# ZC3H15 Correlates with a Poor Prognosis and Tumor Progression in Melanoma

Authors: Qian Li; Jianbing Hou; Chengda Guo; Yanli Zhang; Lichao Liu; Huanrong Hu; Shaomin Shi; Yacong Ji; Leiyang Guo; Yaqiong Shi; Yaling Liu; Hongjuan Cui
Journal: BioMed Research International
 (2021)

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## Abstract

Zinc figure CCCH-type containing 15 (ZC3H15), also called developmentally regulated GTP-binding protein 1 (DRG1) family regulatory protein 1 (DFRP1), is a zinc finger containing protein. Despite playing a role in cellular signaling, it is found overexpressed in acute myeloid leukemia and also an independent prognostic marker in hepatocellular carcinoma patients. However, the biological effect of ZC3H15 in malignant melanoma (MM) remains unexplored. The expression of ZC3H15 in patients was analyzed using the R2: Genomics Analysis and Visualization Platform database. Immunohistochemical analysis, western blot, and qRT-PCR were used to detect ZC3H15 expression in melanoma tissues and cell lines. MTT, BrdU, flow cytometry assay, transwell, and western blot were performed to explore the proliferation, cell cycle, invasion, and migration of melanoma cells. We undertaken colony formation assay in vitro and tumor xenograft in vivo to detect the tumorigenicity of melanoma cells. In the present study, ZC3H15 was demonstrated highly expressed in melanoma tissues and cells. Elevated ZC3H15 impairs the survival of melanoma patients. Meanwhile, attenuation of ZC3H15 in melanoma cells inhibited cell proliferation and induced cycle arrest at G0/G1 phase. Consistently, the expression of cell cycle-related proteins cyclin dependent kinase 4 (CDK4), CDK6, and cyclin D1 (CCND1) was decreased while p21 was upregulated. Furthermore, we found the migration and invasion abilities were inhibited in ZC3H15-knockdown melanoma cells. In addition, downregulation of ZC3H15 resulted in inhibition of colony formation abilities in vitro and tumorigenesis in vivo. ZC3H15 promotes proliferation, migration/invasion, and tumorigenicity of melanoma cells. As a promising biomarker and therapeutic target in MM, ZC3H15 is worthy of further exploration.


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## Body

## 1. Introduction

Malignant melanoma (MM), a malignant transformation of melanocytes located within the deep layer of the epidermis [1], accounts for the fifth most common form of cancer in adults [2]. It can implicate multiple organs, including the eye, gastrointestinal tract, genitalia, sinuses, and meninges, but most commonly arises in the skin, especially in the setting of UV injury [2]. Although MM is less common than other skin cancers and only accounts for less than 5% of all cutaneous malignant neoplasms worldwide [3], it is more lethal and aggressive, responsible for nearly 73% of skin cancer-related deaths [4, 5]. Seriously, the incidence of MM has been arising worldwide [6]. Although early-stage melanoma is treatable with surgical excision at the localized stage with a high 5-year relative survival rate of 98% [5], the advanced/metastatic melanoma has a significantly lower survival rate [7]. Fortunately, the long-term survival outcomes of metastatic melanoma have been dramatically improved by the development of immune checkpoint blockade strategies targeting the PD-1 and CTLA-4 coinhibitory receptors and MAPK molecular targeted therapy directed at oncogenic BRAF and MEK signaling pathways [2, 8]. However, more and more patients have shown resistance to these inhibitors [8], which is a major obstacle to the prognosis of melanoma worldwide [9]. Hence, there is an urgent need to explore new therapeutic target for MM.ZC3H15 is a gene located on chromosome 2q32.1 (GenBank accession no. NM_018471) and highly conserved among eukaryotes [10, 11]. It was originally identified as likely ortholog of mouse immediate early response erythropoietin 4 (LEREPO4) gene, which can be induced by erythropoietin [12]. Meanwhile, ZC3H15 was also termed DFRP1 due to its effect on DRG1, which plays a critical role in cell growth [11, 13, 14]. ZC3H15 can specifically bind with DRG1 and upregulate the expression of DRG1 through blockade of its polyubiquitination [11]. Furthermore, ZC3H15 can enhance the activity of DRG1, promoting a greater thermal stability, and improve DRG1 hydrolysis rate, rendering a more efficient enzyme [15]. In addition, ZC3H15 was found to be upregulated upon HIV infection and promote HIV replication [10]. Given that DRG1 is associated with SCL (TAL-1) oncogenic protein, it is worthy to explore the potential role of ZC3H15 in tumorigenesis. Others had found that ZC3H15 interplays with TRAF-2 protein, which is associated with the NF-κB signal pathway, and is upregulated in AML [16]. In hepatocellular carcinoma (HCC), the expression of ZC3H15 is adverse to the overall survival rate and positively associated with tumor recurrence [17]. However, the biological function of ZC3H15 in MM has not been studied till now.In the present study, we found that ZC3H15 is overexpressed in MM and predicts a poor prognosis. Besides, downregulation of ZC3H15 inhibits melanoma cell proliferation, invasion, and metastasis. This study indicates that ZC3H15 serves as a candidate indicator in MM diagnosis and therapy.


## 2. Materials and Methods

### 2.1. Reagents and Antibodies and Clinical Tissue Samples

Anti-ZC3H15 was purchased from Novus Biologicals (Oklahoma, CO, USA), and anti-α-tubulin antibody was purchased from Proteintech (Wuhan, China). Anti-CDK4 (12790), anti-CDK6 (13331), anti-CCND1 (2922), anti-p21 (2947), anti-E-cadherin (14472), anti-N-cadherin (13116), and anti-vimentin (5741) antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA). MG132 (M7449) and anti-BrdU (ab6326) antibody were obtained from Abcam (Cambridge, MA, USA). After obtaining prior approval, the clinical tissue samples were collected from the Third Hospital of Hebei Medical University, Hebei, China. The ethics committee of the Third Hospital of Hebei Medical University approved the tissue analysis. All the subjects participating in the study provided written informed consent.


