The long non-coding RNA MALAT1 promotes the migration and invasion of hepatocellular carcinoma by sponging miR-204 and releasing SIRT1

Abstract

Increasing evidence supports the significance of long non-coding RNA in cancer development. Several recent studies suggest the oncogenic activity of long non-coding RNA metastasis–associated lung adenocarcinoma transcript 1 (MALAT1) in hepatocellular carcinoma. In this study, we explored the molecular mechanisms by which MALAT1 modulates hepatocellular carcinoma biological behaviors. We found that microRNA-204 was significantly downregulated in sh-MALAT1 HepG2 cell and 15 hepatocellular carcinoma tissues by quantitative real-time polymerase chain reaction analysis. Through bioinformatic screening, luciferase reporter assay, RNA-binding protein immunoprecipitation, and RNA pull-down assay, we identified microRNA-204 as a potential interacting partner for MALAT1. Functionally, wound-healing and transwell assays revealed that microRNA-204 significantly inhibited the migration and invasion of hepatocellular carcinoma cells. Notably, sirtuin 1 was recognized as a direct downstream target of microRNA-204 in HepG2 cells. Moreover, si-SIRT1 significantly inhibited cell invasion and migration process. These data elucidated, by sponging and competitive binding to microRNA-204, MALAT1 releases the suppression on sirtuin 1, which in turn promotes hepatocellular carcinoma migration and invasion. This study reveals a novel mechanism by which MALAT1 stimulates hepatocellular carcinoma progression and justifies targeting metastasis–associated lung adenocarcinoma transcript 1 as a potential therapy for hepatocellular carcinoma.

Introduction

Hepatocellular carcinoma (HCC) is the predominant malignancy in the liver and one of the most common cancers worldwide. In China, the striking high incidence of chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) is responsible for the majority of HCC cases that become the third leading cause of cancer-related death.^1,2 Despite intensive efforts to improve early detection and develop novel therapeutic strategies, the incidence of HCC is still on the rise in China. ¹ Furthermore, the highly aggressive nature of HCC predisposes patients to metastasis and a high rate of recurrence. Therefore, it is critical to continue studies on the molecular mechanisms regulating HCC metastatic behavior to benefit novel therapeutics targeting HCC.

Approximately 80% of the human genome is transcribed into RNA, of which only 2% translated into proteins. ³ The non-coding RNAs (ncRNAs) are further classified into three subtypes based on their sizes: short ncRNAs (20–30 nucleotides) best represented by microRNAs (miRNAs); mid-size ncRNAs (100–300 nucleotides) that include small nucleolar RNAs (snoRNAs), promoter-associated small RNAs (PASRs), and transcription start site (TSS)-associated RNAs; and long non-coding RNAs (lncRNAs) that are typically >200 nucleotides.^4,5 Accumulating evidence reveals the diversified mechanisms of lncRNAs in regulating protein functions on the transcriptional, translational, and post-translational levels and their significance in numerous diseases including cancer.^4–6 For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly abundant and conserved lncRNA in mammals.^3,7 The expression of MALAT1 is dynamically regulated on the transcriptional and post-transcriptional levels by multiple factors and it is frequently upregulated in multiple types of cancers.^7,8 In addition to serving as a cancer biomarker, MALAT1 functionally contributes to the malignant behaviors of tumor cells through variable mechanisms in a cancer-type-dependent manner.^7,9 In HCC, MALAT1 is known to stimulate HCC growth and transformation by upregulating serine-/arginine-rich splicing factor 1 (SRSF1) and activating mechanistic target of rapamycin (mTOR) signaling, ¹⁰ to aggravate the growth of HCC stem cells through enhancing the expression of telomere repeat–binding factor 2 (TRF2), ¹¹ and to mediate multi-drug resistance of HCC by acting on miR-26b. ¹² It is not known whether MALAT1 is critical for regulating the migration and invasion of HCC and if so, what molecular mechanisms are involved.

