Hepatocellular carcinoma (HCC) is a lethal malignancy with few effective options for therapeutic treatment in its advanced stages. While exosomal LINC00161 has been identified as a potential biomarker for HCC, its regulatory function and clinical values remain largely unknown. LINC00161 expressions in serum-derived exosomes from HCC patients and HCC cells were determined by qRT-PCR. The ability of proliferation, migration, and angiogenesis in HUVECs was assessed by MTT, Transwell, and tube formation. Luciferase reporter assay and AGO2-RIP assay were conducted to explore the interactions among LINC00161, miR-590-3p, and ROCK2. The level of ROCK signal-related proteins was examined by Western blotting and immunohistochemistry (IHC) assay. Subcutaneous tumor growth was observed in nude mice, in which in vivo metastasis was observed following tail vein injection of HCC cells. High levels of LINC00161 were detected in both serum-derived exosomes from HCC patients and the supernatants of HCC cell lines and were significantly associated with poor survival. Functional study demonstrated that exosomal LINC00161 derived from HCC-cells were significantly associated with enhanced proliferation, migration, and angiogenesis in HUVECs in vitro, all of which were effectively inhibited when LINC00161 was sliced with shRNA in HCC-cells. In vivo experiment showed that LINC00161 loss inhibited tumorigenesis and metastasis of HCC. Mechanistic study revealed that exosome-carried LINC00161 directly targeted miR-590-3p and induced its downstream target ROCK2, finally activating growth/metastasis-related signals in HCC. Exosome-carried LINC00161 promotes HCC tumorigenesis through inhibiting miR-590-3p to activate the ROCK2 signaling pathway, suggesting that LINC00161 may be used as potential targets to improve HCC treatment efficiency.
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is the second leading cause of cancer-related death globally . Despite advances in both surgical and nonsurgical techniques, the prognosis of HCC still remains poor . Typically, at diagnosis, the majority of patients are present with regional spread and metastasis where surgical resection or liver transplantation is not feasible . Therefore, understanding the molecular mechanisms that regulate HCC metastasis is essential for identifying tumor cell weaknesses that can be targeted for therapy.
Exosomes are extracellular membrane vesicles (30–150 nm) derived from multiple cell types including tumor cells . They are carriers of several molecules, such as DNA, RNA, proteins, and lipids that are biochemically and functionally distinct and can be transferred to recipient cells where they regulate protein expression and signaling pathways [5, 6]. Accumulating evidence suggests that increased secretion of functional tumor exosomes into the extracellular environment plays a critical role in the progression and formation of metastases . Furthermore, the regulatory role of exosomes in HCC has been increasingly recognized . It is thus likely that exosomes can serve as promising biomarkers for HCC diagnosis and even as potential treatment targets for patients with HCC. LncRNAs are non-coding RNAs longer than 200 nucleotides in length whose roles are increasingly implicated in a series of biological processes, including cancer [8, 9]. Exosomal lncRNAs generated from tumor cells have been identified as signaling mediators to organize cell function among neighbor tumor cells . Several deregulated exosomal lncRNAs (such as lncRNA-HEIH, lncRNA TUC339, lncRNA FAL1, lncRNA ENSG00000258332.1, and LUCAT1) have been shown to modulate the growth, migration, invasion, and chemosensitivity of HCC cells [10,11,12,13]. LINC00161 was overexpressed in HCC tissues, and was found to be an independent prognostic factor for overall survival. Moreover, LINC00161 might act as an oncogenic gene and play a central role in promoting HCC migration and invasion . While exosomal LINC00161 was also up-regulated in HCC , the potential biological roles and regulatory mechanisms of exosomal LINC00161 in HCC are poorly understood.
MicroRNAs (miRNAs) are small non-conding RNAs that regulate gene expression primarily at the post-transcriptional levels and regulate different cell activities, including proliferation, differentiation, apoptosis, and angiogenesis which play a crucial part in HCC growth [16,17,18]. A growing number of miRNAs are involved in the regulation of angiogenesis in HCC in a context-dependent manner . miR-590-3p, which is contained within an intron of the eukaryotic translation initiation factor 4H gene , has been shown to serve as a critical modulator on tumorigenesis and malignant progression [21, 22]. Importantly, miR-590-3p was significantly down-regulated in HCC patients, and suppressed cell proliferation, migration, and invasion . While miR-590-3p has been reported to repress angiogenesis of endothelial progenitor cells  and oxLDL-induced angiogenesis , its role on HCC angiogenesis remains to be explored.
Rho-associated coiled-coil containing kinases (ROCKs), consisting of isoforms ROCK1 and ROCK2, are key regulators of focal adhesion, actomyosin contraction, and cell motility . It has been reported that ROCK2 is up-regulated in several types of human cancer, including HCC, and overexpression of ROCK2 is positively correlated with tumor metastasis and poor prognosis . Recently, ROCK has attracted attention for its crucial role in angiogenesis , making it a promising target for new therapeutic approaches. Bioinformatic investigation revealed the binding sites of miR-590-3p in LINC00161 and ROCK2 mRNA, and their potential interplay in HCC angiogenesis and metastasis are thus warranted further investigations.
