| Papillary thyroid carcinoma(PTC)is the most common differentiated malignant thyroid tumor.The incidence of PTC is the fastest growing among all cancer types.Although the overall five-year survival rate after treatment can reach 98%,some patients still have cancer cells infiltrating into surrounding lymph tissues after surgical treatment or chemoradiation,leading to an increase in mortality.Therefore,it is necessary to understand the molecular and genetic mechanisms of thyroid cancer and explore new therapeutic targets.TFF3 belongs to the family of trefoil factors(TFFs).More and more studies have shown that TFF3 is abnormally expressed in a variety of tumor cells and participates in a variety of biological behaviors such as tumor cell proliferation,invasion,and apoptosis.Overexpression of TFF3 can regulate cancer progression through activation of multiple signaling pathways such as MAPK,ERK,PI3K/AKT,while TFF3 has little research on the regulation and signal transduction mechanism of PTC malignant biological behavior.The results of our team’s previous studies showed that when the expression of endogenous TFF3 was knocked down,the growth of TPC-1 cells was sluggish,and the decreased invasiveness was related to the cell arrest in the G0/G1 phase.Based on the previous research results of the research team,further clarify the effect of TFF3 differential expression on the malignant biological behavior of TPC-1 cells and the mechanism of signal transduction,which can provide therapeutic strategies for TFF3 as a potential target gene and effective target inhibitor of PTC treatment.Firstly,we constructed Lentivirus Expression Vectors of TFF3 gene overexpression and knockdown expression,and transfected TPC-1 cellswith lentivirus to harvest TFF3-TPC-1 cells that stably enhanced TFF3 gene expression and corresponding control cells Con TFF3-TPC-1sh RNA-TFF3-TPC-1 cells that inhibited TFF3 gene expression and corresponding control cells sh Con-TPC-1;Real-time PCR(RT-PCR)and Western Blot were used to verify TFF3 overexpression and silencing efficiency.Growth curve and clone formation experiments were used to detect the cell proliferation levels of TFF3-TPC-1,Con TFF3-TPC-1,sh RNA-TFF3-TPC-1,and sh Con-TPC-1;flow cytometry was used to detect the apoptosis rate of the four groups;Western Blot and immunocytochemical staining(ICC)detection of PI3K/AKT,NF-κB pathway protein and apoptosis-related proteins Akt,p-Akt,NF-κB-p65,Bcl-2,Bax,cytochrome C(Cyt-c),cleaved-Caspase-9,cleaved-Caspase-3expression levels.The results showed that(1)the proliferation rate and clonal formation ability of TFF3-TPC-1 cells were significantly higher than those of Con TFF3-TPC-1 cells in the control group(P <0.05 or P <0.01),and the proliferation rate and clonal formation ability of sh RNA-TFF3-TPC-1 cells were significantly lower than those of sh Con-TPC-1 cells in the control group(P <0.01);and sh RNA-TFF3-TPC-1 cell proliferation rate And colony forming ability were significantly lower than the control group sh Con-TPC-1(P <0.01);(2)TFF3-TPC-1 cell apoptosis rate was lower than the control group Con TFF3-TPC-1(0.75%±0.08% VS 5.62%±0.3%,P<0.01),the apoptotic rate of sh RNA-TFF3-TPC-1 was higher than that of control group sh Con-TPC-1(22.2%±1.2% VS 5.34%±0.4%,P <0.01);(3)Compared with the control group,the expression of Bax,Cyt-C,cleaved-caspase-9,cleaved-caspase-3 was down regulated after TFF3 gene expression was enhanced,while the expression of Bcl-2,Akt,p-Akt,NF-κB-p65 was up regulated after TFF3 gene was silenced,and down regulated after TFF3 gene expression was silenced(P <0.05 or P <0.01).In order to further clarify the role of PI3K/AKT/NF-κB pathway in the process of PTC,TFF3-TPC-1 cell line was set as the overexpression group;wild-type TPC-1 cell line without any treatment was set as the control group;TFF3-TPC-1 cell line with different concentrations of PI3K/AKT signal pathway specific inhibitor LY294002(25μmol/L,50μmol/L,75μmol/L)was used as the experimental group for 48 hours.The morphological changes and growth state of cells in each group were observed under inverted microscope;the proliferation level of cells in each group was compared by growth curve;cell invasion and migration ability was detected by Transwell and scratch test;cell apoptosis was detected by flow cytometry;western The expression of p-Akt,p-IκB-α,IκB-α,NF-κB-p65,cleaved-Caspase-3,cleaved-Caspase-9,Bcl-2 and Bax protein were detected by Western Blot..The results showed that:(1)Morphological observation:The cells in the overexpression group and the control group were epithelioid with no significant difference in appearance.With the increase of LY294002 concentration,the intercellular space became larger and the number of round and bright cells increased;(2)Compared with the control group,the cell fusion rate,invasion ability and migration index of the overexpression group were higher than those of the control group,while the apoptosis rate of the overexpression group was lower than that of the control group(0.89%±0.03 VS 2.4%±0.06%,P <0.05 or P <0.01);(3)The cell fusion rate,invasion and migration index of the experimental group were lower than those of the Control group.The apoptosis rate was higher than that in the Control group(6.22%±0.02%,10.8%±0.02%,24%±0.07% VS 2.4%±0.06%,P <0.05 or P <0.01);(4)Western Blot showed: Compared with the Control group,Pathway proteins and anti-apoptotic proteins p-Akt,p-IκB-α,NF-κB-p65,Bcl-2 were down-regulated with increasing concentration of LY294002,pro-apoptotic proteins IκB-α,cleaved-Caspase-3,cleaved-Caspase-9 and Bax expression increased;the expression of pathwayprotein and anti-apoptotic protein in the overexpression group was higher than that in the Control group,while the expression of pro-apoptotic proteins was lower than that in the Control group(P <0.05 or P <0.01).Taken together,TFF3 can promote the proliferation and anti apoptosis malignant biological behavior of TPC-1 cells by affecting the PI3K/AKT/NF-κB signaling pathway,and LY294002,a PI3 K target molecule inhibitor,can effectively inhibit the PI3K/AKT pathway transduction,and its inhibitory effect is to effectively down regulate the NF-κB signaling pathway activity by inhibiting Akt phosphorylation,thereby inhibiting the proliferation of TFF3-TPC-1 cells,Invasion,migration and apoptosis.It can provides theoretical support for target gene therapy of clinical thyroid cancer and development of target molecule inhibitor drugs. |
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