| Objective1.To clarify the expression of REGγ in anaplastic thyroid cancer and its regulatory effect on the proliferation,apoptosis,invasion,migration and epithelial interstitial transformation of anaplastic thyroid cancer and its effect on downstream signaling pathways;2.Preliminary verification of the regulatory effect of REGγ on radiotherapy for anaplastic thyroid cancer.Methods1.To detect the expression of REGγ in anaplastic thyroid cancer.1.1 Detection of REGγ expression in cancer and paracancer tissues of patients with anaplastic thyroid cancer: RT-PCR and Western blot assay were used to detect the expression of REGγ.1.2 Detection of REGγ expression in pathological wax specimens: The expression of REGγ was detected by immunohistochemical test.2.To detect the expression of REGγ in anaplastic thyroid cancer cell lines and its effects on proliferation,apoptosis,invasion,migration,epithelial–mesenchymal transition and AKT/MAPK/β-catenin signaling pathway of anaplastic thyroid cancer cells.2.1 The expression of REGγ in anaplastic thyroid cancer cell lines was detected,and the cell line with the highest expression of REGγ was selected for subsequent tests:The expression levels of REGγ in thyroid epithelial cell lines Nthy-ori-3-1 and anaplastic thyroid cancer cell lines 8305 C,FRO and Cal62 were detected by PCR and Western blot.2.2 The expression of REGγ in the anaplastic thyroid cancer knockout group and the control group was detected,and the experimental group with the lowest expression of REGγ was selected for subsequent experiments: FRO/sh-REGγ of anaplastic thyroid cancer with knockdown REGγ was constructed by sh-RNA technology(the experimental group was randomly divided into sh-REGγ1#,sh-REGγ2#,sh-REGγ3#)and the control group were FRO/sh-NC cell line,and the expression level of REGγwas detected by Western blot.2.3 Detection of the effect of REGγ on the proliferation of anaplastic thyroid cancer cells: CCK-8 and clonal formation assay were used to detect the proliferation of anaplastic thyroid cancer cells in the experimental group and control group.2.4 Detection of the effect of REGγ on apoptosis of anaplastic thyroid cancer cells:Apoptosis of thyroid cancer cells was detected by cell loss cytology in the experimental group and the control group.2.5 Detection of the impact of REGγ on the migration and invasion capacity of anaplastic thyroid most cancers cells: Scratch assay and Transwell assay had been used to observe the modifications in the migration and invasion capability of anaplastic thyroid most cancers cells in the experimental crew and manipulate group.2.6 Detection of the effect of REGγ on epithelial mesenchymal transition of anaplastic thyroid cancer cells: The expression levels of E-cadherin,N-cadherin and Vimentin related to epithelial mesenchymal transition of anaplastic thyroid cancer cells were detected by Western blot.2.7 Detection of the effect of REGγ on the AKT/MAPK/β-catenin signaling pathway of anaplastic thyroid cancer cells: Western blot was used to detect the expression levels of proteins related to the anaplastic thyroid cancer pathway.3.To detect the effect of REGγ on radiotherapy resistance of anaplastic thyroid cancer.3.1 Construct the radiotherapy resistance strain of anaplastic thyroid cancer and prove the successful establishment of its model: construct FRO-IR radiotherapy resistance cell strain of anaplastic thyroid cancer,and draw the fitting survival curve of the experimental group and the control group cells.3.2 Detection of the effect of REGγ on the proliferation of anaplastic thyroid cancer cells under radiotherapy: The proliferation of anaplastic thyroid cancer cells in the experimental group sh-REGγ and the control group sh-NC was detected by clonal formation assay under the same radiation condition.Results1.Expression of REGγ in anaplastic thyroid cancer:1.1 The results of RT-PCR and Western Blot showed that,compared with paracancer tissues,the expression level of REGγ gene and REGγ protein in anaplastic thyroid cancer were significantly up-regulated(P < 0.05).1.2 Immunohistochemical results showed that the expression level of REGγ in anaplastic thyroid cancer was significantly higher than that in paracancer tissue(P <0.05).In conclusion,REGγ expression is up-regulated in tumor tissues of patients with anaplastic thyroid cancer.2.Results of the expression of REGγ in anaplastic thyroid cancer cell lines and its effects on proliferation,apoptosis,invasion,migration,epithelial-mesenchymal transition,and AKT/MAPK/β-catenin signaling pathway:2.1 Compared with Nthy-ori-3-1 cell line of thyroid epithelial cells,REGγ was significantly increased in 8305 C,FRO and Cal62 cell lines,and the expression degree was best in FRO cell line,which was used for follow-up experiments(P < 0.05).2.2 Compared with the sh-NC group,the REGγ protein content in sh-REGγ#1,sh-REGγ#2 and sh-REGγ#3 groups was significantly down-regulated,and the down-regulated content in sh-REGγ#1 group was the most significant,and the cells were subsequently used in functional experiments(P < 0.05).2.3 Compared with the sh-NC group,the CCK-8 OD value of the experimental group decreased with the increase of time,and decreased at 48 h and 72 h,respectively,and the clone formation rate of the experimental group decreased significantly.These results indicated that knockdown REGγ decreased the proliferative ability of anaplastic thyroid cancer cells(P < 0.05).2.4 Compared with sh-NC group,knockdown REGγ significantly increased the number of apoptosis of anaplastic thyroid cancer cells(P<0.05).2.5 Compared with the sh-NC group,the migration distance of cells in the experimental team lowered considerably at 24 h,and the quantity of invasive cells in the experimental team diminished significantly.These results indicated that knockdown REGγ could inhibit the invasion and migration of anaplastic thyroid cancer cells(P < 0.05).2.6 Compared with the sh-NC group,the expression degrees of E-cadherin was once extended in the experimental group,whilst the expression degrees of N-cadherin and Vimentin had been decreased.The results showed that knockdown REGγ inhibited epithelial mesenchymal transition of ATC cells(P < 0.05).2.7 Compared with the sh-NC group,in the experimental team the expression of pathway-related proteins have been considerably decreased.The effects confirmed that down-regulating REGγ inhibited AKT/MAPK/β-catenin signaling pathway(P<0.05).3.Effect of REGγ on radiotherapy resistance of anaplastic thyroid cancer:3.1 The results of fitting survival curve showed that Survive Fraction of FRO cells decreased with the increase of irradiation dose,but the degree of reduction of radiotherapy resistant cells was significantly lower than that of control group,which was much higher after 10 Gy treatment,indicating that the survival ability of radiotherapy resistant FRO cells was improved and radiotherapy resistance was stronger.Radiotherapy resistance cell model was established successfully(P<0.05).3.2 Compared with 0 Gy,the quantity of cell clones decreased with the increase of radiation dose in the sh-NC group and the experimental group.Compared with sh-NC group,the quantity of cell clones in experimental group decreased significantly under the same irradiation condition.These results indicated that knockdown REGγ could decrease the clonal formation ability of tumor cells resistant to radiotherapy and enhance radiotherapy sensitivity(P<0.05).Conclusions1.REGγ is highly expressed in anaplastic thyroid cancer,and regulates the proliferation,apoptosis,invasion,migration,and epithelial-mesenchymal transition(EMT)of anaplastic thyroid cancer through AKT/MAPK/β-catenin signaling pathway.2.REGγ can participate in radiotherapy resistance of anaplastic thyroid cancer cells.In addition,REGγ is a potential marker for clinical diagnosis of anaplastic thyroid cancer and a potent drug target for anaplastic thyroid cancer. |
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