Ginsenoside 20(S)-Rg3 Prevents PKM2-Targeting miR-324-5p from H19 Sponging to Antagonize the Warburg Effect in Ovarian Cancer Cells

Abstract

Background/Aims: The Warburg effect is one of the main metabolic features for cancers, with long non-coding RNA (lncRNA) being involved as a class of crucial regulators. Our previous studies have shown that ginsenoside 20(S)-Rg3, an active saponin monomer extracted from red ginseng, inhibits the Warburg effect in ovarian cancer cells. However, the detailed lncRNA regulatory network modulated by 20(S)-Rg3 to prevent the Warburg effect in ovarian cancer cells has not been explored. Methods: High-throughput sequencing was used to screen out the differentially expressed lncRNAs between 20(S)-Rg3-treated and non-treated SKOV3 cells. The levels of lncRNA H19 and miR-324-5p were manipulated in SKOV3 and A2780, and the glucose consumption, lactate production and PKM2 protein level were detected. Dual-luciferase reporter assay and RIP were utilized to verify the direct binding of H19 to miR-324-5p and miR-324-5p to PKM2. Cell proliferation was examined by CCK8 and colony formation assay. Nude mice subcutaneous xenograft tumor models were established to evaluate the impact of miR-324-5p on tumor growth in vivo. Results: 20(S)-Rg3 downregulated 67 lncRNAs, and H19 was one of the most decreased lncRNAs. Suppression of H19 by siRNA transfection reduced glucose consumption, lactate production and PKM2 expression in ovarian cancer cells, while H19 overexpression in 20(S)-Rg3-treated ovarian cancer cells enhanced glucose consumption, lactate production and PKM2 expression. Dual-luciferase reporter assay and RIP results showed that H19 directly bound to miR-324-5p. Dual-luciferase reporter assay showed that miR-324-5p directly targeted PKM2, and miR-324-5p negatively regulated glucose consumption and lactate production in ovarian cancer cells. miR-324-5p overexpression inhibited cell proliferation in vitro and in vivo. Conclusion: Our study revealed that 20(S)-Rg3 blocked the competitive inhibition of H19 on miR-324-5p, which enhanced the suppression of miR-324-5p on PKM2 and therefore inhibited the Warburg effect and repressed tumorigenesis. In a word, 20(S)-Rg3 inhibited the Warburg effect in ovarian cancer cells via H19/miR-324-5p/PKM2 pathway.

Antitumor effects and molecular mechanisms of action of natural products in ovarian cancer (Review)

“…A previous study reported that 20(S)-Rg3 may block the inhibition of miR-324-5p by H19. Increased miR-324-5p by 20(S)-Rg3 treatment inhibited the activity of pyruvate kinase isozyme type M2, thereby inhibiting the Warburg effect ( 70 ). Furthermore, Lu et al ( 71 ) reported that ginsenoside 20(S)-Rg3 upregulated the expression of miR-603 in ovarian cancer cells by downregulating the DNA methylation mediated by DNA methyltransferase 3 alpha.…”

“…µg/ml Cell line (SKOV3) Downregulated p-STAT, decreased metabolic enzymes in glycolysis,(69) inhibited the Warburg effect, and prevented cancer growth and metabolism.Zheng et al, 2018 20(S)-Rg340, 80 µg/ml Cell lines (SKOV3 and A2780) Blocked the competitive inhibition of H19 on miR-324-5p, increased the(70) and in vivo (BALB/C nude mice) suppression of miR-324-5p on pyruvate kinase isozyme type M2, inhibit Warburg effect, and retarded cancer growth.Lu et al,2019 20(S)-Rg3 40, 80 µg/ml Cell lines (SKOV3 and A2780) Upregulated miR-603, reduced hexokinase-2, inhibited the Warburg effect, Cell lines (SKOV3 and 3AO) Activated ubiquitin-proteasome pathway, decreased HIF-1α, inhibited EMT, (75) upregulated E-cadherin, and decreased the invasion and metastasis of cancer cells. Liu et al, 2017 20(S)-Rg3 80,160 µg/ml Cell lines (SKOV3 and 3AO) Promoted HIF-1α ubiquitin proteasome degradation and inhibited the (76) invasion and metastasis of ovarian cancer cells.…”

“…Liu et al, , phosphorylated signal transducer and activator of transcription; miR, miRNA; LC3, microtubule-associated protein 1 light chain 3; HIF-1α, hypoxia-inducible factor-1α; EMT, epithelial-to-mesenchymal transition; MDR-1, multidrug resistance protein 1.inhibition of miR-324-5p by H19. Increased miR-324-5p by 20(S)-Rg3 treatment inhibited the activity of pyruvate kinase isozyme type M2, thereby inhibiting the Warburg effect(70). Furthermore, Lu et al(71) reported that ginsenoside 20(S)-Rg3 upregulated the expression of miR-603 in ovarian cancer cells by downregulating the DNA methylation mediated by DNA methyltransferase 3 alpha.…”

miR-324-5p Inhibits C2C12 cell Differentiation and Promotes Intramuscular Lipid Deposition through lncDUM and PM20D1

“… 28 Several studies have reported that lncGpr19, lncH19, and lncTPT1-AS1 can absorb miR-324-5p, thereby influencing the migration of cancer cells. 29 , 30 , 31 In this study, we found that miR-324-5p targets lncDum and inhibits the differentiation of C2C12 myoblasts. lncDum can promote the differentiation of skeletal muscle satellite cells by inhibiting the expression of Dppa2 .…”

The Challenges and Opportunities of LncRNAs in Ovarian Cancer Research and Clinical Use

“…In the glycolysis pathway from glucose to pyruvate, the expression of hexokinase 2, catalyzing the first step, is activated by LINC00504 [70]; NRCP activates the second enzyme in the pathway, glucose-6-phosphate isomerase [26]; after that, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 is induced by LINC00092 [31]; in the fourth step catalyzed by aldolase, two forms of the enzyme, A and C, are activated by NRCP [26]. The final step is catalyzed by pyruvate kinase isozyme M2, which is activated by both LINC00504 and H19 [70,132]. Moreover, LINC00504 activates the expression of pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase by phosphorylation [70], thereby inhibiting the Krebs cycle.…”