The Function Of MiRNA-99b-5p In Lung Cancer And The Target Screening And Mechanism Study Of Compound GA13315 In Inhibiting Lung Cancer Cells

Objectives:miRNA plays an important role in regulating the biological behavior of tumor cells.The purpose of this study is to study the effects of miRNA-99b-5p(miR-99b-5p)on the proliferation,invasion and metastasis of lung cancer and explore its target and mechanism.And to investigate its correlation with the survival of lung cancer patients.Method:1.TCGA database(starBase v3.0 project)was used in analysis of miR-99b-5p expression difference between lung cancer tissues and normal tissues and the correlation between its transcription level and survival of lung cancer patients.2.Verify functional mechanism of miR-99b-5p in lung cancer cell lines:after overexpression of miR-99b-5p by cell transfection,the cells were investigated by CCK-8 method,cell scratch assay and cell migration invasion assay(Transwell)technique,respectively.Measure the effects of miR-99b-5p on lung cancer cell proliferation rate,invasion ability and migration ability;3.Verification of miR-99b-5p target and functional mechanism using double luciferase reporter gene detection and Western blotting.Results:Compared with normal lung tissue,miR-99b-5p was under-expressed in tissue samples from patients with lung cancers;Patients with miR-99b-5p high-transcriptional lung cancers had higher survival rates than patients with low transcription of miR-99b-5p,p=0.02;The expression levels of miR-99b-5p in non-small cell lung cancer cells A549 and NCI-H292 were significantly lower than those in human bronchial epithelial cell line 16-HBE(p<0.001);the transcription level of miR-99b-5p in small cell lung cancer strain NCI-H69 was lower than that in human bronchial epithelial cell line.16-HBE(p<0.05);miR-99b-5p has no significant difference in transcription level of human embryonic kidney cell line HEK293T compared with human bronchial epithelial cell line 16-HBE(p=ns).Upregulation of miR-99b-5p transcription level can inhibit the proliferation,invasion and metastasis of lung cancer cells,p<0.01.FZD8 is the binding target of miR-99b-5p.Overexpression of FZD8 can partially reverse the inhibition of proliferation,invasion and metastasis of lung cancer cells by miR-99b-5p,p<0.01.Conclusion:miR-99b-5p inhibits the proliferation,invasion and metastasis of lung cancer cells by targeting FZD8,which is significantly associated with the survival of lung cancer patients.This newly identified miR-99b-5p/FZD8 axis provides new insights into the mechanism of lung cancer progression.Objectives:13-Chlorine-3,15-dioxy-gibberellic acid methylester(GA-13315),a gibberellin derivative,possesses strong anti-tumor activity in vitro and in vivo.The present study aimed to screen targets of the compound GA13315 in inhibiting lung cancer cells,explore related signal transduction pathways,understand the interaction between key molecular targets,interpret the potential tumor inhibition pathways of the compound,and verify key targets to provide new directions of investigating the underlying mechanisms.2.To investigate the underlying mechanisms of GA-13315-induced apoptosis in human non-small cell lung cancer cell lines,and lay a foundation for developing effective,low-toxic,economical new anti-cancer drugs.Materials and methods:Lung cancer cells were treated with different doses of GA-13315 for 48 h,and a CCK8 assay was performed to measure cell viability.Alteration in gene expression was identified using RNA-sequencing(RNA-Seq).Quantitative polymerase chain reaction(qPCR)was used to confrm the differentially expressed genes(DEGs)identifed in RNA-Seq.Gene expression plasmids or small interfering RNA were used to overexpress or silence targeted genes,in order to investigate downstream signals.Chromatin immunoprecipitation was conducted to evaluate the binding of transcription factors to the target genes.A Student’s t-test or one-way analysis of variance followed by Tukey’s honestly signifcant difference post-hoc test were performed to evaluate the significance between groups.P<0.05 was considered to indicate a statistically signifcant difference.Results and Conclusion:GA-13315 signifcantly decreased the number of viable cells and induced apoptosis among lung cancer cells(IC50=11.37±2.23μM).RNA-Seq identifed 250 signifcant DEGs,including 94 upregulated and 156 downregulated genes in A549 cells(P<0.05;fold change≥1.5).Upregulation of TRIM67,NF-κB subunit 2(NF-κB2)and FAS was additionally confrmed using qPCR and western blot analysis in A549 and H460 cells.Apoptosis of A549 cells was significantly decreased following knockdown of TRIM67.GA-13315 promoted TRIM67 expression to increase FAS expression and cell apoptosis.TRIM67 promoted the processing of NF-κB2 into its active form,p52,which then enhanced the NF-κB pathway and GA-13315-induced apoptosis.