ROS-Induced DCTPP1 Upregulation Contributes to Cisplatin Resistance in Ovarian Cancer.

Cisplatin resistance hinders the improvement of the prognosis of patients with ovarian cancer. Cisplatin induces cancer cell apoptosis by inducing reactive oxygen species (ROS). dCTP pyrophosphatase 1 (DCTPP1) is a newly discovered dNTP pyrophosphatase. This study aimed to identify the role of DCTPP1 in oxidative stress and cisplatin response of ovarian cancer. Our results indicates cisplatin-induced ROS generation was responsible for the upregulation of DCTPP1 in ovarian cancer cells, whereas DCTPP1 knockdown significantly enhanced the sensitivity of ovarian cancer cells to cisplatin, reflect in reactive oxygen species (ROS) generation, double-strand DNA breaks, and cell apoptosis. The expression of redox-related genes and the activation of the PI3/Akt signaling pathway were also inhibited by DCTPP1 knockdown. Our data proposes that the development of therapeutic approaches targeting DCTPP1 may be useful in the treatment of ovarian cancer.

Comparatively analyzing the liver-specific transcriptomic profiles in Kunming mice afflicted with streptozotocin- and natural food-induced type 2 diabetes mellitus.

BACKGROUND: Streptozotocin is a classic drug used to induce diabetes in animal models. OBJECTIVE: The aim of this study is to investigate the liver transcriptome of Kunming mice with diabetes induced by either streptozotocin (STZ) or Non-STZ. METHODS: Forty male mice were randomly assigned into four groups: Control (Ctr, standard diet), mHH (high fat and high carbohydrate diet), mHS (high fat and high carbohydrate diet for 4 weeks followed by 60 mg/kg STZ for 3 consecutive days) and mSH (60 mg/kg STZ for 3 consecutive days followed by a high fat and high carbohydrate diet for 12 weeks). All mice injected with STZ were identified as diabetic despite the sequential feeding of high fat and high carbohydrate diets. RESULTS: Only 7 of 13 mice in the mHH group met the diagnostic criteria for diabetes. The asting blood glucose (FBG) of the mHH, mHS, mSH and Ctrl groups was 13.27 ± 1.14, 15.01 ± 2.59, 15.95 ± 4.38 and 6.28 ± 0.33 mmol/L at the 12th week, respectively. Compared with the mHH group, transcription was elevated in 85 genes in the livers of mHS mice, while 21 genes were downregulated and 97 genes were upregulated in the mSH group while 35 genes were decreased. A total of 43 co-expressed genes were identified in the mHS vs mHH and mSH vs mHH groups. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses showed that two corporate GO terms and two KEGG pathways were significantly annotated in the STZ-treated groups. Both the GO term and pathway were related to the metabolism mediated by p53. CONCLUSION: A high fat and high carbohydrate diet combined with a low dose of STZ can effectively induce diabetes in Kunming mice despite the abnormal expressions of genes in the liver. The differentially expressed genes were related to metabolism mediated by p53.

Application of the modified cytosine base-editing in the cultured cells of bama minipig.

Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

Identification of in vitro and in vivo oncolytic effect in colorectal cancer cells by Orf virus strain NA1/11.

Orf virus (ORFV) is a favorable oncolytic viral carrier in research, and ORFV strain NZ2 has been revealed to have antitumor effects in animal models mediated by immunoregulation profile. However, the antitumor effects triggered by the ORFV in colorectal cancer (CRC) cells is poorly characterized. The in vivo and in vitro roles of ORFV in CRC were determined using western blotting, colony formation, CCK­8, wound scratch assay, qPCR, and animal models. Furthermore, cytokine antibody chip assay, flow cytometry, western blotting, and immunohistochemical (IHC) assays were conducted to explore the potential mechanism of ORFV. The present data revealed that ORFV strain NA1/11 infected and inhibited the in vitro growth and migration of CRC cells. By establishing a CRC model in Balb/c mice, it was revealed that ORFV strain NA1/11 significantly inhibited the in vivo growth and migration of CRC cells. A cytokine antibody array was utilized to obtain a more comprehensive profile revealing the differentially expressed cytokines in ORFV infection. Cytokines, such as IL­7, IL­13, IL­15, CD27, CD30, pentraxin 3, and B lymphocyte chemoattractant (BLC), were upregulated. Axl, CXCL16, ANG­3, MMP10, IFN­Î³ R1 and VEGF­B were downregulated. The results indicated that ORFV played roles in the regulation of key factors relevant to apoptosis, autoimmunity/inflammation, angiogenesis, and the cell cycle. Finally, data was presented to validate that ORFV infection induces oncolytic activity by enhancing apoptosis in vivo and in vitro. In conclusion, ORFV could be an oncolytic virus for CRC therapy.

