Mechanism of MicroRNA-708 Targeting BAMBI in Cell Proliferation, Migration, and Apoptosis in Mice With Melanoma via the Wnt and TGF-β Signaling Pathways.

OBJECTIVE: The aim of this study was to evaluate the mechanisms involved with miRNA-708 and its targeting of bone morphogenetic protein and activin membrane-bound inhibitor in cell proliferation, migration, and apoptosis in mice with melanoma via the Wnt and transforming growth factor β signaling pathways.
METHODS: Sixty mice were recruited of which 40 were subsequently assigned into the experimental group (22 mice were successfully established as melanoma model and 18 mice used in tumor xenograft), and the normal control group consisted of 20 mice. B16 cells were assigned to the normal, blank, and negative control, miR-708 mimics, miR-708 inhibitors, si-BAMBI, and miR-708 inhibitors + si-bone morphogenetic protein and activin membrane-bound inhibitor groups. Western blotting and reverse transcription quantitative polymerase chain reaction were employed to detect the expression levels within the tissues and cell lines. TCF luciferase reporter (TOP-FLASH) or a control vector (FOP-FLASH) was applied to detect the activity of the Wnt signaling pathway. MTT3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay, flow cytometry, scratch test, and Transwell assay were conducted, respectively, for cell proliferation, apoptosis, migration, and invasion, while tumor xenograft procedures were performed on the nude mice recruited for the study.
RESULTS: Compared to the normal control group, the model group displayed increased expressions of bone morphogenetic protein and activin membrane-bound inhibitor, Wnt10B, P53, and Bcl-2; TOPflash activity; β-catenin expression; cell proliferation; migration; and invasion capabilities while decreased expressions of miR-708, vascular endothelial growth factor, Fas, Bax, Caspase-3, and cleaved Caspase-3 and apoptosis rate. Compared to the blank and negative control groups, the miR-708 mimics and small-interfering RNA-bone morphogenetic protein and activin membrane-bound inhibitor groups exhibited decreases expressions of bone morphogenetic protein and activin membrane-bound inhibitor, Wnt10B, P53, and Bcl-2 and decreased proliferation, migration, and invasion capabilities, while increases in the apoptosis rate, expressions of vascular endothelial growth factor, Fas, Bax, Caspase-3, and cleaved Caspase-3; however, downregulated levels of TOPflash activity and β-catenin expression were recorded. The miR-708 inhibitors group displayed an opposite trend.
CONCLUSION: Downregulation of miR-708-targeted bone morphogenetic protein and activin membrane-bound inhibitor inhibits the proliferation and migration of melanoma cells through the activation of the transforming growth factor β pathway and the suppression of Wnt pathway.

Introduction

Cutaneous melanoma, the most fatal form of human skin cancer at present, represents a worldwide public health dilemma.[1] Despite exhibiting a relatively steady increase in its prevalence, melanoma mortality rates have not displayed any increased in recent years for a number of reasons, including continuous incremental advances made in the early diagnostic modalities associated with the condition.[2] Melanoma is accompanied by poor prognoses with a median stage IV survival time ranging between 8 and 18 months postdiagnosis.[3] Melanoma is rarely observed among children, with children accounting for approximately 3% of all pediatric malignancies and <1% of all melanomas worldwide.[4] In cases of malignant melanoma, tumor-connected macrophages play many roles in connection to tumor growth, including its involvement in accelerating the transformation of melanocytes under ultraviolet irradiation, increasing melanoma angiogenesis, and acting to repress antitumor immunity.[5] The bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is regarded to be a transmembrane TGF-β/BMP I receptor-associated pseudoreceptor.[6] The BAMBI and Drosophila mothers against decapentaplegic protein 7 (Smad7) have previously been reported to negatively regulate transforming growth factor-β (TGF-β) signaling as well as playing crucial roles in the progression of various malignant tumors.[7] The BAMBI, in particular, can act to promote the migration, invasion, and proliferation of cancer cells as well as repress adiposeness.[8] Previous reports have indicated that BAMBI can act to significantly suppress the expression of carcinoma-related fibroblast markers in human bone marrow mesenchymal stem cells without influencing their stem cell and tumor-tropic–related properties.[9]

Evidence has been provided in various studies highlighting the strong correlation among low microRNA-708 (miR-708) expression, tumor progression, poor survival outcome, and recurrence in patients with prostate cancer.[10] Additionally, descended levels of miR-708 have been detected in cases of hepatocellular carcinoma as well as the repression of tumor migration and invasion.[11] The Wnt signaling pathway has in recent times been shown to play a regulatory growth and patterning role in embryonic development, while its pathological role revealed a relationship between the high frequency of several specific human carcinomas and mutations, which induce the activation of transcription observed in these signaling pathways.[12] The TGF-β signaling pathway has been widely demonstrated to be an essential modulator in a vast array of cellular processes affiliated with carcinogenesis.[13] Recent evidence has indicated that the TGF-β signaling pathway (metazoan-specific intercellular) plays a primary role in a variety of developmental processes in addition to a wide array of cellular processes in both humans and animals.[14] At present, there appears to be a scarcity of studies placing a particular emphasis on correlations shared among miR-708, BAMBI, and Wnt and TGF-β signaling pathways, with regard to their respective roles in the pathological processes of melanoma. Thus, this study aimed to evaluate the interaction between miR-708 and BAMBI and their effects on the development and progression of melanoma through the Wnt and TGF-β signaling pathways.

Materials and Methods

Study Participant and Ethical Statement

Sixty C57BL/6J male mice aged 6 to 8 weeks, weighing 20 ± 2 g (provided by the Institute of Zoology Chinese Academy of Sciences), were recruited for the purposes of this study. All experimental procedures as well as animal conditions were preapproved by the Animal Ethics Committee of our hospital.

