Long non-coding RNA PVT1 regulates the migration of hepatocellular carcinoma HepG2 cells via miR-3619-5p/MKL1 axis

Hepatocellular carcinoma (HCC) is the third most common malignant tumor of the digestive system. Plasma cell tumor heterotopic gene 1 (PVT1) is an intergenic long non-coding RNA that is aberrantly expressed in different cancers. Myocardin-related transcription factor A or megakaryoblastic leukemia 1 (MKL1) is a transcriptional coactivator of serum response factor that has been shown to promote cancer cell migration and invasion. In this study, we investigated the relationship between PVT1 and MKL1 as a novel regulatory mechanism underlying HCC progression. We used HepG2 and Cos-7 cell lines. Transfection experiments with miR-3619-5p mimics/inhibitor, PVT1, siRNA-PVT1, MKL1, or siRNA-MKL1 were performed. RNA and protein levels were analyzed by quantitative reverse transcription PCR and Western blot, respectively. Cell migration was assessed by transwell assay. Luciferase assays, RNA-FISH, RNA immunoprecipitation, and chromatin immunoprecipitation assays were performed to confirm the interaction between PVT1, miR-3619-5p, and MKL1 in HCC cells. Overexpression of PVT1 was positively correlated with MKL1 upregulation, which promoted HepG2 cell migration. miR-3619-5p inhibited MKL1 expression in HCC cells by acting on its 3′-UTR. Furthermore, PVT1 promoted MKL1 expression and migration in HCC cells by directly binding to miR-3619-5p. In a positive feedback loop, MKL1 could activate PVT1 transcription by binding to the CArG box in the promoter region. Our findings may provide a basis for the development of novel targeted therapies in HCC.


INTRODUCTION
Hepatocellular carcinoma (HCC) gradually became one of the most common cancers and the second leading risk around the world [1]. Most HCC patients eventually die due to tumor metastasis-associated deaths [2]. Today, screening novel biomarkers for developing effective therapeutic targets against the metastasis of HCC may make up for the shortcomings of current therapeutic strategies.
In mice, this gene site frequently produces multiple translocations [3]. In addition, the same phenomenon can be detected in one region of the human PVT1 gene in Burkitt' s lymphoma [4]. PVT1, located at 8q24.21, has a complexity in its structure that is revealed gradually, with the development of high flux technology [5]. The existence of an aberrant expression of PVT1 in many human cancers has been reported recently. PVT1 may act as a noninvasive diagnostic biomarker in small cell lung cancer because its overexpression always means a poor prognostic [6]. Moreover, PVT1 can regulate forkhead box M1 (FOXM1) to facilitate gastric cancer growth and invasion [7]. As a lncRNA, PVT1 can also affect miRNAs directly, acting as a molecular sponge to regulate cancer cell development similar to miR-195 in osteosarcoma [8].
Megakaryoblastic leukemia 1 (MKL1) is a transcriptional coactivator of serum response factor (SRF), which can bind to the CArG box (CC(A/T) 6 GG) in the promoters of various immediate-early and muscle-specific genes [9][10][11]. In a previous report, MKL1 could also promote cancer cell migration and invasion [12], which has a similar function as PVT1. However, the relationship between the two factors is still unknown.
Therefore, the aim of this study is to investigate the relationship between PVT1 and MKL1 to reveal new molecular regulatory mechanisms in metastasis of HCC. 188 www.bjbms.org

Cell culture
The human HCC cell line HepG2 and Cos-7 cells used in this study were purchased from ATCC (Manassas, VA, USA; nos. HB-8065 and CRL-1651, respectively). These cells were seeded in Dulbecco' s Modified Eagle Medium (DMEM, Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in humidified air with 5% CO 2 .
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) The total RNA was isolated using TRIzol reagent (Invitrogen, Waltham, MA, USA) and reverse-transcribed using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA). Quantitative PCR was carried out using the Fast SYBR ® Green Master Mix (Applied Biosystems; Thermo Fisher Scientific Inc., Waltham, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as control to show equal loading of the mRNA. The data were shown as relative expression levels after being normalized by GAPDH. For the detection of miR-3619-5p by qRT-PCR, miRNA was extracted using the miRNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA), according to the manufacturer' s instructions. Then, miRNA was reverse-transcribed into firststrand cDNA with miRNA 1 st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme; Nanjing, China). Quantitative PCR was then carried out using the Fast SYBR ® Green Master Mix (Applied Biosystems; Thermo Fisher Scientific Inc., Waltham, MA, USA). The U6 gene was used as the endogenous control gene for normalizing the expression of miR-3619-5p. The primers for the PCR analysis are listed in Table 1. The thermocycling conditions were as follows: 95°C for 5 min followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec, then a melting curve analysis from 60°C to 95°C every 0.2°C for 1.5 min was obtained. Each sample was analyzed in triplicate and quantified using the 2-ΔΔCq method [13].

