miR-3,178 contributes to the therapeutic action of baicalein against hepatocellular carcinoma cells via modulating HDAC10.

Hepatocellular carcinoma (HCC) is the most common type of hepatic malignancies with high mortality and poor prognosis. Baicalein, one of the major and bioactive flavonoids isolated from Scutellaria baicalensis Georgi, which is reported to have anti-proliferation effect in varying cancers, including HCC, whose underlying molecular mechanism is still largely unknown. In this study, we found that baicalein significantly inhibited proliferation and colony formation, blocked cell cycle, and promoted apoptosis in HCC cells MHCC-97H and SMMC-7721 in vitro and reduced tumor volume and weight in vivo. Increased microRNA (miR)-3,178 levels and decreased histone deacetylase 10 (HDAC10) expression were found in cells treated with baicalein and in patients' HCC tissues. HDAC10 was identified as a target gene of miR-3,178 by luciferase activity and western blot. Both baicalein treatment and overexpression of miR-3,178 could downregulate HDAC10 protein expression and inactivated AKT, MDM2/p53/Bcl2/Bax and FoxO3α/p27/CDK2/Cyclin E1 signal pathways. Not only that, knockdown of miR-3,178 could partly abolish the effects of baicalein and the restoration of HDAC10 could abated miR-3,178-mediated role in HCC cells. Collectively, baicalein inhibits cell viability, blocks cell cycle, and induces apoptosis in HCC cells by regulating the miR-3,178/HDAC10 pathway. This finding indicated that baicalein might be promising for treatment of HCC.

MiR-3663-3p participates in the anti-hepatocellular carcinoma proliferation activity of baicalein by targeting SH3GL1 and negatively regulating EGFR/ERK/NF-κB signaling.

Baicalein is a purified flavonoid that exhibits anticancer effects in hepatocellular carcinoma (HCC). However, its underlying molecular mechanisms remain largely unclear. In this study, we found that baicalein inhibited HCC cell growth, induced apoptosis, and blocked cell cycle arrest at the S phase in vitro, as well as reduced HCC tumor volume and weight in vivo. Quantitative reverse transcriptase-PCR (qRT-PCR) results suggested that miR-3663-3p was downregulated in HCC tissues. After baicalein treatment, miR-3663-3p expression was upregulated in HCC cells. Transfection of miR-3663-3p suppressed HCC cell proliferation and colony formation, increased the proportion of apoptotic cells in vitro, and reduced the volume and weight of tumors in vivo. The results of dual-luciferase reporter assay showed that miR-3663-3p could directly bind to the 3'-UTR of SH3GL1. SH3GL1 overexpression partly reduced the growth-inhibiting effect of miR-3663-3p. Both baicalein treatment and miR-3663-3p overexpression downregulated the expression of SH3GL1 and inactivated the Erk1/2, p-NF-κB/p65, and EGFR signaling pathways. Overall, our data suggest that baicalein may act as a novel HCC suppressor, and that the miR-3663-3p/SH3GL1/EGFR/ERK/NF-κB pathway plays a vital role in HCC progression. Thus, baicalein treatment or miR-3663-3p induction may be a promising strategy for HCC therapy.

Silencing of long noncoding RNA HOXD-AS1 inhibits proliferation, cell cycle progression, migration and invasion of hepatocellular carcinoma cells through MEK/ERK pathway.

Accumulating findings reveal that long noncoding RNAs (lncRNAs) as crucial regulatory molecules serve vital functions in the progression of hepatocellular carcinoma (HCC). This study aims to investigate the biological roles and mechanisms of lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) in HCC cells based on transcriptome analysis. The Cancer Genome Atlas data analysis and experimental validation showed that HOXD-AS1 was increased in HCC tissues/cell lines and positively relevant to histologic grade. The subcellular localization results indicated HOXD-AS1 was dispersed both in the nucleus as well as the cytoplasm of HCC cells. In vitro loss-of-function experiments revealed that silencing of HOXD-AS1 could dramatically suppress the proliferation, migration, and invasion, and induce S or/and G2/M phase cell cycle arrest as well as apoptosis of Bel-7402 and MHCC97H cells accompanying the changes in expression levels of cyclin B1, cyclin D1, BCL-2, BAX, and MMP2. In vivo assay also showed that HOXD-AS1 silencing could markedly reduce xenograft tumor volume and weight of HCC cells. Transcriptome and bioinformatic analysis indicated that a total of 1103 genes were significantly altered by HOXD-AS1 silencing, of which 132 genes exhibited a significant correlation with HOXD-AS1 expression in HCC tissues. Gene Ontology (GO) enrichment analysis revealed differentially expressed genes were remarkably enriched in several cancer-related biological processes (cell proliferation, cell cycle, apoptosis, migration, angiogenesis, and hypoxic response). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that HOXD-AS1 has the potential to affect p53, tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK) pathway, and Western blot results further validated that HOXD-AS1 silencing could inhibit the MEK/ERK pathway in Bel-7402 cells. Collectively, HOXD-AS1, as an oncogenic lncRNA, might exert crucial functions in HCC progression and serve as a potential diagnostic biomarker and therapeutic target for HCC.

