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Objective To investigate the effect and mechanism of acteoside (ACT) in inhibiting epithelial-mesenchymal transition (EMT) in human hepatoma HCCLM3 cells by regulating the ERK1/2 pathway. Methods CCK-8 assay was used to detect the effect of hepatocellular carcinoma cell proliferation. The invasion and migration of HCC cells were detected by scratch and Transwell tests. The mRNA and protein expression levels of the ERK1/2 signaling pathway and EMT-related genes (E-cadherin and N-cadherin) were detected by real-time PCR and Western blot analyses. Results ACT reduced the activity of HCCLM3 cells and inhibited the proliferation of HCC cells, and the effects had certain correlation with drug concentration and time. ACT inhibited the migration and invasion process of HCCLM3 cells in a concentration-dependent manner. ACT downregulated the mRNA and protein expression of genes related to the ERK1/2 signaling pathway. It increased the mRNA and protein expression levels of the EMT-related gene E-cadherin but decreased those of N-cadherin. Conclusion ACT could inhibit EMT and the invasion and migration of HCCLM3 cells in human hepatoma, and the underlying mechanism is closely related to the downregulation of the ERK1/2 signaling pathway.
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Increasing evidence suggest that hepatocellular carcinoma (HCC) HCCLM3 cells initially develop pseudopodia when they metastasize, and microRNAs (miRNAs/miRs) and circular RNAs (circRNAs) have been demonstrated to serve important roles in the development, progression and metastasis of cancer. The present study aimed to isolate the cell bodies (CBs) and cell protrusions (CPs) from HCCLM3 cells, and screen the miRNAs and circRNAs associated with HCC infiltration and metastasis in CBs and CPs. The Boyden chamber assay has been confirmed to effectively isolate the CBs and CPs from HCCLM3 cells via observation of microtubule immunofluorescence, DAPI staining and nuclear protein H3 western blotting. Following high-throughput sequencing of the successfully isolated CBs and CPs, 64 pairs of miRNAs, including 23 pairs of upregulated genes and 41 pairs of downregulated genes, and 260 sets of circRNAs, including 127 upregulated genes and 133 downregulated genes, were significantly differentially expressed, using the following criteria: HP/HB ratio, fold change ≥|1.5|, P<0.05). PCR analysis verified that changes in the expression levels of hsa-let-7a-5p, hsa-let-7c-3p, hsa-miR-30c-5p, hsa_circ_0059580, hsa_circ_0067475, hsa_circ_0002100 and hsa_circ_00072309 were consistent with the sequencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze the functions and roles of the differentially expressed miRNAs and circRNAs. The interaction maps between miRNAs and circRNAs were constructed, and signaling pathway maps were analyzed to determine the molecular mechanism and regulation of the differentially expressed miRNAs and circRNAs. Taken together, the results of the present study suggest that the Boyden chamber assay can be used to effectively isolate the somatic CBs and CPs of HCC, which can be used to screen the miRNAs and circRNAs associated with invasion and metastasis of HCC.
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Objective:To explore the anti-tumor molecular mechanism of berberine (BBR)by observing and analyzing its effect on proliferation, apoptosis and autophagy-related gene expression for HCCLM3 cells under high glucose condition. Method:HCCLM3 cells were added into low, medium or high-concentration groups of glucose. It was found in cell counting kit-8(CCK-8)that the high concentration of glucose had the most obvious effect on HCCLM3 cells proliferation. Based on the above experimental result, HCCLM3 cells treated with high concentration of glucose was selected and then different concentrations of berberine (5, 10, 20, 30, 40, 50 μmol·L-1) was added for in vitro intervention for 24 h. Then the effect of each drug group on the proliferation of HCCLM3 cells were studied. At the same time, the control group of metformin was arranged. After that, the changes of apoptosis rate were observed by flow cytometry, and the expression levels of B-cell lymphoma-2(Bcl-2)and autophagy genes Atg5, Beclin1 were detced by Real-time polymerase chain reaction (Real-time PCR). Result:With the increase of glucose concentration, HCCLM3 cell had the strongest migration and proliferation ability in high glucose group (P-1), and the inhibition rate was 33.86%, 40.75% (PPPP PConclusion:BBR could inhibit the proliferation of HCCLM3 cells in high glucose environment. Its inhibition effect for HCCLM3 cells might be achieved by inducing apoptosis of the cells, regulating Bcl-2 and up-regulating the expression levels of autophagy gene Beclin1 and Atg5.Thus BBR plays an anti-tumor role through promoting autophagy in high glucose environment.