Ling Yin

The anti-tumor activities of Neferine on cell invasion and oxaliplatin sensitivity regulated by EMT via Snail signaling in hepatocellular carcinoma

Tumor invasion and chemotherapy resistance, which are associated with epithelial-mesenchymal transition (EMT), remain as major challenges in hepatocellular carcinoma (HCC) treatment. Neferine, a natural component of Nelumbo nucifera, have been proven the antitumor efficiency in cancer, but the effects of Neferine on HCC invasion and chemosensitivity need to be elucidated. Applying multiple assays of cell proliferation, flow cytometry, immunofluorescence staining, qRT-PCR, Western blot, fluorescence molecular tomography imaging, the influences of Neferine on EMT-regulated viability, apoptosis, invasion, and oxaliplatin (OXA) sensitivity were assessed in HCC cells of HepG2 and Bel-7402, as well as in xenograft animal models in vivo. Here, we reported that Neferine had no obvious effects on HCC cells proliferation, but significantly enhanced cytotoxicity and apoptosis caused by OXA in vitro and in vivo. Through an upregulation of E-cadherin and downregulation of Vimentin, Snail and N-cadherin, Neferine suppressed EMT-induced migration and invasion abilities of HCC cells. TGF-β1 cancelled the effects of Neferine on the migration and invasion of HCC cells. Snail overexpression or TGF-β1-induced EMT attenuated Neferine-mediated OXA sensitization in HCC. Together, our data suggest that Neferine enhances oxaliplatin sensitivity through an inhibition of EMT in HCC cells. Neferine may be used as an OXA sensitizer in HCC chemotherapy.

Structure and function of the TIR domain from the grape NLR protein RPV1

The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices (“AE” interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling.

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism

Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3 Mb whole genome sequence of P. viticola isolate ‘JL-7-2’ obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host.

ZEB1 Promotes Oxaliplatin Resistance through the Induction of Epithelial - Mesenchymal Transition in Colon Cancer Cells

Background: Oxaliplatin (OXA) chemotherapy is widely used in the clinical treatment of colon cancer. However, chemo-resistance is still a barrier to effective chemotherapy in cases of colon cancer. Accumulated evidence suggests that the epithelial mesenchymal transition (EMT) may be a critical factor in chemo-sensitivity. The present study investigated the effects of Zinc finger E-box binding homeobox 1 (ZEB1) on OXA-sensitivity in colon cancer cells. Method: ZEB1expression and its correlation with clinicopathological characteristics were analyzed using tumor tissue from an independent cohort consisting of 118 colon cancer (CC) patients who receiving OXA-based chemotherapy. ZEB1 modulation of OXA-sensitivity in colon cancer cells was investigated in a OXA-resistant subline of HCT116/OXA cells and the parental colon cancer cell line: HCT116. A CCK8 assay was carried out to determine OXA-sensitivity. qRT-PCR, Western blot, Scratch wound healing and transwell assays were used to determine EMT phenotype of colon cells. ZEB1 knockdown using small interfering RNA (siRNA) was used to determine the ZEB1 contribution to OXA-sensitivity in vitro and in vivo (in a nude mice xenograft model). Result: ZEB1 expression was significantly increased in colon tumor tissue, and was correlated with lymph node metastasis and the depth of invasion. Compared with the parental colon cancer cells (HCT116), HCT116/OXA cells exhibited an EMT phenotype characterized by up-regulated expression of ZEB1, Vimentin, MMP2 and MMP9, but down-regulated expression of E-cadherin. Transfection of Si-ZEB1 into HCT116/OXA cells significantly reversed the EMT phenotype and enhanced OXA-sensitivity in vitro and in vivo. Conclusion: HCT116/OXA cells acquired an EMT phenotype. ZEB1 knockdown effectively restored OXA-sensitivity by reversing EMT. ZEB1 is a potential therapeutic target for the prevention of OXA-resistance in colon cancer.