Extensive serum cytokine analysis in patients with prostate cancer.

Prostate cancer (CaP) is a common male malignancy. Using prostate specific antigen (PSA) and prostate cancer antigen 3 (PCA3) in the diagnosis of prostate cancer, sensitivity and specificity still require improvement. Additional targets are urgently needed for the diagnosis, prognosis, and prediction of therapeutic response, leading to better treatments in order to reduce the mortality of CaP. Here, we utilized a solid-phase antibody array, which can simultaneously detect 200 proteins, for the screening of novel blood-based biomarkers. The proteins differentially expressed in the pathogenesis of CaP were further analyzed using bioinformatics methods. The identified targets were further validated by the enzyme-linked immunosorbent assay (ELISA). A total of 38 proteins were identified with significantly differential levels in CaP serum compared to healthy control serum, including 21 up-regulated and 17 down-regulated cytokines. ELISA result showed that validated six ones of these differential cytokines were significantly differential between CaP and control, consistent with the antibody array result. The protein-protein interaction (PPI) analysis for these differentially expressed cytokines showed the top five cytokines interacting with most other cytokines were insulin, SDF-1a, CD40L, IL-18 and NCAM-1, suggesting these five targets are important in the pathogenesis of CaP, and more sensitive for the early diagnosis and prognosis of CaP. Targeting these cytokines may be more effective therapies against CaP. Among these differentially expressed cytokines, it was found that AR, BTC, IL-1 F8, IL-31, Marapsin, b-NGF, EDA-A2, MCP-3, MCP-4, MIP-3a, PIGF, and TECK decreased, while Fas, Flt-3L, and NCAM-1 increased in CaP when compared to the controls. Taken together, those 38 differentially expressed cytokines may service as novel serum biomarkers for CaP, which will be further validated with more clinical samples.

Extensive serum biomarker analysis in patients with nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a fast-growing cancer characterized by high occurrences of nodal and distant metastases and poor prognosis. It is therefore important to identify new serum biomarkers for the early diagnosis and prognostic prediction of this disease. The present study identifies biomarkers in NPC patient serum using a solid-phase antibody array detecting the expression profiles of 174 cytokines in a single experiment. ELISA was performed to validate the array results. The levels of TIMP-2, SELL, CCL24, MMP-1, MMP-3, IGF-I and IL-8 were significantly higher in serum from NPC patients, while the levels of MSP-alpha and HCC-4 were lower. Furthermore, the validation results were identical to those obtained from the antibody array. These results indicate that these cytokines might serve as novel biomarkers for the diagnosis and prognostic prediction of NPC.

Identification of host cellular proteins LAGE3 and IGFBP6 that interact with orf virus protein ORFV024.

OBJECTIVE: Orf virus (ORFV) is the pathogen causing contagious pustular dermatitis in goats, sheep and herdsmen. Evidence has confirmed that ORFV can be used as a preventive and therapeutic immunomodulatory agent in several animal models. Our previous data demonstrated that ORFV024 is able to inhibit activation of the NF-κB signaling pathway and act as an important modulator for early immune responses against viral infection. However, the molecular mechanism by which ORFV024 exerting biological function remains unclear. In the present study, we explored and analyzed the function of host cellular proteins that interact with ORFV024. METHODS: The yeast two-hybrid (Y2H) assay was performed to screen proteins interacting with ORFV024 using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). Two of the screened proteins were further confirmed by confocal microscopy, His-tag pull-down assay and CO-Immunoprecipitation (CO-IP) assay. In addition, the ORFV024 interaction network was constructed using the STRING database. RESULTS: In this study, 11 ovine cellular proteins were found to interact with ORFV024. In view of the importance of LAGE3 and IGFBP6 in the ORFV024 functional analysis, we further constructed LAGE3 and IGFBP6 interaction networks. The interactions between ORFV024 and LAGE3 or IGFBP6 were confirmed by confocal microscopy, LAGE3 was further confirmed in the His-tag pull-down assay and CO-IP assay. CONCLUSIONS: Our findings indicate that ORFV024 can interact with ovine cellular proteins LAGE3 and IGFBP6.

