Integrated network pharmacology and metabolomics to reveal the mechanism of Pinellia ternata inhibiting non-small cell lung cancer cells.

Lung cancer is a malignant tumor with highly heterogeneous characteristics. A classic Chinese medicine, Pinellia ternata (PT), was shown to exert therapeutic effects on lung cancer cells. However, its chemical and pharmacological profiles are not yet understood. In the present study, we aimed to reveal the mechanism of PT in treating lung cancer cells through metabolomics and network pharmacology. Metabolomic analysis of two strains of lung cancer cells treated with Pinellia ternata extracts (PTE) was used to identify differentially abundant metabolites, and the metabolic pathways associated with the DEGs were identified by MetaboAnalyst. Then, network pharmacology was applied to identify potential targets against PTE-induced lung cancer cells. The integrated network of metabolomics and network pharmacology was constructed based on Cytoscape. PTE obviously inhibited the proliferation, migration and invasion of A549 and NCI-H460 cells. The results of the cellular metabolomics analysis showed that 30 metabolites were differentially expressed in the lung cancer cells of the experimental and control groups. Through pathway enrichment analysis, 5 metabolites were found to be involved in purine metabolism, riboflavin metabolism and the pentose phosphate pathway, including D-ribose 5-phosphate, xanthosine, 5-amino-4-imidazolecarboxyamide, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Combined with network pharmacology, 11 bioactive compounds were found in PT, and networks of bioactive compound-target gene-metabolic enzyme-metabolite interactions were constructed. In conclusion, this study revealed the complicated mechanisms of PT against lung cancer. Our work provides a novel paradigm for identifying the potential mechanisms underlying the pharmacological effects of natural compounds.

Mechanism Research of PZD Inhibiting Lung Cancer Cell Proliferation, Invasion, and Migration based on Network Pharmacology.

BACKGROUND: A classic Chinese medicine decoction, Pinellia ternata (Thunb.) Breit.-Zingiber officinale Roscoe (Ban-Xia and Sheng-Jiang in Chinese) decoction (PZD), has shown significant therapeutic effects on lung cancer. OBJECTIVE: This study aimed to explore and elucidate the mechanism of action of PZD on lung cancer using network pharmacology methods. METHODS: Active compounds were selected according to the ADME parameters recorded in the TCMSP database. Potential pathways related to genes were identified through GO and KEGG analysis. The compoundtarget network was constructed by using Cytoscape 3.7.1 software, and the core common targets were obtained by protein-protein interaction (PPI) network analysis. Batch molecular docking of small molecule compounds and target proteins was carried out by using the AutoDock Vina program. Different concentrations of PZD water extracts (10, 20, 40, 80, and 160 µg/mL) were used on lung cancer cells. Moreover, MTT and Transwell experiments were conducted to validate the prominent therapeutic effects of PZD on lung cancer cell H1299. RESULTS: A total of 381 components in PZD were screened, of which 16 were selected as bioactive compounds. The compound-target network consisting of 16 compounds and 79 common core targets was constructed. MTT experiment showed that the PZD extract could inhibit the cell proliferation of NCI-H1299 cells, and the IC50 was calculated as 97.34 ± 6.14 µg/mL. Transwell and wound-healing experiments showed that the PZD could significantly decrease cell migration and invasion at concentrations of 80 and 160 µg/mL, respectively. The in vitro experiments confirmed that PZD had significant therapeutic effects on lung cancer cells, mainly through the PI3K/AKT signaling pathway. CONCLUSION: PZD could inhibit the cell proliferation, migration, and invasion of NCI-H1299 cells partially through the PI3K/AKT signaling pathway. These findings suggested that PZD might be a potential treatment strategy for lung cancer patients.

Downregulation of C12orf75 gene inhibits migration and invasion of liver cancer cell via suppressing the Wnt/ß-catenin signaling pathway in vitro.

Hepatic carcinoma (HCC) is the second leading cause of death in cancer patients, and its incidence is on the rise. The prerequisite for the deterioration of liver cancer is the malignant migration and invasion of cancer cells, and in this study the C12orf75 gene was firstly showed that it has high expression characteristics in six HCC cell lines. By bioinformatics analysis, the up-regulation of C12orf75 was negatively correlated with the survival rate of HCC patients. CCK-8 and flow cytometry experiments indicated C12orf75 had no effect on cell proliferation, apoptosis and cycle of HuH-7 and Hep G2 cell lines, respectively. However, transwell assay showed that downregulation of C12orf75 gene could obviously inhibit invasion and migration of liver cancer cell. At the same time, this regulation process has been verified to involve the participation of Wnt/ß-catenin signaling pathway. As a result, all these results showed that C12orf75 gene was a oncogene in liver cancer, which maybe a novel screening and diagnosing biomarker for patients with liver cancer, and this is of great significance for the development of targeted drugs for liver cancer patients.

