Mechanistic Study Of MiR-1293 Targeting ACSL4 To Promote Proliferation,Migration And Cisplatin Resistance Of A549 Cells

Objective:Cancer is a major public health problem in the world.According to the latest statistics of the global cancer burden,lung cancer ranks the second in incidence and the first in mortality,accounting for about one-fifth of cancer deaths.Epigenetic studies have confirmed that miRNA plays an important role in the occurrence,development and prognosis of tumors.This study aims to investigate the expression of miR-1293 in lung cancer and its effect on proliferation,migration and cisplatin resistance of lung cancer cells,laying down the theoretical basis for the diagnosis and treatment of lung cancer patients with miRNA as its target.Methods:(1)Exploration of the expression and function of miR-1293 in lung cancer:GEO data used to analyze the expression of miR-1293 in lung cancer.Realtime Quantitative PCR(qPCR)was used to verify the difference in the expression of miR1293 in human bronchial epithelial cells(BEAS-2B)and human non-small cell lung cancer cells(A549 and H1299).The effects of knocking down or overexpressing miR-1293 on cell proliferation and migration abilities were studied by EdU cell proliferation assay,wound healing assay and Transwell migration assay.Nude mice were subcutaneously injected with A549 cells to construct a subcutaneous xenograft model,and the miR-1293 inhibitor(antagomiR-1293)was injected into the tumor to explore the effect of miR-1293 on tumor size.Hematoxylin-eosin staining(HE)was used to detect the tumorigenesis of nude mice in the control group(NC)and antagomiR-1293 group(antagomiR).The expressions of migration-related proteins in tumors of each group were further analyzed by immunohistochemistry(IHC)and Western Blot.(2)Predict the target genes of miR-1293 and explore its effect on function of lung cancer cells:Targetscan,miRWalk,PITA and DIANA databases were used to predict the key target genes of miR-1293.The mRNA and protein expressions of target gene ACSL4 was analyzed by TCGA,GEO and CPTAC databases,and its expression was verified by qPCR and Western Blot in BEAS-2B,A549 and H1299 cells.Kaplan-Meier Plotter and TIMER2.0 online database were used to analyze the relationship between ACSL4 and the prognosis of lung cancer patients as well as immune cell infiltration,respectively.The binding site of miR-1293 and ACSL4 was predicted by Targetscan database.Dual luciferase reporter gene experiment was used to verify the regulatory relationship between miR-1293 and ACSL4.Western Blot and qPCR verified the effect of knockdown or overexpression of miR-1293 on the expression of ACSL4.Rescue experiments was verified the functional regulation between them.(3)Investigate the effects of miR-1293 and its target gene ACSL4 on cisplatinresistant lung cancer cell:The LUAD dataset of the TCGA database was used to analyze the correlation between miR-1293 and and cisplatin-resistant genes.The change of the cisplatin resistance gene with the knocked-down miR-1293 is verified through qPCR experimens.After treated with different concentrations of cisplatin(2.5,5,10,20,40μmol/L),the cell viability was measured by CCK8 assay and the half maximal inhibitory concentration(IC50)was calculated.A549/DDP cells were co-transfected with miR-1293 inhibitor and siACSL4 to investigate the effects of them on the proliferation,migration and cisplatin resistanceResults:(1)miR-1293 is highly expressed in lung cancer tissues and cells.In vitro and in vivo experiments have shown that knockdown of miR-1293 inhibit the proliferation and migration of lung cancer cells.GEO dataset GSE48414 showed that miR-1293 was highly expressed in lung cancer tissues(P<0.05).Further cell experiments showed that miR-1293 was significantly overexpressed in A549 and H1299 cells compared with BEAS-2B(P<0.05).Knockdown of miR-1293 significantly inhibited the proliferation and migration of A549 cells(P<0.05),on the contrary,overexpression of miR-1293 significantly promoted the proliferation and migration of BEAS-2B cells(P<0.05).Injection of antagomiR-1293 could inhibit the growth of subcutaneous tumor in nude mice and reduce the weight of tumor(P<0.05).The results of IHC and Western Blot showed that compared with NC group,the expression of E-cadherin(CDH1),a characteristic molecular marker of epithelial cells,was significantly increased in the antagomiR group(P<0.05),while the expression of N-cadherin(CDH2)and Vimentin(VIM),characteristic molecular markers of mesenchymal cells,was significantly decreased(P<0.05).(2)The target gene ACSL4 was screened and confirmed to be regulated by miR1293 to affect the proliferation and migration of lung cancer cells.Database analysis showed significantly low expression of A CSL4 in lung cancer(P<0.05)and was associated with poor prognosis and immune cell infiltration in patient.Compared with BEAS-2B cells,the mRNA and protein expressions of ACSL4 in A549 and H1299 cells were significantly down-regulated(P<0.05).The double luciferase reporter gene experiment confirmed the targeting relationship between miR-1293 and ACSL4,and overexpression of miR-1293 could significantly reduce the mRNA and protein expression levels of ACSL4(P<0.05).Knocking down miR-1293 could significantly increase the mRNA and protein expression levels of ACSL4(P<0.05).In addition,knockdown of ACSL4 can rescue A549 cells from the decrease of proliferation and migration abilities caused by knocking down miR-1293(P<0.05),while up-regulate ACSL4 can rescue BEAS-2B cells from the increase of proliferation and migration abilities caused by overexpression of miR-1293(P<0.05).(3)miR-1293 affects the proliferation,migration and cisplatin resistance of A549/DDP cells by targeting ACSL4.Analysis of the LUAD dataset of the TCGA database revealed that miR-1293 was significantly associated with the expression of cisplatin resistance genes,and knocking down miR-1293 could significantly reduce the expression of cisplatin resistance genes RAD23B,RAD51 and STAT1(P<0.05).Compared with A549 cells,miR-1293 was significantly overexpressed in A549/DDP cells(P<0.05),and the mRNA and protein levels of the target gene ACSL4 were significantly down-regulated(P<0.05).The IC50 was 11.40 μmol/L after transfection of inhibitor NC in A549/DDP cells and 6.702 μmol/L after transfection of miR-1293 inhibitor,and IC50 was restored to 11.03μmol/L after co-transfection of miR-1293 inhibitor and siACSL4 in A549/DDP cells.The rescue experiment confirmed that the down-regulate ACSL4 could save A549/DDP cells from the decrease of proliferation and migration abilities caused by knocking down miR1293.Conclusion:(1)miR-1293 is significantly highly expressed in lung cancer tissues and cells,and knock down miR-1293 can reduce proliferation and migration abilities of lung cancer A549 cells as well as inhibit the tumor growth in nude mice.(2)The target gene ACSL4,which is regulated by miR-1293 and affects proliferation and migration of A549 cells.(3)miR-1293 is significantly associated with multiple cisplatin resistance genes.miR1293 can affect proliferation,migration and cisplatin resistance of A549/DDP cells by targeting ACSL4.