Guizhi Fuling pills inhibit the proliferation, migration and invasion of human cutaneous malignant melanoma cells by regulating the molecular axis of LncRNA TPT1-AS1 / miR-671-5p

Bo Zhang

doi:10.14715/cmb/2020.66.5.26

Abstract

The current research was aimed to explore the effects of Guizhi Fuling Pills on the proliferation, migration and invasion of human cutaneous malignant melanoma cells and its regulation on the molecular axis of LncRNA TPT1-AS1 / miR-671-5p. Human cutaneous malignant melanoma cells A375 were cultured in vitro and randomly divided into Con group, Guizhi Fuling pills-L group, Guizhi Fuling pills-M group, Guizhi Fuling pills-H group, Guizhi Fuling pills-H + pcDNA group, Guizhi Poria pills-H + pcDNA-TPT1-AS1 group. MTT was used to detect cell proliferation. The Transwell cell test was used to detect cell migration and invasion. qRT-PCR was used to detect the expression of TPT1-AS1 and miR-671-5p. The dual-luciferase report experiment verified the targeting relationship of TPT1-AS1, miR-671-5p. Western blot was used to detect the expression of Ki-67, PCNA, MMP-2 and MMP-9. Compared with Con group, Guizhi Fuling Pills could inhibit cell proliferation, migration and invasion (p<0.05), and also inhibit the expression of Ki-67, PCNA, MMP-2, MMP-9, TPT1-AS1 (p<0.05), promote the expression of miR-671-5p (p<0.05)., and the differences between the indexes of Guizhi Fuling Pill-L group, Guizhi Fuling Pill-M group and Guizhi Fuling Pill-H group were statistically significant (p<0.05). The dual-luciferase report experiment confirmed that TPT1-AS1 could target and bind to miR-671-5p and could regulate the expression and activity of miR-671-5p. Overexpression of TPT1-AS1 could reduce the inhibitory effect of Guizhi Fuling Pills on proliferation, migration and invasion of A375 cells. Guizhi Fuling Pill may reduce the proliferation, migration and invasion of human cutaneous malignant melanoma cells by down-regulating the expression of TPT1-AS1 and up-regulating the expression of miR-671-5p.

Highlights

Cutaneous malignant melanoma is a clinically common malignant tumor with increasing incidence year by year
Bo Zhang sequence were purchased from Guangzhou Ruibo Biological Technology Co., Ltd.; MTT was purchased from Wuhan Amyjet Scientific Inc.; both Transwell Chamber and Matrigel were purchased from BD, USA; rabbit anti-human Ki-67 and PCNA antibodies were purchased from CST, USA; rabbit anti-human MMP-2 and MMP-9 antibodies were purchased from Santa Cruz, USA; goat anti-rabbit IgG secondary antibody labeled with horseradish peroxidase (HRP) was purchased from Abcam, USA
This study showed that after adding different concentrations of Guizhi Fuling Pills, A375 cells have significantly decreased viability with the increase of the dosage, suggesting that Guizhi Fuling Pills can inhibit the proliferation of cutaneous malignant melanoma cells in a dose-dependent manner

Summary

Introduction

Cutaneous malignant melanoma is a clinically common malignant tumor with increasing incidence year by year. Traditional Chinese medicine plays an important role in regulating tumor cell proliferation, differentiation and apoptosis, but its regulation mechanism in the treatment of cutaneous malignant melanoma has not been fully elucidated [2,3]. The up-regulation of its expression can inhibit the proliferation of gastric cancer cells and promote apoptosis by targeting URGCP [6]. It remains unknown whether Guizhi Fuling Pills can affect the biological behavior of cutaneous malignant melanoma cells by regulating the molecular axis of TPT1-AS1/miR-671-5p. This study mainly investigates the effect of Guizhi Fuling Pills on the biological behavior of cutaneous malignant melanoma cells, and explores its regulatory effect on the molecular axis of TPT1-AS1/miR-671-5p, with a view to laying the experimental foundation for further investigation into the molecular mechanism of Guizhi Fuling Pills in fighting cancer

Methods

Results

Conclusion