Astragalus Polysaccharide Suppresses Cell Proliferation and Invasion by Up-Regulation of miR-195-5p in Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer-related mortality, and it has a high risk of early recurrence and distant metastasis. The prerequisite for the deterioration of NSCLC is the malignant proliferation and migration of cancer cells, and in this study Astragalus membranaceus polysaccharides (APS) was firstly showed that it could decrease the cell proliferation of A549 and NCI-H1299. Through bioinformatics analysis, the up-regulation of miR-195-5p was positively correlated with the survival rate of lung cancer patients. Real-time PCR indicated APS could increase the expression level of miR-195-5p, and the miR-195-5p inhibitor was used to verify that it could reverse the inhibitory effect of Astragalus polysaccharide on lung cancer cell migration and invasion. Therefore, we believe that APS could inhibit the proliferation and migration of NSCLC cells by regulating miR-195-5p, which laid the foundation for further research on the functional mechanism of miR-195-5p in NSCLC.

[EMS mutation of suspension cells and selection of high temperature tolerant mutants from Pinellia ternata].

OBJECTIVE: To determine the optimal concentration and processing time of EMS mutation for suspension cells from Pinellia ternata. METHOD: Under four EMS concentration gradients (0.1% , 0.2%, 0.4%, 0.6%) and three processing time gradients (0.5, 1.0, 2.0 h), the suspension cells of P. ternata were mutagenized. RESULT AND CONCLUSION: The results showed that the survival rate was significantly different under the different concentrations of EMS and the different processing time. In the same processing time, the EMS concentrations were increased, but the suspension cells survival rate decreased gradually. The optimum EMS concentration for the mutagenesis was 0.4% and the best processing time was 1 hour.

Preliminary analysis of the mitochondrial genome evolutionary pattern in primates.

Since the birth of molecular evolutionary analysis, primates have been a central focus of study and mitochondrial DNA is well suited to these endeavors because of its unique features. Surprisingly, to date no comprehensive evaluation of the nucleotide substitution patterns has been conducted on the mitochondrial genome of primates. Here, we analyzed the evolutionary patterns and evaluated selection and recombination in the mitochondrial genomes of 44 Primates species downloaded from GenBank. The results revealed that a strong rate heterogeneity occurred among sites and genes in all comparisons. Likewise, an obvious decline in primate nucleotide diversity was noted in the subunit rRNAs and tRNAs as compared to the protein-coding genes. Within 13 protein-coding genes, the pattern of nonsynonymous divergence was similar to that of overall nucleotide divergence, while synonymous changes differed only for individual genes, indicating that the rate heterogeneity may result from the rate of change at nonsynonymous sites. Codon usage analysis revealed that there was intermediate codon usage bias in primate protein-coding genes, and supported the idea that GC mutation pressure might determine codon usage and that positive selection is not the driving force for the codon usage bias. Neutrality tests using site-specific positive selection from a Bayesian framework indicated no sites were under positive selection for any gene, consistent with near neutrality. Recombination tests based on the pairwise homoplasy test statistic supported complete linkage even for much older divergent primate species. Thus, with the exception of rate heterogeneity among mitochondrial genes, evaluating the validity assumed complete linkage and selective neutrality in primates prior to phylogenetic or phylogeographic analysis seems unnecessary.

[Study on optimization of SRAP-PCR reaction system for Pinellia ternata in Suzhou].

OBJECTIVE: To investigate the optimization system of SRAP-PCR molecular marker technology in the analysis on Pinellia ternata. METHOD: SRAP-PCR reaction system for P. ternata was optimized by L16 (5(4)) orthogonal design with five elements (dNTPs, Mg2+, the template DNA, primers, Taq enzyme) and four standards. RESULT: The most suitable forward primer for SRAP for Pinellia ternata was 5'-TGAGTCCAAACCGGAAG-3', while the reverse primer was 5'-GACTGCGTACGAATTACG-3'. The optimized reaction system contained 70 ng DNA template, 0.9 micromol x L(-1) primer, 0.20 mmol x L(-1) dNTP s, 1.5 - 2.0 mmol x L(-1) Mg2+, and 2 U Taq enzyme. CONCLUSION: SRAP-PCR system for P. ternata is established to lay a foundation for future construction of SRAP genetic map of P. ternata.

[Changes of total RNA and mRNA differential expressions in leaves of Pinellia ternata under high temperature stress].

OBJECTIVE: To study the total RNA and mRNA differential expression in leaves of Pinellia ternata under high temperature, provide more information of the molecular mechanism of the sprout tumble. METHOD: The total RNA and mRNA differential expression in leaves of P. ternata at different stress time was analyzed. RESULT: The results showed that the trend of total RNA content was divided into three descending stages and two ascending stages, the total RNA content was the highest at 0, 6 h, but it was the lowest at and 42 h, as well as when the sprout tumbled. The differential display showed that the polymorphism and type of bands of the sample at 6 h were similar to those at 0 h. But the bands numbers at other time were far less than those at 0, 6 h. And there were some different mRNA differential expression bands between the different samples. CONCLUSION: In the process of the sprout tumble caused by high temperature stress, the RNA and mRNA differential expression in leaves of P. ternata changed.