### 2.2. Patients’ Data Analysis

We followed the methods of Zhang et al. [18]. Gene expression data for melanoma were obtained from R2: microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). Kaplan-Meier analysis was performed, and the resulting survival curves were generated by using GraphPad Prism (version 6.0). All cut-off values for separating the high- and low-expression groups were determined by the online R2 database algorithm.


### 2.3. Immunohistochemistry (IHC) Staining

We followed the methods of Zhang et al. [18]. Paraffin-embedded tissues were cut into slices with a thickness of 5 mm, and then, the sections were dewaxed and rehydrated. Then, paraffin slices were put into citrate buffer (pH 6.0) and heated in a microwave oven at 95°C for 20 min to facilitate antigen retrieval. Then, endogenous peroxidase activity was quenched, which was followed by blocking with normal goat serum. Next, the sections were incubated overnight at 4°C with the antibodies ZC3H15 or Ki67, which was diluted with PBS (1 : 200). Then a horseradish peroxidase-linked secondary antibody was incubated with the sections. Lastly, the sections were counterstained using haematoxylin and visualized with DAB reagent. The results were analyzed under a microscope (Olympus, Japan).


### 2.4. Cell Culture

The human melanoma cell lines A375, MV3, and Skmel28, as well as human immortalized melanocyte cell line PIG1 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were tested mycoplasma negative. A375 cells and PIG1 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA), and MV3 cells and Skmel28 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640, Gibco, New York, NY, USA). Both two media were supplemented with bovine serum (FBS, Gibco), penicillin (100 U/ml), and streptomycin (100ug/ml) (Invitrogen, California, CA, USA), maintained at 37°C in an incubator with a humidified atmosphere of 5% CO2.


### 2.5. Vector Construction, Transfection, and Infection

Short hairpin RNA (shRNA) for ZC3H15 and a negative control shRNA (shGFP) were purchased from Gene Pharma Co. Ltd. (Shanghai, China) and cloned into the pLKO.1 vector. Sequences used were presented as follows: shZC3H15 #1, 5′-CAGATCCCAAGTCTGTAGTAT-3′; shZC3H15#2, 5′-CCTA-GAATCAACAGGATGTTT-3′; and shZC3H15#3, 5′-GCTGACTTCAAAGCAGGGAAA-3′. The recombinant plasmid containing human ZC3H15 full-length cDNA cloned into the pCDH-CMV-MCS-EF1-Hygro vector was purchased from YouBio Company (Changsha, China). Vector encoding of human ZC3H15 was constructed by PCR-based amplification, and the primers used were listed as follows: ZC3H15-F-(EcoRI)—5′-CCGGAATTCATGCCCCCCAAGAAAC-3′; ZC3H15-R-(NotI)—5′-ATTTGCGGCCGCTCATTCTTCTAAATCAAGTGTATTT-3′. For transfection and infection experiments, packaging plasmid including pLP1, pLP2, and VSVG, together with the target plasmid, was transfected into 293FT cells by using the transfection reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Supernatants were collected 48 h later and then used to infect melanoma cells twice with polybrene. The infected cells were screened by treatment with puromycin and hygromycin B for 36 h.


### 2.6. MTT Assay

The cell viability of MV3 and A375 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) assay as previously described. Briefly, 2000 cells in logarithmic phase were seeded in 96-well plates. At designated times, 20μl of MTT (5 mg/ml) solution was added to each well and the cells were incubated in a dark incubator for another 4 h. Finally, 150 μl DMSO was added to dissolve the formazan crystals and absorbance of the plate at 490 nm was measured by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).


### 2.7. Western Blot Analysis

Western blotting was performed as described previously. Briefly, total cell proteins were extracted using lysis buffer. 40μg of protein was used for SDS-PAGE and, subsequently, transferred to onto PVDF membranes. After blocking, the membranes were separately incubated with primary antibody at 4°C overnight. Following incubation with secondary antibodies, the membranes were visualized by an ECL chemiluminescent detection system. Band density was quantified using Syngene-Image System and normalized to β-actin.


### 2.8. Quantitative Reverse Transcription PCR (qRT-PCR)

Total ribonucleic acid (RNA) was extracted from cells using Trizol reagent (Invitrogen, Germany) according to the manufacturer’s instructions. The synthesis of complementary DNA (cDNA), PCR amplification, and calculation of fold change were undertaken as previously described. The primers used were listed as follows: ZC3H15-F—5′-AACAAAATCCACGTCAGGTAGC-3′; ZC3H15-R—5′-TG CACATACTACAGACTTGGGA-3′.


### 2.9. BrdU Staining Analysis

Cell proliferation was monitored by BrdU staining. Firstly,2×104 cells were cultured in 24-well plates. 48 h later, the cells were incubated with 10 μg/ml BrdU for 35 min and subsequently fixed with 4% paraformaldehyde (PFA) for 20 min. Then, the cells were treated with 2 M HCl for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 10% goat serum for 1 h, and incubated a monoclonal rat primary antibody against BrdU (1 : 300, Sigma-Aldrich) overnight at 4°C. Alexa Fluor® 594 secondary antibody (H+L; Invitrogen) was incubated with the cells at room temperature for 2 h, followed by nuclear staining with DAPI (300 nM). Finally, BrdU-positive cells in random fields were counted under the microscopy.


### 2.10. Flow Cytometry Analysis

Cell cycle was detected using flow cytometry. In detail, cells were washed twice with PBS, fixed with 70% ethanol for 24 h at 4°C, and incubated with 200μl PBS containing 1 μl potassium iodide (PI) (BD, San Jose, CA, USA) and 1 μl RNaseA (Sigma-Aldrich, USA) at 37°C for 30 min. Then, cells were detected with a FACS flow cytometer (BD Biosciences, CA, USA), and the results were analyzed using FlowJo software.