MiRNAs are the best-characterized short (~22 nucleotides) ncRNAs that predominantly regulate gene expression through post-transcriptional silencing of messenger RNA (mRNA). ¹³ Emerging evidence suggests the essential role of miRNAs in HCC development, metastasis, and prognosis.^14–16 Jiang et al. ¹⁷ recently showed that microRNA-204 (miR-204), by directly targeting the nicotine adenine dinucleotide (NAD)-dependent deacetylase sirtuin 1 (SIRT1), suppresses the survival, stimulates the apoptosis, and inhibits the invasion and tumorigenesis of HCC cells. In spite of the therapeutic potential of miR-204, its level is downregulated in HCC tissues and negatively correlated with that of SIRT1. ¹⁷ It is not known, however, what controls miR-204 expression in HCC tissues.

In our previous study, we found that MALAT1 was highly expressed in HCC tissues and promoted cell migration and invasion. However, the molecular mechanisms of MALAT1 regulating HCC metastasis are not clear. In this study, we reported for the first time that MALAT1 acts as a sponge RNA for miR-204, which in turn suppresses tumor invasion and metastasis by targeting SIRT1. Thus, these results suggest that the MALAT1-miR-204-SIRT1 axis critically controls the migration and invasion of HCC cells. This study reveals novel mechanisms for HCC therapy.

Materials and methods

Human tissues and HCC cell lines

This study was approved by the Ethics Committee of Xiangya Hospital Central South University (Hunan, China), and all human samples were acquired with patients’ written consents. A total of 20 fresh tumor tissues and matching para-tumor normal tissues were surgically resected from HCC patients admitted to our hospital between May 2014 and January 2017. All tissues were snap frozen in liquid nitrogen till further use.

The normal human liver cell line LO2 and HCC cell lines Bel7404, Huh7, and HepG2 were acquired from the Cell Bank of Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (PS; 100 IU/mL penicillin, 100 μg/mL streptomycin) in a humidified atmosphere containing 5% CO₂ at 37°C.

Reverse transcription followed by quantitative real-time polymerase chain reaction

Total RNA was extracted from the frozen tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Complementary DNA (cDNA) was then synthesized using TaKaRa reverse transcription system (Dalian, China). Quantitative PCR analysis was performed on ABI-7500 using iQTM SYBR^® Green Supermix (Cat no. 170–3884; Bio-Rad, Hercules, CA, USA) reagent. The following primers were used in this study: U6 (internal control) forward primer 5′-CTCGCTTCGGCAGCACA-3′, reverse primer 5′-AACGCTTCACGAATTTGCGT-3′; MALAT1 forward primer 5′-AAAGCAAGGTCTCCCCACAAG-3′, reverse primer 5′-GGTCTGTGCTAGATCAAAAGGC-3′; miR-204 forward primer 5′-TGCGTTCCCTTTGTCATCCT-3′, reverse primer 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGCAT-3′; and SIRT1 forward primer 5′-GACTCTGGCATGTCCCACTA-3′, reverse primer 5′-AGCAGATTAGTAGGCGGCTT-3′. The relative expression of a target gene was calculated using the 2^−ΔΔCt method. ¹⁸

Oligonucleotides and cell transfection

The hsa-miR-204 mimic and hsa-miR-204 inhibitor were purchased from GenePharma (Shanghai, China). The sequences were as follows: hsa-miR-204 mimic: sense 5′-UUCCCUUUGUCAUCCUAUGCCU-3′ and antisense 5′-GCAUAGGAUGACAAAUUUAAUU-3′; hsa-miR-204 inhibitor: 5′-AGGCAUAGGAUGACAAAGGGAA-3′. The following short hairpin RNA (shRNA) was used to target human MALAT1: sense 5′-CACCGCTGTGGAGTTCTTAAATATCTTCAAGAGAGATATTTAAGAACTCCACAGCTTTTTTG-3′ and antisense 5′-GATCCAAAAAAGCTGTGGAGTTCTTAAATATCTCTCTTGAAGATATTTAAGAACTCCACAGC-3′. The sequence of the negative control shRNA was 5′-TTCTCCGAACGTGTCACGT-3′. The sequence of siRNA targeting SIRT1 is as follows: sense 5′-CCCUGUAAAGCUUUCAGAAdtdt-3′ and antisense 5′-UUCUGAAAGCUUUACAGGGdtdt-3′. The transfection of oligonucleotides was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Wound-healing migration assay