A candidate RxLR effector from Plasmopara viticola can elicit immune responses in Nicotiana benthamiana

BackgroundDiverse plant pathogens deliver effectors into plant cells to alter host processes. Oomycete pathogen encodes a large number of putative RxLR effectors which are likely to play a role in manipulating plant defense responses. The secretome of Plasmopara viticola (downy mildew of grapevine) contains at least 162 candidate RxLR effectors discovered in our recent studies, but their roles in infection and pathogenicity remain to be determined. Here, we characterize in depth one of the putative RxLR effectors, PvRxLR16, which has been reported to induce cell death in Nicotiana benthamiana in our previous study.ResultsThe nuclear localization, W/Y/L motifs, and a putative N-glycosylation site in C-terminal of PvRxLR16 were essential for cell death-inducing activity. Suppressor of G-two allele of Skp1 (SGT1), heat shock protein 90 (HSP90) and required for Mla12 resistance (RAR1), but not somatic embryogenesis receptor-like kinase (SERK3), were required for the cell death response triggered by PvRxLR16 in N. benthamiana. Some mitogen-activated protein kinases and transcription factors were also involved in the perception of PvRxLR16 by N. benthamiana. PvRxLR16 could also significantly enhance plant resistance to Phytophthora capsici and the nuclear localization was required for this ability. However, some other PvRxLR effectors could suppress defense responses and disease resistance induced by PvRxLR16, suggesting that it may not trigger host cell death or immune responses during physiological infection under natural conditions.ConclusionThese data demonstrate that PvRxLR16 may be recognized by endogenous proteins in nucleus to trigger immune responses in N. benthamiana, which in turn can be suppressed by other PvRxLR effectors.

BMP4 enhances hepatocellular carcinoma proliferation by promoting cell cycle progression via ID2/CDKN1B signaling

Bone morphogenetic protein‐4 (BMP4) plays a crucial role in carcinogenesis, but the effects and signaling mechanisms of BMP4 in hepatocellular carcinoma (HCC) are not clearly clarified. The present study aimed to identify the roles of BMP4 in the proliferation of human HCC. In this study, BMP4 expression and its correlation with clinicopathological characteristics and the survival of HCC patients were analyzed in two independent cohorts consisting of 310 subjects. Functional analysis of BMP4 on HCC proliferation was performed in vitro and in vivo in human HCC specimens, HCC cells of Bel‐7402 and HCCLM3, and subcutaneous tumor model. The downstream signaling targets of BMP4 in HCC were investigated by PCR Array and Western blot. The results indicated that BMP4 expression was significantly increased in HCC tissues and closely related with unfavorable prognosis of HCC. BMP4 treatment increased cell proliferation and promoted G1/S cell cycle progression. In vivo subcutaneous tumor of nude mice model supported that BMP4 overexpression promoted the growth of HCC cells and BMP4 knockdown hold the opposite trend. Id2 was directly upregulated by BMP4, resulting in the mediated expression of cell cycle regulatory protein of CDKN1B. Blocking of Id2 attenuated BMP4‐induced proliferation, confirming the important roles of Id2 in BMP4‐mediated proliferation in HCC. So BMP4 is overexpressed in HCC tissues and acts as a poor prognostic factor of HCC patients. BMP4‐induced ID2/CDKN1B signaling facilitates proliferation of HCC.

Molecular Characterization of MYB28 Involved in Aliphatic Glucosinolate Biosynthesis in Chinese Kale (Brassica oleracea var. alboglabra Bailey)

