A New Strategy for Secretory Expression and Mixed Fermentation of Recombinant Human Collagen α1 (III) Chain in Pichia pastoris

Lina Wang,Dai Di Fan,Jing He,Zhongcheng Lv,Chen-Hui Zhu 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.5

Recombinant human full-length mature collagenα1 (III) chain (rhCOL3A1) was secreted by Pichia pastorisGS115, using the Saccharmyces cerevisiae á-mating factorprepro signal, and the theoretical molecular weight ofrhCOL3A1 was 95.344 kDa. The gene cloned from humanplacenta, was designed and cloned into expression vectorpPIC9K under the control of a strong inducible promoterAOX1.The expression stage of rhCOL3A1 was sensitiveto different carbon ratios through mixed fermentation. LCMS/MS analysis and western blotting demonstrated thatthe recombinant human full-length mature collagen α1(III) gene was successfully expressed in P. pastoris GS115during the methanol induction stage. Furthermore, aneffective strategy of mixed fermentation was established toexpress rhCOL3A1 in shake flash. Compared to singlecarbon induction, when induced with mixed carbon at theration of 0.8 (glycerol/methanol), the time corresponding tothe highest yield of rhCOL3A1 (1.27 g/L) was drasticallyreduced by 50%. The same conclusion was observed fromRT-qPCR. Consequently, a new strategy which was moretime-saving and effective was provided for the large-scaleproducing the full-length mature rhCOL3A1.

MiR-339 attenuates LPS-induced intestinal epithelial cells inflammatory responses and apoptosis by targeting TLR4

Meiying Xie,Lina Zhang,Luoye Li,Minhuan Fan,Lianjie Hou 한국유전학회 2020 Genes & Genomics Vol.42 No.9

Background Intestinal epithelial cells are important for defending against pathogen infection. LPS is an endotoxin that is highly antigenic and cytotoxic produced by bacteria. LPS disrupts the intestine epithelium integrity and induced the intestinal epithelial cell infammation and apoptosis. Our previous study has predicted the function of exosome miRNAs through bioinformatics analysis, and we found that miR-339 had a potential function in cell infammation response. To our knowledge, no published paper has demonstrated the miR-339 function in protecting the intestine epithelium against bacterial infection. Objective The objective of this study is to evaluate the miR-339 function in regulating intestinal epithelial cells to defend against bacterial infection through biological experiments and bioinformatics analyses. Methods Through the miR-339 transfection experiment and TLR4 interfering experiment, we evaluated the function of miR339 and TLR4 in the process of infammatory responses and apoptosis. Through Bioinformatics analyses and dual-luciferase reporter experiment, we identifed the target gene of miR-339. Results miR-339 attenuates LPS-induced intestinal epithelial cells infammatory responses through the TLR4/NF-κB signaling pathway and inhibited LPS-induced apoptosis through the P53 signaling pathway. TLR4 is the target gene of miR-339. TLR4 reduced LPS-induced proinfammatory responses and apoptosis. Conclusions In conclusion, miR-339 protected the intestine epithelial cells from LPS-induced cell infammation and apoptosis through targeting TLR4. This study expanded our understanding of how miRNAs and genes work collaboratively in regulating intestinal epithelial cells to defend against bacterial infection.

Sex Comb on Midleg Like-2 Accelerates Hepatocellular Carcinoma Cell Proliferation and Metastasis by Activating Wnt/β-Catenin/EMT Signaling

Lei Du,Lina Wang,Hong Yang,Jianping Duan,Jianming Lai,Wei Wu,Shaohua Fan,Xiaoli Zhi 연세대학교의과대학 2021 Yonsei medical journal Vol.62 No.12

Purpose: The purpose of this study was to investigate the influences of sex comb on midleg like-2 (SCML2) on hepatocellular carcinoma (HCC) and potentially related mechanisms. Materials and Methods: SCML2 expression in tumor tissues and cells was analyzed using the TCGA database and/or qRT-PCR. The proliferation of HCC cells was detected by CCK-8, colony formation, and EdU assays. The migration and invasion of HCC cells were detected by transwell and wound healing assays. Apoptosis of HCC cells was determined by flow cytometry. Additionally, qRT-PCR and Western blot were used to detect the expression of SCML2 and Wnt/β-catenin/epithelial–mesenchymal transition (EMT) signaling. A xenograft model in mice was established to verify the in vitro findings. Results: We found that SCML2 was highly expressed in HCC tissues and cells and that high expression of SCML2 was correlated with poor prognosis in HCC patients. SCML2 overexpression promoted proliferation, invasion, and migration and repressed apoptosis of HCC cells. The reverse results were obtained in SCML2-silenced cells. Further, we found that SCML2 activated the Wnt/β-catenin/EMT pathway. SCML2 silencing reduced the protein levels of Wnt3a, β-catenin, N-cadherin, Vimentin, and Snail and enhanced E-cadherin protein expression both in vivo and in vitro. Conclusion: SCML2 silencing inhibits the proliferation, migration, and invasion of HCC cells by regulating the Wnt/β-catenin/EMT pathway.

