Dicer-like2b suppresses the wiry leaf phenotype in tomato induced by tobacco mosaic virus.

Dicer-like (DCL) proteins are principal components of RNA silencing, a major defense mechanism against plant virus infections. However, their functions in suppressing virus-induced disease phenotypes remain largely unknown. Here, we identified a role for tomato (Solanum lycopersicum) DCL2b in regulating the wiry leaf phenotype during defense against tobacco mosaic virus (TMV). Knocking out SlyDCL2b promoted TMV accumulation in the leaf primordium, resulting in a wiry phenotype in distal leaves. Biochemical and bioinformatics analyses showed that 22-nt virus-derived small interfering RNAs (vsiRNAs) accumulated less abundantly in slydcl2b mutants than in wild-type plants, suggesting that SlyDCL2b-dependent 22-nt vsiRNAs are required to exclude virus from leaf primordia. Moreover, the wiry leaf phenotype was accompanied by upregulation of Auxin Response Factors (ARFs), resulting from a reduction in trans-acting siRNAs targeting ARFs (tasiARFs) in TMV-infected slydcl2b mutants. Loss of tasiARF production in the slydcl2b mutant was in turn caused by inhibition of miRNA390b function. Importantly, silencing SlyARF3 and SlyARF4 largely restored the wiry phenotype in TMV-infected slydcl2b mutants. Our work exemplifies the complex relationship between RNA viruses and the endogenous RNA silencing machinery, whereby SlyDCL2b protects the normal development of newly emerging organs by excluding virus from these regions and thus maintaining developmental silencing.

Adjuvant chemotherapy does not improve cancer-specific survival for pathologic stage II/III rectal adenocarcinoma after neoadjuvant chemoradiotherapy and surgery: evidence based on long-term survival analysis from SEER data.

PURPOSE: Adjuvant chemotherapy is controversial in rectal cancer, especially after neoadjuvant chemoradiotherapy (NCRT). This retrospective study aims at evaluating adjuvant chemotherapy's long-term survival benefits in stage II and stage III rectal adenocarcinoma (RC). METHODS: This study obtained data from the Surveillance, Epidemiology, and End Results (SEER) database registered between 2010 and 2015. The survival analyses used the Kaplan-Meier method and were compared by log-rank test. The factors that affect survival outcomes were analyzed by univariate and multivariate Cox regression. The propensity score matching (1:4) was used to ensure the balance of variables between different groups. RESULTS: The median follow-up time for overall patients was 64 months. The 5-year overall survival (OS) and cancer-specific survival (CSS) rates were 51.3% and 67.4% in the adjuvant chemotherapy (-) group and 73.9% and 79.6% in the adjuvant chemotherapy ( +) group (p < 0.001, p = 0.002). However, subgroup analysis showed adjuvant chemotherapy after NCRT improved the 5-year OS but not CSS rates in stage II and stage III RC (p = 0.003, p = 0.004; p = 0.29, p = 0.3). Univariate and multivariate analyses found adjuvant chemotherapy after NCRT was an independent prognosis factor of OS but not CSS (HR 0.8, 95%CI 0.7-0.92, p < 0.001; p = 0.276). CONCLUSION: The survival benefits from adjuvant chemotherapy were associated with the status of NCRT for pathological stage II and III RC. For patients who did not receive NCRT, adjuvant chemotherapy is needed to significantly improve long-term survival rates. However, adjuvant chemotherapy after NCRT did not significantly improve long-term CSS.

Anthocyanins in Plant Food: Current Status, Genetic Modification, and Future Perspectives.

Anthocyanins are naturally occurring polyphenolic pigments that give food varied colors. Because of their high antioxidant activities, the consumption of anthocyanins has been associated with the benefit of preventing various chronic diseases. However, due to natural evolution or human selection, anthocyanins are found only in certain species. Additionally, the insufficient levels of anthocyanins in the most common foods also limit the optimal benefits. To solve this problem, considerable work has been done on germplasm improvement of common species using novel gene editing or transgenic techniques. This review summarized the recent advances in the molecular mechanism of anthocyanin biosynthesis and focused on the progress in using the CRISPR/Cas gene editing or multigene overexpression methods to improve plant food anthocyanins content. In response to the concerns of genome modified food, the future trends in developing anthocyanin-enriched plant food by using novel transgene or marker-free genome modified technologies are discussed. We hope to provide new insights and ideas for better using natural products like anthocyanins to promote human health.

