MiR-498 targets UBE2T to Inhibit Proliferation and Metastasis in Malignant Melanoma


 BackgroundMalignant melanoma (MM) is a common malignant tumor and also one of the fastest growing malignancies. Considering that UBE2T could be a direct target of miR-498, we explore the ability of miR-498 to regulate MM cell progression by targeting UBE2T.MethodsWe detected the expression of miR-498 by quantitative real-time PCR assay. The cell proliferation, invasion and migration in MM cells were measured by cell counting kit-8, clone formation and transwell assays. Flow cytometry assay was used to detect the percentage of apoptotic cells. We explored the correction of miR-498 and UBE2T by dual-luciferase assays.ResultsDuring the study, we found that the overexpression of miR-498 inhibited proliferation, invasion, migration and induced cell apoptosis of M14 and A375 cells. In addition, the expression of epithelial-mesenchymal transition (EMT)-related factors was altered by overexpression of miR-498. Furthermore, UBE2T was differentially expressed in MM and normal tissues and high expression of UBE2T was strongly correlated with poor overall survival in patients with MM. We highlighted that overexpression of UBE2T reversed the inhibitory effects of miR-498 on MM cell progression.ConclusionsAltogether, our findings demonstrated that miR-498 significantly inhibited cell proliferation, invasion, migration and induced apoptosis, whilst confirming that miR-498 regulated MM cell progression by targeting UBE2T.


Introduction
Malignant Melanoma (MM) is a common malignant tumor of the skin, mucous membranes and pigmented membranes. It is also one of the fastest growing malignancies with an annual growth rate of 3-5%(1). Therefore, MM has become a serious threat to the health of our people (2). At present, systemic chemotherapy is still used to treat metastatic MM in clinical practice (3). Despite some effects, the 10-year survival rate of patients is still less than 10% (4). Although the research on the pathogenesis and related signal pathway of MM has made remarkable progress (5), the precise mechanism of the occurrence and development of MM is still not completely clear up to now.
MicroRNA is a short-chain uncoded RNA(6). It mainly roles in regulating the expression of target gene mRNA, which is closely related to the occurrence and development of various diseases. miR-498 is located at 19q13.41 (7). Recently, growing evidence showed that miR-498 plays an important role in the onset of diseases, including ovarian cancer, breast cancer, lung cancer, and colorectal cancer (7)(8)(9).
The ubiquitin-conjugating enzyme E2T (UBE2T) is located on human chromosome 1q32.1 and encodes a total of 197 amino acids. Many studies have con rmed that UBE2T can mediate the ubiquitination of proteins such as FANCD2 and FANCI, and then participate in the repair of DNA damage (14). It has been reported in recent years that UBE2T can be involved in the occurrence and development of many tumors, including breast cancer, lung cancer, prostate cancer, and bladder cancer (14)(15)(16).
In this study, we analyzed the function of miR-498 in MM cells and found that overexpression of miR-498 inhibited MM cells growth, invasion, migration, and induced cell apoptosis. Additionally, the effects of miR-30b as tumor suppressor on MM cells could be attenuated when UBE2T was reintroduced. Our ndings suggested the potential utility of miR-498 for the treatment of MM.

Cell lines and cell treatment
Human malignant melanoma cell lines HEM, A375, M21, M14, and uacc62 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). miR-498 cDNA sequences were inserted into pcDNA3.1 plasmid to generate the pcDNA3-miR-498 plasmid then transfected into M14 and A375 Cell counting kit-8 assay Brie y, 1×10 3 cells were seeded into per well of 96-well plates. After every 24 h, 10 µl CCK8 solution (Solarbio Science & Technology, Beijing, China) was added to detect cell proliferation. After 2 h of incubation, enzyme standard instrument was used to measure OD value at 450 nm to drawn proliferation curve. This assay was performed in triplicate.
Colony formation assay About 3×10 2 transfected cells were planted into a 6 cm dish containing 5 ml medium and maintained at 37°C in 5% CO 2 . When cells formed visible clones, 4% paraformaldehyde solution and 0.1% crystal violet were used to x and stain clones, respectively. Finally, the clones were taken a image and counted. The assay was performed three times.

Transwell assay
The invasion and migration of M14 and A375 cells was determined using matrigel-coated transwell chambers (BD Biosciences). Brie y, 2×10 4 cells with serum-free DMEM medium were transferred to the upper chamber while complete medium was added to the lower chamber. After 24 h of incubation, the invasive and migrated cells were xed with 4 % paraformaldehyde and then stained with 0.1 % crystal violet. Finally, the cells were photographed and counted under a microscope. The migration experiment procedure was similar to the invasion assay, but the cubicles were coated with uncoated lters. Transwell assay included triplicate measurements.

