MiR-124-3p/ZC3H15 Regulates Gastric Cancer Progression by Blocking FBXW7 Mediated Degradation of c-Myc

BACKGROUND: Zinc nger CCCH-type containing 15 (ZC3H15), a highly conserved eukaryotic protein, was involved in tumorigenesis and may be a potential biomarker in hepatocellular carcinoma (HCC) and acute myeloid leukemia (AML). However, the biological role of ZC3H15 in gastric cancer (GC) is unclear. METHODS: The potential correlation between ZC3H15 expression and GC prognosis was assessed based on the patient data analysis. The biological role of ZC3H15 in regulating cell proliferation and metastasis was evaluated in vitro and in vivo. In addition, the potential mechanism of ZC3H15 was investigated. RESULTS: we found that ZC3H15 expression was positively correlated with GC progression, including cell growth, metastasis and cancerogenesis. Through further investigations, we found that ZC3H15 could modulate c-Myc protein stability via suppressing the transcription of FBXW7, which was mainly responsible for c-Myc degradation. In addition, we revealed that miR-124-3p, a tumor suppressor of GC, was negatively associated with ZC3H15. We revealed that miR-124-3p was a critical upstream modulator of ZC3H15 in GC. CONCLUSIONS: Taken together, our studies unearth the important roles of ZC3H15 in GC development and suggest that miR-124-3p/ZC3H15/c-Myc axis may be a potential target for the treatment of GC.

The c-Myc oncoprotein, a transcription factor frequently upregulated in varieties of human neoplasms, is related to many physiological progressions such as cell survival, chemoresistance, and tumorigenesis [19]. Owing to the critical role of c-Myc in modulating cellular pathways, its expression is tightly regulated.
The regulatory mechanisms of c-Myc are majorly included transcriptional regulation, acetylation, phosphorylation and proteasomal degradation [20; 21]. F-box and WD repeat domain containing 7 (FBXW7), a known E3 ubiquitin ligases involved in ubiquitylation and proteasomal degradation of c-Myc, is an important tumor suppressor and is commonly dysregulated in human cancers [22; 23]. Expression of FBXW7 is negatively correlated with the tumor malignancy of human cancers [24; 25]. Therefore, a better understanding of the mechanisms of the regulation of FBXW7 expression may be effective in therapy against cancer.
The overexpression of ZC3H15 was found in GC patients according to our study. In order to identify the underlying mechanism of overexpression in ZC3H15 expression, we put the focus on microRNAs (miRNAs). miRNAs, a kind of small endogenous noncoding RNAs, are crucial mediators for posttranscriptional regulation, and can act as oncogenes or onco-suppressors [26][27][28]. One of the primary ways that miRNAs regulate cancer progression is through mRNA degradation. miRNAs could bind the speci c sequences of the target mRNA and then modulate the gene expression [29; 30]. miR-124-3p has been proven to be a crucial regulator in cancer progression, including breast cancer, cervical cancer, bladder cancer, and gastric cancer [31][32][33][34]. ZC3H15 is a predicted target gene of miR-124-3p according to the starBase. However, the mechanism by which miR-124-3p regulates ZC3H15 in human cells is unknown.
In this study, we demonstrated that ZC3H15 modulated cell proliferation, migration, invasion, and tumorigenesis via a c-Myc-dependent signaling pathway. ZC3H15 increased the protein stability of c-Myc by inhibition of FBXW7 transcription. In addition, we further demonstrated that miR-124-3p was a key upstream regulator of ZC3H15 in GC. Taken together, these data indicated that miR-124-3p/ZC3H15/c-Myc axis may be a potential therapy target for GC.