Relatively few studies have examined the contributions of exosomal lncRNA in HCC progression and metastasis. In the current study, we found that exosomal LINC00161 was up-regulated in serums from HCC tissues and in diverse cell lines, and intercellular transfer of HCC-derived exosomal LINC00161 accelerated proliferation, migration, and angiogenesis of endothelium cells in vitro and stimulated HCC tumorigenesis and metastasis in vivo. Subsequent mechanistic studies uncovered that exosome-carried LINC00161 exerted its oncogenic function by competitively sponging and then inhibiting miR-590-3p to stimulate the ROCK2 signaling pathway. Inhibiting angiogenesis has been used as a strategy in the treatment of HCC, and targeting exosomal LINC00161 that simultaneously inhibit the ROCK pathways involved in angiogenesis and tumor metastasis may thus confer broad and potent antitumor efficacy.
Peripheral blood samples were collected from 56 HCC patients before resection in Shenzhen Hospital of Southern Medical University. Twenty tumor tissues from the HCC patients with CD34 expression and their corresponding normal tissues adjacent to the tumor were included to analyze the expression level of LINC00161, miR-590-3p, and ROCK2. The study protocol was approved by the Medical Ethics Committee of Shenzhen Hospital of Southern Medical University and all patients provided written informed consent.
Cell culture and treatment
The healthy human hepatocyte cell line WRL-68, human umbilical vein endothelium cell line (HUVECs), and HCC cell line Huh-7 were purchased from the American Type Cell Culture Collection (ATCC, Manassas, VA, USA). HCCLM3, MHCC-97L, and MHCC-97H were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology). WRL-68 and HCC cell lines were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone). HUVECs were cultured in endothelial growth medium 2 (EGM-2, Lonza). All cultures were maintained at 37 °C in a 5% CO2 humidified atmosphere. HUVECs were treated with different concentrations of exosomes from HCC cells (10, 20, 40, and 80 µg/mL) for 12, 24, 36, and 48 h.
Exosome isolation, identification, and internalization
Exosomes were isolated for HCC cells by using the Total Exosome Isolation Kit (Invitrogen, Carlsbad, CA, USA). Cells were initially cultured in exosome-free medium for 4 days before exosome isolation. The total exosome isolation reagent (from cell culture media) kit #44578259 (Thermo Fisher Scientific, CA, USA) was then used to isolate and purify the exosomes from the cell supernatants. To remove cells and debris, the serum samples were deforested in a 25˚C water bath and centrifuged at 3000 × g for 25 min. A total of 150 μL exosome isolation reagent was subsequently added into 600 μL serum, and the mixture was vortexed until homogenous. After culture at 4 °C for 45 min, the mixture was centrifuged at 15000 × g for 20 min at room temperature. The exosome pellets are resuspended in 100 μL PBS after carefully removing the supernatant. The protein concentration of the HCC-derived exosomes was then quantified by a determining protein concentration (BCA assay) using the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions.
The exosome suspension was added to an equal volume of 4% paraformaldehyde and a formvar/carbon film-coated transmission electron microscope (TEM) grid (Alliance Biosystems, Osaka, Japan) was applied for exosome identification. The samples were subsequently fixed by harvesting with 1% glutaraldehyde, washed with PBS, contrasted with 1% uranylacetate, embedded in epoxy resin, and polymerized. The Malvern Zetasizer Software v7.11 (Malvern Panalytical Ltd, Malvern, UK) was used to perform the particle size analysis and the green dye PKH-67 (Sigma-Aldrich, St. Louis, MO, USA) was used to track exosome internalization. After incubation for 24 h, the labeled exosomes (40 µg/mL) were added to HUVECs and a Leica TCS SP5 II laser scanning confocal microscope was employed to observe the uptake of labeled exosomes by the recipient HUVECs.
Short hairpin RNAs (shRNA) targeting LINC00161 was purchased from GeneCopoeia (Rockville, MD, USA) and transiently transfected into HCCLM3 and MHCC-97H cells using Lipofectamine 2000 (Invitrogen). The empty vector was used as a negative control. The mimics and inhibitors of miR-590-3p and negative control were purchased from GenePharma Company (Shanghai, China) and incubated with HUVECs. All treated cells were harvested at 48 h after transfection.
HUVECs were harvested in 96-well plates and measured at 0 h, 12 h, 24 h, 36 h, and 48 h after seeding with 20 μL 5 mg/mL MTT solution (Solarbio, Beijing, China). The solution was removed after incubation with MTT solution at 37 °C for 3 h, and dissolved in 100 μL dimethyl sulfoxide with 10 min shaking. A microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) was used to determine the absorbance at 570 nm.
Tube formation assay
Tube formation assay was performed to assess the angiogenic potential. Purified exosomes derived from HCCLM3 and MHCC-97H cell lines were incubated with HUVECs and then transferred onto the 48-well plates precoated with growth factor-reduced Matrigel (BD Biosciences, San Jose, CA, USA). After 2 days of incubation, tube formation was determined in photographs from a microscope. The branches of blood vessels were measured to examine the total tube length by using ImageJ software.
