Negative Control Plasmid Research Articles

MicroRNA-449a suppresses liver cancer migration and invasion through targeting CXC chemokine ligand 5

Objective To explore the expression of microRNA (miRNA, miR)-449a in liver cancer, the effect on migration and invasion, and the possible regulatory mechanism. Methods Eighteen cases of liver cancer tissues and adjacent tissues were collected. The expression of miR-449a in liver cancer tissues and adjacent tissues, 4 kinds of human liver cancer cell lines, and human normal hepatocytes was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The human liver cancer cell lines were transfected with miR-449a mimic and the negative control (NC) plasmid. The expression of miR-449a after transfection was detected by RT-PCR. The cell number of invasion and migration after transfection in the two groups was measured by Transwell assays. The expression of CXC chemokine ligand 5 (CXCL5) protein in the cells was detected by Western blotting. Results As compared with the adjacent tissues, the expression of miR-449a in liver cancer tissues was significantly reduced (0.957±0.165 vs. 0.462±0.221, t=4.137, P=0.021). As compared with the human normal hepatocytes, the expression of miR-449a in four kinds of human liver cancer cell lines was significantly reduced too (P=0.009, 0.008, 0.005, 0.020), the difference being statistically significant (BEL-7402: 6.873±1.293, HepG2: 7.246±1.320, NC: 1.000, P=0.012, 0.017). Two kinds of human liver cancer cell lines with the middle expression of miR-449a were chosen, and the expression of miR-449a after transfection was statistically increased, and the cell number of invasion (BEL-7402 vs. NC: 21.57±13.32 vs. 79.84±11.68; HepG2 vs. NC: 45.32±12.65 vs. 92.21±15.43, t=24.331, 21.109, P=0.011, 0.019) and migration (BEL-7402 vs. NC: 10.25±3.21 vs. 26.63±6.48; HepG2 vs. NC: 23.44±10.08 vs. 65.65±12.26, t=11.039, 12.224, P=0.010, 0.008) after transfection was significantly reduced. The expression of CXCL5 protein after transfection was statistically significantly reduced (BEL-7402 vs. NC: 0.367±0.083 vs. 0.965±0.110; HepG2 vs. NC: 0.163±0.043 vs. 0.949±0.141, t=20.176, 25.158) (P=0.021, 0.016). Conclusion The miR-449a in liver cancer is expressed weakly, and can serve as a tumor suppressor to influence the migration and invasion of liver cancer cells through targeting CXCL5. Key words: MicroRNA-449a; CXC chemokine ligand 5; Liver cancer; Migration; Invasion

Silencing of ANXA3 expression by RNA interference inhibits the proliferation and invasion of breast cancer cells.

The present study aimed to explore the expression of AnnexinA3 (ANXA3) in breast cancer cells and the mechanisms involved in the regulatory effects of ANXA3 on proliferation, invasion and migration of breast cancer cells. Fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were used to measure the expression of ANXA3 mRNA and protein in two breast cancer cell lines (MDA-MB-231 and MCF-7). Three ANXA3 silencing shRNA plasmids (ANXA3-sh1-3) and one negative control plasmid were constructed, and the Lipofectamine transfection method was used for transfecting human breast cancer cell line MDA-MB-231. Flow cytometry was used to measure the transfection efficiency. The expression of ANXA3 protein was measured by western blotting. Cell cycle distribution and apoptosis were assessed by flow cytometry. Migration and invasion of the transfected cells were evaluated using wound healing and Transwell assays, respectively. The expression levels of ANXA3 mRNA and protein were significantly higher in the MDA-MB-231 cells than levels in the MCF-7 cells. Western blotting showed that the ANXA3 protein level was significantly lower in the MDA-MB-231-Sh cells than that in the MDA-MB-231 and MDA-MB-231-NC cells. In addition, the percentage of G0/1 cells and the apoptosis rate were significantly higher, while the cell proliferation rate was significantly lower, in the MDA-MB-231-Sh cells when compared with the MDA-MB-231-NC and MDA-MB-231 cells. The cell migration and invasion abilities were also lower in the MDA-MB-231-Sh cells than these abilities in the MDA-MB-231-NC and MDA-MB-231 cells. The present study investigated the relationships between ANXA3 and proliferation, apoptosis, migration and invasion of breast cancer cells to elucidate the mechanisms involved in the development, progression, invasion and metastasis of breast cancer.