Model Establishment

The mice in the study were housed in a laboratory environment and provided free access to food and drinking mediums (natural lighting, temperature at 18°C-22°C, relative humidity 40%-70%, noise <50 dB). The mice were subsequently assigned by random means into the experimental (40) and normal control (20) groups. In the experimental group, 22 mice successfully underwent melanoma model establishment, while the remaining 18 mice were prepared for a subsequent tumor xenograft assay. The concentration of the B16 melanoma cell suspension was adjusted to 1 × 10[6] cells/mL, and the cell suspension was then subcutaneously injected into the left side of the back of the mice (0.2 mL each). The mice in the normal group were injected with an equal amount of normal saline. The criteria for success model establishment included reduced physical activity, decreased food intake, dry hair, and tumor appearance.[15] The mice were removed from the model group if the aforementioned symptoms were not observed. Eventually, there were 20 mice assigned into the model group.

Hematoxylin–Eosin Staining

The mice were killed on the 25th day posttumor formation. Both melanoma and normal tissues were incised, fixed with 10% formaldehyde for 24 hours, and dehydrated successively with n-butanol and ethanol (80%, 90%, and 100%, respectively). The tissues were then placed into a 60°C wax paraffin box and cut into pieces at a continuous thickness of 5 μm. Next, all the sections were spread and fished at 45°C, baked for 1 hour at 60°C, and dewaxed with xylene. After hydration, the sections were conventionally stained with Hematoxylin–Eosin (HE; Beijing Solarbio Technology Co, Ltd, Beijing, China) for 2 minutes, washed with water for 10 seconds, and color separated using 1% ethanol hydrochloric acid. The sections were rinsed with distilled water for 1 minute, stained with HE solution for 1 minute, and washed again with distilled water for 10 seconds. The sections were dehydrated with gradient alcohol, clarified, and mounted with neutral gum xylene. The morphological changes in the mice in both the model and the normal groups were observed using an optical microscope (XP-330, Shanghai Bing Yu Optical Instrument Co Ltd, Shanghai, China).

Immunohistochemical Staining

The sections were conventionally dewaxed using xylene I and II for 20 minutes, followed by hydration with gradient alcohol (100%, 95%, 80%, and 70%) for 2 minutes, rinsed twice with distilled water (5 minutes each time), and placed onto a shaking table. Next, the sections were immersed in 3% H2O2 for 10 minutes and washed using distilled water. After that, the sections were repaired in high-pressure antigen for 90 seconds and washed with phosphate-buffered saline (PBS) after cooling at room temperature. Five percent bovine serum albumin (BSA) was added to the sections, which were then incubated at 37°C for 30 minutes. Rabbit anti-mouse BAMBI primary antibody (bs-12418R, Jiang Lai biological, Shanghai, China; 1: 100) was added dropwise to the sections and incubated overnight at 4°C. The sections were then rinsed with PBS for a period of 2 minutes. The biotinylated goat antirabbit immunoglobulin G (SF8-0.3 cable, LEYBOLD, Lilac Garden, Beijing, China; 1: 100) was added to the sections, incubated at 37°C conditions for 30 minutes, followed by the addition of SAB working fluid, developed with 3,3′-diaminobenzidine, and counterstained with HE for 5 minutes. The PBS was used as a negative control (NC) in place of the primary antibody. The positive position of the immunohistochemical staining was yellow–brown in color. Five fields of vision with 200 cells per field were randomly selected in each group (200×). The percentage of the number of cells with positive BAMBI expression in relation to the total number of cells was then calculated. The aforementioned experiment was repeated 3 times.

Terminal-Deoxynucleotidyl-Transferase-Mediated dUTP-Nick-End Labeling Assay

One hundred milligram of tissue was removed from the model and normal groups. The paraffin sections were dewaxed using xylene I and II for 10 minutes, dehydrated by gradient alcohol at various concentrations including 100%, 95%, 80%, and 70% (each concentration was maintained for a period of 2 minutes), and irrigated 3 times using PBS (5 minutes each), followed by incubation with protease K solution at 37°C for 30 minutes, 3 PBS washes (5 minutes each), and fixed for 2 hours in 4% paraformaldehyde. After a series of washes with PBS (3 × 5 minutes), the tissues were immersed for 10 minutes in sealing fluid (3% H2O2 dissolved in methanol) at room temperature and then cultured in 20% sucrose phosphate buffer at 4°C overnight. The sections were then cut into 20-μm continuous sections using a cutting machine at a constant temperature of −22°C. Next, the tissue sections were Terminal-Deoxynucleotidyl-Transferase (TdT)-Mediated dUTP-Nick-End Labeling stained (Boehringer-Mannheim, Basil, Switzerland) in accordance to the manufacturer’s instructions (10 sections were randomly selected from each mouse). The sections were observed under an optical microscope (Leica DM4 P; Shanghai Precision Electronic Co Ltd). Ten photographs (200×) of each tissue section were obtained in order to calculate the apoptotic nuclei and sum up the number of total cells. The apoptotic cells were expressed as dark particles. The apoptotic index (AI) of the melanoma tissues as well as the normal tissues was subsequently calculated. The experiment was repeated 3 times, and the average value was calculated accordingly.

Reverse Transcription Quantitative Polymerase Chain Reaction

Three melanoma cell lines, B16, A375, and WM239, were obtained from the Institute of Biochemistry and cell biology, at the Chinese Academy of Sciences. The cells were cultured at 37°C in 5% CO2 with RPMI 1640 medium (SP1355, Shanghai Shifeng Biotechnology Co Ltd, Shanghai, China), supplemented with a mixture of 10% fetal bovine serum (FBS; AAPR123, Xingzhi Biological Technology Co Ltd, Guangzhou, China), with 100 U/mL of penicillin, as well as 100 mg/mL of streptomycin (07500 Hangzhou 100 Biological Technology Co Ltd, Hangzhou, China), and saturated in a humidity incubator (DHP-9162, a Czech experimental instrument; NOKI LTD, Shanghai, China). The culture medium was replaced with a fresh medium at regular 1- to 2-day intervals, and the cells were subcultured in the event that cell confluency reached 80% to 90%.