Transwell inserts with a polycarbonate membrane were used to perform the Transwell assays for migration. In brief, HUVECs were suspended in serum-free media and seeded into the inner chamber, and the complete growth media was contained in the outer chamber. After incubation for 12 h, non-migrating cells were removed with a cotton swab from the inside of the membrane carefully. The cells that had invaded through the membrane to the lower surface were fixed with methanol, stained with 0.1% crystal violet for 10 min, and photographed under a standard bright field microscope. A total of 5–6 microscope fields were selected for analysis and every labeled cell per field was calculated.
Dual-luciferase reporter assay
Serial constructs containing LINC00161 or ROCK2 3′-UTR were generated using the psiCHECK2 plasmid (Promega Corporation, Madison, WI, USA). Point mutations of the miR-590-3p targeting sites in the LINC00161 or ROCK2 3′-UTR were directly synthesized using the QuickChange Multiple Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). Each plasmid construct was subsequently co-transfected with miR-590-3p mimics, or miR-590-3p inhibitor, and a corresponding negative control (NC) into HUVECs seeded in 12-well plates by using the Lipofectamine 2000 method (Invitrogen, Carlsbad, CA, USA). After transfection for 48 h, cells were harvested, and the activities of firefly and renilla luciferases were detected by Progema Dual-Luciferase Assay Kit.
RNA immunoprecipitation (RIP) assay
RIP assay was conducted using the EZMagna RIP Kit (Millipore, Billerica, MA, USA). The Argonaute 2(Ago2)-RIP experiments were carried out in HUVECs treated with blank, HCCLM3- or MHCC-97H-derived exosomal LINC00161 (40 µg/mL) for 24 h. RIP lysis buffer with proteinase and RNase inhibitor were used to lyse HUVECs. Magnetic beads conjugated with human anti-Ago2 antibody or control anti-IgG antibody were performed in incubation of the RIP lysates. The lysates were then harvested at 4 °C for 6 h. The purified RNAs were subjected to qRT-PCR analysis.
Tumor xenografts and lung metastatic experiments
All experimentations involving animals were carried out in full accordance with the institutional animal welfare guideline and approved by Shenzhen Hospital of Southern Medical University. Six-week-old female BALB/c nude (nu/nu) mice were purchased from SJA Laboratory Animal Co., Ltd (Hunan, China) and maintained under pathologic-free conditions. For in vivo tumorigenic assay, stable HCCLM3 cells expressing shNC and shLINC00161 were subcutaneously injected into mice. After nine days of HCCLM3 cells, exosomes isolated from stable HCCLM3 cells expressing shLINC00161 were respectively injected into the xenografts in either side of the same mouse every other day for three times. The diameter of tumor mass was measured with calipers every four days and the mean diameter of the tumor was determined on alternate days. Tumor volume (V) is calculated as follows: V = 0.5 × L × W2, where L and W are defined as the tumor length (L) and width (W). At 32 days post-injection, mice were killed, and the tumor specimens were carefully excised, photographed and tissues were preserved for further immunohistochemical staining. For the tail vein injections experiment, stable HCCLM3 cells expressing shLINC00161 were injected into the tail veins of mice. After 32 days of injection, the mice were killed and the lungs were excised, photographed, and examined histologically. For each lung, the numbers of macroscopic metastatic nodules on the lung surface were counted.
For IHC, 4-μm-thick tumor slides were dewaxed and rehydrated, treated with 3% hydrogen peroxide and incubated in 0.1% citric buffer for antigen retrieval, and blocked with 5% bovine serum albumin. The slides were then incubated overnight with primary rabbit anti-ROCK2 (ab71598, Abcam, Cambridge, MA, USA) and anti-CD34 monoclonal antibodies (ab81289), and subsequently incubated with MaxVisionTM HRP-Polymer IgG complexes (KIT5150; Maixin-Bio, Shenzhen, China). A secondary antibody was then applied for 30 min at room temperature, and the sections were counterstained with hematoxylin and observed under a microscope.
At 32 days after treatment, the mice were killed and lung tissues were harvested, fixed for 48 h in 15% neutral buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E). The tumor slides were then measured microscopically by a trained pathologist.
Total RNA extraction and real-time PCR
Total RNA was extracted from tissues and cell lines using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDAN) was synthesized using random primers and the SuperScript III reverse transcriptase (Quantabio, Beverly, MA). Quantitative real-time PCR (qRT-PCR) analysis was carried out by using Bulge-Loop miRNA qRT-PCR Starter Kit (RiboBio), with U6 serving as an internal normalized reference. For detection of LINC00161 and mRNAs, reverse-transcription reactions were carried out using Prime Script RT reagent Kit (Takara Bio, Shiga, Japan) with GAPDH used as an internal control. The PCR reaction was run in triplicate with the StepOne real-time PCR System (Applied Biosystems, Foster City, CA) using SYBR Premix Ex Taq II (TaKaRa). The amplification comprised of a 5 min denaturation at 90 °C, followed by 45 cycles of denaturation at 90 °C for 10 s, annealing at 60 °C for 40 s, and extension at 60 °C for 1 min. The primer sequences used were as follows: 5′-ACTTGAGTGAGGTGGGTTTC-3′ (forward) and 5′-TTGGTGTTCCTTGGCTTGTA-3′ (reverse) for LINC00161expression, 5′-TAGCCAGTCAGAAATGAGCTT-3′ (forward) and 5′-TGCTGCATGTTTCAATCAGAGA-3′ (reverse) for miR-590-3p expression, 5′-TCAGAGGTCTACAGATGAAGGC-3′ (forward) and 5′-CCAGGGGCTATTGGAAAGG-3′ (reverse) for ROCK2 expression, 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse) for U6 expression, and 5′-CCAGGTGGTCTCCTCTGA-3′ (forward) and 5′-GCTGTAGCCAAATCGTTGT-3′ (reverse) for GAPDH expression. The relative expression levels were calculated using the 2-∆∆Ct method after normalization with reference control.
