Echinacoside inhibits hepatocellular carcinoma progression by targeting the miR-30c-5p/FOXD1/KLF12 axis

Abstract

BACKGROUND:

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-attributed mortality and the primary liver malignancy in the world. Echinacoside is a phenylethanoid glycoside derived from traditional Chinese medicinal herbs which possessed multiple health benefits on humans, including anti-tumor effects.

OBJECTIVE:

This study aimed to demonstrate the function of echinacoside in HCC progression and the involvement of miR-30c-5p/FOXD1/KLF12 axis.

METHODS:

The HepG2 cells were treated by different dose of echinacoside, miR-30c-5p mimic, miR-30c-5p inhibitor, and FOXD1 overexpression lentiviruses or siRNA individually or simultaneously. The cell invasion and migration were measured by transwell assay. RNA and protein levels were tested by RT-PCR and western blot, respectively. The regulatory function of miR-30c-5p on Forkhead box D1 (FOXD1), FOXD1 on Krüppel-like factor 12 (KLF12) was tested by luciferase reporter assay or/and ChIP assay. Meanwhile, a liver cancer lung metastasis mice model was used to examine the functions of echinacoside and miR-30c-5p on HCC metastasis in vivo. Moreover, the correlations among miR-30c-5p, FOXD1, KLF12, and HCC prognosis was analyzed using clinical sample and TCGA database.

RESULTS:

Based on both in vitro and in vivo investigations, we found that echinacoside could inhibit HCC cell migration, invasiveness, and tumor metastasis, and associated with the enhanced miR-30c-5p/FOXD1/KLF12 axis. Furthermore, through analyzing the interactions among intermediate molecules, we revealed that miR-30c-5p, FOXD1, and KLF12üere clinically relevant with each other in HCC patients, correlated with HCC prognosis, and regulated by echinacoside to contribute in the inhibition of HCC progression.

CONCLUSIONS:

These findings suggest that echinacoside could inhibit HCC progression, and the mechanism related to the enhanced miR-30c-5p/FOXD1/KLF12 axis. Moreover, the abovementioned intermediate molecules might serve as prospective biomarkers for HCC prognosis.

Keywords

Echinacoside microRNA FOXD1 KLF12 hepatocellular carcinoma

1. Introduction

Hepatocellular carcinoma (HCC) is the primary hepatic malignancy and the third leading cause of cancer death [1]. It is recognized as the sixth most frequently diagnosed cancer worldwide, and the incidence is still increasing [1, 2]. HCC has an annual diagnosed 865,000 new cases and causes around 758,000 deaths each year in the world, among which over 50% of the cases occurred in China [1, 3]. Liver cancer has become the 2nd leading source of malignancy-relevant death in China, following lung cancer [4]. The primary risk factor of HCC is chronic hepatitis B/C infection, followed by alcohol over-consumption, diabetes, and obesity [5].

Currently, the first-line therapy for unresectable HCC is sorafenib [6, 7]. This multi-kinase inhibitor is effective in inhibiting tyrosine kinase activities that are critical for cell proliferation, tumor progression, and angiogenesis [8]. However, sorafenib therapy requires long-term continuous medication, which induces adverse effects in the patients, such as hand-foot skin reaction, alopecia, abnormalities in digestive and cardiovascular systems, fatigue, hypertension, and weight loss [9, 10]. Moreover, despite the fact that sorafenib is the standard treatment for HCC patients and is regarded as the best option of treatment for advanced HCC at present, it can only extend the survival of patients for a few months [11]. Therefore, proactive studies on locating drug targets and exploring molecular regulatory mechanisms of HCC are crucial for developing efficient HCC therapeutic drugs.

Echinacoside is a natural phenylethanoid glycoside, which is discovered and isolated from the garden plant Echinacea angustifolia DC and Chinese herbal medicines Cistanche and Echinacea [12]. Versatile benefits of echinacoside on human health have been previously demonstrated, including antioxidant, neuroprotective, improving cognitive impairments, liver protection, anti-inflammatory, and anti-tumor, etc. [12]. Moreover, echinacoside is also capable of suppressing various types of human cancers, including HCC [13]. For example, it improved the survival rate of rats by decreasing serum AFP and the number of hepatic nodules [14]; it inhibits HCC cell proliferation through enhancing p21 expression and diminishing protein kinase B (AKT) phosphorylation [15]. The regulatory functions of echinacoside on several tumor-relevant pathways, especially mitogen-activated protein kinase and the phosphoinositide 3-kinase/AKT/HIF-1 $\alpha$ /VEGF pathways, have also been demonstrated [16, 17]. However, little is known about its functions on HCC progression and metastasis, and the molecular mechanism of echinacoside’s anti-tumor effect is still not fully understood.

