Synthetic enhancers including TFREs improve transgene expression in CHO cells.

The human cytomegalovirus major immediate early gene (CMV) promoter is currently the most preferred promoter for recombinant therapeutic proteins (RTPs) production in CHO cells. To enhance the production of RTPs, five synthetic enhancers including multiple transcription factor regulatory elements (TFREs) were evaluated to enhance recombinant protein level in transient and stably transfected CHO cells. Compared with the control, four elements can enhance the report genes expression under both two transfected states. Further, the function of these four enhancers on human serum albumin (HSA) were investigated. We found that the transient expression can increase by up to 1.5 times, and the stably expression can maximum increase by up to 2.14 times. The enhancement of transgene expression was caused by the boost of their corresponding mRNA levels. Transcriptomics analysis was performed and found that transcriptional activation and cell cycle regulation genes were involved. In conclusion, optimization of enhancers in the CMV promoter could increase the production yield of transgene in transfected CHO cells, which has significance for developing high-yield CHO cell expression system.

The Effect of microRNA on the Production of Recombinant Protein in CHO Cells and its Mechanism.

Recombinant protein production by mammalian cells is the initial step in the manufacture of many therapeutic proteins. Chinese hamster ovary (CHO) cells are the most common host system to produce recombinant therapeutic proteins (RTPs). However, it is still challenging to maintain high productivity ensuring the good quality of RTPs produced by CHO cells. MicroRNAs(miRNAs) are short regulatory non-coding RNAs that can regulate cellular behavior and complex phenotypes. It has been found that miRNAs can enhance the expression level of recombinant proteins in CHO cells by promoting proliferation, resisting apoptosis, and regulating metabolism. miRNAs also can affect the quality of RTPs. In this review, we will discuss the effect and mechanism of miRNA on the expression level and quality of recombinant proteins in CHO cells.

miR-181b inhibits chemoresistance in cisplatin-resistant H446 small cell lung cancer cells by targeting Bcl-2.

INTRODUCTION: MicroRNAs (miRNAs) are a group of small non-coding RNAs that affect multiple aspects of tumor biology including chemo resistance. miR-181b has been reported to modulate multidrug resistance in non-small cell lung cancer cells. This study was undertaken to determine the role of miR-181b in chemo resistance of small cell lung cancer cells. MATERIAL AND METHODS: This study was undertaken to determine the role of miR-181b in chemoresistance of small cell lung cancer cells with use of qRt-PCR, WB, bioinformatics analysis, and double luciferase reporter system. RESULTS: Our data showed that miR-181b was significantly downregulated in cisplatin-resistant H446 small cell lung cancer cells, compared to parental cells, compared to parental cells. Ectopic expression of miR-181b inhibited cell proliferation and invasion in cisplatin-resistant H446 cells (p = 0.023). Moreover, overexpression of miR-181b increased the susceptibility of cisplatin-resistant H446 cells to cisplatin. Mechanistic investigations demonstrated that miR-181b inhibited B-cell lymphoma-2 (Bcl-2) expression by binding to the 3'-untranslated region. Overexpression of Bcl-2 reversed miR-181b-mediated chemo sensitization, which is accompanied by a reduced apoptotic response. CONCLUSIONS: Taken together, this work demonstrated that miR-181b might have the ability to overcome chemo resistance of small cell lung cancer cells, and restoration of this miRNA may represent a potential therapeutic strategy for improving chemo sensitivity in small cell lung cancer.

Cytotoxic T lymphocyte-associated antigen-4 +49A>G polymorphism and the risk of non-small cell lung cancer in a Chinese population.

BACKGROUND: CTLA-4 is a potent immunoregulatory molecule and plays a pivotal role in the negative regulation of T-cell proliferation and activation. Previously, the association between CTLA-4 +49A>G polymorphism and the risk of NSCLC has been investigated in several studies, however, their results were inconsistent. Therefore, we aimed to investigated the association between CTLA-4 +49A>G polymorphism and the risk of NSCLC in a Chinese population. METHODS: We recruited 231 NSCLC patients and 250 healthy controls in the present case-control study. PCR-RFLP was used to analyze the polymorphism of CTLA-4. The chi-squared test was used to examine differences between NSCLC patients and controls. The odds ratio (OR) and its 95% confidence interval (95% CI) were obtained by logistic regression methodology to determine correlations between the CTLA-4 polymorphism and the incidence of NSCLC. RESULTS: When the AA genotype was used as the reference group, the GG genotype was significantly associated with increased risk for NSCLC (OR=2.181, 95% CI: 1.244-5.198; P=0.007), however, the AG genotype was not significantly associated with increased risk for NSCLC (OR=2.018, 95% CI: 0.826-3.881; P=0.099). Under the dominant model of inheritance, the AG+GG genotype was significantly associated with increased risk for NSCLC (OR=3.271, 95% CI: 1.827-4.559; P=0.015). In addition, the G allele had a 2.754-fold higher risk of NSCLC in comparison with the A allele (OR=2.754, 95% CI: 1.365-6.891, P=0.005). CONCLUSIONS: Our data provided evidence that the CTLA-4 +49A>G polymorphism is associated with increased risk of NSLCL in Chinese population.

