Research On Sensitization Effect Of Autophagy In Chemotherapy And Radiotherapy On Thyroid Follicular Carcinoma

Autophagy is a cellular decomposition, which decompose the organelles andproteins, it play a imorpant role in maintaining cellular homeostasis. Studies have shownthat autophagy is very important in development, invasion and metastasis in avariety of solid tumor. It induces autophagy occurs, when cells in the face of lack ofoxygen starvation, pathogen infection, ionizing radiation and drugs and other stressconditions. In the studies of the solid tumors, autophagy promotes the tumorsdevelopment by providing nutrients, however, it can also induce cell death whencells can not keep themselves survival. Recently, research results show thatchemotherapy and radiotherapy can induce autophagy in tumor cells. Many studieshave found that abnormal expression of autophagy-related PI3K-AKT-mTORsignaling pathway plays an important role in the incidence, development, invasionand metastasis of thyroid follicular carcinoma, and to some extent, reduce the effectsof radiotherapy and chemotherapy. However, the aspects are few reports. Thisstudy will describe the specific role of autophagy-related PI3K-AKT-mTORsignaling pathway in the pathogenesis of follicular thyroid cancer, and the infjuenceon the radiosensitization and chemosensitizing.Objective:To explore the roles of autophagy in radiosensitization and chemosensitizing offollicular thyroid carcinoma, and the effect of PI3K-AKT-mTOR signalingpathway in IR treatment and doxorubicin treatment combine with autophagyinhibitors of follicular thyroid carcinoma.Methods:(1) Constructe human follicular thyroid carcinoma cell line FTC-133PI3KCAsilence model (2) Cells viability was evaluated by Cell counting Kit-8(3) Colonyassay was used to analysis the radiation sensitivity and drug sensitivity (4) Westernblot was used to analyze protein expression (5) MDC staining was used to monitor autophagy (6) PI staining was used to monitor apoptosis (7) SPSS19.0was used forstatistical purpose (8) Image J software was used to calculate the band valueResults:(1) Establishment of PI3KCA silencing modle of FTC-133According PI3KCA of genetic information in GenBank, the transfer offull-length human cDNA sequence, using the Primer3software for primer design andsequencing primers to construct lentiviral vector with a purine enzyme-resistant andluciferase transfected cells FTC. While establishing transfected with empty vectorinterference target genes control experiment, the eventual establishment of a stablegene silencing model and observe the fluorescence effect after transfection cellstransfected successfully demonstrated by fluorescence microscopy.(2) The mechanisms of PI3KCA genes and Inhibitors LY294002in FTC133In order to further study of the mechanism of and PI3KCA genes andLY294002in FTC133cells, we use the relevant Western Blot method to detect theexpression of the target protein. The results found in both PI3KCA silent group andLY294002inhibited group, the AKT, p-AKT, mTOR, p-mTOR, p-P70proteinexpression were significantly decreased.(3) The LY294002drug sensitivity of FTC133Using CCK8method to detect proliferation activity of FTC133cells ofdifferent concentrations of LY294002. Diluted LY294002by using DMSO into0uM,25uM,50uM,100uM,200uM working solution,and then detect proliferationactivity of FTC133cells by CCK8method. An equal volume of DMSOrespectively FTC133cultured cells as a control group. The results showed that,50uM LY294002was the suitablea concentration in this study.(4) The influence of the PI3KCA gene caused in radiotherapy and chemotherapy inFTC133cellsApplication CCK8method for detecting cell proliferation. The FTC133cellswere divided into control group, DMSO group, LY294002inhibitor group, PI3KNCgroup, PI3KCA silent group, were given IR4Gy after detection. The results showed that16hours after IR4Gy the cell proliferation activity in inhibitor LY294002groupthan in DMSO was significantly lower (P <0.05). The cell proliferation activitybetween DMSO group and the control group had no significant difference.16hoursafter IR4Gy the cell proliferation in PI3KCA silence was significantly lower (P<0.05) compared with PI3KNC group. The cell proliferation activity betweenPI3KNC group and the control group had no significant difference.Application CCK8method for detecting cell proliferation. The FTC133cellswere divided into control group, DMSO group, LY294002inhibitor group, PI3KNCgroup, PI3KCA silent group, were given epirubicin100nM for24hours after testing.The results showed that,100nM epirubicin for24hours appeared inhibitorLY294002group compared with DMSO group significantly decreased cellproliferation (P <0.05). The cell proliferation activity between DMSO group and thecontrol group had no significant difference.100nM epirubicin appears PI3KCAsilent for24hours was significantly lower (P <0.05) compared with cell proliferationPI3KNC group. The cell proliferation activity between PI3KNC cell group and thecontrol group had no significant difference.(5) The sensitization effect of autophagy in chemotherapy and radiotherapy onthyroid follicularMDC fluorescence assay using autophagy situation. In this study, FTC133autophagy background positive rate is4%. First, the positive rate of normal cells inthe cell group FTC133, DMSO group, LY294002inhibitor group, PI3KNC group,PI3KCA the background silence autophagy group was analyzed and found to be4%,5%,9%,34%,52%. Respectively FTC133normal cell group, DMSO group,LY294002inhibitor group, PI3KNC group, PI3KCA silent group4Gy irradiation,after cultured for16hours, take the same approach to calculate the positive rate of cellautophagy, the result was35%,28%,78%,46%,89%. FTC133group on normal cells,DMSO group, LY294002inhibitor group, PI3KNC group, PI3KCA silent group100nM epirubicin treatment, after24hours of incubation, cells adopt the samemethod to calculate the positive rate of autophagy, the result was39%,32%,93%, 52%,97%.Conclusion:1. IR4Gy, epirubicin concentration of100nM are used in this research.2. In thyroid follicular cancer, PI3KCA silence model and application PI3Kinhibitor LY294002were both able to play inhibited autophagy-relatedPI3K-AKT-mTOR signaling pathway role.3. CCK8experiments confirmed, PI3KCA silence model and application PI3Kinhibitor LY294002, increased sensitization effect of autophagy in chemotherapy andradiotherapy in FTC133.4. MDC experiments confirmed the increased sensitization effect of autophagyin chemotherapy mediated by PI3KCA gene was completed by autophagy pathway.