Figure 4.

Targets of the miRNAs in the X‐chromosome cluster show reduced expression. (a) Quantitative PCR analysis of potential targets of the mir‐465 family that are reduced with age in RNA‐seq analysis verifies that expression of GHR, Kitl, PPP2R3C, Vegfa, GBN1, KRAS, and PTEN is downregulated between 27% and 64% at 24 and 36 months of age in liver. Each bar represents the average of five (24 months) or six (5 and 36 months) animals, run in triplicate. (b) GHR, Kitl, and PPP2R3C mRNA levels are significantly reduced by 38%, 25%, and 21%, respectively, in AML12 cells transfected with either pmir465abc or mir‐465c mimics when compared to control cells. mRNA expression levels of the other targets that are reduced with age in liver are not affected by mir‐465. Each bar represents the average expression value of five independent experiments. (c) Western blot analysis revealed a 50% reduction in GHR protein in AML12 cells 48 hr following transfection with pmir465abc or the mir‐465c mimic. The blot image is from a representative experiment, and the bar graph represents the average normalized intensity values of four independent experiments. (d) A 50% reduction in luminescence signal was observed in HEK‐293T cells cotransfected with a reporter plasmid containing the GHR 3′UTR (pGloGHR) along with the mir‐465 family (465abc) when compared to cells transfected with pGloGHR only. A significant reduction in luminescence signal was not seen in cells cotransfected with a reporter plasmid containing a mutated target site (pGloGHRmut) and 465abc, or in cells cotransfected with pGloGHR and a negative control miRNA. Individually, members of the mir‐465 family caused a reduction in luminescence similar to the reduction seen with the combination of all three family members. Each bar represents the average normalized luminescence value from six independent experiments. All error bars represent standard errors. All p‐values were calculated using two‐tailed Student's t test. *p < 0.05, ^† p < 0.01