Background: MicroRNAs (miRNAs), which may function as either tumor suppressor genes or oncogenes, exert considerable regulatory influence on biological processes. Previous studies have shown that miR-455-3p is significantly downregulated in hepatocellular carcinoma (HCC); however, the specific role of miR-455-3p in HCC is unclear.
Methods: We used qRT-PCR to detect the levels of miR-455-3p in HCC cells. We used colony formation assay, EdU assay, and CCK-8 assay to check the effect of miR-455-3p on the proliferation of HCC and HuH-7 cells. Further, transwell and scratch assays were employed to identify the role of miR-455-3p in invasion and migration in these cells. Western blot analysis, angiogenesis assay, dual-luciferase reporter gene assay, and bioinformatic analysis were also performed to investigate the underlying molecular mechanisms.
Results: MiR-455-3p was down-regulated in the HCC cells. In vitro experiments showed that miR-455-3p overexpression inhibits HCC cell proliferation, migration, and invasion. Interestingly, vascular endothelial growth factor C (VEGFC) was identified as a downstream target of miR-455-3p. Additionally, overexpression of miR-455-3p in HCC cells and human umbilical vein endothelial cells (HUVECs) led to a decreased expression of VEGFC and the vascular endothelial growth factor receptor (VEGFR)-2. This subsequently led to a downregulation of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway. Furthermore, in vivo overexpression of miR-455-3p significantly suppressed the tumorigenicity of HuH-7 cells in nude mice and inhibited both angiogenesis and lymphangiogenesis in tumor xenografts.
Conclusions: Our findings suggest that miR-455-3p plays a role in HCC tumorigenesis, at least in part by modulating angiogenesis and lymphangiogenesis by targeting VEGFC and simultaneously blocking the AKT signaling pathway.
As the sixth most ubiquitous cancer worldwide, liver cancer is the fourth leading cause of cancer-related deaths, with nearly one million cases reported in 2016[1, 2]. Survival rates for patients with hepatocellular carcinoma (HCC) are low, with more than 700,000 deaths per year and only 10% survival two years after diagnosis in the UK. According to one study, 27,000 liver cancer patients died in the United States in 2016, with a 5-year survival rate of only 15%. Chronic hepatitis B and C virus infections are the most common risk factors for HCC development, with no curative treatment available for most patients .
MicroRNAs (miRNAs) are endogenous non-coding small RNAs with a length of 19–24 nucleotides. These are known to exert important effects by targeting the 3′-terminal non-coding region of target gene mRNAs, thus causing transcriptional repression or mRNA degradation[7, 8]. Several studies have shown miRNAs to exert considerable regulatory influence on the progression of various diseases, especially cancer[9, 10]. Interestingly, some miRNAs were found to act as tumor suppressors in HCC, like miR-17-5p, miR-20a, miR-202, and microRNA-214. HCC is a typical hypervascular tumor whose growth and metastasis depend on angiogenesis[15-17]. Given that miRNAs function as key modulators of angiogenesis and lymphangiogenesis[18, 19], we studied the role of miRNAs in the pathogenesis of HCC, which are potential targets for early diagnosis and treatment.
Previous reports have shown miR-455-3p to be aberrantly expressed in salivary gland adenoid cystic carcinoma and polymorphous adenocarcinoma. A study from Jiang et al., analyzed RNA microarray data (GEO GSE98269) and found miR-455-3p to be significantly downregulated in HCC, and could be used as a biomarker of HCC. Lan et al., also predicted the low levels of miR-455-3p with a poor prognosis in HCC. Moreover, various other studies have demonstrated miR-455-3p to inhibit the progression of tumors. For example, microRNA-455-3p is known to target eIF4E and function as a tumor suppressor in prostate cancer . By targeting FAM83F, miR-455-3p inhibits the proliferation and invasion of esophageal squamous cell carcinoma cells. . In pancreatic cancer cells, miR-455-3p downregulation is correlated with proliferation and drug resistance via the TAZ pathway. However, the involvement of miR-455-3p in the pathogenesis of HCC remains unclear. Here, we aimed to explore the role of miR-455-3p in the pathogenesis of HCC and its specific mechanism.
Central regulators of vasculogenesis, angiogenesis, and lymphangiogenesis include VEGFs (VEGFD, VEGFC, VEGFB, VEGFA, and PlGF, or placental growth factor), as well as their endothelial tyrosine kinase receptors. VEGFC matures after extensive proteolytic processing within cells. Mature VEGFC binds to both VEGFR-3 and VEGFR-2, which are mostly present in lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) [27-29]. In tumor cells, VEGFC and its cognate receptors are known to partly facilitate tumor progression [30-32].
