FOXO1-Induced miR-502-3p Suppresses Colorectal Cancer Cell Growth through Targeting CDK6

Colorectal cancer (CRC) is the most common tumor of the digestive system and the third most common tumor worldwide. To date, the prognosis of CRC patients remains poor. It is urgent to identify new therapeutic targets for CRC. As a tumor suppresser, microRNA (miRNA) miR-502-5p is downregulated in CRC tissues. Nevertheless, the role of miR-502-3p in CRC is largely unclear. Besides, the transcript factor forkhead box protein O1 (FOXO1) could suppress the CRC cell growth. However, the effect of FOXO1 on miR-502-3p in CRC remains unknown. By contrast, cyclin-dependent kinases 6 (CDK6) promotes the CRC cell growth. Yet the regulatory effect of miR-502-3p on CDK6 in CRC has not been reported. Thus, the primary aim of this study was to investigate whether FOXO1 enhanced miR-502-3p expression to suppress the CRC cell growth by targeting CDK6. Here, RNA level and protein level were detected by quantitative reverse transcription-PCR (qRT-PCR) and western blot (WB), respectively. Besides, the cell growth was detected by Cell Counting Kit 8 (CCK8) assay. Moreover, the regulatory effect of FOXO1 on miR-502-3p or miR-502-3p on CDK6 was determined using dual-luciferase reporter gene (DLR) assay. Results revealed that miR-502-3p and FOXO1 were downregulated in CRC cells. Besides, miR-502-3p suppressed the CRC cell growth. Moreover, FOXO1 could increase the miR-502-3p level through facilitating MIR502 transcription in CRC cells. In addition, miR-502-3p could suppress the CRC cell growth by targeting CDK6. These findings indicated that FOXO1 induced miR-502-3p expression to suppress the CRC cell growth through targeting CDK6, which might provide new therapeutic targets for CRC.


Introduction
CRC is the most common tumor of the digestive system and the third most common tumor worldwide [1][2][3]. In China, 55.5 thousand new CRC cases are reported and 28.6 thousand CRC patients die annually [4][5][6]. More noteworthy is that the incidence rate of CRC is still growing rapidly [4,7]. What is worse is that the prognosis of CRC patients remains poor due to postoperative recurrence and metastasis, and the 5-year survival of stage IV patients with CRC is only 10% [8,9]. Tus, it is urgent to seek new therapeutic targets for CRC to improve the prognosis of patients with CRC.
CDK6 is a recognized cell cycle kinase facilitating cancer cell proliferation to promote cancer progression [21]. Besides, CDK6 is upregulated in CRC cells [22]. Moreover, CDK6 promotes CRC progression. A recent study has revealed that miR-500a-3p suppresses CRC progression through inhibiting aerobic glycolysis by targeting CDK6 [22]. By contrast, lncRNA CASC21 enhances the CRC cell growth by inducing CDK6 expression [23]. Yet the role of miR-502-3p in CDK6 expression in CRC has not been reported.
Terefore, the primary aim of the current study was to investigate whether FOXO1 enhanced miR-502-3p expression to suppress the CRC cell growth by targeting CDK6.

Methods and Materials
2.1. Cell Culture. Normal colonic mucosa cell line FHC cells and CRC cell line HT29 cells were obtained from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). Ten, FHC and HT29 cells were cultured with Dulbecco's modifed eagle's medium (DMEM) and 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA) in a humidifed incubator supplemented with 95% O 2 and 5% CO 2 at 37°C.

Cell Transfection.
In this study, miRNA mimic, inhibitor, and vectors were transfected into HT29 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif, USA). Ten, HT29 cells were collected and used for subsequent experiments at 48 hours post-transfection.

CCK8 Assay.
First, 1 × 10 4 HT29 cells were collected in a well of the 96-well plate. Ten, 10 μL CCK8 solution (#C0038, Beyotime Biotechnology, Shanghai, China) was added into each well at a 1/10 dilution to incubate HT29 cells for 2 hours at 37°C. Next, Multiscan MK3 (Termo Fisher Scientifc, Waltham, MA, USA) was used to read the absorbance at 450 nm. Finally, the rate of HT29 cell proliferation was calculated based on the mean of optical density (OD) at 450 nm.

MiR-502-3p Is Downregulated in CRC Cell
Line. First, the miR-502-3p level in HT29 cells was identifed. Results of qRT-PCR showed that the level of miR-502-3p was reduced in HT29 cells compared to that in FHC cells (Figure 1), suggesting that miR-502-3p was downregulated in the CRC cell line. Tese results were consistent with those found in CRC tissues.
Results of CCK8 assay revealed that miR-502-3p overexpression dramatically decreased the HT29 cell growth rate compared to that of control HT29 cells ( Figure 2). Besides, mimic NC had no efect on the HT29 cell growth rate ( Figure 2). Abovementioned data suggested that miR-502-3p suppressed the CRC cell growth.

