XPA Induces Autophagy And Apoptosis Through PI3K/AKT/mTOR Signaling Pathway To Inhibit Hepatocellular Carcinoma

Object:To explore the potential biological function of XPA in hepatocellular carcinoma(HCC)and the mechanism of XPA regulating HCC autophagy and apoptosis through PI3K/AKT/mTOR signaling pathway,to study whether XPA would become a new therapeutic target,and to provide clinical basis for the development of new therapeutic strategies for HCC.Methods:1.Bioinformatics analysis and XPA related detection analysis:five-year survival curve of HCC prognosis from the database(The Cancer Genome Atlas,TCGA),analyzed by R language analysis software.WB,q RT-PCR and IHC were used to detect the expression of XPA in human HCC tissues with different pathological stages and adjacent non-tumor liver tissues(ANLTs).WB and q RT-PCR techniques were used to detect the protein and m RNA expression of XPA in human liver tumor cell lines Hep3 B,Huh7,SMMC7721,SK-Hep-1,Bel7402,Hep G2,LM9 and human non-tumor liver cell line HL7702.2.Study on the function of cell biology: XPA was overexpressed in Hep3 B and Huh7 HCC cells by LV-XPA infection,and the LV-XPA-Hep3 B and LV-XPA-Huh7 stably expressing XPA were constructed by drug screening.WB and q RT-PCR techniques were detected the expression of XPA protein and m RNA in LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells.Colony formation assay and CCK-8 assay were used to detect the proliferation of LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells.The expression of Epithelial-to-mesenchymal transition(EMT)markers E-cadherin,N-cadherin and vimentin proteins in LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells was detected by WB.The invasion and migration of LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells were detected by Transwell and cell scratch test.3.WB was used to detect the protein expression of autophagy-related molecules LC3 B ?/? and Beclin1 and the expression of apoptosis-related proteins Cleaved-caspase-3,Bax and anti-apoptotic protein Bcl2 in LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells.TUNEL staining detects the apoptotic bodies of LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells.LC3-GFP-m RFP fluorescent double-labeled lentiviral system and laser confocal microscope were used to detect autophagic flux.LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells were treated with RAPA and 3-MA,and the expression levels of autophagy-related proteins LC3 B ?/? and Beclin1 were detected by WB.4.Study on cytological mechanism: the expression levels of autophagy molecules LC3B?/?,Beclin1 and PI3K/AKT/mTOR signal pathway molecules AKT,p-AKT,PI3 K,p-PI3 K,mTOR and p-mTOR in LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells treated with IGF-1 were detected by Western-blot.5.In vivo experiment in nude mice: the experimental model of subcutaneous tumorigenesis of HCC in nude mice was established by injectioning LV-XPA-Hep3 B HCC cells and LV-NC-Hep3 B HCC cells.The volume and weight of the tumor were measured and the growth curve was drawn.The expressions of XPA,LC3B?/?,Beclin1,PI3 K,p-PI3 K,AKT,p-AKT,mTOR and p-mTOR were detected by Wester-blot,XPA and LC3 B were detected by IHC,and apoptosis was detected by TUNEL staining.Results:1.After analyzing the survival data and gene expression information of 349 HCC patients collected in the TCGA database,it is found that the5-year survival rate of HCC patients with high XPA expression is significantly higher than those with low expression.At the same time,we found that XPA expression level is related to age.2.The expression level of XPA in human HCC tissues is lower than that of ANLTs.The higher the expression level of XPA,the higher the overall survival of HCC patients and the better prognosis of the disease.Compared with the human normal liver cell line HL7702,the human liver tumor cell line SMMC7721,Hep G2,SK-Hep-1,LM9,Bel7402,Hep3 B and Huh7 have reduced expression of XPA m RNA and protein levels,of which Hep3 B and Huh7 HCC cells have the lowest expression.3.Overexpress XPA in Hep3 B and Huh7 HCC cells by lentivirus infection,thereby constructing in vitro experimental models of LV-XPA-Hep3 B and LV-XPA-Huh7 HCC cells stably expressing XPA.Compared with the LV-NC group,XPA m RNA and protein expression levels in HCC cells in the LV-XPA group were significantly up-regulated.4.Compared with the LV-NC group,the cell proliferation of HCC in the LV-XPA group was inhibited.In the LV-XPA group of HCC cells,the expression of E-cadherin protein increased,while the expression of N-cadherin and vimentin proteins decreased.The invasion and migration of HCC in the LV-XPA group were significantly inhibited.5.Compared with LV-NC group,the protein expression levels of LC3 B ?