Background
Melanoma is the most aggressive type of skin cancer that originates from melanocytes, accounting for a high mortality rate annually [
1]. It is well known that surgical resection is the most common treatment for melanoma, but it is mostly invalid for patients with advanced melanoma, and the prognosis is poor with a 5-year survival rate < 16% [
2]. For the treatment of advanced malignant melanoma, comprehensive and multidisciplinary approach should be applicable, such as chemotherapy, radiotherapy and immunotherapy [
3,
4]. But little benefit was obtained from these regimens mainly because of the lack of effective biomarkers [
5]. It has attracted increasing attention that looking for new biomarkers and exploring the potential molecular mechanism of melanoma development is pivotal for the treatment of malignant melanoma.
MicroRNAs (miRNAs) are a class of small non-coding RNAs that play an important role in posttranscriptional gene regulation by cleaving or repressing the translation of their target mRNAs [
6]. Accumulating evidence has corroborated that miRNAs are involved in many physiological and pathological processes including cell proliferation, invasion, migration, apoptosis, differentiation, and metabolism [
7,
8]. Some studies have verified that miRNAs are involved in the development and progression of different types of tumors, either as agonist or antagonist [
9‐
11]. Extensive research of functions and the mechanism of miRNAs in melanoma has been carried out in recent years. Ectopic expression of miR-211 in melanoma cell lines has been shown to impose the inhibitory effect on growth, invasion and metastasis of melanocytes [
9‐
12]. Overexpression of miR-196a was shown to reduce the invasive capacity of melanoma cells [
13]. MiR-18b and miR-149 induced the cell apoptosis of melanoma by targeting p53. Reduced expression of miR-210 was indicated to facilitate the escape of melanoma cells from cytotoxic T lymphocytes [
12,
14]. MiR-let-7a, a tumor suppresser, decreased the cellular proliferation by reducing the expression of cyclin-dependent kinases [
15]. MiR-30b, miR-30d, miR-145 and miR-182 were found to regulate cell invasion and metastasis in melanoma [
16‐
18]. Some researchers have demonstrated that miRNAs are involved in epigenetic modification. Methylation of miR-375, miR-34b and miR-182 was shown to be tightly correlated with high stage melanoma, leading to enhance cell invasiveness and motility [
12,
19,
20].
Isoliquiritigenin (ISL), a natural flavonoid isolated from the root of licorice (Glycyrrhiza uralensis), has a chalcone structure (4, 20, 40-trihydroxychalcone) [
21]. ISL has lots of biological properties, such as anti-inflammatory, anti-oxidant, anti-platelet aggregation, vasorelaxant, and estrogenic effects [
22,
23]. Some researchers have found that ISL is an effective mitosis inhibitor and apoptosis inducer [
24,
25]. Studies of the anti-cancer activity of ISL have been conducted for decades, being reported in ovarian cancer, prostate cancer, breast cancer, oral squamous cell carcinoma and colon cancer [
26‐
29]. ISL has been shown to induce the reprogramming of human melanoma cells and inhibits the proliferation of mouse melanoma cells [
30‐
32], but the anti-cancer effect of ISL on human melanoma remains elusive.
In this study, we showed that ISL significantly inhibited the growth and proliferation of melanoma cells, and miR-301b is predicted to be the most relevant miRNA. Next, we confirmed that miR-301b attenuated the anti-cancer effect of ISL on melanoma in vivo and in vitro by functionally targeting LRIG1. Intratumorally silence of LRIG1 mitigated the apoptosis induced by ISL. Finally, we notarized that LRIG1 and miR-301b are negatively correlated in human melanoma progression. These findings provide a proof of concept that ISL exerts anti-proliferation and pro-apoptosis effect on melanoma by suppressing miR-301b and inducing the target LRIG1.
Methods
Cell culture and tissue specimens
Human melanoma cell line(A375, A2058) obtained from American Type Culture Collection (Manassas, VA, US) was maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and cultured at 37 °C in 5% CO2. Primary human melanomas and normal skin specimens were obtained from patients who were diagnosed in Integrated Hospital of Traditional Chinese Medicine, Southern Medical University. All patients who provided melanomas and normal skin specimens provided full consent for the study. Each cancer specimen contained at least 80% tumor cells, as confirmed by microscopic examination. Tissues were preserved by snap-freeze and stored at − 80 °C for subsequent protein and RNA extraction for western blotting and RT-qPCR analysis as per instructions. This study was approved by the ethics committee of the Southern Medical University.