Noncoding RNAs: Potential players in the self-renewal of mammalian spermatogonial stem cells.

Spermatogonial stem cells (SSCs), a unique population of male germ cells with self-renewal ability, are the foundation for maintenance of spermatogenesis throughout the life of the male. Although many regulatory molecules essential for SSC self-renewal have been identified, the fundamental mechanism underlying how SSCs acquire and maintain their self-renewal activity remains largely to be elucidated. In recent years, many types of noncoding RNAs (ncRNAs) have been suggested to regulate the SSC self-renewal through multiple ways, indicating ncRNAs play crucial roles in SSC self-renewal. In this paper, we mainly focus on four types of ncRNAs including microRNA, long ncRNA, piwi-interacting RNA, as well as circular RNAs, and reviewed their potential roles in SSC self-renewal that discovered recently to help us gain a better understanding of molecular mechanisms by which ncRNAs perform their function in regulating SSC self-renewal.

Baicalein sensitizes hepatocellular carcinoma cells to 5-FU and Epirubicin by activating apoptosis and ameliorating P-glycoprotein activity.

Hepatocellular carcinoma (HCC) has a dismal prognosis in part because of multi-drug resistance (MDR). Baicalein is a flavonoid extracted from Radix Scutellariae with anti-HCC activity. We tested the effects of Baicalein on multi-drug resistant HCC cells (Bel7402/5-FU) known to be resistant to the anticancer drugs 5-FU and Epirubicin. Flow cytometry analysis showed that treatment with 5??g/ml and 10??g/ml Baicalein resulted in increases in the intra-cellular concentrations of Rho123 and Epirubicin in the corresponding group of cells compared to untreated cells, illustrating that Baicalein reverses MDR in Bel7402/5-FU cells. Bel7402/5-FU cells displayed increased P-glycoprotein (P-gp)-mediated drug efflux. However, this efflux was inhibited in cells pre-incubated in Baicalein for 48?h. Moreover, Baicalein induced apoptosis and autophagy and decreased P-gp and Bcl-xl expression levels. All of these results indicate that Baicalein can reverse P-gp-mediated MDR in HCC and may thus be useful for the treatment of drug-resistant HCC.

Emodin and baicalein inhibit sodium taurocholate-induced vacuole formation in pancreatic acinar cells.

AIM: To investigate the effects of combined use of emodin and baicalein (CEB) at the cellular and organism levels in severe acute pancreatitis (SAP) and explore the underlying mechanism. METHODS: SAP was induced by retrograde infusion of 5% sodium taurocholate into the pancreatic duct in 48 male SD rats. Pancreatic histopathology score, serum amylase activity, and levels of tumour necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-10 were determined to assess the effects of CEB at 12 h after the surgery. The rat pancreatic acinar cells were isolated from healthy male SD rats using collagenase. The cell viability, cell ultrastructure, intracellular free Ca2+ concentration, and inositol (1,4,5)-trisphosphate receptor (IP3R) expression were investigated to assess the mechanism of CEB. RESULTS: Pancreatic histopathology score (2.07 ± 1.20 vs 6.84 ± 1.13, P < 0.05) and serum amylase activity (2866.2 ± 617.7 vs 5241.3 ± 1410.0, P < 0.05) were significantly decreased in the CEB (three doses) treatment group compared with the SAP group (2.07 ± 1.20 vs 6.84 ± 1.13, P < 0.05). CEB dose-dependently reduced the levels of the pro-inflammatory cytokines IL-6 (466.82 ± 48.55 vs 603.50 ± 75.53, P < 0.05) and TNF-α (108.04 ± 16.10 vs 215.56 ± 74.67, P < 0.05) and increased the level of the anti-inflammatory cytokine IL-10 (200.96 ± 50.76 vs 54.18 ± 6.07, P < 0.05) compared with those in the SAP group. CEB increased cell viability, inhibited cytosolic Ca2+ concentration, and significantly ameliorated intracellular vacuoles and IP3 mRNA expression compared with those in the SAP group (P < 0.05). There was a trend towards decreased IP3R protein in the CEB treatment group; however, it did not reach statistical significance (P > 0.05). CONCLUSION: These results at the cellular and organism levels reflect a preliminary mechanism of CEB in SAP and indicate that CEB is a suitable approach for SAP treatment.