Abstract 1376: Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency (<0.1%) was also detected in the same reaction. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for exon19 deletion, and 0.9959 for T790M, respectively. CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1376.

Inhibitory effect of RNA-mediated knockdown of zinc finger protein 91 pseudogene on pancreatic cancer cell growth and invasion.

Worldwide, human pancreatic cancer is a rare malignancy with a poor prognosis. Long non-coding RNAs (lncRNAs) are known to have a crucial role in cancer occurrence and progression; however, the role of pseudogene-expressed lncRNAs, a major type of lncRNA, have not been thoroughly analyzed in cancer. Therefore, the present study focused on zinc finger protein 91 pseudogene (ZFP91-P). ZFP91-P expression was initially detected in two pancreatic cancer cell lines by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the highest expression of ZFP91-P was found in the BXPC-3-H cell line. Subsequently, BXPC-3-H cells were transfected with ZFP91-P short hairpin RNA (shRNA) using a plasmid vector and termed shZFP91-P. Cells transfected with negative control plasmid vector were termed shCon. MTT and Transwell assays were performed to analyze the proliferation and migration of BXPC-3-H cells, respectively, and western blotting was used to detect epithelial-mesenchymal transition markers, including vimentin and β-catenin. The present study showed that depletion of ZFP91-P markedly decreased pancreatic cancer cell proliferation and inhibited cell migration capacity. In addition, the expression of β-catenin increased while vimentin expression decreased. The current findings suggest that high expression of ZFP91-P promotes the migration of BXPC-3-H cells and may be a novel marker for early diagnosis for pancreatic cancer.

Knockdown of MACC1 expression increases cisplatin sensitivity in cisplatin-resistant epithelial ovarian cancer cells.

Abnormal expression of metastasis-associated in colon cancer 1 (MACC1) was found to be closely associated with several types of malignant tumors. The present study aimed to verify the relationship between MACC1 and cisplatin resistance in ovarian cancer cells and the possible mechanisms, which was implemented by inhibition of the expression of MACC1 in cisplatin-resistant human ovarian cancer cell lines A2780/DDP and COC1/DDP. MACC1 shRNA eukaryotic plasmids and negative control plasmids were transfected into A2780/DDP and COC1/DDP cells, respectively, while A2780/DDP and COC1/DDP cells were used as blank controls. Western blotting and sqRT-PCR were used to detect the expression of MACC1 in the different cell groups. Different concentrations of cispaltin (0, 10, 20, 30, 40, 50 and 60 µmol/l) were used to treat the cell groups, respectively, and then the chemosensitivity of cisplatin and cell apoptosis were examined by MTT and flow cytometry, respectively. The activity of caspase-3 was determined by spectrophotometry. Expression levels of p-ERK1/2, permeability glycoprotein (P-gp), B-cell lymphoma 2 (Bcl-2), Bcl-XL, Bax and Bad protein were detected in the different ovarian cancer cells by western blotting. After MACC1 knockdown, the chemosensitivity of cisplatin in the ovarian cancer cells was enhanced, and the cell growth inhibition and apoptosis rates were increased. The expression levels of Bax and Bad were upregulated, the activity of caspase-3 was increased, while the expression levels of p-ERK1/2, P-gp, Bcl-2 and Bcl-XL were downregulated as a result of MACC1 inhibition. These results indicate that inhibition of MACC1 improves the chemosensitivity of cisplatin in epithelial ovarian cancer cells, through the regulation of the ERK1/2 signaling pathway on P-gp and its downstream apoptosis proteins.

Construction of a Hep-2 cell line stably transfected with Livin shRNA.