Cell culture and transfection
The GES-1 cells, GC cell lines, and embryonic renal cell line 293FT were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The HGC -27 cells was cultured in MEM (Minimum Essential Medium) supplement with penicillin and streptomycin(P/S) and fetal bovine serum (FBS); The other GC cell lines and GES-1 cells were cultured in RPMI-1640 (Roswell Park Memorial Institute-1640) mediums supplement with P/S and FBS. 293FT cell line was cultured as previously described [35]. The MEM, RPMI-1640 and DMEM meida, FBS, and antibiotics were obtained from Thermo Fisher Scienti c, Inc. (Waltham, MA, USA).
Sequences of the shZC3H15 and shFBXW7 were obtained from GenePharma Co., Ltd (Shanghai, China), and were listed as below:

Immunohistochemistry staining
Tumor specimen was embedded in para n and sectioned into 5μm thick sections, and then depara nized and hydrated. The sections were performed by microwave heating for antigen retrieval, and then incubated with endogenous peroxidase and blocking with goat serum. After quenching for primary antibodies at 4℃ and secondary antibodies at room temperature, sections were covered with DAB (diaminobenzidine) for visualizing the staining.
Cell viability and proliferation assays MTT assay was performed to examine the cell viability of indicated GC cell lines. Cells (1x10 3 cells/ well) were cultured in the 96-well plates, and then were detected according to the manufacture's protocol.

BrdU staining
For BrdU staining, indicated cells were seeded into 24-well plates. After incubated with BrdU (Sigma) and xed in 4% PFA, cells were treated with 1 mol/l HCl and 5% goat serum. Then, cells were incubated sequentially with primary antibody against BrdU, and Alexa FluorR 594 secondary antibody. DAPI (4',6diamidino-2-phenylindole) was used for nuclear staining.
Western blot analysis and Co-IP For Co-IP assay, cells were lysed in IP lysis buffer (Sigma) and then incubated on a rocker with antibody as well as IgG at 4 ℃ overnight. After incubation with Protein A/G PLUS-Agarose, cell lysate were washed by PBS and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Western blotting was performed as previously described [36].

Ubiquitination assay
For the ubiquitination assay, indicated plasmids were transfected into the 293FT cells. MG132 (50 μg/ml, Selleck, Houston, TX, USA) was added into the cells for 6h before harvesting. Cells were lysed and then performed following the same protocol used in Co-IP.

Turnover assay
The cells were transfected with indicated plasmids, and then a nal concentration (100μg/ml) of CHX was added into the media. After harvesting at the indicated time points, cell were lysed and analyzed by Western blotting.
Quantitative and RT-PCR Total RNA was harvested from the indicated cells and then reversely transcribed into cDNA by iScript cDNA Synthesis Kit (BioRad, #170-8891). The expression of mRNA was measured by using a Roche LightCycler Real-Time PCR System. Primers for RT-PCR assays were listed as Table II. Luciferase reporter assay Cells were transfected with shZC3H15, ZC3H15 or miR-124-3p mimics together with the indicated reporter (FBXW7, ZC3H15-WT, or ZC3H15-Mut) or control plasmid. Dual luciferase assay was performed by using the Dual-Luciferase® Reporter Assay System (Promega, #E1910). The promoter fragments of FBXW7, ZC3H15-WT, and ZC3H15-Mut were purchased from Wuhan GeneCreate Biological Engineering Co., Ltd.

