Exploring the mechanism of bioactive components of Prunella vulgaris L. in treating hepatocellular carcinoma based on network pharmacology.

In traditional Chinese Medicine, Prunella vulgaris L. (PVL) is potentially effective in the treatment of some human malignancies including hepatocellular carcinoma (HCC). However, the detailed mechanism of action remains unclear. The purpose of this study was to decipher the constitutes of the bioactive ingredients of PVL, and its mechanism against HCC using network pharmacology and in vitro experiments. The bioactive components of PVL were obtained by Traditional Chinese Medicine System Pharmacology Database and Analysis platform database, and the targets of bioactive components of PVL was investigated by Swiss Target Prediction database. HCC related targets were obtained from GEO database, GeneCards database and DisGeNET database, and the gene ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted for annotating the biological function of gene targets. A protein-protein interaction network was constructed using STRING database. Molecular docking of key bioactive ingredients was performed using AutoDock Vina. Cell proliferation and apoptosis were detected by cell counting kit-8 and flow cytometry, respectively. The expression level of the target genes of PI3K/Akt pathway were detected by qPCR. In the present work, 11 bioactive components of PVL were screened out, which acted on 177 potential targets. In addition, 13,517 genes were strongly associated with HCC pathogenesis, of which 158 targets are overlapped with PVL's targets. KEGG results identified 39 signaling pathways closely associated with the 158 targets. Molecular docking showed that the main bioactive components of PVL, kaempferol, morin, quercetin, luteolin, and spinasterol, had good binding activity with the core proteins in cancer biology such as AKT1, EGFR, SRC, ESR1, and PPARG. In vitro assays showed that quercetin, one of the main components of PVL extracts effectively inhibited HCC cell proliferation, and promoted apoptosis, which may be associated with PI3K/AKT signaling pathway. In summary, PVL may regulate HCC progression by regulating core targets such as AKT1, EGFR, SRC, ESR1, and PPARG, and acting on PI3K-Akt signaling pathway.

ß-Patchoulene represses hypoxia-induced proliferation and epithelial-mesenchymal transition of liver cancer cells.

Hepatocellular carcinoma (HCC) is a malignant tumor originating from liver epithelial cells with a high clinical mortality rate. ß-Patchoulene (ß-PAE) is a compound extracted from patchouli, which has analgesic, anti-inflammatory and antioxidant effects. This research aims to probe the impacts of ß-PAE on hypoxia-induced HCC cell proliferation and epithelial-mesenchymal transition (EMT). Firstly, hypoxic injury models were constructed in HCC Huh-7 and MHCC97 cells, and the hypoxic injury cell models were then treated with different concentrations of ß-PAE. The cell viability, proliferation, migration, invasion and apoptosis were checked by the cell counting kit-8 (CCK-8) assay, colony formation assay, Transwell assay, flow cytometry and terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. The expression of Survivin protein, EMT markers and the NF-κB/HIF-1α pathway was gauged by Western blot (WB) or cellular immunofluorescence or reverse transcription-polymerase chain reaction (RT-PCR). The in-vivo experiment was conducted to confirm the anti-tumor role of ß-PAE. As a result, ß-PAE abated hypoxia-induced HCC cell growth, proliferation, migration, invasion and EMT and facilitated apoptosis in vitro and in vivo dose-dependently. Further mechanism studies displayed that ß-PAE inactivated the NF-κB/HIF-1α pathway, and HIF-1α activation significantly reversed the ß-PAE-mediated tumor inhibition. ß-PAE repressed the proliferation and EMT of hypoxia-induced HCC cells by choking the NF-κB/HIF-1α pathway, suggesting that ß-PAE was a potential drug for HCC treatment.

LncRNA DARS-AS1 aggravates the growth and metastasis of hepatocellular carcinoma via regulating the miR-3200-5p-Cytoskeleton associated protein 2 (CKAP2) axis.