Protein extraction and Western blotting
The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were immunoblotted with anti-MKL1 antibody (dilution 1:1,000; cat. no. 14760; Cell Signaling Technology Inc., Danvers, MA, USA) or anti-GAPDH antibody (dilution 1:2,000; cat. no. 97166; Cell Signaling Technology Inc., Danvers, MA, USA) overnight at 4°C and then incubated with IRDye TM -800 conjugated anti-mouse secondary antibody (dilution 1:5,000; cat. nos. 115-005-146; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 60 min at room temperature. The specific proteins were visualized using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). GAPDH expression was used as control to show the equal loading of the protein samples.

Transwell assay
The invasion assay was performed using transwell chambers (Corning Incorporated, Corning, NY, USA) that had Matrigel (50 μL; BD Biosciences, New Jersey, USA) precoated polycarbonate membranes (8.0 μm pore size). A total of 1 × 10 4 cells were added to the upper chamber with FBSfree culture medium. The lower chamber was filled with 500 μL complete medium. Following incubation for 24 h, the cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The cells were counted in five random microscopic fields (magnification, ×20). Finally, the crystal violet was washed with 33% acetic acid and measured at 570 nm.

RNA fluorescence in situ hybridization (RNA-FISH)
A Cy3-labeled lncRNA-PVT1 complementary DNA probe was synthesized and used for RNA-FISH (GeneChem Co. Ltd., Shanghai, China). The procedures were performed as previously described with slight modification [14].

RNA immunoprecipitation (RIP)
RNA immunoprecipitation experiments were performed as previously described [15]. For anti-protein argonaute-2 (anti-Ago2) RNA immunoprecipitation (RIP), 2 × 10 7 HepG2 cells were used with the RIP kit (Cell Signaling Technology Inc., Danvers, MA, USA). Normal rabbit immunoglobulin G (IgG) antibody was used as negative control. The final results were detected by qRT-PCR. The gene-specific primer used for detecting lncRNA PVT1 is provided in Table 1. MS2-RIP MS2 (Addgene, Watertown, Massachusetts, MA, USA), MS2-PVT1, and MS2-PVT1-Mut were used for this test. The three plasmids were transfected into HepG2 cells. After 48 h of transfection, the HepG2 cells were used to perform RIP experiments as previously described [16]. The RNA fraction isolated by RIP was analyzed using qRT-PCR. The gene-specific primer used for detecting miR-3619-5p is provided in Table 1.
MiR-3619-5p mimics and miR-3619-5p inhibitor MiR-3619-5p mimics, miR-3619-5p inhibitor, and the negative control (control or NC) were purchased from RiboBio. The cells in the six-well plates were transfected with the mimics at 100 nM. The transfection was performed using riboFECTTM CP Reagent (Guangzhou RiboBio Co. Ltd., Guangzhou, China) according to the manufacturer' s instructions.

Chromatin immunoprecipitation (ChIP) assay
The researchers used a commercial ChIP assay kit (Merck KGaA, Darmstadt, Germany) following the manufacturer' s instructions. Each test group was incubated with 1% formaldehyde to cross-link DNA-protein complexes. After washing with ice-cold phosphate buffer saline (PBS) for 3 times, the cells were lysed in SDS lysis buffer. The lysate was then sonicated to shear DNA to 200-1000 bp fragments. Then, anti-MKL1 antibody (cat. no. 14760; Cell Signaling Technology Inc., Danvers, MA, USA) was used to immunoprecipitate the cross-linked protein at 4°C overnight. IgG acted as negative control. The DNA was used as a template for qRT-PCR and utilized the MKL1 binding sites. The PCR primer sequences were forward, 5′-AAATGTGTTTGTGTGTGGTGG-3′ and reverse, 5′-TCAACTTGTCGCTG CCAGTAT-3′.

