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Knockdown of the Sonic Hedgehog (SHH) Gene Inhibits Proliferation of Hep3B and SMMC-7721 Hepatocellular Carcinoma Cells via the PI3K/Akt/PCK1 Signaling Pathway.

ABSTRACT: BACKGROUND The PCK1 gene encodes phosphoenolpyruvate carboxykinase (PEPCK), which has been shown have a role in metabolic events in hepatocellular carcinoma (HCC). This study aimed to investigate the role of the SHH gene and its encoded protein, sonic hedgehog (SHH), in two human hepatocellular carcinoma (HCC) cell lines. MATERIAL AND METHODS The human HCC cell lines Hep3B and SMMC-7721 were cultured. Cells were transfected with plasmids carrying specific SHH gene short-hairpin RNA (shRNA) and negative control (NC) shRNA. The effects of knockdown of expression levels of the SHH gene were studied on cell survival, cell apoptosis, the cell cycle, gluconeogenesis, and the expression of PCK1. Anchorage-independent growth, a characteristic of transformed cells, was detected by the colony formation assay. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot were performed 24 h after transfection. RESULTS Knockdown of expression levels of the SHH gene reduced cell proliferation and growth of HCC cells and induced cell apoptosis and G1 cell cycle arrest in both HCC cell lines. Knockdown of the SHH gene decreased the levels of glycolysis products and increased the production of glucose and reduced the phosphorylation of PI3K and Akt but induced the expression of PCK1. CONCLUSIONS Knockdown of the SHH gene reduced cell survival of HCC cells by increasing apoptosis, reducing cell proliferation, inducing G1 cell cycle arrest, and restoring gluconeogenesis, and was associated with the inhibition of the PI3K/Akt axis and induced the expression of PCK1.

SUBMITTER: Chen Y

PROVIDER: S-EPMC6703083 | biostudies-other | 2019 Aug

REPOSITORIES: biostudies-other

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Knockdown of the Sonic Hedgehog (SHH) Gene Inhibits Proliferation of Hep3B and SMMC-7721 Hepatocellular Carcinoma Cells via the PI3K/Akt/PCK1 Signaling Pathway.

Chen Youping Y Zhu Weifang W

Medical science monitor : international medical journal of experimental and clinical research 20190813

BACKGROUND The PCK1 gene encodes phosphoenolpyruvate carboxykinase (PEPCK), which has been shown have a role in metabolic events in hepatocellular carcinoma (HCC). This study aimed to investigate the role of the SHH gene and its encoded protein, sonic hedgehog (SHH), in two human hepatocellular carcinoma (HCC) cell lines. MATERIAL AND METHODS The human HCC cell lines Hep3B and SMMC-7721 were cultured. Cells were transfected with plasmids carrying specific SHH gene short-hairpin RNA (shRNA) and n ...[more]

PMID: 31406102

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Project description:BackgroundCircular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions.MethodsCDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs.ResultsGF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms.ConclusionsOur results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC. Video abstract.

Dataset Information

Knockdown of the Sonic Hedgehog (SHH) Gene Inhibits Proliferation of Hep3B and SMMC-7721 Hepatocellular Carcinoma Cells via the PI3K/Akt/PCK1 Signaling Pathway.

Publications

Knockdown of the Sonic Hedgehog (SHH) Gene Inhibits Proliferation of Hep3B and SMMC-7721 Hepatocellular Carcinoma Cells via the PI3K/Akt/PCK1 Signaling Pathway.

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