# Feedback Modulation between Human IN080 Chromatin Remodeling Complex and miR-372 in HCT116 Cells
## 2 Results  
DNA Microarray Analysis of Gene Expression from HeLa Cells Indicated That INO80 Complex Mainly Regulates Cell Proliferation and Cell Viability  
The INO80 complex contains six metazoan-specific subunits, which all assemble on the N-terminus of the INO80 protein and form an N-terminal regulatory module (Figure 1A). To investigate the target genes of the INO80 complex, subunits of the INO80 complex, including _INO80_, _Arp5_, _Arp8_, _Ies2_, and _Ies6_, were knocked down with specific siRNAs in HeLa cells. The total RNA from HeLa cells was extracted, and the knockdown efficiency was measured by RT-qPCR (Figure 1B). After that, the total RNA samples were sent for DNA microarray analysis. The Illumina microarray datasets are accessible from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) data repository (accession#: GSE68655). As shown in Figure 1C, 9478 genes were differentially expressed among INO80, Arp8, Arp5, hles6, or hles2 and non-targeting (NT) siRNA knockdown HeLa cells. To further identify the function of genes regulated by INO80, Arp8, Arp5, Ies2, and Ies6, enriched Gene Ontology (GO) terms were analyzed, as shown in Figure 1D. GO terms enriched by INO80-, Arp8-, Arp5-, Ies2-, and Ies6 represented housekeeping functions that were related to cell proliferation, cell growth, cell adhesion, cell migration, cell motion, cell division, and cell motility, suggesting the important roles of INO80 complex in regulating these basic cell biological functions. In addition, hundreds of overlapping genes were found to be regulated by INO80, Arp8, Arp5, hles6, and hles2, which are components of the HSA and SNF2 modules (accession#: GSE68655). The Silencing of INO8 Caused HeLa Cells to Grow in Clusters and Inhibited the Colony-Forming Ability of HCT116 Cells  
To further confirm the reliability of DNA microarray analysis results, we decided to establish INO80-inducible silencing cell lines. Using the pSingle-tTS-shRNA system, we first constructed the pSingle-tTS-shINO80 plasmid, then the HeLa cells were transfected with pSingle-tTS-shINO80 plasmids and selected using neomycin to obtain _INO80_-inducible knockdown HeLa cells. As shown in Figure 2A, when the selected pSingle-tTS-shINO80 cells were treated with doxycycline (Dox), it was observed that the level of INO80 protein decreased gradually with the increase in Dox concentration, which proved that Dox could induce the silencing of _INO80_ gene in cells. Moreover, to observe the effect of _INO80_ silencing on cell growth status, 2 ug/mL Dox was used to induce INO80 silencing for 2 weeks. Compared with cells before Dox-induction, silencing of _INO80_ by Dox caused the cells to grow in clusters and resulted in changes in cell morphology. This phenomenon became obvious over time (comparing the cells at 1 and 2 weeks), suggesting the involvement of the INO80 complex in regulating the normal cell growth process (Figure 2B). In addition, using the pLVX-shRNA lentivirus knockdown system, we established a stable knockdown _INO80_ cell line in HCT116 colorectal cancer cells. Stably expressing pLVX-shINO80-1 and pLVX-shINO80-2 cells were confirmed by western blotting (Figure 2C). To further verify whether the knockdown of _INO80_ affects cell proliferation of the HCT116 cells, the colony formation assay was performed using pLVX-shNT- and pLVX-shINO80- plasmids transfected HCT116 cells. As shown in Figure 2D, downregulation of _INO80_ significantly inhibited the proliferation of HCT116 cells (upper), and the quantified number of colonies in each group was shown in (lower) (***\(p<0.001\)). Additionally, fluorescence-activated cell sorting (FACS) analysis was performed for pLVX-shNT and pLVX-shINO80 transfected HCT116 cells and observed cell cycle arrest at the G2/M phase as compared to the control empty vector, as shown in Figure 2E. Figure 1: The INO80 complex was mainly involved in regulating cell proliferation and cell viability. (**A**) Functional modules of INO80 complex.