The cell lines were screened with the content of miR-708 as the standard. Total RNA was extracted from B16, A375, and WM239 cell lines using Trizol reagent (15596-018; Invitrogen, New York) in accordance with the protocols of the manufacturer. The purity and concentration of the RNA among the tissues and cells were detected by means of ultraviolet spectrophotometry (UV1901; Shanghai Olympic scientific instrument Co Ltd, Shanghai, China). The concentration of each sample consisting of a purity of 0.1 A260/A280 = 1.8 ∼ 2.0 g/L was adjusted to 0.1 g/μL. Next, total RNA was reverse transcribed using a PrimeScript RT reagent Kit (Takara, RR047A; Beijing Zhijie far Technology Co, Ltd, Beijing, China) in order to obtain the complementary DNA (50 ng/μL) using a 10-μL reverse transcription system. The reaction conditions were coordinated in accordance with the following criteria: 3 times × 15 minutes at 37°C (reverse transcriptase reaction), 5 seconds at 85°C (reverse transcriptase inactivation reaction), and −80°C freezing. The primers were designed by Premier 5.0 software and synthetized by Beijing Xinye Qing Department Biological Technology Co Ltd[16] (Table 1). The procedure was performed in ABI 7900HT real-time quantitative polymerase chain reaction using a 2-step method, while U6 and GAPDH were used as an internal reference. This procedure was conducted in accordance with the following reaction conditions: predenaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 15 seconds, and 40 cycle repetitions. The relative values of miR-708, BAMBI, VEGF, Wnt10B, Fas, P53, Bax, Bcl-2 and Caspase-3 mRNA were recorded using 2−ΔΔCT. Genes from each sample were organized for 3 repetitions. The experiment was repeated 3 times.

Table 1.

RT-qPCR Primer Sequences.

Genes	Primer sequences
miR-708	Forward: 5′-GGCGCGCAAGGAGCTTACAATC-3′
	Reverse: 5′-GTGCAGGGTCCGAGGTAT-3′
BAMBI	Forward: 5′-GCAATTATCGAGGACTGCATGAC′
	Reverse: 5′-GCGGAACCACAGTTCTTTGGAG-3′
VEGF	Forward: 5′-ATGAACTTT CTGCTGTCTTGG-3′
	Reverse: 5′-TCACCGCCTCGGCTTGTCACA-3′
Wnt10B	Forward: 5′-CATCCAGGCACGAATGCCAATC-3′
	Reverse: 5′-AGGCTCCAGAATTGCGGTTGTG-3′
Fas	Forward: 5′-TATCACCACTATTGCTGGAGTCATG-3′
	Reverse: 5′-TCAATGTGTCATACGCTTCTTTCTT-3′
P53	Forward: 5′-ACGGTGACACGCTTCCCTGGATTGG-3′
	Reverse: 5′-CTGTCAGTGGGGAACAAGAAGTGGAGA-3′
Bax	Forward: 5′-GGCGAATTGGAGATGAACTG-3′
	Reverse: 5′-GTCACTGTCTGCCATGTGGG-3′
Bcl-2	Forward: 5′-GTGGGATACTGGAGATGAAGACT-3′
	Reverse: 5′-CAGCCAGGAGAAATCAAACAG-3′
Caspase-3	Forward: 5′-AAGAACTTAGGCATCTGTGGGCA-3′
	Reverse: 5′-TTCAGGACAAACATCACAAAACCA-3′
GAPDH	Forward: 5′-AACGGATTTGGTCGTATTG-3′
	Reverse: 5′-GGAAGATGGTGATGGGATT-3′
U6	Forward: 5′-CTCGCTTCGGCAGCACA-3′
	Reverse: 5′-AACGCTTCACGAATTTGCGT-3′

Abbreviations: BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Western Blotting

Total protein was extracted from 30 g of melanoma and normal tissues. The protein concentrations were determined using a bicinchoninic acid protein assay kit (P0012-1; Shanghai Beyotime Biotech Co Ltd, Shanghai, China). The protein samples (50 µg) were mixed with 2 × sodium dodecyl sulfate (SDS) loading buffer, boiled for 5 minutes, and separated by 10% acrylamide denaturing gels [SDS-polyacrylamide gel electrophoresis (SDS-PAGE)], followed by transfer onto a polyvinyldifluoride transfer membrane (PVDF). The membrane was blocked for a 1-hour period with 5% nonfat milk at room temperature and incubated with rabbit-derived primary antibody (BAMBI, 1: 200, ab57043; VEGF, 1: 300, ab32152; Wnt10B, 1: 100, ab66721; Fas, 1: 1000, ab15385; P53, 1: 200, ab26; Bax, 1: 500, ab2503; Bcl-2, 1: 100, ab119506; Caspase-3, 1: 300, ab2171; cleaved Caspase-3, 1: 500, ab32042; Abcam Inc, Cambridge, Massachusetts) for 1=hour at room temperature. Next, the membrane was washed with Tris-buffered Saline with Tween 20 and incubated for an hour, followed by the addition of horse radish peroxidase-marked goat anti-rabbit secondary antibody (1: 5000) at room temperature. The membrane was washed again 3 times with TBST (5 minutes each) and developed with enhanced chemiluminescence (ECL) solution. The membrane was then exposed and photographed using an X-ray film. The respective absorbance of each band was acquired using the gel imaging analysis system, and the relative content of the sample protein was calculated, equating to the average absorbance of the sample divided by that of the corresponding reference. The relative content of all protein was compared by statistical analyses. The experiment was repeated 3 times.

