# Feedback Modulation between Human IN080 Chromatin Remodeling Complex and miR-372 in HCT116 Cells
## 2 Results
### Mimics miR-372 Upresulated the p53/p21 Pathway in HCT116 Cells  
CDKN1A (p21) is a universal inhibitor of cyclin kinases that controls the cell cycle by activating and inactivating the cyclin-dependent kinases (CDKs) [38]. In addition, p21 carries out its diverse biological functions by detecting and responding to multiple signals through the p53-dependent pathway and p53-independent pathway. Its primary role as a tumor suppressor is attributed to its ability to impede cell cycle progression and facilitate DNA damage repair. Nevertheless, considerable evidence suggests that p21 can also exhibit oncogenic properties primarily by suppressing apoptosis [39]. We previously demonstrated that the IN080 complex negatively regulates the _p21_ expression [35]. Based on the published literature, there are two binding sites (\(-\)2.5-2.2 kb and \(-\)1.3-1.0 kb) upstream of the _p21_ transcriptional start site (TSS) for the tumor suppressor gene _p53_, which is a regulator of p21 [40]. Thus, to understand the enrichment of IN080 near the _p21_ transcription start site (TSS) as the cell cycle changes, we designed six pairs of qPCR primers, including two p53 binding sites, in the _p21_ locus to amplify ChIP DNA (Table 2) (Figure 6A). First, HCT116 cells were synchronized with 1 mM hydroxyurea (HU) at the G1/S phase. Then, cells were released from G1/S arrest by HU. We harvested cells at different times to collect G1, S, and G2/M cells (Figure 6B, upper). ChIP assays were then performed using INO80 antibody in different cell cycle phases HCT116 cells. As expected, INO80 occupied the p53 binding sites (\(-\)2.2 kb and \(-\)1.0 kb upstream of the p21 TSS) at G1 and S phases but not at the G2/M phase (Figure 6B, lower). It suggests that INO80 may activate _p21_ transcriptional expression through p53. In line with this prediction, after knocking down _INO80_ with Dox induction, increased p53 and p21 protein levels were observed. In addition, the knockdown of _INO80_ resulted in decreased Arp8 protein in cells (Figure 6C). Interestingly, HCT116 cells that mimic _miR_-372 expression not only caused a reduction of INO80 protein expression but also led to the increase of p53 and p21 protein levels (Figure 6D), suggesting that miR-372 may post-transcriptionally inhibit the expression of _INO80_ through binding to INO80-3\({}^{\prime}\)-UTR, thereby reducing the expression of INO80 targeted gene _p21_. Our subsequent experimental results also support this assumption. In Dox-induced _INO80_ knockdown HCT116 cells, mimic _miR_-372 significantly upregulated the p21 and p53 protein levels (lane 3) compared to those in _INO80_ knockdown-only group (lane 2) (Figure 6E). The above data suggest that miR-372 may post-transcriptionally regulate the stability of the INO80 complex and play roles through co-regulating the p53/p21 pathway in cells.  
Figure 5: miR-372 post-transcriptionally regulated the expression of INO80/Arp8 by binding to their 3\({}^{\prime}\)-UTR in HCT116 cells. (**A**) Binding sites of miR-372-3p on the INO80 and Arp8 3\({}^{\prime}\)-UTR. The 3\({}^{\prime}\)-UTR fragments of human INO80 (wild type, wt; mutant, mt) and Arp8 (wt) were cloned downstream of the luciferase between the MluI and HindIII sites. (**B**) Relative luciferase activities. HCT116 cells were transfected with a plasmid containing pMIR-REPORT-Arp8-3\({}^{\prime}\)-UTR and pMIR-REPORT-INO80 3\({}^{\prime}\)-UTR with or without miR-372. **\({}^{**}p<0.01\), ***\(p<0.001\), vs. pMIR-REPORT-Arp8-3\({}^{\prime}\)-UTR or pMIR-REPORT-INO80 3\({}^{\prime}\)-UTR group. (**C,D**) Effects of miR-372 on the Arp8 and INO80 protein expression in HCT116 cells. Cells were co-transfected with pMIR-REPORT-Arp8-3\({}^{\prime}\)-UTR and pMIR-REPORT-INO80 3\({}^{\prime}\)-UTR plasmids with or without miR-372, and the protein levels were analyzed with a western blot. GAPDH was used as an internal control. (**E**) Mutant sites in pMIR-INO80-3\({}^{\prime}\)-UTR lost their response to miR-372. Lane 1 was transfected with an empty vector. (**F**) Possible working diagram.