| Purpose:The incidence of thyroid cancer is increasing year by year worldwide.Papillary thyroid cancer is the most common subtype of differentiated thyroid malignant cancer.Because it maintains the features of thyroid cells,they are usually curable with surgery and radioactive iodine(131I)therapy with good overall prognosis.However,some patients have had local invasion or distant metastasis at diagnosis,or recurrence after treatment.The low iodine-uptaking ability of the tumor leads to poor 131I treatment,resulting in disappointing disease-free survival rate and poor prognosis.At present,there is no reliable means for the effective treatment of refractory thyroid cancer.Therefore,looking for a novel biomarker that can regulate the growth process of thyroid cancer and as a therapy target may benefit the diagnosis and treatment of thyroid cancer.The alteration in energy metabolism in cells is one of the hallmarks in tumor cells.It has been confirmed that many malignant cells have an aerobic glycolysis(also known as the Warburg effect)and an increase in glutamine metabolism.Although glutamine is a non-essential amino acid,it is the most abundant amino acid in human blood,and glutamine is an important nutrient for many tumor cells to maintain their growth and vitality.Although some scholars have found that there are changes in glutamine metabolism in various types of blood and solid tumors,there is no research article focusing on the function of glutamine and glutamine metabolism-related proteins in papillary thyroid cancer.Therefore,this study was designed to investigate whether thyroid cancer cells require glutamine for survival and growth,and whether glutamine metabolism-related proteins are aberrantly expressed in thyroid cancer cells,and identify the functions of glutaminolysis in thyroid cancer cells,as well as looking for biological targets that may affect the growth and progression of thyroid cancer.Methods:First,four papillary thyroid cancer cells(K1,IHH4,BCPAP,TPC-1)and thyroid non-cancerous Nthy-ori 3-1 were cultured in glutamine-free cell culture medium and normal medium,respectively.The growth viability and proliferation ability of cells in different culture conditions were compared,and detection methods include trypan blue staining,CCK8 assays and CellTiter-LumiTM Luminescent Cell Viability Assay Kit.The aberrant expression of amino acid metabolism-related genes in thyroid cancer specimens was detected and screened using commercially available RT2-PCR arrays.Testing samples include thyroid tissues and thyroid cell lines(6 pairs of freshly collected human thyroid cancer tissues and paired paracancerous tissue specimens),as well as IHH4 and BCPAP cells).According to the results of PCR arrays,glutaminase(GLS)is abnormally upregulated in thyroid cancer and need further confirmation.Methods for confirming includes immunohistochemical staining,Real-time PCR and Western-blotting.Quantitative and locating analysis with immunohistochemical staining would contribute to the identification of expression levels of GLS in thyroid cancer,and the combination of clinical and pathological factors of patients would be helpful to certify possible associations between GLS levels and related clinical factors.Real-time PCR was used to detect the difference of GLS expression in 90 pairs of thyroid cancer tissues and matched paracancerous tissues,and thyroid cells,including K1?IHH44?BCPAP?TPC-1 and Nthy-orn 3-1.Meanwhile,western-blotting was used to detect the protein levels of GLS in thyroid cells.Targeting inhibition of GLS expression levels in the above four PTC cells using GLS specific chemical inhibitors CB-839,BPTES and genetic knockdown.Furthermore,glutamate and a-KG detection kit were used for measuring glutamine metabolites,and seahorse energy metabolic analyzers were applied to determine changes in mitochondrial respiration and oxygen consumption in PTC cells.Trypan blue staining,CCK8 assays and CTL luminescence cell viability assay kit were used to detect the effect of GLS expression level on the growth viability and proliferation of PTC cells.The transwell assays measured changes in migration and invasion ablity of PTC cells after inhibition of GLS.Western-blotting assays were used to detect the alteration of mTORCl signaling pathway and autophagy marker molecules,including p-p70s6k,p70s6k,p-4EBP1,4EBP1,p-ULK1,ULK1,p-mTOR,mTOR,p62 and LC3B before and after GLS inhibition in PTC cells.Annexin V-FITC and PI double staining flow cytometry were used to determine the effect of GLS inhibition on PTC cell apoptosis.Real-time PCR was used to detect the difference of SLC1A5,SLC7A5 and SLC6A14 expression in 92 pairs of thyroid cancer tissues.Quantitative and locating analysis with immunohistochemical staining would contribute to the identification of expression levels of the three transporters mentioned above in thyroid cancer,and the combination of clinical and pathological factors of patients would be helpful to certify possible associations between SLC1A5,SLC7A5 and SLC6A14 expression levels and related clinical factors.Meanwhile,expression levels of biological invasive markers in papillary thyroid cancer,including E-cad,?