Cell proliferation assay
CCK-8 assay was performed to evaluate the viability and proliferation of melanoma cells after Isoliquiritigenin (ISL) treatment. To evaluate the viability of melanoma cells after Isoliquiritigenin (ISL) treatment, A375/A2058 cells were inoculated in 96-well plate and treated with indicated concentration of ISL for 24 h, then the supernatant was removed and complete media containing 10% CCK-8 was added to each well, the 96-well plate was incubated at 37 °C for 2 h, the optical density was read by Varioskan LUX Multimode Microplate Reader (Thermofisher, USA). For the detection of cell proliferation of melanoma cells, A375/A2058 cells were treated with ISL for 24, 48 and 72 h, then the supernatant was removed and complete media containing 10% CCK-8 was added, the plate was incubated at 37 °C for 2 h and read the optical density.
For colony formation assays, 600 cells were inoculated into 6-well plates with 2 mL Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. After 14 days, the resulting colonies were rinsed with PBS, and then fixed with 4% paraformaldehyde for 10 min, and stained with Giemsa (Sigma, USA) for 40 min, then rinsed with PBS again. Only the visible colonies (diameter > 50 mm) were counted.
Flow cytometry
Cell apoptosis was evaluated by flow cytometry(BD Biosciences, USA), A375 cells and A2058 cells were treated with indicated concentration of ISL for 24 h, respectively. 195 μL Annexin V-fluorescein isothiocyanate (Beyotime, China) and 5 μL propidium iodide were added according to the manufacturer’s protocol, and samples were then incubated for 10 min in dark place at room temperature prior to flow cytometric analysis (BD Accuri C6; software version 1.0.264.21; BD Biosciences).
High-throughput genome sequencing
Total RNA was extracted with RNeasy Mini Kit (Cat# 74106, Qiagen) according to the instruction. An Agilent Bioanalyzer and a NanoDrop ND-2000 spectrophotometer 2100 (Agilent Technologies, Santa Clara, CA, USA) were used to determine the RNA quality. Total RNA(initial volume 1 μL) was used to establish a MiRNA library using the TruSeq Small RNA Sample Prep kit (Illumina, Inc). Sample preparation was performed using the acceptable miRNA library according to the method described in the Illumina HiSeq 2500 User Guide and the ultimate concentration for each sample was 10 pM.
Lentiviral transfection
Lentivirus containing pre-miRNA-301b expressed in a lentiviral vector (pLKO.1-puro) were generated in 293 T cells as previously described [
33]. Briefly, the coding oligo-nucleotides of antisense human miR-301b mimics and NC sequence were cloned and inserted into a lentivirus expression vector, pLKO.1-puro. The miR-301b mimics and NC viral particles were produced in 293 T cells (CRL-3216; ATCC, Manassas, VA, USA) via lentivirus expression vector co-expressed with pPACK packaging system (Systems Biosciences, Palo Alto, CA, USA). A375 and A2058 cells were transfected by miRNA 301b-GFP-expressing lentiviruses with the multiplicity of infection or MOI equals to 40. Transfection monitoring was performed by observing the GFP positive cells under a Fluorescent microscopy.
In vivo tumor model
8-week-old immunocompromised mice were injected with 3 × 106 A2058 cells into the flanks. The growth of the tumors was observed by naked eyes. After the tumor reached 80 mm3 in volume, the mice were divided into 3 treatment group at random: PBS + miR-301b angomir control (5 nmoL), ISL(20 mg/kg) + miR-301b angomir control (5 nmoL), ISL(20 mg/kg) + miR-301b angomir (5 nmoL). For the treatment of Si-LRIG1, mice were divided into 3 treatment group: PBS + si-NC, ISL + si-NC, ISL + si-LRIG1. MiR-301b/angomir and Si-LRIG1/NC was injected into the tumors while ISL into abdominal cavity every other day after the tumors reached the desired size. The animals were sacrificed after 6 weeks treatment and the tumors were excised for pathological examination.
