MicroRNA-21 Regulates Non-Small Cell Lung Cancer Cell Invasion and Chemo-Sensitivity through SMAD7.

BACKGROUND/AIMS: SMAD7 is a key inhibitor of transforming growth factor ß (TGFß) receptor signaling, which regulates the alteration of cancer cell invasiveness through epithelial-mesenchymal cell conversion. Carboplatin is a commonly used drug in the chemotherapy for non-small cell lung cancer (NSCLC). Nevertheless, the molecular mechanisms underlying its suppressive effects on the NSCLC cell invasion are not completely understood. In the current study, we addressed this question by analyzing the effects of Carboplatin on microRNA-regulated SMAD7. METHODS: We used Carboplatin to treat NSCLC cell lines. We performed bioinformatics analyses on the binding of microRNA-21 (miR-21) to the 3'-UTR of SMAD7 mRNA, and verified the biological effects of this binding using promoter luciferase reporter assay. The effects of Carboplatin or miR-21-modification on NSCLC cell invasion were evaluated in either a transwell cell invasion assay, or a scratch wound healing assay. RESULTS: We found that Carboplatin inhibited the NSCLC cell invasion, in either a transwell cell invasion assay, or a scratch wound healing assay. Moreover, Carboplatin increased the levels of SMAD7 protein, but not mRNA, in NSCLC cells, suggesting presence of post-transcriptional control of SMAD7 by Carboplatin. Furthermore, expression of miR-21 was found to be inhibited by Carboplatin, and bioinformatics analyses showed that miR-21 targeted the 3'-UTR of SMAD7 mRNA to inhibit its translation, which was confirmed by luciferase reporter assay. CONCLUSION: Carboplatin may upregulate SMAD7 through suppression of miR-21 to inhibit TGFß receptor signaling mediated NSCLC cell invasion.

The approach for action recognition based on the reconstructed phase spaces.

This paper presents a novel method of human action recognition, which is based on the reconstructed phase space. Firstly, the human body is divided into 15 key points, whose trajectory represents the human body behavior, and the modified particle filter is used to track these key points for self-occlusion. Secondly, we reconstruct the phase spaces for extracting more useful information from human action trajectories. Finally, we apply the semisupervised probability model and Bayes classified method for classification. Experiments are performed on the Weizmann, KTH, UCF sports, and our action dataset to test and evaluate the proposed method. The compare experiment results showed that the proposed method can achieve was more effective than compare methods.

The complex action recognition via the correlated topic model.

Human complex action recognition is an important research area of the action recognition. Among various obstacles to human complex action recognition, one of the most challenging is to deal with self-occlusion, where one body part occludes another one. This paper presents a new method of human complex action recognition, which is based on optical flow and correlated topic model (CTM). Firstly, the Markov random field was used to represent the occlusion relationship between human body parts in terms of an occlusion state variable. Secondly, the structure from motion (SFM) is used for reconstructing the missing data of point trajectories. Then, we can extract the key frame based on motion feature from optical flow and the ratios of the width and height are extracted by the human silhouette. Finally, we use the topic model of correlated topic model (CTM) to classify action. Experiments were performed on the KTH, Weizmann, and UIUC action dataset to test and evaluate the proposed method. The compared experiment results showed that the proposed method was more effective than compared methods.

[Construction of a trans-splicing ribozyme for restoring EGFP truncation mutation].

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.

A novel mutation of estrogen receptor gene detected in girls with precocious puberty.