The aim of this study was to construct a eukaryotic expression plasmid with a short hairpin RNA (shRNA) targeting Livin in order to obtain a stably transfected Hep-2 cell line with a reduced expression of Livin. The shRNA targeting Livin mRNA was designed, and a shRNA plasmid and a negative control plasmid were constructed. After amplification in E. coli, restriction endonuclease digestion and sequence confirmation, the plasmids were transfected into Hep-2 cells using Lipofectamine 2000. The stably transfected cell line was screened using G418, and inhibition of Livin mRNA and protein levels were detected using real-time PCR and western blotting, respectively. pGenesil-Livin-shRNA eukaryotic expression plasmid was successfully constructed and identified by sequencing. Green fluorescent protein (GFP) expression was observed in Hep-2 cells transfected with shRNA plasmids by fluorescence microscopy. The expression levels of Livin mRNA and protein decreased significantly in Hep-2 cells transfected with the shRNA recombinant plasmid. The mRNA level was reduced by 47.17 %, and the protein level was reduced by 34.25 %. The shRNA eukaryotic expression plasmid targeting Livin was successfully constructed, which could significantly inhibit the expression of Livin in laryngeal cancer Hep-2 cells. This provides a basis for future research on the function of Livin in Hep-2 cells, and gene therapy for laryngeal cancer.

Abstract 4908: Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of EGFR tyrosine kinase inhibitor (TKI) treatment. Three most common EGFR mutations (L858R, exon19 deletion, and T790M) are known to be major selection marker for EGFR-TKI therapy. Noninvasive techniques for tumor genotyping may be needed to fully realize the potential of genotype-directed cancer care as a “Liquid biopsy”. The low abundance of mutants and limited amount and quality of available cell-free DNA (cfDNA) in plasma samples render it difficult to reliably detect multiple mutations with current platforms and methods. Here, we develop a multiplex dPCR assay to detect three common EGFR mutations in one reaction using picodroplet digital PCR (ddPCR) technology, which enable us to provide 3-fold throughput and save the cost as well as DNA samples. METHODS: Positive and negative control plasmids for the EGFR assay were prepared by cloning DNA fragments containing wild-type or the EGFR mutations. The appropriate concentration of plasmid DNA was determined empirically to yield a mixture in which the number of copies of mutant DNA was ca. 0.01-1.00% of the number of wild-type EGFR fragments. Genomic DNA from the lung tumor cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. RESULTS: Serial dilutions experiments using plasmid as well as genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9983for L858R, 0.9911 for exon19 deletion, and 0.9781 for T790M. The limit of blank (LOB) was defined by the frequency of positive droplets measured in 400 ng of wild-type DNA control samples. The number of false events of mutant droplets detected per analysis is: 8 for L858R, 6 for Exon 19 deletion, 6 for T790M from human normal gDNA, and 9 for L858R, 4 for Exon 19 deletion, 2 for T790M from A549 gDNA. Thus, the LOB was determined as a 10 events/assay. CONCLUSIONS: Using ddPCR, we established multiplex and ultra-sensitive genotyping platform for three common EGFR mutations. Results of a proof-of-principle study using preclinical model indicated that the clinical utility of multiplex ddPCR to detect for multiple EGFR mutations. Further evaluation using clinical samples is warranted. Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Masaru Watanabe, Takashi Kikuchi, Masanori Nakanishi, Kazuto Matsunaga, Nobuyuki Yamamoto. Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4908. doi:10.1158/1538-7445.AM2015-4908

Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.

Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NC- PANC-1, and si-PANC-1 cells, respectively. After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.

Silencing the wild-type and mutant K-ras increases the resistance to 5-flurouracil in HCT-116 as a colorectal cancer cell line.

Colon cancer is the second to third common cancer worldwide. Several efforts have been made to reveal the pathways responsible for drug resistance in this type of cancer. We aimed to investigate the effect of silencing both mutant and wild-type Kristen Rous sarcoma (k-ras) on the response of human colorectal tumor 116 (HCT-116) as a colon cancer cell line to the cytotoxic effect of 5-flurouracil (5-FU). One oligonucelotide against mutant k-ras (12th codon, namely 207) and two against wild-type k-ras (namely 535 and 689) were cloned into pSilencer neo2.1. The linearized vectors besides the negative control plasmid were stably transfected into HCT-116. The proliferation rates of these cells in different concentrations of 5-FU and the apoptosis rates of the cells after treatment with lethal doses of 5-FU were studied. Moreover, the cell cycle in these cells was also analyzed by staining the cells with propidium iodide. Stably transfected cells were named HCT207ks, HCT535ks, HCT689ks, and HCT-Sc (transfected with the negative control plasmid). Decreased expression of k-ras in HCT207ks, HCT535ks, and to a lesser extent in HCT689ks was proved by quantitative real-time PCR. Although in HCT207ks the cells were mostly in G0/G1 and G2/M phases, in HCT535ks and HCT689ks, the cells in the S phase were higher in comparison with nontransfected HCT-116. Lethal doses of 5-FU in HCT-116 and HCT-Sc were 2.5-3 and 3-3.5 µmol/l, whereas in HCT207ks, HCT535ks, and HCT689ks, they were 35-40, 37.5-40, and 22.5-25 µmol/l. In conclusion, silencing mutant and wild-type k-ras would increase the resistance of HCT-116 cell line as a model of colorectal cancer to 5-FU. The degree of resistance was related directly to the k-ras mRNA level. Therefore, both mutant and wild-type k-ras may play a role in sensitizing colorectal cancer cells to 5-FU as a common chemotherapeutic drug.

