The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

Objective:To clarify the function and molecular mechanisms of serpin family E member 2 (SERPINE2) in cellular migration and invasion of esophageal squamous cell carcinoma (ESCC).Methods:The expression of SERPINE2 in ESCC was analyzed by using online databases TCGA (http: //gepia.cancer-pku.cn/detail.php and http: //ualcan.path.uab. edu/index.html). The expressions of SERPINE2 mRNA in normal human esophageal epithelial cell line NE2, human ESCC cell lines KYSE30 and KYSE150 were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). SERPINE2-konckdown or SERPINE2-overexpressed plasmid was transfected into KYSE30 cells, and the efficiencies of the knockdown and overexpression system were tested by qRT-PCR. The relationships of SERPINE2 and ESCC migration and invasion were determined by migration and invasion assays in vitro. The associations between SERPINE2 expression and β-catenin as well as its target genes including c-Myc, cyclin D1 and CD44 were analyzed by immunofluorescence, qRT-PCR and western blot, respectively.Results:The expressions of SERPINE2 were significantly upregulated in both esophageal cancer (ESCA) and ESCC tissues compared to normal tissues by analyzing 182 and 95 cases, respectively ( P<0.01). SERPINE2 is highly expressed in both KYSE30 and KYSE150 cells ( P<0.05). The number of migrating and invading cells in control group were (212.66±24.11)/field and (136.00±14.42)/field, while were (88.33±9.71)/field and (77.00±9.53)/field in SERPINE2-knockdown 1 group, and (66.00±8.00)/field and (45.66±3.78)/field in SERPINE2-knockdown 2 group, respectively, and the differences were dramatically significant compared with the control group ( P<0.01). The number of migrating and invading cells in control group were (250.00±30.00)/field and (203.33±15.27)/field, while were (383.33±35.11)/field and (246.66±25.16)/field in SERPINE2-overpressed group, and the differences were strikingly significant compared with the control group ( P<0.01). The protein expression of β-catenin was upregulated while phosphorylated β-catenin protein expression was downregulated in SERPINE2-overexpressed KYSE30 cells when compared to control cells.The transcription activity of β-catenin was significantly upregulated and the mRNA expressions of its target genes including c-Myc, cyclin D1 and CD44 were all increased. After treated with 25 μM iCRT14, the number of migrated cells in the control and SERPINE2-overpressed groups were (200.00±36.05)/field and (258.33±22.54)/field, and the number of invaded cells were (160.00±17.32)/field and (188.33±25.65)/field, respectively, the differences were dramatically significant compared with the group without iCRT14 treatment ( P<0.01). Conclusion:SERPINE2 is significantly upregulated in ESCC cells and can promote cellular migration and invasion by activating β-catenin, which may provide a potential therapeutic target for patients with ESCC.

Objective:To clarify the function and molecular mechanisms of serpin family E member 2 (SERPINE2) in cellular migration and invasion of esophageal squamous cell carcinoma (ESCC).Methods:The expression of SERPINE2 in ESCC was analyzed by using online databases TCGA (http: //gepia.cancer-pku.cn/detail.php and http: //ualcan.path.uab. edu/index.html). The expressions of SERPINE2 mRNA in normal human esophageal epithelial cell line NE2, human ESCC cell lines KYSE30 and KYSE150 were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). SERPINE2-konckdown or SERPINE2-overexpressed plasmid was transfected into KYSE30 cells, and the efficiencies of the knockdown and overexpression system were tested by qRT-PCR. The relationships of SERPINE2 and ESCC migration and invasion were determined by migration and invasion assays in vitro. The associations between SERPINE2 expression and β-catenin as well as its target genes including c-Myc, cyclin D1 and CD44 were analyzed by immunofluorescence, qRT-PCR and western blot, respectively.Results:The expressions of SERPINE2 were significantly upregulated in both esophageal cancer (ESCA) and ESCC tissues compared to normal tissues by analyzing 182 and 95 cases, respectively ( P<0.01). SERPINE2 is highly expressed in both KYSE30 and KYSE150 cells ( P<0.05). The number of migrating and invading cells in control group were (212.66±24.11)/field and (136.00±14.42)/field, while were (88.33±9.71)/field and (77.00±9.53)/field in SERPINE2-knockdown 1 group, and (66.00±8.00)/field and (45.66±3.78)/field in SERPINE2-knockdown 2 group, respectively, and the differences were dramatically significant compared with the control group ( P<0.01). The number of migrating and invading cells in control group were (250.00±30.00)/field and (203.33±15.27)/field, while were (383.33±35.11)/field and (246.66±25.16)/field in SERPINE2-overpressed group, and the differences were strikingly significant compared with the control group ( P<0.01). The protein expression of β-catenin was upregulated while phosphorylated β-catenin protein expression was downregulated in SERPINE2-overexpressed KYSE30 cells when compared to control cells.The transcription activity of β-catenin was significantly upregulated and the mRNA expressions of its target genes including c-Myc, cyclin D1 and CD44 were all increased. After treated with 25 μM iCRT14, the number of migrated cells in the control and SERPINE2-overpressed groups were (200.00±36.05)/field and (258.33±22.54)/field, and the number of invaded cells were (160.00±17.32)/field and (188.33±25.65)/field, respectively, the differences were dramatically significant compared with the group without iCRT14 treatment ( P<0.01). Conclusion:SERPINE2 is significantly upregulated in ESCC cells and can promote cellular migration and invasion by activating β-catenin, which may provide a potential therapeutic target for patients with ESCC.