In this study, we found miR-455-3p to be downregulated in HCC cells, and miR-455-3p to function as a tumor suppressive miRNA by targeting VEGFC. In addition to VEGFC and VEGFR-2 expression, ectopic expression of miR-455-3p led to inhibition of cell invasion, migration, and proliferation. In an HCC xenograft treatment model, miR-455-3p overexpression inhibited tumor growth, angiogenesis, and lymphangiogenesis. In summary, we propose that miR-455-3p plays a major role in HCC progression, and provides a new potential target for therapy in patients with HCC.
Five HCC cell lines (HB611, HHCC, H-97, HuH-7, Li-7), a normal liver cell line (THLE-3), and human umbilical vein endothelial cells (HUVECs) were acquired from Procell (Wuhan, China). We verified the authenticity of each cell line using short tandem repeat analysis. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS; Gibco, USA), in a 5% CO2 humidified incubator at 37 °C.
qRT-PCR (Quantitative reverse transcriptase-polymerase chain reaction)
Total RNA from cells was extracted using the RNeasy Mini Kit (Qiagen, Germany). Reverse transcription was performed using the Quantitect Reverse Transcription Kit (Qiagen, Germany), according to the manufacturer’s instructions. Amplification was performed in triplicates on a LightCycler 480 platform (Roche, Basel, Switzerland) for each sample. Relative expression levels of genes were normalized to GAPDH levels. Following are the primer sequences used:
MiR-455-3p: 5′-ACACTCCAGCTGGGGCAGTCCATGGGCAT-3′ (F),
Western blot analysis
Protein lysates were prepared using RIPA buffer (Thermo Scientific Inc., Waltham, MA, USA) containing 1% Thermo Scientific Halt Protease Inhibitor Cocktail (Product No. 78410). Western blotting was performed in three independent experiments. Protein lysates were subjected to electrophoresis on a 4% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were electrotransferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked using 5% non-fat dry milk in TBS and probed with anti-MMP-9 (1:1000, ab76003; Abcam, Shanghai, China), anti-VEGFC, anti-VEGFR-2 (1:1000, sc-374628, sc-6251 respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-FoxO1, anti-pFoxO1, anti-pAKT, anti-AKT, and anti-β-actin (1:1000, #3195, #13116, #46173, #2880, #2486, #4056, #9272, #4970, respectively; Cell Signaling Technology, USA) primary antibodies diluted in TBST (0.1% Tween 20 in TBS). Immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG (1:1000, Cell Signaling Technology, USA) using chemiluminescence (Pierce® ECL kit).
Cell proliferation assays
Cell proliferation assay using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan), was performed according to the manufacturer’s instructions. We seeded 1 × 103 cells per well and cultured them for 1, 2, 3, 4, and 5 days. Ten microlitres of the CCK-8 solution was added to each well and incubated at 37 °C for 4 h. The absorbance was measured at 450 nm using a microplate reader (MultiSkan Spectrum).
For the EdU assay, we used an EDU-labeled solution (KeyGen Biotech, Nanjing, China) for cell culture for 2 h. As recommended by the manufacturer, EdU staining was performed after 15 min of cell fixation in 4% paraformaldehyde.
For colony-formation assay with trypsin–collagenase, we treated transfected HCC cells to separate them into a single cell suspension, and seeded 400 cells/well into 12-well plates before culturing them at 37 °C for 14 days in a 5% CO2 incubator. The cells were then washed twice with PBS, stained with 2% crystal violet for 15 min, and dried at room temperature. The number of clones formed on the plate were counted. Here we report measurements were taken over three independent experiments, with the mean number of clones obtained in each experiment.
Cells were seeded in six-well plates and cultured till they reached confluence. These confluent monolayer cells were then scratched and washed thrice with PBS to clear suspended cells and cell debris. Fresh serum-free medium was added and cells were allowed to close the wound for 24 h. Comparable images were taken keeping the wound position same using a computer-assisted microscope (Nikon).
