# Feedback Modulation between Human IN080 Chromatin Remodeling Complex and miR-372 in HCT116 Cells
## 2 Results
### Elevated pri-miR-372 Expression Level Was Detected in INO80-Complex Knockdown HCT116 Cells
\begin{table}
\begin{tabular}{c c c c c c c} \hline
**Target** & **Representative** & **3P-seq** & **Total** & **7 mer-m8** & **7 mer-A1** & **Representative** \\
**Gene** & **Transcript** & **tags + 5** & **Sites** & **Sites** & **Sites** & **miRNA** \\ \hline INO80 & ENST00000361937.3 & 279 & 2 & 1 & 1 & has-miR-372-3p \\ YY1 & ENST00000262238.4 & 1803 & 3 & 1 & 2 & has-miR-372-5p \\ INO80D & ENST00000403263.1 & 5 & 2 & 1 & 1 & has-miR-372-5p \\ ARP8 & ENST00000335754.3 & 327 & 2 & 1 & 1 & has-miR-372-5p \\ MCRS1 & ENST00000546244.1 & 914 & 1 & 1 & 0 & has-miR-372-5p \\ \hline \end{tabular}
\end{table} 
Table 1: Complementary sequences of miR-372 were found in the 3′-UTR of the multi-subunits of the INO80 complex. Figure 4: Pri-miR-372 suppressed the expression levels of INO80 and Arp8 in both mRNA and protein in HCT116 cells. (**A**) Validation of pmR-mCherry-miR-372 plasmid expression efficiency. *** \(p\) < 0.001, compared to vector only group. (**B**) The relative pri-_miR_-372 levels in the presence or absence of pri-miR-372-inhibitors. **_p_ < 0.01, compared to the negative control group. (**C**) IN080 and Arp8 protein levels were estimated in miR-372 transfected HCT116 cells compared to the pcDNA3.1 group. The protein levels were analyzed by western blot with specific antibodies. Dose-dependent decrease of IN080 (**D**) and Arp8 (**E**) mRNA expression levels was observed in pmR-mCherry-miR-372 transfected HCT116 cells. (***_p_ < 0.01, compared to pcDNA3.1 group). (**F**) miR-372 mimics inhibited HCT116 cell proliferation. The cell proliferation abilities were estimated with colony-formation assays in HCT116 cells (upper) (magnification, \(\times\)200). Quantified numbers of colonies for each group are displayed as a bar graph (lower). Data are presented using mean \(\pm\) standard deviation values. ***_p_ < 0.001, vs. the vector groups, ***_p_ < 0.001, vs. the miR-372 group. MiR-372 Post-Transcriptionally Inhibited IN080/Arp8 Expression by Targeting Their 3\({}^{\prime}\)-Ultr in HCT116 Cells  
The previous experimental results suggest that Arp8 and IN080 may be miR-372 targeted regulatory genes. To confirm whether miR-372 can bind to the 3\({}^{\prime}\)-UTR of IN080 and Arp8 and post-transcriptionally regulate their expression levels, we predicted possible binding sites in the 3\({}^{\prime}\)-UTR of _INO80_ and _Arp8_. We found two possible binding sites in the 3\({}^{\prime}\)-UTR of _INO80_ (153-159 and 4644-4650) and _Arp8_ (457-463 and 1594-1601), respectively, and introduced the DNA fragments containing the binding sites into the pMIR-REPOIRT-Luc vector to construct the luciferase reporter gene plasmid. Concurrently, the mutagenic plasmid of the miR-372 binding site in the IN080-3\({}^{\prime}\)-UTR was designed and constructed (Figure 5A). First, pMIR-REPORT-INO80-3\({}^{\prime}\)-UTR and pMIR-REPORT-Arp8-3\({}^{\prime}\)-UTR plasmids (a vector carrying the wild-type 3\({}^{\prime}\)-UTR of _Arp8_ and _INO80_) were transiently transfected into HCT116 cells, and the luciferase activities were detected in the presence or absence of miR-372. As shown in Figure 5B, the luciferase activities were significantly inhibited by co-transfection with miR-372 compared to the no-miR-372 group (**_p_ < 0.01, ***_p_ < 0.001). As expected, co-transfection of pMIR-REPORT-INO80-3\({}^{\prime}\)-UTR or pMIR-REPORT-Arp8-3\({}^{\prime}\)-UTR with miR-372 reduced Arp8 (Figure 5C, lanes 2 and 4) or IN080 (Figure 5D, lanes 3 and 4) protein levels compared those to the transfection of pMIR-REPORT-INO80-3\({}^{\prime}\)-UTR or pMIR-REPORT-Arp8-3\({}^{\prime}\)-UTR only group, suggesting that IN080 and Arp8 may be the targets of miR-372. Furthermore, while the miR-372 target sites in the 3\({}^{\prime}\)-UTR of IN080 linked to the luciferase reporter were mutagenized, both mutant sites lost their response to miR-372, and the IN080 protein level no longer decreased due to the co-transfection of miR-372 (Figure 5E, lanes 5 and 6), indicating the site-specificity of the repression.