MiR-26a targets the 3′UTR of PDHX mRNA directly. A(I) The miR-26a matches the eight nucleotide sequences (468-475 nt, UACUUGAA) of the 3′ UTR of the PDHX mRNA; A(II) The 3′UTR of PDHX mRNA was amplified and the cDNA fragment was cloned to construct the wild type recombinant plasmid pwt-PDHX, which contains the eight nucleotide sequences (…TACTTGAA…); A(III) The relevant eight nucleotides (…TACTTGAA…) were mutated to a random sequence (…TCACCAAT…) to construct the mutant recombinant plasmid pmt-PDHX. B. The miR-26a targets the 3′ UTR of PDHX mRNA analyzed by the luciferase reporter assays. Both of the two luciferase signals were measured and the activity of the Renilla luciferase was normalized to the firefly luciferase to generate the normalized Renilla luciferase activity. In the case of pwt-PDHX (left), the expression of miR-26a reduced luciferase activity effectively, while the luciferase activity was not inhibited in the case of the pmt-PDHX (right). Data are shown as the mean ± the standard error of the mean (SEM) of three replicates. P-value was computed using the Student’s t-test. *p < 0.05. pwt-PDHX: wild type recombinant plasmid of 3′UTR of PDHX mRNA; pmt-PDHX: mutant recombinant plasmid of 3′UTR of PDHX mRNA; pENTR-miR-26a: the miR-26a expression plasmid; pENTR-MIRNA: the empty vector without miR-26a.