[Expressions of NF-κBp65 and IκBα in gestational trophoblastic disease and clinical significance].

AIM: To investigate the roles of nuclear factor κB p65 (NF-κBp65) and inhibitor of nuclear factor κB α (IκBα) in the development and metastasis of gestational trophoblastic neoplasia (GTN) by analyzing the expressions of NF-κBp65 and IκBα in normal early pregnancy villi and gestational trophoblastic diseases, and to reveal the relationship of NF-κBp65 and IκBα with age and clinical stage of GTN patients. METHODS: The expressions of NF-κBp65 and IκBα were detected by immunohistochemistry in normal pregnancy villi (20 cases), hydatidiform moles (HM, 30 cases), invasive hydatidiform moles (IHM, 13 cases) and chorionic carcinomas (CCA, 15 cases). RESULTS: NF-κBp65 expression was statistically different (P<0.05) between normal pregnancy villi and IHM (P=0.013), normal pregnancy villi and CCA(P=0.018), HM and IHM(P=0.026), HM and CCA (P=0.035). Differences in IκBα expression were statistically significant between normal pregnancy villi and IHM, normal pregnancy villi and CCA, HM and IHM, HM and CCA (P<0.01). The expressions of NF-κBp65 and IκBα were correlated to clinical stage (P=0.043, 0.042, P<0.05), but not to patients' ages. Spearman correlation analysis revealed that there was a negative association between the protein expressions of NF-κBp65 and IκBα in GTN (r=-0.403, P=0.034, P<0.05). CONCLUSION: Up-regulated expression of NF-κBp65 and down-regulated expression of IκBα may be related to the development, invasion and metastasis of GTN. The expressions of NF-κBp65 and IκBα are negatively correlated in gestational trophoblastic tumor tissues.

[Expression of caveolin-1 and the invasion of choriocarcinoma].

OBJECTIVE: To investigate the association between the expression of caveolin-1(CAV-1) and the invasion of choriocarcinoma, and to explore the effect of CAV-1 small interfering RNA(siRNA) on the invasion of choriocarcinoma cell line JEG-3. METHODS: (1) Matrigel invasion assay and 3-(4,4)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumormide (MTT) assay were used to examine the difference in invasion and proliferation ability between JEG-3 cells and JAR cells;(2) Expression of caveolin-1 gene in the human chorionic villi tissues and chorionicnoma cell lines (JEG-3 cells and JAR cells) were detected by semi-quantitative RT-PCR. (3) The effect of CAV-1 siRNA transfection on the expression of CAV-1 mRNA, and the invasion and proliferation ability of JEG-3 cells were measured by RT-PCR, Matrigel invasion assay and MTT assay. RESULTS: (1) The invasion ability of JEG-3 cell line was stronger than that of JAR cell line (P<0.05), but the difference in proliferation ability between JAR and JEG-3 was not obvious (P>0.05);(2) The expression of caveolin-1 gene in chorionicnoma cell lines was significantly stronger than that in the human normal chorion(P<0.05), and the expression of caveolin-1 gene in JEG-3 cells was stronger than that in the JAR cells (P<0.05). The data suggested that there was significantly positive correlation between caveolin-1 and the invasiveness of chorionicnoma cells (r=0.086,P<0.05);(3) CAV-1 siRNA could knock-out the expression of CAV-1 mRNA, and inhibit the invasion and proliferation ability of chorionicnoma cells. CONCLUSION: CAV-1 can promote the invasion ability of chorionicnoma cells. CAV-1 siRNA can inhibit the invasion and proliferation ability of chorionicnoma cells.

[Intrathecal administration of HSV-I amplicon vector-mediated HPPE gene therapy of nocicepion in rats with formalin pain].