Western blot analysis
Total proteins were isolated from tissues and cell lines by RIPA buffer and the concentrations were measured by BCA protein assay kit. Protein (30 μg) was separated by 10% SDS-PAGE and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h in TBS buffer containing 1% BSA and primary antibodies were incubated at 4 °C. The membrane was washed with 1× TBST and incubated with secondary antibody conjugate in 1×TBS for 1 h at room temperature. The membrane was washed with 1× TBST and the relative concentrations of each protein were measured with Quantity One software (Bio-Rad, California, USA). GAPDH quantified on the same blot served as a loading control. The primary antibodies were obtained from Abcam as following: ROCK2 (ab71598), LIMK1 (ab81046), p-LIMK1 (ab194798), MLC (ab79935), p-MLC (ab2480, MMP-2 (ab37150), MMP-9 (ab38898), VEGF-A (ab51745), CD63 (ab59479), CD9 (ab223052), TSG101 (ab30871) and HSP70 (ab2787), and Calnexin (ab22595).
All values are presented as the mean ± standard deviation (SD). Statistical analyses were conducted by using GraphPad Prism 6 (GraphPad Software, Inc.). Unpaired two-tailed students’ t-test was employed to compare the difference between the two groups. One-way analysis of variance (ANOVA) followed by Tukey post hoc test was used for multiple comparisons. Kaplan-Meier method was used to calculate survival curves, and the significance was analyzed by log-rank test. P < 0.05 was considered significant.
Exosomal LINC00161 is overexpressed in HCC and is associated with poor survival
A previous study demonstrated that exosomal LINC00161 was up-regulated in HCC cells , so we first sought to confirm the expression levels of exosomal LINC00161 in HCC. To this end, we detected the expression of LINC00161, miR-590-3p, and ROCK2 in HCC tissues positively expressing CD34. We found an enhancement of LINC00161 and ROCK2 and an attenuation of miR-590-3p expression (Fig. S1A–C). Moreover, the expression levels of LINC00161 and ROCK2 were inversely correlated with that of miR-590-3p (Fig. S1D–F). We found that exosomes purified from serum of patients with HCC by differential centrifugation displayed a typical cup-shaped morphology according to electron microscopy (Fig. 1A). Size of the purified exosomes ranged between 30 and 150 nm and peaked around 70 nm, suggesting their high quality and purity (Fig. 1B). These vesicles were further confirmed by Western blotting analysis of the well-established exosome markers including CD63, CD9, TSG101and HSP70, and the results suggested the relatively higher expression level of exosomal markers in HCC patients (Fig. 1C). On the other hand, we found expression of Calnexin in HCC cell lines but not in the HCC-derived exosomes, excluding the possibility of cellular contamination when preparing the exosomes (Fig. 1C). We then evaluated the expression levels of LINC00161 in exosomes derived from serum of patients with HCC by qRT-PCR. The enrichment of exosomal LINC00161 in patients with HCC was observed (Fig. 1D), and high exosomal LINC00161 expression was associated with significantly worse survival (Fig. 1E). Additionally, LINC00161 was dramatically up-regulated in HCC cells compared to WRL-58 cells, especially in HCCLM3 and MHCC-97H cells, two cell lines with higher metastasis potential (Fig. 1F). More importantly, exosomal portions from HCC cells contained LINC00161 that were akin to the intracellular levels of LINC00161 (Fig. 1G). Overall, these results demonstrate the high expression level of LNC00161 in exosomes of HCC, suggesting a possible regulating function of exosomal LINC00161 in the HCC development.
