Analysis of the Expression of PRDX6 in Patients with Hepatocellular Carcinoma and its Effect on the Phenotype of Hepatocellular Carcinoma Cells.

Objectives: This research aimed to study the expression of PRDX6 mRNA in hepatocellular carcinoma (HCC) and its effect on the prognosis of HCC. Moreover, the effect of PRDX6 gene knockdown on the proliferation, migration, and invasion of HepG2 cells mediated by lentivirus was also examined. This study offers a theoretical and experimental basis for further research on the mechanism of PRDX6 in liver cancer and new methods for clinical diagnosis and treatment. Methods: RNA sequence data of 369 HCC patients were screened through the TCGA database, and the expression and clinical characteristics of PRDX6 mRNA were analyzed based on high-throughput RNA sequencing data. HepG2 cells were divided into WT, sh-NC and sh-PRDX6 groups. Real-time PCR and Western blot were used to detect the expression levels of the PRDX6 gene and protein, respectively. CCK8 method was used to detect the proliferation activity of HepG2 cells, scratch healing test was used to detect the migration ability, Transwell chamber was used to detect the invasion ability, and Western blot was used to detect the expression levels of PI3K/Akt/mTOR signaling pathway and Notch signaling pathway-related proteins. Results: The expression of PRDX6 was significantly correlated with the gender, race, clinical stage, histological grade, and survival time of HCC patients (P < 0.05). Compared with that in WT and sh-NC groups, the expression level of PRDX6 protein in HCC patients was significantly lower (P < 0.01), the proliferation activity of HCC cells was significantly decreased (P < 0.05), and the migration and invasion ability was significantly decreased (P < 0.05) in the sh-PRDX6 group. The expression levels of PI3K, p-Akt, p-mTOR, Notch1, and Hes1 proteins in the sh-PRDX6 group were significantly lower than those in WT and sh-NC groups (P < 0.05). Conclusion: The expression of PRDX6 may be closely related to the prognosis of HCC. Lentivirus-mediated PRDX6 knockdown can inhibit the proliferation, migration and invasion of HCC cells, which may be related to its regulating the PI3K/Akt/mTOR and Notch1 signaling pathways. PRDX6 is expected to be a new target for the diagnosis and treatment of liver cancer.

Effect of RNAi-Mediated Survivin and Hypoxia-Inducible Factor 1α Gene Silencing on Proliferation, Invasion, Migration and Apoptosis of Gastric Cancer BGC-823 Cells.

In order to investigate the effects of RNAi-mediated survivin and hypoxia-inducible factor 1α (HIF-1α) gene silencing on the proliferation and apoptosis of gastric cancer BGC-823 cells, small interfering RNAs (siRNAs) targeting survivin and HIF-1α mRNAs, respectively, as well as scrambled siRNAs (SCRs) were designed and synthesized, namely siRNA-survivin group, siRNA-HIF-1α group, and SCR group. The hypoxia-sensitive gastric cancer BGC-823 cells were identified and transfected in vitro with Hifectin II under hypoxic conditions, and the expression of survivin and HIF-1α was assessed by RT-PCR and Western blotting assays, respectively. The ability of apoptosis, proliferation, invasion, and migration was measured, and the results showed that HIF-1α expression was significantly increased in BGC-823 cells under hypoxic conditions, and survival-targeted siRNA transfection decreased the expression of survivin under hypoxic conditions, while co-transfection of survivin-targeted siRNA and HIF-1α-targeted siRNA down-regulated both survivin and HIF-1α expression. Compared with the blank control group, the co-transfected siRNA group exhibited distinct characteristics, with decreased invasion and migration ability, increased apoptosis, and significantly decreased cell proliferation under hypoxic conditions. It was confirmed that the downregulation of survivin and HIF-1α in BGC-823 cells may induce anticancer effects by enhancing apoptosis and decreasing proliferation, migration, and invasion ability. The novelty lies in the application of RNAi technology to silence the expression of both survivin and HIF-1α genes in gastric cancer BGC-823 cells by single and combined interference in an established gastric cancer cell model and observed the mechanism of its effect on the proliferation and apoptosis of gastric cancer cells. Concerning the success of this highly active antiretroviral therapy of gene disruption therapies, which is the first of its kind in the world, we wonder whether we can find other better gene targets for more kinds of tumor therapy.

The Effect of GPX2 on the Prognosis of Lung Adenocarcinoma Diagnosis and Proliferation, Migration, and Epithelial Mesenchymal Transition.