MicroRNA (miRNA) is a class of naturally occurring, non-coding single-stranded RNA. miRNA acts as a key gene expression regulator and participates in almost all biological processes, including HCC invasion and metastasis [18]. And several miRNAs were reported as novel biomarkers for hepatocellular carcinoma [19]. In our preliminary experiments, we have examined the expression of miRNA biomarkers including miR-17-5p, miR-miR-125a, miR-200a, miR-223-3p, miR-30c-5p, and miR-302c-3p in echinacoside treated HepG2 cells. The expression of miR-30c-5p changes along with the echinacoside dose, while other miRNAs remain unchanged (Fig. S1). Further, miR-30c-5p was predicted to binding the 3’ UTR of FOXD1. And elevated FOXD1 had worse predictions and clinicopathological parameters in most cancers [20]. As a member of the forkhead box (FOX) family of transcription factors, the analysis based on JASPAR database revealed that FOXD1 could potentially regulate the expression of Kruppel-like factor 12 (KLF12). KLFs such as KLF15, KLF4, and KLF10 were reported played crucial role in HCC progression [21, 22, 23]. There was no study about KLF12 function in HCC. Thus, this study designated to examine the efficacy and molecular basis of echinacoside on HCC progression through both in vitro and in vivo investigations. And we investigated whether miR-30c-5p/FOXD1/KLF12 axis participated in HCC progression and echinacoside mediated therapeutic effect.

2. Methods

2.1 Patient information

HCC tumor tissues and the adjacent normal liver tissues were collected from 30 patients who had received surgery treatment at The Seventh People’s Hospital (Shanghai, China) between 2020 and 2021.

2.2 Cell culture and treatment

HepG2 cells were purchased from Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) $+$ 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) $+$ 100 $\mu$ g/mL streptomycin $+$ 100 units/mL penicillin under standard conditions at 5% CO₂, 90% humidity, and 37^∘C. Trypsin was used to harvest cells, which were routinely passaged when $\sim$ 90% confluent at a 1:3 ratio. Media was replaced every 2–3 days. The treatments of 0, 20, 50, and 100 $\mu$ g/mL Echinacoside (Aladdin Biochemical Technology, Shanghai, China) for 24 h were performed on HepG2 cells.

2.3 MicroRNA interference and overexpression

Approximately 2 $\times$ 10⁵ cells/well HepG2 cells in exponential growth phase were transferred to six-well plates. Then, the cells were transfected with miR-30c-5p mimic (50 nM, 5’-UGUAAACAUCCUACACUCUCAGC-3’), miR-30c-5p inhibitor (50 nM, 5’-GCUGAGAGUGUAGGAUGUUUACA-3’), or negative control (NC) RNA (50 nM, 5’-CAGUACUUUUGUGUAGUACAA-3’) using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in accordance with the instructions from the manufacturer.

2.4 Forkhead box D1 (FOXD1) interference

Short interference RNAs (siRNAs) targeting FOXD1 (siFOXD1-1: 5’-GAGCACUGAGAUGUCCGAU TT-3’; siFOXD1-2: 5’-GGAAACAGACAUCGACGUGTT-3’; and siFOXD1-3: 5’-GUCGAGAACUUUA CUGCUATT-3’) and control siRNA (siNC: 5’-UUCUCCGAACGUGUCACGUTT -3’) were obtained from Genepharma (Shanghai, China).

2.5 Construction of lentiviruses

Human FOXD1 cDNA was cloned into pLVX-puro (Clontech, Palo Alto, CA, USA). The 293T cells (cell bank of Chinese Academy of Science, Shanghai) were transfected with the constructed vector and lentiviral packaging vector (psPAX2 and pMD2G, 10:9:1, Addgene) using Lipofectamine2000 (Thermo Fisher Scientific, Inc.) to generate stable cell lines. The third-generation system was used and a MOI of $\sim$ 10 was used to infect cells. Subsequently, the 293T cells were cultured with DMEM-F12 containing 10% FBS, 100 U/ml penicillin and 100 $\mu$ g/ml streptomycin at 37^∘C with 5% CO2 for 48 h. The lentiviral supernatants were then harvested (centrifuged at 3,000g, 4^∘C for 10 min). The HepG2 cells were infected with lentiviral supernatant, incubated at 37^∘C overnight and selected with 1.5 $\mu$ g/ml puromycin for 14 days and maintained with 0.625 $\mu$ g/ml puromycin in the subsequent experiments.