To perform migration assays, ¹⁹ target cells were plated into 24-well cell culture plate and allowed to grow in DMEM medium to confluence, washed with serum-free medium, and serum-starved for 16 h. A 1-mm-wide scratch was made across the cell layer using a sterile pipette tip. Plates were photographed immediately (0 h) and at 24 h after scratching at an identical location, respectively, with the width (W) of the scratch measured. The migration rate was calculated as (W_0h − W_24h)/W_0h × 100%. All experiments were performed in triplicates for at least three times.

Transwell invasion assay

To assess cell invasion, Transwell insert (−8.0 µm; Corning) was coated with Matrigel (BD Biosciences, San Jose, CA, USA). The single-cell suspension of target cells was seeded into the top well at 1 × 10⁵ cells/well and cultured in serum-free DMEM medium at 37°C. In the lower chamber, we added 500 μL of DMEM containing 10% FBS. After 24 h, the non-invaded cells from the upper side of the membrane were gently removed with cotton swabs, and the invaded cells on the lower side of the membrane were fixed in 95% methanol and stained with crystal violet for 5–10 min. The invaded cells were counted and photographed under an inverted microscope (100×).

Luciferase reporter assay

We used miRanda software (http://www.microrna.org/microrna/getGeneForm.do) and predicted the potential binding sites of miR-204 on MALAT1 and SIRT1. The 3′-untranslated region (3′-UTR) of human MALAT1 and SIRT1 gene containing the potential miR-204-binding sites was cloned into pRL-CMV luciferase reporter plasmid, and the mutated sequence was used as the mutated control. HepG2 cells were co-transfected with the luciferase reporter plasmid and hsa-miR-204 mimic or miR-204 inhibitor or control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 48 h following the transfection, luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

RNA-binding protein immunoprecipitation assay

RNA-binding protein immunoprecipitation (RIP) assay was performed using Magna RIP Kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, HepG2 cells treated as indicated were lysed in RIP lysis buffer, and the cell lysate was incubated with magnetic beads conjugated to human anti-Ago2 antibody (Millipore) or isotype-matched control antibody (normal mouse IgG; Millipore). Following the recovery of antibody using protein A/G beads, quantitative real-time polymerase chain reaction (qRT-PCR) was performed on the precipitates to detect MALAT1 and miR-204 levels.

RNA pull-down assay

To examine the potential association of MALAT1 with miR-204, RNA pull-down assay was performed as described before. ²⁰ Briefly, purified RNAs were biotin-labeled with the Pierce RNA 3′ End Desthiobiotinylation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Positive control (biotin-labeled wild-type miR-204, miR-204-Bio), negative control (mutant miR-204, miR-204-Bio-mut), and biotinylated RNAs (NC-Bio) were mixed and incubated with HepG2 cell lysates. Then, magnetic beads were added to each binding reaction and incubated at room temperature. Finally, the beads were washed and the eluted RNA was detected by qRT-PCR.

Western immunoblot

Total proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) buffer and separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Following the transfer of separated proteins onto a polyvinylidene difluoride membrane, the membrane was blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST; 10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) at room temperature for 1 h, washed three times in TBST, and incubated with primary antibodies against SIRT1 (Abcam, Cambridge, MA, USA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After three washes with TBST, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies at room temperature for 2 h. The signal was developed using the enhanced chemiluminescence (ECL) system (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions.

Statistical analysis

All data were analyzed by SPSS 13.0 software and presented as mean ± standard deviation (SD). Student’s t-test was used to compare the mean values between two groups. A p value of less than 0.05 was considered statistically significant.