Chromatin immunoprecipitation
Chromatin was isolated from 2x10 7 293FT/Vector and 293FT/Flag-ZC3H15. ChIP assays were performed using the EZ-ChIP TM kit (Millipore, CA, USA), and then detected according to the manufacture's protocol. The primers used in ChIP assays are listed as Table II. Soft agar assay For the soft agar assay, 0.4 × 10 3 cells were mixed with 0.6% agarose (Sigma-Aldrich, USA) in RPMI-1640 medium and then plated into 12-well plates containing a solidi ed bottom layer (0.3% agarose in medium).
Animal experimental procedures, tumour xenograft experiment, and lung metastasis assay All animal studies were approved by the Institutional Animal Care and Use Committee of Southwest University. Four-week-old female nude mice were purchased from Beijing Animal Research Center and were housed in the SPF room. For the tumor xenograft experiment, mice were randomly divided into three groups. HGC-27 cells (1×10 6 ) stably transfected with shGFP, shZC3H15-1 and shZC3H15-2 were subcutaneously injected into the mice in 18 November, 2019. Iso urane anaesthesia system, which could help animals enter an anaesthetised state faster and recover quickly, was used reduce the pain of the mice. Iso urane anaesthesia, is an inhalation general anesthesia, and the anesthesia-induction is stable, rapid, comfortable, fast recovery, good muscle relaxation, no sympathetic nervous system excitatory effect. In addition, iso urane has a low metabolic rate in the liver, so it has little toxicity to the liver, and repeated use has no effect obvious side effects. Iso urane was purchased from Reyward Life Technology Co., Ltd. (Shenzhen, China), and the concentration was MAC 1.6%. After subcutaneous injection, the mice were sterilised with 75% medical alcohol. The mice were observed and weighed every 3 days, and the feeding conditions were strictly standardized. The volume of tumors was calculated as follows: V = (length × width2)/2. Before the tumors were collected, the iso urane anaesthesia system was also used to reduce mice's pain, and then the mice were killed by cervical dislocation and the tumors were harvested. The bodies of mice were frozen at -20 °C and then transferred to Laibite Biotech Inc.
For the lung metastasis model, mice were randomly divided into three groups. HGC-27 cells (5×10 5 cells/ml) stably transfected with shGFP, shZC3H15-1 and shZC3H15-2 were injected subcutaneously into the tail vein of the mice in 18 November, 2019. Iso urane anaesthesia system was used to reduce the mice's pain during this experiment. The mice were observed and weighed every 3 days. Before the lungs were collected, the iso urane anaesthesia system was also used to reduce mice's pain, and then the mice were killed by cervical dislocation and the lungs were harvested. The bodies of mice were frozen at -20 °C and then transferred to Laibite Biotech Inc. (Chongqing, China) for incineration. The lungs were xed with paraformaldehyde for H&E staining.

Transwell assay
For the transwell assay, cells in serum-free MEM or RPMI-1640 Mediem were seeded into the 24-well Boyden chambers (8μm pore size, Corning). MEM or RPMI-1640 Mediem with 10% FBS was added to the lower chamber. Cells were xed in 4% paraformaldehyde (PFA) and then stained with crystal violet. Then, Cells were imaged and calculated.

Patient data analysis and patient tumor tissues
Bioinformatics analyses were performed using these speci c programs: TCGA

Statistical analysis
All experiments were performed at least three independent experiments, and the quantitative data were expressed as mean ± SD. Two-tailed Student's t-test was performed to calculate signi cance, and a value of P < 0.05 was considered statistically signi cant, *P<0.05, **P<0.01, ***P<0.001.

Results
ZC3H15 is up-regulated in GC and high expression of ZC3H15 correlates with poor patient prognosis Overexpression of ZC3H15 was found in 8 of 20 cancer types through Oncomine data-mining analysis ( Fig. 1 A). In DErrico, Cho and Chen's dataset from Oncomine database, we found that expression of ZC3H15 mRNA was signi cantly increased from normal stomach tissues to gastric cancer tissues ( Fig. 1  B-D). Then, we analyzed the expression data and survival information from the Gene Expression Omnibus (GEO) (GSE14210, GSE15459, and GSE22377), which was available from the Progression-free survival Kaplan-Meier database. We found that ZC3H15 high expression was signi cantly correlated with poor survival of GC patients (Fig. 1 E-G). To con rm the role of ZC3H15 in GC, we performed the univariate cox regression analyses based on the TCGA database and the results indicated that ZC3H15 expression was signi cantly correlated with age, depth of invasion, and histologic grade of gastric cancer (Table I). Moreover, multivariate cox regression analysis con rmed that age (P=0.039), depth of invasion (P=0.005), and histologic grade (P=0.022) as independent prognostic factors for the overall survival of GC patients (Fig. 1 H). To con rm the role of ZC3H15 in GC, we performed immunohistochemistry analysis (IHC) using primary tissue samples from GC patients. The results demonstrated that ZC3H15 expression was signi cantly higher in GC tissues ( Fig. 1 I and J). Then, we detected ZC3H15 expression at the mRNA and protein level in human GC cell lines and normal gastric epithelial cells (GES-1). We found that ZC3H15 expression was commonly expressed in GC cell lines ( Fig. 1 K). Therefore, these data indicated that ZC3H15 was upregulated in GC and high levels of ZC3H15 was correlated with the poor prognosis of patients with GC.

