AIM:To construct eukaryotic expression plasmids encoding Sox2 and Sox2 K247R and identify their expression and SUMOylation. METHODS: With gift plas-mid encoding Sox2 gene as a template, Sox2 K247R was obtained by overlapping extension PCR, followed by construction of pCMV-HA-Sox2 and pCMV-HA-Sox2 K247R. After enzyme digestion analysis and DNA sequencing, these two constructs were transfected or co-transfected with pC-MV-Myc-SUMO1 into 293 FT cells by lipofectin method. Western blot was employed to analyze expression and SUMO ylation of Sox2. RESULTS: It was revealed that eukaryotic expression vectors were constructed with correct sequence, where in mutant Sox2, the AAG codon was switched to CGG codon. Western blot results showed that good expression of both wt and mut Sox2, of which the latter could not be modified by SUMO1. CONCLUSION: Successful construction and expression of Sox2 and Sox2 K247R. Sox2 could be SUMO lyated in vitro but Sox2 K247R not.

Objective To explore the optimal dose ratio set in the hip digital tomosynthesis (DTS),and to reduce patient's examination dose as low as possible.Methods Ninety patients who received hip DTS examination were randomly divided into 3 groups,with the dose ratio as 6,7 and 8,respectively.One-way ANOVA test was used to compare.the image quality and radiation dose among 3 groups.Results The values of entrance surface dose (ESD),dose-area product (DAP) and image quality score of 3 groups were [(3.76 ± 1.89) mGy,(18.41 ± 11.71) dGy·cm2,3.03 ± 0.24],[(5.24±2.76)mGy,(26.99±13.34)dGy·cm2),3.60±0.11],and [(6.39±1.75)mGy,(36.96± 22.49) dGy· cm2,3.64 ± 0.09],respectively.The difference among three groups was statistically significant (F =10.94,9.45,139.26,P ＜ 0.05).The S-N-K test showed that both ESD values and DAP values in 6,7,and 8 time-dose ratio group was from small to large.The image quality score of 6 time-dose ratio group was lower than that of the other groups with no significant difference.Conclusions Dose ratio is one of the important parameters to result in the radiation dose of DTS.Dose ration 7 is optimal in hip DTS,which could match the image quality and radiation dose best and finally protect patients from unnecessary radiation damage.

Hepatitis B virus X protein (HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma (HCC). However, due to different transfection efficiency levels and experimental approaches, it is difficult to correlate the exact functions of HBx to HBV-associated HCC. In this study, we constructed two prokaryotic expression vectors, pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM, which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively. Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens. In addition, EGFP was expressed as a reporter reflecting the transduction efficiency of TLM. The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by AKTA Purifier system was incubated with AML12 and SMMC-7721 cells. Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm. Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. In conclusion, we expressed a cell-permeable HBx, which could provide a new method to study the functions of HBx.
Amino Acid Motifs ; genetics ; Cell-Penetrating Peptides ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; genetics ; Protein Precursors ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Trans-Activators ; biosynthesis ; genetics

Background/Aims: Ubiquitination is widely involved in the progression of hepatocellular carcinoma (HCC) by regulating various cellular processes. However, systematic strategies for screening core ubiquitin-related genes, clarifying their functions and mechanisms, and ultimately developing potential therapeutics for patients with HCC are still lacking. Methods: Cox and LASSO regression analyses were performed to construct a ubiquitin-related gene prediction model for HCC. Loss- and gain-of-function studies, transcriptomic and metabolomics analysis were used to explore the function and mechanism of UBE2S on HCC cell glycolysis and growth. Results: Based on 1,423 ubiquitin-related genes, a four-gene signature was successfully constructed to evaluate the prognosis of patients with HCC. UBE2S was identified in this signature with the potential to predict the survival of patients with HCC. E2F2 transcriptionally upregulated UBE2S expression by directly binding to its promoter. UBE2S positively regulated glycolysis in a HIF-1α-dependent manner, thus promoting the proliferation of HCC cells. Mechanistically, UBE2S enhanced K11-linkage polyubiquitination at lysine residues 171 and 196 of VHL independent of E3 ligase, thereby indirectly stabilizing HIF-1α protein levels by mediating the degradation of VHL by the proteasome. In particular, the combination of cephalomannine, a small molecule compound that inhibits the expression of UBE2S, and PX-478, an inhibitor of HIF-1α, significantly improved the anti-tumor efficacy. Conclusions UBE2S is identified as a key biomarker in HCC among the thousands of ubiquitin-related genes and promotes glycolysis by E3 enzyme-independent ubiquitination, thus serving as a therapeutic target for the treatment of HCC.