Baicalein: A review of its anti-cancer effects and mechanisms in Hepatocellular Carcinoma.

Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is a flavonoid compound derived from the roots of Scutellaria baicalensis. It has historically been used in anti-oxidant, anti-virus, anti-bacteria, anti-inflammatory and anti-allergic therapies. Recently, baicalein has been found to possess anti-cancer activities via its effect on a variety of biological processes involving cell proliferation, metastasis, apoptosis and autophagy and so on. Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and high fatality rate worldwide. Noteworthy, treatment protocols of HCC include conventional resection and chemotherapy, all of which may result in enormous mortality rate. Therefore, there is extreme interest to find a relatively non-toxic medicine which may reduce side effects without compromising therapeutic efficacy. Many studies have showed that baicalein is one such potential candidate. In this review, we summarized the various anti-cancer effects of baicalein on HCC and their underlying molecular mechanisms based on in vitro and in vivo experimental evidences discovered so far. Taken together, baicalein may be developed as a potential, novel anticancer drug for HCC treatment.

Baicalein, a Natural Anti-Cancer Compound, Alters MicroRNA Expression Profiles in Bel-7402 Human Hepatocellular Carcinoma Cells.

BACKGROUND/AIMS: Baicalein has been shown to possess significant anti-hepatoma activity by inhibiting cell proliferation. Whether the anti-proliferative effect of baicalein is related to its modulation of miRNA expression in hepatocellular carcinoma (HCC) is still unknown. METHODS: The anti-proliferative effects of baicalein on HCC cell line Bel-7402 was assessed by detecting the proliferation activity, cell cycle distribution, expression changes of p21/CDKN1A, P27/CDKN1B, total Akt and phosphoryted AKT. Microarray analysis was conducted to determine the miRNA expression profiles in baicalein-treated or untreated Bel-7402 cells and then validated by qRT-PCR in two HCC cell lines (Bel-7402 and Hep3B). The gain-of-function of miR-3127-5p was performed by detecting anti-proliferative effects after transfecting miRNA mimics in cells. Finally, the expression level of miR-3127-5p in different HCC cell lines was determined by qRT-PCR. RESULTS: Baicalein was able to inhibit the proliferation of Bel-7402 cells by inducing cell cycle arrest at the S and G2/M phase via up-regulating the expression of p21/CDKN1A and P27/CDKN1B and suppressing the PI3K/Akt pathway. Baicalein could alter the miRNA expression profiles in Bel-7402 cells. Putative target genes for differentially expressed miRNAs could be enriched in terms of cell proliferation regulation, cell cycle arrest and were mainly involved in MAPK, PI3K-Akt, Wnt, Hippo and mTOR signaling pathways. MiR- 3127-5p, one of up-regulated miRNAs, exhibits low expression level in several HCC cell lines and its overexpression could inhibit cell growth of Bel-7402 and Hep3B cell lines by inducing S phase arrest by up-regulating the expression of p21and P27 and repressing the PI3K/Akt pathway. CONCLUSIONS: Modulation of miRNA expression may be an important mechanism underlying the anti-hepatoma effects of baicalein.

Micro-trichome as a class I homeodomain-leucine zipper gene regulates multicellular trichome development in Cucumis sativus.

Plant trichomes serve as a highly suitable model for investigating cell differentiation at the single-cell level. The regulatory genes involved in unicellular trichome development in Arabidopsis thaliana have been intensively studied, but genes regulating multicellular trichome development in plants remain unclear. Here, we characterized Cucumis sativus (cucumber) trichomes as representative multicellular and unbranched structures, and identified Micro-trichome (Mict), using map-based cloning in an F2 segregating population of 7,936 individuals generated from a spontaneous mict mutant. In mict plants, trichomes in both leaves and fruits, are small, poorly developed, and denser than in the wild type. Sequence analysis revealed that a 2,649-bp genomic deletion, spanning the first and second exons, occurred in a plant-specific class I homeodomain-leucine zipper gene. Tissue-specific expression analysis indicated that Mict is strongly expressed in the trichome cells. Transcriptome profiling identified potential targets of Mict including putative homologs of genes known in other systems to regulate trichome development, meristem determinacy, and hormone responsiveness. Phylogenic analysis charted the relationships among putative homologs in angiosperms. Our paper represents initial steps toward understanding the development of multicellular trichomes.