OBJECTIVE: To investigate the antinociceptive effect of intrathecal administration of HSV-I amplicon vector-mediated HPPE. METHODS: Sprague Dawley rats (290+/-30) g were randomly divided into pHSVIRES-HPPE-LacZ (SHPZ) group, pHSVIRES-LacZ (SHZ) group, and saline group (NS), and 3 d, 1 week, 2 weeks, 3 weeks, 4 weeks, and 5 weeks group,which were anesthetized with 10% chlroral hydrate 300- 350 mg/kg. A microspinal catheter was inserted into the lumbar subarachnoid space. Rats were intrathecally delivered with recombinant HSV-I amplicon vector SHPZ, SHZ or NS. The HPPE expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and radioimmune assay. Formalin 50 microL (5%) was injected into the left hindpaw, pain intensity scoring (PIS) was used to assess the antinociceptive effect. RESULTS: After in vivo transferring,neurocyte demonstrated strong positive signals with X-gal immunohistochemical staining. RT-PCR and L-enkephalin radioimmune assay found that the neural cells transferred foreign gene (HPPE) had effective expression. Intrathecal delivery of SHPZ showed antinociceptive effects on formalin induced pain for 5 weeks compared with SHZ. CONCLUSION: This amplicon virus can transfer HPPE into rat central nerve system neural cells and express efficiently, suggesting SHPZ is satisfactory treatment for gene therapy for chronic pain. Intrathecal delivery SHPZ demonstrated antinociceptive effects on formalin induced pain.

[Expression of heparanase and angiopoietin-2 mRNA in endometriosis].

OBJECTIVE: To investigate the relationship between heparanase (Hpa) and angiopoietin-2 (Ang-2) gene expression and the development of endometriosis (EM). METHODS: Expression of Hpa gene and Ang-2 mRNA in the eutopic and ectopic endometrium collected from 86 patients with endometriosis (EM group) and the normal endometrium from 30 women without endometriosis (control group) was determined by RT-PCR. RESULTS: (1) Hpa gene expression was detected in 53 ectopic endometrium and 47 eutopic endometrium specimens in the EM group and 8 normal endometrium specimens in the control group. There was no significant difference in Hpa gene expression between ectopic and eutopic endometrium in the EM group (P > 0.05). However, the Hpa gene expression in normal endometrium in the control group was significantly lower than that in ectopic and eutopic endometrium in the EM group (P < 0.05). (2) Ang-2 gene expression was detected in 61 ectopic endometrium and 56 eutopic endometrium specimens in the EM group and 10 normal endometrium specimens in the control group. There was no significant difference in Ang-2 gene expression between ectopic and eutopic endometrium in the EM group (P > 0.05), however, the Ang-2 positive rate in normal endometrium in the control group was significantly lower than that in ectopic and eutopic endometrium in the EM group (P < 0.05). (3) The expression of Hpa gene in stage III-IV endometriosis was significantly higher than that in stage I-II endometriosis (P < 0.05). (4) The difference of Ang-2 mRNA expression between stage III-IV and stage I-II endometriosis was not significant (P > 0.05). (5) The expression of Hpa gene was shown to be significantly correlated to the expression of Ang-2 gene in ectopic endometrium specimens from patients with endometriosis (P < 0.05). CONCLUSION: Hpa and Ang-2 genes are highly expressed in both ectopic and eutopic endometrium of patients with endometriosis. Over-expression of these two genes may play a role in the development and progression of endometriosis.

[Maternal blood and amniotic fluid insulin-like growth factor detection and amniotic cavity drug delivery for early diagnosis and management of fetal growth restriction].

OBJECTIVE: To explore the relationship between insulin-like growth factors (IGFs) and fetal growth restriction (FGR) and early treatment of FGR. METHODS: The levels of IGF-I and IGF-II were detected with radioimmunoassay in maternal blood and amniotic fluid samples of 44 pregnant women with FGR and 36 normal gravidas. The 44 gravidas with FGR were randomized into treatment group with amino acid by a pediatric administration to the amniotic cavity formula and control group with intravenous infusion of compound amino acid. The therapeutic effects were compared between the two groups on the basis of B-type ultrasound findings. RESULTS: Maternal blood IGF-I and amniotic fluid IGF-I and IGF-II levels in the pregnant women with FGR were significantly lower than those in normal gravidas (P<0.01). After therapy, IGF-I and IGF-II levels were significantly increased in the treatment group (P<0.01), but no obvious changes in IGF-I and IGF-II levels were observed in the control group (P>0.05). Compared with the control group, IGF-I and IGF-II levels increased significantly in the amniotic fluid in the treatment group, with also marked elevation of IGF-I levels in maternal blood (P<0.01). Better therapeutic effects were achieved in the treatment group than in the control group (P<0.01), and the birth weights of the neonates in the treatment group were basically normal. CONCLUSIONS: Detection of amniotic fluid IGF-I and IGF-II and maternal blood IGF-I can help predict the condition and facilitate early diagnosis of FGR. Injection of pediatric amino acid into the amniotic cavity can be effective for treatment of FGR.