-cat,MMP-9 and VEGFA were investigated,and analyze possible correlations with SLC1A5,SLC7A5 and SLC6A14 expression levels.Results:The results of trypan blue staining,CCK8 assays and CTL cell viability assays consistently showed that the cell viability and growth proliferative ability of four PTC cell lines K1,IHH4,BCPAP and TPC-1 was impaired in the absence of glutamine(p<0.05),and the ability of thyroid non-cancerous cell Nthy-ori 3-1 showed no significant change(p>0.05).Among the differentially expressed genes screened by RT2-PCR,GLS was the only up-regulated gene in PTC,which was statistically different(p=0.0015).The Real-time PCR results of 90 pairs of papillary thyroid cancer confirmed that the mRNA expression of GLS was significantly increased compared with matched paracancerous tissues(p<0.001).Immunohistochemical staining identified that GLS was expressed in cytoplasm in PTC,and the immunoreactive scores were higher than that of non-cancerous thyroid tissues(p<0.001).Besides,analysis of clinical pathological data of patients indicated that high GLS staining was associated with extra-thyroidal extension in PTC patients(p=0.005).The results of real-time PCR and western blotting assays suggested that GLS mRNA levels in the four PTC cells were higher than those in Nthy-ori 3-1 cells(P<0.01),and the protein expression level of GLS was also significantly higher than that of non-cancerous thyroid cells.The concentrations of glutamine metabolites glutamate and a-KG in K1,IHH4,BCPAP and TPC-1 cells were decreased after inhibiting the expression of GLS in PTC cells,suggesting that glutamine metabolism process was impeded.Mitochondrial respiration capacity of PTC cells was decreased after GLS inhibition,including both basal oxygen consumption and maximal oxygen consumption levels.Meanwhile,growth viability,proliferation,migration and invasion ability of PTC cells were significantly attenuated after the expression of GLS was decreased.Western blotting assays confirmed that levels of mTORCl signaling pathway markers(p-p70s6k,p70s6k,p-4EBP1,4EBP1,p-ULK1,ULK1,p-mTOR and mTOR)were decreased.The protein levels of autophagy marker molecules p62 and LC3B-I were down-regulated,and LC3B-II was up-regulated,suggesting that inhibition of GLS expression leads to decreased activity of mTORCl signaling pathway and induced autophagy.Flow cytometry results showed that the proportion of apoptosis in PTC cells was significantly increased after the expression of GLS was decreasedThe Real-time PCR results of 92 pairs of papillary thyroid cancer confirmed that the mRNA expression of SLC1A5,SLC7A5 and SLC6A14 were significantly increased compared with matched paracancerous tissues(p<0.001).Immunohistochemical staining results identified that the immunoreactive scores of SLC1A5,SLC7A5 and SLC6A1 were higher than that of non-cancerous thyroid tissues(p<0.001).Besides,analysis of clinical pathological data of patients indicated that male PTC patients have higher SLC7A5 staining compared with female patients(p=0.003).The protein expression levels of SLC1A5 and SLC6A14 were associated with ?-cat.SLC7A5 and SLC6A14 levels were found to be positively correlated with MMP9 and VEGFA levelsConclusions:This study mainly confirmed the presence of glutamine-dependence of thyroid papillary cancer cells,and found that glutaminase and some glutaminolysis-associated transporters,including SLC1A5,SLC7A5,and SLC6A14 were aberrantly up-regulated in human thyroid papillary cancer with RT2-PCR arrays.The above results were further confirmed in subsequent Real-time PCR,Western blotting and immunohistochemical staining experiments.We found that the expression level of glutaminase was significantly associated with extra-thyroidal extension of thyroid cancer.The expression levels of SLC1A5?SLC7A5 and SLC6A14 were found to be positively correlated with tumor invasive markers ?-cat,MMP9 and VEGFA.In addition,we investigated the role of GLS in thyroid papillary cancer and its possible mechanisms.Experiments have confirmed that the glutamine metabolism process and mitochondrial respiratory level in PTC cells was significantly inhibited after decreasing the expression level of GLS.We used both chemical inhibitors and genetic tools to inhibit the expression of GLS in PTC cells.Both treatments reduced the proliferation,survival,migration and invasion ability of thyroid papillary cancer cells.In terms of possible mechanism of action,inhibition of glutaminase promotes the inactivation of the rapamycin complex 1(mTORCl)signaling pathway,thereby promoting autophagy and apoptosis in cells.Collectively,the above findings suggest that glutaminase-mediated glutamine dependence may be a potential therapeutic target for papillary thyroid cancer. |
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