BACKGROUND: To explore the anti-tumor and anti-virus key active ingredients of Sini Decoction Plus Ginseng Soup (SNRS) and their mechanisms.
METHODS: The main ingredients of SNRS were analyzed by network pharmacology, and quercetin was identified as the key active ingredient. Then, we obtained the targets of quercetin by using Drugbank, PharmMapper, and SwissTargetPrediction databases. Then, the targets of HBV-related hepatocellular carcinoma (HBV-related HCC) were obtained by using Genecards database. In addition, using the gene expression profiles of HBV-related HCC patients in GEO database and the genes with the greatest survival difference in GEPIA 2 database identified the potential targets of quercetin. In addition, the mechanism of potential genes was studied through GO, KEGG analysis, and PPI network. Using AUC and survival analysis to evaluate the diagnostic and prognostic value of cyclin-dependent kinase 1 (CDK1) and CCNB1. Finally, the effects of quercetin on proliferation of Hep3B and HepG2215 cells and the level of CDK1 and CCNB1 were verified in vitro. ELISA was used to measure the expression levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) after the intervention by quercetin for 24 h and 48 h in HepG2215 cell.
RESULTS: The first 10 key ingredients of SNRS were identified, and quercetin was the most key ingredient. The 101 potential quercetin targets were identified for the treatment of HBV-related HCC. GO and KEGG showed that 101 potential target enrichment in cancer and cell cycle regulation. By Venn analysis, CDK1 and CCNB1 were intersection targets, which could be used as potential targets for the action of quercetin on HBV-related HCC. Moreover, the expression of CDK1 and CCNB1 was highly expressed in the high-risk group, while the OS rate was low. The 1-year, 3-year and 5-year area under the curve (AUC) curves of CDK1 and CCNB1 were 0.724, 0.676, 0.622 and 0.745, 0.678, 0.634, respectively. Moreover, experimental results also showed that quercetin inhibited cell proliferation and reduced CDK1 expression in Hep3B and HepG2215 cells. The expressions of HBsAg and HBeAg in HepG2215 cell supernatant and cell gradually decreased with the increase of intervention time of quercetin and CDK1 inhibitor.
CONCLUSIONS: Quercetin is a key ingredient of anti-HBV-related HCC activity and inhibits HBV replication in SNRS by inhibiting CDK1.

OBJECTIVE: The cell cycle-related proteins cyclin B1 (CCNB1) and cyclin B2 (CCNB2) are potentially involved in the underlying mechanisms of psoriasis. The present study aimed to explore this possibility using bioinformatics approaches.
METHODS: CCNB1 and CCNB2 protein levels were evaluated in 14 psoriasis patients and five healthy controls by enzyme-linked immunosorbent assays, and their mRNA levels were evaluated using data from four publicly available datasets (GSE53552, GSE41664, GSE14905, and GSE13355). Comparison of high- and low-expressing groups were performed to reveal CCNB1- and CCNB2-related differentially expressed genes, which were then assessed based on gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Correlation analyses between CCNB1 and CCNB2 levels and immune infiltration, as well as typical targets of psoriasis, were also performed.
RESULTS: Overall, 12 CCNB1 and CCNB2 common immune-related targets potentially involved in psoriasis were identified. These could regulate the cell cycle of through multiple pathways. In addition, CCNB1 and CCNB2 were found to potentially support the release of key molecular targets of psoriasis through the regulation of mast cell activation and macrophage polarization.
CONCLUSIONS: These findings suggest that CCNB1 and CCNB2 may represent valuable molecular biomarkers of psoriasis, contributing to its onset and progression.

To study the effect of integrin αvβ6 on the proliferation and apoptosis of thyroid carcinoma cells.
The experiment was conducted on 3 groups : the control group, the positive observation group (in which the ανβ6 on the surface of the thyroid carcinoma cell line SW579 was blocked by monoclonal antibody 10D5) and the negative observation group (in which the ανβ6 was dealt with the negative placebo of 10D5-the IgG2a). Cell proliferation was detected by MTT assay, apoptosis by flow cytometry and the protein levels in Caspase-3, CyclinB1 and Bcl-xl as well as the protein levels in ERK, p-ERK, JNK, p-JNK, p38 and p-p38 were detected by Western blot.
The cell survival rates of the control group and the negative observation group were prominently higher than those of the positive observation group, following decrease in the apoptosis rates, and the differences were statistically significant (p<0.05). The protein levels in CyclinB1 and Bcl-x1 of the control group and the negative observation group were prominently higher than those of the positive observation group, whereas the levels in Caspase-3 were decreased; the differences were statistically significant (p<0.05). The protein levels in p-ERK, p-JNK and p-p38 of the control group and the negative observation group were prominently higher than those of the positive observation group, while the protein levels of ERK, JNK and p38 showed no difference.
Integrin ανβ6 can mediate the MAPK signal pathway of the cells and regulate the expression of CyclinB1 and the apoptosis-related proteins like Bcl-x1 and Caspase-2, thus affecting the process of the proliferation and apoptosis of thyroid carcinoma cells.

