Figure 4. miR-228 Affects Longevity and Stress Response in C. elegans(A) miR-228 antagonizes longevity in C. elegans because
mir-228 mutants are long lived compared to N2 wild-type (p < 0.01). Three lines of
mir-228 overexpressors coinjected with
myo-3::GFP are shorter lived than N2 or
myo-3::GFP (p < 0.05). (B)
mir-228 mutants are resistant to heat stress. When day 1 adult animals grown on solid media are subjected to a 4 hr heat shock at 35C, the percentage of
mir-228 animals surviving this stress is significantly higher than N2 (*p < 0.05). Conversely, the percentage of
mir-228 overexpressor animals surviving this stress is significantly lower than N2 or
myo-3::GFP (*p < 0.05). This indicates that the longevity and stress response phenotypes of this miRNA are highly correlated. Error bars show SEM. (C and D) miR-228 is dynamically expressed during aging.
mir-228::GFP expression during aging (head region) is shown (C). GFP expression was quantified as a readout of
mir-228 levels, which peak in mid-adulthood. This same pattern holds true when quantifying
mir-228 in the head region (amphid and excretory cells), where expression is brightest, or quantifying expression across the entire animal. Expression of mature miR-71, miR-228,
pha-4, and
skn-1 in wild-type animals over multiple aging time points via qRT-PCR is shown (D).
pha-4 follows the same pattern as shown by Lund et al. [49], in which levels peak at about day 6 adulthood before decreasing; miR-228 exhibits the same expression profile, indicating that PHA-4 promoting expression of
mir-228 is the stronger side of the feedback loop. As miR-71 levels are increasing,
pha-4 levels are lower than miR-71, but when miR-71 levels decrease,
pha-4 levels are higher, consistent with the model that miR-71 targets
pha-4. Finally,
skn-1 levels remain constant during all aging time points, indicating that the opposing interactions of miR-228 targeting and downregulating
skn-1 while SKN-1 targets
mir-228 may effectively cancel each other out so that
skn-1 levels do not change. Error bars show SEM.