Glucosinolates are Brassicaceae-specific secondary metabolites that act as crop protectants, flavor precursors, and cancer-prevention agents, which shows strong evidences of anticarcinogentic, antioxidant, and antimicrobial activities. MYB28, the R2R3-MYB28 transcription factor, directly activates genes involved in aliphatic glucosinolate biosynthesis. In this study, the MYB28 homology (BoaMYB28) was identified in Chinese kale (Brassica oleracea var. alboglabra Bailey). Analysis of the nucleotide sequence indicated that the cDNA of BoaMYB28 was 1257 bp with an ORF of 1020 bp. The deduced BoaMYB28 protein was a polypeptide of 339 amino acid with a putative molecular mass of 38 kDa and a pI of 6.87. Sequence homology and phylogenetic analysis showed that BoaMYB28 was most closely related to MYB28 homologs from the Brassicaceae family. The expression levels of BoaMYB28 varies across the tissues and developmental stages. BoaMYB28 transcript levels were higher in leaves and stems compared with those in cotyledons, flowers, and siliques. BoaMYB28 was expressed across all developmental leaf stages, with higher transcript accumulation in mature and inflorescence leaves. Over-expression and RNAi studies showed that BoaMYB28 retains the basic MYB28 gene function as a major transcriptional regulator of aliphatic glucosinolate pathway. The results indicated that over-expression and RNAi lines showed no visible difference on plant morphology. The contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes increased in over-expression lines and decreased in RNAi lines. In over-expression lines, aliphatic glucosinolate contents were 1.5- to 3-fold higher than those in the wild-type, while expression levels of aliphatic glucosinolate biosynthesis genes were 1.5- to 4-fold higher than those in the wild-type. In contrast, the contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes in RNAi lines were considerably lower than those in the wild-type. The results suggest that BoaMYB28 has the potential to alter the aliphatic glucosinolates contents in Chinese kale at the genetic level.

Molecular Cloning, Expression Pattern and Genotypic Effects on Glucoraphanin Biosynthetic Related Genes in Chinese Kale (Brassica oleracea var. alboglabra Bailey)

Glucoraphanin is a plant secondary metabolite that is involved in plant defense and imparts health-promoting properties to cruciferous vegetables. In this study, three genes involved in glucoraphanin metabolism, branched-chain aminotransferase 4 (BCAT4), methylthioalkylmalate synthase 1 (MAM1) and dihomomethionine N-hydroxylase (CYP79F1), were cloned from Chinese kale (Brassica oleracea var. alboglabra Bailey). Sequence homology and phylogenetic analysis identified these genes and confirmed the evolutionary status of Chinese kale. The transcript levels of BCAT4, MAM1 and CYP79F1 were higher in cotyledon, leaf and stem compared with flower and silique. BCAT4, MAM1 and CYP79F1 were expressed throughout leaf development with lower transcript levels during the younger stages. Glucoraphanin content varied extensively among different varieties, which ranged from 0.25 to 2.73 µmol·g−1 DW (dry weight). Expression levels of BCAT4 and MAM1 were high at vegetative–reproductive transition phase, while CYP79F1 was expressed high at reproductive phase. BCAT4, MAM1 and CYP79F1 were expressed significantly high in genotypes with high glucoraphanin content. All the results provided a better understanding of the roles of BCAT4, MAM1 and CYP79F1 in the glucoraphanin biosynthesis of Chinese kale.

Knockdown of Beclin-1 impairs epithelial-mesenchymal transition of colon cancer cells

Activation of autophagy significantly affects cancer cell behaviors, such as proliferation, differentiation, and invasiveness. Epithelial‐to‐mesenchymal transition (EMT) as an initial step of malignant transformation of cancer cells was linked to the activation of autophagy, but the detailed molecular mechanisms are still unknown. The present study investigates the effects of Beclin‐1, a key molecule involved in activation of autophagy, on EMT of colon cancer cells. The normal colon epithelia cell line of CCD‐18Co and six colon cancer cell lines with different expression levels of Beclin‐1 were used in this study. The activation of autophagy and EMT markers of cancer cells were monitored by Western blotting and quantitative real‐time PCR assay in the presence or absence of rapamycin (autophagy activator) and 3‐MA (autophagy inhibitor). The expression of Beclin‐1 in selected cell lines was modulated using small interfering RNA, and consequentially EMT markers, and cancer cell behaviors including migration and invasion, were also explored. Activation or inhibition of autophagy in colon cancer cells had positive or negative impacts on the expression of EMT markers and malignant behaviors such as cell migration and invasion. Knockdown of beclin‐1 by siRNA apparently inhibited the activation of autophagy induced by rapamycin, consequentially resulted in suppression of EMT and attenuation of invasiveness of colon cancer cells. The results in this study demonstrated an association between activation of autophagy and EMT in colon cancer cells. The results showed suppression of Beclin‐1 expression significantly reduced EMT and invasive behaviors in colon cancer cells.

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.