The Effect And Mechanism Of ASAP1 In Autophagy Of Papillary Thyroid Carcinoma Cells

Background and ObjectivePapillary thyroid carcinoma(PTC)is the most common thyroid cancer,and its incidence is increasing.Although most patients with PTC have a better prognosis through surgery and iodine 131,some patients have had severe lymph node metastasis and invasion at an early stage.These patients with PTC cannot achieve satisfactory treatment results through surgery and iodine 131.Therefore,finding effective targets related to PTC metastasis is a new strategy for the treatment of PTC patients.ASAP1 is a GTPase-activating protein of ADP-ribosylation factor and has multiple domains such as SH3,BAR,PH,GAP,and ANK.ASAP1 has been established as a regulator of the actin cytoskeleton and focal adhesion complex.ASAP1 plays an important role in the regulation of cell movement and intercellular adhesion.AS AP1 is highly expressed in a variety of cancer tissues and is considered a potential therapeutic target for various tumor treatments.By inhibiting or knocking out the ASAP1 gene,biological behaviors such as tumor invasion and migration will be inhibited,thereby achieving the effect of treating tumors.Our previous work has found that ASAP1 is significantly overexpressed in PTC tissues and is related to clinical staging.Studies have shown that the activation of SH3 and PH domains of ASAP1 can regulate autophagy and promote tumor cell invasion and migration.At present,autophagy is a targeted treatment for cancer,which plays an important role in the treatment of cancer.Autophagy plays a dual role in tumor,not only inhibiting tumor growth,but also maintaining the survival of cancer cells.The role of autophagy in cancer depends on the pathological type and stage of the tumor.In the early stages of tumorigenesis,autophagy may inhibit tumor development.However,autophagy can promote tumor growth after tumor formation.Previous studies have shown that autophagy is closely related to the proliferation and metastasis of tumor cells.The proliferation and migration of tumor cells can be regulated by autophagy.Therefore,regulating the autophagy level of PTC is likely to be a potential treatment for PTC.The purpose of this study is to investigate whether ASAP1 has a regulatory role and mechanism in papillary thyroid carcinoma autophagy.Materia and Methods1.Fresh specimens of cancer tissue and its adjacent tissues were collected from 60 patients with PTC.The expression of ASAP1 and LC3 in cancer and adjacent tissues was detected by tissue immunofluorescence.2.In this study,MDA-T32 and MDA-T85 cell lines were selected.The stably expressed cell lines were constructed by MDA-T32 and MDA-T85 cell that knocked out of ASAP1 by CRISPR/cas9 gene technology.3.The expression of autophagy in ASAP1 knockout group and control group was detected by Western Blot and cellular immunofluorescence methods.By combining chloroquine(CQ),we confirmed that knockout of ASAP1 improved the autophagy level of MDA-T32 and MDA-T85 cells.4.In order to explore the specific mechanism of autophagy inhibition,the expression of mTOR pathway activity related protein(p-p70S6K,p-mTOR)was detected by Western Blot,5.SPSS 21.0 statistical software was used for analysis.The graphs were drawn using GraphPad Prism 8 software.The differences between the variables were analyzed by chi-square test and t-test.P<0.05 means that the difference is statistically significant.Results1.Compared with adjacent cancer tissues,the expression of ASAP1 in PTC tissues is increased and related to the poor prognosis of patients.2.Compared with adjacent cancer tissues,the expression level of autophagy marker protein LC3B in PTC tissues was reduced.3.Knockout of ASAP1 increased the level of autophagy in MDA-T32 and MDA-T85 cells.4.Knockout of AS AP1 inhibited the mTOR signaling pathway.Conclusions1.ASAP1 inhibits autophagy of thyroid papillary cancer cells.2.ASAP1 down-regulates the autophagy level of thyroid papillary cancer cells through the mTOR signaling pathway,which may be one of the mechanisms that ASAP1 participates in the regulation of metastasis of thyroid papillary cancer cells.