Transcriptomic profiles of human foreskin fibroblast cells in response to orf virus.

Orf virus has been utilized as a safe and efficient viral vector against not only diverse infectious diseases, but also against tumors. However, the nature of the genes triggered by the vector in human cells is poorly characterized. Using RNA sequencing technology, we compared specific changes in the transcriptomic profiles in human foreskin fibroblast cells following infection by the orf virus. The results indicated that orf virus upregulates or downregulates expression of a variety of genes, including genes involved in antiviral immune response, apoptosis, cell cycle and a series of signaling pathways, such as the IFN and p53-signaling pathways. The orf virus stimulates or inhibits immune gene expression such as chemokines, chemokine receptors, cytokines, cytokine receptors, and molecules involved in antigen uptake and processing after infection. Expression of pro-apoptotic genes increased at 8 hours post-infection. The p53 signaling pathway was activated to induce apoptosis at the same time. However, the cell cycle program was promoted after infection, which may be due to the immunomodulatory genes of the orf virus. This presents the first description of transcription profile changes in human foreskin fibroblast cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus. These data offer new insights into the understanding of the mechanisms of infection by orf virus and identify potential targets for future studies.

Are Sertoli cells a kind of mesenchymal stem cells?

OBJECTIVE: Sertoli cells (SCs) are a major component of testis which secrete a variety of cytokines and immunosuppressive factors, providing nutritional support and immune protection for sperm growth and development. The purpose of this study was to investigate the relationship between SCs and bone marrow mesenchymal stem cells (BMSCs) in order to provide a theoretical basis for better application of SCs. METHODS: We used the adherence method to isolate Sprague-Dawley rat SCs and BMSCs. Cells surface markers were detected by flow cytometry. The capacity of cells to differentiate was determined by osteogenic and adipogenic induction. Assessment of cell proliferation was performed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide] assay. Changes in the nucleus were analyzed by Hoechst nuclear staining. Cell aging was observed with ß-galactosidase, which is a biological marker of senescence. RT-PCR was employed to detect the expression of cytokines. RESULTS: From the aforementioned experiments, we found that the surface markers of SCs and BMSCs were almost exactly the same. Proliferation of SCs, as well as osteogenic and adipogenic differentiation, were weaker than in BMSCs. Compared with BMSCs, Hoechst nuclear staining showed that the chromatin of SCs began to aggregate and was slightly larger. ß-galactosidase staining showed that SCs were in a slightly aging state. The secretion of cytokines from SCs was slightly less than the secretion from BMSCs. CONCLUSION: SCs are a kind of mesenchymal stem cells which have begun the process of differentiation.

[Oxidative stress and semen parameters in the serum and seminal plasma of infertile men with chronic viral hepatitis].

OBJECTIVE: To investigate the malondialdehyde (MDA) level and paraoxonase-1 (PON-1) activity in the serum and seminal plasma of infertile men with chronic viral hepatitis and their influence on the semen parameters of the patients. METHODS: We collected serum and semen samples from 42 infertile men, 45 infertile males with chronic viral hepatitis, and 50 healthy fertile men as controls. We measured the MDA level in the serum and seminal plasma by spectrophotometry, detected the PON-1 activity by spectrophotometry, and determined the sperm DNA fragmentation index (DFI) by acridine orange fluorescence staining. RESULTS: The MDA level was significantly higher but the PON-1 activity remarkably lower in the serum and seminal plasma of the infertile males with chronic viral hepatitis than in the healthy controls and infertile patients (P <0.01 or P <0.05). Total sperm motility and sperm survival rate were significantly lower while the sperm DFI markedly higher in the former than in the latter two groups (P <0.01 or P <0.05). No statistically significant difference was found among the three groups in sperm concentration (P >0.05). The WBC counts in the semen of the infertile and infertile with chronic viral hepatitis groups were significantly higher than that in the health controls (P <0.05). The MDA level and PON-1 activity in the seminal plasma were positively correlated with those in the serum in the infertile males with chronic viral hepatitis (r=0.57 or 0.48, P <0.01). CONCLUSION: Virus-induced chronic active hepatitis enhances oxidative stress in the reproductive system, aggravates sperm damage, and affects sperm quality parameters.