A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

A tomato receptor-like cytoplasmic kinase, SlZRK1, acts as a negative regulator in wound-induced jasmonic acid accumulation and insect resistance.

Jasmonates accumulate rapidly and act as key regulators in response to mechanical wounding, but few studies have linked receptor-like cytoplasmic kinases (RLCKs) to wound-induced jasmonic acid (JA) signaling cascades. Here, we identified a novel wounding-induced RLCK-XII-2 subfamily member (SlZRK1) in tomato (Solanum lycopersicum) that was closely related to Arabidopsis HOPZ-ETI-DEFICIENT 1 (ZED1)-related kinases 1 based on phylogenetic analysis. SlZRK1 was targeted to the plasma membrane of tobacco mesophyll protoplasts as determined by transient co-expression with the plasma membrane marker mCherry-H+-ATPase. Catalytic residue sequence analysis and an in vitro kinase assay indicated that SlZRK1 may act as a pseudokinase. To further analyse the function of SlZRK1, we developed two stable knock-out mutants by CRISPR/Cas9. Loss of SlZRK1 significantly altered the expression of genes involved in JA biosynthesis, salicylic acid biosynthesis, and ethylene response. Furthermore, after mechanical wounding treatment, slzrk1 mutants increased transcription of early wound-inducible genes involved in JA biosynthesis and signaling. In addition, JA accumulation after wounding and plant resistance to herbivorous insects also were enhanced. Our findings expand plant regulatory networks in the wound-induced JA production by adding RLCKs as a new component in the wound signal transduction pathway.

A Y(III)-based Metal-Organic Framework as a Carrier in Chemodynamic Therapy.

A biocompatible Y(III)-based metal-organic framework [Y4(TATB)2]·(DMF)3.5·(H2O) (ZJU-16, H3TATB= 4,4',4''-(1,3,5-triazine-2,4,6-triyl) tribenzoic acid) was synthesized, and it was adopted to load Mn2+ for chemodynamic therapy. Meanwhile, ibuprofen sodium (IBUNa), an anti-inflammatory drug, was introduced to increase the amount of Mn2+ (about 5.66 wt %) due to the low loading capacity of Mn2+. Mn&IBUNa@ZJU-16 which was loaded by Mn2+ and IBUNa exhibited significant effects of chemodynamic therapy and excellent inhibition of the 4T1 tumor cell growth, implying its long-term prospects in chemodynamic therapy and its possibility in bimodal cancer therapy.

Analysis of long-non-coding RNAs associated with ethylene in tomato.

Long-Non-Coding RNAs (LncRNAs) are a class of non-coding endogenous RNAs contributing to numerous biological processes. LeERF1 is a tomato ethylene response factor (ERF) near the end of the ethylene signal transduction pathway. To identify lncRNAs in tomato and elucidate their roles in ethylene signaling, deep sequencing was deployed in over-expression and repression LeERF1 transgenic and control tomato fruits. A total of 397 lncRNAs were identified, including 169 tomato lncRNAs that had not previously been identified. Among these, 12 were differentially expressed between the transgenic and control tomato fruits. Numerous lncRNA target genes were identified including many associated with ethylene signaling including auxin response factors and auxin-induced proteins, F-box proteins, ERFs and MADS-box proteins. In addition, two lncRNAs were found to be the precursor of three miRNAs and four lncRNAs could be targeted by five miRNAs. We propose a regulatory model highlighting the relationships between lncRNAs and their targets involved in ethylene signal transduction which establishes a foundation for addressing the role of LncRNAs in ethylene response.