### 2.11. Migration and Invasion

We followed the methods of Zhang et al. [18]. Cell migration and invasion were explored using Transwell Chambers (8 μm pore size, Corning, Beijing, China). Referring to invasion experiment, the membranes were covered with matrigel (BD Biosciences). In the upper chamber, cells were cultured in serum-free medium, and in the lower chamber, mediums supplemented with 10% FBS were added. After culturing at 37°C for 24 h for the migration assay and 48 h for the invasion assay, respectively, cells were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet. The mean numbers of cells were calculated from at least six randomly chosen microscopic images.


### 2.12. Colony Formation Assay

A total of2×103 cells/mL was seeded into a six-well plate. All cells were incubated at 37°C for 9–12 days when the number of colonies showed more than 50. The cells were fixed with formalin for 30 minutes and stained with 1% crystal violet. Colony formation number was subsequently calculated.


### 2.13. Animal Experimental Procedures and Tumor Xenograft Experiment

We followed the methods of Zhang et al. [18]. Animal experiments were approved by the Committee for Animal Protection and Utilization of Southwest University. All experiments were conducted according to the Guidelines for Animal Health and Use (Ministry of Science and Technology, China, 2006). Four-week-old male nude mice were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China) and were placed in SPF rooms for feeding and observation. Human A375 cells (1×106 cells) stably transfected with shGFP, shZC3H15, shZC3H15/GFP, or shZC3H15/ZC3H15 were injected into the right dorsal side of five-week-old female nude mice (n=3). The mice were sterilized with 75% medical alcohol after subcutaneous injection. Then, tumor growth was measured by a Vernier caliper every three day, and the tumor volume was calculated by smalldiameter2×largediameter/2. Two weeks after injection, the mice were sacrificed, and the tumors were excised, weighed, photographed, and subjected to immunohistochemical staining.


### 2.14. Statistical Analysis

All experiments were performed at least three times. Values are presented as themean±standarddeviation (SD). Student’s t test was used to evaluate significant differences between two samples. P values < 0.05 were considered to indicate a statistically significant difference.


## 2.1. Reagents and Antibodies and Clinical Tissue Samples

Anti-ZC3H15 was purchased from Novus Biologicals (Oklahoma, CO, USA), and anti-α-tubulin antibody was purchased from Proteintech (Wuhan, China). Anti-CDK4 (12790), anti-CDK6 (13331), anti-CCND1 (2922), anti-p21 (2947), anti-E-cadherin (14472), anti-N-cadherin (13116), and anti-vimentin (5741) antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA). MG132 (M7449) and anti-BrdU (ab6326) antibody were obtained from Abcam (Cambridge, MA, USA). After obtaining prior approval, the clinical tissue samples were collected from the Third Hospital of Hebei Medical University, Hebei, China. The ethics committee of the Third Hospital of Hebei Medical University approved the tissue analysis. All the subjects participating in the study provided written informed consent.


## 2.2. Patients’ Data Analysis

We followed the methods of Zhang et al. [18]. Gene expression data for melanoma were obtained from R2: microarray analysis and visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). Kaplan-Meier analysis was performed, and the resulting survival curves were generated by using GraphPad Prism (version 6.0). All cut-off values for separating the high- and low-expression groups were determined by the online R2 database algorithm.


## 2.3. Immunohistochemistry (IHC) Staining

We followed the methods of Zhang et al. [18]. Paraffin-embedded tissues were cut into slices with a thickness of 5 mm, and then, the sections were dewaxed and rehydrated. Then, paraffin slices were put into citrate buffer (pH 6.0) and heated in a microwave oven at 95°C for 20 min to facilitate antigen retrieval. Then, endogenous peroxidase activity was quenched, which was followed by blocking with normal goat serum. Next, the sections were incubated overnight at 4°C with the antibodies ZC3H15 or Ki67, which was diluted with PBS (1 : 200). Then a horseradish peroxidase-linked secondary antibody was incubated with the sections. Lastly, the sections were counterstained using haematoxylin and visualized with DAB reagent. The results were analyzed under a microscope (Olympus, Japan).


## 2.4. Cell Culture

The human melanoma cell lines A375, MV3, and Skmel28, as well as human immortalized melanocyte cell line PIG1 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were tested mycoplasma negative. A375 cells and PIG1 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA), and MV3 cells and Skmel28 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640, Gibco, New York, NY, USA). Both two media were supplemented with bovine serum (FBS, Gibco), penicillin (100 U/ml), and streptomycin (100ug/ml) (Invitrogen, California, CA, USA), maintained at 37°C in an incubator with a humidified atmosphere of 5% CO2.


## 2.5. Vector Construction, Transfection, and Infection

Short hairpin RNA (shRNA) for ZC3H15 and a negative control shRNA (shGFP) were purchased from Gene Pharma Co. Ltd. (Shanghai, China) and cloned into the pLKO.1 vector. Sequences used were presented as follows: shZC3H15 #1, 5′-CAGATCCCAAGTCTGTAGTAT-3′; shZC3H15#2, 5′-CCTA-GAATCAACAGGATGTTT-3′; and shZC3H15#3, 5′-GCTGACTTCAAAGCAGGGAAA-3′. The recombinant plasmid containing human ZC3H15 full-length cDNA cloned into the pCDH-CMV-MCS-EF1-Hygro vector was purchased from YouBio Company (Changsha, China). Vector encoding of human ZC3H15 was constructed by PCR-based amplification, and the primers used were listed as follows: ZC3H15-F-(EcoRI)—5′-CCGGAATTCATGCCCCCCAAGAAAC-3′; ZC3H15-R-(NotI)—5′-ATTTGCGGCCGCTCATTCTTCTAAATCAAGTGTATTT-3′. For transfection and infection experiments, packaging plasmid including pLP1, pLP2, and VSVG, together with the target plasmid, was transfected into 293FT cells by using the transfection reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Supernatants were collected 48 h later and then used to infect melanoma cells twice with polybrene. The infected cells were screened by treatment with puromycin and hygromycin B for 36 h.