Results

MiR-204 is negatively regulated by MALAT1 and affects HCC migration/invasion

Cumulative evidence suggests that by acting as sponge RNA and through competitive binding, lncRNA releases the inhibition on miRNA targets. ²¹ To characterize the molecular mechanisms underlying the effects of MALAT1 on HCC migration/invasion, we resorted to bioinformatic approach to identify potential miRNAs that could interact with MALAT1. First, we detected significantly higher MALAT1 levels in HCC cell lines, Bel7404, Huh7, and HepG2, compared with normal hepatocytes LO2 cells (Figure 1(a)), and we focused on HepG2 cells for further functional and mechanistic studies. Then, we applied the loss-of-function approach and knocked down the endogenous MALAT1 level in HepG2 cells using sh-MALAT1. As shown in Figure 1(b), sh-MALAT1 effectively reduced the endogenous MALAT1 by approximately 50%, when compared to cells transfected with sh-NC.

Figure 1.

MiR-204 is negatively regulated by MALAT1 and affects HCC migration/invasion. (a) The expression level of MALAT1 was examined by qRT-PCR and compared between human HCC cell lines Bel7404, Huh7, HepG2, and normal hepatocytes LO2 (*p < 0.05 and **p < 0.01 when compared to LO2 cells). (b) The transfection of HepG2 cells with sh-MALAT1 significantly reduced its endogenous level when compared to cells transfected with sh-NC. (c) The expression levels of indicated miRNAs were examined by qRT-PCR and compared between sh-MALAT1 and sh-NC cells. (d) The expression of miR-204 was examined and compared between HCC tissues (n = 15) and the matching para-tumor normal tissues (**p < 0.01). (e) The correlations between MALAT1 level and miR-204 level in HCC tissues (n = 15) were examined by Pearson test. (f) HepG2 cells were treated with NC, miR-204 inhibitor, or miR-204 mimic, and the expression of MALAT1 was examined and compared among different treatments. (g) HepG2 cells were treated with NC, miR-204 inhibitor, or miR-204 mimic, and the migration of these cells was examined by wound-healing migration assay. Left panels: representative images of wounds made on indicated cell layers at 0 and 24 h; right graph: the migration rate was significantly increased in miR-204 inhibitor–treated cells and reduced in miR-204 mimic–treated cells. (h) The invasion of indicated cells was compared using the Transwell invasion assay. Left panels: representative images of invaded cells after 24-h invasion; right graph: the number of invaded cells was significantly enhanced following the treatment with miR-204 inhibitor and reduced upon the treatment with miR-204 mimic (**p < 0.01 when compared to cells treated with NC).

Using the miRNA software, we focused on five potential MALAT1-interacting miRNA candidates: miR-17-5p, miR-338, miR-200C, miR-204, and miR29c. qRT-PCR analysis showed that all miRNAs, except for miR29c, were significantly higher in sh-MALAT1 cells than in sh-NC cells (p < 0.05), of which, miR-204 presented the most robust upregulation (approximately 2.7-fold; Figure 1(c)) and was thus focused on in this study. Further analysis showed that miR-204 level was significantly lower in HCC tissues than in normal tissues (p < 0.01; Figure 1(d)). Interestingly, MALAT1 expression correlated negatively with miR-204 levels (r = −0.81, p < 0.01; Figure 1(e)). Moreover, we treated HepG2 cells with either miR-204 inhibitor or mimic. As shown in Figure 1(f), when compared to cells treated with Ctrl, the level of MALAT1 was significantly upregulated in cells treated with miR-204 inhibitor and downregulated in those treated with miR-204 mimic (p < 0.05), suggesting a reciprocal negative regulation between MALAT1 and miR-204. Meanwhile, we examined the migratory and invasive behaviors of HepG2 cells in response to treatment with miR-204 inhibitor or mimic. As shown in Figure 1(g) and (h), miR-204 inhibitor significantly stimulated migration/invasion of HepG2 cells, while miR-204 mimic reduced the migration/invasion of HepG2 cells, as compared to those treated with Ctrl (p < 0.05). Taken together, the data suggest that MALAT1 promotes HCC migration and invasion through the regulation on miR-204.