Heat shock protein A12B decreases lipopolysaccharide-induced permeability of human umbilical vein endothelial cells

Objective To explore the lipopolysaccharide(LPS)-induced changes of heat shock protein A12B(HSPA12B)expression and its effect on the permeability of human umbilical vein endothelial cells(HUVECs).Methods The HUVECs cultured in vitro were divided into four groups: control group(without any treatment); LPS group(1 μg / m L LPS); LPS + p IRES2-EGFPHSPA12B-3Flag group(The HSPA12 B gene overexpression plasmid was transiently transfected into HUVECs and then LPS of 1 μg / m L was added); and LPS + p IRES2-EGFP-3Flag group(The negative control plasmid was transiently transfected into HUVECs and then LPS of 1 μg / m L was added).The transendothelial electrical resistance(TEER) of HUVECs was measured by MERSST × 01 Electrode.The expression of VE-cadherin was studied by flow cytometry.RT-PCR and Western blotting analysis were used to examine the expression changes of HSPA12 B mRNA and protein in HUVECs stimulated with LPS at various time points.Results It was showed that after stimulated with LPS,HSPA12 B mRNA and protein were gradually upregulated,and peaked at 12 h.HSPA12 B significantly increased the TEER value of HUVECs(P 0.001),and it could completely offset the decline of TEER value induced by LPS(P 0.001).Besides,HSPA12 B could cause upregulation of VE-cadherin expression.Conclusion HSPA12 B can reduce the permeability of vascular endothelial cells by up-regulating the expression of VE-cadherin,thus protecting the vascular endothelial barrier function of endothelial cells.

The noninvasive detection of RARβ2 promoter methylation for the diagnosis of prostate cancer.

Prostate cancer is a kind of commonly diagnosed male malignancy. With the aging population in China, both incidence and mortality of prostate cancer are expected to keep increasing in the future. The methylation of RARβ2 gene promoter is a common molecular event in prostate cancer. Thus, we aimed at establishing a high-performance noninvasive DNA methylation assay based on pyrosequencing for screening of prostate cancer in this article. The assay is designed to detect aberrant promoter methylation of RARβ2 gene in ejaculate samples. The negative and positive control plasmids were constructed with different treatments by direct bisulfite conversion or conversion after Sss I Methylase methylation to establish quality control standard. The ejaculate and tissue samples were collected from patients with histologically confirmed adenocarcinoma of prostate (n = 43) and benign prostatic hyperplasia (n = 40). Significant correlation was observed between prostate cancer and methylation level of RARβ2 gene promoter. In addition, the results of pyrosequencing in ejaculate samples were compared with that of DNA sequencing in tissue samples from the same patients. There is no significant difference in the detection of RARβ2 promotor methylation between these two methods (p < 0.05). In conclusion, we have developed a high-performance noninvasive DNA methylation assay based on pyrosequencing which is more suitable for high-throughput detection of aberrant promoter methylation in ejaculate samples. Moreover, the acceptive degree of this noninvasive method makes it potentially promising for future screening of prostate cancer.