Objective: To investigate the expression of glutathione peroxidase 2 (GPX2) in human lung adenocarcinoma tissues and its effect on the biological function of lung adenocarcinoma A549 cells. Methods: The expression of GPX2 in lung adenocarcinoma and its effect on survival were analyzed by the TCGA database and the GEPIA 2 database. A total of 45 cases of primary lung adenocarcinoma tissue specimens and 45 cases of their paracancerous tissue specimens were collected, and the expression of GPX2 in the two types of tissues was detected by immunohistochemistry. Lung adenocarcinoma A549 cells were divided into the GPX2 overexpression group (GPX2), the GPX2 knockdown group (si-GPX2), the empty vector group (Vector), the siRNA negative control group (si-NC), and the WT group; the mRNA level and protein expression of GPX2 in each group of A549 cells were detected by real-time fluorescence quantitative PCR and Western blotting; the proliferation activity of each group of cells was detected by the CCK-8 assay; the effect of GPX2 on cell migration and invasion ability was detected by the scratch assay and the Transwell invasion assay; the apoptosis of each group of cells was detected by flow cytometry; Western blotting was performed to detect the expression levels of Bax, Bcl-2, E-cadherin, vimentin, and MMP2 and MMP9 proteins in each group of cells. Results: Bioinformatics analysis showed that the expression of GPX2 was strongly correlated with the prognosis of lung adenocarcinoma patients (P < 0.01). The positive expression rates of GPX2 in lung adenocarcinoma and its paracancerous tissues were 66.0% and 15.7%, respectively (P < 0.05). The results of RT-qPCR and Western blotting showed that the expression level of GPX2 mRNA and protein in A549 cells in the GPX2 group increased, which was significantly higher than that in the WT group (P < 0.05); the expression levels of GPX2 mRNA and protein in A549 cells in the si-GPX2 group were the same, that is, significantly lower than the WT group (P < 0.05). GPX2 overexpression promoted the proliferation, migration, and invasion of A549 cells and inhibited their apoptosis; the results in the si-GPX2 group were opposite to those in the GPX2 group. Compared with the WT group, the expression of Bcl-2, vimentin, and MMP2 and MMP9 protein in the GPX2 group increased (P < 0.05), while the expression of Bax and E-cadherin protein decreased in the GPX2 group (P < 0.05); the results in the si-GPX2 group were opposite to those in the GPX2 group. Conclusion: The expression of GPX2 in lung adenocarcinoma is related to the prognosis of patients. It is proved that GPX2 can promote the migration and invasion of lung adenocarcinoma cells and is related to the EMT/ß-catenin pathway. Thus, GPX2 is expected to be an important target for the diagnosis and treatment of lung adenocarcinoma.

Effect of lentivirus-mediated peroxiredoxins 6 gene silencing on the phenotype of human gastric cancer BGC-823 cells.

Aims: Peroxiredoxins (PRDX6) regulates the occurrence and progression of cancer. The aim of this study is to investigate the effect of PRDX6 knockdown on the biological behavior of human gastric cancer cell line BGC-823 cells. Settings and Design: Research article. Subjects and Methods: The differential expression of PRDX6 in gastric cancer and normal gastric tissues was tested by immunohistochemistry. Ribonucleic acid plasmid of PRDX6 gene was packaged using a lentivirus, and BGC-823 cells were transfected with the lentivirus to obtain a BGC-823 cell line in which the expression of PRDX6 was stably silenced. Statistical Analysis Used: The proliferation activity of BGC-823 cells was detected using the cell counting kit-8 method. The effect of PRDX6 on the migration and invasion of BGC-823 cells was evaluated using the scratch test and Transwell assay, and the expression of related proteins was detected by western blot. Results: The expression of PRDX6 in gastric cancer was significantly increased (P < 0.05). Compared with those in the untransfected and negative control groups. The proliferation, migration, and invasion of gastric cancer BGC-823 cells were significantly inhibited, and the apoptotic rates were significantly increased in the lentivirus-transfected (short hairpin-PRDX6) group. Western blot analysis showed that the expression of Bax protein increased, whereas that of proliferating cell nuclear antigen, Bcl-2, PI3K, phospho (p-Akt), and phosphorylated-mammalian target of rapamycin (mTOR) decreased significantly compared with that in WT and vector groups (P < 0.05). Conclusion: The knockdown of PRDX6 gene expression in BGC-823 cells can inhibit the proliferation, migration, and invasion of gastric cancer cells and promote apoptosis, thereby affecting gastric cancer cells.

Effects of Peroxiredoxin 6 and Its Mutants on the Isoproterenol Induced Myocardial Injury in H9C2 Cells and Rats.