2.6 Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted by using the TRIzol reagent (Invitrogen). Complementary DNA was synthesized with 1 $\mu$ L of total RNA, a special stem-loop primer for microRNA, and random primer for gene expression, using the Revert Aid^TM First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Franklin, MA, USA). Then, qRT-PCR was performed to quantify the expression levels of target genes and microRNAs using the SYBR Green Master Mix (Thermo Fisher Scientific), following the instructions from the manufacturer. GAPDH and $U6$ served as the internal reference genes for measuring target gene and microRNA expression, respectively. The primers are listed in Table S1. The qRT-PCR was conducted on an ABI 7300 thermocycler (Applied Biosystems, Foster City, CA, USA). The thermocycling conditions were as follows: Denaturation 95^∘C for 5 min, followed by 40 cycles of denaturation at 95^∘C for 10 sec, annealing at 60^∘C for 20 sec and extension at 72^∘C for 20 sec. The quantification of gene expression was carried out by using the 2^{- ΔΔCt} method.

2.7 Western blot analysis

Total protein was extracted by using the radio-immunoprecipitation assay buffer (Solarbio, Shanghai, China). Then, the concentration of protein was determined by using the bicinchoninic acid assay (BCA) kit (Thermo Fisher Scientific). Approximately 30 $\mu$ g protein was loaded into each well of SDS-PAGE, followed by transferring onto nitrocellulose membranes (Millipore, Bredford, USA). The membrane was blocked in 5% skimmed milk and probed with primary antibodies at 4^∘C overnight (Table S2). After washed three times using TBST, the membranes were incubated with secondary antibodies at 37^∘C for 1 h. Target protein expressions were detected with the enhanced chemi-luminescence system (Bio-Rad, Richmond, CA, USA). The analysis for band densitometry was conducted with the software of Image J (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA), with the loading control as GAPDH.

2.8 Cell invasion and migration assays

Cell migration was assessed by using an 8 $\mu$ m-pore filter-containing chamber (Corning, New York, NY, USA). The 50% confluent HepG2 cells with the desired siRNAs transfection were maintained in serum-free medium for 24 h, followed by trypsinization. Approximately 5 $\times$ 10⁴ cells were subsequently collected and transferred into the upper chamber. The medium containing 10% FBS was transferred into the lower chamber. The cells were incubated at 37^∘C for 24 h, followed by completely removing all the non-migrating cells. Then, the migrated cells were fixed with 4% paraformaldehyde, followed by staining with 0.5% crystal violet, visualization under microscope, and counting.

For cell invasiveness assay, 1 mg/ml Matrigel was pre-coated in the upper chamber (BD Biosciences, San Jose, CA, USA). The rest procedures of the assay were following those of the cell migration assay.

2.9 Luciferase reporter assay

Complete FOXD1 3’UTR, either at the wild-type (WT) or mutated state, was inserted into pGL3 vector (Promega, Madison, WI, USA). Subsequently, pGL3-WT-FOXD1 3’UTR or pGL3-mutant-FOXD1 3’UTR was transfected into HepG2 cells with miR-30c-5p inhibitor, mimic, or NC. The full-length Krüppel-like factor (KLF) 12 promoter was inserted into pGL3 vector (Promega). The pGL3-KLF12 promoter, siFOXD1-1/siNC, and/or infected with lentivirus expressing FOXD1 (oeFOXD1)/pLVX-puro (Vector) were transfected into HepG2 cells. Firefly luciferase activity was assessed by using the Luciferase assay kit (Promega), normalized to the corresponding Renilla luciferase activity.

2.10 Chromatin immunoprecipitation (ChIP) assay

HepG2 cells were treated by 1% formaldehyde on a rocking platform at room temperature for chromatin crosslinking. Then, the chromatin solution was incubated with anti-FOXD1 (Santa Cruz, sc-293238) or control IgG (Proteintech, 30000-0-AP) antibody overnight with rotation at 4^∘C. The binding was assessed by PCR using the following primers: 5’-GTGCCGCAGCAAAGTGG-3’ (forward) and 5’-AGCAGGCGCTAAGTCGC-3 (reverse).

