Study on the Mechanism of Cancer/Testicular Antigen FSIP1 in Regulating Autophagy and Promoting Survival of Lung Cancer Cells

Abstract

Objective — To investigate the molecular mechanism of FSIP1 gene expression and autophagy inhibition in patients with lung adenocarcinoma. Methods — TTP expression in lung adenocarcinoma H1299, H1975 and normal lung forming cells was detected by QRT-PCR and Western Bloting. Quantitative analysis and Western Bloting were performed by transfecting pCMV-FSIPI at 24h, 48h and 72h respectively to analyze the expression of autophagy related factors like P62, LC3II/LCI and BecIinl. The transient transfection of overexpressed and empty plasmid was performed by adding 10 mg/ml of actinomycin D. After the termination of this transcription, RNA extraction was performed at different time points to detect the expression of Beclin1m RNA at different time points. After adding TNF-a by transfection plasmid, lung adenocarcinoma cells were divided into different groups, including no-load group, FSIPI group, no-load + TNF-A group, and FSIPI + TNF-a group. The gene expression of P50, c-Rel and NF-KBp65 in the nucleus was analyzed by immunofluorescence and Western Bloting. Lung adenocarcinoma was divided into Ikba-mut no-load group, FSIPI no-load group, 1Ikba-mut group, FSIPI group and FSIPI+ Ikba-mut group. FSIPI expression and autophagy gene expression were detected by quantitative analysis and Western Bloting. Results — In lung adenocarcinoma cells, FSIP1 RNA and protein levels were RELATIVELY low, and autophagy Becline and LC 3II/I had lower RNA and protein levels after overexpression of FSIPI compared with the no-load group. Transcription is terminated by the addition of actinomycin D. There was no significant difference in the expression of the autophagy-related gene BEC LINEM between FSIP1 and the no-load group. After overexpression of FSIPI, according to Western Bloting results, nuclear C-Rel and P65 proteins were less than those in the no-load +TNF-a group and the no-load group, whileP50 protein did not change significantly. Combined with immunofluorescence studies, it was found that the expressions of c-Rel and P65 were significantly decreased after FSIP1 overexpression compared with the no-load group, while the expression of P50 was not significantly changed. Conclusion — FSIP1 is usually low expressed in lung adenocarcinoma. Overexpression of FSIP1 can effectively inhibit the nuclear metastasis of C-Rel and NF-KBp65, thus inhibiting autophagy of the cells.