MicroRNA-34a inhibits the proliferation and promotes the apoptosis of non-small cell lung cancer H1299 cell line by targeting TGFßR2.

MicroRNAs (MiRNAs) are small non-coding RNA molecules which act as important regulators of post-transcriptional gene expression by binding 3'-untranslated region (3'-UTR) of target messenger RNA (mRNA). In this study, we analyzed miRNA-34a (miR-34a) as a tumor suppressor in non-small cell lung cancer (NSCLC) H1299 cell line. The expression level of miR-34a in four different NSCLC cell lines, H1299, A549, SPCA-1, and HCC827, was significantly lower than that in the non-tumorigenic bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34a expression level was also significantly decreased in pT2-4 compared with the pT1 group. Moreover, miR-34a mimic could inhibit the proliferation and triggered apoptosis in H1299 cells. Luciferase assays revealed that miR-34a inhibited TGFßR2 expression by targeting one binding site in the 3'-UTR of TGFßR2 mRNA. Quantitative real-time PCR (qRT-PCR) and Western blot assays verified that miR-34a reduced TGFßR2 expression at both mRNA and protein levels. Furthermore, downregulation of TGFßR2 by siRNA showed the same effects on the proliferation and apoptosis as miR-34a mimic in H1299 cells. Our results demonstrated that miR-34a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the downregulation of its target gene TGFßR2.

[Tumor-targeting expression of a new tumor suppressor gene HCCS1 and its tumor-selective inhibitory effects on hepatocellular carcinoma].

OBJECTIVE: To construct a tumor-targeting recombinant adenovirus vector containing hepatocellular carcinoma suppressor gene HCCS1 to enhance the safety of tumor treatment. METHODS: CCK-8 assay was used to observe different inhibitory effects on normal and malignant liver cells with high expressions of HCCS1 protein. The relative transcriptional activity of PEG-3p was quantified by luciferase assay. Recombinant adenovirus Ad-PEG-3p-HCCS1 was packaged with AdEasy system and confirmed by PCR. The tumor-targeted expression of HCCS1 protein in cells infected with Ad-PEG-3p-HCCS1 was determined by Western blot. Crystal violet assay and MTT assay were applied to observe the selective anti-tumor effects of the newly constructed virus in vitro. RESULTS: A higher inhibitory rate of about 60% was found in BEL-7404 and SW-620 than that in L02 and NHLF 96 h after the high expression of HCCS1. Luciferase assay showed 3.9-, 4.7-, and 1.5-fold transcriptional activity in BEL-7404, BEL-7405 and QGY-7703 respectively, in comparison with that in L02. Ad-PEG-3p-HCCS1 was constructed successfully and was verified by PCR. Western blot indicated that high expression of HCCS1 could be induced in BEL-7404 and QGY-7703 but not in L02. Crystal violet assay and MTT assay showed that it remarkably reduced the toxicity to L02 but still had enough antitumoral effect on Ad-CMV-HCCS1. CONCLUSIONS: With high expression of HCCS1 the tumor cells we used are being inhibited more. PEG-3p has the tumor-selective driving function in malignant liver cells. Our recombinant adenovirus Ad-PEG-3p-HCCS1 can tumor-targeting induce HCCS1 expression in tumor cells, which can improve the safety of gene therapy with HCCS1.

[The anti-tumour effect of Wuxing soup and its mechanism in inducing apoptosis of tumour cells mediated by calcium].

OBJECTIVE: To confirm the anti-cancer effect and mechanism of Wuxing soup. METHODS: Inhibition of cellular growth under Wuxing soup treatment was observed by MTT; Apoptosis was detected by gel electrophoresis, transmission electron microscopy and FACS; The concentration of calcium was measured by fluorescence probe. RESULTS: After SGC-7901 cell being treated by Wuxing soup, it showed that: 1) Wuxing soup could specifically inhibit cancer cells proliferation in a time and dose dependent manner; 2) Typical apoptotic morphological changes and DNA ladder of SGC-7901 cells were observed; 3) calcium inhibitor Bapta AM could reduce the apoptotic rate and protect SGC-7901 cells in a dose dependent manner. CONCLUSION: Wuxing soup has an effective inhibition on cancer cells, and can induce SGC-7901 cells to apoptosis by calcium.

The Principle of the Micro-Electronic Neural Bridge and a Prototype System Design.

