Mechanism Of TREM2 Regulating Autophagy And Apoptosis In Papillary Thyroid Carcinoma Via AKT/mTOR Signaling Pathway

Background:Accumulating evidence has shown that triggering receptor expressed on myeloid cells 2(TREM2)plays essential roles in tumorigenesis,progression and tumor therapy.TREM2 plays different or even opposite roles in different tumors.However,there are no relevant studies or case reports on the role of TREM2 in papillary thyroid carcinoma.Object:To investigate the biological function of TREM2 in vivo and in vitro;to explore whether TREM2 could be a potential novel target for the treatment of PTC by constructing the tumor-bearing nude mouse model of PTC cells.Method:1.Bioinformatics analysis and TREM2-related detection analysis:Single-cell RNA sequencing(scRNA-seq)was used to screen genes that are highly expressed in PTC,and the survival analysis based on data in TCGA database was analyzed.WB and qRT-PCR was used to detect the expression of TREM2 in cancer tissues and adjacent normal thyroid tissues of PTC patients as well as the expression in human papillary throid cancer cell lines TPC1,K1,BCPAP,KTC-1 and human normal thyroid cell line Nthy-ori 3-1.2.Cell biological function research:Stable TREM2 overexpression or interference model cell lines was established by lentiviral infection:TREM2-shRNA TPC-1,TREM2OE KTC-1 and their negative control cells TREM2-NC TPC-1,REM2-NC KTC-1.The protein and mRNA expression levels of TREM2 in TREM2-shRNA TPC-1 and TREM2-OE KTC-1 were detected by WB and qRT-PCR.Cell migration and invasion were tested by scratch and Transwell assays,and cell proliferation was detected by CCK-8 assay.3.Mechanism of autophagy and apoptosis:WB detected the expressions of autophagyrelated molecules ATG7,P62 and LC3B Ⅱ/Ⅰ and apoptosis-related proteins Caspase3,Cleaved Caspase3,Bcl2 and Bax.Cell apoptosis was assessed by flow cytometry assay.Rescue experiments were applied to verify conclusions.Fluorescence microscopy was used to observe the level of autophagic flux in cells infected with Stub-RFP-Sens-GFP-LC3 double fluorescent lentivirus.WB was applied to detect the expression of ATG7,P62 and LC3B Ⅱ/Ⅰ in TREM2-OE KTC-1 cells treated with RAPA and in TREM2-shRNA TPC-1 cells treated with 3-MA.4.Cytological mechanism study:The autophagy-related proteins ATG7,P62 and LC3BⅡ/Ⅰ as well as AKT/mTOR signaling pathway molecules AKT,p-AKT,mTOR and p-mTOR were detected using WB.5.Subcutaneous-tumor-bearing nude mouse model:The nude mice were injected with TREM2-shRNA TPC-1 cells and TREM2-NC TPC-1 cells subcutaneously to establish the model.Tumor growth was assessed via measuring the tumor volume.WB was used for detecting expressions of TREM2,autophagy-related proteins and pathway-related molecules.Results:1.We analyzed TCGA data of 564 patients with available clinical and pathological information,it was found that the five-year survival rate of patients with elevated TREM2 expression levels was lower when compared with that of patients with low TREM2 expression levels,and the level of TREM2 was correlated with the histological type and TNM stage.2.Compared with para-carcinoma normal tissues,TREM2 protein and mRNA expression levels were significantly increased in PTC tissues.Compared with the human normal thyroid cell line Nthy-ori 3-1,the expression of TREM2 was generally up-regulated in TPC-1,K1 and BCPAP,the TPC-1 cell line had the highest TREM2 expression level among them,while the expression of KTC-1 was down-regulated.3.PTC cells stably overexpressing or interference TREM2 were constructed by lentiviral transduction:TREM2-shRNA TPC-1 and TREM2-OE KTC-1.Compared with the TREM2-NC TPC-1 group,the mRNA and protein levels of the TREM2-shRNA TPC-1 group were significantly down-regulated;compared with the TREM2-NC KTC-1 group,the expression of the TREM2-OE KTC-1 group was significantly up-regulated.4.Compared with the control group,the migration,invasion and proliferation of PTC cells were inhibited in the TREM2-shRNA group,while in the TREM2-OE KTC-1 group,the results showed the opposite changes.5.Compared with the control group,the level of P62 and Bcl2 protein in the TREM2shRNA TPC-1 group was down-regulated,while the expressions of ATG7,LC3B Ⅱ/Ⅰ,Bax and Cleaved caspase3/caspase3 were increased,meanwhile,flow cytometric analysis showed apoptotic cells were increased.The apoptosis level of TREM2-shRNA TPC-1 group co-cultured with 3-MA was reversibly inhibited.The fluorescent spots were increased than control group,as well as the number of red merging-blot.It is suggested that interfering with TREM2 expression could promote cell apoptosis and autophagy.The results of the overexpression group were opposite to those of the interference group.6.Compared with the TREM2-OE KTC-1 group,treatment with RAPA could promote the P62 protein level of PTC cells while suppressed ATG7 and LC3B Ⅱ/Ⅰ.Also,treatment with 3-MA could suppressed the P62 protein level of PTC cells while promote atg7 and LC3B Ⅱ/Ⅰ compared with the TREM2-shRNA TPC-1 group.7.Compared with the TREM2-shRNA group,the TREM2-shRNA group after SC79 treatment had increased P62,p-AKT and p-mTOR protein level,decreased ATG7 and LC3BⅡ/Ⅰ.The data indicated that the Akt-mTOR signal pathway was involved in the TREM2 induced autophagy.8.Subcutaneous-tumor-bearing nude mouse model showed that compared with the control group,the tumor volume of the nude mice in the TREM2-shRNA group were smaller.The protein expression of ATG7,LC3B Ⅱ/Ⅰ was increased;while TREM2,P62 and p-AKT and p-mTOR was decreased.It follows that TREM2 inhibits the autophagy through the AKTmTOR signaling pathway.Conclusion:1.TREM2 may be a poor prognostic marker for PTC patients;2.The expression of TREM2 in human PTC tissues was significantly higher than that in paracancerous tissues;3.It was confirmed that interfering with TREM2 inhibited cell proliferation,invasion and migration,while promoting autophagy and apoptosis,by stably transfecting the PTC cell line.Overexpression of TREM2 could produce the opposite result;4.The activation of SC79 on pathway could be reversed by TREM2-shRNA,as well as the inhibitory effect of SC79 on autophagy.The inhibitory effect of RAPA on pathway could be reversed by TREM2-OE,as well as the activation of RAPA on autophagy.We speculated that TREM2 might modulate AKT-mTOR signaling via regulating autophagy.This conclusion could also be obtained in the nude mice experiment.