 cell lines was examined by real time quantitative RT-PCR and Western Blot analysis.3.3. Tca8113 cells transfected with the Gadd45a-siRNA and the control cells were irradiated in the dose range from 0 to 10 Gy. After 24 h exposed to IR, MTT colorimetric assay was used to analyse the viability of the cells and flow cytometry measurement was used to quantify the percentage of apoptotic cells in the total cell population.Results1. The expression status and clinical significance of Gadd45a in human oral squamous cell carcinomaThe Immunohistochemistry results showed that Gadd45a was expressed with cytoplasm-dominant and nucleus-dominant patterns. All of 60 cases showed significant nucleus-dominant expression pattern in adjacent tissue. But in oral squamous cell carcinoma,60 out of 106 cases showed Gadd45a expression with nucleus-dominant patterns. The expression patterns of Gadd45a showed a significant difference in age, clinical stage, histological grade and lymph node metastasis (p<0.05).2. The effect of Gadd45a-siRNA on cell growth and invasion of Tca8113 cells2.1. Tca8113 cells are well-differentiated tongue squamous cell cancer cell lines and the expression of Gadd45a protein was found to mainly locate in nucleus of Tca8113 cells.2.2. Real time quantitive RT-PCR and Western Blot showed that Gadd45a expression was blocked efficiently by using siRNAs in Tca8113 cells.2.3. MTT assay showed that the proliferation of Tca8113 cells two days later after RNAi was significantly higher than that of the control (p<0.05).2.4. Flow cytometry detection confirmed that the percentage of G2/M phase cell in Gadd45a-siRNA group significantly decreased compared with the control groups (p<0.05).2.5. The migrating ability of Tca8113 cells in the group transfected with Gadd45a-siRNA increased dramatically in comparison to the other two groups(p<0.05).3. The effect of Gadd45a-siRNA on radiosensitivity of Tca8113 cells3.1. The effect of IR on biological behavior of Tca8113 cells. We observed the effect of IR on the morphology of Tca8113 cell by HE staining and inverted microscope. We found that Tca8113 cells irradiated with high doses of IR became larger and the space between cells became wider.3.2. Basal and IR-induced Gadd45a expression in Tca8113 cells. We initially examined the effect of IR on Gadd45a expression in human Tca8113 cell line by real-time quantitative RT-PCR and Western Blot analysis. Gadd45a induction was assessed by comparing the basal level to that present 24 h following treatment with 4Gy or 10Gy IR. We showed IR obviously induced Gadd45a mRNA expression compared with the basal level (0.000965±0.00005 vs 0.000387±0.00002 and 0.001644±0.000065 vs 0.000387±0.00002) in the dose range examined. Consistently, Gadd45a protein level detected by Western Blot was also induced by lOGy IR (P=0.0028) although Gadd45a protein level observed in Tca8113 cells treated with 4Gy IR had no statistical significance in comparison with the basal level (P=0.0566).3.3. The effect of Gadd45a gene silencing combining IR on the biological behavior of Tca8113 cells. To research the the influence of Gadd45a gene silencing on the radiotherapy sensitivity of Tca8113 cells, we firstly detected the effectiveness of Gadd45a-siRNA using real-time quantitative RT-PCR and Western Blot analysis. The results showed that Gadd45a siRNA could effectively inhibit the expression of Gadd45a at the mRNA and protein level in Tca8113 cells (p<0.05). Then using MTT and Flow Cytometry, we inspected the surviving fraction and apoptosis status of Tca8113 cells when receiving different dose of IR. The result showed that the surviving fraction of Gadd45a-siRNA group was obviously higher than the control group (p<0.05) when the Tca8113 cells receiving 4Gy or more dose of irradiation. The result indicated that Gadd45a-siRNA impeded the down of surviving fraction of Tca8113 cells induced by IR. The Flow Cytometry result showed that when receiving 4Gy and10Gy IR, the Gadd45a-siRNA group's apoptosis was inhibited and the apoptotic fraction was obviously descended (p< 0.05). Conclusion1. Gadd45a was expressed with cytoplasm-dominant and nucleus-dominant patterns in human oral squamous carcinoma, and the expression patterns of Gadd45a showed a significant difference in age, clinical stage, histological grade and lymph node metastasis.2. Gadd45a gene silencing could distinctly enhance the proliferation and migrating ability of Tca8113 cells. The mechanism might be the interference of cell cycle and inhibition of apoptosis.3. Gadd45a overexpression induced by IR could enhance the radiotherapy efficacy of human oral squamous cell carcinoma.Originality1. We demonstrate, for the first time, that Gadd45a was expressed with cytoplasm-dominant and nucleus-dominant patterns in human oral squamous carcinoma, and the expression patterns of Gadd45a showed significant difference in age, clinical stage, histological grade and lymph node metastasis. And we found that Gadd45a gene silencing could distinctly enhance the proliferation and migrating ability of Tca8113 cells.2. We demonstrate, for the first time, that Gadd45a overexpression induced by IR could enhance the radiotherapy efficacy of human oral squamous cell carcinoma.
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