ZC3H15 is negatively regulated by miR-124-3p
By using an online bioinformatics database (starBase, http://www.starbase.sysu.edu.cn/), miR-124-3p was identi ed as a putative miRNA targeting ZC3H15. Downregulation of miR-124-3p was found in gastric cancer compared with normal stomach tissues (Fig. 2 A). And MTT assays demonstrated that miR-124-3p inhibited cell proliferation in HGC-27 cells (Fig. 2 B). By analysis of the data from the starBase database, we found that miR-124-3p expression was negatively correlated with ZC3H15 expression (R=-0.135, P=9.00e-03) (Fig. 2 C). Then, miR-124-3p mimic, miR-124-3p inhibitor or control miRNA was then transfected into HGC-27 and MKN-45 cells to evaluate the in uence of miR-124-3p on the ZC3H15 expression. Transfection with miR-124-3p mimics signi cantly decreased ZC3H15 mRNA and protein expression in GC cells. In contrast, miR-124-3p inhibitor increased ZC3H15 expression in the cells (Fig. 2 D and E). By analysis of starBase database, we found that miR-124-3p has a seed region contains 6 nucleotides that match the 3′UTR of human ZC3H15. To verify whether ZC3H15 is a direct target of miR-124-3p, we constructed luciferase reporter plasmids carrying wild-type ZC3H15 3′-UTR or mutant ZC3H15 3′-UTR and then transfected 293FT cells together with miR-124-3p mimics or control. The relative luciferase activity of the reporter was inhibited by the mimics; however, there was no signi cant change in the luciferase activity of mutagenesis reporter (Fig. 2 F). Taken together, these data indicate that miR-124-3p reduces ZC3H15 expression by directly targeting the 3'-UTR of ZC3H15 mRNA.

ZC3H15 promotes cell proliferation, migration, and invasion in vitro
To investigate the biological function of ZC3H15 in GC cells, we established stably transfected ZC3H15knockdown and ZC3H15-overexpressing cells for further investigation. Western blot and RT-PCR analysis was conducted to con rm the e ciency of the knockdown and overexpression system (Fig. 3 A). GESA using TCGA datasets showed positive association with cell cycle and metastasis in ZC3H15 high expression GC (Fig. 3 B and C). Then, MTT and BrdU assays demonstrated that silencing of ZC3H15 in HGC-27 cells signi cantly inhibited cell proliferation (Fig. 3 D and Fig-S1). Conversely, ectopic ZC3H15 overexpression enhanced cell proliferation in MKN-45 cells (Fig. 3 E). In addition, ZC3H15 increased the colony formation of GC cells (Fig. 3 F and G). Then, the transwell assays were performed and the results demonstrated that ZC3H15 knockdown in HGC-27 cells dramatically suppressed cell migration and invasion (Fig. 3 H). However, the metastatic effect was signi cantly elevated in ZC3H15-overexpressing MKN-45 cells (Fig. 3 I). Therefore, these results indicated that ZC3H15 accelerates cell proliferation, migration, and invasion of GC cells in vitro.

ZC3H15 promotes tumor growth and lung metastasis in vivo
To investigate the role of ZC3H15 in tumor growth of GC cells, we performed the subcutaneous xenograft experiment and then found that ZC3H15 knockdown signi cantly retarded the tumor growth of GC cells (Fig. 4 A and B). Immunohistochemical staining revealed that expression ZC3H15 was dramatically reduced in the ZC3H15-knockdown tumors, and the expression of Ki-67 was also decreased in the shZC3H15 tumors (Fig. 4 C). To determine whether ZC3H15 in uences GC metastasis in vivo, the lung metastasis models were used to evaluate the metastatic effect of ZC3H15. The number and size of lung nodules was signi cantly reduced by ZC3H15 knockdown in HGC-27 cells (Fig. 4 D). Taken together, these results demonstrated that ZC3H15 promotes tumor growth and lung metastasis of GC cells in vivo.