Accumulating signs have found that long noncoding RNAs (lncRNAs) contribute to hepatocellular carcinoma (HCC). Here, we probed the effect and mechanism of lncRNA DARS-AS1 in HCC. The profiles of DARS-AS1 and Cytoskeleton associated protein 2 (CKAP2) in 50 HCC tissues and non-tumor tissues were examined by real-time quantitative polymerase chain reaction (RT-qPCR). DARS-AS1 and CKAP2 overexpression and/or knockdown cell models were established. The proliferation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) were determined. CKAP2, and focal adhesion kinase (FAK)-extracellular signal-regulated kinase (ERK) was tested by Western blot (WB). The relationship between DARS-AS1 and CKAP2 was predicted by Bioinformatics, and the dual-luciferase reporter assay was applied to verify the targeting association between miR-3200-5p and DARS-AS1 and CKAP2. DARS-AS1 was overexpressed in HCC tissues (vs. that in non-tumor tissues) and was closely correlated with the patients' tumor stage. DARS-AS1 facilitated HCC cell proliferation and hampered apoptosis. HCC cell migration and EMT were enhanced by DARS-AS1. DARS-AS1 up-regulated CKAP2, which aggravated HCC. Further investigation illustrated that either DARS-AS1 or CKAP2 activated FAK-ERK pathway, and miR-3200-5p was competitively restrained by DARS-AS1. miR-3200-5p exerted tumor-suppressive effects in HCC and inactivated CKAP2 and FAK-ERK pathway. All in all, this study corroborates that DARS-AS1 facilitates HCC proliferation and metastasis by regulating miR-3200-5p-mediated CKAP2, which provides a potential target for HCC diagnosis and treatment.Abbreviations: CCK-8: cell counting kit-8; CKAP2: Cytoskeleton associated protein 2; cDNA:complementary DNA; DAPI: 4',6-diamidino-2-phenylindole; DARS-AS1: DARS1 antisense RNA 1; DEPC: diethyl pyrocarbonate; DMEM-F12: Dulbecco's minimal essential medium/Ham's-F12; EMT: epithelial-mesenchymal transition; ERK: extracellular signal-regulated kinase; FAK: focal adhesion kinase; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HE: hematoxylin-eosin; IHC: Immunohistochemistry; LIHC: Liver hepatocellular carcinoma; lncRNAs: long noncoding RNAs; MIAT: lncRNA myocardial infarction-related transcripts; MT: Mutant; NC: negative control; PBS: phosphate-buffered saline; PMSF: Phenylmethylsulfonyl fluoride; PVDF: polyvinylidene difluoride; RT: room temperature; RT-qPCR: real-time quantitative polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPF: specific pathogen-free; TMAP: tumor-associated microtubule-associated protein; TUNEL: TdT-mediated dUTP nick end labeling; V: volume; WT: wild type.

Downregulation of thymopoietin by miR-139-5p suppresses cell proliferation and induces cell cycle arrest/apoptosis in pancreatic ductal adenocarcinoma.

MicroRNAs (miRNAs) serve a pivotal role in tumor development and progression, in which miRNA (miR)-139-5p functions as a tumor suppressor. However, the functions and mechanisms of miR-139-5p in pancreatic ductal adenocarcinoma (PDAC) remain unclear. In the present study, it was found that miR-139-5p was markedly decreased in PDAC tissues and cell lines. Noticeably, thymopoietin (TMPO) was predicted and confirmed as a direct target of miR-139-5p using a luciferase reporter system. The expression level of miR-139-5p was inversely associated with the expression of TMPO in PDAC specimens. A series of gain-of-function assays elucidated that the overexpression of miR-139-5p suppressed cell proliferation, and induced cell cycle arrest and cell apoptosis, determined with a Cell Counting Kit-8, colony formation assays and flow cytometry, respectively. Furthermore, the re-expression of TMPO eliminated the effects of miR-139-5p on cell proliferation, cell cycle progression and apoptosis. In summary, these findings demonstrated that miR-139-5p may be a tumor suppressor in PDAC, which may be useful in developing promising therapies for PDAC.

MicroRNA-212 inhibits hepatocellular carcinoma cell proliferation and induces apoptosis by targeting FOXA1.

MircroRNA-212 (miR-212) is proposed as a novel tumor-related miRNA and has been found to be significantly deregulated in human cancers. In this study, the miR-212 expression was found to be obviously downregulated in hepatocellular carcinoma (HCC) tissues as compared with adjacent nontumor tissues. Clinical association analysis indicated that low expression of miR-212 was prominently correlated with poor prognostic features of HCC, including high AFP level, large tumor size, high Edmondson-Steiner grading, and advanced tumor-node-metastasis tumor stage. Furthermore, the miR-212 expression was an independent prognostic marker for predicting both 5-year overall survival and disease-free survival of HCC patients. Our in vitro studies showed that upregulation of miR-212 inhibited cell proliferation and induced apoptosis in HepG2 cells. On the contrary, downregulation of miR-212 promoted cell proliferation and suppressed apoptosis in Huh7 cells. Interestingly, we found that upregulation of miR-212 decreased FOXA1 expression in HepG2 cells. Significantly, FOXA1 was identified as a direct target of miR-212 in HCC. FOXA1 was downregulated in HCC tissues as compared with noncancerous tissues. An inverse correlation between FOXA1 and miR-212 expression was observed in HCC tissues. Notably, FOXA1 knockdown inhibited cell proliferation and induced apoptosis in HepG2 cells. In conclusion, miR-212 is a potent prognostic marker and may suppress HCC tumor growth by inhibiting FOXA1 expression.