Intercellular transfer of LINC00161 by HCC-derived exosomes to HUVECs stimulates cell proliferation, migration, and angiogenesis
Exosomes derived from tumor cells are important factors influencing cancer progression and metastasis, whereby these exosomes can transfer bioactive molecules between different cell types within the tumor microenvironment . A few lncRNAs have been implicated in angiogenesis that drives tumor metastasis [29, 30]. We first asked whether LINC00161 carried by exosomes was expressed in HUVECs. To this end, we treated the HUVECs with different doses of exosomes (i.e., 10, 20, 40, and 80 µg/mL) at different time points (i.e., 12, 24, 36, and 48 h). As expected, we found that the expression of HCC-derived LINC00161 increased in a dose- and time-dependent manner in HUVEC cells (Fig. 2A, B). The expression level of LINC00161 was significantly increased at the treatment 40 µg/mL exosome after 24 h, and this doses and time point were used for the following experiments. We then investigated whether exosomes induced the transcription of endogenous LINC00161 in HUVECs. We pre-treated HUVECs with RNA polymerase inhibitor α-amanitin and subsequently treated these HUVECs with HCC-derived exosomes. We then performed qRT-PCR to detect the expression levels of LINC00161, miR-590-3p, and ROCK1 in HUVECs. We found no significant difference in LINC00161 expression before and after treatment with RNA polymerase inhibitor α-amanitin, suggesting that exosomes will not induce the transcription of endogenous LINC00161 in HUVECs (Fig. 2C). To determine that HCC-derived LINC00161 can be transferred to endothelial cells via exosomes to promote angiogenesis, PKH-67 labeled exosomes secreted from HCCLM3 and MHCC-97H cells, two cell lines with relatively high LINC00161 levels, were cultivated with HUVECs. Results from confocal microscopy revealed the successful internalization of exosomes into HUVECs (Fig. 2D), indicating the successful transfer of exosomal LINC00161 to HUVECs. To investigate the impact of exosome-carried LINC00161 on HUVECs, we performed in vitro cell viability assays. We found that exosomal LINC00161 internalization markedly increased HUVECs proliferation (Fig. 2E). We then performed Transwell migration assays to examine whether LINC00161-containing exosomes internalized by HUVECs are able to trigger cell migration. We found that the addition of exosomal LINC00161 significantly increased cell migration (Fig. 2F, G). Moreover, angiogenesis of HUVECs was evaluated. We found that in vitro model of angiogenesis in which HUVECs were induced with exosomal LINC00161 formed networks of cord-like structures (Fig. 2H). Quantitative evaluation of cord formation revealed that coincubation with exosomal LINC00161 increased the branches of the endothelial tubular structures compared to that seen with the blank group (Fig. 2I). Taken together, these data suggest that HCC-derived exosomes transfer LINC00161 to HUVECs and promote migration and angiogenesis of HUVECs.
Exosomes derived from HCC cells expressing shLINC00161 inversely correlated with reduced cell proliferation, migration, and angiogenesis in HUVECs
Based on the above findings, we sought to figure out whether inhibition of LINC00161 in HCC cells would lead to suppression of proliferation, migration, and angiogenesis in HUVECs. To this end, we transplanted HUVECs with exosomes derived from HCCLM3 or MHCC-97H cells expressing inducible shLINC00161 or shRNA. Efficient LINC00161 knockdown was confirmed by qRT-PCR (Fig. 3A). As expected, shLINC00161 treatment was significantly associated with decreased cell viability and reduced cell migration potential of HUVECs (Fig. 3B–D). Importantly, we found that endothelial tube formation was attenuated in HUVECs in the presence of exosomes derived from the shLINC00161-expressing HCC cells (Fig. 3E&F). Collectively, our data strongly suggest the involvement of exosome-carried LINC00161 in the progression and angiogenesis of HUVECs.
Exosome-carried LINC00161 binds to miR-590-3p and negatively regulates its expression in HUVECs
Accumulating evidence has shown that lncRNAs can function as competitive endogenous RNAs (ceRNAs) and sequester miRNAs (“miRNA sponge”) , targeting chromatin modification complexes or RNA-binding proteins to alter gene expression programs . As miR-590-3p has been implicated in HCC tumorigenesis  as well as angiogenesis of endothelial progenitor cells , we thus asked if there is any interaction between LINC00161 and miR-590-3p in HUVECs progression and angiogenesis. Our qRT-PCR designed to check the expression of miR-590-3p in HCC-derived exosomes with or without LINC00161 knockout revealed no obvious miR-590-3p expression in either condition, thus ruling out the possibility that miR-590-3p was transported by exosomes. Furthermore, we found that miR-590-3p level was dramatically decreased in HUVECs treated with exosomal LINC00161 derived from HCCLM3 or MHCC-97H cells (Fig. 4A). And treatment of HUVECs with α-amanitin, a specific RNA polymerase inhibitor, could further promote low expression of miR-590-3p, suggesting that exosomal LINC00161 regulates endogenous miR-590-3p expression in HUVECs (Fig. S2A). Accordingly, depletion of LINC00161 in HCCLM3 and MHCC-97H cells significantly up-regulated the expression of miR-590-3p in HUVECs transfected with the corresponding exosomes (Fig. 4B). This suggested the inversely correlated expression of exosomal LINC00161 and miR-590-3p in HUVECs. The effects of miR-590-3p mimics and inhibitors on the expression level of miR-590-3p were then confirmed by qRT-PCR (Fig. 4C). Bioinformatic analysis revealed the potential target sequence of LINC00161 and miR-590-3p (Fig. 4D). We then constructed luciferase report assay with wild type LINC00161 and mutated LINC00161 in the target sequence. We found that miR-590-3p mimics reduced the luciferase signal of wild type LINC00161, whereas miR-590-3p inhibitor displayed the opposite effect. Both miR-590-3p mimics and inhibitors showed no effect on the mutated LINC00161 (Fig. 4E). Finally, RIP assay was performed using anti-Ago2 in the HUVECs extract. Our results showed that in HUVECs without exosome treatment, LINC00161 and miR-590-3p were enriched preferentially in miRNA ribonucleoprotein complexes containing Ago2 compared with anti-IgG immunoprecipitates. However, the enrichment reduced dramatically after treatment with HCC-derived exosomes (Fig. 4F). Collectively, these results provide strong evidence for the direct targeting miR-590-3p by LINC00161 in HUVECs.
