LncRNA GAS8-AS1 Inhibits Cell Proliferation Through ATG5-Mediated Autophagy In Papillary Thyroid Cancer

Objective: Thyroid cancer is the most common endocrine system malignancy,representing 1–2% of total cancer cases,and the worldwide incidence of thyroid cancer has been increasing rapidly over the past few decades.Papillary thyroid cancer(PTC)is the most common type of thyroid cancer,and comprises the vast majority(>80%)of all thyroid cancers.Although most of PTCs could be managed successfully with a combination of radioiodine treatment and levothyroxine suppression therapy after complete surgical intervention,a certain proportion of PTCs remain irresponsive to treatment and result in comorbidity and mortality.Therefore,it is crucial to further elucidate the pathogenesis of PTC regarding tumor behavior at the molecular level.Long non-coding RNA GAS8 antisense RNA 1(lncRNA GAS8-AS1)has been reported to be a novel tumor suppressor gene that affects tumor cell proliferation in PTC,but how GAS8-AS1 regulates tumor cell function remains unclear.Autophagy is a dynamic response to maintain cellular ecological balance,and modulates cell status by degrading abnormal proteins and organelles.The change of the regulation level of autophagy affects the occurrence and development of cancer,leading tumor cell to death by destroying essential cellular components,tipping cells into apoptosis.The molecular function and autophagy activation of GAS8-AS1 in PTC cells will be intensively studied in this research.Methods: The human PTC cell line,TPC1 and BCPAP,were selected to use in experiments.Construction of GAS8-AS1 overexpressing cell lines and low expression cell lines by transfecting with plasmids and short interfering RNAs(siRNAs).Cell Counting Kit-8(CCK-8)was selected as cell proliferation assay to detect the proliferation of cells after cell transfection.qRT-PCR and western blot were used to determine changes of genes expression at mRNA and protein level.Immunofluorescence microscopy demonstrated high magnification of punctate aggregates of LC3 staining and transmission electron microscopy was used to observe the formation of autophagosome.Results: GAS8-AS1 expression was decreased in PTC cell lines,TPC1 and BCPAP,comparing with Nthy-ori-3-1.Further assays revealed that overexpression of GAS8-AS1 can inhibit cell proliferation and significantly accumulate the ratio of LC3II/LC3 I and reduce p62 expression,whereas GAS8-AS1 knockdown shown the opposite appearance.Bafilomycin A1 markedly blocked GAS8-AS1 induced autophagy flux and increased the LC3 II accumulation,indicating the accelerated conversion of LC3 I to LC3 II.LC3immunofluorescence staining demonstrated high magnification of punctate aggregates of LC3 staining and transmission electron microscopy also revealed more characteristic autophagosomes in the GAS8-AS1 overexpression group,indicating the formation of autophagosomes.We found that autophagy-related gene(ATG)5 were markedly upregulated by GAS8-AS1 overexpression and downregulated by GAS8-AS1 decreased expression at mRNA level and protein level.Lastly,silencing of ATG5 attenuated autophagy activation and cell proliferation inhibition caused by GAS8-AS1,indicating the important role of ATG5 in GAS8-AS1 induced autophagy.Conclusion: As a new tumor suppressor gene,GAS8-AS1 can inhibit the proliferation of tumor cells,and promote the activation of autophagy and increase the expression of ATG5.ATG5 can reverse GAS8-AS1 mediated autophagy activation and cell death,revealing a novel mechanism of GAS8-AS1-ATG5 axis in PTC cells.This provides a new experimental basis for the treatment of thyroid cancer with lncRNA effects of autophagy.