Selumetinib overcomes gefitinib primary and acquired resistance by regulating MIG6/STAT3 in NSCLC

Xiaoping Song,Lina Wang,Wei Tang,Luyao Yuan,Qingchao Liu,Jing Li,Daidi Fan 대한약학회 2023 Archives of Pharmacal Research Vol.46 No.12

Gefitinib, as the first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has achieved great advances in the treatment of non-small cell lung cancer (NSCLC), but drug resistance will inevitably occur. Therefore, exploring the resistance mechanism of gefitinib and developing new combination treatment strategies are of great importance. In our study, the results showed that selumetinib (AZD6244) synergistically inhibited the proliferation of NSCLC with gefitinib. Selumetinib also enhanced gefitinib-induced apoptosis and migration inhibition ability in gefitinib-resistant lung cancer cell lines. Subsequently, the negative regulation between MIG6 and STAT3 was observed and verified through the STRING database and western blotting assays. Sustained activation of STAT3 was significantly downregulated when co-treatment with selumetinib in gefitinib-resistant cells. However, the downregulation of p-STAT3, resulting from the combination of selumetinib and gefitinib was counteracted by the deletion of MIG6, suggesting that selumetinib enhanced gefitinib sensitivity by regulating MIG6/STAT3 in NSCLC. In contrast, p-STAT3 was further inhibited after treatment with gefitinib and selumetinib when MIG6 was overexpressed. Furthermore, the combined administration of selumetinib and gefitinib effectively promoted the sensitivity of lung cancer xenografts to gefitinib in vivo, and the tumor inhibition rate reached 81.49%, while the tumor inhibition rate of the gefitinib monotherapy group was only 31.95%. Overall, MIG6/STAT3 negative regulation plays an important role in the sustained activation of STAT3 and the resistance to EGFR-TKIs. Our study also suggests that EGFR-TKIs combined with MEK1/2 inhibitors, such as selumetinib, may be beneficial to those NSCLC patients who develop a primary or acquired resistance to EGFR-TKIs, providing theoretical support for combining TKIs and selumetinib in clinical cancer treatment.

The effects of cigarettes and alcohol on intestinal microbiota in healthy men

Lin Renbin,Zhang Yawen,Chen Luyi,Qi Yadong,He Jiamin,Hu Mengjia,Zhang Ying,Fan Lina,Yang Tao,Wang Lan,Si Misi,Chen Shujie 한국미생물학회 2020 The journal of microbiology Vol.58 No.11

Human intestinal microbiota is affected by the exogenous microenvironment. This study aimed to determine the effects of cigarettes and alcohol on the gut microbiota of healthy men. In total, 116 healthy male subjects were enrolled and divided into four groups: non-smoking and non-drinking (Group A), smoking only (Group B), drinking only (Group C), and smoking and drinking combined (Group D). Fecal samples were collected and sequenced using 16S rRNA to analyze the microbial composition. Short-chain fatty acid (SCFAs) levels in feces were determined by gas chromatography. We found that cigarette and alcohol consumptions can alter overall composition of gut microbiota in healthy men. The relative abundances of phylum Bacteroidetes and Firmicutes and more than 40 genera were changed with cigarette and alcohol consumptions. SCFAs decreased with smoking and alcohol consumption. Multivariate analysis indicated that when compared with group A, group B/C/D had higher Bacteroides, and lower Phascolarctobacterium, Ruminococcaceae_ UCG-002, Ruminococcaceae_UCG-003, and Ruminiclostridium_ 9 regardless of BMI and age. Additionally, the abundance of Bacteroides was positively correlated with the smoking pack-year (r = 0.207, p < 0.05), the abundance of predicted pathway of bacterial toxins (r = 0.3672, p < 0.001) and the level of carcinoembryonic antigen in host (r = 0.318, p < 0.01). Group D shared similar microbial construction with group B, but exerted differences far from group C with lower abundance of Haemophilus. These results demonstrated that cigarette and alcohol consumption separately affected the intestinal microbiota and function in healthy men; furthermore, the co-occurrence of cigarette and alcohol didn’t exacerbate the dysbiosis and cigarette played the predominated role on the alteration.