Background: Peroxiredoxin 6 (PRDX6) is an important antioxidant enzyme, with a potential application value in the treatment of diseases caused by oxidative damage. Methods: PRDX6 and a mutant (mPRDX6) were heterologously expressed by using an E.coli expression system and purified by Ni-affinity chromatography. Isoproterenol (ISO) was used to induce a myocardial cell injury model and an animal myocardial injury model. After the treatment with PRDX6 and mPRDX6, the proliferation activity of H9C2 cells was detected by Cell Counting Kit-8 (CCK8) method; the apoptosis was evaluated by flow cytometry, and the histological changes of myocardial cells were observed by hematoxylin and eosin (H&E) staining, the levels of catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), and superoxide dismutase (SOD) in ISO-treated H9C2 cells as well as in the heart tissue and serum of rats treated with ISO were detected, and the expression levels of Bax, Bcl-2 and peroxisome proliferators-activated receptors-γ (PPAR-γ) proteins were detected by Western blot. Results: PRDX6 and mPRDX6 were successfully expressed and purified. The results of efficacy study showed that the mutant mPRDX6, in which the phospholipaseA2 (PLA2) activity of PRDX6 was deleted by site directed mutation, had a better protective effect against the myocardial injury than PRDX6. CCK8 results showed that compared with that in ISO group, the proliferation activity of H9C2 cells was significantly enhanced (P < 0.01), the apoptosis rate was significantly decreased (P < 0.01), and the fluorescence intensity of reactive oxygen species (ROS) was significantly decreased (P < 0.01) in mPRDX6 group. The results of H&E staining showed that the myocardial injury was alleviated to a certain extent in mPRDX6 group. Compared with those in ISO group, the activities of CAT, GPX, and SOD in H9C2 cells and the heart tissue and serum of rats were significantly increased (P < 0.05), while the contents of MDA were significantly decreased (P < 0.05). Western blot analysis showed that the expression level of Bcl-2 in H9C2 cells was significantly decreased (P < 0.01), and that of Bax and PPAR-γ was significantly increased (P < 0.05). Conclusion: mPRDX6 has a protective effect against the myocardial injury induced by ISO, and the mechanism may be related to its antioxidation. This study may provide a theoretical basis for the research and development of drugs used for the treatment of myocardial injury.

RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo.

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF-C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF-C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF-C expression, cleaved caspase-3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme-linked immunosorbent assay were used to measure the expression of VEGF-C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF-C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF-C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase-3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF-C expression and increased induction of apoptosis. The present study therefore indicated that VEGF-C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

Characterization of peptidoglycan hydrolase in Cag pathogenicity island of Helicobacter pylori.

The Cag Type IV secretion apparatus proteins in Helicobacter pylori can mediate the injection of effector CagA protein into eukaryotic target cells. Although this apparatus forms an important pathway for bacterium-host interaction, its assembly process in vivo is poorly understood, and the proteins which contribute to break the bacterial cell walls in Cag-PAI have not yet been identified. The cagγ gene in Cag-PAI is a unique member that contains a conserved SLT catalysis domain, which makes it an attracting question whether cagy gene has the capacity to digest the bacterial cell wall. In the current study, therefore, the cagγ gene was cloned from the H. pylori NCTC 11637 and expressed in Escherichia coli, and its lytic effect on cell walls in vitro was observed. Results indicated that Cagγ protein has a lytic activity against bacterial cell walls. An allelic-exchange mutant (Δcagγ) was further constructed to investigate the relationship between Cagγ and effector CagA translocation. These results suggested that Cagγ contributed to the assembly of Cag Type IV secretion apparatus by digesting the peptidoglycan meshwork of bacterial cell walls.

[Construction of hp0523 gene mutant in Helicobacter pylori Cag-PAI and the influence on the ability of CagA protein translocation].

OBJECTIVE: To construct the hp0523 gene mutant of Helicobacter pylori and investigate the function of hp0523 gene. METHODS: We designed and amplified the upstream homologous fragment and downstream homologous fragment of hp0523 gene via PCR method. We constructed the suicide plasmid pBlueKM40-deltahp0523 based on allelic exchange. We introduced the suicide plasmid pBlueKM40-deltahp0523 into Helicobacter pylori 11637 by electroporation and screened the mutant based on antibiotic selection. We checked the mutant using the PCR and gene sequenced. We performed the coculture of Helicobacter pylori and gastric cell BGC-823 and detected the ability of CagA's translocation and expression via Western blot. RESULTS: We constructed the suicide plasmid pBlueKM40-deltahp0523 successfully and got the hp0523 deletion mutant. PCR and gene sequenced results showed the gene hp0523 was deleted. The results of CagA translocation assay showed that hp0523 interrupted the translocation of CagA. The comparison between wild-type and mutant showed that hp0523 affected the expression of CagA. CONCLUSIONS: We constructed the hp0523 deletion mutant of Helicobacter pylori NCTC11637. This study suggests that hp0523 gene is an important virulence factor, which may be a component of the apparatus for CagA's translocation.