Dual-Luciferase Reporter Gene Assay

The miR-708 target gene was predicted using the website microRNA.org, and the double-luciferase reporter gene assay was used to verify the predicted result, providing verification that BAMBI was in fact the direct target gene of miR-708. The full length of the 3′UTR region of BAMBI gene was cloned into the polyclonal site of pmirGLO (E1330, Promega Biotechnology Co, Ltd, Madison, Wisconsin), a downstream of the luciferase gene, referred to as pBAMBI-Wt. Site-directed mutagenesis was conducted on the BAMBI gene binding sites for miR-708 according to the bioinformatics prediction, and the pBAMBI-Mut vector was constructed. The pRL-TK vector (E2241, Promega Corporation) expressing sea pansy fluorescein luciferase was used as an internal reference for the regulation of the difference in transfection efficiency and the number of cells. MiR-708 mimics or NC oligos and luciferase reporter gene vectors were cotransfected into melanoma cells. A double-luciferase activity assay was performed following the method provided by Promega Corporation. The experiment was repeated 3 times.

Cell Culture, Grouping, and Transfection

Normal HEK293 cells (31800-022; Tong Pai Biological Technology Co, Ltd, Shanghai, China) were cultured using Dulbecco’s Modified Eagle Medium (190040, Gibco Company, Carlsbad, California) supplemented with 10% FBS, and B16 melanoma cells were cultured in 1640 RPMI (22400089, Gibco Company). A total of 100 U/mL penicillin and 100 mg/mL streptomycin were supplemented in both culture mediums, while the plated cells were cultured in conditions of 37°C and 5% CO2. The culture medium was changed at 2- to 3-day intervals in accordance with the relative cell growth conditions. The cells were subcultured when the spread reached an approximate value of between 80% and 90% of the culture plate. The third-generation cells were used for subsequent experiments.

Besides the normal HEK293 cells group, the melanoma cells were classified into 6 groups: the blank group (blank control group, melanoma B16F10 cell, without any transfection of any sequence), NC group (with transfection of miR-708 NC sequence), miR-708 mimics group (with transfection of miR-708 mimics), miR-708 inhibitors group (with transfection of miR-708 inhibitors), si-BAMBI group (with the transfection of siRNA-BAMBI), and miR-708 inhibitors + si-BAMBI group (with co-transfection of miR-708 inhibitors and siRNA-BAMBI). Cells at the logarithmic growth phase were implanted into 6-hole plates and transfected based on the instruction of the lipofectamin 2000 (Invitrogen, Carlsbad, California) when the cell density increased at a rate of 30% to 50%. In a succinct manner, the corresponding freeze-dried oligos (Invitrogen) were prepared with RNase-free water; equal amounts of oligos were diluted with 250 µL serum-free MEM culture medium (Gibco Company) and mixed with 250 µL MEM containing 5 µL lipofectamin 2000 transfection regent and incubated for 20 minutes at room temperature. The transfection solution was then placed into the cell plate. After culturing for 6 to 8 hours, the medium was changed and replaced with a complete medium, and 24 to 48 hours later, the cells were collected for subsequent experimentation.

TOP/FOPflash Luciferase Reporter Assay

The cells in each group were seeded into a 24-well plate at a density of 5 × 104 cells/well. After 20 hours, based on the lipofectamine 2000 instructions (11668019; Thermo Fisher Scientific, Sunnyvale, California), the cells in the NC group were cotransfected with pRL-TK (served as the internal reference), TOP/FOPflash (Promega), and miR-708 NC sequences; cells in the miR-708 mimics group were cotransfected with pRL-TK, TOP/FOPflash, and miR-708 mimics; cells in the si-BAMBI group were cotransfected with pRL-TK, TOP/FOPflash, and siRNA-BAMBI; cells in the miR-708 inhibitors + si-BAMBI group were cotransfected with pRL-TK, TOP/FOPflash, miR-708 inhibitors, and siRNA-BAMBI; cells in the normal and blank groups were cotransfected with pRL-TK and TOP/FOPflash. The mixture (0.5 μL lipofectamine 2000 and 100 μL L-DMEM) was then prepared and left idle for 5 minutes. The original culture medium containing L-DMEM was removed, and a fresh mixture was added prior to a 6-hour period of transfection. Next, a complete medium employed for cell incubation and subsequently abandoned 24 to 48 hours later. Dual-Luciferase Reporter Assay System (E1910; Promega) was applied in order to detect the values of the firefly luciferase as well as the application of a renilla luciferase in each well. The ratio of TOP/FOPflash in each group was representative of the transcriptional activity of the Wnt signaling pathway.

Immunofluorescence Staining

After conventional digestion and transfection, the cells were cultured in an immunofluorescence chamber with 2 × 105 cells each well. When cell confluency reached 90%, the cells were washed 3 times with PBS on ice, fixed and added with 1 mL 4% paraformaldehyde, and left idle for 15 minutes at room temperature. After another 3 subsequent washes with PBS, the cells were punched with 0.3% Triton, 10 minutes later, rewashed 3 times with PBS, and blocked in Lowlenthal serum for 30 minutes. The primary antibodies prepared by PBS were used to incubate the cells at 4°C overnight, followed by the addition of secondary antibodies, followed by an hour of cell incubation in dark conditions at room temperature. After another 3 PBS washes, the cells were stained with 4′,6-diamidino-2-phenylindole 2hci (a ; DAPI) for 15 minutes in dark conditions, rewashed 3 times with PBS, and sealed with a fluorescence quencher. A fluorescence microscope was used for observation and photographical purposes.