Objective:To explore the structure and diversity of intestinal flora in patients with community acquired pneumonia (CAP) before and after treatment with cefotaxime combined with levofloxacin.Methods:From October to December 2018, 6 patients with CAP in the Department of Infection, Zhejiang Provincial Hospital of Tongde, were treated with cefotaxime injection 2.0 g (once/8 h) combined with 0.5 g levofloxacin injection (once a day). A total of 12 fecal samples were collected before and after 7 days of treatment. The stool samples before and after treatment were analyzed by 16S rRNA sequencing.Results:⑴ The structure of intestinal flora before and after treatment : at the phylum level: Firmicutes 59.2% vs 40.8%, Proteobacteria 18.6% vs 35.5%, Bacteroidetes 14.8% vs 20.8%, Actinobacteria 5.6% vs 1.2%; At the family level: Ruminococcaceae 34.5% vs 13.0%, Lachnospiraceae 15.9% vs 9.7%, Veillonellaceae 1.8% vs 3.3%, Lactobacillaceae 0.3% vs 8.0%, Streptococcaceae 2.9% vs 1.1%, Enterococcaceae 0.02% vs 5.2%, Enterobacteriaceae 16.4% vs 34.6%, Bacteroidaceae 13.3% vs 16.8%, Porphyromonadaceae 0.3% vs 3.4%, Adlercreutzia 4.4% vs 0.5%. There was no significant difference in the composition and structure of intestinal flora before and after treatment ( P>0.05). ⑵ The diversity of intestinal flora before and after treatment: operational taxonomic units (OTU) mean (150.5±59.0) vs (93.2±34.1), t=2.72, P=0.04; Chao1 index (169.25±49.61) vs (117.92±35.06), t=3.22, P=0.02; shannon index (3.61±0.83) vs (2.31±0.73), t=4.54, P=0.01; simpson index (0.80±0.10) vs (0.61±0.20), t=2.76, P=0.04. There were significant differences in the diversity of intestinal flora before and after treatment ( P<0.05). ⑶ There was significant difference in desulfovibrio between the two groups before and after treatment (LDA=2.03, P=0.02). Conclusions:After intravenous infusion of cefotaxime combined with levofloxacin for one week , the diversity of intestinal flora was significantly reduced after treatment. Desulfovibrio was the flora with statistical differences between before and after treatment.

Schizophrenia is one of the most serious mental diseases found in humans. Previous studies indicated that the single nucleotide polymorphism (SNP) rs1344706 in the gene ZNF804A encoding zinc finger protein 804A was associated with schizophrenia in Caucasian population but not in Chinese Han population. However, current results are conflicting in Asian population. In the present study, a meta-analysis was performed to revisit the association between rs1344706 and the risk of schizophrenia in Asian, Caucasian and other populations. Electronic search of PubMed database identified 25 case–control studies with available genotype frequencies of rs1344706 for the meta-analysis, involving a total of 15,788 cases and 22,654 controls. A pooled odds ratio (OR) with 95% confidence interval (CI) was used to assess the association. The current meta-analysis showed an association between rs1344706 and schizophrenia in Caucasian populations (P= 0.028, OR= 1.138, 95% CI: 1.014–1.278; P = 0.004 for heterogeneity) and Asian populations (P = 0.008, OR = 1.092, 95% CI: 1.023–1.165; P = 0.001 for heterogeneity), but not in other populations (P= 0.286, OR= 1.209, 95% CI: 0.853–1.714, P = 0.120 for heterogeneity). Egger’s test (P > 0.05) and Begg’s test (P>0.05) are both suggestive of the lack of publication bias for the included studies. Thus, the absence of association in other populations suggests a genetic heterogeneity in the susceptibility of schizophrenia and demonstrates the difficulties in replicating genome-wide association study findings regarding schizophrenia across different ethnic populations. To validate the association between rs1344706 and schizophrenia, further studies with larger participant populations worldwide are needed.