# 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 mentioned in Figure 1C, 9478 genes were differentially expressed in INO80 complex knockdown HeLa cells. Hundreds of overlapping genes were found to be co-regulated by INO80, Arp8, Arp5, hles6, and hles2. Interestingly, miR-372 and miR-373 also exist in co-regulated genes (Figure 3A), suggesting the INO80 complex may regulate the expression of miR-372/miR-373. Therefore, to address this speculation, we designed RT-PCR primers to detect _miR-372_ expression. At first, HCT116 cells were transiently transfected with 20 pmol of siNT, siles2, and siArp8 for 48 h. After confirming the knockdown efficiency (Figure 3B), pri-_miR-372_ levels were then measured by RT-qPCR. As a result, pri-_miR-372_ levels were significantly increased by knocking down _les2_ (\"_p_ < 0.05) and _Arp8_ (***_p_ < 0.001) (Figure 3C). Next, HCT116 cells were transiently transfected with 20 pmol of non-targeting siRNA (siNT) and 10 or 20 pmol of siYY1; 48 h later, cells were harvested, and the whole cell lysate and total RNAs were prepared. The knockdown efficiency of YY1 was detected through western blot (Figure 3D), and pri-_miR-372_ was significantly upregulated by knocking down _YY1_ in a dose-dependent manner (Figure 3E). Similar results were obtained in pLVX-shYY1-1 or pLVX-shYY1-2 transfected HCT116 cells. As shown in Figure 3F, the knockdown efficiency of YY1 was measured using the western blot method, and a significant increase in  
Figure 2: INO80 knockdown induced abnormal cell growth and proliferation in HeLa and HCT116 cells. (**A**) Dox-induced reduction of INO80 protein levels in HeLa cells. pSingle-tTS-shINO80 inducible HeLa cells were grown without or presence of Dox (0, 0.5, 1, and 2 \(\upmu\)g/mL) for 72 h. GAPDH was used as an internal control (upper). INO80 protein levels were quantified by normalizing to the corresponding internal protein GAPDH using ImageJ software Version 1.8.0 (lower). * \(p\) < 0.05 or **_p_ < 0.01, compared to the no-Dox group. (**B**) Growth of wild-type (lower) and induced INO80 knockdown (upper) HeLa cells under an inverted microscope (magnification, \(\times\)200). (**C**) The western blot method confirmed decreased INO80 protein levels by pLVX-shINO80-1 and pLVX-shINO80-2. GAPDH was used as an internal control (upper). Normalized INO80 protein levels were shown in the lower panel. ***_p_ < 0.001, compared to the shNT group. (**D**) Knockdown of INO80 inhibited HCT116 cell proliferation (upper) (magnification, \(\times\)200). Each group’s quantified numbers of colonies are displayed as a bar graph. ***_p_ < 0.001, compared to the shNT group (lower). (**E**) Flow cytometry analysis of shINO80 knockdown HCT116 cell cycle. pri-_miR_-372 level in _YY1_ knocked down HCT116 cells was observed (Figure 3G). Finally, we detected pri-_miR_-372 level in _INO80_ knockdown HCT116 cells. To silence IN080, HCT116 cells were transfected with pSingle-tTS-shlNO80 plasmids with or without 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL Dox treatment. The experiment design is shown in Figure 3H--Dox-induced reduction of IN080 protein levels. Simultaneously, the pri-_miR_-372 levels were increased in a dose-dependent manner (Figure 3I). In conclusion, the elevated _miR_-372 expression was observed by knocking down multiple subunits of the IN080 complex, confirming that the IN080 complex regulates _miR_-372 transcription. Mimics miR-372 in HCT116 cells, in Turn, Suppressed IN080 and Arp8 Expression in Both mRNA and Protein  
The above results suggest that the IN080 complex can transcriptionally regulate the expression of miR-372. To further clarify the mutual function between the IN080 complex and miR-372, we searched the possible targeted regulatory genes of miR-372 using the Target Scan Human ([[https://www.targetscan.org](https://www.targetscan.org)]([https://www.targetscan.org](https://www.targetscan.org)), accessed on 5 March 2021) website.