Proteins that promote epithelial to mesenchymal transition (EMT) are associated with cancer metastasis. Inhibition of EMT regulators may be a promising approach in cancer therapy. In this study, Thymoquinone (TQ) was used to treat cancer cell lines to investigate its effects on EMT-regulatory proteins and cancer metastasis. We show that TQ inhibited cancer cell growth, migration and invasion in a dose-dependent manner. At the molecular level, TQ treatment decreased the transcriptional activity of the TWIST1 promoter and the mRNA expression of TWIST1, an EMT-promoting transcription factor. Accordingly, TQ treatment also decreased the expression of TWIST1-upregulated genes such as N-Cadherin and increased the expression of TWIST1-repressed genes such as E-Cadherin, resulting in a reduction of cell migration and invasion. TQ treatment also inhibited the growth and metastasis of cancer cell-derived xenograft tumors in mice but partially attenuated the migration and invasion in TWIST1-overexpressed cell lines. Furthermore, we found that TQ treatment enhanced the promoter DNA methylation of the TWIST1 gene in BT 549 cells. Together, these results demonstrate that TQ treatment inhibits TWIST1 promoter activity and decreases its expression, leading to the inhibition of cancer cell migration, invasion and metastasis. These findings suggest TQ as a potential small molecular inhibitor of cancer growth and metastasis.

As an edible fruit and source of traditional medicine, D. longan is grown in most areas of Southern China. Identification of D. longan cultivars by using molecular markers is important genetically. In this study, we cloned fragments from improved randomly amplified polymorphic DNA (RAPD), and developed stably diagnostic sequence-characterized amplified region (SCAR) markers. The specific RAPD bands of D. longan cultivars from Guangxi, with size ranging from 500 bp to 900 bp were gel-purified, cloned and sequenced. Four clones named LY2-1, LY4-7, LY4-8 and LY5-2 were identified. In order to investigate whether the fragments were specific for the species, four pairs of SCAR primers were then designed. PCR amplifications were conducted to analyze 18 samples including different D. longan cultivars and other species. The specific bands with expected sizes were amplified in five D. longan samples but not in others. To identify and characterize the difference between D. longan and D. confinis, PCR amplifications were performed again. The specific bands with expected sizes were found in D. longan but not in D. confinis by SCAR markers LY2-1, LY4-7 and LY5-2, respectively. These results showed that our developed SCAR markers could be very useful as a specific D. longan variety authentication. Therefore, our study provides an effective and precise PCR-based diagnostic method and markers to identify D. longan species.

ABSTRACT. Dimocarpus longan Lour. is an edible and traditional herb in China, commonly referred to as longon. An improved randomly amplified polymorphic DNA (RAPD) protocol was here developed in order to determine the geographical origins of D. longan samples collected from 5 provinces in the southern and southwestern areas of China, including Sichuan, Hainan, Fujian, Guangdong, and Guangxi. Generally, the improved RAPD method generated good fingerprinting of the 5 samples using the selected 17 primers. In particular, primers SBS-A5, SBS-A13, SBS-I9, SBS-I20, SBS-M1, and SBS-Q12 produced distinguishable bands that clearly separated all 5 cultivars, suggesting that there are variations in RAPD genetic sites among the samples. The similarity index ranged from 0.69 to 0.76. The Sichuan and Hainan clades clustered together with a 0.73 similarity index. The Guangxi and Fujian clades clustered together with a 0.76 similarity index, and they formed the sister clade to the Sichuan/Hainan clade with a 0.71 similarity index. The Guangdong clade was in a basal polytomy with a 0.70 similarity index. Based on the abundant DNA polymorphisms, these longan accessions are distinguishable using our improved RAPD technique. Therefore, RAPD analysis is an effective technique in distinguishing the geographical origins of D. longan. Moreover, the improved method could also be employed for a variety of applications including genetic diversity and fingerprinting analyses.

Litchi (Litchi chinensis Sonn., L. chinensis), a type of tree growing in most areas of southern China, produces an edible fruit that is also a source of traditional medicine. Genetic identification of litchi species or cultivars using molecular markers is very important. In this study, a total of six litchi samples from Fujian, Hainan, Guangdong, Guangxi and Sichuan province, as well as one wild Dimocarpus confinis (D. confinis) sample from Guangxi province were collected for genetic analysis. The cluster dendrograms were constructed for genetic analysis on the basis of DNA amplification results by RAPD and ISSR. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 176 bands were found, indicating a 72.7 % polymorphism in L. chinensis DNA samples. Significant genetic distances were found among the different species or cultivars, with an index of similarity coefficient ranging from 0.59 to 0.87. Similar to RAPD results, ISSR analysis of the L. chinensis DNA samples showed a range of 0.70-0.93 similarity coefficients. The genetic distance between Hainan sample and Sichuan samples was the farthest, which is consistent with their geographic distance. Furthermore, the index of similarity coefficient between D. confinis and L. chinensis was 0.35-0.41 by RAPD and 0.38-0.48 by ISSR, indicating that these two species have significant genetic difference. This study reveals the high level of genetic differences between different litchi species or cultivars, and confirms the significance of the improved RAPD method in genetic characterization of organisms. Taken together, the improved RAPD combined with ISSR analysis can be used frequently for the genetic diversity, germplasm resources preservation, molecular-assisted breeding, and genetic characterization of various organisms.

Background: Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results: The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions: This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.