Figure 4.

Functional validation of miR-128-3p targets in SH-SY5Y cell line and proposed Wnt inhibition model. (a–g) Bar diagram represents the relative expression of Wnt3, Wnt5b, Lrp6, Gskip, Dvl1, Lef1, and Gsk3b transcripts in SH-SY5Y cell line transiently transfected with mimic miR-128-3p overexpression oligo (mimic-128) or anti-miR-128-3p overexpression oligo (anti-128) and compared with control cells. GAPDH was used as a normalizer for target gene expression. Values are represented as mean ± SEM. (e1) Diagram representing predicted target site interaction between rat miR-128-3p seed region and Dvl1 3’ untranslated region (UTR). (e2) Dvl1 gene expression showed significant (P = .06) change when the mimic-128 group was compared with control. (e3) Immunoblot analysis of Dvl1 protein expression in SH-SY5Y cell line miR-128-3p gain (mimic-128) and loss (anti-128) of function mutations. A significant (P = .017) decrease in Dvl1 protein level was noted in the mimic-128 group compared with control. Conversely, a marked increase in Dvl1 protein expression was identified in the anti-128 group compared with control. (f) A significantly (P = .002) higher expression of Lef1 gene was observed in anti-128 transfected cell line compared with the control group. (g) Expression of Gsk3b gene was significantly (P = .01) lower in anti-128 transfected cell line compared with control. (h) Expression level of miR-128-3p in SH-SY5Y cell line individually transfected with miR-128-3p oligo (mimic-128) and miR-128-3p anti-sense oligo (anti-128). Compared with the control group, the mimic-128 transfected cell line showed a significant (P < .001) increase in miR-128-3p expression, whereas significantly (P < .001) reduced miR-128-3p expression was observed in the anti-128 transfected group. (i) The cellular model of canonical Wnt pathway inhibition partially mediated via miR-128-3p overexpression under stress-induced maladaptive changes.