Regulation Of LncRNA BACE1-AS On Hepatocellular Carcinoma Progression And Related Molecular Mechanisms

Objective: To evaluate the expression characteristics a clinical significance of BACE1-AS in hepatocellular carcinoma(HCC)and its relationship with tumor immunity.To further explore its role in hepatocellular carcinoma and related molecular mechanism.Methods: In this study,the expression level of lncRNA BACE1-AS in HCC and pan carcinoma,the significance of prognosis,and the influence of clinicopathological parameters were analyzed from the perspective of bioinformatics analysis and clinical tissue samples.The associations between lnc RNA BACE1-AS and tumor immunity were analyzed.The effects of lnc RNA BACE1-AS on the proliferation,apoptosis,invasion,and metastasis of HCC cells and autophagy were analyzed by cell proliferation assay,flow cycle,and apoptosis detection,transwell invasion and migration assay,wound-healing assay,western blot assay,double luciferase reporter assay,tumor formation in nude mice and imaging in vivo.The potential molecular mechanism of BACE1-AS promoting HCC progression was discussed from the perspective of ce RNA.Results: The expression of lnc RNA BACE1-AS was up-regulated in hepatocellular carcinoma,which was negatively correlated with the prognosis of patients.In HCC patients,the expression level of BACE1-AS was higher in C1 and C2 immune subtypes,positively correlated with M0-type macrophages,B cells and NK/T cells,and negatively correlated with neutrophils,M1-type and M2-type macrophages.There was the strongest correlation between the expression level of BACE1-AS and the genes of CD276,TNFSF15 and TNFSF4.In HCC cells,overexpression of BACE1-AS promoted cell proliferation,invasion and migration,autophagy,and apoptosis.BACE1-AS overexpression promoted the expression level of autophagy related proteins,thus activating autophagy to promote HCC malignant biological behavior,which could be reversed by the autophagy inhibitor.Silencing BACE1-AS inhibited the expression level of autophagy related proteins,thereby inhibiting autophagy to curb the HCC malignant biological behavior,which could be reversed by the autophagy enhancer.Up-regulated BACE1-AS promoted the expression of p-ERK and p-P38 proteins,while silencing BACE1-AS inhibited the expression of p-ERK and p-P38 proteins.BACE1-AS could competitively bind with IKBKE to let-7c-5p,and IKBKE partially reversed the role of BACE1-AS in HCC.Conclusions: The expression of lnc RNA BACE1-AS in HCC was up-regulated,which was negatively correlated with the prognosis of patients and could be used as an independent prognostic factor of HCC.BACE1-AS activated MAPK/ERK signaling pathway in HCC to promote autophagy of HCC cells.BACE1-AS could induce HCC proliferation,invasion and inhibit cell apoptosis in vivo and in vitro through regulation of the let-7c-5p/IKBKE axis.Part Ⅰ: The Expression and Clinical Significance of Lnc RNA BACE1-AS in Hepatocellular CarcinomaObjective: To investigate the expression level of lnc RNA BACE1-AS in HCC and to evaluate the relationship between the expression level of BACE1-AS and clinicopathologic parameters of HCC patients,its effect on the prognosis of HCC patients,and its relationship with immune cell infiltration.The correlation of immune subclassification.Methods:(1)The expression level of BACE1-AS in tumor and normal tissues was analyzed by the bioinformatics method.(2)The prognostic significance of BACE1-AS expression level in HCC patients was analyzed.(3)The effects of BACE1-AS expression level on immune typing,immune cell infiltration,and immune-related functions of liver cancer cells were analyzed.(4)The relative expression levels of BACE1-AS were compared between five human hepatocellular carcinoma cell lines and normal human liver cells.(5)40 pairs of clinical liver cancer tissues and adjacent non-tumor normal tissues were collected to measure the expression level of BACE1-AS in HCC and para-cancer tissues.(6)The correlation between clinicopathologic parameters and BACE1-AS expression level in cases of HCC was analyzed.(7)The molecular pathway that BACE1-AS involved in the regulation of hepatocellular carcinoma was predicted.