Effects Of Leptin Regulating GRP78 Expression On Biological Characteristics Of Lung Cancer Cells

Background: Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world,which poses a huge threat to human survival.Obesity has been shown to be associated with a variety of cancers,but the relationship between obesity and lung cancer remains unclear due to confounding factors such as smoking,pathological wasting etc.Leptin,as the most studied adipokine,is synthesized and secreted by white adipose tissue,which can participate in key steps such as tumor cell proliferation,migration and invasion,and regulate the tumor microenvironment.GRP78 is a key chaperone protein in endoplasmic reticulum stress,which can promote tumor development and drug resistance.Studies have found that leptin and GRP78 are highly expressed in breast cancer,colon cancer,lung cancer and other tissues.As a chronic stress,obesity can lead to endoplasmic reticulum stress,but the relationship among obesity,endoplasmic reticulum stress and lung cancer is not clear.Objective:(1)TCGA and GTEx databases were used to explore the relationship between leptin and GRP78 transcriptome data and lung cancer,and to explore the correlation between leptin,GRP78 and the prognosis of lung cancer.(2)To explore the relationship between leptin and GRP78 and its effect on lung cancer cell migration ability and possible mechanisms.(3)To explore the effect of GRP78 on the proliferation and apoptosis of lung cancer cells and its possible mechanisms.Methods: The expressions of GRP78 in lung adenocarcinoma,lung squamous cell carcinoma,and healthy people were analyzed by the UALCAN website,and the difference of overall survival of patients with high expression and low expression of GRP78 and leptin in lung cancer were analyzed by the GEPIA website.The effects of leptin and GRP78 on the migration of lung cancer cells A549 and H460 were detected by wound healing test;the effect of leptin on the expression of GRP78 in A549 and H460 cells was detected by immunofluorescence;the effect of leptin on activation of PI3K/mTOR/STAT3 pathway and GRP78 in A549 cells was detected by Western blot;immunohistochemistry was used to detected the expression of GRP78 in lung tissues of lung cancer mice induced by urethane and normal mice.After targeting inhibition of GRP78 with HA15,Cell Counting Kit-8(CCK-8)was used to detect lung cancer cells A549,H460 and H1975 abilities;EdU assay was used to detect the proliferations of A549,H460 and H1975 cells;flow cytometry was used to detect the apoptosis rates of A549,H460 and H1975 cells;Western blot was used to detect apoptosis,endoplasmic reticulum stress and autophagy-related proteins in A549 cells;RT-PCR was used to detect endoplasmic reticulum stress and autophagy-related mRNAs in A549 cells;immunofluorescence was used to detect the expression of LC3 B expression in A549 cells;electron microscopy was used to observe the morphology,endoplasmic reticulum morphology and number of autophagosomes in A549 cells.Results:(1)GRP78 is not only highly expressed in lung adenocarcinoma and squamous cell carcinoma,but also significantly higher in lung tissue of urethane induced lung cancer mice than that of normal mice.The overall survival rate of lung cancer patients with high expressions of GRP78 and leptin is significantly lower than that of normal mice.(2)Leptin significantly promoted the migration of lung cancer cells.After 100ng/mL leptin treatment for 48 h,wound healing test showed that compared with the 0ng/mL control group,the wound healing rates of A549 and H460 cells increased significantly.HA15(GRP78 inhibitor)significantly reduced the promotion effect of leptin on wound healing rate of A549 cells.(3)Leptin up-regulated the protein expressions of mTOR,STAT3 and GRP78 in A549 cells.The protein expressions of mTOR,STAT3 and GRP78 in the combined treatment group of leptin and Ly294002(PI3K inhibitor)was significantly lower than that of leptin group.The protein expressions of STAT3 and GRP78 in the combined treatment group of leptin and rapamycin(mTOR inhibitor)was significantly lower than that in the leptin group.(4)HA15 significantly inhibited the viabilities of lung cancer cells A549,H460 and H1975 in a dose-and time-dependent manners.After 10?M HA15 treatment,the apoptosis rates of A549,H460 and H1975 cells increased significantly compared with the control group,while the proliferations decreased.Compared with the control group,A549 cells treated with HA15 showed apoptosis characteristics,such as cell collapse and the nuclear membrane shrinkage.The protein expressions of Bcl-2 and p53 decreased,while the protein expression of Bax increased in A549 cells after HA15 treatment.(5)Endoplasmic reticulum stress occurred in A549 cells after HA15 treatment.The mRNA expressions of ATF4,Cleaved-ATF6,XBP1 and IRE1 increased significantly compared with the control group.The mRNA and protein expressions of CHOP increased significantly,and the endoplasmic reticulum expanded.(6)Autophagy occurred in A549 cells after HA15 treatment.Autophagosomes appeared in the cells,the mRNA expressions of Atg4,Atg7,Atg12,LC3 and ULK1 increased significantly compared with the control group,the protein expressions of Beclin1 and LC3 B increased,while the protein expression of p62 decreased;the autophagosomes were observed in A549 cells.Conclusions:(1)Lung cancer patients with high expressions of leptin and GRP78 have a poor prognosis and overall survival rate declines.(2)Leptin up-regulates GRP78 expression by activating PI3K/mTOR/STAT3 pathway;GRP78 participates in leptin-induced lung cancer cell migration.(3)HA15 targeted inhibition of GRP78 can significantly reduce cell viability,promote lung cancer cells apoptosis and inhibit lung cancer cells proliferation,and trigger autophagy and endoplasmic reticulum stress in A549 cells.