6'-O-galloylpaeoniflorin regulates proliferation and metastasis of non-small cell lung cancer through AMPK/miR-299-5p/ATF2 axis

Abstract

Background: Recent studies have shown 6'-O-galloylpaeoniflorin (GPF), a nature product extracted from the roots of paeoniflorin exerts anti-oxidant and anti-inflammatory activities. However, the effects of GPF on the proliferation and invasion in non-small cell lung cancer (NSCLC) cells have not been clarified.

Methods: MTT assay was performed to determine the cytotoxicity of GPF treatment on NSCLC cells. Colony formation assay, cell scratch test and transwell assay were performed to determine the proliferation and invasion of NSCLC cells in vitro, respectively. An A549 cell xenograft mouse model was performed to confirm the growth of NSCLC cells in vivo. Western blotting was used to measure the levels of activating transcription factor 2 (ATF2), AMP-activated protein kinase (AMPK) and phosph-AMPK (p-AMPK). Luciferase assay was used to validate the binding of miR-299-5p on the 3' untranslated region (UTR) of ATF2.

Results: Administration of GPF (50 or 100 μM) was significantly cytotoxic to A549 cells and H1299 cells, as well as inhibited the clonality, invasion and metastasis of NSCLC cells in vitro. GPF treatment also inhibited the tumor growth of NSCLC cell mouse xenografts in vivo. Exotic expression of miR-299-5p significantly inhibited the growth of NSCLC cells in vitro and in vivo. Downregulation of miR-299-5p expression attenuated the inhibition of the proliferation and metastasis of non-small cell lung cancer cells by GPF treatment. miR-299-5p significantly decreased ATF2 mRNA and protein levels in A549 cells (p < 0.05). Overexpression of ATF2 blocked the inhibitory effect of miR-299-5p on the proliferation and invasiveness of A549 cells.

Conclusions: GPF regulates miR-299-5p/ATF2 axis in A549 cells via the AMPK signalling pathway, thereby inhibiting the proliferation and metastasis of non-small cell lung cancer cells.

Keywords: ATF2; metastasis; Non-small cell lung cancer; Proliferation; microRNA.

Fig. 1

A549, H1299, 16-HBE and Beas 2B cells were treated with different concentrations of GPF, and MTT assays were performed. a The chemical structural formula of GPF. b GPF did not inhibit the activity of Beas 2B or 16-HBE human normal epithelial cell lines at 50 or 100 μM. c GPF had significantly cytotoxicity activity on A549 or H1299 human non-small cell lung cancer cell lines in vitro at GPF concentrations of 50 or 100 μM. The volume of tumours in the control group was significantly larger than that in treatment groups from day 21

Fig. 2

Effects of GPF on the formation and growth of non-small cell lung cancer cells in vivo. a GPF (50 or 100 μM) inhibited the cloning ability of A549 and H1299 cells in vitro. b The volume of tumours in the control group was significantly larger than in treatment groups from day 21. c The weight of tumours was also higher in the control group than in treatment groups from day 21

Fig. 3

Effects of GPF on migration and invasion of non-small cell lung cancer cells in vitro. a The results of scratch tests of non-small cell lung cancer cells treated or untreated with GPF. b The results of Transwell assays of non-small cell lung cancer cells treated or untreated with GPF

Fig. 4

a Comparison of differentially expressed microRNAs in stimulated and unstimulated cells by deep sequencing. b Expression of miR-299-5p at different concentrations of GPF (50 and 100 μM) at different timepoints assessed by qRT-PCR. c GPF (100 μM) stimulation significantly activates the AMPK pathway in A549 cells. d Levels of microRNA-299-5p in A549 cells pretreated with Compound C are significantly lower than in untreated cells after GPF stimulation

Fig. 5

a Absorption changes of A549 and H1299 cells transfected with mimics at different timepoints assessed by MTT assays. b The number of clones of A549 and H1299 cells transfected with miR-299-5p is significantly fewer than that of the negative control group in vitro. c, d Effect of microRNA-299-5p on the growth rate of subcutaneous non-small cell lung cancer in nude mice

Fig. 6

Effect of microRNA-299-5p on migration and invasion of non-small cell lung cancer cells in vitro. a The results of scratch test of non-small cell lung cancer cells in mimics or scramble group. b, c The results of Transwell assays of non-small cell lung cancer cells in mimics and scramble group

Fig. 7

a-c The proliferation and cloning ability of A549 cells transfected with microRNA-299-5p inhibitor are significantly increased in vitro, and the ability of migration and invasion is also significantly increased in vitro. d, e The growth rate of A549 cells transfected with microRNA-299-5p inhibitor is also significantly higher than that in the GPF group in vivo

Fig. 8

ATF2 may be a target of miR-299-5p

Fig. 9

a ATF2–3′-UTR-wt and ATF2–3′-UTR-mut were cloned into the luciferase reporter vector. The results of luciferase assays showed that the fluorescence intensity of the ATF2–3′-UTR-wt group was significantly lower than that of the Scramble group after transfection of microRNA-299-5p mimics, but there was no significant difference between ATF2–3′-UTR-mut, empty vector or Scramble groups. b Western blotting showing that microRNA-299-5p mimics could significantly reduce ATF2 protein levels in A549 cells. c qRT-PCR showing that microRNA-299-5p mimics downregulate ATF2 expression in A549 cells. All results are expressed as mean ± SD (*p < 0.05 compared to the Scramble group, ** < 0.01 compared to the Scramble group)

Fig. 10

a After transfection of microRNA-299-5p mimics, the proliferation of ATF2-overexpressing A549 cells was significantly higher than that in the negative control group. b The cloning ability of ATF2-overexpressing A549 cells (mimics + ATF2) was significantly higher than that of negative control cells (mimics + vector). c, d The growth rate and weight of implanted tumours in the Agomir + ATF2 group were significantly higher than those in the AgomiR + Vector group. All results are expressed as mean ± SD. = (*p < 0.05 compared to the mimics + vector group, **p < 0.01 compared to the mimics + vector group)

Fig. 11

a The migration rate of A549 cells in the mimics + ATF2 group was significantly higher than the negative control (mimics + vector) group. b, c Transwell assays showed that that the migration and invasion ability of cells in the mimics + ATF2 group was significantly higher than in the negative control group. All results are expressed as mean ± SD (*p < 0.05 compared to the mimics + vector group, **p < 0.01 compared to the mimics + vector group)