miR-590-3p directly targets ROCK2 to inhibit ROCK signaling pathway in HUVECs
ROCK signaling pathway is a key regulator in the process of angiogenesis, including endothelial cell migration, survival, and cell permeability . Next, we explored the effects of exosomal LINC00161 and miR-590-3p on the ROCK signaling pathway in HUVECs. ROCK2 mRNA level was measured in HUVECs with HCC-derived exosomes and the results showed that exosomes from HCC cells improved the ROCK2 mRNA level (Fig. 5A). And α-amanitin, a specific RNA polymerase inhibitor, could significantly inhibitROCK2 expression induced by exosomes from HCC cells, suggesting that exosomal LINC00161 promote endogenous ROCK2 transcription in HUVECs (Fig. S2B).In contrast, knockdown of LINC00161 level by shRNA reduced the ROCK2 mRNA level (Fig. 5B). miR-590-3p mimics showed inhibition effect similar to LINC00161 shRNA treatment on the ROCK2 mRNA level while miR-590-3p inhibitor displayed promoting effect in a manner akin to exosomes containing LINC00161 (Fig. 5C). The complementary sequence was also found between miR-590-3p and 3’-UTR of ROCK2 (Fig. 5D). The wild type ROCK2 luciferase reporter and mutated luciferase reporter were constructed. The miR-590-3p mimics inhibited the wild type luciferase signal and the miR-590-3p inhibitor increased it (Fig. 5E), suggesting the sequence-specific target of ROCK2 by miR-590-3p. Finally, we evaluated the ROCK2 proteins level and related proteins in ROCK2 pathway by Western blotting. The results revealed that miR-590-3p mimics suppressed the ROCK2 expression level and the phosphorylation of its downstream proteins LIMK1 and MLC while the miR-590-3p inhibitor showed the opposite effect (Fig. 5F&G). Overall, these results demonstrate that ROCK2 pathway is directly targeted by miR-590-3p in HUVECs.
miR-590-3p inhibits HUVECs migration and angiogenesis through repressing ROCK signaling pathway
To further investigate the biological consequence of miR-590-3p-mediated functions in HUVECs, we performed a series of functional studies using both gain- and loss-of-function experiments. Transwell analysis showed that overexpression of miR-590-3p led to a dramatic decrease in migration potential of HUVECs, whereas miR-590-3p loss exerted an opposite effect (Fig. 6A, B). Moreover, ROCK signaling pathway inhibitor Y-27632reduced cell migration to the same levels present in miR-590-3p overexpressing cells, and drastically reduced cell migration induced by miR-590-3p loss (Fig. 6A, B). Next, we determined the impact of miR-590-3p/ROCK2 axis in angiogenesis in HUVECs. As expected, HUVECs transfected with miR-590-3p mimics showed attenuated network cord formation and conversely, depletion of miR-590-3p increased tube formation ability in HUVECs (Fig. 6C, D). Similarly, inhibition of ROCK signaling pathway decreased tube formation to the same extent as observed in HUVECs treated with miR-590-3p mimics, and treatment of Y-27632 efficiently counteracted the tube formation induced by miR-590-3p depletion (Fig. 6C, D). In support of these results, miR-590-3p enhancement or Y-27632 treatment led to a marked reduction in MMP-2, MMP-9 and VEGFA protein expression, and simultaneous Y-27632 treatment antagonized the argument in these angiogenesis-related proteins induced by miR-590-3p inhibition (Fig. 6E, F). Collectively, our data demonstrate that miR-590-3p modulates the cell migration and angiogenesis of HUVECs by regulating ROCK signaling pathway.
miR-590-3p/ROCK2 axis mediates the migration and angiogenesis of HUVECs induced by exosome-carried LINC00161
Toclarifythat exosome LINC00161 regulates endothelial cell angiogenesis and migration by regulating miR-590-3p/ROCK axis, we first performed gain-of-function experiments by treated HUVECs cells with HCCLM3-derived exosomal LINC00161. As expected, LINC00161 significantly enhanced the migratory ability of HUVECs (Fig. 7A&B). Moreover, angiogenesis of HUVECs were increased remarkably (Fig. 7C&D). In order to functionally confirm that LINC00161 promotes and migration and angiogenesis of HUVECs by miR-590-3p, we carried out rescue experiments by treated exosomal LINC00161 and miR-590-3p mimics or ROCK signaling inhibitor Y-27632 in HUVECs. We found that overexpression of miR-590-3p and Y-27632 treatment effectively reversed the promoting effects of LINC00161 overexperssion on cell migration and angiogenesis (Fig. 7A–D). Moreover, while exosome-carried LINC00161 increased the expression level of MMP-2, MMP-9, and VEGFA, overexpression of miR-590-3p and Y-27632 suppressed the effect of LINC00161 on these biomarkers (Fig. 7E&F). Overall, these results indicate that miR-590-3p/ROCK2 axis mediates the migration and angiogenesis of HUVECs induced by exosome-carried LINC00161.