Active components, derived from Kai-xin-san, a herbal formula, increase the expressions of neurotrophic factor NGF and BDNF on mouse astrocyte primary cultures via cAMP-dependent signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Kai-Xin-San (KXS), an ancient formula composed of Ginseng Radix et Rhizoma, Polygalae Radix, Acori Tatarinowii Rhizoma and Poria, was frequently applied for Alzheimer's disease and major depression disorders for thousands of years. However, its active components and molecular mechanism have not clearly been investigated. AIM OF THE STUDY: We aimed to reveal the active components of KXS on regulating neurotrophic factor NGF and BDNF expressions and its mechanisms on mouse astrocyte primary cultures. MATERIALS AND METHODS: Extracts of KXS had been prepared by water reflux and chemical standardization was carried out by HPLC-MS/MS. Various ethanol elution components were prepared by eluting ethanol on macro pore resin column and compound identification was carried out by high-resolution mass spectrometry. KXS extract, elution components and identified chemicals were applied on mouse astrocytes and expressions of NGF and BDNF and related metabolic enzymes were analyzed by qPCR and western blotting analysis. RESULTS: One compatible ratio of KXS named D-652 exerted the best effect on stimulation of NGF and BDNF expressions on mouse astrocytes. 70% ethanol elution fraction of D-652 exerted the highest increase tendency on expressions of NGF and BDNF by activating cAMP-dependent signaling pathway as well as stimulating enzymes accounting for neurotrophic factor synthesis. Combined with compound identification by high-resolution mass spectrometry, ginsenoside Rg1 and Rb1 might be the active compounds of this fraction on increasing NGF and BDNF expressions. CONCLUSIONS: The active compounds of KXS on increasing NGF and BDNF expressions might be the ginsenosides via activating cAMP-dependent signaling pathway as well as stimulating enzymes accounting for neurotrophic factor synthesis, which partly reveal the target of this formulae supported the clinically usage of this decoction.

A Viral Satellite DNA Vector (TYLCCNV) for Functional Analysis of miRNAs and siRNAs in Plants.

With experimental and bioinformatical methods, numerous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), have been found in plants, and they play vital roles in various biological regulation processes. However, most of these small RNAs remain to be functionally characterized. Until now, only several viral vectors were developed to overexpress miRNAs with limited application in plants. In this study, we report a new small RNA overexpression system via viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV) vector, which could highly overexpress not only artificial and endogenous miRNAs but also endogenous siRNAs in Nicotiana benthamiana First, we constructed basic TYLCCNV-amiRPDS(319L) vector with widely used AtMIR319a backbone, but the expected photobleaching phenotype was very weak. Second, through comparing the effect of backbones (AtMIR319a, AtMIR390a, and SlMIR159) on specificity and significance of generating small RNAs, the AtMIR390a backbone was optimally selected to construct the small RNA overexpression system. Third, through sRNA-Seq and Degradome-Seq, the small RNAs from AtMIR390a backbone in TYLCCNV-amiRPDS(390) vector were confirmed to highly overexpress amiRPDS and specifically silence targeted PDS gene. Using this system, rapid functional analysis of endogenous miRNAs and siRNAs was carried out, including miR156 and athTAS3a 5'D8(+). Meanwhile, through designing corresponding artificial miRNAs, this system could also significantly silence targeted endogenous genes and show specific phenotypes, including PDS, Su, and PCNA These results demonstrated that this small RNA overexpression system could contribute to investigating not only the function of endogenous small RNAs, but also the functional genes in plants.

Transcriptome Analysis Provides a Preliminary Regulation Route of the Ethylene Signal Transduction Component, SlEIN2, during Tomato Ripening.

Ethylene is crucial in climacteric fruit ripening. The ethylene signal pathway regulates several physiological alterations such as softening, carotenoid accumulation and sugar level reduction, and production of volatile compounds. All these physiological processes are controlled by numerous genes and their expression simultaneously changes at the onset of ripening. Ethylene insensitive 2 (EIN2) is a key component for ethylene signal transduction, and its mutation causes ethylene insensitivity. In tomato, silencing SlEIN2 resulted in a non-ripening phenotype and low ethylene production. RNA sequencing of SlEIN2-silenced and wild type tomato, and differential gene expression analyses, indicated that silencing SlEIN2 caused changes in more than 4,000 genes, including those related to photosynthesis, defense, and secondary metabolism. The relative expression level of 28 genes covering ripening-associated transcription factors, ethylene biosynthesis, ethylene signal pathway, chlorophyll binding proteins, lycopene and aroma biosynthesis, and defense pathway, showed that SlEIN2 influences ripening inhibitor (RIN) in a feedback loop, thus controlling the expression of several other genes. SlEIN2 regulates many aspects of fruit ripening, and is a key factor in the ethylene signal transduction pathway. Silencing SlEIN2 ultimately results in lycopene biosynthesis inhibition, which is the reason why tomato does not turn red, and this gene also affects the expression of several defense-associated genes. Although SlEIN2-silenced and green wild type fruits are similar in appearance, their metabolism is significantly different at the molecular level.