3-(4,5-Dimethylyhiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide Assay

Forty-eight hours after transfection, the cells were digested, resuspended, and transferred into a 96-well plate with a concentration of 1 × 105/mL. Eight repetitions were set in each group with 100 μL complete medium placed in each hole. The cells were then incubated in a 5% CO2 culture box at 37°C. Ten microliter of 3-(4,5-Dimethylyhiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide (MTT; 5 mg/mL; Sigma, San Francisco) was added into each hole at 24, 48, and 72 hours, respectively. After a subsequent 4-hour period of incubation, the supernatant was replaced with 150 μL dimethyl sulfoxide (DMSO). Ten minutes of vortex was employed in order to fully dissolve the DMSO. The optical density (OD) of each well was read at wavelength of 490 nm in an automatic enzyme-linked immunosorbent assay reading instrument (BIO-RAD, California). The experiment was repeated 3 times.

Flow Cytometry

The cells were collected 48 hours after transfection and then treated with 0.25% trypsin. The cell number was adjusted to 1 ×106 cells/mL. Cell suspension of 1 mL was centrifuged at 1500 r·min−1 for 10 minutes and washed with 2 mL of PBS. The cells were then fixed with precooled 70% ethanol at 4°C overnight. One hundred microliters of cell suspension was collected, respectively, after washing the cells with PBS twice, followed by the addition of 50 μg PI dye liquor containing RNAase (40710ES03; Shanghai on Biological Technology Co Ltd, Shanghai, China). The cells were filtered with a 100-purposes nylon filter after 30 minutes of incubation in conditions void of light. The cell life cycle was detected by means of flow cytometry (BD, Franklin Lakes, New Jersey) at a wavelength excitation of 488 nm.

Next, Annexin V-FITC/PI double-staining method was used to detect apoptosis. The procedure was conducted as follows: The treated cells were initially cultured at 37°C in 5% CO2 for 48 hours, and the cells were suspended in a 200-μL binding buffer and washed twice with PBS followed by a centrifugation process. Ten microliters of Annexin V-FITC (ab14085, Abcam Inc, Cambridge, Massachusetts) and 5 μL of PI were then added and mixed gently. After a 15-minute period had lapsed permitting a reaction (room temperature, protecting from light), 300 μL of binding buffer was added, and cell apoptosis was detected by means of flow cytometry at 488 nm. The experiment was repeated 3 times.

Scratch Test

Forty-eight hours posttransfection, the transfected cells in each group were plated in a 6-well plate at a concentration of 5 × 105 per hole. The cells were then gently streaked across the entire axle wire using sterile tips when the cell density had reached approximately 90%. The cells were cultured in a serum-free medium for 0.5 to 1 hour in order to restore the growth ability after the floating cells had been removed by PBS washing. The cell culture plates were photographed at 0 and 24 hours, and the cell migration distance was measured using Image-Pro Plus Analysis software (Media Cybernetics company, Rockville, Maryland). Finally, the respective average values of the data were obtained, and the experiment was repeated 3 times.

Transwell Assay

Matrigel (356234; BD) was dissolved at 4°C overnight followed by dilution with serum-free medium (1: 3). The upper chamber of the Transwell chamber was treated with 50 μL of matrigel solution and placed in a culture box for 30 minutes. After 48 hours of cell transfection, the cells were digested, washed 3 times with serum-free medium, counted, and employed in the production of cell suspension. The matrigel was rinsed once with serum-free medium, and 1 × 105/ml cell suspensions were inoculated in the upper chamber. Following a 24-hour period of incubation at 37°C, the lower chamber was subsequently added with serum-free medium containing 10% FBS. The lower chamber was then withdrawn, washed twice with PBS (5 minutes per wash), fixed with 5% glutaraldehyde at 4°C, and followed by the addition of 0.1% crystal violet for staining purposes for a period of 30 minutes. The chamber was wiped and observed under a microscope following 2 PBS washes (5 minutes per wash). The number of cells that passed through the matrigel was counted and was regarded as an indicator of cell invasion. The experiment was conducted 3 times.

Tumor Xenograft Assay

Eighteen housed nude mice were divided into 6 groups, namely, the blank, NC, miR-708 mimics, miR-708 inhibitors, siRNA-BAMBI, and miR-708 inhibitors + siRNA-BAMBI groups (n = 3). The nude mice from each group were then anaesthetized and inoculated with 1 × 106 (200 L) B16 melanoma cells in the right hind leg. The mice were housed under identical conditions and observed every 7 days in order to maintain an accurate record of the length and width of the respective tumors. The tumor volume was calculated according to the following formula: tumor volume = length × (width)2/2. On the 36th day, the mice were killed, and the tumors were removed. Three tumors were collected from each group.

Statistical Analysis

All data were analyzed using SPSS version 21.0 software (SPSS Inc, Chicago, Illinois). Measurement data were presented as mean ± standard deviation (SD). Comparisons between the 2 groups were measured by t test. Comparisons among multiple groups were conducted using 1-factor analysis of variance (ANOVA). P < .05 was considered to be of statistical significance.

Results

Pathological Changes in Melanoma and Normal Tissues of Mice in the Normal and Model Groups

In the model group, the nuclei of cells were deeply stained. Observations of inflammatory cell infiltration, consisting mainly of lymphocytes, were made. However, in the normal group, the nuclei were lightly staining, and no obvious variation was displayed (Figure 1).

Figure 1.

Pathological changes in melanoma and normal tissues of mice in the normal and model groups by HE staining. HE indicates hematoxylin–eosin.

Positive Expressions of BAMBI in Melanoma and Normal Tissues of Mice in the Normal and Model Groups

The immunohistochemical staining results displayed in Figure 2 revealed that when compared to the normal group, the stained melanoma tissues in the model group were deeper, with a greater degree of brown granules observed. The positive BAMBI expression rate among the melanoma tissues of the model group was notably higher than that of the normal tissues in the normal group (P < .05).

Figure 2.

Immunohistochemistry staining results (A, ×200) and positive expression rate (B) of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) in melanoma tissues and normal tissues of mice in the normal and model groups. *P < .05, compared with the normal group.

Cell Apoptosis in Melanoma and Normal Tissues of Mice in the Normal and Model Groups

As shown in Figure 3, the stained melanoma particles in the model group as well as the apoptotic cell number were both less than that of the normal group. Compared to the normal group, the number of positive cells as well as the rate of cell apoptosis both exhibited significant reductions (P < .05).