(8)The upstream regulatory transcription factors of BACE1-AS were predicted and verified by the JAPSPAR database.Results:(1)The expression of BACE1-AS in HCC tissues was significantly higher than that in normal tissues by bioinformatics analysis,cell level,and clinical tissue detection.(2)BACE1-AS was mainly expressed in the cytoplasm,but rarely distributed in the nucleus,ribosome,cytoplasm,and exosome.(3)Univariate and multivariate Cox analysis showed that BACE1-AS expression was a risk factor for HCC patients independent of other clinical factors(including age,sex,grade,stage,T,M,N).(4)The expression level of BACE1-AS was negatively correlated with the prognosis of HCC patients.(5)BACE1-AS was significantly related to the expression of immune checkpoint genes and the content of infiltrating immune cells.(6)The results of KEGG pathway enrichment analysis showed that the expression level of BACE1-AS affected the activation of DNA-binding transcription activator,amino acid metabolism,neuroactive ligand receptor interaction and participates in autophagy regulation.(7)Analysis and verification confirmed that the up-regulation of BACE1-AS in HCC may be regulated by the transcription factor MAZ.Conclusions: Lnc RNA BACE1-AS was highly expressed in patients with HCC,which was negatively correlated with the prognosis of HCC patients.BACE1-AS could be used as an independent prognostic factor in patients with HCC.MAZ activated the transcription of BACE1-AS and promoted its expression level.Part Ⅱ: Effects of Lnc RNA BACE1-AS on Growth,Apoptosis,Invasion and Metastasis of HCCObjective: To investigate the effects of high-and low-BACE1-AS expression on proliferation,apoptosis,invasion,and migration of HCC in vitro and in vivo.Methods:(1)Overexpressed plasmids and si RNA of BACE1-AS had been transfected into MHCC-97 H and Hep G2 cell lines,respectively.Quantitative real-time PCR (q RT-PCR)was used to detect BACE1-AS expression in transfected HCC cells.(2)The CCK-8 cytotoxicity experiment and EDU proliferation experiment were performed to test cell proliferation ability on groups of high-and low-expression BACE1-AS.(3)Transwell cell migration and invasion experiments were conducted to compare the migration and invasion abilities of hepatocellular carcinoma cells under different BACE1-AS expression levels.(4)Cell apoptosis and cycle distribution were detected by flow cytometry.(5)Western blot assay was used to investigate the effects of BACE1-AS knockdown or overexpression on apoptosis and autophagy-related protein expression levels in HCC cells.(6)Western blot assay was used to investigate the influence of BACE1-AS overexpression or knockdown on the expression levels of key protein molecules of the MAPK pathway.(7)The model of subcutaneous transplanted tumor in nude mice was established to study the effect of BACE1-AS expression on tumor proliferation in vivo observed by imaging techniques of small animals.(8)The effect of BACE1-AS knockdown on Ki-67,LC3 B,and IKBKE expression in transplanted tumor tissues of nude mice was evaluated by hematoxylin-eosin staining and immunohistochemical staining.Results:(1)HepG2 and MHCC-97 H were successfully transfected with BACE1-AS overexpression plasmid or si-BACE1-AS.(2)In vitro studies showed that overexpression of BACE1-AS enhanced the proliferation,invasion,and metastasis of hepatocellular carcinoma cells,and significantly inhibited cell apoptosis.(3)Down-regulated BACE1-AS expression significantly inhibited the proliferation and in vitro metastasis of hepatocellular carcinoma cells,and promoted cell apoptosis.(4)When BACE1-AS expression was up-regulated,the protein expression levels of BCL2,LC3 B,Beclin-1,ATG5 and the phosphorylation levels of P38 and ERK1/2 proteins were significantly increased,while the expressions of PARP1,BAX and P62 proteins were down-regulated.(5)When BACE1-AS expression was interfered in vitro,the expression levels of BCL2,LC3 B,Beclin-1,ATG5 protein and protein phosphorylation levels of P38 and ERK1/2were significantly decreased,and the expressions of PARP1,BAX and P62 protein were up-regulated.(6)In vivo,when the BACE1-AS expression level was decreased,the growth rate of subcutaneous transplanted tumor in nude mice was significantly slowed down.