The Research Of TRAF2siRNA In The Enhancement Of Radiotherapy Sensitivity Of The Oral Squamous Cell Carcinoma

Objectives:Oral squamous cell carcinoma (OSCC) is one of most common malignant tumors in head and neck region. An integrated approach has been used to treat OSCC, in which surgeries are introduced in combination with radiation treatments, chemical treatments and/or biological treatments. Although certain positive curative effects can be attained via the integrated approach, the prognosis is still not predictable for patients with OSCC in moderate to late stages, in the other word, the integrated approach hardly brings a higher survival rate of this patient population than the simple surgery approach does. Compared to the radio therapy and chemotherapy, the targeted therapy shows advantages of high specificity, low dosage, low side effects and good tolerance. In recent years, quite a few targeted therapies have been introduced into clinical application and achieved good results. Since the proliferation of tumor cells are controlled by multiple pathways, the long-term prognosis of those targeted therapies aiming at single pathway is not assured and comes with tumor reoccurrence. Therefore it is of important clinical value to find new targets, especially those as key signal molecules in the crosstalk of multiple pathways. Tumor necrosis factor receptor- associated factor2(TRAF2) is a cytoplasmic adapter protein and has been reprted to be highly expressed in quite a few malignant tumors. It plays a key role in multiple anti-apoptotic signal pathways and occupies a key position in the crosstalk between the intrinsic and extrinsic apoptotic pathways. Since to date, there is scarcely reports in the topic of the expression and biological effects of TRAF2in the oral maxillofacial malignant tumors, the present study aims (1) to reveal the expression of TRAF2in paraffin sections of oral malignant tumor tissues, especially OSCC, and to correlate the levels of TRAF2expression with the clinical grades of the tumors;(2) to manifest the biological behavior changes (cell proliferative capacity and cell apoptotic ratio) of OSCC cell line after TRAF2siRNA transfection in vitro;(3) to manifest the biological behavior changes (cell proliferative capacity, cell apoptotic ratio and cell cycle distribution) of TRAF2siRNA transfected OSCC cells after radiation in vitro;(4) to manifest the anticancer effect of TRAF2siRNA transfection introduced together with nano-granules and in combination with X-ray radiation in an OSCC mouse model. The ultimate purpose of the present study is to prove that TRAF2is a potential, effective and efficient curative target for OSCC and TRAF2siRNA is of particular effect in the enhancement of radiotherapy sensitivity of OSCC.Methods:1. Fifty-seven oral malignant tumor tissue samples (50OSCC and7others) and30normal mucosa samples2+cm adjacent to tumor were collected and their paraffin-embedded sections were immunohistochemically stained to manifest the expression levels of TRAF2. Immunochemical score (IHS) and mean optical density (MOD) were calculated and statistically analyzed to reveal the correlation between the TRAF2expression and the clinical and pathological characteristics, including the tumor size, TNM grade, pathological grade and lymphatic metastasis. 2. An OSCC cell line (CAL27) was transfected with TRAF2siRNA in which the down-regulation of TRAF2expression at the protein level was confirmed by Western blot. In the TRAF2siRNA transfected OSCC cells (experimental group), the control siRNA transfected OSCC cells (negative control) and the blank OSCC cells (blank control), the cell proliferative capacity (MTT tests, CCK-8tests) and cell apoptotic ratio cell circle distribution (flow cytometry methods via Annexin V/PI double staining) were assessed. These biological behavior data of the three groups were statistically analyzed. Furthermore, the protein level of RIP1, a signal downstream of TRAF2in NF-κB pathway was also manifested by Western blotting technique.3. Radiation of various dosages (2Gy,4Gy,6Gy,8Gy) was done with TRAF2siRNA transfected OSCC cells, control siRNA transfected OSCC cells (negative control) and blank OSCC cells (blank control). All the above-mentioned biological behavior parameters (cell proliferative capacity and cell apoptotic ratio) were collected via the same methods and moreover, cell circle distribution of the three groups were analyzed via flow cytometry methods (PI staining).4. An OSCC nuke mouse model was constructed by subcutaneous injection of suspension of cultured CAL27cells and at the20th day post-injection, the constructed tumors in the mice were transfected with TRAF2siRNA blended in EntransterTM-in vivo transfection reagent as the experimental group (radiation+transfected group) and with saline as controls (radiation group and non-radiation group). At48h post-transfection,6Gy radiation were done. All the mice were sacrificed at10d post-radiation, and tumors in different groups were harvested. The sizes and weights of tumor masses were measured, and the tumor growth inhibition rates were calculated and compared.Results:1. TRAF2expression was92%positive in50cases of OSCC samples, including 26cases of weak positive (52.0%) and20cases of positive (40.0%). So was the TRAF2expression in other types of oral malignant tumors within the present study. In contrast, TRAF2was quite low expressed in normal oral mucosal tissues. TRAF2expression levels were statistically correlated with the pathological grades and lymphatic metastasis (P<0.05).2. The CAL27OSCC cells were effectively transfected with TRAF2siRNA and the protein levels of TRAF2and its downstream signal RIP1were down-regulated as shown in Western blot. Compared with the control siRNA transfected group and the blank group, the cell proliferative capacity in the TRAF2siRNA transfected group was not changed significantly, but the early apoptotic rate was increased significantly (P<0.05).3. The cells in the TRAF2siRNA transfected group, the control siRNA transfected group and the blank group behaved differently after radiation of various dosages. After radiation of6Gy or8Gy, the cell proliferative capacity in the TRAF2siRNA transfected group were lower than those of the other two groups, while the apoptotic rate were higher. In the cell circle distribution analysis, the cells in all the three groups exhibited G2/M phase retardation, especially prominent in the TRAF2siRNA transfected group. Compared with all the other groups, the6Gy group showed statistically the most prominent changes in the above-mentioned parameters (P<0.05).4. The CAL27tongue squamous cell carcinoma nuke mouse model was established successfully. After6Gy radiation the tumor sizes and weights of the radiation+transfected group were the smallest (statistically significant) in the three groups. The tumor inhibition rate in the radiation+transfected group was88.2%, more than that of the radiation group by18.1%.Conclusions:1. TRAF2was specifically expressed in OSCC tissue, in contrast to normal oral mucosal tissue.2. Transfection of TRAF2siRNA could effectively down-regulate the protein level of TRAF2in OSCC cell line. There was no significant change in the cell proliferative capacity after TRAF2siRNA transfection, but the cell apoptotic rate increased with the down-regulation of TRAF2expression.3. Radiation of various dosages made the TRAF2siRNA transfected OSCC cells exhibite G2/M phase retardation (the proportion of the cells in the G2/M phase increased accordingly). At the same time, their apoptotic rate increased and their proliferative capacity decreased. It is demonstrated that the TRAF2siRNA can enhance the radiotherapy sensitivity of OSCC cells in vitro.4. TRAF2siRNA transfection carried by nano-granules could enhance the radiotherapy sensitivity of OSCC nuke mouse models. TRAF2siRNA transfection plus radiation could reduce the size and weight of OSCC tumor and increase the tumor inhibition rate efficiently and effectively.