Histological analysis
Samples were fixed in 4% paraformaldehyde and then embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed as previously described [
34]. For immunohistochemistry analysis, the paraffin sections were deparaffinized, and cooked in citrate buffer (2.1 M citric acid, pH 6.0) at 120 °C for 30 min for antigen retrieval followed by incubation in 5% BSA(Sigma Aldrich, USA) to block nonspecific binding. And the sections were incubated in primary antibody overnight at 4 °C. The sections were then washed with PBS and incubated for 15 min at room temperature in a solution of anti-rabbit IgG (Abcam, UK). For detection of apoptosis index, sections were stained with TdT-mediated dUTP Nick-End Labeling(KeyGen BioTECH, China) according to the manufacturer’ instructions. Nucleus were stained with DAPI and the sections were processed using Histostain Plus and DAB kits, and the pictures were captured using a light microscope.
Prediction of target genes of miR-301b
Melanoma-associated miRNA Microarray dataset GSE46517 and GSE15605 was downloaded from Gene Expression Omnibus (GEO) database (
http://www.ncbi.nlm.nih.gov/geo/). The dataset GSE46517 and GSE15605, based on the platform as GPL96 and GPL570, respectively. GSE46517 included thirty-one tumor samples from melanoma patients and eight normal skin samples without melanoma as control, GSE15605 included forty-six tumor samples from melanoma patients and sixteen normal skin samples without melanoma as control. Differentially expressed genes (DEGs) were screened using the online tool GEO2R/R package limma, and DEGs between melanoma group and control group were screened and selected by the cut-off point of
P < 0.05 and the [Log FC(fold change)] ≥ 1.5. TargetScan (
http://www.targetscan.org) was used to predict the target genes of miR-301b. The common target genes predicted by TargetScan and GEO database were selected as potential target genes of miR-301b.
Luciferase reporter assay
MiR-301b binding sites of LRIG1 3’UTRs were amplified by PCR from human total cDNA. The PCR product was then subcloned into NheI and SalI sites of pMIR-Report vector to generate the pmiR-LRIG1-Luc reporter constructs as described previously [
35,
36]. Wild-type and mutagenic binding sequence of the target gene LRIG1 are listed in Additional file
1: Figure S2B. Constructed wild-type luciferase reporter (WT), mutant luciferase reporter(MUT) or empty vectors were co-transfected with miR-301b mimics or its corresponding negative control into MSC. The luciferase activity was assessed with a Double-Luciferase Reporter Assay Kit, purchased from Promega Biotech Co., Ltd. (Beijing, China), using the Dual-Light Chemiluminescent Reporter Gene Assay System (Berthold, Germany), which was normalized to firefly luciferase activity.
RT-qPCR
Total RNA extraction was performed using the Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instruction. The cDNAs were synthesized using a PrimeScript RT reagent kit (TaKaRa, Japan) with the following conditions: 37 °C for 15 min, 85 °C for 5 s, hold at 4 °C. For validation of the differentially expressed miRNAs, miRNA was reverse-transcribed using specific RT primers provided with the TaqMan MicroRNA Assay (Applied Biosystems). The reverse transcription was performed at following conditions: 16 °C for 30 min; 42 °C for 30 min, and 85 °C for 5 min. The miRNA cDNAs were amplified using the TaqMan Universal PCR master mix II (Applied Biosystems) with specific probe provided in the TaqMan Small RNA Assay (Applied Biosystems). This process was performed using ABI Prism 7500 HT sequence detection system (Applied Biosystems, Foster City, CA) at the conditions: 3 min at 95 °C, 15 s at 95 °C and 30 s at 60 °C for 40 cycles. The β-actin and U6 were used as loading controls for quantitation of mRNA and miRNAs, respectively. Sequences of mRNA primers used for RT-qPCR in this study were listed in Additional file
2: Table S1.
Western blot
Proteins were extracted from cultured A375 and A2058 cells using RIPA solution (Beyotime, China) with protease inhibitor and phosphatase inhibitor. And protein concentration was measured using BCA Protein Assay Kit (Thermofisher, USA). Next, protein sample was separated via SDS-PAGE and electro-transferred onto PVDF membranes. Following blockade with 5% BSA, membranes were incubated with the following antibodies (all purchased from Abcam, UK) overnight at 4 °C:bcl-2 (1:1000), bax (1:1000), cleaved-parp (1:1000), cleaved-caspase-3 (1:1000),LRIG1 (1:1000), β-actin (1:1000). The membrane was then incubated with HRP-conjugated IgG (1:5000) for 2 h at room temperature. Immunoblots were quantified using Image Lab (version 2.0) software.
