The Study Of The Roles Of TRIB2 In Radioresistant Lung Adenocarcinoma Cells By Affecting NF-?B-related Signals

Lung cancer has become one of the greatest threats to human health in the world. As an important treatment for lung cancer, radiotherapy could enhance the local restriction and survival rate of patients with lung cancer. However, the radioresistance from the lung cancer cells seriously attenuated the effects of radiotherapy. Therefore, exploring how to sensitize lung cancer cells to radiation is the major problem. TRIB2 have been found to be closely related to progression of lung cancer. This study aimed to elucidate the radioresistant roles of TRIB2 and its related signals in lung cancer with radiation treatment. There were two parts in this study.1. TRIB2, NF-?B ( P65 ) and Smad3 expression in lung adenocarcinoma tissues and para-carcinoma tissues.Lung adenocarcinoma tissues (n=20) and para-carcinoma tissues (n=20) were collected after clinical operation, then TRIB2, NF-?B (P65) /p-NF-KB(p-P65), and Smad3/p-Smad3 proteins were detected by western blot. Our results showed that the expression of TRIB2, p-Smad3, Smad3 increased in lung adenocarcinoma tissues compared with controls, while NF-?B ( P65 )/p-NF-KB(p-P65) decreased. The data of TRIB2, NF-?B ( P65) and Smad3 was obtained from ONCOMINE database and anzlyzed with Pearson test. TRIB2 expression was found to be negatively correlated to NF-?B (P65) expression (P < 0.05), while the expression between Smad3 and TRIB2 has been found positively correlated (P<0.05).2. The roles of TRIB2 in radioresistant lung adenocarcinoma cells by affecting NF-KB-related signals(1)The gene expression of TRIB2 in radioresistant lung adenocarcinoma cellsFirstly, the radioresistant lung adenocarcinoma cells (A549-34R and A549-40R) were established with radiation treatment. Then TRIB2 mRNA and protein in the radioresistant cells were analyzed with fluorescence quantitative PCR, western blot and immunofluorescence. The results indicated that expression of TRIB2 was higher in the A549-34R and A549-40R cells compared with A549 cells.(2) The effects of TRIB2 on the proliferation, apoptosis and radiosensitivity of radioresistant lung adenocarcinomaAfter A549-34R and A549-40R were transfected with TRIB2-overexpressed vector and siRNA specific to TRIB2 respectively, cell proliferation was detected by MTT and cell apoptosis was analyzed by Annexin V - FITC/PI. The results suggested that overexpression of TRIB2 would significantly promote the cell proliferation and inhibit the cell apoptosis, while siRNA treatment could inhibit the cell proliferation and promote the cell apoptosis. The results of X-ray irradiation assay showed that the inhibition of TRIB2 could enhance the radiosensitivity of radioresistant lung adenocarcinoma.(3)TRIB2 regulating NF-?B-related signal factorWestern blot results showed that the expression of NF-?B (P65), p-NF-?B(p-P65), TNF-a and IFN-? decreased in A549-34R and A549-40R cells compared with A549 cells, while the expression of IL-10 didn't change significantly. In order to further study the regulating role of TRIB2 to NF-?B signals in radioresistant lung adenocarcinoma cells, TRIB2-overexpressed vector was transfected into cells. After overexpression of TRIB2, NF-?B (P65), p-NF-KB(p-P65), TNF-a,IFN-y significantly decreased, while siRNA treatment led to the opposite results. The p-NF-KB luciferase reporter was used to detect whether TRIB2 could regulate the p-NF-?B(p-P65)transcriptional activity. The results showed that the activity was significantly reduced after overexpression of TRIB2, while increased after inhibiting the expression of TRIB2 .(4)The mechanism of TRIB2 in regulating NF-?B-related signalsFirstly, we hypothesized that TRIB2 regulates NF-?B signals by interacting with the NF-?B(P65). But the result of Co-IP showed that there was no interaction. Secondly, we detected the expression of I?B-? and p-I?B-a A549-34R and A549-40R cells by Western blot. We found that the expression of p-?B-? decreased while I?B-? increased in radioresistant lung adenocarcinoma cells.And overexpression of TRIB2 reduced the expression of p-I?B-? and increased the expression ofIKB-a. But knockdown of TRIB2 led to the opposite results. In the end, Co-IP assay was performed to evaluate the interaction among TRIB2 with I?B-?, ?-Trcp, IKK or p100. our results indicated that TRIB2 interacts with p100, but not with I?B-?, P-Trcp or IKK.In summary1. TRIB2 expression negatively correlates with NF-?B(P65), but positively correlates with Smad3 in lung cancer tissues (P < 0.05).2. Inhibition of TRIB2 inhibits cell proliferation, promotes apoptosis and enhances the radiosensitivity of lung adenocarcinoma cells,the mechanism of which might involve that TRIB2 negatively regulates the expression of NF-?B(P65), TNF-a and IFN-y.3. In radioresistant lung adenocarcinoma cells, TRIB2 interacts with p100, which may further regulates NF-?B signals.