| Objective:Lung cancer is a widely diagnosed cancer worldwide.Lung cancer-related mortality and morbidity are increasing every year in China[1].Although in addition to conventional treatment methods such as surgery,radiotherapy and chemotherapy,comprehensive treatment methods such as molecular diagnosis and targeted drugs have been applied to lung adenocarcinoma,the prognosis of patients with lung adenocarcinoma is still poor,and the five-year survival rate is only about 15%[2].Although there are currently some markers that can predict the prognosis of lung adenocarcinoma,patients with the same pathological type may still have different prognosis due to biological diversity and individual differences.There is still much room for urgently affecting the mechanism of lung adenocarcinoma prognosis.explore.Factors and biomarkers that affect the prognosis of lung adenocarcinoma have been one of the main directions of lung cancer research.Revealing the markers that affect the prognosis of lung adenocarcinoma can not only help us better predict the survival rate of patients,but also provide new ideas and new directions for accurate treatment and personalized treatment of lung adenocarcinoma.Radiotherapy plays a key role in the treatment of lung adenocarcinoma.It is part of the standard treatment for lung adenocarcinoma.Stereotactic radiotherapy can be used for early or oligometastatic disease.Locally advanced stage III lung adenocarcinoma will receive chest local radiotherapy and systemic Chemotherapy[3].Radiosensitivity is one of the main reasons affecting the efficacy of radiotherapy for lung adenocarcinoma.Tumor resistance to radiation therapy is a common phenomenon,and the mechanism is not fully understood.At the same time,the treatment is limited by the protection of normal tissues,and the radiation dose cannot be increased too much.How to overcome the radioresistance of tumors,improve radiosensitivity,and kill tumor cells to the greatest extent is a major research direction of current radiotherapy.The TEDC2 gene was originally named C16ORF59.The ORF refers to an open reading frame?Open Reading Frame,ORF?starting from the start codon,a DNA base sequence without a stop codon interruption,and has the ability to encode a protein.The TEDC2 gene contains 10 exons,6 transcripts,and encodes the protein tedc2.However,little research has been done on the role of tedc2 protein.In 2018,David and other scholars discovered that tedc2 and tedc1,?-tubulin,and?-tubulin together form a tetramer,which has a certain effect on maintaining the stability of centrioles,and is an important component of fibril[4].The HUGO Gene Naming Committee officially renamed it TEDC2?tubulin epsilon and delta complex 2?.However,the specific biological function of this gene in somatic cells and tumor cells still needs further study.In this study,258 key genes affecting the prognosis of lung adenocarcinoma were identified through bioinformatics analysis,including TEDC2.However,whether it really has an impact on the prognosis of lung adenocarcinoma,can it be a new indicator for evaluating the prognosis of lung adenocarcinoma Further research is needed.At the same time,in order to explore the effect of TEDC2 on the radiosensitivity of lung adenocarcinoma and its mechanism of action,we conducted the following studies.Methods:1.Download the gene expression and clinical data of lung adenocarcinoma patients in the TCGA database,analyze the data using R software,analyze the expression level of TEDC2 and judge its correlation with the clinicopathological parameters and survival prognosis of lung adenocarcinoma patients.2.Collect surgical specimens of lung adenocarcinoma patients?lung adenocarcinoma tissue and paired normal lung tissue?and related clinical data.Real time PCR and Western blot were used to detect the expression of TEDC2 in lung adenocarcinoma tissues and matched normal lung tissues.Immunohistochemical methods were used to detect the protein differences in lung adenocarcinoma tissues and matched normal lung tissues of surgical patients,and to analyze their correlation with clinical pathological parameters and survival prognosis of patients.3.Human lung adenocarcinoma cell lines A549 and H1299 were cultured in vitro.Lentivirus was transfected into overexpressing and silencing