## 2.6. MTT Assay

The cell viability of MV3 and A375 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) assay as previously described. Briefly, 2000 cells in logarithmic phase were seeded in 96-well plates. At designated times, 20μl of MTT (5 mg/ml) solution was added to each well and the cells were incubated in a dark incubator for another 4 h. Finally, 150 μl DMSO was added to dissolve the formazan crystals and absorbance of the plate at 490 nm was measured by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).


## 2.7. Western Blot Analysis

Western blotting was performed as described previously. Briefly, total cell proteins were extracted using lysis buffer. 40μg of protein was used for SDS-PAGE and, subsequently, transferred to onto PVDF membranes. After blocking, the membranes were separately incubated with primary antibody at 4°C overnight. Following incubation with secondary antibodies, the membranes were visualized by an ECL chemiluminescent detection system. Band density was quantified using Syngene-Image System and normalized to β-actin.


## 2.8. Quantitative Reverse Transcription PCR (qRT-PCR)

Total ribonucleic acid (RNA) was extracted from cells using Trizol reagent (Invitrogen, Germany) according to the manufacturer’s instructions. The synthesis of complementary DNA (cDNA), PCR amplification, and calculation of fold change were undertaken as previously described. The primers used were listed as follows: ZC3H15-F—5′-AACAAAATCCACGTCAGGTAGC-3′; ZC3H15-R—5′-TG CACATACTACAGACTTGGGA-3′.


## 2.9. BrdU Staining Analysis

Cell proliferation was monitored by BrdU staining. Firstly,2×104 cells were cultured in 24-well plates. 48 h later, the cells were incubated with 10 μg/ml BrdU for 35 min and subsequently fixed with 4% paraformaldehyde (PFA) for 20 min. Then, the cells were treated with 2 M HCl for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 10% goat serum for 1 h, and incubated a monoclonal rat primary antibody against BrdU (1 : 300, Sigma-Aldrich) overnight at 4°C. Alexa Fluor® 594 secondary antibody (H+L; Invitrogen) was incubated with the cells at room temperature for 2 h, followed by nuclear staining with DAPI (300 nM). Finally, BrdU-positive cells in random fields were counted under the microscopy.


## 2.10. Flow Cytometry Analysis

Cell cycle was detected using flow cytometry. In detail, cells were washed twice with PBS, fixed with 70% ethanol for 24 h at 4°C, and incubated with 200μl PBS containing 1 μl potassium iodide (PI) (BD, San Jose, CA, USA) and 1 μl RNaseA (Sigma-Aldrich, USA) at 37°C for 30 min. Then, cells were detected with a FACS flow cytometer (BD Biosciences, CA, USA), and the results were analyzed using FlowJo software.


## 2.11. Migration and Invasion

We followed the methods of Zhang et al. [18]. Cell migration and invasion were explored using Transwell Chambers (8 μm pore size, Corning, Beijing, China). Referring to invasion experiment, the membranes were covered with matrigel (BD Biosciences). In the upper chamber, cells were cultured in serum-free medium, and in the lower chamber, mediums supplemented with 10% FBS were added. After culturing at 37°C for 24 h for the migration assay and 48 h for the invasion assay, respectively, cells were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet. The mean numbers of cells were calculated from at least six randomly chosen microscopic images.


## 2.12. Colony Formation Assay

A total of2×103 cells/mL was seeded into a six-well plate. All cells were incubated at 37°C for 9–12 days when the number of colonies showed more than 50. The cells were fixed with formalin for 30 minutes and stained with 1% crystal violet. Colony formation number was subsequently calculated.


## 2.13. Animal Experimental Procedures and Tumor Xenograft Experiment

We followed the methods of Zhang et al. [18]. Animal experiments were approved by the Committee for Animal Protection and Utilization of Southwest University. All experiments were conducted according to the Guidelines for Animal Health and Use (Ministry of Science and Technology, China, 2006). Four-week-old male nude mice were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China) and were placed in SPF rooms for feeding and observation. Human A375 cells (1×106 cells) stably transfected with shGFP, shZC3H15, shZC3H15/GFP, or shZC3H15/ZC3H15 were injected into the right dorsal side of five-week-old female nude mice (n=3). The mice were sterilized with 75% medical alcohol after subcutaneous injection. Then, tumor growth was measured by a Vernier caliper every three day, and the tumor volume was calculated by smalldiameter2×largediameter/2. Two weeks after injection, the mice were sacrificed, and the tumors were excised, weighed, photographed, and subjected to immunohistochemical staining.


## 2.14. Statistical Analysis

All experiments were performed at least three times. Values are presented as themean±standarddeviation (SD). Student’s t test was used to evaluate significant differences between two samples. P values < 0.05 were considered to indicate a statistically significant difference.