(7)The expression levels of Ki-67,LC3 B and IKBKE protein in the transplanted tumor tissue of BACE1-AS silencing group were decreased.Conclusions: The up-regulated BACE1-AS in HCC could enhance the ability of cell proliferation,migration and invasion in vitro,inhibit apoptosis and induce tumor autophagy.Silencing BACE1-AS inhibits the growth of transplanted tumor in nude mice.Part Ⅲ: The Relationship between BACE1-AS and Autophagy and the Regulatory Mechanism in HCCObjective: To evaluate the relationship between BACE1-AS and autophagy,and to further investigate the molecular mechanism of BACE1-AS as a cancer-promoting factor in HCC.Methods:(1)Autophagy enhancer Rapamycin(RAPA)and autophagy inhibitor3-Methyladenine(3-MA)were introduced to treat human hepatocellular carcinoma cell lines with BACE1-AS knockdown and overexpression,respectively.(2)CCK-8 assay,cell scratch assay,transwell assay,and flow cytometry were used to analyze the changes in cell proliferation,apoptosis,migration,and invasion ability after the introduction of autophagy enhancer or inhibitor of BACE1-AS at different expression levels.(3)RFP-GFP-LC3 lentivirus was used to trace the changes in autophagy flow.(4)Western blot assay was used to analyze the effects of apoptosis-related and autophagy-related proteins.(5)The lentivirus infection technique was used to construct BACE1-AS silencing and overexpressing stable cell lines.The effects of different BACE1-AS expression levels on subcutaneous tumor formation in nude mice were evaluated by imaging techniques of small animals.(6)The expressions of Ki-67,LC3 B,and IKBKE protein were analyzed by immunohistochemical staining.(7)The mi RNA with binding sites of BACE1-AS and downstream target genes directly bound to mi RNA were identified by DIANA and RNAhybrid databases.(8)q RT-PCR and double luciferase reporter gene experiments were used to verify the interaction between molecules.(9)The expression and prognosis of downstream target genes in HCC were detected by bioinformatics analysis and western blot.(10)MHCC-97 H cells were co-transfected with BACE1-AS si RNA and overexpressed IKBKE plasmid(si-BAC + oe-IKBKE).Hep G2 cells were co-transfected with BACE1-AS overexpressed plasmid and IKBKE si RNA(p LVX-BAC + si-IKBKE)to compare changes in cell proliferation,apoptosis,invasion,and metastasis in each group.Results:(1)The experiment showed that BACE1-AS overexpression significantly promoted HCC autophagy,and 3-MA partially reversed the proliferation,migration,and invasion of HCC promoted by BACE1-AS overexpression.(2)BACE1-AS silencing significantly inhibited liver cancer autophagy,and RAPA could effectively reverse the weakening of the proliferation,migration,and invasion of HCC cells caused by BACE1-AS knockdown.(3)In vivo experiments of nude mice showed that when BACE1-AS expression was increased,the rate of tumor growth was significantly enhanced.Moreover,intraperitoneal injection of 3-MA could reverse the growth acceleration of subcutaneous tumors caused by BACE1-AS overexpression.(4)When BACE1-AS was silenced,the expression of p-ERK1/2 and p-P38 was down-regulated,and when combined with RAPA(si-BAC + RAPA),the expression of p-ERK1/2 and p-P38 increased.(5)TCGA database and q RT-PCR results showed that BACE1-AS was negatively correlated with the m RNA expression level of let-7c-5p;BACE1-AS was positively correlated with IKBKE m RNA expression level;IKBKE was negatively correlated with the m RNA expression level of let-7c-5p.(6)The results showed increased let-7c-5p expression level and decreased IKBKE expression level in the group of knockdown of BACE1-AS expression level.However,when BACE1-AS expression was up-regulated,the expression level of let-7c-5p was significantly reduced,while the expression level of IKBKE was significantly increased.(7)The results of the double luciferase reporter gene experiment showed that there were binding sites between let-7c-5p and BACE1-AS and IKBKE.(8)BACE1-AS could competitively bind let-7c-5p with IKBKE,thereby relieving the interference of let-7c-5p on IKBKE expression and promoting IKBKE expression.(9)IKBKE could partially reverse the malignant biological behavior of human HCC cells caused by the change of BACE1-AS expression level.Conclusions: BACE1-AS promoted the progression of HCC and regulated autophagy through MAPK/ERK signaling pathway.BACE1-AS might up-regulate the expression of autophagy regulatory gene IKBKE through the adsorption of let-7c-5p,and promote the proliferation,migration,and invasion of HCC cells.