A viral satellite DNA vector-induced transcriptional gene silencing via DNA methylation of gene promoter in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mß vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes.

Efficient Virus-Induced Gene Silencing in Solanum rostratum.

Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

Attenuation of EGFL7 inhibits human laryngocarcinoma cells growth and invasion.

AIM: To investigate its effect on the proliferation and invasion of laryngeal carcinoma and understand the potential underlying mechanisms to provide new targets for the diagnosis and treatment of recurrent laryngeal cancer metastasis. METHODS: We constructed a lentiviral vector expressing EGFL7 specific shRNA, and introduced it in EGFL7 functions were attenuated by a lentiviral vector harboring shRNA targeting at EGFL7 in laryngeal carcinoma cell line Hep-2. Prolifereation and invasion assays were carried out in vitro. And in vivo tumor burden assay was done in nude mice. RESULTS: The expression of EGFL7 was knocked-down by 80% in hep-2 cells transfected by the lentiviral EGFL7 shRNA vector and EGFL7 gene expression was detected by realtime PCR and Western blotting analysis respectively. The flow cytometric analysis showed that arrested the cell cycle in G1 phase, In tumor burden assay, to parental And vector control cells, the survival rates Of nude mice in EGFL7 shRNA group dropped down from the first day after implantation as indicated by MTT assay (P < 0.05). The formation and growth rate of xenograft tumor in mice transfected with siRNA against Bmi-1 slowed down significantly. CONCLUSION: Attenuation of EGFL7 function significantly suppresses tumor growth and induces apoptosis, both in vitro and in vivo. EGFL7 may be play a key role in invasion and metastasis of Laryngeal squamous cell carcinoma (LSCC), thus would to be a new target for gene therapy in LSCC.

In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1.

microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 reconstitution assays. The protocol comprises three major parts (Figure 1): 1) Preparation of pri- and pre-miRNA transcripts (Procedures A-C); 2) Purification of the recombinant Arabidopsis DCL1 machinery from Nicotiana benthamiana (N. benthamiana) through immunoprecipitation (IP) (Procedures D and E); and 3) in vitro processing of radioisotope-labeled pri- or pre-miRNAs using the isolated DCL1 complexes (Procedure F). It is our desire that the protocol be a powerful tool for the RNAi community to study mechanistic issues or to develop RNA silencing technologies.

Silencing Bmi-1 expression by RNA interference suppresses the growth of laryngeal carcinoma cells.

The aim of this study was to investigate the effect of a B-cell-specific MLV integration site-1 (Bmi-1) RNA interference (RNAi) expression vector on the proliferation and invasiveness of laryngeal carcinoma. We constructed a lentiviral vector expressing Bmi-1-specific short hairpin RNA (shRNA), and transfected it into HEp-2 cells. Bmi-1 gene expression was detected by real-time RT-PCR and western blot analysis. We used flow cytometry and TUNEL assay to analyze the apoptosis of transfected cells, and examined cellular growth in vitro by MTT assay. We established an animal model and evaluated the therapeutic effects of small interfering RNA (siRNA) against Bmi-1. siRNA against Bmi-1 significantly knocked down Bmi-1 expression in HEp-2 cells, induced cell cycle arrest at the G1 phase, inhibited cell proliferation and promoted cell apoptosis. Lentiviral Bmi-1-shRNA vector transfection also significantly reduced cell migration. The formation and growth rate of xenograft tumors in mice transfected with siRNA against Bmi-1 was significantly reduced. The loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, and the increased activity of caspase-3, -8 and -9 occurred concomitantly with the inhibition of Bmi-1. Our data indicate that siRNA against Bmi-1 significantly suppresses tumor growth and induces apoptosis in vitro and in vivo.

[Effects of Bmi-1 RNAi gene on laryngeal carcinoma Hep-2 cells].