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2\'-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

The CCNB1 and CDK1 genes encode the proteins of CyclinB1 and CDK1 respectively, which interact with each other and are involved in cell cycle regulation, centrosome duplication and chromosome segregation. This study aimed to investigate whether the genetic variants in these two genes may affect breast cancer (BC) susceptibility, progression, and survival in Chinese Han population using haplotype-based analysis. A total of ten tSNPs spanning from 2kb upstream to 2kb downstream of these genes were genotyped in 1204 cases and 1204 age-matched cancer-free controls. The haplotype blocks were determined according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. For CCNB1, rs2069429 was significantly associated with increased BC susceptibility under recessive model (OR=2.352, 95%CI=1.480-3.737), so was the diplotype TAGT/TAGT (OR=1.947 95%CI=1.154-3.284, P=0.013). In addition, rs164390 was associated with Her2-negative BC. For CDK1, rs2448343 and rs1871446 were significantly associated with decreased BC risk under dominant models, so was the haplotype ATATT. These two SNPs also showed a dose-dependent effect on BC susceptibility. Using stratified association analysis, we found that women with the heterozygotes or minor allele homozygotes of rs2448343 had much less BC susceptibility among women with BMI<23. In CDK1, three closely located SNPs, rs2448343, rs3213048 and rs3213067, were significantly associated with tumor\'s PR status: the heterozygotes of rs2448343 were associated with PR-positive tumors, while the minor allele homozygotes of rs3213048 and heterozygotes of rs3213067 were associated with PR-negative BC tumors. In survival analysis, rs1871446 was associated with unfavorable event-free survival under recessive model, so was the CDK1 diplotype ATATG/ATATG, which carried the minor allele homozygote of rs1871446. Our study indicates that genetic polymorphisms of CCNB1 and CDK1 are related to BC susceptibility, progression, and survival in Chinese Han women. Further studies need to be performed in other populations as an independent replication to verify these results.

BACKGROUND: The imprinted gene dlk1 has been recognized as a cancer related gene since it aberrantly expressed in a series of cancer tissues, but its role in lung cancer is still unknown. The aim of this study is to examine dlk1\'s expression in non-small cell lung cancers (NSCLCs) and investigate the molecular mechanism by which dlk1 could accelerate the proliferation of the cells in lung cancer cell lines (H520).
METHODS: The relative expression of dlk1 among 30 NSCLC specimens and their adjacent normal lung tissues were analyzed by RT-PCR. A cell model that stably expressed exogenous dlk1 was established following that the dlk1 gene was cloned into a eukaryotic expression vector and then transfected into the lung cancer cells H520. CCK8 analysis and colony forming assay were employed to investigate the effect of dlk1 on cell proliferation. The expression of CyclinB1 was detected by Western blot.
RESULTS: dlk1 aberrantly expressed in 36.7% (11/30) of the tumor tissues of NSCLC compared with their adjacent cancer lung tissues. CCK8 analysis showed that overexpression of dlk1 could promote the proliferation of H520 cells (P < 0.05) and the results was further confirmed by colony forming assay. Western blot analysis found that over expression of dlk1 could up-regulate the expression of CyclinB1 (P < 0.05).
CONCLUSIONS: dlk1 aberrantly expressed in NSCLCs. The Overexpression of dlk1 could accelerate the proliferation of lung cancer cells H520 in vitro, probably through up-regulating the expression of cell cycle protein CyclinB1.

BACKGROUND: Stage IIIA non-small cell lung cancer (NSCLC) with ipsilateral mediastinal lymph node metastases (N2) is a heterogeneous disease with differing prognoses. In this study, we retrospectively investigated the prognostic value of the expression of 10 molecular markers in 87 patients with stage IIIA pN2 NSCLC treated with radical surgery.
METHODS: Primary tumor tissue microarrays (TMAs) were constructed and sections used for immunohistochemical analysis of epidermal growth factor receptor, ErbB-2, c-kit, cyclooxygenase-2, survivin, bcl-2, cyclin D1, cyclin B1, metalloproteinase (MMP)-2, and MMP-9. Univariate and multivariate analyses and unsupervised hierarchical clustering analysis of clinical pathologic and immunostaining data were performed.
RESULTS: Bcl-2 (p < 0.0001) and cyclin D1 (p = 0.015) were more highly expressed in squamous cell carcinoma (SCC), whereas MMP-2 (p = 0.009), MMP-9 (p = 0.005), and survivin (p = 0.032) had increased expression in other histologic subtypes. In univariate analysis, SCC histology and cyclin D1 expressions were favorable prognostic factors (p = 0.015 and p < 0.0001, respectively); by contrast, MMP-9 expression was associated with worse prognosis (p = 0.042). In multivariate analysis, cyclin D1 was the only positive prognostic factor (p < 0.0001). Unsupervised hierarchical clustering analysis of TMA immunostaining data identified five distinct clusters. They formed two subsets of patients with better (clusters 1 and 2) and worse (clusters 3, 4, and 5) prognoses, and median survival of 51 and 10 months, respectively (p < 0.0001). The better prognosis subset mainly comprised patients with SCC (80%).
CONCLUSIONS: Hierarchical clustering of TMA immunostaining data using a limited set of markers identifies patients with stage IIIA pN2 NSCLC at high risk of recurrence, who may benefit from more aggressive treatment.