2.11 Lung metastasis assay

Experimental protocols were approved by the Animal Experimentation Ethics Committee of The Seventh People’s Hospital (approval number: 2018-35). A total of 24 BALB/c male nude mice at six-week-old were raised under the condition of specific pathogen-free. Approximately 1 $\times$ 10⁶ HepG2 cells were intravenously injected into the mice via lateral tail veins to induce experimental lung metastasis. The mice were further randomly and evenly divided by 4 groups ( $n=$ 6 per group): Group 1 (Control), mice were intraperitoneally administered with NC microRNA; Group 2 (ECH-low) and Group 3 (ECH-high), mice were daily intraperitoneally administered with 20 and 50 mg/kg echinacoside ( $\geqslant$ 98%; Aladdin), respectively, for 4 weeks; and Group 4 (mimic), mice were intraperitoneally administered with miR-30c-5p for 4 weeks. The mice were sacrificed after 4 weeks, followed by counting lung metastasis numbers under a dissecting microscope. Then, the lung tissues collected were fixed with 10% formalin and embedded with paraffin. Histological examination was conducted by staining the tissues with hematoxylin and eosin.

2.12 Bioinformatic analysis

The mRNA level of FOXD1 in liver hepatocellular carcinoma (LIHC) and its correlation with LIHC prognosis were analyzed based on The Cancer Genome Atlas (TCGA) LIHC dataset through UALCAN (http://ualcan.path.uab.edu/) [24].

2.13 Statistical analyses

The results were presented as the mean $\pm$ standard deviation of three independent experiments performed in duplicate. The statistical analyses were conducted using the GraphPad Prism software (version 6.0, San Diego, CA, USA). One-way analysis of variance (ANOVA) was applied for analyzing the differences among groups. $P<$ 0.05 was considered as statistically significant.

3. Results

3.1 Echinacoside inhibits HepG2 cell invasiveness and migration

To examine the effect of echinacoside on invasiveness and migration of HepG2 cells, the cells were treated with 0, 20, 50, and 100 $\mu$ g/mL echinacoside for 24 h. We observed that 20, 50, and 100 $\mu$ g/mL echinacoside could significantly ( $P<$ 0.001) reduce the proportions of both migrated and invasive cells in a dose-dependent manner (Fig. 1).

Figure 1.

Effects of echinacoside on HepG2 cell invasiveness and migration. HepG2 cells were treated with 0, 20, 50, and 100 $\mu$ g/mL echinacoside for 24 h. Cell migration and invasion were assessed based on Transwell system. Magnification: 200 $\times$ . ^*** indicates $P<$ 0.001 in related to 0 $\mu$ g/mL echinacoside; ^### indicates $P<$ 0.001 in related to 20 $\mu$ g/mL echinacoside; ⁺ indicates $P<$ 0.05, and ⁺⁺ indicates $P<$ 0.01 in related to 50 $\mu$ g/mL echinacoside.

Figure 2.

Effects of miR-30c-5p inhibitor on HepG2 cell invasiveness and migration. (A) The miR-30c-5p level in HepG2 cells treated with echinacoside (0, 20, 50, and 100 $\mu$ g/mL) for 24 h. ^** and ^*** represent $P<$ 0.01 and $P<$ 0.001, respectively, in related to 0 $\mu$ g/mL echinacoside; ^# and ^### represent $P<$ 0.05 and $P<$ 0.001, respectively, in related to 20 $\mu$ g/mL echinacoside; ⁺⁺ indicates $P<$ 0.01 in related to 50 $\mu$ g/mL echinacoside. (B–D) HepG2 cells transfected with (inhibitor) or without (Control or NC) miR-30c-5p inhibitor were treated by DMSO or 10 $\mu$ g/mL echinacoside (ECH). (B) MiR-30c-5p level was quantified with qRT-PCR. Cell migration (C) and invasion (D) were assessed based on Transwell system. Magnification: 200 $\times$ . ^* and ^*** indicate $P<$ 0.05 and $P<$ 0.001, respectively, in related to Control or NC; ^### represents $P<$ 0.001 in related to inhibitor.

Figure 3.

