# 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
As a result, the complementary sequences of miR-372 were found in the 3\({}^{\prime}\)-UTR of the multiple subunits of the IN080 complex, including IN080, YY1, IN080D, Arp8, and MCRS1 (Table 1), suggesting that the expression of _miR_-372 is not only transcriptionally regulated by IN080 complex, but in turn, miR-372 may affect the expression level of IN080 complex by binding to the 3\({}^{\prime}\)-UTR of the above genes; this may further affect the stability and biological function of the IN080 complex. We constructed the pmR-mCherry-miR-372 plasmid to prove this assumption, which can express miR-372 in mammalian cells. To confirm _miR_-372 expression, HCT116 cells were transfected with pmR-mCherry empty vector and pmR-mCherry-miR-372 plasmids for 48 h, cells were then collected, and the  
Figure 3: Elevated pri-_miR_-372 expression level was detected in IN080-complex knockdown HCT116 cells. (**A**) miR-372/373 were identified in IN080 complex knockdown HeLa cells. Downregulation: \(\downarrow\), Upregulation: \(\uparrow\). (**B**) Knockdown efficiency of les2 and Arp8 in HCT116 cells. ***\(p<0.001\), compared to the siNT group. (**C**) Pri-_miR_-372 expression levels in siles2 or siArp88 transfected HCT116 cells. *\(p<0.05\) or ***\(p<0.001\), compared to the siNT group. (**D**) Knockdown efficiency of siYY1. GAPDH was used as an internal control. (**E**) Pri-_miR_-372 expression levels in siYY1 transfected HCT116 cells. ***\(p<0.001\) compared to the siNT group. (**F**) The YY1 protein level in pLVX-shYY1-transfected HCT116 cells. GAPDH was used as an internal control. (**G**) Pri-_miR_-372 expression levels in pLVX-shYY1 transfected HCT116 cells. ***\(p<0.001\), compared to the siNT group. (**H**) Dox-induced IN080 knockdown efficiency. \(\alpha\)-Tubulin was used as an internal control. (**I**) Pri-_miR_-372 expression levels in Dox-induced IN080 knockdown HCT116 cells. *\(p<0.01\) or ***\(p<0.001\), compared to the no-Dox induction group. total RNA was extracted. The pri-_miR_-372 levels were analyzed by RT-qPCR using specific primers. Compared with the empty vector group, a significant dose-dependent increase of _miR_-372 was observed (*** \(p\) < 0.001) (Figure 4A), and this expression was effectively inhibited by co-transfecting with miR-372-3p inhibitors (Figure 4B). Thus, we examined the effect of miR-372 on the expression level of key subunits of the INO80 complex. As shown in Figure 4C, after transfection of pmR-mCherry-miR-372 plasmids for 48 h in HCT116 cells, INO80 and Arp8 protein levels were decreased in mimic _miR_-372 expression cells. In addition, the relative mRNA levels of INO80 and Arp8 in HCT116 cells were significantly decreased by the expression of _miR_-372 dose-dependent (Figure 4D-E). By examining multiple vector concentrations, we aimed to determine the optimal dosage that would yield the most significant and consistent effects of miR-372 on our target. In the colony formation assay, the clonogenic ability of HCT116 cells was suppressed by transfecting with miR-372-3p, and this effect was antagonized by inhibitors (Figure 4F upper). The quantified number of colonies in each group is shown in (Figure 4F lower). This result is consistent with inhibiting colony-forming ability by knocking down the _INO80_ gene. Combined with the previous experimental results, undoubtedly, miR-372 regulates the INO80 and Arp8 expression levels in both mRNA and proteins in HCT116 cells. Therefore, it can be speculated that miR-372 may regulate the function of the INO80 complex by targeting multi-subunits in the complex.