MALAT1 directly binds to and controls the expression of miR-204 through post-transcriptional regulation

To examine whether the crosstalk between MALAT1 and miR-204 is through direct interaction, we mutated the potential binding site within MALAT1 3′-UTR region (Figure 2(a)), cloned either wild-type (MALAT1-WT) or mutant MALAT1 (MALAT1-MUT) sequence into a luciferase reporter plasmid, and transfected the plasmids into HepG2 cells. As shown in Figure 2(b), miR-204 mimic significantly reduced the luciferase activity from cells transfected with MALAT1-WT, but not from those with MALAT1-MUT, indicating the capability of MALAT1 to directly interact with miR-204. Meanwhile, we compared the levels of primary miR-204 (Pri-miR-204), precursor miR-204 (Pre-miR-204), and mature miR-204 between sh-NC and sh-MALAT1 cells. We found that only the mature miR-204, but not Pri-miR-204 or Pre-miR-204, was significantly upregulated in sh-MALAT1 cells (Figure 2(c)), suggesting that MALAT1 downregulates miR-204 on the post-transcriptional level. To examine whether MALAT1 functions as a sponge RNA for miR-204 (i.e. through base-paring with miR-204 and subjecting the latter to Ago2-mediated cleavage), we used RIP assay and found that MALAT1 and miR-204 were strikingly more abundant in Ago2 pellet than in IgG pellet (Figure 2(d)). RNA pull-down assay using biotinylated miR-204 (miR-204-bio) probe yielded a significantly higher level of MALAT1 than the assay using control (NC-bio) or miR-204 probes containing mutations within the MALAT1-binding site (miR-204-bio-mut; p < 0.01; Figure 2(e)). Collectively, these data supported that miR-204 was a primary target of MALAT1 in HCC cells.

Figure 2.

MALAT1 directly binds to and controls the expression of miR-204 through post-transcriptional regulation. (a) Sequence alignment showing the binding site between miR-204 and MALAT1 and the mutations made in MALAT1 sequence that disrupts the interaction between miR-204 and MALAT1. (b) HepG2 cells were transfected with luciferase reporter gene driven by either the wild-type MALAT1 3′-UTR sequence containing the miR-204 binding site (MALAT1-WT) or the mutant MALAT1 3′-UTR sequence (MALAT1-MUT). The luciferase activity was measured and compared between cells treated with NC and miR-204 mimic. (c) While MALAT1 shRNA induced a significant upregulation of mature miR-204, it had no effect on pri-miR-204 or pre-miR-204, implying that this negative regulation might be through a post-transcriptional mechanism. (d) The association between MALAT1 and Ago2 was determined by RIP assay. Cellular lysates of HepG2 cells were immunoprecipitated using Ago2 antibody or IgG. MALAT1 level was determined using qRT-PCR. SNRNP70 level was determined as a positive control. (e) RNA pull-down assay revealed the direct interaction between miR-204 and MALAT1. Cellular lysates of HepG2 cells were pulled down using biotinylated control (NC-Bio), miR-204 (miR-204-Bio), or miR-204 probe containing mutations in the MALAT1-binding site (miR-204-Bio-mut), and the level of MALAT1 in precipitated samples was determined by qRT-PCR (**p < 0.01).