[Cloning and expressing cagT gene of type IV secretion system in Helicobacter pylori and influence of cytokine secretion and cell proliferation on SGC-7901 cell].

OBJECTIVE: To detect the influence of the CagT protein on interleukin-8 secretion and proliferation in SGC-7901 cells. METHODS: Helicobacter pylori cagT gene was amplified by PCR with the genomic DNA of H. pylori NCTC 11637 as template, then it was inserted into an expression vector pQE30. The recombinant plasmid was transformed into E. coli M15. Recombinant protein was expressed by Isopropylthio-beta-D-Galacgoside (IPTG) induction and confirmed by Western blot. Fusion protein with 6xHis tag was purified using Ni(2+)-NTA agarose. Interleukin-8 mRNA expression of SGC7901 cells was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Cell viability was determined by methyl thiazolyl tetrazolium assay (MTT). RESULTS: The GenBank accession number of the amplified sequence is EF114758. The sequence analysis for cagT showed that it shares 97%-99% homology with other strains of H. pylori in Gene bank. The molecular mass of the product is 32kDa, and its purity is 98% analyzed by SDS-PAGE. After the protein was dialyzed, it can stimulate SGC7901 cells to express interleukin-8 and the growth of cells was inhibited in a dose- and time-dependent manner. CONCLUSION: It is indicated that we have obtained the correct cagT gene and expressed in E. coli M15. The protein can stimulate the cells to express cytokine interleukin-8 and inhibit proliferation of cells, which posed a basis for further research on its biological function.

The nanos gene of Bombyx mori and its expression patterns in developmental embryos and larvae tissues.

The nanos gene encodes a zinc-finger protein which is required for the migration and differentiation of primordial germ cells as well as for their fate maintenance. In this study, a 1913 bp nanos gene was cloned and characterized in silkworm (Bombyx mori). RT-PCR and Western blot analysis showed that the nanos was expressed in developing embryos and various silkworm larval tissues. The expression patterns of Nanos and Vasa in silkworm larval gonads were analyzed using immunohistochemistry. It was found that, in silkworm larval ovaries, the Nanos and Vasa proteins were expressed in oocytes. While in testes, high expression of Nanos and Vasa was detected in spermatogonia and relatively weaker expression was found in spermatocytes at latter stages.

Identification and characterization of a novel 1-Cys peroxiredoxin from silkworm, Bombyx mori.

Peroxiredoxins (Prxs) are believed to play an important role in insects for protection against the toxicity of reactive oxygen species (ROS). A gene encoding a novel 1-Cys Prx was firstly identified and characterized from an expressed sequence tag database (EST) in the lepidopteran insect, Bombyx mori. The 1-Cys Prx of B. mori (BmPrx) cDNA contained an open reading frame of 672 bp encoding a protein of 223 amino acid residues with calculated molecular mass of 25 kDa and included conserved cysteine residues signature motifs (PVCT) in the N-terminus amino acid. Sequence comparison showed that BmPrx shared 53 to 64% identity with other species 1-Cys proteins. RT-PCR revealed that the BmPrx transcripts were present in all tissues and developmental stages. The coding sequence was cloned and expressed as a 30-kDa protein in Escherichia coli. The purified enzyme acted as a catalyst in ferrithiocyanate system and protected supercoiled form of plasmid DNA from damage in metal-catalyzed oxidation (MCO) system in vitro. In addition, real-time PCR analysis indicated that significant rise of the transcripts level of BmPrx was induced by temperature stress including low and high temperature stimuli.

Type IV secretion system in Helicobacter pylori: a new insight into pathogenicity.

OBJECTIVE: To review the research progress on Type IV secretion system (T4SS) in Helicobacter pylori. DATA SOURCES: The data used in this review were identified by searching of PUBMED (1995 - 2007) online resources using the key terms 'Type IV secretion system' and 'Helicobacter pylori'. STUDY SELECTION: Mainly original articles and critical reviews written by major pioneer investigators of this field were selected. RESULTS: The research progress on T4SS in Helicobacter pylori was summarized. The structure and function was discussed. CONCLUSIONS: T4SS is not only involved in toxin secretion and injection of virulence factors into eukaryotic host target cells, but also involved in horizontal DNA transfer to other bacteria and eukaryotic cells, through DNA uptake from or release into the extracellular milieu. It provides a new insight into the pathogenicity of Helicobacter pylori and a novel target for antimicrobials development. However, many challenges remain for us in understanding the biological role of T4SS in Helicobacter pylori.