Stable knockdown of protein kinase CK2-alpha (CK2α) inhibits migration and invasion and induces inactivation of hedgehog signaling pathway in hepatocellular carcinoma Hep G2 cells

Protein kinase CK2-alpha (CK2α), one isoform of the catalytic subunits of serine/threonine kinase CK2, has been indicated to participate in tumorigenesis of various malignancies, including hepatocellular carcinoma (HCC). In the present study, in order to explore the potential role of CK2α in human HCC, we employed short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology to inhibit the endogenous CK2α expression in HCC cells and established a Hep G2 cell line with stable knockdown of CK2α. Results from wound healing and transwell invasion assays indicated that stable knockdown of CK2α markedly inhibited Hep G2 cell migration and invasion as compared with those transfected with a negative control plasmid. This alteration was accompanied with expression down-regulation of matrix metalloproteinase (MMP)-2, MMP-9, Snail, Slug, Vimentin, and up-regulation of epithelial cadherin (E-cadherin). Moreover, CK2α silencing also induced inactivation of Hedgehog signaling pathway by inhibiting Gli1 and Patched homolog 1 (PTCH1) expressions in HCC cells. Collectively, these results demonstrate that knockdown of CK2α can suppress cell migration and invasion, reduces expression of MMPs, inhibits epithelial-mesenchymal transition (EMT) process and induces inactivation of Hedgehog pathway in HCC cells in vitro. Our study provides in vitro evidence to demonstrate that the pathogenesis of human HCC may be correlated with the high expression of CK2α.

Transcription factor Snai1-1 induces osteosarcoma invasion and metastasis by inhibiting E-cadherin expression

Osteosarcoma (OS) is a type of primary malignant bone tumor with a high propensity for local recurrence and distant metastasis. A previous study showed Snail-1 is highly expressed in OS cells. The present study aimed to investigate the association between the transcription factor Snai1 and E-cadherin in OS. SaOS2 OS cells were transfected either with a plasmid expressing short hairpin RNA (shRNA) specific for the Snai1-1 gene (SaOS2-shRNA) or a negative control plasmid (SaOS2-Mock). The expression levels of E-cadherin and Snai1-1 in the transfected and control cells were determined by quantitative polymerase chain reaction and western blot analysis. In addition, the study was extended to evaluate the migratory and invasive properties of the cells through a Transwell experiment. The results show that E-cadherin was expressed at a high level in the SaOS2-shRNA cells, which were much less migratory and invasive than the control cells. Overexpression of Snai1-1 in OS is associated with tumor progression, possibly through the suppression of E-cadherin expression and induction of the process of epithelial-mesenchymal transition, which contributes to the proceeding invasion and metastasis of OS cells.

EphB4 regulates the growth and migration of pancreatic cancer cells.

Pancreatic cancer is a serious threat to human life. Moreover, its treatment is complicated and its prognosis is very poor. Therefore, a new method for the diagnosis and treatment of pancreatic cancer is very essential. In this study, a eukaryotic expression plasmid targeting EphB4 was constructed and transfected into PANC-1 pancreatic cancer cells to investigate the inhibition of cell growth and the progression of iRNA against EphB4. This study provides the basis for the gene therapy of pancreatic cancer. The recombinant eukaryotic EphB4 expression plasmid, pSIREN-RetroQ-ZsGreen-EphB4 and a negative control plasmid, pSIREN-RetroQ-ZsGreen-N, were constructed. At 48 h after transfection, the relative messenger RNA (mRNA) and protein levels of EphB4 were measured by RT-PCR and western blot. The proliferation of the transfected cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while cell migration ability was analyzed using the scratch migration assay. At 48 h after transient transfection, EphB4 mRNA expression was significantly decreased in transfected PANC-1 cells as compared to the control group (P < 0.01). In vitro, inhibition of EphB4 expression weakened the proliferation and cell migration ability of PANC-1 cells compared to the control group. The small interfering RNA (siRNA) eukaryotic expression plasmid vector targeting EphB4 was successfully constructed and effectively transfected into PANC-1 cells. The recombinant plasmid can inhibit the expression of EphB4 mRNA and protein in PANC-1 cells, as well as cell growth and migration.

Effect of ATG initiation codon context motifs on the efficiency of translation of mRNA derived from exogenous genes in the transgenic silkworm, Bombyx mori.