Cell invasion experiments were performed using a transwell chamber (8-µm pore size; Corning, USA). In the upper chamber, HCC cells were seeded at a density of 105 cells/well with DMEM containing 1% FBS. The upper chamber was precoated with Matrigel (Corning, USA, dilution ratio: 1:6), and 600 μL of DMEM containing 10% FBS was used in the lower chamber. Cells that had migrated or invaded the lower surface of the membrane were fixed with 4% methanol. After incubation at 37 °C for 24 h, they were stained with crystal violet. Reported here are results obtained from three independent experiments.
Generation of stable cell lines expressing miR-455-3p
A genomic DNA fragment containing miR-455-3p was amplified from the human normal liver cell line (THLE-3), and cloned into the pcDNA-copGFP vector (System Biosciences, USA). The lentivirus vector expressing miR-455-3p was named Lv-miR-455-3p. Lentiviral vectors Lv-miR-455-3p or Lv-miR-NC (used as a negative control) and lentiviral packaging plasmids were co-transfected into 293FT packaging cells using Lipofectamine2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. Forty-eight hours after transfection, the lentivirus in the supernatant was collected, filtered, and used to infect the HCC cells and HUVECs. After antibiotic selection for two weeks, stable clones were obtained, and the expression of mature miR-455-3p was confirmed by qRT-PCR.
Tumor xenograft treatment model
Experimental procedures for animals were approved by the Institutional Animal Care and Use Committee of Guangxi Medical University. A suspension of Lv-miR-NC or Lv-miR-455-3p transfected HuH-7 cells (1 × 107) was injected subcutaneously into the left flank of each BALB/c nude mouse (5–6 weeks of age, 20–22 g, n=3 in each group). All the mice were purchased from the Laboratory Animal Center (Shanghai, China) and maintained under specific pathogen-free conditions in laminar flow cabinets. The volume of the tumor was calculated using the formula: V = 0.5 × L (length) × W2 (width).
Dual-luciferase reporter gene assay
We cloned the 3′-UTR region of human VEGFC mRNA downstream of the luciferase reporter gene. We first amplified the region using PCR and then transferred it into a pGL3-basic vector (Promega, Madison, WI, USA). The construct was named as VEGFC Wt. Using PCR and a site-directed mutagenesis kit (Takara), which acted as a template, the mutated 3′-UTR was generated. We inserted the mutated sequence into the luciferase reporter and named it VEGFC-Mut. Human HHCC and HuH-7 cells were co-transfected with mock Lv-miR-NC or Lv-miR-455-3p vector and a firefly luciferase reporter - VEGFC Wt or VEGFC-Mut for 48h. Cells were then harvested and treated with the Dual Luciferase Assay Kit l.
In vitro angiogenesis assays
We determined the in vitro angiogenic activity of HUVECs using the tube formation assay. After transfection, HUVECs were serum-starved for 24 h in endothelial basal medium (EBM; Clonetics, CA, USA) with 0.2% BSA. Post serum starvation, HUVECs were harvested and seeded in a 12-well plate with 8 × 104 cells/well, which were coated with Matrigel basement membrane matrix (BD Biosciences, USA). Tube formation was observed after 8 h of incubation using a computer-assisted microscope (Nikon). Tube formation is defined by a tube-like structure with a length four times its width. We captured images of tube morphology at 100× magnification and measured the number of tubes using the LAS software (Leica).
Statistical analyses were performed using the SPSS statistical software (16.0 for Windows). Experimental data are presented as mean ± standard deviation (SD). For normally distributed data with equal variance, the difference was evaluated by the two-tailed Student t test (for 2‐group comparisons) or ANOVA followed by the post hoc Bonferroni test (for multigroup comparisons), as appropriate. For non-normally distributed data or data with unequal variances, the difference was evaluated using a nonparametric Mann-Whitney U test (for 2‐group comparisons) or the Kruskal-Wallis test followed by the post hoc Bonferroni test (for multigroup comparisons). Values of P < 0.05 were considered statistically significant. Data presented here was obtained from at least three biologically independent experiments.
miR-455-3p expression is down-regulated in human HCC tumor cells
RNA microarray data (GEO GSE98269) analyzed by Wen et al., and further studies establish that miR-455-3p was downregulated in HCC . Using qRT-PCR, we assessed miR-455-3p levels in human HCC cell lines (HB611, HHCC, H-97, HuH-7, and Li-7) and normal liver cell line (THLE-3) to determine the involvement of miR-455-3p in the regulation of human HCC progression. Compared to that in THLE-3 cell, miR-455-3p was downregulated in HCC cell lines, as shown in Fig. 1.