Interaction between FOXD1 and miR-30c-5p in HepG2 cells. (A) The predicted binding site of miR-30c-5p on FOXD1 3’UTR based on Targetscan analysis. (B) The miR-30c-5p and FOXD1 mRNA levels in HepG2 cells transfected with miR-30c-5p inhibitor, miR-30c-5p mimic, or negative control (NC) were measured with qRT-PCR. ^** and ^*** represent $P<$ 0.01 and $P<$ 0.001, respectively, in related to NC. (C) FOXD1 protein expression in HepG2 cells transfected with NC, miR-30c-5p mimic, or miR-30c-5p inhibitor was analyzed by western blot. (D) The luciferase activity of FOXD1 3’UTR in wild type (WT) or FOXD1 3’UTR-mutated (mutant) HepG2 cells with indicated transfection. ^** and ^*** represent $P<$ 0.01 and $P<$ 0.001, respectively, in related to NC. (E–G) HepG2 cells with (oeFOXD1) or without (Vector) FOXD1 overexpression were co-transfected with NC or miR-30c-5p mimic. (E) FOXD1 protein expression was analyzed by western blot. Cell migration (F) and invasion (G) were assessed based on Transwell system. Magnification: 200 $\times$ . ^** indicates $P<$ 0.01 and ^*** indicates $P<$ 0.001 in related to Vector $+$ NC; ^# indicates $P<$ 0.05 and ^## indicates $P<$ 0.01.

Figure 4.

Effects of FOXD1 on HepG2 cell invasiveness and migration. (A) The mRNA level and (B) the protein level of FOXD1 in HepG2 cells treated with 0, 20, 50, and 100 $\mu$ g/mL echinacoside for 24 h. ^** and ^*** represent $P<$ 0.01 and $P<$ 0.001, respectively, in related to 0 $\mu$ g/mL echinacoside; ^## and ^### represent $P<$ 0.01 and $P<$ 0.001, respectively, in related to 20 $\mu$ g/mL echinacoside; ⁺⁺indicates $P<$ 0.01 in related to 50 $\mu$ g/mL echinacoside. (C–E) HepG2 cells with (oeFOXD1) or without (Vector) FOXD1 overexpression were treated by DMSO or 10 $\mu$ g/mL echinacoside. (C) FOXD1 protein expression was analyzed by western blot. Cell migration (D) and invasion (E) were assessed based on Transwell system. Magnification: 200 $\times$ . ^** and ^**** indicate $P<$ 0.01 and $P<$ 0.001, respectively, in related to Vector; ^### represents $P<$ 0.001 in related to oeFOXD1.

Figure 5.

FOXD1 regulates KLF12. HepG2 cells was transfected with siNC/siFOXD1 or Vector/oeFOXD1 for FOXD1 knockdown or overexpression. (A) Cell migration and invasion were assessed based on Transwell system. Magnification: 200 $\times$ . ^** and ^*** indicate $P<$ 0.01 and $P<$ 0.001, respectively, in related to siNC. (B) FOXD1 and KLF12 protein expression levels. (C) The transcriptional activity of KLF12 promoter assessed by luciferase assay. ^* and ^*** indicate $P<$ 0.05 and $P<$ 0.001, respectively, in related to siNC $+$ Vector. (D) Chromatin immunoprecipitation of FOXD1 and KLF12 promoter.

Figure 6.

Effects of echinacoside and miR-30c-5p on lung metastasis. Echinacoside (ECH-low at 20 mg/kg or ECH-high at 50 mg/kg), miR-30c-5p mimic, or placebo (Control) was administrated by mice with experimental lung metastasis induction. (A) Hematoxylin and eosin staining for pulmonary nodules. Magnification: 100 $\times$ . (B) Quantification of pulmonary nodules. (C) The miR-30c-5p level in pulmonary nodules. (D) The mRNA level of FOXD1 in pulmonary nodules. (E) The mRNA level of KLF12 in pulmonary nodules. ^*, ^**, or ^*** represent $P<$ 0.05, $P<$ 0.01, or $P<$ 0.001, respectively, in related to Control.

Figure 7.

Clinical relevance among miR-30c-5p, FOXD1, KLF12, and hepatocellular carcinoma (HCC). (A) miR-30c-5p level and the mRNA levels of FOXD1 and KLF12 in paired tumorous and adjacent normal tissues from HCC patients. ^*** indicates $P<$ 0.001. (B) Pearson correlations among miR-30c-5p level and the mRNA levels of FOXD1 and KLF12.

3.2 MiR-30c-5p inhibitor counteracts the effects of echinacoside on HepG2 invasiveness and migration

We next examined the effect of echinacoside on microRNAs that were downregulated in HepG2 cells. Echinacoside treatment up to 100 $\mu$ g/mL induced numerical but statistically insignificant ( $P>$ 0.05) alteration on miR-17-5p, miR-125a, miR-200a, miR-233-3p, and miR-302c-3p levels (Fig. S1). Whereas, the cellular level of miR-30c-5p was significantly upregulated by 50 ( $P<$ 0.01) and 100 ( $P<$ 0.001) $\mu$ g/mL echinacoside treatment, and the effect of echinacoside on miR-30c-5p was in a dose-dependent manner (Fig. 2A).