## 3. Results

### 3.1. ZC3H15 Is Upregulated in Human Melanoma and Is a Prognostic Indicator for Melanoma Patients

To explore whether ZC3H15 could be a prognosis marker for melanoma, IHC was performed to detect the expression of ZC3H15. The results showed in melanoma tissues, the ZC3H15 expression was significantly higher compared with that observed in the benign nevus tissues (Figures1(a) and 1(b)). To further determine whether the ZC3H15 expression level is associated with the clinical prognosis of melanoma patient, we evaluated the prognostic value of ZC3H15 in the Tumor Melanoma-Jonsson-214 database from the R2 platform (genomics analysis and visualization platform). The data indicates that high expression of ZC3H15 is strongly associated with poor overall survival in melanoma patients (Figure 1(c)). Then, in order to further detect the role of ZC3H15 in melanoma cells, qRT-PCR and western blot were performed to detect ZC3H15 expression at the mRNA and protein level in melanoma cell lines A375, MV3, and Skmel28, as well as human immortalized melanocyte cell line PIG1. As shown in Figures 1(d) and 1(e), ZC3H15 expression was significantly increased in A375 and MV3 cell lines at both mRNA and protein levels. Taken together, ZC3H15 is upregulated in melanoma cell lines and tissues and is associated with poor prognosis of melanoma patients.Figure 1
ZC3H15 is upregulated in human melanoma and is a prognostic indicator for melanoma patients. (a) Representative immunohistochemical staining assays of ZC3H15 expression in human melanoma (right) and benign tissue (left). (b) Immunohistochemistry analyses of ZC3H15 expression levels in 8 benign tissue samples and 15 melanoma samples. (c) Kaplan-Meier analysis of progression-free survival using data from the Tumor Glioma-kawaguchi-50 database and Tumor Glioma-French-284 database with the log-rank testP values indicated. (d, e) Quantitative PCR assays and western blot assays were performed to detect the expression of ZC3H15 in melanoma cell lines A375, MV3, and Skmel28, as well as human melanocyte cell line PIG1. The data are represented as the mean±SD; ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)


### 3.2. ZC3H15 Promotes Cell Proliferation in Melanoma Cells

In order to evaluate the effect of ZC3H15 on the proliferation of melanoma cells, three independent short hairpin RNAs (shRNAs) against ZC3H15 were utilized to knock down expression of ZC3H15 in A375 and MV3 cells, and shGFP was used as a control. Based on the results of qRT-PCR and western blot (Figure2(a)), we choose shZC3H15#1 which showed stronger knockdown efficiency in subsequent experiments. Next, we examined the proliferation abilities of MV3 and A375 using an MTT assay, and the results revealed that knocking down ZC3H15 significantly inhibited the growth of melanoma cells (Figure 2(b)). Additionally, BrdU incorporation experiments showed that ZC3H15-knockdown led to a significant reduction in DNA synthesis compared with that of the control cells (Figure 2(c)). To further validate ZC3H15 is helpful in proliferation of melanoma cells, we recovered ZC3H15 through transfection of a full-length CSN6 sequence resistant to shRNA#1 targeting into ZC3H15-knockdown melanoma cell, and the efficiency was confirmed by western blot and RT-PCR (Figure 2(d)). MTT assay revealed the cell viability was obviously increased following recovery of ZC3H15 expression in MV3 and A375 cells (Figure 2(e)). Consistently, BrdU assay indicated the DNA synthesis was also significantly increased after restoration of ZC3H15 in ZC3H15-knockdown cells (Figure 2(f)). These results indicated ZC3H15 is positively associated with the proliferation and viability of MV3 and A375 cells.Figure 2
ZC3H15 promotes cell proliferation in melanoma cells. (a) Western blot analyses of ZC3H15 in cells with ZC3H15-knockdown. (b) MTT assay was performed to examine the viability of ZC3H15- knockdown melanoma cells. (c) BrdU incorporation assays were performed to detect the amount of DNA synthesis in ZC3H15-knockdown melanoma cells. (d) After restoration of ZC3H15 in ZC3H15-knockdown cells, the expression of ZC3H15 at protein and mRNA level was detected using quantitative PCR assays and Western blot, respectively. (e) MTT assay was performed in ZC3H15-rescued cells. (f) BrdU was carried out after ZC3H15 was rescued in ZC3H15-knockdown cells. The data are represented as themean±SD; ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


### 3.3. ZC3H15 Is Involved in the G1-to-S Phase Transition of Melanoma Cell Cycle

The aforementioned results indicate silencing ZC3H15 inhibits cell proliferation in melanoma cells. The inhibition of cell proliferation can result from cell cycle arrest, so we undertake flow cytometry analysis to explore whether ZC3H15-knockdown caused cell cycle arrest in melanoma cells. The results displayed ZC3H15-knockdown induced cell cycle arrest at the G0/G1 phase (Figure3(a)) in A375 and MV3 cells. To further verify these results, the expression of G0/G1 phase-related proteins was analyzed by western blot. We found that in ZC3H15-knockdown cells, the key regulated proteins of G0/G1 phase were distinctly altered, with CDK4, CDK8, and CCND1 being downregulated, while p21 was upregulated (Figure 3(b)). In addition, we found the expression of these cell cycle-related proteins was altered in opposite trend when ZC3H15 was restored (Figure 3(c)). In summary, ZC3H15 is critical for the G1-to-S phase transition in the cell cycle of MV3 and A375 cells.Figure 3
ZC3H15 is involved in the G1-to-S phase transition of melanoma cell cycle. (a) The cell cycle of MV3 and A375 cells after ZC3H15 silencing was analyzed by flow cytometry, and the percentage of indicated cells in different phases was determined. (b, c) The expression of proteins related to G0/G1 phase including CDK4, CDK6, CCND1, and p21 was detected by Western blot in ZC3H15-knockdown and ZC3H15-rescued cells. The data are represented as themean±SD; ∗∗P<0.01.
(a)(b)(c)


