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

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. 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. 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. 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.

Zheng X ，Zhou Y ，Chen W ，Chen L ，Lu J ，He F ，Li X ，Zhao L ... -  《-》

Ginsenoside 20(S)-Rg3 Inhibits the Warburg Effect Via Modulating DNMT3A/ MiR-532-3p/HK2 Pathway in Ovarian Cancer Cells.

The Warburg effect is one of the main energy metabolism features supporting cancer cell growth. 20(S)-Rg3 exerts anti-tumor effect on ovarian cancer partly by inhibiting the Warburg effect. microRNAs are important regulators of the Warburg effect. However, the microRNA regulatory network mediating the anti-Warburg effect of 20(S)-Rg3 was largely unknown. microRNA deep sequencing was performed to identify the 20(S)-Rg3-influenced microRNAs in SKOV3 ovarian cancer cells. miR-532-3p was overexpressed by mimic532-3p transfection in SKOV3 and A2780 cells or inhibited by inhibitor532-3p transfection in 20(S)-Rg3-treated cells to examine the changes in HK2 and PKM2 expression, glucose consumption, lactate production and cell growth. Dual-luciferase reporter assay was conducted to verify the direct binding of miR-532-3p to HK2. The methylation status in the promoter region of pre-miR-532-3p gene was examined by methylation-specific PCR. Expression changes of key molecules controlling DNA methylation including DNMT1, DNMT3A, DNMT3B, and TET1-3 were examined in 20(S)-Rg3-treated cells. DNMT3A was overexpressed in 20(S)-Rg3-treated cells to examine its influence on miR-532-3p level, HK2 and PKM2 expression, glucose consumption and lactate production. Deep sequencing results showed that 11 microRNAs were increased and 9 microRNAs were decreased by 20(S)-Rg3 in SKOV3 cells, which were verified by qPCR. More than 2-fold increase of miR-532-3p was found in 20(S)-Rg3-treated SKOV3 cells. Forced expression of miR-532-3p reduced HK2 and PKM2 expression, glucose consumption and lactate production in SKOV3 and A2780 ovarian cancer cells. Inhibition of miR-532-3p antagonized the suppressive effect of 20(S)-Rg3 on HK2 and PKM2 expression, glucose consumption and lactate production in ovarian cancer cells. Dual-luciferase reporter assay showed that miR-532-3p directly suppressed HK2 rather than PKM2. miR-532-3p level was controlled by the methylation in the promoter region of its host gene. 20(S)-Rg3 inhibited DNMT3A expression while exerted insignificant effect on DNMT1, DNMT3B and TET1-3. 20(S)-Rg3 reversed DNMT3A-mediated methylation in the promoter of the host gene of miR-532-3p, and thus elevated miR-532-3p level followed by suppression of HK2 and PKM2 expression, glucose consumption and lactate production. 20(S)-Rg3 modulated microRNAs to exert the anti-tumor effect in ovarian cancer. 20(S)-Rg3 lessened the DNMT3A-mediated methylation and promoted the suppression of miR-532-3p on HK2 to antagonize the Warburg effect of ovarian cancer cells.

Zhou Y ，Zheng X ，Lu J ，Chen W ，Li X ，Zhao L ... -  《-》

Ginsenoside 20(S)-Rg3 upregulates HIF-1α-targeting miR-519a-5p to inhibit the Warburg effect in ovarian cancer cells.

The Warburg effect, one of the metabolic hallmarks of cancer, is responsible for rapid energy production through a high rate of aerobic glycolysis. Ginsenoside 20(S)-Rg3 antagonizes the Warburg effect in ovarian cancer cells by upregulating some microRNAs, including miR-519a-5p, that target key enzymes involved in aerobic glycolysis. How 20(S)-Rg3-upregulated miR-519a-5p influences the Warburg effect in ovarian cancer cells remains poorly defined, however. Here we report that while overexpression of miR-519a-5p in ovarian cancer cells inhibited the Warburg effect, inhibition of miR-519a-5p negated the suppressive action of 20(S)-Rg3 against the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and HK2 expression. We identified HIF-1α as a direct target of miR-519a-5p via luciferase reporter assays and demonstrated the counteraction by overexpressed HIF-1α of 20(S)-Rg3-suppressed Warburg effect. Further, 20(S)-Rg3 decreased DNMT3A-mediated DNA methylation in the promoter region of its precursor gene, leading to an increase in the level of miR-519a-5p. In conclusion, 20(S)-Rg3 upregulates miR-519a-5p via reducing DNMT3A-mediated DNA methylation to inhibit HIF-1α-stimulated Warburg effect in ovarian cancer.

Lu J ，Chen H ，He F ，You Y ，Feng Z ，Chen W ，Li X ，Zhao L ... -  《-》

Oviductus ranae protein hydrolysate (ORPH) inhibits the growth, metastasis and glycolysis of HCC by targeting miR-491-5p/PKM2 axis.

Oviductus Ranae (OR) is a valuable Chinese crude drug and has been reported to have a range of biological activities. Protein hydrolysate extracted from OR (ORPH) was previously found to have immune regulatory effect and anti-glioma activity. This study was aimed to investigate the effects of ORPH on hepatocellular carcinoma (HCC) progression. MTT, BrdU, colony formation and transwell assays were used to determine proliferation and mobility of HCC cells in vitro. Glucose consumption and lactate production assays were carried out to measure the glycolysis of HCC cells. The subcutaneous tumor model and lung metastasis model in nude mice were established to detect tumor growth and metastasis of HCC in vivo. The direct binding of miR-491-5p to 3'UTR of pyruvate kinase M2 (PKM2) was confirmed by luciferase reporter assay. In vitro experiments showed that ORPH significantly inhibited proliferation, migration, invasion, epithelial-to-mesenchymal transition (EMT) and glycolysis of HCC cells. Moreover, ORPH treatment prominently suppressed HCC growth and metastasis in mice. We demonstrated that ORPH effectively decreased the expression of PKM2 in HCC cells. Forced expression of PKM2 abrogated the inhibitory effects of ORPH on HCC cells. Mechanically, ORPH reduced PKM2 expression in a post-transcriptional manner by up-regulating miR-491-5p. miR-491-5p exhibited a similar tumor suppressive effects with ORPH in HCC cells. Moreover, ORPH exerted its inhibitory effects on HCC cells through regulating miR-491-5p/PKM2 axis. Lastly, decreased miR-491-5p level and increased PKM2 expression were correlated with unfavorable clinical features and poor prognosis of HCC patients. In all, this study reveals that ORPH inhibits the growth, metastasis and glycolysis of HCC cells by targeting miR-491-5p/PKM2 axis. ORPH may be a potential effective anti-tumor agent for HCC.

Xu Q ，Dou C ，Liu X ，Yang L ，Ni C ，Wang J ，Guo Y ，Yang W ，Tong X ，Huang D ... -  《-》

Ginsenoside 20(S)‑Rg3 inhibits the Warburg effect through STAT3 pathways in ovarian cancer cells.

Li J ，Liu T ，Zhao L ，Chen W ，Hou H ，Ye Z ，Li X ... -  《-》