The Expression And Mechanism Study Of MiR-4458 And Its Target Gene HMGA1 In Non-Small Cell Lung Cancer

Objective:Lung cancer is one of malignant neoplasms with high mortality around the world.Non-small cell lung cancer?NSCLC?accounts for approximately 80%of lung cancer.Although considerable advancements have been made in diagnosis and targeted therapy for NSCLC,the prognosis is still poor.Therefore,it is crucial to have a better understanding of the exact mechanism for the development and progression of NSCLC,which could provide more individualized and effective therapeutic strategies for NSCLC patients.The high mobility group protein A1?HMGA1?serves as a regulator of the chromatin structure via direct binding to A/T-rich DNA sequences.More studies find that HMGA1 plays a carcinogenic role in various cancer types,such as breast cancer,colon cancer and thyroid cancer.Increasing evidence shows that HMGA1 is involved in the development of cell proliferation,cell cycle and metastasis.However,the molecular mechanism of HMGA1 in NSCLC has not been fully understood.MicroRNAs?miRNAs?are a class of small?22-24 nucleotides?non-coding RNAs that plays a pivotal role in the diagnosis and prognosis of malignant neoplasm.Previous studies have already found that miRNAs could inhibit the transcription of target mRNAs via binding to complementary 3'-untranslated region?3'-UTR?.Previous studies have shown that multiple miRNAs can participate in biological processes such as proliferation and metastasis of NSCLC,and play an important role in regulating targeted genes.miR-4458 plays a biological role in hepatocellular carcinoma and colon cancer cells.miR-4458 is in the low expression level in NSCLC,but its specific mechanism in NSCLC is not clear.The aim of this study is to investigate the expression and biological behavior of miR-4458 and its target gene HMGA1 in NSCLC and explore the exact mechanism to provide evidence for molecular targeted therapy.Methods:1.RT-PCR assay was used to analyze the expression of HMGA1 in NSCLC cells.The expression of HMGA1 was down-regulated by siRNA:CCK-8 assay and colony assay were used to analyze the proliferation capacity in NSCLC cells.Transwell assay and wound healing assay were used to analyze the migration ability in NSCLC cells.Western blot assay was used to analyze the expression of the EMT and glycolysis related genes in NSCLC cells.Lactic acid test was used to analyze the products of glycolysis in NSCLC cells.2.Bioinformatics softwares were used to predict miRNAs binding to HMGA1 and luciferase reporter assay was used to verify the relationship between miR-4458 and HMGA1.The up-regulation and down-regulation of miR-4458 were investigated with the transfection of miR-4458 mimic or inhibitor in NSCLC cells:RT-PCR assay and western blot assay were used to analyze the expression of miR-4458 in NSCLC cells.CCK-8 assay and colony assay were used to analyze the proliferation capacity in NSCLC cells.Transwell assay and wound healing assay were used to analyze the migration ability in NSCLC cells.Western blot assay was used to analyze the expression of the EMT and glycolysis related genes in NSCLC cells.Lactic acid test was used to analyze the products of glycolysis in NSCLC cells.3.Co-transfection cell lines were investigated with the transfection of miR-4458 inhibitor and si-HMGA1 in NSCLC cells simultaneously:Transwell assay was used to analyze the migration ability in NSCLC cells.Western blot assay was used to analyze the expression of the EMT related genes in NSCLC cells.4.The xenograft model in nude mice was constructed successfully and dynamic monitoring of tumor weight and volume were conducted.¹⁸F-FDG Micro-PET was used for the molecular imaging evaluation.The inhibitory effect of miR-4458 on the growth of NSCLC was verified in vivo.Results:1.The expression and biological behavior of HMGA1 in NSCLC?1?RT-PCR results showed that HMGA1 was highly expressed in NSCLC cell lines A549,H1299,HCC827 and PC9.?2?The down-regulation of HMGA1 inhibited the proliferation in NSCLC cells.?3?The down-regulation of HMGA1 inhibited the metastasis in NSCLC cells.?4?The down-regulation of HMGA1 inhibited the EMT process in NSCLC cells.?5?The down-regulation of HMGA1 affected the formation of lactic acid and glucose metabolism in NSCLC cells.2.The study of miR-4458 was conducted in NSCLC about identification and biological behavior?1?miRwalk,TargetScan,MicroT-CDS and miRTarBase databases were used to predict miRNAs and then miR-4458 was screened out.?2?Dual luciferase assay demonstrated that miR-4458 was binding to 3'UTR of HMGA1.?3?The up-regulation of miR-4458significantly inhibited the proliferation,migration and EMT process in NSCLC cells,and affected the formation of lactic acid and glucose metabolism.?4?The down-regulation of miR-4458 significantly enhanced the proliferation,migration and EMT process in NSCLC cells,and regulated the formation of lactic acid and glucose metabolism.?5?miR-4458 affected migration and EMT process by targeting HMGA1:Knockdown of HMGA1 abolished the role of downregulated miR-4458 in the migration and EMT process.3.The inhibitory effect of miR-4458 on the growth of tumors in NSCLC nude mice?1?Compared with the NC group,the volume of miR-4458 agomir group were reduced;?2?Compared with the NC group,the weight of miR-4458 agomir group were reduced;?3?Compared with the NC group,the results suggest that SUVmax in the miR-4458 agomir group is significantly reduced using ¹⁸F-FDG Micro-PET.Conclusion:The expression of HMGA1 is up-regulated in NSCLC cells,and its expression level is correlated with the proliferation ability,migration ability,EMT process and glucose metabolism process in NSCLC cells.miR-4458 is binding to the3'UTR of HMGA1,and can affect the proliferation ability,migration ability and EMT process,glucose metabolism process in NSCLC cells.miR-4458 regulates the migration and EMT process in NSCLC cells by targeting HMGA1.miR-4458 inhibits the growth of NSCLC cells in nude mice.