### 3.4. ZC3H15 Promotes the Migration and Invasion of Melanoma Cells

In order to investigate whether ZC3H15 is associated with migration and invasion in melanoma cells, a transwell assay was performed. The results showed that ZC3H15-knockdown melanoma cells migrated slower than cells of the control group (Figure4(a)). Matrix gel was then added to detect invasion, and we found a similar results as migration assay (Figure 4(b)). In addition, we performed western blot analysis to detect the expression of proteins which are essential for migration and invasion. The results indicated N-cadherin and vimentin were obviously decreased, while E-cadherin was significantly increased in ZC3H15-knockdown MV3 and A375 cells (Figure 4(c)). To further verify the effect of ZC3H15 on migration and invasion, we restored the expression of ZC3H15 in ZC3H15-knockdown melanoma cells. As expected, restoration of ZC3H15 expression rescued migratory and invasive abilities in ZC3H15-knockdown cells (Figures 4(d)–4(f)). Collectively, ZC3H15 plays a positive role in the migration and invasion of melanoma cells.Figure 4
ZC3H15 promotes the migration and invasion of melanoma cells. (a, b) Migration and invasion assays were performed in ZC3H15-knockdown A375 and cells. (c) The expression of metastasis-related proteins was explored using Western blot. (d, e) Migration and invasion assays were performed after overexpression of ZC3H15 in ZC3H15-knockdown cells. (f) Western blot was conducted to evaluate the protein expression in ZC3H15-rescued cells. The data are represented as themean±SD; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


### 3.5. ZC3H15 Is Required for Colony Formation and Tumorigenesis of Melanoma Cells

To further confirm the effects of ZC3H15 expression on self-renewal of melanoma cells in vitro, we performed colony formation assay and demonstrated colonies were significantly fewer and smaller in the ZC3H15-knockdown group than in the control group, and the colony number can be rescued by overexpression of ZC3H15 (Figure5(a)). Next, to evaluate the role of ZC3H15 in tumorigenesis of melanoma cells in vivo, subcutaneous xenograft experiments using ZC3H15-knockdown A375 cells and control cells were carried out with nude mice. Consistent with the in vitro results, tumor formation was significantly slower and tumors were obviously smaller in the ZC3H15-knockdown group (Figures 5(b) and 5(c)). Immunohistochemical staining showed that the percentage of ZC3H15-positive cells was dramatically decreased in the ZC3H15-knockdown tumor samples. In addition, the expression levels of Ki-67, a well-known cell proliferation marker, were also substantially reduced in the shZC3H15 tumor samples (Figure 5(d)). When expression of ZC3H15 was restored, the growth rate, volume and weight of tumors, and the expression of Ki67 were all partially rescued (Figures 5(b)–5(d)). Taken together, ZC3H15 was indispensable for the cloning and tumorigenesis of melanoma cells.Figure 5
ZC3H15 is required for colony formation and tumorigenesis of melanoma cells. (a) Colony formation assay was carried out in A375 and MV3 cells after ZC3H15-knockdown and ZC3H15 restoration. (b) The growth curve of xenograft tumors was analyzed, andP value is indicated. (c) The size and weight of xenograft tumors were analyzed. (d) Immunohistochemical staining was performed to detect the expression of ZC3H15 and Ki67 in ZC3H15-knockdown and ZC3H15 restoration tumor tissues. The data are represented as the mean±SD; ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)


## 3.1. ZC3H15 Is Upregulated in Human Melanoma and Is a Prognostic Indicator for Melanoma Patients

To explore whether ZC3H15 could be a prognosis marker for melanoma, IHC was performed to detect the expression of ZC3H15. The results showed in melanoma tissues, the ZC3H15 expression was significantly higher compared with that observed in the benign nevus tissues (Figures1(a) and 1(b)). To further determine whether the ZC3H15 expression level is associated with the clinical prognosis of melanoma patient, we evaluated the prognostic value of ZC3H15 in the Tumor Melanoma-Jonsson-214 database from the R2 platform (genomics analysis and visualization platform). The data indicates that high expression of ZC3H15 is strongly associated with poor overall survival in melanoma patients (Figure 1(c)). Then, in order to further detect the role of ZC3H15 in melanoma cells, qRT-PCR and western blot were performed to detect ZC3H15 expression at the mRNA and protein level in melanoma cell lines A375, MV3, and Skmel28, as well as human immortalized melanocyte cell line PIG1. As shown in Figures 1(d) and 1(e), ZC3H15 expression was significantly increased in A375 and MV3 cell lines at both mRNA and protein levels. Taken together, ZC3H15 is upregulated in melanoma cell lines and tissues and is associated with poor prognosis of melanoma patients.Figure 1
ZC3H15 is upregulated in human melanoma and is a prognostic indicator for melanoma patients. (a) Representative immunohistochemical staining assays of ZC3H15 expression in human melanoma (right) and benign tissue (left). (b) Immunohistochemistry analyses of ZC3H15 expression levels in 8 benign tissue samples and 15 melanoma samples. (c) Kaplan-Meier analysis of progression-free survival using data from the Tumor Glioma-kawaguchi-50 database and Tumor Glioma-French-284 database with the log-rank testP values indicated. (d, e) Quantitative PCR assays and western blot assays were performed to detect the expression of ZC3H15 in melanoma cell lines A375, MV3, and Skmel28, as well as human melanocyte cell line PIG1. The data are represented as the mean±SD; ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)