We further transfected miR-30c-5p inhibitor into HepG2 cells to examine the function of miR-30c-5p on cell invasiveness and migration. The transfection of miR-30c-5p inhibitor substantially downregulated the level of miR-30c-5p ( $P<$ 0.05; Fig. 2B) and reduced the proportions of both migrated ( $P<$ 0.001; Fig. 2C) and invasive ( $P<$ 0.001; Fig. 2D) HepG2 cells. Furthermore, it could also substantially ( $P<$ 0.001) counteract the effects of echinacoside on upregulating miR-30c-5p level (Fig. 2B) and inhibiting the migration (Fig. 2C) and invasion (Fig. 2D) of HepG2 cells.

3.3 MiR-30c-5p inhibits HepG2 cell invasiveness and migration by regulating FOXD1

Targetscan analysis predicted the binding between miR-30c-5p and FOXD1 3’UTR (Fig. 3A). Hence, we further transfected both miR-30c-5p mimic and its inhibitor into HepG2 cells (Fig. 3B), to examine the regulatory function of miR-30c-5p on FOXD1. We found that miR-30c-5p mimic could significantly ( $P<$ 0.01) downregulate FOXD1 at both the mRNA and protein levels, while miR-30c-5p inhibitor significantly ( $P<$ 0.001) upregulated it, in HepG2 cells (Fig. 3C). However, neither miR-30c-5p mimic nor its inhibitor could alter the luciferase activity of FOXD1 3’UTR in HepG2 cells with pGL3-mutant-FOXD1 3’UTR transfection (Fig. 3D), which confirmed that miR-30c-5p could regulate FOXD1 through interacting with FOXD1 3’UTR.

To further examine the mechanism of miR-30c-5p’s effects on cell migration and invasion, we next overexpressed FOXD1 in HepG2 cells. The overexpression of FOXD1 substantially ( $P<$ 0.001) increased the proportions of both migrated (Fig. 3F) and invasive (Fig. 3G) HepG2 cells, while on the contrary, miR-30c-5p mimic downregulated the protein level of FOXD1 (Fig. 3E) and substantially ( $P<$ 0.001) inhibited HepG2 cell migration (Fig. 3F) and invasiveness (Fig. 3G). Meanwhile, miR-30c-5p mimic could also significantly counteract the promotive effects of FOXD1 overexpression on HepG2 cell migration (Fig. 3F) and invasiveness (Fig. 3G). These results indicated that miR-30c-5p suppressed HepG2 cell migration and invasion through downregulating FOXD1.

3.4 Echinacoside can downregulate FOXD1 level in HepG2 cells

We further explored the connection between echinacoside and FOXD1, and found that echinacoside treatment (0–100 $\mu$ g/mL) could notably downregulate FOXD1 in HepG2 cells following a dose-dependent manner at both the mRNA (Fig. 4A) and protein (Fig. 4B) levels. Moreover, the overexpression of FOXD1 in HepG2 cells noticeably reversed the effectiveness of echinacoside on downregulating FOXD1 protein expression (Fig. 4C) and inhibiting cell migration ( $P<$ 0.01; Fig. 4D) and invasiveness ( $P<$ 0.01; Fig. 4E), indicating that the suppressive effects of echinacoside on HepG2 cell invasiveness and migration were achieved by downregulating FOXD1.

3.5 FOXD1 promotes HepG2 cell invasiveness and migration via regulating KLF12 transcription

In order to confirm the effects of FOXD1 on the invasiveness and migration of HepG2 cells, we next interfered FOXD1 expression in HepG2 cells (Fig. S2). The proportions of both migrated ( $P<$ 0.001 for both siFOXD1-1 and 2) and invasive ( $P<$ 0.001 for siFOXD1-1 and $P<$ 0.01 for siFOXD1-2) FOXD1-deficient HepG2 cells were substantially smaller compared with those of the control cells (Fig. 5A), which demonstrated the promotive effects of FOXD1 on HepG2 cell invasiveness and migration.