The context sequence motif surrounding the ATG initiation codon influences mRNA translation efficiency and affects protein production; however, the optimal sequence differs among species. To determine the optimal sequence for production of recombinant proteins in a transgenic silkworm, we compared 14-nucleotide context motifs around the ATG (ATG-context) in 50 silkworm genes and found the following consensus: (A/T)AN(A/T)ATCAAAatgN. We were also able to define the least-common motif: CCN(C/G)CGN(C/T/G)(G/C/T)(T/G)atgC, which served as a negative control. To examine the regulatory role of these motifs in protein expression, we constructed reporter plasmids containing different ATG-context motifs together with either the luciferase gene or an enhanced green fluorescent protein (EGFP) gene. These constructs were then used for comparison of luciferase reporter activity and EGFP production in BmN4 cells in vitro as well as in transgenic silkworms in vivo. We detected 10-fold higher luciferase activity in BmN4 cells transfected with the consensus ATG-context motif construct, compared to the negative control plasmid. ELISA measurements of EGFP translation products with the corresponding constructs in BmN4 cells showed consistently similar results. Interestingly, the translation efficiency of the novel consensus ATG-context motif did not show the highest activity in the transgenic silkworms in vivo, except for the fat body. The highest efficiency in the middle and posterior silk glands was produced by the sericin 1 context. Our results show that the ATG-context motifs differ among silkworm tissues. This result is important for the further improvement of the transgenic silkworm system for the production of recombinant proteins.

Enhanced sensitivity of breast cancer MDA-MB-231 cells to chemotherapy by silencing forkhead box C2 gene with RNA interference technology

Objective To observe the effects of forkhead box C2 gene (FOXC2) short hairpin RNA on docetaxel chemotherapy sensitivity of human breast cancer MDA-MB-231 cells.Methods MDA-MB-231 cells were transfected with recombinant expression plasmid FOXC2-small interfering RNA (siRNA) by lipofectamine 2000,and the expression of FOXC2 mRNA and protein was detected at 48 h after transfection.The morphological changes of apoptotic cells were observed under fluorescent microscope at 72 h after transfection.Methyl thiazol tetrazolium (MTT) assay was used to examine the inhibitory rate and the 50％ inhibitory concentration (IC50).Flow cytometry was applied to examine apoptosis and cell cycle at 72nd h after transfection.Results As compared with negative control plasmid and FOXC2-siRNA non-transfection group,the typical apoptotic morphology of MDA-MB-231 cells was observed,and the mRNA and protein expression levels of FOXC2 were obviously reduced in FOXC2-siRNA transfection group (P ＜ 0.05).Silencing FOXC2 increased sensitivity of MDA-MB-231 cells to docetaxel (P ＜ 0.05),and IC50 was decreased from (6.08 ±1.23) mg/L to (0.65 ±0.01) mg/L.Meanwhile,the apoptosis rate of MDA-MB-231 cells transfected with pFOXC2-shRNA was (20.01 ± 0.19)％,which was significantly higher than other three groups,and the proportion of cells in S phase and G0/G1 phase was decreased and that in G2/M phase was increased (P ＜0.05).Conclusion Down-regulation of FOXC2 expression by siRNA in breast cancer MDA-MB-231 cells could effectively induce apoptosis of MDA-MB-231 cells and increase their sensitivity to docetaxel. Key words: Breast cancer; Docetaxel; Apoptosis; Sensitizing effect

Role of Wnt/β-catenin signaling pathway in proliferation of neural stem cells of neonatal rats in vitro after hyperbaric oxygen therapy

Objective To investigate the role of Wnt/β-catenin signaling pathway in hyperbaric oxygen (HBO)therapy for hypoxic-ischemic brain damage(HIBD). Methods The neural stem cells (NSCs) of neonatal rats were randomly divided into control group and 8 treatment groups, then the treatment groups were cultured in the supernatant of brain homogenate to simulate micro-environment of HIBD and normal brain respectively.Through electroporation β-catenin siRNA and negative control plasmid was transfected into NSCs respectively, then HBO therapy was performed.Immunocytochemical staining was performed simultaneously in the 9 groups of NSCs on precoated chamber slides to detect the differentiation of NSCs.Quantitative reverse transcriptase(RT)-PCR was used to detect the relative content of Ngn1 mRNA and bone morphogenetic protein(BMP4) mRNA in the of NSCs.Western blot was used to detect the relative content of Ngn1 protein and BMP4 protein in the NSCs. Results In vitro, HBO alone promoted NSCs infected with negative control siRNA (ncNSCs) to differentiate into neurons and oligodendrocytes and depressed astrogliosis; HIBD or normal brain tissue extract cultures promoted ncNSCs to differentiate into neurons and oligodendrocytes but depressed astrogliosis, and the effect of HIBD brain extract cultures was superior to the latter and could be further increased by HBO; β-catenin siRNA decreased the NSE-positive neurons and increased glial fibrillary acidic protein-po-sitive astrocytes in the siNSCs (NSCs infected with β-catenin siRNA) in vitro, the effect could not be inversed by HBO, but could be alleviated; HBO increased the level of Ngn1 mRNA and Ngn1 protein, decreased BMP4 mRNA and BMP4 protein of ncNSCs, transfection of β-catenin siRNA could down-regulate the expression of Ngn1 mRNA and up-regulate BMP4 mRNA of NSCs in vitro respectively. Conclusions HBO can promote NSCs cultured with HIBD brain extract cultures to differentiate into neuronal or Oligodendrocyte, and inhibit them to differentiate into astrocytes.HBO therapy promotes the proliferation of NSCs in vitro, an effect which is correlated with β-catenin protein.HBO therapy can promote neurogenesis by β-catenin-induced activated Ngn1, and repress astrocytogenesis by β-catenin-induced down-regulated BMP4.There are potential cooperative actions of BMP4 and Ngn1 on differentiating rat NSCs in cerebral ischemic brain.The ability of Ngn1 to promote neurogenesis may allow Ngn1 to act as a potent neuronal commitment factor. Key words: β-catenin; RNA interference; Hyperbaric oxygen therapy; Hypoxic-ischemic brain damage; Electroporation