Female precocious puberty is caused by premature activation of the hypothalamic-pituitary-gonadal axis, exposure to exogenous sex steroid hormones, and the presence of endogenous sex steroids caused by various factors. Estrogen is the final key factor to start onset of puberty. However,in some cases of precocious puberty in girls estrogen elevation could not be detected. The raised sensitivity of estrogen receptor, which may caused by ESR1 mutation or polymorphism, has been frequently mentioned for interpreting the etiology of sporadic low estrogen type cases. But no case evidence has been found in clinical practice. For the purpose of screening possible mutations in estrogen receptor gene, leukocyte genomic DNA were collected from 16 girls with precocious puberty of sporadic low estrogen,and exons of ESR1 were amplified and analysized using PCR-SSCP/silver staining method. A single strand conformation change in exon 8 was found in one of the patients (No. 14). The suspected fragment were cloned to a T vector and sequenced for analysis. Sequencing of these clones revealed that this conformation change is caused by a C to T transition. This mutation results in the replacement of arginine by cystine at position 548 of ESR1 protein. The mutation created an extra Btsl digest site and made it can be readily identified by PCR-PFLP method. Further detection using this method, and sequencing of cloned exon8 colonies from patients proved that the patient No. 14 is Arg548/Cys548 heterozagous in genotype. This mutation increased hydrophobility of the area dramatically. The position and the conservative of this residue in vertebrates suggested Arg548 may play an important role in ESR1 function. For study the role of this mutation in the onset of precocious puberty, a firefly luciferase reporter plasmid pGL3-promoter-ERE was constructed,and a pCR3. 1-hermut pisimid expressing Cys548 ER was constructed based on wild type pCR3. 1her. Co-transfection of reporter and pCR3. 1 -hermut in CMF-7 cell strain proved that Cys548 mutant can significantly increase the transcription activity over the Arg548 wild type.

Identification of an E-box motif as a transcriptional repressor element in the proximal promoter region of the GCLC gene in rat lung epithelial L2 cells.

Glutathione (GSH) is a critical antioxidant for protecting the airway epithelium from oxidant injury and its levels are mainly controlled by glutamate-cysteine ligase (GCL), which is the rate-limiting enzyme in GSH synthesis. A full understanding of the gene regulation mechanism of this important enzyme may disclose the role it plays in respiratory diseases. GCL is made up of two differentially regulated subunits, a catalytic or heavy subunit (GCLC) and a modifier or light subunit (GCLM). Many studies in this field led to the findings of important positive regulatory regions of the GCLC promoter. For a detailed analysis of this gene regulation in the respiratory system, we cloned a 1.76-kb 5'-flanking region of the rat GCLC gene, inserted into a luciferase reporter vector. Exonuclease III was used to cut the 5'-flanking region of the rat GCLC gene unidirectionally into deletion mutants of different lengths. Sequential deletion analysis revealed that regions from -403 to -111 and from -705 to -613 are involved in positive regulation and the region from -745 to -705 is involved in negative regulation of the GCL gene in rat lung epithelial L2 cells. Specific proteins binding to these regions were confirmed by electrophoretic mobility-shift assays (EMSAs) and antibody supershift assays. An E-box motif was found in the negative regulatory region -745 to -705. Site-directed mutagenesis proved that the functional element in this negative regulatory region was a putative E-box element. EMSA and supershift assays showed that USF1 and USF2 can specifically bind to the E-box element. Overexpression of USFs in L2 cells led to a decreased activity of the GCLC promoter. Western blotting demonstrated that the expression of GCLC protein was decreased in the retroviral USFs-expressing cells than in nontransfected (no DNA added) cells, suggesting that USF binding to the E-box at -729/-724 serves to trans-repress GCLC gene expression. These findings indicate that the E-box is an important transcriptional suppressor element in the GCLC promoter in rat lung epithelial L2 cells. Inhibition of interaction between the E-box and the USF may provide an effective means of ameliorating oxidant injury of the lung.

[Prokaryotic expression of human cTnI and preparation of rabbit anti-hcTnI antibody].