## 3.2. ZC3H15 Promotes Cell Proliferation in Melanoma Cells

In order to evaluate the effect of ZC3H15 on the proliferation of melanoma cells, three independent short hairpin RNAs (shRNAs) against ZC3H15 were utilized to knock down expression of ZC3H15 in A375 and MV3 cells, and shGFP was used as a control. Based on the results of qRT-PCR and western blot (Figure2(a)), we choose shZC3H15#1 which showed stronger knockdown efficiency in subsequent experiments. Next, we examined the proliferation abilities of MV3 and A375 using an MTT assay, and the results revealed that knocking down ZC3H15 significantly inhibited the growth of melanoma cells (Figure 2(b)). Additionally, BrdU incorporation experiments showed that ZC3H15-knockdown led to a significant reduction in DNA synthesis compared with that of the control cells (Figure 2(c)). To further validate ZC3H15 is helpful in proliferation of melanoma cells, we recovered ZC3H15 through transfection of a full-length CSN6 sequence resistant to shRNA#1 targeting into ZC3H15-knockdown melanoma cell, and the efficiency was confirmed by western blot and RT-PCR (Figure 2(d)). MTT assay revealed the cell viability was obviously increased following recovery of ZC3H15 expression in MV3 and A375 cells (Figure 2(e)). Consistently, BrdU assay indicated the DNA synthesis was also significantly increased after restoration of ZC3H15 in ZC3H15-knockdown cells (Figure 2(f)). These results indicated ZC3H15 is positively associated with the proliferation and viability of MV3 and A375 cells.Figure 2
ZC3H15 promotes cell proliferation in melanoma cells. (a) Western blot analyses of ZC3H15 in cells with ZC3H15-knockdown. (b) MTT assay was performed to examine the viability of ZC3H15- knockdown melanoma cells. (c) BrdU incorporation assays were performed to detect the amount of DNA synthesis in ZC3H15-knockdown melanoma cells. (d) After restoration of ZC3H15 in ZC3H15-knockdown cells, the expression of ZC3H15 at protein and mRNA level was detected using quantitative PCR assays and Western blot, respectively. (e) MTT assay was performed in ZC3H15-rescued cells. (f) BrdU was carried out after ZC3H15 was rescued in ZC3H15-knockdown cells. The data are represented as themean±SD; ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


## 3.3. ZC3H15 Is Involved in the G1-to-S Phase Transition of Melanoma Cell Cycle

The aforementioned results indicate silencing ZC3H15 inhibits cell proliferation in melanoma cells. The inhibition of cell proliferation can result from cell cycle arrest, so we undertake flow cytometry analysis to explore whether ZC3H15-knockdown caused cell cycle arrest in melanoma cells. The results displayed ZC3H15-knockdown induced cell cycle arrest at the G0/G1 phase (Figure3(a)) in A375 and MV3 cells. To further verify these results, the expression of G0/G1 phase-related proteins was analyzed by western blot. We found that in ZC3H15-knockdown cells, the key regulated proteins of G0/G1 phase were distinctly altered, with CDK4, CDK8, and CCND1 being downregulated, while p21 was upregulated (Figure 3(b)). In addition, we found the expression of these cell cycle-related proteins was altered in opposite trend when ZC3H15 was restored (Figure 3(c)). In summary, ZC3H15 is critical for the G1-to-S phase transition in the cell cycle of MV3 and A375 cells.Figure 3
ZC3H15 is involved in the G1-to-S phase transition of melanoma cell cycle. (a) The cell cycle of MV3 and A375 cells after ZC3H15 silencing was analyzed by flow cytometry, and the percentage of indicated cells in different phases was determined. (b, c) The expression of proteins related to G0/G1 phase including CDK4, CDK6, CCND1, and p21 was detected by Western blot in ZC3H15-knockdown and ZC3H15-rescued cells. The data are represented as themean±SD; ∗∗P<0.01.
(a)(b)(c)


## 3.4. ZC3H15 Promotes the Migration and Invasion of Melanoma Cells

In order to investigate whether ZC3H15 is associated with migration and invasion in melanoma cells, a transwell assay was performed. The results showed that ZC3H15-knockdown melanoma cells migrated slower than cells of the control group (Figure4(a)). Matrix gel was then added to detect invasion, and we found a similar results as migration assay (Figure 4(b)). In addition, we performed western blot analysis to detect the expression of proteins which are essential for migration and invasion. The results indicated N-cadherin and vimentin were obviously decreased, while E-cadherin was significantly increased in ZC3H15-knockdown MV3 and A375 cells (Figure 4(c)). To further verify the effect of ZC3H15 on migration and invasion, we restored the expression of ZC3H15 in ZC3H15-knockdown melanoma cells. As expected, restoration of ZC3H15 expression rescued migratory and invasive abilities in ZC3H15-knockdown cells (Figures 4(d)–4(f)). Collectively, ZC3H15 plays a positive role in the migration and invasion of melanoma cells.Figure 4
ZC3H15 promotes the migration and invasion of melanoma cells. (a, b) Migration and invasion assays were performed in ZC3H15-knockdown A375 and cells. (c) The expression of metastasis-related proteins was explored using Western blot. (d, e) Migration and invasion assays were performed after overexpression of ZC3H15 in ZC3H15-knockdown cells. (f) Western blot was conducted to evaluate the protein expression in ZC3H15-rescued cells. The data are represented as themean±SD; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


## 3.5. ZC3H15 Is Required for Colony Formation and Tumorigenesis of Melanoma Cells

To further confirm the effects of ZC3H15 expression on self-renewal of melanoma cells in vitro, we performed colony formation assay and demonstrated colonies were significantly fewer and smaller in the ZC3H15-knockdown group than in the control group, and the colony number can be rescued by overexpression of ZC3H15 (Figure5(a)). Next, to evaluate the role of ZC3H15 in tumorigenesis of melanoma cells in vivo, subcutaneous xenograft experiments using ZC3H15-knockdown A375 cells and control cells were carried out with nude mice. Consistent with the in vitro results, tumor formation was significantly slower and tumors were obviously smaller in the ZC3H15-knockdown group (Figures 5(b) and 5(c)). Immunohistochemical staining showed that the percentage of ZC3H15-positive cells was dramatically decreased in the ZC3H15-knockdown tumor samples. In addition, the expression levels of Ki-67, a well-known cell proliferation marker, were also substantially reduced in the shZC3H15 tumor samples (Figure 5(d)). When expression of ZC3H15 was restored, the growth rate, volume and weight of tumors, and the expression of Ki67 were all partially rescued (Figures 5(b)–5(d)). Taken together, ZC3H15 was indispensable for the cloning and tumorigenesis of melanoma cells.Figure 5
ZC3H15 is required for colony formation and tumorigenesis of melanoma cells. (a) Colony formation assay was carried out in A375 and MV3 cells after ZC3H15-knockdown and ZC3H15 restoration. (b) The growth curve of xenograft tumors was analyzed, andP value is indicated. (c) The size and weight of xenograft tumors were analyzed. (d) Immunohistochemical staining was performed to detect the expression of ZC3H15 and Ki67 in ZC3H15-knockdown and ZC3H15 restoration tumor tissues. The data are represented as the mean±SD; ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)


