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 ... -  《-》

LNCAROD enhances hepatocellular carcinoma malignancy by activating glycolysis through induction of pyruvate kinase isoform PKM2.

Mounting evidence has suggested the essential role of long non-coding RNAs (lncRNAs) in a plethora of malignant tumors, including hepatocellular carcinoma. However, the underlyling mechanisms of lncRNAs remain unidentified in HCC. The present work was aimed to explore the regulatory functions and mechanisms of LncRNA LNCAROD in HCC progression and chemotherapeutic response. The expression of LNCAROD in HCC tissues and cell lines were detected by quantitative reverse transcription PCR (qPCR). Cancer cell proliferation, migration, invasion, and chemoresistance were evaluated by cell counting kit 8 (CCK8), colony formation, transwell, and chemosensitivity assays. Methylated RNA immunoprecipitation qRCR (MeRIP-qPCR) was used to determine N6-methyladenosine (m6A) modification level. RNA immunoprecipitation (RIP) and RNA pull down were applied to identify the molecular sponge role of LNCAROD for modulation of miR-145-5p via the competing endogenous RNA (ceRNA) mechanism, as well as the interaction between LNCAROD and serine-and arginine-rich splicing factor 3 (SRSF3). The interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and LNCAROD was also identified by RIP assay. Gain- or-loss-of-function assays were used to identify the function and underlying mechanisms of LNCAROD in HCC. We found that LNCAROD was significantly upregulated and predicted a poorer prognosis in HCC patients. LNCAROD upregulation was maintained by increased m6A methylation-mediated RNA stability. LNCAROD significantly promoted HCC cell proliferation, migration, invasion, and chemoresistance both in vitro and in vivo. Furthermore, mechanistic studies revealed that pyruvate kinase isoform M2 (PKM2)-mediated glycolysis enhancement is critical for the role of LNACROD in HCC. According to bioinformatics prediction and our experimental data, LNCAROD directly binds to SRSF3 to induce PKM switching towards PKM2 and maintains PKM2 levels in HCC by acting as a ceRNA against miR-145-5p. The oncogenic effects of LNCAROD in HCC were more prominent under hypoxia than normoxia due to the upregulation of hypoxia-triggered hypoxia-inducible factor 1α. In summary, our present study suggests that LNCAROD induces PKM2 upregulation via simultaneously enhancing SRSF3-mediated PKM switching to PKM2 and sponging miR-145-5p to increase PKM2 level, eventually increasing cancer cell aerobic glycolysis to participate in tumor malignancy and chemoresistance, especially under hypoxic microenvironment. This study provides a promising diagnostic marker and therapeutic target for HCC patients.

Jia G ，Wang Y ，Lin C ，Lai S ，Dai H ，Wang Z ，Dai L ，Su H ，Song Y ，Zhang N ，Feng Y ，Tang B ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

In vitro and in vivo anti-metastatic effect of the alkaliod matrine from Sophora flavecens on hepatocellular carcinoma and its mechanisms.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer with high metastasis and recurrence rates. Hypoxia-induced miRNAs and HIF-1α are demonstrated to play essential roles in tumor metastasis. Matrine (C15H24N2O), an alkaloid extracted from Sophora flavescens Aiton, has been used as adjuvant therapy for liver cancer in China. The anti-metastasis effects of matrine on HCC and the underlying mechanisms remain poorly understood. We aimed to investigate the effects of matrine on metastasis of HCC both in vitro and in vivo, and explored whether miR-199a-5p and HIF-1α are involved in the action of matrine. MTT method, colony formation, wound healing and matrigel transwell assays were performed to evaluate the effects of matrine on cell proliferation, migration and invasion. Nude mice xenograft model and immunohistochemistry (IHC) assay were employed to investigate the anti-metastatic action of matrine in vivo. Quantitative real-time PCR, western blot and dual luciferase reporter assay were conducted to determine the underlying mechanisms of matrine. Matrine exerted stronger anti-proliferative action on Bel7402 and SMMC-7721 cells under hypoxia than that in normoxia. Both matrine and miR-199a-5p exhibited significant inhibitory effects on migration, invasion and EMT in Bel7402 and SMMC-7721 cells under hypoxia. Further study showed that miR-199a-5p was downregulated in HCC cell lines, and this microRNA was identified to directly target HIF-1α, resulting in decreased HIF-1α expression. Matrine induced miR-199a-5p expression, decreased HIF-1α expression and inhibited metastasis of Bel7402 and SMMC-7721 cells, while miR-199a-5p knockdown reversed the inhibitory effects of matrine on cell migration, invasion, EMT and HIF-1α expression. In vivo, matrine showed significant anti-metastatic activity in the nude mouse xenograft model. H&E and IHC analysis indicated that lung and liver metastasis nodules were reduced, and the protein expression of HIF-1α and Vimentin were significantly decreased by i.p injection of matrine. Matrine exhibits significant anti-metastatic effect on HCC, which is attributed to enhanced miR-199a-5p expression and subsequently impaired HIF-1α signaling and EMT. These findings suggest that miR-199a-5p is a potential therapeutic target of HCC, and matrine may represent a promising anti-metastatic medication for HCC therapy.

Dai M ，Chen N ，Li J ，Tan L ，Li X ，Wen J ，Lei L ，Guo D ... -  《-》

Long non-coding RNA AGAP2-AS1, functioning as a competitive endogenous RNA, upregulates ANXA11 expression by sponging miR-16-5p and promotes proliferation and metastasis in hepatocellular carcinoma.

Liu Z ，Wang Y ，Wang L ，Yao B ，Sun L ，Liu R ，Chen T ，Niu Y ，Tu K ，Liu Q ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

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 ... -  《-》