Statistical analysis
Data were expressed as mean ± SEM, accompanied by the number of experiments performed independently. Statistical analysis of the differences between the two groups was performed using Student' s t-test. A one-way analysis of variance followed by Tukey test was performed to compare differences among multiple groups. The differences at p < 0.05 were considered statistically significant.

PVT1 can promote migration of HepG2 cells via MKL1
To illustrate the possible function of PVT1 in HepG2 cells, the researchers facilitated PVT1 overexpression or silencing in HepG2 cells. The transwell results showed that PVT1 can inhibit the migration of HepG2 cells ( Figure 1A-D). Moreover, qRT-PCR and Western blot were used to reveal the potential relationship between PVT1 and MKL1. The data showed that the expression of MKL1 was upregulated when PVT1 was overexpressed. Similarly, when PVT1 was silenced, the expression of MKL1 was depressed, suggesting that PVT1 promoted the migration of HepG2 cells, which may be due to the fact that MKL1 expression had a positive correlation with the expression of PVT1 ( Figure 1E-J). Further experiments confirmed our results. When MKL1 was knocked down, PVT1 lost its ability to regulate the migration of HepG2 cells ( Figure 1K-N). These results indicate that PVT1 can promote the migration of HepG2 cells via MKL1.

MKL1 can be regulated by miR-3619-5p
The FISH experiment proved that PVT1 can be localized in the cytoplasm, suggesting that PVT may act as a competing endogenous RNA (ceRNA) on some miRNAs regulating the expression of MKL1 (Figure 2A). Based on the results, and following the bioinformatics analysis (using online TargetScan Software, available at http://www.targetscan.org), we found that miR-3619-5p may be a potential target. Then, qRT-PCR

PVT1 can promote MKL1 expression and migration of HepG2 cells through miR-3619-5p
To explore whether miR-3619-5p is the target of PVT1, qRT-PCR and Western blot testing were used to explore the expression of MKL1 in HepG2 cells with knocked-down miR-3619-5p. The results showed the overexpression or silencing of PVT1 in HepG2 cells with miR-3619-5p knocked down cannot regulate MKL1 expression ( Figure 3A-C). Moreover, the transwell test showed that PVT1 lost the ability to affect HepG2 cell migration after miR-3619-5p was knocked down (Figure 3D,E). Evidently, these results suggest that PVT1 can promote MKL1 expression and migration of HepG2 cells through miR-3619-5p.

PVT1 can bind to miR-3619-5p directly
In order to explore the interaction between PVT1 and miR-3619-5p, the sequences of PVT1 and miR-3619-5p were compared through bioinformatics (using online starBase v2.0 software, available at http://starbase.sysu.edu.cn), and the potential binding site was detected ( Figure 4A). Then, two luciferase plasmids containing PVT1 (pmirGLO-PVT1-WT) and the binding site mutated form (pmirGLO-PVT1-mut) were transfected. The luciferase assay results revealed that miR-3619-5p can bind to the pmirGLO-PVT1-WT instead of pmirGLO-PVT1-mut ( Figure 4B). In addition, RIP assay was performed with Ago2 antibody to determine whether PVT1 associated with the form of RNA-induced silencing complex. Data showed that PVT1 can be enriched by Ago2 antibody ( Figure 4C). Furthermore, the researchers tested whether PVT1 can pull down miR-3619-5p and the MS2 RIP test was used. Subsequently, with an MS2 vector (MS2) as control, a plasmid containing the PVT1 sequence (MS2-PVT1) and its mutation type with mutated miR-3619-5p binding site (MS2-PVT1-MUT) were engineered. The qRT-PCR results showed that miR-3619-5p can be significantly enriched in the MS2-PVT1 group compared with MS2 and the MS2-PVT1-MUT group ( Figure 4D). Therefore, PVT1 can bind to miR-3619-5p directly.

