| In this study,the non-small cell lung cancer cell lines and the tumor xenograft model of nude mice were used as the research object to study the effect of 6’-O-Galloylpaeoniflorin(GPF)on the biological function of NSCLC and the underlying molecular mechanism.This experiment is divided into 3 parts:1 GPF inhibits the proliferation and metastasis of non-small cell lung cancer cellsGPF is a galloylated paeoniflorin extracted from the root of Paeonia lactiflora and consists of D-glucose,galloyl and benzoyl 3 moieties.Studies have shown that GPF has significant antioxidant capacity,but its effect on tumor cell growth and metastasis is not yet fully understood.In this study,we found for the first time that GPF concentrations at 100 μM or 50 μM did not significantly inhibit the activity of human normal epithelial cell line Beas 2B and 16-HBE cells,but significantly inhibited human non-small cell lung cancer cells in vitro.The proliferation of A549 and H1299 cells.In vitro administration of GPF(100 μM or 50 μM)also inhibited the clonality of A549 and H1299 cells.Furthermore,administration of GPF(5 mg/kg or 10 mg/kg)significantly inhibited the growth of nude mice bearing A549.In addition,GPF(100 μM or 50 μM)also inhibited in vitro invasion and metastasis of A549 and H1299 cells.This result shows that GPF has an inhibitory effect on the proliferation and metastasis of non-small cell lung cancer cells and has potential value for the development of new drugs for the treatment of non-small cell lung cancer.2 miR-299-5p inhibits proliferation and metastasis of non-small cell lung cancer cellsIn the first part of the experiment,we tested the anticancer effect of GPF on non-small cell lung cancer.In this study,we further studied its anticancer mechanism of action.Micro RNAs(miRNAs)are small,non-coding RNAs of 18-25 nucleotides,and there is increasing evidence that miRNAs are key regulators in various types of malignancies,including NSCLC.The miRNA expression profile in A549 cells stimulated by GPF was detected by Deep Sequencing.After 12 hours of GPF stimulation,the levels of approximately 67 miRNAs,including miR-299-5p,were upregulated by more than 2 fold compared with 34.The levels of these miRNAs were down-regulated by 2 fold.The results of q RT-PCR showed that miR-299-5p was upregulated 12 hours after GPF stimulation,and the expression was highest at about 18 hours after stimulation,and then decreased after 24 hours of stimulation.At 24 hours,miR-299-5p was still higher than cells not stimulated by GPF.GPF stimulation also significantly activates the AMPK pathway in A549 cells.After pretreatment with AAMP cell specific AMPK inhibitor Compound C,the expression of miR-299-5p induced by GPF stimulation was significantly lower than that of A549 cells without Compound C treatment.This result indicates that AMPK is a signaling pathway by which GPF stimulates upregulation of miR-299-5p expression.After miR-299-5p agonist was transfected into A549 and H1299 cells,its ability to proliferate,clone and metastasize in vitro was significantly inhibited.At the same time in vivo experiments also confirmed that miR-299-5p transfection can significantly inhibit the growth rate of non-small cell lung cancer in nude mice.Finally,we also observed the effect of inhibition of miR-299-5p on the proliferation and metastasis of GPF against lung cancer cells.The results showed that the ability of GPF to proliferate and transfect A549 cells was significantly decreased after miR-299-5p expression was inhibited.In summary,GPF can upregulate the expression of miR-299-5p in A549 cells by activating the AMPK signaling pathway.However,miR-299-5p can inhibit the growth,migration and invasion of non-small cell lung cancer cells A549 and H1299.But what is the molecular biological basis of this role of miR-299-5p? To solve this problem,we searched for potential targets of miR-299-5p through Pictar and miRBase databases.The specific targets and mechanisms of action will be performed in the third part of the experimental study.3 miR-299-5p exerts its influence on the biological characteristics of non-small cell lung cancer by inhibiting the expression of target ATF2To study the mechanism of miR-299-5p affecting the biological characteristics of NSCLC,we used miRbase,Targetscan,and micro RNA.org software to predict the target of miR-299-5p and found that the 3’UTR end of ATF2 m RNA.Contains an 8-mer size sequence that matches miR-299-5p.To determine that these sequences bind to miR-299-5p and down-regulate the translation of ATF2 m RNA,we constructed the ATF2 m RNA 3’UTR containing this site into the luciferase reporter system(ATF2-3’UTR-wt).The ATF2 m RNA 3’UTR was also inserted into the luciferase reporter system(ATF2-3’UTR-mut).miR-299-5p was then co-transfected into luciferase empty vector or ATF2-3’ UTR-wt plasmid or ATF2-3’ UTR-mut plasmid into HEK-293 cells,and the fluorescence in each group of cells was detected.strength.Furthermore,we transfected miR-299-5p and its negative Scramble into A549 cells and H1299 cells.And detected ATF2 protein and m RNA levels by western blot and q RT-PCR.The results showed that the fluorescence intensity of ATF2-3’UTR-wt group was significantly lower than that of the negative control group(Scramble),while the fluorescence intensity of ATF2-3’UTR-mut group or empty plasmid group had no significant difference with its negative control;miR-299-5p Mimics significantly decreased ATF2 protein and m RNA levels in A549 cells and H1299 cells(p<0.05).To determine if ATF2 is involved in miR-299-5p’s effect on the biological behavior of NSCLC cells,we also constructed A549 non-small cell lung cancer cells over-expressing ATF2,and further studied miR-299-5p over-expression of ATF2.Effects of Biological Behavior of A549 Cells The results showed that miR-299-5p transfection,ATF2 overexpression A549 cell proliferation and metastasis was significantly higher than its negative control(p <0.05).In summary,this study found that miR-299-5p can bind to the 3’UTR end of ATF2 m RNA and inhibit gene expression,thereby inhibiting the proliferation and metastasis of non-small cell lung cancer cells.This study provides a new complement to the study of the relationship between miR-299-5p and the growth and metastasis of non-small cell lung cancer,providing a potential clinical therapeutic target for non-small cell lung cancer. |
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