Reduction of Osteopontin In Vivo Inhibits Tubular Epithelial to Mesenchymal Transition in Rats With Chronic Allograft Nephropathy

ObjectiveChronic allograft nephropathy (CAN) is an important etiological factor causing graft loss. However, the mechanism of CAN is unclear. Osteopontin (OPN), a proinflammatory and profibrosis molecule, plays a key role in late stages of renal diseases. We investigated the potential role of OPN in the pathogenesis of CAN. MethodsUsing a F344 to Lewis rat CAN model, we injected short hairpin RNA (shRNA) constructs targeting OPN or negative control plasmids through the renal vein following electroporation. At 12 weeks after the transplantation, we determined interstitial fibrosis (IF) and tubular atrophy (TA) of the tubular epithelial cells (TECs). OPN expression was examined using Western blots and immunohistochemistry (IHC). Molecules involved in epithelial to mesenchymal transition (EMT) of TECs were examined using IHC and Western blots. ResultsOPN expression in kidney grafts was decreased by the RNA interference (RNAi) group. Histology observations showed IF and TA to be mild with stable renal function in the RNAi-treated group. EMT of TECs was significantly lessened after reducing OPN. ConclusionReduction of OPN in vivo inhibited progression of CAN. OPN may be of therapeutic value in transplantation settings.

Efficient nonviral gene therapy with FasL and Del1 fragments in mice

The expression of FasL in cancer cells is currently being explored as a potential cancer therapy. Because high levels of FasL are necessary for effective treatment, current methods typically rely on the use of highly efficient viral vectors. However, because viral vector-based gene therapy is associated with certain risks, the development of effective nonviral routes for gene delivery would be useful. The present study aimed to improve FasL gene therapy with a nonviral vector by taking advantage of the E3 and C1 domains of Del1 protein, which induces apoptosis and localizes to the extracellular matrix. Mouse explanted tumors derived from a human oral squamous cell carcinoma cell line, SCCKN, were treated with plasmids encoding FasL (pFasL), E3C1 (pE3C1), and a fusion of FasL and E3C1 (pFasL-E3C1). The plasmids were injected locally every 7 days along with a transfection reagent, Jet-PEI (PolyPlus-transfection, San Marcos, CA, USA). All mice treated with a negative control plasmid or pFasL died within 49 days. By contrast, 83% of mice treated with pFasL-E3C1 survived longer than 49 days. Histochemical studies revealed that the fusion protein is localized to the stroma and induces apoptosis in stromal cells and adjacent parenchymal cells. The results obtained in the present study suggest that the protein deposition-based approach described, which makes use of the E3 and C1 domains of Del1, could comprise a novel method for cancer gene therapy with nonviral vectors.

HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module

To investigate the possible mechanism by which hepatitis B virus X protein (HBx) mediates apoptosis of HepG2 cells. HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high-expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX (HBx antagonist) were cotransfected into HepG2 cells to establish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation levels of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases (MAPKs), were measured in each group. Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and JNKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and JNKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group. HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.