## 4. Discussion

MM, as one of the most aggressive and deadliest forms of skin cancers worldwide [4], has an increasing incidence rate year by year [19]. While it is usually curable by surgical excision when detected at early stage, the outcome of advanced/metastatic melanoma is poor [5, 8]. Over the past decades, we have achieved significant improvement in the therapy of patients with unresectable or metastatic melanoma, primarily due to the advent of molecular targeted therapy, immunotherapy, and the incorporation of palliative care services into the management scheme [2, 20]. As we know, the molecular targeted therapy mainly focused on BRAF and MEK signaling pathways, and immune checkpoint blockade strategies targeted the PD-1 and CTLA-4 [2]. However, the treatment has met new challenges because of resistance to these inhibitors, which push the need to explore novel effective therapies [8, 21, 22]. Therefore, discovering the molecular mechanism involved in melanoma progression is of paramount importance for developing alternative treatment options for this devastating disease.ZC3H15, also termed LEREPO4 or DFRP1 [11–13], is a protein that shows ubiquitous expression among various normal human tissues and diffuse cytosolic localization [16]. It was shown that DFRP1 (ZC3H15) can bind to DRG1 specifically and upregulate its expression by inhibiting its polyubiquitination [11] and promoting its thermal stability [15]. DRG1 is highly conserved in almost all tissues [23] and plays a role in regulating cell growth [24, 25]. A growing body of studies have demonstrated that ectopic expression of DRG1 is related to the occurrence and development of cancers and has contradictory effects in diverse type of cancers. While DRG1 acts as a cancer-promoting effector in lung adenocarcinoma, melanoma, hepatocellular carcinoma, and cervical adenocarcinoma [26–29], its lower expression was associated with poor survival in breast cancer and colorectal cancer [30, 31]. With regard to ZC3H15, previous studies about its role in tumorigenesis are limited. In the present study, we investigate the relationship between ZC3H15 and MM.In the present study, we demonstrated ZC3H15 served as a tumor promoter in melanoma. The expression of ZC3H15 was noticeably elevated in tumor tissue, compared with the benign tissues, indicating ZC3H15 may be regarded as a biomarker in diagnosis of melanoma. Similar results were also found in melanoma cell lines using western blot and RT-PCR when compared with normal human melanocytes. Moreover, melanoma patients with a high level of ZC3H15 experienced a poorer prognosis and a lower survival rate, suggesting ZC3H15 may be utilized as a prognostic marker in melanoma. Consistent with our results, expression of ZC3H15 is found unregulated in acute myeloid leukemia [16] and hepatocellular carcinoma [17].Meanwhile, we studied the biological function of ZC3H15 at the cellular level. Imbalance between cell proliferation and cell death (apoptosis) leads to tumorigenesis [32]. MTT and BrdU assay indicated suppression of ZC3H15 in MV3 and A375 melanoma cells markedly inhibited cell proliferation, which was similar to others’ results in hepatocellular carcinoma [17]. The cell cycle progresses sequentially through G0/G1, S, G2, and M phases for normal cell proliferation. In this study, silencing the expression of ZC3H15 decreased the percentage of cells in S phase but increased the percentage of cells in G0/G1 phase. Correspondingly, we found the expression levels of biomarkers of the G1-to-S phase transition, including CDK4, CDK5, and CCND1, were all positively correlated to ZC3H15 expression. To our knowledge, our study is the first to confirm ZC3H15 has an effect on cell cycle and related protein expression. p21, a well-established CDK inhibitor, was found to play an important role in controlling cell cycle progression [32, 33]. Recently, despite its tumor-suppressor function, oncogenic/antiapoptotic function of p21 has been under scrutiny [32, 34]. In this study, the expression of p21 was upregulated by downregulation of ZC3H15, implying p21 maybe plays an antitumor role here. However, the precise mechanism of ZC3H15 regulating p21 is not explored in this study and we will focus on this point in a further study.It is widely known that the poor prognosis and increasing mortality of melanoma are mainly attributed to metastasis, which can occur even in patients with thin small primary MM [35]. To our knowledge, no studies have investigated the relationship between ZC3H15 and metastasis of tumor. E-cadherin, N-cadherin, and vimentin are key biomarkers of epithelial-mesenchymal transition (EMT) [36], which is a process promoting cancer cells to obtain metastatic properties [37]. Importantly, we found silencing of ZC3H15 successfully inhibited the migration or invasion of melanoma cells and promoted the expression of E-cadherin, while attenuating the expression of N-cadherin and vimentin. These indicate that ZC3H15 might promote metastasis of melanoma cells via enhancing EMT.Anchorage-independent growth represents the tumorigenic ability and metastatic potential of tumor cells in vivo [27]. By the use of colony formation assay, we found ZC3H15 promoted anchorage-independent growth of melanoma cells in vitro. At the meanwhile, we found ZC3H15 interference significantly weakened the growth of xenograft tumors in vivo, which further highlighted the important role of ZC3H15 in MM progression. However, the deeper mechanism underlying the oncogenic role of ZC3H15 is not explored in this study. According to previous studies, ZC3H15 can interact with TRAF2 functionally within the NF-κB pathway [16, 17]. With the constant-depth study, we will demonstrate more experimental values about ZC3H15.


## 5. Conclusions

Collectively, all the above mentioned results indicate ZC3H15 promote proliferation, migration and invasion of melanoma cells, and it may represent a promising biomarker and therapeutic target for melanoma. Further research is needed to uncover the mechanism underlying the action of ZC3H15 in MM.



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