Research On Effect Of Autophagy In Pathogenesis And Sensitization Of Radiothrapy And Chemothrapy In Anaplastic Thyroid Carcinoma

Autophagy is a phagocytosis of cytoplasmic protein or organelles on itself andallowed to enter the vesicles, formate autophagic lysosomal lysosome fusionde andgrade the contents of its package, thereby maintaining intracellular homeostasis.When cells face with hypoxic, hunger, ionizing radiation, chemicalsother and otherexternal stimuli, they will induce autophagy occurs, thereby activateautophagy-related signaling pathways. Research has reported that thyroid cancer cellsunder stress (eg: ionizing radiation or chemicals, etc.) can trigger autophagy andautophagy-related PI3K-AKT-mTOR signaling pathway abnormal expression playsan important role in differentiated thyroid cancer during, but the pathogenesis of thesignaling pathway in anaplastic thyroid carcinoma is not yet clear. The study hasfound that the anaplastic thyroid carcinoma present PI3KCA abnormal amplificationand mutations,and the targeted therapy for this pathway inhibitors is likely to becomean emerging treatment strategies for anaplastic thyroid carcinoma. Research for PI3Ksignaling pathway inhibitors with chemoradiotherapy to treat a variety of malignanttumors has become the focus of the current reports, but the relevant research inanaplastic thyroid carcinoma is not much. Therefore, this experiment by studyingautophagy on anaplastic thyroid carcinoma pathogenesis and chemotherapysensitizing effect is to deepen the theory understanding of PI3K-AKT-mTORsignaling pathway inhibitor combined with chemotherapy to treat anaplastic thyroidcarcinoma and provide a new idea of full complement anaplastic thyroid carcinomatreatment strategy.Objective:To investigate the effects of autophagy on anaplastic thyroid carcinomapathogenesis and chemotherapy sensitizing impact, and study the toxic effect on tumor cells of chemotherapy combined with autophagy inhibitors in anaplastic thyroidcarcinoma.Methods:(1) To detect the transfection efficiency of PI3KCA gene silence model in anaplasticthyroid carcinoma cell8505c with fluorescence microscope assay;(2) To detectPI3K-AKT-mTOR signaling pathway protein levels with western blot;(3) To detectanaplastic thyroid carcinoma cell8505c proliferation activity with cell proliferationCCK-8assay;(4) To detect the autophagy in anaplastic thyroid carcinoma cell8505cwith MDC assay;(5) To analyze the data with the SPSS19.0software，and P <0.05isa statistically significant difference.Results:(1) Construction the PI3KCA silence model of anaplastic thyroid carcinoma cell8505cAccording to the GenBank PI3KCA human full length cDNA sequenceinformation, design primers by Primer3software and sequencing, transfection withluciferase lentivirus vector to8505c cells, establish stable PI3KCA gene silencemodel, while setting up the empty vector as a control experimental group, detecttransfection efficiency with fluorescence microscopy assay, and culture aftersuccessful verification.(2) Mechanisms of PI3KCA gene and LY294002inhibitors in cancer cell8505cDetect the expression of PI3K-AKT-mTOP signaling pathway related proteinswith Western Blot assay. AKT、p-AKT、mTOR、p-mTOR、p-P70protein expressionin cancer cell8505c were detected in PI3KNC group and PI3KCA silence group,control group and LY294002group, results showed that the expression of theseproteins in PI3KCA silent group were significantly lower than PI3KNC group, whileLY294002group compared with the control group, also significantly reduced theexpression of these proteins.(3)8505c on chemotherapy sensitivity Detect8505c cancer cell proliferation effect on radiotherapy and chemotherapywith CCK-8assay. Using0Gy,2Gy,4Gy,6Gy different doses of ionizing radiation totreat8505c cell and detect cell proliferation activity after24h, and the results showed4Gy irradiation group siginificantly decreased cell proliferation activity (P <0.05),with dose increasing, cell proliferation reduced and cytotoxicity increased, so wechoose2Gy as an experimental radiation dose. Using0nmol/L,1nmol/L,10nmol/L,100nmol/L,1umol/L,10umol/L different concentrations of epirubicin to treat8505c cell and detect cell proliferative activity after24h, and the results showed100nmol/L significantly inhibited cell proliferative activity (P <0.05), with theincrease of concentration, cell proliferation decreased, and cytotoxicity increased, sowe choose10nmol/L as an experimental chemotherapy concentration.(4)8505c on drug sensitivity with LY294002inhibitorsDetect cancer cell proliferation activity treated with different concentrations ofLY294002with CCK-8assay. Using LY294002dissolved in DMSO, and dubbed0umol/L,2.5umol/L,5umol/L,10umol/L,30umol/L,50umol/L differentconcentrations working solution to treat8505c cells and detect cell proliferationactivity after24h, and the results found30μmol/L LY294002significantly decreasedcell proliferation activity (P <0.05), so we choose10μmol/L as the drug testconcentration.(5) Effects of PI3KCA gene on cancer radiotherapy in8505cIn cancer cell8505c to detect cell proliferation activity of PI3KNC group,PI3KCA silent group, DMSO group and LY294002group treated with2Gy radiationunder4h,8h,12h,24h,48h in respectively with CCK-8assay, the results showed thatPI3KNC group cell proliferation at12h was significantly reduced, and with timegoing, the activity decreased, while PI3KCA silent group showed the same trend, butcompared with PI3KNC group, reducing cell proliferation at the same time was moresignificantly (P <0.05). LY294002group consistented with the role of PI3KCA silentgroup, and its inhibition compared with DMSO group was significantly (P <0.05).(6) Influence of PI3KCA gene on cancer chemotherapy in8505cIn cancer cells8505c to detect cell proliferation activity of PI3KNC group, PI3KCA silent group, DMSO group and LY294002group treated with10nmol/Lepirubic under12h,24h,36h,48h,72h in respectively with CCK-8assay, the resultsshowed that PI3KNC group cell proliferation at36h significantly decreased, and withtime going, the activity decreased, whereas PI3KCA silent group showed the sametrend, but cell proliferation at the same time was significantly lower than PI3KNC (P<0.05). PLY294002group consistented with PI3KCA group, and the inhibitory effectwas significantly higher than DMSO group (P <0.05).(7) Autophagy impact on chemotherapy sensitizing effect in8505cIn cancer cell8505c to detect autophag of untreated8505c group, PI3KNC group,PI3KCA silent group, DMSO group and LY294002group with MDC fluorescenceassay. Giving2Gy radiatio for12h and10nmol/L epirubicin for36h separately toeach group, the results showed that PI3KCA silent group could more significantlyincrease autophagy than PI3KNC group and untreated8505c group, while cellautophagy treated with LY294002group was also significantly higher than the DMSOgroup and untreated8505c group.Conclusion:1, In anaplastic thyroid carcinoma cell8505c, silencing PI3KCA gene and applicationof PI3K inhibitor LY294002inhibit autophagy related PI3K-AKT-mTOR signalingpathway.2, In anaplastic thyroid carcinoma cell8505c chemoradiotherapy study, the minimumeffective dose is2Gy, the minimum effective concentration of epirubicin is10nmol/L, and the minimum effective concentration of the LY294002drug is10umol/L.3, CCK-8experiments confirmed that silencing PI3KCA gene and application ofPI3K inhibitor LY294002can increase sensitivity to radiotherapy and chemotherapyfor anaplastic thyroid carcinoma.4, MDC experiments confirmed that the chemotherapy sensitizing effect of PI3KCAgene and PI3K inhibitor LY294002for anaplastic thyroid carcinoma is mediated byautophagy.