ZC3H15 stabilizes c-Myc by mediating its ubiquitination degradation
To further con rm the effect of ZC3H15 on GC cells, some proteins linked to cell proliferation and metastasis were analyzed by western blot. We found that silencing of ZC3H15 signi cantly decreased the protein levels of c-Myc, CyclinD1, CDK4, CDK6, MMP7, and N-cadherin of HGC-27 cells. In addition, overexpression of ZC3H15 in MKN-45 cells could increase the protein levels of these proteins (Fig. 5 A). Interestingly, quantitative PCR analysis revealed that downregulation of ZC3H15 slightly reduced c-Myc mRNA levels, suggesting that ZC3H15 may regulate c-Myc levels post-transcriptionally (Fig. 5 B). To further con rm that ZC3H15 modulates c-Myc ubiquitination, ZC3H15-knockdown HGC-27 cells were treated with the proteasome inhibitor MG-132, and the results demonstrated that c-Myc downregulation could be rescued by MG-132 (Fig. 5 C). We then examined the turnover rate of c-Myc, and we found that silencing of ZC3H15 in HGC-27 cells signi cantly increased the turnover rate of c-Myc (Fig. 5 D). Conversely, ZC3H15 overexpression could reduce the turnover rate of c-Myc (Fig. 5 E). Moreover, the ubiquitination of c-Myc was detected by the ubiquitination assay, and the results indicated that upregulation of ZC3H15 could reduce the ubiquitination levels of c-Myc (Fig. 5 F). Then, we examine and con rm the relationship between ZC3H15 and c-Myc in human cancers, we performed IHC staining on clinical tumor tissues of GC patients to assess the expression of ZC3H15 and c-Myc. The results demonstrated that ZC3H15 and c-Myc showed a signi cant positive correlation in staining intensity (Fig-S2). Taken together, these data suggested that ZC3H15 regulated the stability of c-Myc through reduction of c-Myc ubiquitination degradation.

ZC3H15 directly inhibits the transcription of FBXW7 in GC cells
FBXW7 is an important tumor suppressor, and is responsible for the ubiquitylation and proteasomal degradation of c-Myc. ZC3H15 is a classical CCCH-type zinc nger protein, suggesting it may be function as a transcription factor role in cell signaling. Thus, we speculated that ZC3H15 might modulate the protein stability of c-Myc by targeting FBXW7. Then, we performed quantitative PCR and western blot analysis and found that the mRNA and protein expression levels of FBXW7 were negatively correlated with ZC3H15 in HGC-27 and MKN-45 cells (Fig. 6 A and B). Then, we performed the dual-luciferase reporter assay found that FBXW7 promoter activity was signi cantly enhanced in ZC3H15-knockdown cells and was reduced in ZC3H15-overexpressing cells, indicating that the promoter activity of FBXW7 was inhibited by ZC3H15 (Fig. 6 C). To further determine whether ZC3H15 bind the promoter of FBXW7, we performed the ChIP assay and found that ZC3H15 bind the region P3 (-1020 to -804 bp) of FBXW7 promoter (Fig. 6 D). These data indicated that ZC3H15 could suppress FBXW7 transcription.

