The compound 3β, 16β, 17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1→3)-(2-O-acetyl-α-L-arabinopyranoside (OSW-1) is a member of the cholestane saponin family that was created in the bulbs of Ornithogalum saudersiae. OSW-1 has previously been shown as cytotoxic against numerous types of malignant cells, however, its antitumoral mechanisms remain unclear. The present study aimed to examine the potential changes in the gene expression of a hepatocellular carcinoma (HCC) cell line (Hep3B) incubated with OSW-1 in vitro. The results showed that OSW-1 inhibited tumors through invasiveness, angiogenesis, cell polarity and cell adhesion (as shown by Roche NimbleGen gene expression analysis), in addition to inducing apoptosis through the mitochondrial pathway. This affected the expression of a number of core genes in a number of signaling pathways, including WNT, MAPK, VEGF and P53. To the best of our knowledge, the present study is the first to report that OSW-1, as a molecular compound, induces necroptotic death in hepatocellular carcinoma (HCC).

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MicroRNAs (miRNAs) are a large family of post‑transcriptional regulators of gene expression that control a number of developmental and cellular processes in eukaryotic organisms and are ~23 nucleotides in length. miRNA‑122 is an abundant liver‑specific miRNA, implicated in fatty acid and cholesterol metabolism, as well as in hepatitis C viral replication and is frequently suppressed in primary hepatocellular carcinomas. In the current study, the Hep3B cell line with stable overexpression of miR‑122 was successfully established through gene transfection methods and drug screening. miR‑122 was observed to alter cell morphology in vitro by stable overexpression in Hep3B cells. This alteration was viewed by light microscopy and transmission electron microscopy. These alterations included increases in the cell volume, the appearance of lipid granules and vacuoles, thickening of nuclear membrane, swelling of the mitochondria, cytoplasm vacuolization and a more prominent nucleolus. Furthermore, the study provided novel evidence that miR‑122 function was dependent upon its expression level. In addition, it was observed to negatively regulate mitochondria.

To investigate the effect of mitochondrial DNA deletion and OSW-1 on PI3K-AKT signaling pathway PCR Array in SK-Hep1 hepatocellular carcinoma cells, we prepared SK-Hep1 cells with mtDNA deletion, that is, p 0 SK Hep. Then the OSW-1 of 100 ng/L was used to intervene SK-Hep1 and p 0 SK-Hep1. RT-qPCR was used to detect the difference of gene expression on PI3K-AKT signaling pathway PCR Array in four groups of cells. The gene expression of TLR4, FOS and TSC2 markers in SK-Hep1 cells treated with OSW-1 were significantly increased. The gene expressions of PDPK1, GJA1, TLR4 and TSC2 markers were significantly increased in p 0 SK-Hep1 cells, and the gene expressions of IRAK1 and GJA1 markers were significantly increased in p 0 SK-Hep1 cells treated with OSW-1. OSW-1 mainly affects the genes related to TLR4 pathway on PI3K-AKT signaling pathway PCR Array in SK-Hep1 HCC cells. P 0 SK-Hep1 mainly affects the upstream PDK1 gene and downstream TSC2 gene of Akt on PI3K-AKT signaling pathway PCR Array, and also affects the gene expression of gap junction at the same time.

RAC1 is a small-molecule G protein that regulates multiple cell cycle, cytoskeletal reorganization, cell migration, and apoptosis. FADD-dependent TRAIL can promote tumor metastasis through RAC1 and PI3K, and down-regulating RAC1 expression can reduce FasL-induced apoptosis. In addition, RIP1 bound to GTP acts as an activating protein for RAC1 and is involved in cytoskeletal reorganization. TRAF6 promotes migration and metastasis by regulating the RAS pathway in tumors. Thus, it is necessary to understand the interaction between RAC1 and TRAF6 as well as FADD and RIP1. In this study, we cultured hepatoma SK-Hep1 cells in vitro, specifically blocked the necroptosis pathway with Nec-1, and silenced FADD, RIP1 and TRAF6 gene expression using RNAi technology. At the same time, the expression of RAC1 was evaluated separately using RT-PCR and Western blot. The hepatoma SK-Hep1 cells survival rate was highest when the concentration of Nec-1 was 60 μM and the concentration of Z-vad-fmk was 20 μM. And the apoptosis rate of the transfected RAC1 siRNA cells was 3.59% compared with transfected siRNA cells 10.01% which was significantly decreased (P < 0.01). RAC1 could promote the occurrence of apoptosis in SK-Hep1 cells. RAC1 expression was suppressed in both protein and gene level in SK-Hep1 cells when the TRAF6 gene was silenced, but there was no significant change in RAC1 gene and protein expression when FADD and RIP1 genes were silenced. TRAF6 affects RAC1 expression and apoptosis in SK-Hep1 cells, while the FADD and RIP1 genes do not affect the role of RAC1. The TRAF6 gene is an important target in liver cancer cells.