OBJECTIVE: To construct the Bmi-1 RNAi expression vector and investigate its influence on the proliferation and invasiveness of laryngeal carcinoma Hep-2 cells. METHOD: The recombinant vshRNA-Bmi-1 plasmid of Bmi-1 RNAi was constructed by the lentiviral expression system, pHelper1.0/pHelper2.0/pGCL2GFP. Bmi-1mRNA and protein expressions of stably transfected laryngeal carcinoma cells were identified tespectively by real-time PCR and Western blot analyses. The changes of the proliferation and invasiveness of laryngeal carcinoma were detected by clone formation test and an invasion assay. RESULT: The Bmi-1 mRNA expressions of stably transfected laryngeal carcinoma Hep-2 cells were significantly decreased. The expression of Bmi-1 protein in laryngeal carcinoma Hep-2 cells was significantly decreased. And the inhibitory rates were 79% and 88% respectively. Whereas the proliferation and invasiveness of Hep-2 cells were significantly reduced. CONCLUSION: The Bmi-1 RNAi expression vector were constructed successful. Reduced Bmi-1 expression of Hep-2 cells demonstrated the role of Bmi-1 RNAi in restraining proliferation and invasiveness of laryngeal carcinoma cells.

[Toll-like receptor 2 and Toll-like receptor 4 participates in mediation of acute otitis media and mortality in pneumococcal infections in mice].

OBJECTIVE: To investigate the roles of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in host defense against Streptococcus pneumoniae infection in the middle ear. METHODS: Wild-type (WT) C57BL/6J, TLR2-deficient (TLR2(-/-)) and TLR4-deficient (TLR4(-/-)) mice were inoculated with Streptococcus pneumoniae (1 × 10(6)CFU) through the tympanic membrane. All animals were tested the mouse ABR thresholds and tympanometry measurement before, and 1 day, 3 days and 7 days following pneumococcal challenge. Blood bacterial titer were determined by plating 50 µl volumes of 10-fold diluted blood. Histological analysis of middle ear and inner ear were performed by fixation, decalcification, embedded section, and counterstained with hematoxylin/eosin and toluidine blue staining. Semi-quantitative RT-PCR was applied to determine mRNA accumulation of TLR2 and TLR4 related genes. RESULTS: Forty of 68 TLR2(-/-) mice and twenty-one of 59 TLR4(-/-) mice showed bacteremia and died within 3 days after the pneumococcal challenge, however, only 9 of 52 WT mice died. The survive mice were shown have more severe hearing loss in the TLR2(-/-) and TLR4(-/-) mice than in the WT mice, indicated by ABR thresholds, at 3 or 7 days postinoculation. The histological pathology was characterized by effusion and tissue damage in the middle ear, and in the TLR2(-/-) and TLR4(-/-) mice, the outcome of infection became more severe at 7 days. At both 3 and 7 days after challenge, the TLR2(-/-) mice had higher blood bacterial titers than WT mice (P < 0.05). Temporal bone histopathologic change indicated that 3 days after the pneumococcal challenge, the TLR2(-/-) and TLR4(-/-) mice showed effusion and tissue damage in the middle ear, and the infection became more severe at 7 days postinoculation. TLR2(-/-) mice showed severe inflammatory cell infiltration in the cochlear, the organ of Corti showed the outer hair cells damage, the tectorial membrane swelling, degeneration of the stria vascularis, and severe loss of spiral ganglion cells; However, the WT mice was not found the cell infiltration and tissue damage in the cochlear, the organ of Corti shown normal of outer hair cells. Mast cells were not found in the middle ear mucosa of TLR2(-/-) mice, but in the TLR4(-/-) and WT mice, more mast cells were found in the middle ear mucosa of effusion ear by 3 and 7 days postchallenge. Moreover, by 3 days postchallenge, the mRNA accumulation levels of NF-κB, tumor necrosis factor alpha (TNFα), interleukin1ß, MIP-1α, MUC5AC and MUC5B were significantly lower in the ears of TLR2(-/-) mice than that in WT and TLR4(-/-) mice. CONCLUSIONS: TLR2(-/-) mice may produce relatively low levels of proinflammatory cytokines following pneumococcal challenge, thus hindering the clearance of bacteria from the middle ear and leading to sepsis and high mortality rate. This study indicated that TLR2 and TLR4 are important in the molecular pathogenesis and host response to otitis media.

[Construction of lentivirus vector of interference of EGFL7 gene and its inhibitive role on the invasion of laryngeal cancer cell].