Downregulation of FBXW7 in ZC3H15-knockdown cells abrogates the effects induced by ZC3H15 silencing
To further con rm that ZC3H15 regulates the ubiquitination degradation of c-Myc by targeting FBXW7. We knockdown FBXW7 expression with the highly effective shFBXW7#2 in ZC3H15-knockdown HGC-27 cells, and found that c-Myc expression was increased after FBXW7 knockdown in ZC3H15-knockdown cells (Fig. 7 A and Fig. S3). MTT assays were performed and indicated that the cell proliferation of ZC3H15-knockdown cells were clearly increased after FBXW7-knockdown treatment (Fig. 7B). In addition, silencing of FBXW7 also could promote cell migration and invasion of ZC3H15-knockdown cells (Fig. 7  C). These data demonstrated that the ZC3H15-FBXW7-c-Myc axis might play a critical role in the cell proliferation and tumorigenesis of GC cells (Fig. 7 D).
GC exhibits the high rates of proliferation and metastasis, is a seriously threatens for human health. Gastrectomy is currently considered to be the mainstay radical treatments. If the tumor is detected and treated in early diagnosis, the 5-year survival rate of GC can reach 90% [37]. However, the overall survival is extremely poor, with an average 5-year survival rate of less than 20% [38]. Therefore, a better understanding of the relationships between cancerogenesis, development and prognosis will help to improve the diagnosis and treatment of GC. ZC3H15, a highly conserved eukaryotic protein widely expressed in various normal human tissues, contains a DFRP domain and two CCCH-type zinc nger domains. DFRP domain was responsible for interacting with DRG1 and then blocking the polyubiquitination and degradation of DRG1. In addition, ZC3H15 is also a classical CCCH-type zinc nger protein, suggesting it may function as a putative transcription factor role in cell signaling. To date, dysregulation of ZC3H15 has been reported in HCC and AML. However, the biological roles of ZC3H15 in GC remain unclear.
In the present study, we found that ZC3H15 was up-regulated in the patients with GC according to the immunohistochemistry and western blot analysis. Moreover, we observed that silencing of ZC3H15 inhibited cell proliferation, metastasis, and tumorigenesis of GC cells, and ZC3H15 overexpression could accelerate these progressions. These data suggest that ZC3H15 plays as an oncogene in GC cells.
The biological mechanism of ZC3H15 in human cancers remains largely unclear. Bei et al. used a microarray to evaluate the functional role of ZC3H15, and they found that ZC3H15 was involved in several critical signaling pathways, such as WNT pathway, NF-κB pathway, EGF pathway, TGF-β pathway, and PDGF pathway [18]. Here, we demonstrated that silencing of ZC3H15 reduced the protein expression levels of c-Myc and its downstream molecules such as CDK4, CDK6, and CyclinD1 in GC cells. However, c-Myc was not obviously changed at the mRNA level in ZC3H15-silencing GC cells. Subsequently, we performed the ubiquitination assay and turnover assay and found that ZC3H15 positively regulated c-Myc protein levels through reducing c-Myc degradation. We then found that the mRNA expression of FBXW7, a well-known E3 ubiquitin ligase of c-Myc, was signi cantly elevated in ZC3H15-knockdown GC cells. In addition, we performed the Dual-luciferase reporter assay and ChIP assay, and found that ZC3H15 could inhibit the transcription of FBXW7 by binding to the promoter-proximal region P3 of FBXW7 promoter.
There are few studies about the mechanism in regulation of ZC3H15 expression. To identify the mechanism in the up-regulation of ZC3H15 in GC, we put the focus on miRNAs. Based on bioinformatics analysis, ZC3H15 is a predicted target of miR-124-3p. miR-124-3p has been proven to be a tumor suppressor in gastric cancer progression [34]. We found that overexpression of miR-124-3p inhibited the expression of ZC3H15, and further revealed that miR-124-3p degraded ZC3H15 by directly targeting its 3′UTR.
In conclusion, our results demonstrated that ZC3H15 promoted cell proliferation, migration, invasion, and tumorigenesis of GC cells, and this function was associated with transcriptional repression of FBXW7, which was responsible for the ubiquitination and degradation of c-Myc. Additionally, our data revealed that ZC3H15 was directly target of miR-124-3p in GC. These results provide new insights into the functions of ZC3H15 and suggest that miR-124-3p/ZC3H15 may be a potential target for the treatment of

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