SIRT1 is a direct target of miR-204

A recent study showed that miR-204 targets SIRT1 and regulates HCC progression. ¹⁷ Bioinformatic analysis showed that the binding site of SIRT1 on miR-204 greatly overlaps with the binding site of MALAT1 on miR-204 (Figure 3(a)), which prompted us to examine the significance of SIRT1 in MALAT1-mediated regulations on HCC migration/invasion. We first cloned SIRT1 3′-UTR region containing the miR-204-binding site into luciferase reporter plasmid and transfected the plasmid into HepG2 cells. The luciferase activity was significantly reduced following the treatment with miR-204 mimic, but enhanced in response to miR-204 inhibitor (p < 0.05; Figure 3(b)). Similarly, the endogenous mRNA and protein levels of SIRT1 were reduced in response to miR-204 mimic and increased to miR-204 inhibitor (p < 0.05; Figure 3(c) and (d)). To address the significance of MALAT1 in regulating SIRT1 level, we transfected the cells with sh-MALAT1 and found that in sh-MALAT1 cells, not only the luciferase activity but also the endogenous levels of SIRT1 mRNA and protein were reduced (Figure 3(e)–(g)). More importantly, although miR-204 inhibitor significantly boosted the promoter activity and the expression level of SIRT1 in sh-NC cells, it failed to perform in sh-MALAT1 cells (Figure 3(e)–(g)), suggesting that MALAT1 might increase SIRT1 by inhibiting miR-204 in HCC.

Figure 3.

SIRT1 is a direct target of miR-204. (a) Sequence alignment showing the binding site between miR-204 and MALAT1 and that between miR-204 and SIRT1. (b) HepG2 cells were transfected with luciferase reporter gene driven by the wild-type MALAT1 3′-UTR sequence containing the miR-204 binding site. The luciferase activity was measured and compared among cells treated with NC, miR-204 mimic, and miR-204 inhibitor. The expression of SIRT1 was measured on the (c) mRNA and the (d) protein levels in HepG2 cells following treatment with NC, miR-204 mimic, or miR-204 inhibitor. (e) The luciferase activity of luciferase reporter gene driven by the wild-type MALAT1 3′-UTR sequence containing the miR-204 binding site was measured in HepG2 cells treated with sh-NC, sh-MALAT1, miR-204 inhibitor, and miR-204 inhibitor + sh-MALAT1. The expression of SIRT1 was measured on the (f) steady-state mRNA and (g) protein level in HepG2 cells following treatment with sh-NC, sh-MALAT1, miR-204 inhibitor, and miR-204 inhibitor + sh-MALAT1 (*p < 0.05 when compared to sh-NC or NC).

SIRT1 is essential for the migration and invasion of HCC cells

To assess the biological significance of SIRT1 in HCC behavior, we transfected HepG2 cells with si-SIRT1 and robustly reduced the endogenous level of SIRT1 (Figure 4(a)). By wound-healing migration and Transwell invasion assays, we found that knocking down SIRT1 significantly reduced the migration and invasion of HepG2 cells (p < 0.01; Figure 4(b) and (c)). Furthermore, knocking down SIRT1 significantly decreased the level of Twist1 (by approximately 8-fold) and vimentin (by approximately 2-fold) and increased that of E-cadherin by approximately 1.7-fold (Figure 4(d)). These data suggest that SIRT1 is essential for the migration, invasion, and epithelial–mesenchymal transition (EMT) of HCC cells.

Figure 4.

SIRT1 is essential for the migration/invasion of HCC cells. (a) The endogenous level of SIRT1 was detected by western blot after transfecting HepG2 cells with si-Ctrl or si-SIRT1. GAPDH was detected as the loading control. (b) The migration of si-SIRT1 and si-Ctrl cells was compared using the wound-healing migration assay. Left panels: representative images of wounds made on indicated cell layers at 0 and 24 h; right graph: the migration rate was significantly reduced in si-SIRT1 cells than in si-Ctrl cells (**p < 0.01 when compared to si-Ctrl cells). (c) The invasion of si-SIRT1 and si-Ctrl cells was compared using the Transwell invasion assay. Left panels: representative images of invaded cells after 24 h invasion; right graph: the number of invaded cells was significantly lower in si-SIRT1 cells than in si-Ctrl cells (**p < 0.01 when compared to si-Ctrl cells). (d) The expression levels of Twist1, E-cadherin, and vimentin were detected by western blot and compared between si-Ctrl and si-SIRT1 cells. GAPDH was detected as the loading control. The relative expression of indicated proteins in si-Ctrl cells was arbitrarily defined as 1.0 and that in si-SIRT1 cells was labeled below the corresponding western blot signal.

