AbstractBackgroundGinsenoside Rh2 (G‐Rh2) exerts anti‐tumor activity in non‐small cell lung cancer (NSCLC). microRNAs (miRNAs, miRs) play pivotal roles in NSCLC. We aimed to investigate whether G‐Rh2 inhibited NSCLC progression by targeting miRNA.MethodsCell viability, apoptosis and cycle were determined by Cell Counting Kit‐8, 6‐diamidino‐2‐phenylindole (DAPI) staining and flow cytometry. The potential target miRNAs of G‐Rh2 were screened by real‐time quantitative polymerase chain reaction (RT‐qPCR). The difference in miR‐28‐5p expression between lung adenocarcinoma (LUAD) tissues and normal tissues or lung squamous cell carcinoma (LUSC) tissues and normal tissues was retrieved from TCGA‐LUAD and TCGA‐LUSC, respectively. Kaplan–Meier Plotter was conducted to analyze the survival rate for different serine/threonine‐protein kinase 4 (STK4) expressions with different prognostic risks. immunohistochemistry of STK4 expression in non‐tumor and tumor tissues was analyzed from the HPA database. RT‐qPCR and Western blot were adopted for detecting mRNA and protein expression. TargetScan V7.2, miRanda and PITA were adopted for predicting targets of miR‐28‐5p, overlapped genes were subjected to GO analysis. The interactions of miR‐28‐5p‐Wnt and miR‐28‐5p‐STK4 were detected by TOP/FOP luciferase reporter assay and dual luciferase reporter assay, respectively.ResultsCurrent study observed that G‐Rh2 reduced miR‐28‐5p expression in NSCLC cells dose‐dependently. miR‐28‐5p was upregulated in NSCLC tissues and cells. The target genes of miR‐28‐5p were enriched in negative regulation of Wnt signaling. miR‐28‐5p inhibitor inactivated Wnt signaling, inhibited cell viability and cell cycle, while enhanced cell apoptosis of NSCLC cells by targeting STK4. G‐Rh2 exerted the similar effects with miR‐28‐5p inhibitor by reducing miR‐28‐5p. G‐Rh2 and miR‐28‐5p inhibitor exerted a synergistic effect on inhibiting NSCLC tumor growth.ConclusionIn conclusion, G‐Rh2 attenuates NSCLC development by affecting miR‐28‐5p/STK4 axis and inactivating Wnt signaling. Taken together, we project out a novel therapeutic target for NSCLC.