Figure 3.

TUNEL staining results (A, ×200) and cell apoptosis rate (B) in melanoma and normal tissues of mice in the normal and model groups. *P < .05, compared with the normal group.

Melanoma Cell Line Selection Process

The comparisons of expression content of miR-708 and BAMBI in B16, A375, and WM239 cell lines are illustrated in Figure 4. The miRNA-708 expression in descending order was as follows: B16 > A375 > WM239. The BAMBI expression in descending order was as follows: WM239 > A375 > B16. Therefore, B16 cell line was selected for the subsequent experiment as it exhibits the highest expression in miR-708 and lowest expression in BAMBI. Significant differences were detected among the 3 melanoma cell lines (all P < .05).

Figure 4.

Selection of melanoma cell line. A, Expression histogram of miR-708. B, messenger RNA (mRNA) expression histogram of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI). C, protein expression histogram of BAMBI. D, Western blotting map of BAMBI protein expression; *P < .05, compared with the B16 cell line.

The miR-708 expression as well as mRNA and protein expressions of BAMBI, Wnt10B, P53, Bcl-2, VEGF, Fas, Bax, Caspase-3, and cleaved Caspase-3 in the normal and model groups

As illustrated in Figure 5, the expressions of miR-708 and cleaved Caspase-3, as well as mRNA and protein expressions of VEGF, Fas, Bax, and Caspase-3, were all lower in the model group than that of the normal group; however, greater mRNA and protein expressions of BAMBI, Wnt10B, P53, and Bcl-2 were detected in the model group when compared to the normal group (all P < .05).

Figure 5.

Expressions of miR-708, bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), Wnt10B, P53, Bcl-2, VEGF, Fas, Bax, caspase-3, and cleaved caspase-3 in each group. A, Histogram of mRNA expressions. B, Electrophoretograms of protein expressions. C, Histogram of protein expressions. *P < .05, compared to the normal group.

Target Relationship Between miR-708 and BAMBI Gene

The specific binding region was observed between 3′ untranslated regions (3′UTR) of the BAMBI gene, while the miR-708 sequences were assessed using online software analysis (Figure 6A). Luciferase reporter gene assay was used to confirm that BAMBI was in fact a target gene of miR-708 (Figure 6B). Compared to NC group, the luciferase activity of wild-type 3′UTR of BAMBI gene displayed clear signs of inhibition in the miR-708 in the miR-708 mimics group (P < .05). However, no significant difference was found between the miR-708 mimics and NC groups when Mut-miR-708 plasmid was cotransfected with BAMBI. This demonstrates that miR-708 was specifically bound to the 3′-UTR of BAMBI gene.

Figure 6.

Verification of target relationship between bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) and miR-708 gene. A, predicted binding site of miR-708 and 3′UTR. B, detection of luciferase activity. wt indicates wild-type; mut: mutant. *P < .05, as compared with the NC group; NC, negative control.

The miR-708 expression as well as mRNA and protein expressions of BAMBI, Wnt10B, P53, Bcl-2, VEGF, Fas, Bax, and Caspase-3 in cells after transfection in each group

Compared to the normal HEK293 cells, the mRNA and protein expression levels of BAMBI, Wnt10B, P53, and Bcl-2 were far greater among the melanoma cells while miR-708, VEGF, Fas, Bax, Caspase-3, cleaved Caspase-3 RNA, and protein expression levels all displayed notably decreased levels (all P < .05). When compared to the blank and NC groups, an increased rate of miR-708 expression was detected in the miR-708 mimics group; in the miR-708 mimics and siRNA-BAMBI groups, the mRNA and protein expressions of BAMBI, Wnt10B, P53, and Bcl-2 displayed decreased levels, while the mRNA and protein expressions of VEGF, Fas, Bax, Caspase-3, and cleaved Caspase-3 all displayed increased levels (all P < .05); in the miR-708 inhibitors group, an opposite trend was observed, by which the expressions of miR-708, VEGF, Fas, Bax, Caspase-3, and cleaved Caspase-3 were remarkably decreased; however, the expressions of BAMBI, Wnt10B, P53, and Bcl-2 all exhibited increased levels (all P < .05); a decline in the expression of miR-708 was observed in the miR-708 inhibitors + si-BAMBI group (P < .05); meanwhile, no significant statistical difference was detected in relation to the expressions of BAMBI, Wnt10B, P53, Bcl-2, VEGF, Fas, Bax, Caspase-3, and cleaved Caspase-3 in the miR-708 inhibitors + si-BAMBI group (P > .05; Figure 7).

Figure 7.

Expressions of miR-708, BAMBI, Wnt10B, P53, Bcl-2, VEGF, Fas, Bax, Caspase-3, and cleaved Caspase-3 after transfection in each group. A, Histogram of mRNA expressions. B, Histogram of protein expressions. C, electrophoretograms of protein expressions. *P < .05, compared to the normal group; # P < .05, compared with the blank and NC groups. NC indicates negative control.