[Clinical value of seminal paraoxonase-1 activity evaluation in the diagnosis of male infertility].

OBJECTIVE: To investigate the changes in seminal paraoxonase-1 (PON-1) activity in infertile male patients and assess the clinical value of seminal PON-1 examination in the diagnosis of male infertility. METHODS: Seminal PON-1 activity was detected by spectrophotometric method in the semen samples from 270 infertile male patients and 50 health fertile males (control), and the semen parameters were analyzed using a computer-assisted semen analysis system. RESULTS: In the male infertility group, seminal PON-1 activity was 1.22∓0.76 U/L in the patients with normal semen parameters and 0.64∓0.54 in the patients with abnormal semen parameters, both significantly lower than that of the control group (3.17∓0.89 U/L, P<0.01). In patients with asthenospermia, the declined sperm motility was associated with decreased seminal PON-1 activity, which showed significant differences between patients with mild, moderate, and severe asthenospermia. Seminal PON-1 activity was positively correlated with the percentage of sperm viability (P<0.01), but inversely with the percentage of morphologically abnormal sperm (P<0.01). According to ROC curves, the area of seminal PON-1 activity under the curve was 0.907, showing a statistical significance (P<0.01). CONCLUSION: The detection of seminal PON-1 activity can provide a laboratory evidence for the diagnosis of male infertility.

[Diagnostic value of serum CEACAM1 in patients with pancreatic cancer].

OBJECTIVE: To investigate the diagnostic value of serum CEACAM1 in patients with pancreatic cancer. METHODS: Fifty patients with pancreatic cancer and 50 with chronic pancreatitis were examine for serum levels of CEACAM1 by enzyme-linked immunosorbent assay (ELISA). The cut-off values and area under curve (AUC) of CEACAM1 was obtained by receiver operating characteristic (ROC) curve. The diagnostic efficiency of the tumor markers for pancreatic cancer was assessed by the fourfold table. RESULTS: The serum level and positivity rate of CEACAM1 in pancreatic cancer patients were higher than those in chronic pancreatitis patients (P<0.05). Based on the ROC curve, the cut-off values and AUC of CEACAM1 were 13.835 ng/ml and 0.780, respectively (P<0.05). In pancreatic cancer patients, the diagnostic sensitivities of the tumor markers decreased in the order of CEACAM1 < CA242 < CA19-9 (P<0.05), and the specificity in the order of CA242 < CA19-9 < CEACAM1 (P<0.05). CONCLUSION: CEACAM1 shows a higher diagnostic sensitivity than CA19-9 and CA242 for pancreatic cancer, but due to its low specificity this marker alone is not sufficient for diagnostic purposes.

[Effects of survivin antisense RNA and HSP70 double gene transfection on MCF-7 cells in vitro].

OBJECTIVE: To explore the effects of survivin antisense RNA and HSP70 double gene transfection on breast cancer cell line MCF-7. METHODS: MCF-7 cells was transfected with the double-gene vector pIRES2-EGFP-survivin antisense RNA/HSP70 via liposome. After a 72-h transfection, the cells were collected for observation under inverted fluorescent microscope. The changes of survivin mRNA and HSP70 protein expressions in the cells were detected with real-time PCR and Western-blot before and after the cell transfection, and the apoptotic rate of the transfected MCF-7 cells was detected by flow cytometry analysis with Annexin-V-cy5/7AAD double staining. RESULTS: Green fluorescence was detected in MCF-7 cells transfected with the double-gene expression vector and the empty vector under inverted fluorescent microscope. The expression level of survivin mRNA in the cells was reduced effectively after the transfection with the double-gene expression vector, which also induced obvious cell apoptosis and enhanced the expression level of HSP70 protein as compared with those in MCF-7 cells transfected with the empty vector and the untransfected MCF-7 cells. CONCLUSION: Survivin antisense RNA can interfere with the expression of endogenous survivin and induce apoptosis of MCF-7 cells. HSP70 can increase the expression of HSP70 protein in MCF-7 cells.