MALAT1 promotes HCC migration/invasion via miR-204

Since MALAT1 negatively regulates the expression of miR-204, which in turn releases the silencing effect on SIRT1 (Figure 3), and SIRT1 is functionally critical for the migration/invasion of HCC cells (Figure 4), we aim to assess the effect of concomitant targeting MALAT1 and miR-204 on the migration/invasion of HCCs. As shown in Figure 5, the wound-healing assay and Transwell assay showed that knocking down MALAT1 alone significantly reduced migration/invasion of HCC cells, while inhibiting miR-204 increased the migration/invasion of HCC cells (p < 0.01, when compared to cells treated with sh-NC). Importantly, decreasing the expression of miR-204 in HepG2 cells could abolish the weakening effects of sh-MALAT1 on cell migration and invasion. Thus, these results indicated that MALAT1 promoted HCC cell migration and invasion by operating as a competitive endogenous RNA for the miR-204.

Figure 5.

MALAT1 promotes HCC migration/invasion by acting as a ceRNA. (a) HepG2 cells were treated as indicated and the migration of different cells was examined using the wound-healing migration assay. Left panels: representative images of wounds made on indicated cell layers at 0 and 24 h; right graph: the migration rate was quantified and compared among different treatments (**p < 0.01 when compared to sh-NC cells). (b) The invasion of HepG2 cells upon indicated treatments was compared using the Transwell invasion assay. Left panels: representative images of invaded cells after 24 h invasion; right graph: the number of invaded cells was quantified and compared among different treatments (**p < 0.01 when compared to sh-NC cells).

Discussion

The high prevalence of HBV and HCV infection in Asia and particularly in China makes HCC a leading cause of cancer death. The intrahepatic tumor growth and even the recurrence of intrahepatic lesions are susceptible to complete treatment. It is the extrahepatic metastasis developed from local migration/invasion and distant dissemination of tumor cells that constitutes the lethal event of HCC. ²² Therefore, targeting the migratory/invasive behaviors of HCC cells becomes a therapeutic goal for developing novel HCC treatments. In this study, we revealed that the miR-204 is significantly downregulated in HCC tissues and cells, and it was negatively regulated by MALAT1. Furthermore, we showed that MALAT1, through competitive binding to miR-204, releases the silencing on SIRT1 by miR-204. Since SIRT1 is critical for mediating HCC migration/invasion, targeting MALAT1 significantly inhibits the aggressive behavior of HCCs and thus becomes a promising therapeutic strategy.

Although not encoding any protein products, lncRNAs are not junky bystanders in human genome; instead, they are active and crucial players in regulating a variety of physiological and pathological processes.^5,21 Studies from the past decade reveal limited yet important information on the mechanisms and functions of lncRNAs. First, through the association with RNA polymerase II or by guiding chromatin-modifying complexes, lncRNAs modulate gene transcription on both genetic and epigenetic levels. ²³ Second, lncRNAs may act as endogenous sponge RNA or mimic miRNA targets, contributing to mRNA or miRNA degradation or inhibiting their activities. ²⁴ Third, lncRNAs regulate protein functions by acting as scaffolds, guiding molecule (in cis or in trans), or decoys.^25,26

Of the limited number of lncRNAs examined so far, MALAT1 is well demonstrated to stimulate tumor progression.^7,27 Robust upregulation of MALAT1 is detected in a variety of cancers (including HCC) or cancers with higher metastatic potential. The elevation of MALAT1 is achieved through the following: (1) transcriptional activation on the epigenetic level such as demethylation of histone H3K9 ²⁸ or on the genetic level such as through transforming growth factor-β (TGF-β), ²⁹ cyclic AMP–responsive element binding (CREB), ³⁰ Wnt/β-catenin pathway, ³¹ or SOX17; ³² (2) post-transcriptional regulation, such as miRNA-mediated silencing, as demonstrated by miR-125, ³³ miR-9, ³⁴ miR-101, and miR-217. ³⁵ In addition to functioning as a marker for malignancy, MALAT1 upregulation significantly correlates with the poor prognosis of cancer patients, rendering this molecule a prognostic indicator.^36–38 In this study, we found that MALAT1 and miR-204 mutually and negatively regulated the expression of each other, and their levels negatively correlated with each other in HCC tissues, suggesting that although miR-204 could control the expression of MALAT1, the upregulation of MALAT1 in HCC tissues would downregulate the level of miR-204, which in turn further elevates MALAT1 and exacerbates the phenotypes induced by MALAT1.