Overexpression of miR-455-3p suppressed the proliferation, migration, and invasion of HCC cells
We used HHCC and HuH-7 cells to probe the function of miR-455-3p. We overexpressed miR-455-3p and studied its effects on cell invasion, migration, and proliferation. The transfection efficiency is shown in Fig. 2A. Cell proliferation was examined using CCK-8, EdU, and colony formation assays. The growth rates of HHCC and HuH-7 cells were markedly decreased upon miR-455-3p overexpression (Fig. 2B-D). We then used the scratch assay to investigate the influence of miR-455-3p overexpression on cell migration. Compared to control cells, miR-455-3p-overexpressing cells showed a significantly slower migration, as shown in Fig. 2E. In addition, the invasiveness of miR-455-3p overexpressed cells (as measured using the transwell assay) was significantly lower than that of control cells (Fig. 2F). Next, we investigated the potential mechanisms by examining the expression levels of MMP-9 (a crucial matrix metalloproteinase in HCC cell metastasis) [33-35]. The over-expression of miR-455-3p decreased MMP-9 protein levels in HCC cells (Fig. 2G). As epithelial–mesenchymal transition (EMT) has a vital role in HCC cell migration and invasion, we further explored the effect of miR-455-3p on EMT. The expression of epithelial marker E-cadherin, and mesenchymal markers N-cadherin and Vimentin were detected. As shown in Fig. 2G, over-expression of miR-455-3p remarkably reduced the expression of N-cadherin and Vimentin, conversely induced E-cadherin expression at protein levels.
miR-455-3p directly targets the VEGFC gene
Using miRDB and starBase, we searched for candidate target genes of miR-455-3p. We then selected potential targets from the overlap between miRDB (Score>80) (Supplementary Table 1) and starBase (the intersection of PITA, miRmap, microT, TargetScan) (Supplementary Table 2). Through literature review, we identified VEGFC as a potential target of miR-455-3p. The binding sequences of miR-455-3p and VEGFC are shown in Fig. 3A. To verify the targeted binding between miR-455-3p and VEGFC we used a dual-luciferase reporter gene assay in HHCC and HuH-7 cells (Fig. 3B). The relative fluorescence activity in the miR-455-3p group was dramatically decreased in the cells transfected with VEGFC Wt. However, cells co-transfected with VEGFC-Mut did not show this effect. Further, in miR-455-3p-overexpressing HCC cells, the protein level of VEGFC was significantly lower, as shown by western blot analysis (Fig. 3C). These results suggested that VEGFC is a downstream target of miR-455-3p.
Studies have indicated that VEGFC could perform multiple biological functions to promote tumor progression, such as having autocrine stimulation effects on the expression of VEGFR-2, which is required for tumor angiogenesis and lymphangiogenesis[36, 37]. Thus, we tested whether miR-455-3p–mediated VEGFC inhibition could affect the expression of VEGFR-2 in HCC. Compared to Lv-miR-NC cells, the expression of VEGFR-2 protein in miR-455-3p-overexpressing HCC cells was significantly reduced (Fig. 3C). The phosphatidylinositol 3-kinase (PI3K)-AKT pathway, which is critical for cellular survival and growth, is activated by VEGFRs both in cancer cells and in endothelial cells[38-41]. Hence, we further examined whether this pathway is involved in miR-455-3p-mediated growth suppression. We found that over-expression of miR-455-3p in HCC cells significantly decreased the extent of AKT and FoxO1 phosphorylation. This confirmed that the effect of miR-455-3p on cell survival and growth involves the AKT signaling pathway (Fig. 3D).
MiR-455-3p reduced tumor growth, angiogenesis and lymphangiogenesis in tumor xenografts
Using HuH-7 cells, we performed in vivo xenograft experiments to investigate whether the expression of miR-455-3p has an effect on tumor growth. Xenograft tumors are shown in Fig. 4A. Mice treated with Lv-miR-455-3p showed a reduced tumor volume and weight when compared to those treated with Lv-miR-NC (Fig. 4B, C). Next, in the Lv-miR-455-3p-treated mice, VEGFC protein levels were seen to be significantly lower than in the Lv-miR-NC treated mice (Fig. 4D). Further, mice treated with Lv-miR-455-3p show remarkably lower the Ki-67 levels than those shown by Lv-miR-NC groups, as indicated by immunohistochemical staining (Fig. 4E). Subsequently, HuH-7 cells were injected into nude mice via the tail vein to establish the lung metastasis model. Six weeks later, lung metastasis was found to be suppressed by over-expression of miR-455-3p, as confirmed by hematoxylin and eosin (H&E) staining of excised lungs (Fig. 4F, G).