OBJECTIVE: To construct a lentivirus vector of RNA interference (RNAi) of EGFL7 gene and observe its inhibitive role on the invasion of laryngeal cancer cell. METHOD: The effective sequence of siRNA targeting EGFL7 gene was confirmed. Both sense and antisence Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pLV vector,which contained H1 promotor and green fluorescent protein (GFP). The resulting lentivirus vector containing EGFL7 shRNA was called LV-sh EGFL7,and it is confirmed by PCR and sequencing. After that, EGFL7 shRNA was transfected into Hep-2 cells and Western blot was used to test the expression of EGFL7. At last, boyden chamber was used to observe the invasion of the Hep-2 cells. Colony formation assay using a EGFL7 gene silencing on the Hep-2 cell colony forming ability. RESULT: PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of EGFL7 (LV-sh EGFL7) producing EGFL7 shRNA was constructed successfully. The titer of concentrated virus was 5 x 10(8) TU/L. Western blot showed that the expression of EGFL7 was negative in the EGFL7 siRNA Hep-2 cells. And boyden chamber showed the invasive capability of Hep-2 cells transfected EGFL7 siRNA were obviously decreased. EGFL7 gene silencing of cell colony formation rate of cloned Hep-2 cells and compared with empty vector cells, cell cloning and colony formation was significantly reduced. CONCLUSION: The lentivirus RNAi vector of EGFL7 was constructed successfully. And EGFL7 silence can inhibit invasion of laryngeal cancer in vitro. After silence EGFL7, Hep-2 cell colony formation was significantly lower, that is, gene expression can be down EGFL7 some extent laryngeal cancer cells inhibited anchorage independent growth capacity.

[Inhibitory effect of silencing hTERT gene on growth of human squamous cell carcinoma xenograft in nude mice].

OBJECTIVE: This study is to explore the inhibitory effect of silencing hTERT gene by short-hairpin RNA on growth of nasopharyngeal carcinoma xenograft in nude mice with RNAi technique. METHOD: Construction and expression of hTERT cDNA sequence according to the specific hTERT mRNA, including fluorescein eukaryotic expression vector, packaged into a lentivirus. qPCR and Western blot analyzed hTER.T mRNA and protein levels in transfected cells. Proliferation rate of transfected cells was determined by MTT assay in vitro. Cell growth cycle was detected by flow cytometry. The invasiveness of each group was compared using in vitro cell invasion assay. RESULT: RT-PCR and Western blot analysis showed that, hTERT siRNA significantly reduced hTERT mRNA and protein levels, especially hTERT siRNA1. siRNA treatment inhibited tumor cell proliferation, and cell migration and invasiveness were significantly lower. Tumor cell growth rate was significantly different between control group and siRNA group (P < 0.01) while tumor cell growth rate in empty virus group(NC group) and control group was not significantly different (P > 0.05). CONCLUSION: Lentivirus containing specific sequences of hTERT gene could significantly inhibit the growth of nasopharyngeal carcinoma cells line. hTERT siRNA expression vector can effectively inhibit NPC cell proliferation, migration and invasion, which may provide a novel molecular targets for gene therapy of nasopharyngeal carcinoma.

[Inhibitory effect of lentiviral vectors expressing small interfering RNA targeting survivin gene on Hep-2 cell growth in vitro and in nude mice].

OBJECTIVE: To observe the effect of the lentiviral vectors expressing small interfering RNA (siRNA) for survivin gene knockdown in inhibiting Hep-2 cell growth in vitro and its tumorigenicity in nude mice. METHODS: The tumorigenicity of Hep-2 cells transfected with the siRNA mediated by the lentiviral vectors was tested in nude mice. The expression of survivin gene of the transfected cells at the mRNA and protein levels were detected by RT-PCR and Western blotting, respectively, and the cell cycle changes were analyzed by flow cytometry. RESULTS: Transfection of the siRNA targeting survivin significantly decreased the expression of survivin mRNA and protein in Hep-2 cells in vitro by 60%-85% and 70%, respectively, resulting also in increased cell apoptosis as shown by flow cytometry (P<0.01). The transfection significantly lowered the tumorigenicity of the cells in nude mice. CONCLUSION: The lentiviral vectors expressing survivin siRNA can significantly inhibit survivin gene expression in Hep-2 cells and induce the cell apoptosis in vitro, and suppress the tumorigenicity of the cells in nude mice.