The heavy metals lead (Pb) and mercury (Hg) pose potential risks to sustainability of environment and thus to our future generations. General objective of this in vitro study was to examine the secretory activity of porcine ovarian granulosa cells after Pb and Hg administration and to outline the potential intracellular mediators of its effects. For this purpose, release of insulin-like growth factor I (IGF-I) and steroid hormone progesterone (P(4)), expression of proliferation- related (cyclin B1) and apoptosis-related (caspase-3) peptides was examined in porcine ovarian granulosa cells after heavy metals administration. Obtained data indicate Pb-induced inhibition of IGF-I release at lower doses (0.063 mg/mL and 0.046 mg/mL) by ovarian granulosa cells. However, P(4) release was not influenced by Pb addition, while the expression of cyclin B1 and caspase-3 was induced by Pb addition. These results indicate that Pb can affect the pathway of proliferation and apoptosis of porcine ovarian granulosa cells through intracellular substances such as cyclin B1 and caspase-3. On the other hand, the P(4) release by ovarian granulosa cells of pregnant gilts was stimulated by experimental Pb administration at doses of 0.25 mg/mL and 0.063 mg/mL and experimental Hg administration at doses 0.25 mg/mL and 0.083 mg/mL. P(4) release by ovarian cells of pregnant gilts was not influenced by a combinatory dose of FSH (1.0 ng/mL) + Pb (0.083 mg/mL) + Hg (0.083 mg/mL) but it was inhibited by experimental administration of FSH (10 ng/mL) + Pb (0.25 ng/mL) + Hg (0.25 ng/mL). Possible involvement of heavy metals - Pb and Hg and pituitary hormone FSH, in the regulation of P(4) release by porcine ovarian granulosa cells of pregnant gilts was noted. Data obtained from in vitro studies suggest the dose dependent association of heavy metals administration with the hormonal release by porcine ovarian granulosa cells. This association also depended on pregnancy of the gilts.

The chronic usage of nifedipine is associated with the appearance of gingival overgrowth (GO). The frequency of GO associated with chronic nifedipine therapy remains controversial and the possible subclinical effects of this drug on the gingival epithelium should be investigated. We investigated the epithelial proliferation index and apoptosis rate, and their association with epithelial enlargement. Proliferation (Ki67 and Cyclin B1) and apoptosis (BCL2, Bax and p53) markers were identified by immunohistochemistry in twenty-one samples of gingival tissue from patients undergoing chronic treatment with nifedipine and in eleven samples of gingival tissue from healthy patients who did not use drugs associated with GO (control). Our results show that the epithelial tissue of nifedipine users has considerably longer rete pegs compared to control (P = 0.01). However, the density of Ki67(+) and Cyclin B1(+) cells was similar in both groups. Regarding apoptosis, we found more BCL2(+) cells in the nifedipine group when compared to controls (P = 0.12). An increase in Bax(+) cells in the nifedipine group compared to control (P = 0.003) was also seen, and slightly lower levels of p53(+) expression were observed (P = 0.51). Our results suggest that the chronic use of nifedipine is not associated with subclinical changes in gingival tissue.

OBJECTIVE: Previous researches showed that down-regulating the expression of cyclin B1 in tumor cells by RNA interference may inhibit tumorigenesis, but the mechanism remains to be clarified. This study was to reveal the molecular mechanism of antisense cyclin B1 in tumorigenesis inhibition by comparative proteomic technique.
METHODS: A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin B1 was transfected into mouse colon carcinoma cell line CT26. Total proteins of transfected cells and control cells were extracted and separated by two-dimensional gel electrophoresis (2-DE). The differential expression proteins were analyzed with PDQuest software, and identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Mascot database searching. The 2 differential proteins with the highest confidence of the peptides were selected and verified by Western blot.
RESULTS: Seven differentially expressed proteins were identified: Axin2, CCTtheta, DR5, and HPCM27 were up-regulated in transfected cells, while RFP17, mKIAA1195, and LOC77035 were down-regulated. The expression abundance differences of Axin2 and DR5, with the highest confidence, were verified by Western blot.
CONCLUSIONS: Several proteins expressed differentially in CT26 cells after transfection of antisense cyclin B1, which take part in some signal pathways in cell proliferation, differentiation, migration, apoptosis, and transcriptional control. The antitumor effect of antisense cyclin B1 may relate to the interplay of the above proteins.