The analysis based on JASPAR database revealed that FOXD1 could potentially regulate the expression of KLF12 (data not shown), which led us to further investigate the interaction between these two molecules. We observed that FOXD1 knockdown in HepG2 cells downregulated the protein level of KLF12 (Fig. 5B). Meanwhile, the transcriptional activity of KLF12 promoter was considerably elevated ( $P<$ 0.001) by FOXD1 overexpression while decreased ( $P<$ 0.05) by FOXD1 knockdown (Fig. 5C). Based on ChIP assay, we also confirmed that FOXD1 could directly bind to KLF12 promoter (Fig. 5D). These results demonstrated that FOXD1 could regulate KLF12 expression by binding with its promoter and promoting its transcription.

3.6 Both echinacoside and miR-30c-5p suppress lung metastasis by regulating FOXD1 and KLF12

To examine the functions of echinacoside and miR-30c-5p on HCC metastasis in vivo, specific pathogen-free mice were intravenously injected with HepG2 cells and intraperitoneally administered with placebo, 20 mg/kg echinacoside, 50 mg/kg echinacoside, or miR-30c-5p. We observed that HepG2 cell injection induced lung metastasis in the control mice, whereas, both echinacoside and miR-30c-5p administration inhibited lung metastasis in vivo (Fig. 6A). The numbers of lung modules were significantly ( $P<$ 0.001) reduced in the mice administered with 20, 50 mg/kg echinacoside, and miR-30c-5p (Fig. 6B). The levels of miR-30c-5p in pulmonary nodules were significantly upregulated by 20 ( $P<$ 0.05), 50 ( $P<$ 0.001) mg/kg echinacoside, and miR-30c-5p ( $P<$ 0.001) administration (Fig. 6C). Additionally, the transcript levels of FOXD1 and KLF12 in pulmonary nodules were significantly downregulated by 20 ( $P<$ 0.01), 50 ( $P<$ 0.001) mg/kg echinacoside, and miR-30c-5p ( $P<$ 0.001) administration (Fig. 6D and E). These results illustrated that echinacoside and miR-30c-5p administration could inhibit lung metastasis in vivo through upregulating miR-30c-5p, thereby downregulating FOXD1 and KLF12 expressions.

3.7 MiR-30c-5p, FOXD1, and KLF12 are clinically relevant and differentially expressed between HCC and adjacent normal tissues

To confirm that the abovementioned intermediate molecules are clinically related to HCC, we further investigated the correlations among miR-30c-5p, FOXD1, KLF12, and HCC prognosis based on both patient cohort and TCGA database. The miR-30c-5p level in HCC tumorous tissues collected from HCC patients was considerably ( $P<$ 0.001) lower than that in the adjacent normal tissues, while the mRNA levels of FOXD1 and KLF12 were considerably ( $P<$ 0.001) higher in HCC tumors than in the normal tissues (Fig. 7A). Furthermore, we observed that the mRNA level of FOXD1 was negatively ( $r=-$ 0.6315, $P=$ 0.0002) correlated with miR-30c-5p level while positively ( $r=$ 0.6015, $P=$ 0.0004) correlated with that of KLF12 (Fig. 7B). Additionally, bioinformatic analyses of TCGA LIHC dataset showed that the transcript level of FOXD1 in primary LIHC tumors was found numerically higher than that in the normal tissues (Fig. S3A); meanwhile, the expression of FOXD1 was significantly ( $P=$ 0.014) correlated with the survival probability of LIHC patients (Fig. S3B), indicating the correlation between FOXD1 expression and LIHC prognosis.

4. Discussion

Echinacoside, as a phenylethanoid glycoside, has been associated with hepato-protective and HCC-counteractive effects [15]. For example, it inhibits tumor growth and prevents tumor invasion in vivo [14]. Here, we focused on the function of echinacoside in HCC progression. The present study suggested that Echinacoside is effective in suppressing HCC cell invasiveness and migration in vitro and preventing HCC metastasis in vivo.

The invasive growth and migration of cells are recognized as the primary manifestations of tumor progression and cancer malignancy [25]. It was reported that dysregulation of microRNAs associated with HCC angiogenesis, endothelial cell permeability, tube formation, and metastasis to pulmonary tissues [26]. The present study investigated whether these microRNAs are responsible for the anti-HCC activities of echinacoside and observed that miR-30c-5p was substantially elevated by echinacoside treatment (Fig. 2, Fig. S1). In addition, we observed that miR-30c-5p inhibitor could restrict the effects of echinacoside on HCC invasiveness and migration (Fig. 2), which further confirms that echinacoside suppresses HCC progression via upregulating miR-30c-5p. As a matter of fact, though the connection between phenylethanoid glycoside and microRNAs has been rarely mentioned, the neuro-protective effect of echinacoside was demonstrated to be partially relied on its regulatory function on microRNA expression [27]. For the first time, our study systematically reported the regulatory role of echinacoside on the expression of microRNA, especially miR-30c-5p.

