The Influence And Mechanism Of SIRT6 On The Biological Function Of Non-small Cell Lung Cancer

Background:Lung cancer has become the most common malignancy in the world with the highest incidence and mortality.The type of lung cancer is mainly divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC)and others.This subject is mainly focused on the study of the biological function of NSCLC cells.With the continuous development of clinical techniques,radiotherapy has become one of the main treatments for NSCLC.Although radiotherapy has achieved certain clinical efficacy,it has not significantly improved the prognosis of patients with lung cancer.Although radiotherapy has achieved certain clinical efficacy,it has not significantly improved the prognosis of patients with lung cancer.Invasion and metastasis is the main cause of poor prognosis in NSCLC patients.At the same time,radiation-induced lung injury(RILI)is the most important dose-limiting toxicity factor in NSCLC radiotherapy.SIRT6 which owns NAD+-dependent protein deacetylase,ADP-ribose transferase and other activities,is a regulatory protein that plays an important role in the development of tumors.Recent studies have shown that SIRT6 plays an important role in tumor invasion and metastasis,inflammatory response and radiotherapy resistance,but the mechanism of SIRT6 in NSCLC is still limited.Therefore,this study preliminarily explored the effects of regulating SIRT6 expression on invasion,metastasis and RILI in NSCLC and its possible mechanism.Simultaneously detected the survival of NSCLC cells after radionuclide irradiation after SIRT6 regulation,and defined the role of SIRT6 in radiotherapy resistance of NSCLC.It provides new ideas and theoretical basis for the clinical treatment of lung cancer from basic to clinical transformation.Methods:1.Western blot was used to detect the expression of SIRT6 protein in lung cancer tissues and normal lung tissues,and construct the SIRT6 overexpression and knockout vector in NSCLC cells.A549 and H1299 cell strains were infected with lentivirus by packaging and SIRT6-overexpression stable cell lines were selected.SIRT6 in A549 cells was knocked out by CRISPR/Cas9 gene editing technology,and SIRT6 knockout stable cell lines were selected.2.The effects of SIRT6 on the invasion and metastasis of NSCLC cells were investigated by cell scratch healing assay and transwell invasion assay.RT-PCR was used to detect changes of EMT surface marker mRNA after NSCLC cell overexpression and knockout of SIRT6.Western blot was used to screen SIRT6 signaling pathways that affect the invasion and metastasis of NSCLC.3.RT-PCR was used to detect SIRT6 gene overexpression and knockout cell line inflammatory cytokines mRNA.Western blot was used to screen out the signal pathways that influence the inflammatory response under cisplatin drug stimulation.To investigate the specific mechanism of SIRT6 in influencing the inflammation of NSCLC cells.4.The CCK-8 assay was used to detect the effect of SIRT6 on the survival curve of different A549 cell lines after irradiation,and to investigate the effect of SIRT6 on the cell survival rate after irradiation of A549 cells,and to clarify the role of SIRT6 in radiotherapy resistance of lung cancer.Results:1.Western blot analysis showed that the expression of SIRT6 protein in NSCLC tissues was lower than that in adjacent tissues.Successfully constructed NSCLC SIRT6 overexpression and knockout cell lines.The results of sequencing alignment showed that SIRT6 was successfully overexpressed in A549 cells and H1299 cells,and SIRT6 designed target was successfully knocked out in A549 cells.Western blot results showed that SIRT6 protein was significantly up-regulated in overexpression cell lines,and no SIRT6 protein was expressed in knockout cell lines.2.After overexpression of SIRT6,there was no significant change in the migration of A549 cells.However,the overexpression of SIRT6 in H1299 cells significantly inhibited cell migration.After SIRT6 was knocked out,the morphology of A549 cells changed significantly,and the shape was changed from polygonal or oval cells to elongated ones.Fibroid cells are the main features of epithelial-mesenchymal transition(EMT),and cell migration is accelerated.Invasion experiments showed that the number of invasion of A549 cells was significantly reduced after SIRT6 was overexpressed While knockout of SIRT6 was opposite.After knock-down of SIRT6,the expression of E-cadherin mRNA was down-regulated in A549 cells,and the expression of N-cadherin and vimentin mRNA was significantly up-regulated.For mechanism studies,SIRT6 inhibited the phosphorylation level of ERK1/2 signaling pathway activator EGF by Western blotting,but there was no significant change in the phosphorylation level of smad2/3 signaling pathway.The above results suggest that SIRT6 may inhibit the invasion and metastasis of NSCLC cells through ERK1/2 signaling pathway.3.The inflammatory response was induced by the addition of cisplatin to SIRT6 overexpression and knockout cell lines.RT-PCR was performed to detect the expression of related inflammatory cytokines.IL-1A and IL-1B were detected in A549 cells after overexpression of SIRT6.IL-12 mRNA expression was down-regulated,whereas knockout of SIRT6 showed opposite results.After overexpression of SIRT6,cisplatin was added to A549 cells and the expression of p-NF-κB protein was down-regulated 30 min later.Based on this,we speculated that SIRT6 may inhibit inflammatory responses through NF-κB signaling pathway in lung cancer cells.4.NSCLC cells were irradiated with 6 MV-X rays generated by a Simens ARTISTE linear accelerator in vitro.The source skin distance was SSD 100 cm,and the injection field was 10×10 cm.The dose rate was 600 MU/min.Cell irradiation was performed according to dose gradients of 0 Gy,5 Gy,10 Gy,15 Gy,and 20 Gy,and cell viability was measured using CCK-8 reagent 48 hours after irradiation.The results showed that the survival rate of A549 cells after over-expression of SIRT6 with irradiation dose was not observed.Significant changes occurred,and the survival rate of A549 cells was significantly increased after knockout of SIRT6.Conclusions:SIRT6 protein expression in NSCLC tissue was lower than that in paracancerous tissue;SIRT6 may effectively inhibit the invasion and migration of NSCLC cells through ERK1/2 signaling pathway;SIRT6 may inhibit the inflammatory response of A549 cells through NF-κB signaling pathway;SIRT6 may reduce NSCLC Cell survival rate after radiotherapy can help solve the radiotherapy resistance problem of lung cancer.