miR-708 Targets BAMBI to Inhibit the Activity of the Wnt Signaling Pathway

The TOPflash plasmid was comprised of the firefly luciferase reporter gene, and the luciferase promoter containing 3 repeats of TCF binding sequence, which was capable of modulating the expression of the downstream luciferase according to β-catenin activity. The TCF binding sequence in FOPflash plasmid was in correspondence with the mutant variety, while the other sequences consistent with TOPflash were unaffected by the activity of β-catenin. As a result, TOP/TOPflash is often used as an index in the evaluation of the activation of the Wnt/β-catenin signaling pathway. The key point is that Wnt/β-catenin in nucleus binds with TCF/LEF to regulate gene expression. During this experiment, the TOP/FOPflash luciferase reporter gene assay revealed that the other groups exhibited an increased amount of TOP/FOPflash activity when compared to the normal group (P < .05). Compared to the blank and NC groups, TOP/FOPflash activity was decreased in the miR-708 mimics and siRNA-BAMBI groups and increased in the miR-708 inhibitors group (all P < .05; Figure 8A). The immunofluorescence staining results revealed upregulated levels of the fluorescent expression of β-catenin protein in the other groups when compared to that of the normal group (P < .05). Compared to the blank and NC groups, the fluorescent expression of β-catenin protein was decreased in nucleus of the miR-708 mimics and siRNA-BAMBI groups; however, increases in the cell nucleus of the miR-708 inhibitors group were observed; no significant difference in the miR-708 inhibitors+ si-BAMBI group was noted (all P < .05; Figure 8B). The aforementioned obtained results demonstrated that miR-708 could target BAMBI to inhibit the activity of the Wnt signaling pathway.

Figure 8.

Wnt signaling pathway activities in each group. A, Activity of TOP/FOPflash in each group. B, β-catenin expression detected by immunofluorescence staining. *P < .05 compared to the normal group; # P < .05 compared with the blank and NC groups. NC indicates negative control.

Cell Proliferation in the Normal, Blank, NC, miR-708 Mimics, miR-708 Inhibitors, siRNA-BAMBI, and miR-70 8inhibitors + siRNA-BAMBI Groups

The MTT assay demonstrated that the cell proliferation rate was enhanced in the cell-transfected melanoma groups more so than the normal group (all P < .05; Figure 9). Compared to the blank and NC groups, the cell proliferation rate was decreased in the miR-708 mimics and siRNA-BAMBI groups; however, increases in the miR-708 inhibitors group were recorded (all P < .05); there were no changes in the miR-708 inhibitors + siRNA-BAMBI group (all P > .05).

Figure 9.

Melanoma cell proliferations in different groups. *P < .05, compared with the normal group; # P < .05, compared with the blank and NC groups. OD value indicates optical density value; NC, negative control.

Cell Cycle and Apoptosis Rates of Melanoma Cells in Each Group

Cell cycle was detected by PI single staining (Figure 10A and B). The number of cells at the G0/G1 phase was reduced; however, a greater number of cells at the S phase were observed in the transfected melanoma cells when compared to the normal HEK293 cells (all P < .05). There was no statistical significance detected between the blank and the NC groups (P > .05). Compared to the blank and NC groups, the number of cells arrested at the G0/G1 phase was increased; however, there was a decrease in cells at the S phase in the miR-708 mimics and siRNA-BAMBI groups; on the contrary, the cell number arrested at the G0/G1 phase declined but an increase in the S phase in the miR-708 inhibitors group was observed (all P < .05); there was no apparent difference in relation to cell cycle in the miR-708 inhibitors + siRNA-BAMBI group (P > .05).

Figure 10.

Cell cycle distribution (A) and cell cycle (B, %) of melanoma cell in each group. *P < .05, compared to the normal group; # P < .05, compared to the blank and NC groups. NC indicates negative control.

Cell apoptosis rate was measured by Annexin V-FITC/PI double staining (Figure 11A and B). The cell apoptosis rate in transfected melanoma cells displayed a marked increase in comparison to the normal group (all P > .05). There was no statistical significances with regard to the cell apoptosis rate between the blank and the NC groups (P > .05). Compared to the blank and NC groups, a sharp elevation in the rate of cell apoptosis was detected in the miR-708 mimics and siRNA-BAMBI groups while a decline was detected in the miR-708 inhibitors (P < .05); there was no obvious difference in relation to cell apoptosis in the miR-708 inhibitors + siRNA-BAMBI group.

Figure 11.

Cell apoptosis (A) and apoptosis rate (B, %) of melanoma cell in each group. *P < .05, compared to the normal group; # P < .05, compared to the blank and NC groups. NC indicates negative control.

Cell Migration in the Normal, Blank, NC, miR-708 Mimics, miR-708 Inhibitors, siRNA-BAMBI, and miR-70 8inhibitors + siRNA-BAMBI Groups

Cell migration abilities were assessed by means of a scratch test (Figure 12). No notable differences were observed in relation to cell migration between the blank and the NC groups (P > .05). Compared to the blank and NC groups, cell migration underwent a significant decrease in the miR-708 mimics and siRNA-BAMBI groups (all P < .05), while there was an increase in the miR-708 inhibitors group (P < .05). No difference was exhibited with regard to cell migration abilities between the miR-708 inhibitors + siRNA-BAMBI, blank, and NC groups (P > .05).

Figure 12.

Cell migration (A) and migration distance (B) in each group. *P < .05, compared to the normal group; # P < .05, compared to the blank and NC groups. NC indicates negative control.

Cell Invasion in the Normal, Blank, NC, miR-708 Mimics, miR-708i Inhibitors, siRNA-BAMBI, and miR-708 Inhibitors + siRNA-BAMBI Groups

As shown in Figure 13, there was no remarkable difference detected in relation to the cell invasion between the blank and the NC groups (P > .05). Compared to the blank and NC groups, cell invasion was significantly decreased in the miR-708 mimics and siRNA-BAMBI groups (all P < .05), while an increase in the miR-708 inhibitors group was detected (P < .05). No difference was recorded with regard to cell invasion ability between the miR-708 inhibitors + siRNA-BAMBI, blank, and NC groups (P > .05).

Figure 13.

Transwell results (A, × 200) and cell invasion (B) in each group. *P < .05, compared to the normal group; # P < .05, compared to the blank and NC groups. NC indicates negative control.