Long non-coding RNAs: critical players in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a complex disease with multiple underlying pathogenic mechanisms caused by a variety of etiologic factors. Emerging evidence showed that long non-coding RNAs (lncRNAs), with size larger than 200 nucleotides (nt), play important roles in various types of cancer development and progression. In recent years, some dysregulated lncRNAs in HCC have been revealed and roles for several of them in HCC have been characterized. All these findings point to the potential of lncRNAs as prospective novel therapeutic targets in HCC. In this review, we summarize known dysregulated lncRNAs in HCC, and review potential biological roles and underlying molecular mechanisms of lncRNAs in HCC. Additionally, we discussed prospects of lncRNAs as potential biomarker and therapeutic target for HCC. In conclusion, this paper will help us gain better understanding of molecular mechanisms by which lncRNAs perform their function in HCC and also provide general strategies and directions for future research.

Ectopic expression of CsCTR1, a cucumber CTR-like gene, attenuates constitutive ethylene signaling in an Arabidopsis ctr1-1 mutant and expression pattern analysis of CsCTR1 in cucumber (Cucumis sativus).

The gaseous plant hormone ethylene regulates many aspects of plant growth, development and responses to the environment. Constitutive triple response 1 (CTR1) is a central regulator involved in the ethylene signal transduction pathway. To obtain a better understanding of this particular pathway in cucumber, the cDNA-encoding CTR1 (designated CsCTR1) was isolated from cucumber. A sequence alignment and phylogenetic analyses revealed that CsCTR1 has a high degree of homology with other plant CTR1 proteins. The ectopic expression of CsCTR1 in the Arabidopsis ctr1-1 mutant attenuates constitutive ethylene signaling of this mutant, suggesting that CsCTR1 indeed performs its function as negative regulator of the ethylene signaling pathway. CsCTR1 is constitutively expressed in all of the examined cucumber organs, including roots, stems, leaves, shoot apices, mature male and female flowers, as well as young fruits. CsCTR1 expression gradually declined during male flower development and increased during female flower development. Additionally, our results indicate that CsCTR1 can be induced in the roots, leaves and shoot apices by external ethylene. In conclusion, this study provides a basis for further studies on the role of CTR1 in the biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.

Tuberculate fruit gene Tu encodes a C2 H2 zinc finger protein that is required for the warty fruit phenotype in cucumber (Cucumis sativus L.).

Cucumber fruits that have tubercules and spines (trichomes) are known to possess a warty (Wty) phenotype. In this study, the tuberculate fruit gene Tu was identified by map-based cloning, and was found to encode a transcription factor (TF) with a single C2 H2 zinc finger domain. Tu was identified in all 38 Wty lines examined, and was completely absent from all 56 non-warty (nWty) lines. Cucumber plants transgenic for Tu (TCP) revealed that Tu was required for the Wty fruit phenotype. Subcellular localization showed that the fusion protein GFP-Tu was localized mainly to the nucleus. Based on analyses of semi-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and mRNA in situ hybridization, we found that Tu was expressed specifically in fruit spine cells during development of fruit tubercules. Moreover, cytokinin (CTK) content measurements and cytological observations in Wty and nWty fruits revealed that the Wty fruit phenotype correlated with high endogenous CTK concentrations. As a result of further analyses on the transcriptomic profile of the nWty fruit epidermis and TCP fruit warts, expression of CTK-associated genes, and hormone content in nWty fruit epidermis, Wty fruit warts and epidermis, and TCP fruit warts and epidermis, we found that Tu probably promoted CTK biosynthesis in fruit warts. Here we show that Tu could not be expressed in the glabrous and tubercule-free mutant line gl that contained Tu, this result that futher confirmed the epistatic effect of the trichome (spine) gene Gl over Tu. Taken together, these data led us to propose a genetic pathway for the Wty fruit trait that could guide future mechanistic studies.