Apoptosis detecting assay
Apoptosis of cells was determined using Annexin V-FITC Apoptosis Detection kit I (Thermo Fisher Scienti c, Shanghai, China) according to the manufacturer's instructions. Brie y, after 48 h of incubation, the cells were collected and mixed with 10 µl Annexin V-FITC and 5µl propidium lodide (PI) at room temperature in the dark for 5 min. Apoptosis of cells were meansured using ow cytomertry. This assay was performed three times.

Western blot assay
After 48 h of incubation, cells were centrifuged at 4°C and the protein was extracted with RIPA lysis buffer. Subsequently, 50 µg proteins were separated using SDS-PAGE and then transferred to PVDF membrane. Then, the membrane and antibody to Bcl-2, Bax, cleaved caspase3, E-cad, N-cad, Vimentin, UBE2T, GAPDH were incubated overnight. Finally, the membrane was incubated with HRP-conjugated secondary antibodies. Each experiment included triplicate measurements.

Dual-luciferase assay
The pmirGLO-UBE2T-3'UTR wild type (wt) or mutant (mut) reporter plasmid were co-transfected into cells using Lipofectamine 2000. After 48 h of transfection, cells were lysed and luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA). Renilla luciferase activity was used for standardization. Each experiment was performed three times.

Statistical analysis
Data was obtained from three independent experiments and analyzed using SPSS 18.0. The comparison of two groups and multiple groups was analyzed using Student's t-test and ANOVA test, respectively. Differences with P values of less than 0.05 was considered statistically signi cant.
miR-498 expression levels in four human MM cell lines (A375, M21, M14, and uacc62) and primary HEM cells were analyzed by qRT-PCR assay. As shown in Fig. 1A, miR-498 expression was signi cantly downregulated in MM cell lines compared to normal HEM cells (P < 0.05). We further explored the function of miR-498 in M14 and A375 cells. As shown in Fig. 1B, miR-498 mRNA level was signi cantly increased by miR-498 mimics in M14 and A375 cells (P < 0.05). Moreover, the results in Fig. 1C and 1D showed that the OD value of M14 and A375 cells infected with miR-498 was signi cantly decreased (P < 0.05). Clone formation assay also showed similar results in Fig. 1E that the clonogenic abilities of M14 and A375 cells transfected with miR-498 was signi cantly decreased compared with NC (P < 0.05).

miR-498 inhibits MM cell invasion and migration and induces cell apoptosis
The effect of miR-498 on MM cell invasion and migration was detected using transwell assay. As shown in Fig. 2A, the number of invaded and migrated M14 cells in miR-498 group was signi cantly decreased compared to those in NC group (P < 0.05). Then as shown in Fig. 2B, transwell assay also showed similar results that the number of invaded and migrated A375 cells in miR-498 group was signi cantly declined in comparison to those in NC group (P < 0.05). The apoptosis of M14 and A375 cells transfected with miR-498 were detected by ow cytometry assay. As shown in Fig. 2C, the percentage of apoptotic cells was signi cantly increased from 14.38% or 10.28-25.00% or 19.03% in M14 and A375 cells respectively in miR-498 group compared to NC (P < 0.05). Western blot was further used to detect cell apoptosis. As shown in Fig. 3A, the expression of anti-apoptotic protein Bcl2 in miR-498 group was strongly decreased compared with the NC group, while the expression of pro-apoptotic proteins Bax, cleaved caspase3 was signi cantly increased (P < 0.05).