We predicted and detected that FOXD1 is a target of MiR-30c-5p (Fig. 3). FOXD1 belongs to the forkhead box family, which participates in embryogenesis and cellular homeostasis by functioning as a transcription factor [20]. It also acts as an oncogene and participates in the tumorigenesis of HCC [28], and the expression of FOXD1 was upregulated in HCC tissues [29]. In line with the previous report that miR-30 family could directly target FOXD1 3’UTR [30], we also detected that miR-30c-5p downregulated FOXD1 through interacting with its 3’UTR (Fig. 3). Furthermore, the fact that both echinacoside treatment and miR-30c-5p mimic could counteract FOXD1’s promotive effects on HCC cell invasiveness and migration (Figs 3 and 4) further illustrates that echinacoside or miR-30c-5p suppresses HCC progression through downregulating FOXD1.

Further, we also identified the potential interaction between FOXD1 and KLF12 (Fig. 5). The KLF family represents a group of transcription factors critical for cancer cell proliferation, apoptosis, and differentiation [31]. The aberrant expression of KLF12 has been recognized in different types of human cancers such as lung cancer and colorectal cancer [32, 33]. In the present study, we demonstrated the regulatory role of FOXD1 on KLF12 through binding to its promoter and promoting its transcriptional activity (Fig. 5). Previously, several KLF family members have been reported to promote HCC cell proliferation, invasion, and tumor metastasis [33, 34]. Accordantly, we also revealed the contribution of KLF12 in HCC metastasis (Fig. 6). Based on our observations in the mouse model, we further confirm that echinacoside could upregulate miR-30c-5p while downregulate FOXD1 and KLF12, thereby suppressing the metastasis of HCC in vivo (Fig. 6). Collectively, we propose that echinacoside could inhibit HCC progression through regulating the miR-30c-5p/FOXD1/KLF12 axis.

The correlation and relevance among miR-30c-5p, FOXD1, and KLF12 were revealed by clinical data as well (Fig. 7), which supported their dynamic interplays and simultaneous contribution in echinacoside’s effect on HCC. In fact, miR-30c-5p was suggested as an independent parameter for predicting the overall survival of HCC patients [35]. Echinacoside upregulates miR-30c-5p, which further downregulates FOXD1. FOXD1 can bind to the promoter of KLF12 and enhance its transcriptional activity, thus contributing to the inhibitory effects of echinacoside on HCC cell invasiveness and migration. Furthermore, miR-30c-5p, FOXD1, and KLF12 are clinically relevant in HCC patients, differentially expressed between HCC tumors and adjacent normal tissues, and correlated with HCC prognosis. Overall, the therapeutic efficacy of echinacoside in suppressing HCC progression, together with the uncovered miR-30c-5p/FOXD1/KLF12 axis, suggests its future clinical application for HCC treatment. The deficiencies of the present study were that we did not validate gene knockout mice, and this issue would be resolved in subsequent experiments.

5. Conclusion

Echinacoside is effective in suppressing HCC cell invasiveness and migration in vitro and preventing HCC metastasis in vivo. The mechanism is related to upregulating miR-30c-5p, which further downregulates FOXD1, and then decreases the transcriptional activity of KLF12. Over all, this study suggests the efficacy of echinacoside on restricting HCC progression, and demonstrates that the miR-30c-5p/FOXD1/KLF12 axis is the molecular mechanism underlying the anti-HCC activities of echinacoside.

Funding

The study was supported by National Natural Science Foundation of China (81873178), the Budgetary Fund of Shanghai University of TCM (2019LK097), the Key Discipline Construction Project of Pudong Health Burea of Shanghai (PWZxk2022-04), General project of Shanghai Municipal Health Commission (202040183).

Data availability

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

Ethical approval and consent to participate

The study was approved by the Ethics Committee of The Seventh People’s Hospital of Shanghai University of Traditional Chinese Medicine (approval number: 2018-HIRB-045). Signed written informed consents were obtained from the patients and/or guardians. The animal experimental protocols were approved by the Animal Experimentation Ethics Committee of The Seventh People’s Hospital (approval number: 2018-35).

Supplementary data

The supplementary files are available to download from https://dx-doi-org.web.bisu.edu.cn/10.3233/THC-241449.

Footnotes

Acknowledgments

The authors have no acknowledgments.

Conflict of interest

The authors declare that they have no conflict of interest.

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