The Experimental Study Of LncRNA HOXA11-AS Promoting Lung Adenocarcinoma Proliferation And Glycolysis Through Regulation Of MiR-148b-3p/PKM2

Objective:Lung adenocarcinoma accounts for the highest proportion of non-small cell lung cancers.Studies have shown that the lnc RNA HOXA11-AS is aberrantly expressed in many tumors and highly expressed in lung adenocarcinoma.The role of HOXA11-AS in glycolysis of lung adenocarcinoma is of great interest but has not been fully elucidated.This study aims to investigate the role and mechanism of HOXA11-AS in lung adenocarcinoma proliferation and glycolysis through in vivo and in vitro experiments,and to visualize the changes in ¹⁸F-FDG uptake in lung adenocarcinoma transplantation tumors of nude mice after silencing of HOXA11-AS by Micro-PET molecular imaging,thus reflecting the changes of glycolysis in lung adenocarcinoma transplantation tumors.This study will provide an imaging basis for future evaluation of the efficacy of targeting HOXA11-AS.Methods:In this study,the expression of HOXA11-AS and pyruvate kinase M2（PKM2）in lung adenocarcinoma in the TCGA database was analyzed by bioinformatics,and prognostic analysis was performed.The bioinformatics analysis predicted that HOXA11-AS might promote proliferation and glycolysis of lung adenocarcinoma through micro RNA-148b-3p（mi R-148b-3p）/PKM2,followed by in vivo and in vitro experiments to verify this prediction.Human bronchial epithelial-like cell lines（HBE）and lung adenocarcinoma cell lines（A549,HCC827,H1299）were used as the study subjects.The m RNA levels of HOXA11-AS,mi R-148b-3p,and PKM2 were detected by q RT-PCR,and the expression of PKM2 protein was detected by Western Blot.Two selected cell lines with the highest and lowest expression of HOXA11-AS for subsequent experiments（A549,H1299）.HOXA11-AS,mi R-148b-3p,and PKM2 were silenced or overexpressed,respectively,and transfection efficiency was detected.CCK-8,Ed U,and colony formation assay were used for proliferation assays.Changes in glycolysis were assessed by lactate production assay,ATP production assay,and ¹⁸F-FDG uptake assay.Using bioinformatic analysis and dual-luciferase reporter assays to identify the binding relationship among HOXA11-AS,mi R-148b-3p,and PKM2.And rescue experiments were used to verify the intermolecular regulatory relationships.In vivo experiments were performed by inoculating lung adenocarcinoma cells under the skin of nude mice to form lung adenocarcinoma transplantation tumors.After the animal model was constructed,si RNA was injected to downregulate HOXA11-AS,followed by Micro-PET scanning,immunohistochemical staining,q RT-PCR,and Western Blot to analyze the expression of HOXA11-AS,mi R-148b-3p and PKM2,as well as changes in proliferation and glycolysis.Results:1.Bioinformatics analysis of lung adenocarcinoma data from the TCGA database showed that HOXA11-AS and PKM2 were highly expressed in lung adenocarcinoma compared to normal tissues and correlated with poor prognosis.The differences were statistically significant（P<0.05）.2.Expression of HOXA11-AS was found to be increased in all three lung adenocarcinoma cell lines by q RT-PCR or Western Blot compared to HBE cells,with the highest expression in A549 cells and the lowest expression in H1299 cells;mi R-148b-3p expression was decreased in lung adenocarcinoma cell lines;PKM2 expression was increased in lung adenocarcinoma cell lines.The differences were statistically significant（P<0.05）.3.Silencing or overexpression of HOXA11-AS,mi R-148b-3p,and PKM2 in A549 and H1299,respectively.q RT-PCR or Western Blot results showed successful silencing or overexpression.The differences were statistically significant（P<0.05）.4.The proliferative capacity of transfected A549 and H1299 cells was examined by CCK-8,Ed U,and colony formation assays,which showed reduced proliferation of silenced HOXA11-AS compared to the negative control;on the contrary,the proliferation ability