Knockdown of LINC00161 suppresses tumorigenesis and metastasis of HCC in vivo
Finally, we investigated the impact of LINC00161 in tumorigenesis and metastasis in vivo by subcutaneously transplanted HCCLM3 cells stably expressing shLINC00161 into nude mice. As expected, shLINC00161 treatment slowed down the tumor growth in nude mice by size and weight (Fig. 8A–C). In addition, qRT-PCR analysis confirmed the up-regulation of miR-590-3p and the down-regulation of ROCK2 in tumor tissue from the nude mice treated with HCCLM3 cells stably expressing shLINC00161 (Fig. 8D). Consistent with results from cell experiments, we found a marked decrease in protein levels of ROCK2, MMP-2, MMP-2, and VEGFA in the shLINC00161 treatment group (Fig. 8E&F). We sought to confirm these results by immunohistochemistry. Our results showed that ROCK2 and CD34 protein levels were lower in tumor tissue from the nude mice treated with HCCLM3 cells stably expressing shLINC00161 (Fig. 8G). Most importantly, lungs in nude mice were harvested and metastasis was examined. The results showed LINC00161 loss reduced the metastatic nodules of the lungs (Fig. 8H&I). The H&E staining of lung slides also suggested that a markedly reduction in the area of lung metastasis in the shLINC00161 knockdown group (Fig. 8J). Additionally, enhanced tumor growth in nude mice by size and weight, as well as higher protein levels of ROCK2 CD34, MMP2, MMP-9, and VEGFA were observed in tumor tissue from the nude mice treated with HCCLM3-derived exosomes (Fig. 9A–F). Opposite effects were found in tumor tissue from the nude mice treated with HCCLM3-derived exosomes with LINC00161 knockdown (Fig. 9A–F). Taken together, these findings suggest that LINC00161 is associated with tumorigenesis and increased lung metastasis of HCC in vivo.
HCC has a high mortality rate, which is mainly owing to the late manifestation at an advanced stage, a high incidence of distant metastasis, and recurrence after surgical resection of HCC . Notably, metastasis is the predominant cause of HCC recurrence without promising therapies. Recently, angiogenesis has become an established therapeutic approach to fight solid tumor growth in patients with cancer , and exosomes have gain increasing interest for their role in promoting angiogenesis and metastasis . In this study, we showed that LINC00161 from HCC-derived exosomes could promote angiogenesis and tumor metastasis. Our study pioneered the study of non-coding RNA and exosome in the HCC metastasis and provided important biomarker and drug target for the diagnosis and treatment of HCC.
Exosomes have been broadly studied for their biological function in intercellular communications between the tumor and the tumor microenvironment. A few independent studies have demonstrated the involvement of exosomal lncRNAs in the growth, migration, invasion, and chemosensitivity of HCC cells. Li et al.  reported that HCC-derived exosomal lncRNA TUC339 was involved in macrophage activation and M1/M2 polarization. Li et al.  reported that circulating exosomes-mediated transfer of lncRNA FAL1 increased HCC cell proliferation and migration by acting as a ceRNA of miR-1236. Gramantieri et al.  reported that higher circulating exosomal CASC9 were associated with tumor size and HCC recurrence after surgery. While LINC00161 is dramatically overexpressed in several human cancers, such as osteosarcoma , ovarian cancer , and HCC , the role of exosomal LINC00161 in tumors remains largely clear . In the present study, we confirmed the up-regulation of exosomal LINC00161 expression in HCC. Furthermore, we found that LINC00161 level in exosomes was significantly correlated with poor survival in patients with HCC.
Tumor angiogenesis is a distinct feature of malignant tumors that enables the expansion and metastasis of tumors to distant organs. HCC cell-derived exosomes can regulate the angiogenesis of endothelial cells by transfer of their biologically active lncRNAs and proteins to endothelial cells . In our study, we first used HUVECs to examine the influence of exosomal LINC00161 on tumor angiogenesis. Consistent with previous studies , we discovered that HCC cell-derived exosomes transferred LINC00161 to HUVECs, markedly promoted cell viability and migration features, and facilitated angiogenesis as evidenced by the formation of the tube-like structure in endothelial cells. HCC-derived exosomes can also transfer their pro-tumorigenic RNAs and proteins to normal hepatocyte and facilitate tumorigenesis in normal hepatocytes . In our study, the functional role of LINC00161 in stimulating tumor metastasis was further supported by the in vivo experimental metastasis assay, in which knockdown of LINC00161 in HCCLM3 cells that secret exosomes significantly reduced tumorigenesis and lung metastasis of HCC in nude mice. Further studies are warranted to demonstrate if the effects LINC00161 on angiogenesis are uniquely attributable to the exosome-carried LINC0016, which may be verified by inhibiting this lncRNA in HUVEC before exosome treatment.