The micro-electronic neural bridge (MENB) aims to rebuild lost motor function of paralyzed humans by routing movement-related signals from the brain, around the damage part in the spinal cord, to the external effectors. This study focused on the prototype system design of the MENB, including the principle of the MENB, the neural signal detecting circuit and the functional electrical stimulation (FES) circuit design, and the spike detecting and sorting algorithm. In this study, we developed a novel improved amplitude threshold spike detecting method based on variable forward difference threshold for both training and bridging phase. The discrete wavelet transform (DWT), a new level feature coefficient selection method based on Lilliefors test, and the k-means clustering method based on Mahalanobis distance were used for spike sorting. A real-time online spike detecting and sorting algorithm based on DWT and Euclidean distance was also implemented for the bridging phase. Tested by the data sets available at Caltech, in the training phase, the average sensitivity, specificity, and clustering accuracies are 99.43%, 97.83%, and 95.45%, respectively. Validated by the three-fold cross-validation method, the average sensitivity, specificity, and classification accuracy are 99.43%, 97.70%, and 96.46%, respectively.

MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2.

Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3'-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2.

[Experimental induction of immunity by anti-idiotypic vaccines against small-cell-lung cancer].

OBJECTIVE: To investigate the efficacy of anti-idiotype antibody 3F6 and its single-chain variable fragment (3F6 ScFv) to induce humoral and cellular immune responses against small-cell-lung cancer (SCLC). METHODS: 3F6 and 3F6 ScFv (Ab2) were used to immunize BALB/c mice. The reaction of antibodies (Ab3) with specific antigen on NCI-H128 cells was tested by ELISA and Western blot, and the antibody binding inhibition assays were performed by competitive Western blot. Cellular immunity against SCLC induced by Ab2 was detected by a delayed-type hypersensitivity response and mouse lymphocyte proliferation assay. RESULTS: The sera immunized with Ab2 showed significant reaction (P < 0.001, as compared to control sera) with SCLC-specific antigen on NCI-H128 cells and specifically competed the binding of 2F7 (Ab1) to the specific antigen. DTH responses challenged with NCI-H128 cells were significantly (P < 0.001) stronger in mice immunized with Ab2 as compared to mice immunized with normal mouse IgG. T cell proliferation was significantly higher in Ab2-immunized mice (P < 0.05) than in control mice. CONCLUSION: The two kinds of anti-idiotypic antibodies successfully mimic the SCLC-specific antigen on NCI-H128 cell and induce strong humoral and cellular immune responses to SCLC-specific antigen in syngeneic mice. They may become novel vaccines against human small-cell-lung cancer and worthy of further investigation.

Investigation of Protein-DNA Interactions in Enhancer II and Core Promoter of HBV by in vivo Footprinting.

The protein-DNA interactions in the enhancer II (ENII) and core promoter regions of hepatitis B virus (HBV) were investigated by the ligation-mediated polymerase chain reaction (LMPCR) in vivo footprinting in HepG2.2.15 cell line. Major footprints are evident at the enhancer II and core promoter regions. In particular, proteins appear to interact with the sites for Sp1, C/EBP, HNF4, HNF3, HNF1 and hB1F, as well as the TATA-like element. It confirmed that these protein-DNA interactions defined in vitro play important roles in the regulation of 3.5 kb RNAs transcription of HBV in vivo. Notably, three novel sites nt 1774-1789, nt 1801-1818 and nt 1835-1843 were found to be protected or hypersensitive, indicating that they were also involved in protein-DNA interactions in vivo. Functional analysis using CAT reporter gene demonstrated that the site from nt 1801 to 1818 was important for the function of HBV core promoter.

Modification of YACs by Homologous Recombination and Transfer of a 330 kb YAC into Mammalian Cells.

YAC clones were modified via homologous recombination by transfecting yeast cells containing YAC with plasmid PRAN4 DNAs. The integration of neo gene into YAC DNA was identified using Southern blotting and PCR methods. A modified YAC that carried 330 kb human DNA was introduced into L929 cells through PEG mediated spheroplast fusion, and G418 resistant colonies were obtained.

miR-150, p53 protein and relevant miRNAs consist of a regulatory network in NSCLC tumorigenesis.

microRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. Tumor protein p53, a transcriptional factor, plays an important role in the progression of tumorigenesis. miR-150 was the only miRNA predicted to target 3'-UTR of p53 by Targetscan. In order to investigate the function of miR-150, p53 and relevant miRNAs in non-small cell lung cancer (NSCLC), we constructed two expression vectors of p53 (pcDNA3.1-p53 and pcDNA3.1-p53-3'-UTR) and two report vectors (pGL3-p53-3'-UTR and pGL3-p53-3'-mUTR). The activity of luciferase transfected with miR-150 mimics was lower by 30% when compared to that of the miRNA-negative control (miRNA-NC). Moreover, the p53 protein was downregulated by at least 50% when miR-150 mimics were cotransfected with pcDNA3.1-p53-3'-UTR when compared to miRNA-NC. We also determined the expression of miR-150 and p53 in NSCLC patient tissue samples. The expression of miR-150 in T2 stage tissue samples was higher than that in T1 stage tissue samples. The corresponding target gene p53 was correlated with miR-150 expression. In the present study, we further analyzed the cell cycle distribution. The cells transfected with pcDNA3.1-p53 were significantly arrested in the G1 phase when compared to the control cells. When miR-150 mimics were cotransfected with pcDNA3.1-p53-3'-UTR, the percentage of cells in the G1 phase was significantly lower by 4% when compared to miRNA-NC. To identify miRNAs that are regulated by the p53 protein, qRT-PCR was performed after pcDNA3.1-p53 transfection. miR-34a, miR-184, miR-181a and miR-148 were upregulated significantly. However, there was no distinct difference in the expression of miR-10a, miR-182 and miR-34c. Our results showed that miR-150 targets the 3'-UTR of p53, and p53 protein promotes the expression of miRNAs which affect cell cycle progression. These findings suggest that miR-150, p53 protein and relevant miRNAs are members of a regulatory network in NSCLC tumorigenesis.

[Preparation of anti-human integrin beta 3 monoclonal antibody and study on its antitumor activity].

AIM: To prepare the monoclonal antibody (mAb) against human integrin beta 3, and explore its role in tumor therapy. METHODS: Total RNA was isolated from human lymphocytes, and the DNA fragment encoding extra-cellular domain of human integrin beta 3 was amplified by RT-PCR. The integrin beta 3 gene was cloned into the prokaryotic expression vector pQE30, and the expression plasmid pQE30-beta 3 was transformed into E.coli M15. The expression of beta 3 protein was induced by IPTG, and the expressed beta 3 protein was purified by Ni-affinity agarose. BALB/c mice were immunized with the purified beta 3 protein, and mAb was prepared by hybridoma technique. The specificity of the mAbs was identified by Western blot. The mAbs were screened by their inhibitory effect on the tumor growth in vivo. The inhibition of the mAb to HUVEC cell growth was detected by MTT assay. The role of the mAb in inducing HUVEC apoptosis was analyzed by flow cytometry (FCM). RESULTS: The extra-cellular gene fragment of human integrin beta 3 was amplified by RT-PCR, and the expression plasmid pQE30-beta 3 was constructed. Human integrin beta 3 protein was expressed and purified, and used for immunization. Eight clones of mAb against human integrin beta 3 were successfully prepared. The mAb 4F12 was found to inhibit tumor growth in vivo and reduce blood vessels in tumor. Furthermore, the mAb 4F12 could inhibit HUVEC growth and induce apoptosis in vitro. CONCLUSION: The human integrin beta 3 protein was obtained and the mAbs against it have been prepared successfully. The mAb 4F12 can inhibit tumor growth in vivo and reduce blood vessels in tumor. One of the mechanisms of the antitumor effect is inhibition of growth and induction of apoptosis of endothelial cells of blood vessels in tumor.

A new combination of mutated loxPs in a vector for construction of phage antibody libraries.

In the construction of large antibody libraries by in vivo recombination, two non-homogeneous loxP sites are required for the exchange of V genes between phagemids to create many new VH-VL combinations. The mutated loxP511 was designed not to recombine with the wild-type loxP (loxPwt) in early studies and a combination of the two has been used to construct antibody libraries. But recent reports have shown that recombination occurs between loxPwt and loxP511. This suggests that the combinational use of loxP511 and loxPwt might lead to the loss of the V gene diversity of antibody libraries. Therefore, it is necessary to find a new combination of loxPs to avoid the excision recombination in the antibody library. In this study, we found that the excision recombination between loxP511 and loxP2272, another mutated loxP sequence, was undetectable within one phagemid, while the excision recombination between loxP511 and loxPwt occurred at a frequency of 40%, higher than that reported previously. Furthermore, the in vivo recombination of different phagemids with loxP511 and loxP2272 showed that the V gene exchange was efficiently mediated to produce new VH-VL combinations. It was concluded that the loxP511 and loxP2272 combination was more favorable for reducing the excision recombination and constructing large phage antibody libraries with high diversity.