to overexpress HOXA11-AS was increased.Silencing of mi R-148b-3p increased proliferative ability;in contrast,overexpression of mi R-148b-3p decreased proliferative ability.The difference was statistically significant（P<0.05）.5.Changes in glycolysis were assessed by lactate production assay,ATP production assay,and ¹⁸F-FDG uptake assay.The results showed that silencing of HOXA11-AS or PKM2 decreased the glycolytic capacity compared to the negative control;in contrast,overexpression of HOXA11-AS or PKM2 glycolytic capacity was increased.Silencing mi R-148b-3p glycolytic capacity was increased;in contrast,overexpression of mi R-148b-3p glycolytic capacity was decreased.The difference was statistically significant（P<0.05）.6.The Akt pathway was examined in cells after overexpression or silencing of PKM2,the outcome of the Western Blot showed that in comparison to the negative control group,the level of Akt phosphorylation was decreased after silencing PKM2,and increased after overexpression of PKM2.Further studies revealed that the application of the inhibitor LY294002 could reverse the promotion of proliferation after overexpression of PKM2.7.Using bioinformatic analysis and dual-luciferase reporter assays to identify the binding relationship among HOXA11-AS,mi R-148b-3p,and PKM2.The results showed that there was targeted binding between mi R-148b-3p and HOXA11-AS and PKM,respectively.The differences were statistically significant（P<0.05）.8.Using rescue experiments to verify the intermolecular regulatory relationship,downregulation of HOXA11-AS along with downregulation of mi R-148b-3p or upregulation of PKM2 revealed that downregulation of mi R-148b-3p or upregulation of PKM2 could reverse the inhibitory effect of downregulation of HOXA11-AS on tumor proliferation and glycolysis compared with the control group.The difference was statistically significant（P<0.05）.9.Two lung adenocarcinoma cell lines,A549 and H1299,were inoculated subcutaneously in nude mice and injected with si-NC and si-HOXA11-AS（5 nmol per mouse,3 times per week for 2 weeks）.The results showed that the transplanted tumors injected with si-HOXA11-AS were smaller in size and increased in volume more slowly compared to the si-NC group.Micro-PET scans were performed,and the uptake of ¹⁸F-FDG was found to be lower in the si-HOXA11-AS group than in the negative control group.The difference was statistically significant（P<0.05）.10.Immunohistochemical results showed that staining for Ki67 and glycolysis-related proteins were significantly lower in both A549 and H1299 transplanted tumors with in vivo silencing of HOXA11-AS compared with controls,and the scores were statistically significant（P<0.05）.11.Finally,we detected the expression of HOXA11-AS,mi R-148b-3p,and PKM2 in tumor tissues by q RT-PCR and Western Blot.The results showed that HOXA11-AS and PKM2 expression was decreased and mi R-148b-3p expression was increased in transplanted tumors in the si-HOXA11-AS group compared with the si-NC group.The differences were statistically significant（P<0.05）.Conclusions:1.Lnc RNA HOXA11-AS is highly expressed in lung adenocarcinoma and promotes proliferation and glycolysis of lung adenocarcinoma.mi R-148b-3p is low expressed in lung adenocarcinoma and inhibits proliferation and glycolysis of lung adenocarcinoma.PKM2 promotes glycolysis of lung adenocarcinoma and promotes proliferation of lung adenocarcinoma through PI3K/Akt pathway.2.Lnc RNA HOXA11-AS regulates the expression of PKM2 by targeting mi R-148b-3p to regulate PKM2expression,which in turn promotes proliferation and glycolysis of the lung adenocarcinoma.3.In vivo and in real-time,the reduced uptake of ¹⁸F-FDG and reduced glycolysis levels in a nude mouse transplantation tumor model after HOXA11-AS silencing could be shown by ¹⁸F-FDG Micro-PET molecular imaging.The results of this study may provide an imaging basis for monitoring the therapeutic effects of targeting HOXA11-AS.