FOXO1 Elevates miR-502-3p
Level through Facilitating MIR502 Transcription in CRC Cells. Te pri-miR-502 is a transcript from MIR502. Moreover, potential FOXO1 binding site was found on the promoter of MIR502 by bioinformatics analysis (Figure 3(a)). Consistent with the miR-502-3p level, the protein level of FOXO1 was also reduced in HT29 cells compared to that in FHC cells (Figure 3(b)). Tese results suggested that FOXO1 might regulate the miR-502-3p level in HT29 cells.
Next, results of qRT-PCR confrmed that FOXO1 overexpression by transfection of FOXO1 expression vector upregulated the miR-502-3p level in HT29 cells (Figure 3(c)). Besides, results of DLR assay indicated that the luciferase activity of HT29 cells transfected with pGL3-basic vectors containing the promoter of MIR502 was increased by transfection of FOXO1 expression vector, while the blank expression vector had no efect on the luciferase activity of HT29 cells (Figure 3(d)). Tus, these data suggested that FOXO1 could elevate the miR-502-3p level through facilitating MIR502 transcription in CRC cells.
To further identify the regulatory efect of miR-502-3p on CDK6, DLR assay was performed. Results showed that the luciferase activity of HT29 cells transfected with pGL3basic vector containing the WT CDK6 mRNA 3′ UTR was reduced by miR-502-3p mimic whereas elevated by miR-502-3p inhibitor (Figure 4(c)). However, the luciferase activity of HT29 cells transfected with pGL3-basic vector containing the MUT CDK6 mRNA 3′ UTR with mutated miR-502-3p binding site was not regulated by miR-502-3p mimic or inhibitor (Figure 4(c)). Moreover, the luciferase activity of HT29 cells transfected with pGL3-basic vector was not afected by mimic NC and inhibitor NC (Figure 4(c)). As   Journal of Oncology CDK6 could promote the CRC cell growth [23], these results together suggested that miR-502-3p should suppress the CRC cell growth through targeting CDK6.
Te current study had indicated that the FOXO1 protein level was downregulated in CRC cells. Nevertheless, the mechanism regulating the FOXO1 protein level in CRC remains unclear. Several studies have revealed that FOXO1 protein expression is reduced by miRNAs in CRC. For example, miR-544 facilitates CRC progression through decreasing FOXO1 protein expression [25]. Besides, miR-135b decreases sensitiveness of oxaliplatin by reducing the FOXO1 protein level in CRC cells [17]. Moreover, miR-183-5p stimulates angiogenesis through suppressing FOXO1 protein expression in CRC [26]. In addition, miR-96 promotes CRC cell proliferation via downregulating the FOXO1 protein level [27]. Tus, the FOXO1 protein level might be reduced by miRNAs in CRC cells.
CDK6 could be regulated by miRNAs in various cancers. A previous study has revealed that miR-204 suppresses nonsmall cell lung cancer (NSCLC) progression through downregulating the CDK6 level [28]. Similarly, a recent study has indicated that miR-370-3p restrains the progression of ovarian cancer by reducing CDK6 expression [29]. Moreover, miR-576 represses CDK6 expression to promote bladder cancer cell proliferation [30]. In addition, miR-186 decreases the CDK6 level to inhibit the prostate cancer cell growth [31]. In CRC, both miR-539-5p and miR-500a-3p depress CRC progression via targeting CDK6 [22,23]. Nevertheless, the efect of miR-502-3p on CDK6 is largely unknown. Terefore, this study for the frst time revealed the inhibitory role of miR-502-3p in CDK6 during CRC progression.
However, there are still some limitations in the current study. For example, the association between FOXO1 and MIR502 promoter should be further identifed by chromatin immunoprecipitation (ChIP), while the interaction of miR-502-3p and CDK6 mRNA should be further determined using miRNA pulldown. In addition, the efect of miR-502-3p on the CRC cell growth should be confrmed by in vivo study performed in nude mice.

Conclusion
In summary, the current study revealed that downregulated miR-502-3p suppressed the CRC cell growth. Besides, FOXO1 could increase the miR-502-3p level through facilitating MIR502 transcription in CRC cells. Moreover, miR-502-3p should suppress the CRC cell growth by targeting CDK6. Tese fndings indicated that FOXO1 induced miR-502-3p expression to suppress the CRC cell growth through targeting CDK6, which might provide novel therapeutic targets for CRC.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.