Objective To clone and express cathepsin B gene of Echinococcus granulosus(EgCatB)and analyze EgCatB protein by using bioinformatics tools and online databases. Methods The total RNA of E. granulosus was extracted and reverse?ly transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In?Fusion PCR cloning method and expressed by a wheat germ cell?free system,and then the recombinant protein was identified by Western blotting. The signal peptide,transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online soft?ware SignalP 4.1,TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently,the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally,the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam,SMART,Predictprotein,Swiss?model,NetOGlyc 4.0 and NetNGlyc 1.0 approaches. Results The EgCatB gene was successfully amplified from cDNA of E. granulosus and ex?pressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a cata?lytic active center was formed through Gln106,Cys112,His282 and Asn302. It was found that there were nine O?glycosylation sites in the EgCatB sequence,but no N?glycosylation sites. Conclusions The EgCatB gene is cloned and expressed successfully,and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful informa? tion for further functional study of the EgCatB protein.

Objective:To study the influence of long non-coding RNA (LncRNA) plasmacytoma variant translocation 1 (PVT1) in glucose transporter 3 (GLUT3) expression, and cell proliferation and invasion in glioma.Methods:(1) The correlation between PVT1 and GLUT3 gene expressions and their influences in overall survival (OS) were analyzed using data from 222 cases of primary gliomas from Chinese Glioma Genome Atlas mRNAseq_325 data set. (2) Fifteen glioma specimens, including 8 from patients with low-grade glioma (LGG group) and 7 from patients with glioblastoma (GBM group), were collected in our hospital from January 2019 to December 2019; the PVT1 expression was detected by fluorescence in situ hybridization (FISH); the GLUT3 protein expression was detected by immunohistochemistry. (3) Normal human astrocyte (NHA) and glioblastoma cell lines U87, LN229 and U251 (NHA group, U87 group, ln229 group and U251 group) were cultured in vitro; real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the PVT1 and GLUT3 mRNA expressions; Western blotting was used to detect the GLUT3 protein expression; U87 and LN229 cells were divided into PVT1 overexpression plasmid group and blank plasmid group, PVT1 short hairpin RNA (shRNA) group and negative control shRNA group; the GLUT3 mRNA and protein expressions were detected by RT-qPCR and Western blotting. (4) In U87 and LN229 cells of negative control shRNA group and PVT1 shRNA group, CCK-8 assay and colony formation assay were used to detect the cell proliferation and Transwell assay was used to detect the cell invasion. (5) Ten female BALB/c-nu nude mice were randomly divided into experimental group and control group ( n=5); the U87 cells from PVT1 shRNA group and negative control shRNA group were transplanted into the mice to establish subcutaneously transplanted tumor models. The animals were sacrificed and the tumors were weighed and measured 4 weeks after transplantation; the Ki-67 and GLUT3 protein expressions were detected by immunohistochemistry. Results:(1) The gene expressions of PVT1 and GLUT3 were positively correlated in the 222 cases of primary glioma from mRNAseq_325 data set ( r=0.514, P=0.000); the OS of patients in the PVT1 high-expression group or GLUT3 high-expression group was significantly shorter as compared with that in the PVT1 low-expression group or GLUT3 low-expression group, respectively ( P<0.05). (2) As compared with the low-grade glioma group, the glioblastoma group had significantly increased PVT1 and GLUT3 protein expressions ( P<0.05). (3) As compared with NHA cells, the U87, LN229 and U251 cells had significantly increased PVT1 and GLUT3 mRNA and protein expressions ( P<0.05). As compared with those in the blank plasmid group, the GLUT3 mRNA and protein expressions were significantly increased in the U87 and LN229 cells of PVT1 overexpression plasmid group ( P<0.05); as compared with those in the negative control shRNA group, the GLUT3 mRNA and protein expressions were significantly decreased in the U87 and Ln229 cells of PVT1 shRNA group ( P<0.05). (4) As compared with negative control shRNA group, PVT1 shRNA group had significantly reduced optical density value, significantly smaller numbers of clone formation and invasive cells in U87 and LN229 cells ( P<0.05). (5) As compared with those in the control group, the subcutaneous transplanted tumor volume was significantly smaller, the subcutaneous transplanted tumor mass and Ki-67 and GLUT3 protein expressions were significantly lower in the experimental group ( P<0.05). Conclusion:Down-regulation of PVT1 can decrease the GLUT3 expression, therefore, inhibit the proliferation and invasion of glioma cells.