Tumor Growth of Nude Mice Among Each Group

As shown in Figure 14, no significant difference was detected between the blank and the NC groups (P > .05). Compared to the blank and NC groups, there were much larger tumors observed as well as a much faster rate of tumor growth in the miR-708 inhibitors group (all P < .05). However, the volume of tumor and its growth rate in the miR-708 mimics and siRNA-BAMBI groups was found to be minor comparatively speaking (P < .05). No difference in relation to the tumor volume and growth rate in the miR-708 inhibitors + siRNA-BAMBI group was noted (all P > .05).

Figure 14.

Tumor volume (A) and tumor size (B) of nude mice among each group. *P < .05, compared to the blank and NC groups. NC indicates negative control.

Discussion

The miRNAs have been widely confirmed to be key regulators in a variety of pathological processes as well as that of tumor cell proliferation and migration including that of melanoma, of which miR-34a and miR-137 have been demonstrated to play a role.[17,18] In this study, the role of miR-708 was highlighted with regard to its status as a crucial tumor-suppressive miRNA, playing a primary role in melanoma. Several previous studies have provided evidence elucidating the benefits of miR-708 in various conditions including renal cancer, prostate cancer, and breast cancer.[10,19,20] However, this study took a novel position placing a particular emphasis demonstrating that miR-708 targeting BAMBI inhibits the proliferation and migration of melanoma cell by activating the TGF-β signaling pathway as well as suppressing the Wnt signaling pathway.

Thus far, no evidence has been reported asserting that BAMBI is the target gene of miR-708, and no research study has highlighted the correlation between miR-708 and both WNT and TGF-β signaling pathways. During this study, the miR-708 inhibitors group exhibited distinctive BAMBI gene increases as well as that of mRNA and protein expressions of Wnt10B, P53 and Bcl-2; as also, descended levels of the BAMBI gene and the expressions of VEGF, Fas, Bax, Caspase-3 and cleaved Caspase-3 were observed. Moreover, miR-708 mimics group saw a significant decrease in BAMBI gene as well as in the expressions of Wnt10B, P53, and Bcl-2, while distinctive elevations in BAMBI gene and the expressions of VEGF, Fas, Bax, Caspase-3, and cleaved Caspase-3 were recorded. Therefore, miR-708 was confirmed to play a contributory role in the activation of the TGF-β signaling pathway as well as in the suppression of the Wnt signaling pathway by downregulating BAMBI.

The Wnt signaling pathway commands central functions in adult tissue homeostasis, stem cell maintenance, cardiac development and differentiation, angiogenesis, cardiac hypertrophy, cardiac failure, and aging.[21] Furthermore, Wnt proteins possess multiple functions on a cellular level, including in the regulation of gene expression via different mechanisms (and consequently that of differentiation), cell cycle and proliferation as well as other nongenomic responses such as cell migration and cilia formation.[22] The TGF-β signaling pathway is an essential regulator of cellular proliferation, differentiation, apoptosis, and extracellular matrix remodeling.[23] Additionally, this signaling pathway is also involved in both angiogenesis and inflammation.[24] The TGF-β signaling pathway has the ability to mediate the intracellular actions of proinflammatory cytokines, including the activation of nuclear factor-kappa B (NFκB). Deficiency in TGF-β has been witnessed in the progression of extensive inflammatory conditions.[13] In our study, the mRNA and protein expressions of Wnt10B, P53, and Bcl-2 were significantly upregulated, while the expressions of VEGF, Fas, Bax, and Caspase-3 all exhibited downregulated levels among the melanomas. These results were largely suggestive of the notion that the activation of the WNT signaling pathway in addition to the repression of the TGF-β signaling pathway could lead to melanoma occurrence.

A key finding of our study involved the observation that the expression of miR-708 was significantly downregulated in human melanoma cell specimens, which was consistent with the melanoma cell lines. Numerous published studies have demonstrated BAMBI to be an indispensable regulator of cell proliferation and differentiation that acts to represses TGF-β while enhancing the WNT signaling pathway in various cell types.[8,25,26] Our study suggests that miR-708 is indeed a regulator of BAMBI that selectively induces apoptosis in tumor-derived cell types through activation of the WNT signaling pathway, highlighting its promise as an antitumor agent. The relative mRNA and protein expressions of the Wnt signaling pathway, including Wnt10B, P53, and Bcl-2, all recorded upregulated levels among the melanoma tissues, while the relative mRNA and protein expressions of TGF-β pathway, including VEGF, Fas, Bax, and Caspase-3, displayed downregulated levels among the tissues in our experiments. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting results both revealed that the BAMBI expressions of mRNA and protein were remarkably increased in the model group. Moreover, the knockdown of the BAMBI gene was accompanied by a decline in cell proliferation and Wnt10B, P53, and Bcl-2. The BAMBI gene was demonstrated to be highly expressed in melanoma tissues, as well as demonstrating its ability to accelerate the progression of melanoma through activation of the Wnt signaling pathway and the inhibition of TGF-β signaling pathway.

Apoptosis is a well-orchestrated cellular mechanism that balances cell proliferation and cell death.[19,27] During our study, the essential proapoptotic role of miR-708 was investigated. The expression of miR-708 leading to the dramatic induction of cell apoptosis in melanoma cell lines was observed. In the present study, we asserted that miR-708 was a novel miRNA that is capable of regulating apoptosis in melanoma cells. Our results suggested that miR-708 re-expression induces apoptosis in addition to eliciting a decrease in both migration and invasion. Due to the predominant and dramatic proapoptotic effects of miR-708, decreased migratory and invasive properties may be caused partly by the effects involved with the events of apoptosis.

In conclusion, our study identified miR-708 to be an essential regulatory miRNA that controls melanoma cell through the activation of the TGF-β signaling pathway as well as the repression of the WNT signaling pathway by downregulating BAMBI. These findings provide a basis for the potential treatment of melanoma. However, there is a lack of substantial experimental data, and miRNA knockout mice with or predicted to have cancer have not yet been reported currently. Further studies are needed to investigate the detailed mechanism among miR-708, Wnt, and TGF-β signaling pathway and melanoma to verify our study.