AIM: To construct the prokaryotic expression vector pET-21a(+)-hcTnI and prepare the rabbit anti-hcTnI antibody using hcTnI expressed in E.coli as immunogen. METHODS: The full-length gene encoding human cardiac troponin I (hcTnI) was synthesized chemically and inserted into expression plasmid pET-21a(+) to construct recombinant plasmid pET-21a(+)-hcTnI. The recombinant plasmid was transformed into E.coli BL21 (DE3) plysS which then expressed hcTnI under IPTG induction. The immunological activity of the expressed hcTnI was analyzed by Western blot. A rabbit was immunized with purified hcTnI to prepare anti-hcTnI antibody and the Ab's properties were identified. RESULTS: Human cTnI gene was synthesized and confirmed by DNA sequencing. Positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing. After induction with IPTG, hcTnI with M(r) being 24 000 was expressed in E.coli BL21 (DE3) plysS, and the expressed hcTnI accounted for 28% of total bacterial protein. Western blot analysis showed that the hcTnI protein could be recognized by an anti-hcTnI antibody. Rabbit polyclonal antibody with a good specificity was obtained. The titer of the polyclonal antibody was 3x10(-4). CONCLUSION: The recombinant expression plasmid of hcTnI was constructed successfully and expressed in E.coli. The prepared rabbit anti-hcTnI antibody had a high titer and specificity.

[Experimental study of E-box, a transcriptional suppressor element in gamma-GCS gene].

OBJECTIVE: To validate that the E-box element in the -745-705 region of rat gamma-GCS catalytic subunit gene (GCLC gene), already found to be a negative regulatory region, is an important transcriptional suppressor element. METHODS: Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to confirm the specific binding of USF, a transcription factor that binds the regulatory sequence -745 - 705, and E-box element. Rat alveolar epidermal cells of the line CCL-149 were cultured and then divided into 4 groups: group 1 to be transfected with GCLC-Luc, group 2 to be co-transfected with pGCLC-Luc and pCMV-USF1, group 3 to be co-transfected with pGCLC-Luc and pCMV-USF2, and group 4 to be co-transfected with pGCLC-Luc and blank vector pCMV. Recombinant retrovirus vector PLXSN-USF1 and recombinant retrovirus vector PLXSN-USF2 were constructed. Another CCL-149 cells were divided into 4 groups: group A to be infected with recombinant USF1 virus, group B to be infected with recombinant USF2 virus, group C to be infected with blank vector, and group D not to be infected with any plasmid. Western blotting was used to detect the concentrations of GCLC protein. RESULTS: EMSA showed that only the probes with complete E-box element could be bound by the USFs. Supershift assay showed that the transcription factor binding the probe was USF. The luciferase activity of the CCL-149 cells co-transfected with pCMV-USF1/USF2 and GCLC-Luc were decreased by 66.4% and 63.2% respectively in comparison with those transfected with GCLC-Luc only (both P < 0.05) and decreased by 54.5% and 61.1% respectively in comparison with those of the cells of the blank vector group (both P < 0.05). Western blotting showed that the expression of GCLC protein of the CCL-149 cells transfected with PLXSN-USF1 and PLXSN-USF2 were decreased in comparison with that of the control cells. CONCLUSION: The interaction between E-box and USF suppresses the expression of GCLC gene. E-box is an important transcriptional suppressor element of gamma-GCS gene.

[Preliminary analysis of the 5'-flanking region of rat gamma-glutamylcysteine synthetase catalytic subunit gene].