miR-498 regulates epithelial-mesenchymal transition (EMT) of MM cells
The expression of EMT-related proteins was detected using western blot. We observed signi cant upregulation of E-cad in miR-498 group in M14 and A375 cells in comparison to that in NC group (P < 0.05), while N-cad and Vimentin were signi cantly down-regulated in miR-498 group (Fig. 3B), which suggested that miR-498 regulated EMT process in MM cells.
Higher level of UBE2T is associated with poor prognosis in MM cells TCGA is initiated by the National Cancer Institute (NCI) and the National Human Genome Institute (NHGRI), and has now generated genomic data sets for 33 cancers. Based on the TCGA database, we analyzed and screened the poor prognosis genes for MM, and found that UBE2T was a new poor prognosis index for patients with MM. As shown in Fig. 4A, UBE2T expression was markedly higher in SKCM compared with normal samples (P < 0.05). The result showed in Fig. 4B that the survival rate of SKCM patients with high expression of UBE2T was signi cantly lower compared to those with low expression of UBE2T (P < 0.05). It suggested that UBE2T might play a role in promoting MM cell progression.
UBE2T is a novel target gene of miR-498 and overexpression of UBE2T reverses the inhibitory effects of miR-498 on MM cell progression Based on TargetScan, we predicted UBE2T could be a direct target of miR-498 (Fig. 5A). In order to further determine whether UBE2T was a direct target of miR-498, we performed dual-luciferase assay. As shown in Fig. 5B, miR-498 signi cantly inhibited the luciferase activity of reporter genes wild-type of 3'-UTR of UBE2T (P < 0.05). Then western blot results in Fig. 5C con rmed that UBE2T expression was signi cantly decreased in M14 and A375 cells transfected with miR-498 (P < 0.05). Subsequently, cell counting kit-8 and transwell assays were used to identify whether miR-498 regulated proliferation and migration of MM cells by targeting UBE2T. The result showed in Fig. 5D that UBE2T reversed the effect of miR-498 on inhibiting tumor growth of M14 and A375 cells (P < 0.05). As shown in Fig. 5E, UBE2T reversed the effect of miR-498 on inhibiting tumor migration of M14 and A375 cells (P < 0.05). Together, these results indicated that miR-498 could inhibit tumor cell growth, invasion, and migration by directly targeting UBE2T in human MM.

Discussion
EMT is an important factor leading to an increase in the malignancy of tumors (17,18). When EMT occurs, the expression of epithelial-derived markers E-cadherin and α1-catenin is reduced, and the expression of mesenchymal-derived markers N-cadherin, bronectin and vimentin is increased (19). Previous studies have shown that when EMT occurs in tumor cells, due to changes in the expression of cytoskeletal proteins and decreased expression of interstitial adhesion proteins such as E-cadherin, the morphology of tumor cells will change, and the ability of cell migration and invasion will increase (20). Here, we found that the overexpression of miR-498 signi cantly inhibited the activation of EMT including upstream protein E-cad and downsream proteins N-cad and Vimentin, and inhibited cell proliferation, invastion and migration in M14 and A375 cells.
miR-498 is a tumor suppressor miRNA, which can regulate a variety of tumor suppressor genes or proteins related to the regulation of tumor growth, invasion and metastasis, thereby regulating the occurrence and development of various tumors (7,21,22). As shown in previous study, miR-498 expression is reduced in colon cancer tissues, and miR-498 can inhibit the proliferation of colon cancer cells (9). It has been reported that miR-498 can inhibit the proliferation and migration of triple-negative breast cancer cells by down-regulating the BRCA1 gene (7). Consistent with the above ndings, we used M14 and A375 cells transfected with miR-498 mimics and found that miR-498 inhibited the proliferation, invastion and migration of MM cells, suggesting that miR-498 was a tumor suppressor miRNA in MM.
Previous studies have found that UBE2T is highly expressed in multiple tumors tissues, and its expression level is signi cantly related to patient survival (23). In breast cancer, UBE2T can not only be used as a molecular marker to predict the prognosis of breast cancer patients, but also promote the occurrence and development of tumors by participating in the ubiquitination degradation of breast cancer susceptibility protein 1. In gastric cancer, interfering with UBE2T expression not only causes gastric cancer cell cycle arrest and apoptosis, thereby inhibiting the growth of tumor cells, but also inhibits the migration and invasion of tumor cells through EMT. Clinically, high expression of UBE2T often indicates a poor prognosis in patients with gastric cancer. In hepatocellular carcinoma (HCC), previous studies analyze TCGA and Oncomine database data and nd that the expression level of UBE2T in HCC tissues is signi cantly higher than that in adjacent tissues. The high expression of UBE2T in HCC is related to intrahepatic metastasis of the tumor, and can promote the migration and invasion of HCC cells (24). In this study, we found that UBE2T mRNA expression in SKCM tissues was signi cantly higher than in normal tissues by searching on GEPIA. We explored the relationship of miR-498 and UBE2T and rst found that overexpression of UBE2T reversed the inhibitory effects of miR-498 on MM cell progression.

Conclusion
In summary, the results of our study indicate that miR-498 can inhibit the EMT of M14 and A375 cells by targeting UBE2T, providing new ideas for the treatment of MM.

Consent for publication
Not applicable.

Data statement
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Con ict of Interests Statement
The authors declare that they have no competing interests, and all authors should con rm its accuracy.
Authors' contributions ND performed the experiments, analyzed the data and wrote the paper. WC and PL assisted with the experiments and data analysis. SH helped analyzed the data and modify the paper. All authors have edited and approved the nal manuscript.