interfering RNA.Puromycin selection was used to obtain cells that were stably transfected with overexpressing and silencing TEDC2.Transfection efficiency was measured by PCR and Western blot;the effect of TEDC2 on cell viability was evaluated by CCK-8 experiment;clonal formation experiment was used to evaluate the colony formation rate of cells in different transfection groups under different irradiation doses,and survival was fitted by a multiple target click model Curve to calculate the radiobiological indicators such as D0,Dq,SF2,k,N to evaluate the effect of silent TEDC2 on cell radiosensitivity;use flow PI single staining to analyze the effect of TEDC2 cell cycle distribution;use flow double staining to analyze the effect of TEDC2 on Effects of apoptosis.4.Predict TEDC2 upstream transcription factor as SP1 by software.SP1 wild type,mutant plasmid and TEDC2 upstream promoter expression plasmids were constructed.SP1 was used as an upstream transcription factor to verify the expression of TEDC2 by double luciferase reporter assay.5.Using Tandem Mass Tags?TMT?to detect the relative content of protein in stable transfected A549 cells overexpressing TEDC2 and A549 cells overexpressing the control group.Statistical analysis of differential proteins;GO analysis of the screened differential proteins to obtain functional annotation information for each protein;use KEGG database to annotate the identified proteins at the pathway level.6.Statistical analysis of data was performed using SPSS 22.0 and GraphPad Prism5software.All experimental data were obtained from at least three independent experiments and presented as Mean±SD.The data conformed to the normal distribution.Student's t test was used to compare the differences between the groups;one-way ANOVA was used to compare at different time points/doses within the same group.Differences were considered statistically significant when P<0.05.Results:1.The expression level of TEDC2 mRNA in patients with lung adenocarcinoma in the TCGA database correlates with the prognosis of patientsThe TCGA database collected data from 504 patients with lung adenocarcinoma.Analysis of TEDC2 mRNA expression in lung adenocarcinoma was higher than that in adjacent tissues?TEDC2:logFC=3.538177,P=9.69E-76?.In lung adenocarcinoma,the survival rate of the TEDC2 mRNA high expression group was low,and the survival rate of the low expression group was high?P=0.00085?,indicating that the high expression of TEDC2 gene was negatively correlated with the prognosis of lung adenocarcinoma.The clinical stage,T,N,M stage,and TEDC2 mRNA expression in patients with lung adenocarcinoma were correlated with prognosis?P<0.05?.The results showed that clinical stage and TEDC2 mRNA expression were independent prognostic factors for lung adenocarcinoma?P<0.05?The relative risk of clinical stage and TEDC2 mRNA expression were 2.433 and 1.441,respectively,indicating that the risk of death in patients with high TEDC2 expression was 1.441 times that of patients with low expression,and the risk of death in patients with clinical stages III and IV was clinical stages I and II.Patients were 2.433 times.2.The expression levels of TEDC2 mRNA and protein in LUAD tissue specimens are higher than those in normal lung tissuesReal time PCR method was used to detect the differential expression of TEDC2mRNA in 28 pairs of lung adenocarcinoma tissues and paired normal lung tissues.The results showed that TEDC2 mRNA was significantly increased in lung adenocarcinoma tissues?P=0.0001?,and TEDC2 mRNA was significantly increased in lung adenocarcinoma tissues.The expression level was about 23.99±5.570 times that of the adjacent tissues.Western blot results showed that the expression of tedc2 protein in 28paired lung adenocarcinoma tissues was significantly higher than that in normal lung tissues,and the difference was statistically significant?P<0.001?.?The expression of tedc2 protein relative to actin in lung adenocarcinoma tissue:0.7260±0.04170;normal lung tissue:0.2727±0.03822?3.The expression level of TEDC2 in LUAD tissues is correlated with the prognosis of patientsThe analysis of the correlation between the expression level of TEDC2 and the clinicopathological factors in 93 surgical specimens showed that the expression of TEDC2 protein was correlated