OBJECTIVE: To analyze the characteristic of regulatory elements and corresponding transcriptional factors in the 5'-flanking region of rat gamma-glutamylcysteine synthetase (gamma-GCS) catalytic subunit (GCLC) gene. METHODS: A 1 760 bp 5'-flanking region of the rat GCLC was cloned and constructed into pGL-3 enhancer vector which includes luciferase reporter gene. Exonuclease III was used to cut the 5'-flanking region of rat GCLC gene unidirectionally into deletion mutants of different length, GCLC-Luc and its deletion mutants were used to transfect rat alveolar epithelium cells CCL-149, then the regulatory region of the gene was determined by luciferase activity assay of the transfected cells. Analysis of the transcription-factor-binding site was done using Transcription Factor Search software to indicate possible transcriptional factors. Electrophoresis mobility shift assay (EMSA) was used to determine the cis-acting elements and transcriptional factors in these regulatory regions. RESULTS: The experiment cloned the upstream regulatory sequence of rat GCLC gene and its reporter vector GCLC-Luc, as well as 11 deletion mutants of GCLC-Luc. Luciferase activity assay of the cells transfected by GCLC-Luc (-1 758/+2-Luc), mutant 1 (-1 231/+2-Luc), mutant 2 (-1 108/+2-Luc), mutant 3 (-1 087/+2-Luc), mutant 4 (-876/+2-Luc), mutant 5 (-745/+2-Luc), mutant 6 (-705/+2-Luc), mutant 8 (-613/+2-Luc), mutant 9 (-595/+2-Luc), mutant 10 (-403/+2-Luc) and mutant 11(-111/+2-Luc) were (90 012 +/- 2 445), (77 652 +/- 840), (149 927 +/- 4 915), (71 588 +/- 1 108), (99 283 +/- 2 612), (75 443 +/- 1 438), (282 772 +/- 7 046), (96 891 +/- 2 275), (148 917 +/- 5 966), (258 991 +/- 5 015) and (895 +/- 49) U, respectively. EMSA proved that activated protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) can bind to the region of -403 to -111 bp; nuclear factor-1 (NF-1) and CCAAT/enhancer binding protein (C/EBP) can bind to the region of -705 to -613 bp; and upstream stimulatory factor (USF) can bind to the region of -745 to -705 bp. CONCLUSIONS: Two DNA regions -403 to -111 bp and -705 to -613 bp of GCLC gene, which can be bound by transcriptional factors such as NF-1, C/EBP, AP-1, and NF-kappaB on EMSA, are involved in positive gene regulation. A newly identified region -745 to -705 bp of GCLC gene, which can be bound by USF, is involved in negative gene regulation, suggesting that the interaction between E-box and USF can inhibit the expression of gamma-GCS.

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3',5'-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (), having 61.7% beta-sheet and no alpha-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD(50) of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5+/-160.5%. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.

Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E. coli.

Three full-length phospholipase A(2) (PLA(2)) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study. In order to investigate their biological functions, we established a fusion expression system for PLA(2)-9 in E. coli. The open reading frame encoding mature peptide of PLA(2)-9 was subcloned into the vector pTRX. The Trx-PLA(2)-9 fusion protein was expressed as a soluble protein by IPTG induction at 23 degrees C. The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase. The mature recombinant PLA(2)-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA(2)-9 from 1l of bacteria culture was obtained. The catalytic activity of recombinant PLA(2)-9 (rPLA(2)-9) was measured and found to be similar to native enzyme. As the Austrelaps superbus PLA(2), which shares 90% nucleotide sequence similarity to PLA(2)-9, the rPLA(2)-9 displayed the anti-platelet aggregation effect. Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA(2)-9 may be dissociated.

Cloning and characterization of an acidic cytolysin cDNA from sea anemone Sagartia rosea.

A full-length cDNA of cytolysin (Src I) was isolated from the tentacle of Sagartia rosea (a representative species in China) by reverse transcription polymerase chain reaction. The cDNA with an open reading frame of 648 bp encodes a precursor protein of 216 amino acids, which contains a prepropeptide of 38 amino acids including a signal peptide of 19 amino acids and a propart of 19 amino acids. Lys-Pro at C-terminus of propart is a cleavage site for proline-endopeptidase-like protease. The mature cytolysin has a molecular mass of 19.6 kDa and a pI value of 4.8. Src I is an acidic cytolysin found in sea anemone and shares 75% amino acid sequence similarity to equinatoxin II (Eqt II). The predicted secondary structure of the mature cytolysin comprises 15% alpha-helix, 45% beta-sheet, and 40% random coil. The characteristic amphiphilic alpha-helix of cytolysin is located at the N-terminus of the processed Src I.

[The construction of cDNA expression library from the tentacles of Sagartia rosea].

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.