Conclusion
miR-34a targets and negatively regulates Netrin1 to mediate the proliferation, apoptosis, apoptosis, migration, and invasion of drug-resistant gastric cancer cells via the MEK/ERK pathway, and change the chemosensitivity in GC cells. miR-34a/Netrin1/MEK/ERK axis may serve as a novel therapeutic target for chemoresistance in GC, it is of great significance for overcoming drug resistance and developing new therapeutic strategies for GC.
Methods
The Cancer Genome Atlas (TCGA), Differentially Expressed MicroRNAs (miRNAs) in human cancers (dbDEMC), and Starbase online databases were used to analyze the correlation between miR-34a and Netrin-1 and prognosis in GC, and to predict and verify the targeted binding of miR-34a to Netrin-1. The experimental methods including Cell transfection, real-time polymerase chain reaction (RT-PCR), Cell-Counting-Kit-8 (CCK8) assay, flow cytometry, wound scratch assay, transwell assay, and western blotting were used to investigate the effects of miR-34a and Netrin1 on chemotherapy resistance and biological characteristics in cisplatin-resistant GC cells (HGC27/DDP), and to analyze the molecular mechanism of cisplatin resistance.
Objective
To explore the mechanism of action of MicroRNAs-34a (miR-34a) and Eurite growth guiding factor 1 (Netrin1) in cisplatin resistance in gastric cancer (GC), providing new clues for overcoming tumor resistance and optimizing anti-tumor therapy for GC.
Results
miR-34a expression was downregulated in gastric cancer clinical samples and cisplatin-resistant cells, while Netrin1 was upregulated, and was related to overall survival (OS). Upregulation of miR-34a can significantly reduce the IC50 value of cisplatin(0.65 vs 1.6 ng/mL) and Multidrug Resistance 1 (MDR-1) protein level, inhibit the proliferation activity, reduce the expression levels of proliferating cell nuclear antigen (PCNA) and ki-67 protein, and induce the increase of apoptosis rate and the enhancement of cycle arrest. Upregulation of miR-34a can also significantly reduce the expression level of Matrix metalloproteinase 9 (MMP9) protein, promote the expression of E-cadherin protein, reduce the wound healing rate and invasion number to inhibit migration and invasion ability in drug-resistant gastric cancer cells. Moreover, overexpression of Netrin1 on the basis of upregulation of miR-34a can weaken the above changes caused by upregulation of miR-34a. In addition, upregulation of miR-34a can significantly inhibit the Mitogen-activated protein kinase kinase (MEK) / Extracellular regulated protein kinases (ERK) pathway, while overexpression of Netrin1 can activate the MEK/ERK pathway, and inhibition of MEK/ERK pathway can effectively counteract the protein expression of Netrin1, and reverse changes in the expression of cisplatin IC50 and MDR-1 proteins caused by co-upregulation of miR-34a/Netrin1 in HGC27/DDP, as well as changes in proliferation, apoptosis, migration and invasion. In addition, upregulation of miR-34a can significantly inhibit the MEK/ERK pathway, while overexpression of Netrin1 can activate the MEK/ERK pathway. If the MEK/ERK pathway was inhibited, it can effectively counteract the protein overexpression of Netrin1, and reverse the changes in the expression of cisplatin IC50 and MDR-1 proteins in HGC27/DDP induced by co-upregulation of miR-34a / Netrin1, as well as changes in proliferation, apoptosis, migration and invasion.