We then analyzed angiogenesis and lymphangiogenesis in tumors using immunohistochemical staining with anti-LYVE-1, anti-CD34 and anti-CD31 antibodies. Compared with Lv-NC, the Lv-miR-455-3p treated groups showed remarkable reductions in the lymph and blood vessels’ formations, which was identified by quantitative analysis (Fig. 4H). These results indicated that miR-455-3p expression inhibits tumor progression in vivo, and that miR-455-3p regulates tumorigenesis by inhibiting VEGFC-mediated angiogenesis and lymphangiogenesis.
Overexpression of miR-455-3p inhibited angiogenic tube formation
Because capillary tube formation on Matrigel is a significant angiogenic property of HUVECs, the effect of miR-455-3p on tube formation was further investigated. After transfection with Lv-miR-455-3p or Lv-miR-NC for 48 h, we serum-starved HUVECs for 24 h before seeding them on a Matrigel-coated 12-well plate. Serum-starved Lv-miR-NC-transfected HUVECs plated on Matrigel coated wells for 8 h show well-organized capillary-like structures, as shown in Fig. 5A. However, the tube-forming activity was significantly impaired upon transfection with Lv-miR-455-3p. These results illustrate that miR-455-3p exerts a negative effect on angiogenesis in HUVECs. The AKT pathway is vital downstream of VEGF/VEGFR signaling. We noted that in miR-455-3p-overexpressing HUVECs, VEGFC and VEGFR-2 were negatively regulated (Fig. 5B). Given the importance of AKT pathway and the downregulation of VEGFC and VEGFR-2, we checked the effect of miR-455-3p overexpression on the AKT pathway. Compared to Lv-miR-NC-transfected HUVECs, AKT phosphorylation was significantly reduced in miR-455-3p-transfected HUVECs (Fig. 5C). These results indicated that the inhibition of VEGFC protein by miR-455-3p suppresses VEGFR-induced activation of the AKT signaling pathway, as evidenced by a reduction in AKT phosphorylation.
As shown in Fig. 6, downregulation of miR-455-3p upregulated VEGFC, VEGFR-2, and promoted the phosphorylation of AKT. This was accompanied by an increase in cell proliferation, migration, invasion, angiogenesis, and lymphangiogenesis.
Increasing evidence has demonstrated that miRNAs regulate tumor phenotypes by regulating the expression of signaling pathways and critical genes involved in tumorigenesis and other malignant processes[42, 43]. Our study clearly shows that in HCC cell lines miR-455-3p expression was significantly down-regulated. Further investigations with patient tissues are necessary to confirm the clinical importance of miR-455-3p in HCC.
Lentivirus-mediated miR-455-3p-overexpression in HHCC and HuH-7 cells was established to study the functions of miR-455-3p in HCC. We then conducted a series of in vivo and in vitro experiments. to demonstrate that HCC cell proliferation, colony formation, migration, and invasion was significantly suppressed by miR-455-3p in vitro, and tumor growth was suppressed in nude mice HCC xenografts. These results implicate miR-455-3p as an inhibitor of HCC tumorigenesis.
Through bioinformatics analysis, western blot assay, and dual-luciferase reporter gene assay, we found that miR-455-3p targets the 3-UTR of VEGFC. In addition, VEGFC, VEGFR-2, and AKT phosphorylation, angiogenesis, and lymphangiogenesis were decreased by miR-455-3p overexpression in HCC cells. Our study provides a basis for novel anti-HCC therapies, with particular emphasis on anti-angiogenic and anti-lymphangiogenic approaches.
In conclusion, we show that in HCC, miR-455-3p is downregulated and functions as a tumor suppressor by directly targeting VEGFC. Ectopic expression of miR-455-3p inhibited tumor progression, angiogenesis, and lymphangiogenesis, and simultaneously inhibited the AKT signaling pathway. In summary, these conclusions provide a strategy for targeting the interaction between miR-455-3p and VEGFC, as well as a novel treatment module for HCC.
The data used to support the findings of this study are included within the article.
The authors declare that there is no conflict of interest regarding the publication of this paper.
We have submitted this manuscript in a preprint version on https://www.researchsquare.com/article/rs-632152/v1.
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