Plasmid puri cation kit

We synthesized oligonucleotides in OTUB1 mRNA 3'untranslated region (3'UTR) containing targeting sequences with miR-103a. Meanwhile, miR-103a overexpression vector, inhibitor vector and vector containing miR-103a mutation were designed. The targeting sites were mutated to construct miR-103a mutant. The pGLO vector was selected to construct the uorescence reporter vector pGLO-OTUB1. The vectors were extracted using a plasmid puri cation kit (Invitrogen). 293T cells were cultivated in a 24-well plate. The 200 ng pGLO-OTUB1 plasmid and 20 nM miR-103a mimic, inhibitor or mutant were cotransfected for a total of 24 h before the cell lysates were collected and the luciferase activity was determined by the dual luciferase reporter system (E1910, Promega). Fire y luciferase activity was used as a normalizer.
Statistics SPSS 22.0 statistical software (IBM Corp. Armonk, N.Y., USA) was applied for all statistical analysis. All data are displayed as the mean ± standard deviation. Comparisons between two groups were analyzed by paired t test (between normal tissues and tumor tissues) or unpaired t test (other two groups), while comparisons among multiple groups were assessed by one-way or two-way analysis of variation (ANOVA), followed by Tukey's post hoc test. The survival of patients was evaluated using Kaplan-Meier analysis. p value < 0.05 was symbolic of a signi cant difference.

We synthesized oligonucleotides in OTUB1 mRNA 3'untranslated region (3'UTR) containing targeting sequences with miR-103a. Meanwhile, miR-103a overexpression vector, inhibitor vector and vector containing miR-103a mutation were designed. The targeting sites were mutated to construct miR-103a mutant. The pGLO vector was selected to construct the uorescence reporter vector pGLO-OTUB1. The vectors were extracted using a plasmid puri cation kit (Invitrogen). 293T cells were cultivated in a 24-well plate. The 200 ng pGLO-OTUB1 plasmid and 20 nM miR-103a mimic, inhibitor or mutant were cotransfected for a total of 24 h before the cell lysates were collected and the luciferase activity was determined by the dual luciferase reporter system (E1910, Promega). Fire y luciferase activity was used as a normalizer.
Statistics SPSS 21.0 statistical software (IBM Corp. Armonk, N.Y., USA) was applied for all statistical analysis. All data are displayed as the mean ± standard deviation. The data accorded with the normal distribution were analyzed by unpaired t test, one-way or two-way analysis of variation (ANOVA). p value < 0.05 was symbolic of a signi cant difference.

Oligonucleotides in OTUB1 mRNA 3'untranslated region (3'UTR) containing targeting sequences with miR-103a (5'-UGGCUCCAGCCCGCUGCUCUG-3') were synthesized by GenePharma (Shanghai, China). Meanwhile, miR-103a overexpression vector, inhibitor vector and vector containing miR-103a mutation were designed. The targeting sites were mutated to construct miR-103a mutant (5'-AGCGUGGACCCCGUCACGACG-3'). The pGLO vector was selected to construct the uorescence reporter vector pGLO-OTUB1. The vectors were extracted using a plasmid puri cation kit (Invitrogen). 293T cells were cultivated in a 24-well plate. The 200 ng pGLO-OTUB1 plasmid and 20 nM miR-103a mimic, inhibitor or mutant were co-transfected for a total of 24 h before the cell lysates were collected and the luciferase activity was determined by the dual luciferase reporter system (E1910, Promega). Fire y luciferase activity was used as a normalizer.