/? and Beclin1 in LV-XPA group increased significantly,while the protein expression level of p62 decreased,the protein expression levels of Cleaved-caspase-3 and Bax increased and the expression level of BCL2 protein decreased.TUNEL staining showed that the number of apoptotic bodies of HCC cells in LV-XPA group was much higher than that in LV-NC group,indicating that the activation of autophagy led to the increase of apoptosis.The results of LC3-GFP-m RFP autophagy double labeling lentivirus system showed that the number of fluorescent spots in HCC cells in LV-XPA group was significantly higher than that in LV-NC group.The number of red and yellow merging-blot were increased significantly,suggesting that autophagy flow was enhanced,suggesting that overexpression of XPA would effectively enhance autophagy flow.6.LV-XPA-Hep3 B,LV-XPA-Huh7 HCC cells were treated with RAPA and 3-MA,and the results showed that when RAPA and XPA act on HCC cells alone,they can effectively induce the lethal autophagy of HCC cells,LC3?/? and Beclin1 Increased protein expression level.When RAPA and XPA act together on HCC cells,the autophagy induction function is stronger.When 3-MA acts on HCC alone,the expression of LC3?/? and Beclin1 protein is partially inhibited.However,when 3-MA and XPA act together on HCC cells,compared with the LV-XPA group,the expression levels of LC3?/? and Beclin1 protein decreased,indicating that 3-MA inhibited XPA-induced autophagy activation.7.HCC cells were treated with different concentrations of IGF-1.The expression level of p-AKT in HCC cells was up-regulated in a dose-dependent manner of IGF-1,and had the strongest effect at the concentration of 200ng/m L and 300 ng/m L,there was no statistical difference between the two.The expression levels of LC3 B ?/? and Beclin1 proteins increased in HCC cells in LV-XPA group,while IGF-1 inhibited the expression of these proteins.When only XPA was overexpressed in HCC cells,the expression levels of p-PI3 K,p-AKT and p-mTOR protein decreased.Compared with IGF-1 alone,the protein expression levels of p-PI3 K,p-Akt and p-mTOR were decreased when IGF-1 and XPA acted together on HCC cells.The results showed that the activation of HCC autophagy by up regulating XPA may be dependent on the regulation of PI3K/AKT/mTOR signaling pathway.8.By constructing a subcutaneous tumor model of HCC nude mice,the study found that compared with nude mice in the LV-NC group,the average tumor volume and tumor weight of the nude mice in the LV-XPA group were smaller,and the survival time of the nude mice was prolonged.The expression levels of XPA,LC3?/? and Beclin1 proteins in tumor tissues of nude mice in the LV-XPA group increased,and the expression levels of p-PI3 K,p-AKT and p-mTOR proteins decreased.IHC staining showed that XPA expression was significantly increased in tumor tissues of nude mice in the LV-XPA group,and LC3 B expression also increased.TUNEL staining found that compared with the LV-NC group,the number of apoptotic cells in the tumor tissue of nude mice in the LV-XPA group increased.All these data indicate that XPA can induce autophagy and promote apoptosis in HCC nude mice subcutaneous tumor tissues,and this effect may be achieved through the PI3K/AKT/mTOR signaling pathway.Conclusions:1.The high expression of XPA may be a potential marker of good prognosis for clinical HCC patients.2.The expression level of XPA was significantly lower in human HCC tissues and human HCC cell lines compared to human ANLTs and human normal liver cell lines.3.Successfully constructed a HCC cell model overexpressing XPA and up-regulated XPA expression in HCC cells,confirming that XPA inhibits EMT of HCC cells by increasing E-cadherin protein expression and reducing N-cadherin and vimentin protein expression.Up-regulating the expression of XPA in HCC cells significantly inhibited the proliferation,invasion and migration of HCC cells.4.Overexpression of XPA induces autophagy in HCC cells by increasing LC3 B ?/?,Beclin1 and reducing the expression level of p62 protein.The expression level of autophagy-related proteins and the number of autophagosomes are positively correlated with the expression level of XPA.At the same time,the expression level of apoptosis-related proteins Cleaved-caspase-3 and Bax increased,and the expression level of Bcl2 protein decreased.The expression level of apoptosis-related protein and the number of apoptotic bodies are positively correlated with the expression level of XPA,and the expression level of anti-apoptotic protein is negatively correlated with the expression level of XPA.XPA-induced autophagy in HCC cells can be enhanced by RAPA and reversed by 3-MA,indicating that XPA can induce the activation of lethal autophagy,which may promote cell apoptosis,indicating that lethal autophagy is in the process of XPA inhibiting HCC cells Has an important role.5.XPA can reverse the activation of IGF-1 on signaling pathways,and XPA can reverse the inhibitory effects of IGF-1 on autophagy,indicating that XPA may inhibit the activation of PI3K/AKT/mTOR signaling pathways to achieve the purpose of inhibiting HCC.This One effect may occur by inducing lethal autophagy in HCC.6.Successfully constructed the HCC nude mouse subcutaneous tumor model.Compared with the LV-NC group,the growth of the nude mouse subcutaneous tumor in the LV-XPA group was significantly inhibited,and the survival time of the nude mice was significantly prolonged.XPA may induce HCC lethal autophagy and promote HCC cell apoptosis by inhibiting PI3K/AKT/mTOR signaling pathway,and ultimately inhibit the occurrence and development of HCC.