Based on our bioinformatics data, we assumed that there was a binding site of LINC00161 and miR-590-3p, and luciferase reporter assay and RIP analysis confirmed that LINC00161 could directly bind to miR-590-3p. Additionally, in our RIP analysis, enrichment of both miR-590-3p and LINC00161 decreased markedly in miRNA ribonucleoprotein complexes containing Ago2 after treatment with HCC-derived exosomes LINC00161. This might be because exosome-carried LINC0016 suppressed the expression of miR-590-3p, which in turn dampened the binding affinity of miR-590-3p to Ago2, thus likely reducing the enrichments of both LINC00161 and miR-590-3p. A previous study also indicated that interfering miRNA expression could affect the level of Ago2 enriched genes . Several studies have demonstrated that miR-590-3p can functions as both a tumor-promoter and a tumor-suppressor. For example, miR-590-3p accelerated cell proliferation and invasion in ovarian cancer by inhibiting FOXA2 and facilitating VCAN signaling pathway . In contrast, miR-590-3p inhibits proliferation and migration in bladder cancer cells by inhibiting PHF14 . In terms of HCC, miR-590-3p has been found to be down-regulated in HCC and acts as an antitumor miRNA by inhibiting polycomb protein EED . Moreover, miR-590-3p has been implicated in angiogenesis of endothelial progenitor cells  and oxLDL-induced angiogenesis . However, whether (or how) miR-590-3p is involved in tumor angiogenesis was unclear prior to this study. Here we provide the first strong evidence and suggest that miR-590-3p plays a critical role in angiogenesis in HCC, whereby miR-590-3p overexpressing HUVECs displayed lower proliferation, migration ability, and decreased tube-like formation in vitro. Loss of miR-590-3p, on the other hand, exerted a completely opposite effect.
It has been reported that ROCK2 is up-regulated in several types of human cancer, including HCC [27, 44], and overexpression of ROCK2 is positively correlated with tumor metastasis and/or poor prognosis [27, 44]. According to our observations on the antimetastatic functions of miR-590-3p in this study, intertwining with the significance of ROCK2 in the invasion of HCC, we wondered whether the antimetastatic function of miR-590-3p in HCC was attributable to its suppressing effect on ROCK2 expression. We that miR-590-3p bound the complementary sites of 3’-UTR of ROCK2, and dramatically decreased the expression of ROCK2 as well as the phosphorylation of its downstream genes LIMK1 and MLC. Of note, silencing ROCK2 largely mimicked the migration and angiogenesis-inhibiting effect of miR-590-3p overexpression. In addition, miR-590-3p was dramatically attenuated while ROCK mRNA and protein expression was significantly enhanced upon LINC00161 knockdown in vivo. This is further accompanied by the down-regulation of metalloproteinases MMP-2, MMP-9, and VEGFA. ROCK signaling is reportedly essential for VEGF-dependent in vivo angiogenesis and in vitro capillary formation [33, 45] that play critical roles in angiogenesis and lymphangiogenesis . MMPs, on the other hand, play key roles in the regulation of extracellular matrix turnover, cancer cell migration, cell growth, inflammation, and angiogenesis . It does appear, therefore, that in HCC cells, exosome-carried LINC00161 regulates ROCK-meditated angiogenesis and HCC metastasis via targeting miR-590-3p. Nevertheless, this may be only one of the possible mechanisms given that an inhibition mediated by a miRNA is not transcriptional and is independent of the activity of the polymerase, and that the exosomes may carry other elements acting on ROCK at the transcriptional level, which needs to be further explored in the future. In addition, given that knockdown of LINC00161 has been reported to significantly inhibit liver cancer cell migration and invasion in previous work , more studies are needed to investigate the direct effects of LINC00161 on cancer cell and to uncover the underlying molecular mechanisms.
In summary, to the best of our knowledge, we reported here for the first time that HCC-derived exosomes can transfer LINC00161 to HUVECs to promote angiogenesis. This is mainly through the maintenance of cell survival, the promotion of cell migration, and the induction of tube formation. Moreover, intracellular LINC00161 stimulates HCC tumorigenesis and metastasis by activating the ROCK signaling pathway throuh sponging miR-590-3p. Our findings provide new evidence that exosome-carried LINC00161 plays an important role in HCC metastasis, suggesting that LINC00161 may serve as a novel prognostic marker and therapeutic target for HCC.
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This work was supported by Scientific Research and Innovation Fund of Xinjiang Medical University (NO.XJC2013118).
The study protocol was approved by the Medical Ethics Committee of Shenzhen Hospital of Southern Medical University and all patients provided written informed consent. All experimentations involving animals were carried out in full accordance with the institutional animal welfare guideline and approved by Shenzhen Hospital of Southern Medical University.
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You, LN., Tai, QW., Xu, L. et al. Exosomal LINC00161 promotes angiogenesis and metastasis via regulating miR-590-3p/ROCK axis in hepatocellular carcinoma. Cancer Gene Ther (2021). https://doi.org/10.1038/s41417-020-00269-2