Background/Aims: Ubiquitination is widely involved in the progression of hepatocellular carcinoma (HCC) by regulating various cellular processes. However, systematic strategies for screening core ubiquitin-related genes, clarifying their functions and mechanisms, and ultimately developing potential therapeutics for patients with HCC are still lacking. Methods: Cox and LASSO regression analyses were performed to construct a ubiquitin-related gene prediction model for HCC. Loss- and gain-of-function studies, transcriptomic and metabolomics analysis were used to explore the function and mechanism of UBE2S on HCC cell glycolysis and growth. Results: Based on 1,423 ubiquitin-related genes, a four-gene signature was successfully constructed to evaluate the prognosis of patients with HCC. UBE2S was identified in this signature with the potential to predict the survival of patients with HCC. E2F2 transcriptionally upregulated UBE2S expression by directly binding to its promoter. UBE2S positively regulated glycolysis in a HIF-1α-dependent manner, thus promoting the proliferation of HCC cells. Mechanistically, UBE2S enhanced K11-linkage polyubiquitination at lysine residues 171 and 196 of VHL independent of E3 ligase, thereby indirectly stabilizing HIF-1α protein levels by mediating the degradation of VHL by the proteasome. In particular, the combination of cephalomannine, a small molecule compound that inhibits the expression of UBE2S, and PX-478, an inhibitor of HIF-1α, significantly improved the anti-tumor efficacy. Conclusions UBE2S is identified as a key biomarker in HCC among the thousands of ubiquitin-related genes and promotes glycolysis by E3 enzyme-independent ubiquitination, thus serving as a therapeutic target for the treatment of HCC.

Background/Aims: Ubiquitination is widely involved in the progression of hepatocellular carcinoma (HCC) by regulating various cellular processes. However, systematic strategies for screening core ubiquitin-related genes, clarifying their functions and mechanisms, and ultimately developing potential therapeutics for patients with HCC are still lacking. Methods: Cox and LASSO regression analyses were performed to construct a ubiquitin-related gene prediction model for HCC. Loss- and gain-of-function studies, transcriptomic and metabolomics analysis were used to explore the function and mechanism of UBE2S on HCC cell glycolysis and growth. Results: Based on 1,423 ubiquitin-related genes, a four-gene signature was successfully constructed to evaluate the prognosis of patients with HCC. UBE2S was identified in this signature with the potential to predict the survival of patients with HCC. E2F2 transcriptionally upregulated UBE2S expression by directly binding to its promoter. UBE2S positively regulated glycolysis in a HIF-1α-dependent manner, thus promoting the proliferation of HCC cells. Mechanistically, UBE2S enhanced K11-linkage polyubiquitination at lysine residues 171 and 196 of VHL independent of E3 ligase, thereby indirectly stabilizing HIF-1α protein levels by mediating the degradation of VHL by the proteasome. In particular, the combination of cephalomannine, a small molecule compound that inhibits the expression of UBE2S, and PX-478, an inhibitor of HIF-1α, significantly improved the anti-tumor efficacy. Conclusions UBE2S is identified as a key biomarker in HCC among the thousands of ubiquitin-related genes and promotes glycolysis by E3 enzyme-independent ubiquitination, thus serving as a therapeutic target for the treatment of HCC.

Background/Aims: Ubiquitination is widely involved in the progression of hepatocellular carcinoma (HCC) by regulating various cellular processes. However, systematic strategies for screening core ubiquitin-related genes, clarifying their functions and mechanisms, and ultimately developing potential therapeutics for patients with HCC are still lacking. Methods: Cox and LASSO regression analyses were performed to construct a ubiquitin-related gene prediction model for HCC. Loss- and gain-of-function studies, transcriptomic and metabolomics analysis were used to explore the function and mechanism of UBE2S on HCC cell glycolysis and growth. Results: Based on 1,423 ubiquitin-related genes, a four-gene signature was successfully constructed to evaluate the prognosis of patients with HCC. UBE2S was identified in this signature with the potential to predict the survival of patients with HCC. E2F2 transcriptionally upregulated UBE2S expression by directly binding to its promoter. UBE2S positively regulated glycolysis in a HIF-1α-dependent manner, thus promoting the proliferation of HCC cells. Mechanistically, UBE2S enhanced K11-linkage polyubiquitination at lysine residues 171 and 196 of VHL independent of E3 ligase, thereby indirectly stabilizing HIF-1α protein levels by mediating the degradation of VHL by the proteasome. In particular, the combination of cephalomannine, a small molecule compound that inhibits the expression of UBE2S, and PX-478, an inhibitor of HIF-1α, significantly improved the anti-tumor efficacy. Conclusions UBE2S is identified as a key biomarker in HCC among the thousands of ubiquitin-related genes and promotes glycolysis by E3 enzyme-independent ubiquitination, thus serving as a therapeutic target for the treatment of HCC.