MKL1 can activate the expression of PVT1 by combining with CarG box
As reported, MKL1 can regulate the expression of the target gene in the nucleus by binding to CarG box in its promoter Luciferase activity in Cos-7 cells cotransfected with miR-3619-5p mimics, and luciferase reporters containing PVT1 or Mut-PVT1. Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity (n = 3, *p < 0.05, # p > 0.05). (C) Anti-protein argonaute-2 (anti-Ago2) RNA immunoprecipitation (RIP) was performed in HepG2 cells followed by quantitative reverse transcription polymerase chain reaction to detect PVT1 association with Ago2 (n = 3, **p < 0.01). (D) MS2-RIP assay was used to prove the direct binding between miR-3619-5p and PVT1 in HepG2 cells (n = 3, **p < 0.01, # p > 0.05).
A B D C www.bjbms.org  region. Interestingly, the researchers found a CarG box in the promoter region of PVT1 (the CarG box sequences: 5′-CCTATTTT GC-3′; position: −48-−38 of the PVT1 promoter region). Subsequently, the results of qRT-PCR confirmed that MKL1 can activate the expression of PVT1 ( Figure 5A-D). Furthermore, the luciferase assay showed that MKL1 can activate the transcription activity of the PVT1 promoter, while MKL1 cannot activate the transcription of PVT1 with CArG box mutation ( Figure 5E and F). Moreover, the ChIP assay also confirmed that MKL1 can combine with the CArG box of the PVT1 promoter region ( Figure 5G). These results indicate that MKL1 can activate the expression of PVT1 by combining with CArG box.

DISCUSSION
China is a high-incidence area of liver cancer, and the incidence of new cases accounts for about half of the global cases every year [17]. Hepatectomy is still the most important treatment for HCC in China. However, the overall curative effect after hepatectomy is still poor. The overall recurrence rate of 5 years after hepatectomy is over 80%, and the overall survival rate of 5 years is only 30-70% [18]. Intrahepatic and extrahepatic metastases are important factors affecting the long-term effect of hepatectomy. Therefore, an in-depth study of the new molecular mechanism of increased metastasis of HCC is of great significance for the design of more effective drugs to improve or supplement the surgical efficacy.
In this study, the researchers revealed a new pathway to regulate the migration of HCC cells driven by PVT1. Our results showed that MKL1 was significantly increased when PVT1 was overexpressed, resulting in enhancing the ability of migration and invasion of HepG2 cells. However, when MKL1 was silenced, PVT1 lost the ability to regulate HepG2 cell migration. These data may suggest that PVT1 can regulate the migration of HepG2 cells through MKL1. In addition, PVT1 regulated MKL1 expression by adsorption of miR-3619-5p.
The function and mechanism of lncRNAs are complex. There is a lot of evidence showing that lncRNAs can cause target gene overexpression by functioning as a ceRNA sponge to miR-NAs, suppressing the binding of miRNAs to the target mRNAs and regulating their function [50,51]. For example, lncRNA small nucleolar RNA host gene 20 (SNHG20) induces gastric cancer progression via upregulating zinc finger protein X-linked (ZFX) expression by absorbing miR-495-3p [52]. LncRNA activated by transforming growth factor-β (ATB) contributes to Twist1 www.bjbms.org expression and breast cancer epithelial-mesenchymal transition (EMT) through inhibiting miR-200c [53]. For C5orf66-AS1, miR-637 is a target miRNA in the process of cervical cancer growth [54]. In this study, the FISH results have indicated that PVT1 was partly located in the cytoplasm of HepG2 cells, suggesting that PVT1 may work as a ceRNA for some miRNAs. Through bioinformatics analysis, the researchers identified a potential target miR-3619-5p, which may be a regulator of MKL1. Finally, we showed that PVT1 can sponge miR-3619-5p from MKL1 mRNA to enhance metastasis of HCC cells.
Collectively, our data revealed a new kind of regulation mechanism led by PVT1. PVT1 can upregulate the expression of MKL1 via competitive sponging miR-3619-5p to exhibit oncogenic activity, which may be a novel signaling pathway for HCC treatment. Finally, the study confirmed that MKL1 can reverse-activate the transcription of PVT1 ( Figure 6) and MKL1 and PVT1 formed a positive feedback loop that contributed to tumor migration.

ACKNOWLEDGMENTS
This work was supported by The Third Central Hospital of Tianjin.