Statistical analysis
Data with normal distribution were presented as mean ± SD. Student’s t-test was applied for comparison between two groups, and One-way ANOVA followed by Tukey comparison test was used for comparison between at least three groups. All statistical analyses were performed using SPSS 21.0 software. P < 0.05 was considered statistically significant.
Discussion
Isoliquiritigenin (ISL) is the active medicinal component isolated from the root of licorice, which possesses great therapeutic value in the treatment of various human diseases based on its biological properties such as anti-oxidant, anti-inflammation and anti-cancer activities. In particular, the anti-cancer effect of ISL has been researched in wide-spectrum of malignant tumors. For instance, the cycle of prostate cancer cells was arrested and the apoptosis was increased after ISL application [
37]. Application of ISL also showed desirable efficacy on the treatment of human brain glioma by stimulating the differentiation of glioma stem cells [
38]. And ISL was deemed to enhance WIF1 gene expression via promoting the demethylation of its promoter, which exerted the anti-cancer activity against breast cancer [
39]. And an alternative study has proposed that ISL inhibited breast cancer metastasis by downregulating COX-2 and CYP4A signaling [
40]. In human lung cancer cells, activation of wild type or mutant EGFR was suggested to mediate the ISL induced apoptosis, p53 and p21 upregulation was also verified to be part of the mechanism [
41,
42]. In malignant melanoma, ISL induced reprogramming of melanoma cells by activating mTORC2-AKT-GSK3β signaling,and it has the same effect on mouse melanoma cells [
30,
31]. In accordance with these findings, we showed that ISL significantly suppressed the cell growth and induced apoptosis of two human melanoma cell lines. Our data confirmed the inhibitory role of ISL played in the treatment of melanoma. We used high-throughput sequencing to screen out the most differentially expressed miRNAs in response to the ISL treatment, and miR-301b demonstrated the most dramatic expression alteration.
Cumulative researches of the functions of miR-301b in tumor progression have been carried out in the past few years. MiR-301b has been shown to promote the proliferation, migration and aggressiveness of human bladder cancer cells by targeting EGR1 or through FAK and Akt phosphorylation by regulating PTEN [
43,
44]. The underlying mechanism of the malignant pancreatic cancer aggressiveness driven by migration inhibitory factor was through upregulation of miR-301b [
45]. And the diagnostic indicative value of miR-301b has also been extensively interrogated, miR-301b along with other miRNAs were abundant in plasma of patients diagnosed with Acute myelocytic leukemia(AML), revealing that it may be a sensitive therapeutic signature of AML. Consistent with these studies, we provided proofs in favor of the crucial role of miR-301b in mediating the anti-cancer effect of ISL on melanoma. Relative expression of miR-301b in A375 and A2058 cells was significantly restrained by the ISL administration, and ectopic introduction of miR-301b partially abolished the ISL elicited suppression on melanoma cells. Intratumoral overexpression of miR-301b ablated the growth inhibition and apoptosis promotion induced by ISL in vivo.
Mechanistically, we traced the pathological role of miR-301b through identification of the candidate target genes. Based on GEO and bioinformatics analysis, we confirmed that LRIG1 exhibited the most significant expression change upon the treatment with miR-301b. LRIG1 belongs to LRIG gene family and functions as a tumor suppressor, and its high expression is associated with an increased survival in various tumor types including breast cancer, ovarian cancer, uterine cervical cancer, cutaneous squamous cell carcinoma, nasopharyngeal, oropharyngeal cancer, non-small cell lung cancer and hepatocellular carcinoma [
46,
47]. Our data demonstrated that LRIG1 is a potential modulator in anti-cancer activity of ISL against melanoma, as specific knockdown of LRIG1 decreased cell apoptosis exerted by ISL. Invalidation of the relationship between LRIG1 and miR-301b suggested that the expression of LRIG1 in melanoma cells declined drastically upon the ectopic expression of miR-301b, and intratumoral expression level of LRIG1 was significantly restored when miR-301b was silenced. At the genetic level, we found that miR-301b had a binding site on the 3’UTR of LRIG1. Clinically, the expression of LRIG1 in tumor samples obtained from melanoma patients was decreased compared to normal skin samples, while the expression of miR-301b showed the contrary tendency, indicating a negative correlation between LRIG1 and miR-301b. This experimental evidence denoted that LRIG1 is a functional target of miR-301b and mediates the biological behavior of ISL in the treatment of melanoma.