with the tumor size,clinical stage,and T stage of patients?P<0.05?.Kaplan-Meier survival analysis found that patients with low TEDC2expression had better prognosis than patients with high TEDC2 expression?median survival time of patients with low TEDC2 expression was significantly shortened,and the 5-year survival rate was significantly reduced,P<0.05?.Cox proportional hazards regression analysis showed that the clinical stage and TEDC2 expression of patients with LUAD were correlated with the prognosis of patients with lung adenocarcinoma?P<0.05?.TEDC2 expression was an independent prognostic factor for patients with LUAD?P<0.05?.The relative risk is 1.768.4.Up-regulation of TEDC2 enhances the proliferation of LUAD cells,and down-regulation of TEDC2 inhibits the proliferation of LUAD cellsThe cck-8 experiment showed that compared with cells that did not up-regulate TEDC2 expression,TEDC2 over-expressed cells had improved cell proliferation ability,which was statistically different?P<0.05?.Down-regulating TEDC2 expression could significantly inhibit cell proliferation ability,with statistics.Difference?P<0.05?.5.Up-regulation of TEDC 2 expression reduces radiosensitivity of LUAD cells,and down-regulation of TEDC 2 expression enhances radiosensitivity of LUAD cellsThrough experiments,it can be seen that the activity of cells that up-regulate TEDC2 expression was significantly higher than that of cells that did not up-regulate TEDC2 expression after irradiation with different doses of radiation?P<0.05?.Comparing the cell survival curve and related parameters fitted by applying the multi-target click model,it can be found that the cell survival rate of cells that down-regulate TEDC2 expression was significantly reduced when receiving 2Gy irradiation,and the quasi-threshold dose was also significantly reduced?P<0.05?.This shows that the radiosensitivity of cells that down-regulate TEDC2 expression is significantly enhanced.Conversely,up-regulating TEDC 2 expression enhances the radioresistance of LUAD cells.6.TEDC2 expression affects cell cycle distributionThrough flow cytometry analysis,it was found that high expression of TEDC2reduced the proportion of cells in G1 phase,increased the proportion of cells in S phase,and there was no significant change in G2/M phase.Low expression caused more cells in G1 phase,decreased cells in S phase,and cells in G2/M phase increase?P<0.05?.7.Up-regulating TEDC2 expression can reduce LUAD cell apoptosis,down-regulating TEDC2 expression can increase LUAD cell apoptosisFlow cytometry assay showed that the apoptosis rate of LUAD cells that up-regulated TEDC2 expression was significantly reduced,and the apoptosis rate of LUAD cells that down-regulated TEDC2 expression was significantly increased.There was a statistical difference?P<0.05?.8.TEDC2 may affect radiosensitivity by affecting cell functions such as ciliary/microtubule stability,cell cycle,and multiple signal pathways such as P53,Hh,and homologous recombination.Through proteomic analysis,it was found that differentially expressed proteins after TEDC2 overexpression were mainly concentrated in cell functions such as cilia/microtubule stability and cell cycle.At the same time,differential proteins are mainly concentrated in multiple signal pathways such as Hh,P53,and Wnt,which affects the radiosensitivity of LUAD cells.Conclusion:1.The expression levels of TEDC2 mRNA and protein in tissues of LUAD patients were higher than those in normal lung tissues.2.TEDC2 is negatively correlated with the prognosis of LUAD patients.The prognosis of TEDC2 high expression is poor,the prognosis of TEDC2 low expression is good.3.Overexpression of TEDC2 enhances cell proliferation,reduces apoptosis,increases the proportion of cells in the S phase,decreases the proportion of cells in the G1 phase,and increases the radioresistance of LUAD cell lines.Silencing TEDC2 reduces cell proliferation,increases apoptosis,decreases the proportion of S-phase cells,increases the proportion of G1 and G2/M phases,and improves the radiosensitivity of LUAD cell lines.4.TEDC2 may change the radiosensitivity of tumor cells by regulating ciliary function,centromere stability,Hh signaling pathway and other signaling pathways in LUAD cells. |
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