| Objective: The study was carried out to investigate the effects of LASS2 on the autophagy activity of BCPAP and epithelical-mesenchymal transition(EMT)in Papillary thyroid carcinoma(PTC)cells.The expression of LASS2 and SQSTM1/p62 in PTCs cancer tissues and adjacent normal tissues and its correlation with clinicopathological features were verified by tissue specimen detection.Methods: 1.Adv-LASS2-GFP recombinant adenovirus infected BCPAP cells to construct LASS2 overexpression cell model.Real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)and Western blotting(WB)experiments verified the successful construction of BCPAP cell model with LASS2 overexpression.2.At 48 h after infection,the autophagy-related structures of each group were observed by transmission electron microscopy,and the expressions of autophagy-related proteins SQSTM1/ p62,LC3A/B and Beclin-1 in each group were detected by q RT-PCR and WB.3.The invasion and migration abilities of cells in each group were detected by scratch test and transwell chamber test,and the expression of EMT-related proteins MMP2,MMP9,E-cadherin,N-cadherin and Vimentin were detected by WB.4.co-IP/LC-MS screened and identified LASS2 interacting proteins,carried out biogenic analysis by GO Pathway,STRING and other software and constructed protein-protein interaction network,and further verified interaction relationship by co-IP WB.5.Immunohistochemistry(IHC)assay detects the expression differences of LASS2 and SQSTM1/ p62 in PTC tissue specimens and their correlation with invasion and metastasis.Results: 1.BCPAP cell model of thyroid papillary carcinoma with overexpression of LASS2 gene was successfully constructed.q RT-PCR and WB experiments showed that overexpression of LASS2 significantly increased the relative expression levels of LASS2 m RNA and protein in the experimental group(Adv-LASS2-GFP group).2.More autophagosomes and lysosomes were observed in Adv-LASS2-GFP group than in Adv-GFP group after transfection 48 h.The results of q RT-PCR and WB showed that overexpression of LASS2 significantly promoted the relative expression of Beclin-1 and LC3 B m RNA and protein(P < 0.05),but overexpression of LASS2 did not affect the m RNA expression of SQSTM-1/ p62(P > 0.05).WB results showed that overexpression of LASS2 significantly down-regulated the relative expression level of SQSTM-1/ p62 protein in BCPAP cells(P < 0.001).3.The results of scratch test and transwell test showed that overexpression of LASS2 significantly inhibited the invasion and migration of BCPAP cells(P < 0.01),and changed the expression of EMT process-related proteins at the protein level(P < 0.001).4.LASS2 interacting proteins were isolated by co-IP /LC-MS,and bioinformatics analysis was performed by GO Pathway,STRING and other software to screen the key autophagy proteins interacting with LASS2 and construct protein interaction network diagram.The results suggest that LASS2 may interact directly or indirectly with p62 /CSNK2A1/HSP90/HSP70.co-IP WB analysis showed no direct binding between LASS2 and p62,suggesting that LASS2 and p62 may interact with each other through other intermediate proteins.5.The relative expressions of LASS2 and SQSTM1/ p62 proteins in 30 pairs of PTC tissue samples were analyzed by IHC.The results showed that the expression level of LASS2 in cancer tissues was lower than that in adjacent normal tissues,and was related to lymph node metastasis(P < 0.01).The expression level of p62 in cancer tissues was significantly higher than that in adjacent normal tissues(P < 0.01).The expression of p62 was correlated with the breakthrough of tumor capsule and lymph node metastasis(P < 0.01).Conclusion: 1.LASS2 may interact with p62 /CSNK2A1/HSP90/HSP70 to activate autophagy and inhibit the invasion of BCPAP cells;2.The expression of LASS2 and p62 was different in PTC tissues and adjacent normal tissues and correlated with lymph node metastasis.3.LASS2 may interact with p62 indirectly through other proteins,thereby regulating PTC autophagy and mediating PTC tumor metastasis. |
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