Phenotypically, MALAT1 stimulates tumor growth, inhibits apoptosis, and promotes metastasis via distinct mechanisms. In bladder cancer, MALAT1 interacts with suz12 to reduce the expression of E-cadherin and to increase that of N-cadherin and fibronectin, contributing to EMT and stimulating tumor metastasis. ²⁹ In colorectal carcinoma, MALAT1 activates SRPK1-SRSF1 axis and stimulates AKAP-9 expression, promoting tumor cell proliferation, migration, and invasion. ³⁹ Another study showed that in colorectal carcinoma, the tumor-promoting activities of MALAT1 are accomplished through the interaction with the splicing factor SFPQ and the subsequent releasing of SFPQ-interacting protein PTBP2. ⁴⁰ Other studies showed that MALAT1 acts as a sponge RNA for different miRNAs in cancer development.^11,41,42 In this study, we used bioinformatic approach and identified five potential miRNAs that could interact with the 3′-UTR of MALAT1. Confirmation analysis showed that miR-204 presented the most robust response to MALAT1 knockdown in HCC cells, suggesting that miR-204 is a target for MALAT1 in HCC. RIP and RNA pull-down assays further revealed that MALAT1 was associated with Ago2 and functioned as a sponge RNA for miR-204, as supported by the change in miR-204 only on the mature miRNA level, but not on the pri- or pre-miRNA level. Functionally, applying miR-204 mimic in HCC cells presented similar inhibition on cell migration/invasion as targeting MALAT1, implying that MALAT1 may signal through miR-204 to regulate HCC migration/invasion.

Except for a few types of cancers, including insulinoma and acute lymphocytic leukemia,^43,44 miR-204 level is significantly lower in most solid tumors than in normal tissues and functions as a tumor suppressor by promoting apoptosis through the activation of pro-apoptotic protein such as p53 and the inhibition of anti-apoptotic protein such as Bcl-2, ⁴⁵ inhibiting EMT and cancer stemness ⁴⁶ and enhancing sensitivity of cancer cells to chemotherapy.^47,48 Recent studies showed that SIRT1 is a target for miR-204 and is functionally critical for multiple cancer phenotypes such as migration/invasion, EMT, and resistance to anoikis.^17,49,50 Consistent with these studies, we showed that both MALAT1 and SIRT1 bound to the same site on miR-204, and therefore, in addition to targeting miR-204 to Ago2-mediated silencing, MALAT1 may also compete with SIRT1 for binding to miR-204, both mechanisms releasing SIRT1 from the negative control by miR-204. In addition, SIRT1 is functionally critical for HCC migration/invasion, supporting that miR-204-SIRT1 axis mediates the activity of MALAT1 on HCC migration/invasion. Consistently, targeting MALAT1 together with inhibiting miR-204 potently maintained the migration/invasion of HCC at the basal level.

In summary, we reveal a dual mechanism by which MALAT1 promotes HCC migration/invasion, by sponging miR-204 and releasing SIRT1 from miR-204. These findings not only corroborate that targeting MALAT1 is a promising therapy for HCC but also suggest that fine-tuning the balance between miR-204 and SIRT1 would alter the metastatic behavior of HCCs harboring MALAT1 upregulation. Thus, miR-204 may function as a useful small-molecule drug for the treatment of HCC. However, it still needed more and further research to explore the upregulation mechanisms of MALAT1 in HCC. For instance, we can first take the 5′-UTR of MALAT1 for analysis, and future efforts may be directed toward transcription factor regulations. Furthermore, it is also important to identify the downstream signaling of SIRT1, which will provide more different targets for therapy of HCC.