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MicroRNA-769-5p contributes to the proliferation, migration and invasion of hepatocellular carcinoma cells by attenuating RYBP
Biomedicine & Pharmacotherapy 118 (2019) 109343
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Original article
MicroRNA-769-5p contributes to the proliferation, migration and invasion of hepatocellular carcinoma cells by attenuating RYBP ⁎⁎
Yao Xiana, Liang Wangb, Bowen Yaob, Wei Yangb, Huanye Mob, Lei Zhangc, , Kangsheng Tub,
T
⁎
a
Department of Nutrition, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province 710061, China Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province 710061, China c Department of Geriatric Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi Province 710061, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma miR-769-5p RYBP Proliferation Tumor metastasis
Hepatocellular carcinoma (HCC) is the commonest primary liver cancer with highly aggressive features. MicroRNAs (miRNAs) are demonstrated to play important roles in the tumorigenesis and progression of HCC. miR-769-5p is a recently identified cancer-associated miRNA. But, the expression level of miR-769-5p and its function in HCC are unexplored. In this study, we found that miR-769-5p expression was obviously increased in HCC samples compared to adjacent noncancerous liver tissues. Additionally, we revealed that miR-769-5p was over-expressed in HCC cells as compared with LO2 cells. Notably, HCC tissues from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages showed higher levels of miR-769-5p compared to those from corresponding controls. Interestingly, our data indicated that HCC patients highly expressing miR-769-5p had significant shorter survivals. Next, functional experiments verified that miR-769-5p knockdown markedly suppressed HCC cell proliferation, migration and invasion. Conversely, ectopic expression of miR-769-5p promoted these biological behaviors of Hep3B cells. Furthermore, depletion of miR-769-5p repressed the growth and metastasis of HCCLM3 cells in vivo. Importantly, miR-769-5p inversely modulated RING1 and YY1 binding protein (RYBP) by directly binding to 3’ untranslated region (UTR) in HCC cells. The expression of RYBP mRNA was down-regulated in HCC tissues and negatively correlated with miR-769-5p level. RYBP overexpression remarkably inhibited the proliferation, migration and invasion of HCCLM3 cells. Accordingly, knockdown of RYBP partially abolished miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells. In summary, our study revealed the up-regulated expression of miR-769-5p, which contributed to HCC progression possibly by targeting RYBP.
1. Introduction Hepatocellular carcinoma (HCC) is the commonest primary liver cancer with high mortality and one of the leading cause of cancer-related deaths worldwide [1]. In the last decades, surgical resection, liver transplantation, radiofrequency ablation (RFA), transarterial chemoembolization (TACE) and sorafenib effectively improved the prognosis of HCC patients [2]. However, high recurrence rate in patients with HCC within two years after surgery causes an unsatisfied long-
term survival [3]. Thus, investigating the mechanisms involved in HCC progression may be essential to improve the clinical outcomes. MicroRNAs (miRNAs) are crucial regulators in various biological and pathological processes viadirectly binding to 3’ untranslated region (UTR) of mRNAs to induce degradation and/or translation inhibition [4,5]. A large number of studies suggest that miRNAs are widely deregulated in HCC and regulate tumor growth and metastasis [6–12]. Our study reports that miR-187-3p functions as an anti-metastatic factor by repressing S100 calcium binding protein A4 (S100A4) in HCC
Abbreviations: HCC, hepatocellular carcinoma; RFA, radiofrequency ablation; TACE, transarterial chemoembolization; miRNAs, microRNAs; 3’UTR, 3’-untranslated region; S100A4, S100 calcium binding protein A4; SRPK1, SRSF protein kinase 1; PI3K, phosphatidylinositol 3-kinase; OSCC, oral squamous cell carcinoma; CRC, colorectal cancer; HEY1, hes related family bHLH transcription factor with YRPW motif 1; CDK1, cyclin-dependent kinase 1; NSCLC, non-small cell lung cancer; TGFBR1, transforming growth factor beta receptor 1; EGFR, epidermal growth factor receptor; lncRNA, long noncoding RNA; RYBP, RING1 and YY1 binding protein; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; KLF4, Kruppel like factor 4 ⁎ Corresponding author at: Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi Province 710061, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (L. Zhang), [email protected] (K. Tu). https://doi.org/10.1016/j.biopha.2019.109343 Received 15 June 2019; Received in revised form 1 August 2019; Accepted 7 August 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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[12]. miR-1296 is frequently under-expressed in HCC, and suppresses tumor metastasis by inhibiting SRSF protein kinase 1 (SRPK1) and inactivating phosphatidylinositol 3-kinase (PI3K)/AKT pathway [11]. Additionally, miR-671-5p and miR-204 are deregulated under hypoxia condition and participate in regulating the growth and metastasis of HCC [7,8]. Recently, miR-769-5p is identified as a cancer-associated miRNA. miR-769-5p expression is down-regulated in oral squamous cell carcinoma (OSCC) tissues and has a promising diagnosis potential [13]. miR-769-5p suppresses colorectal cancer (CRC) progression viadirectly targeting hes related family bHLH transcription factor with YRPW motif 1 (HEY1) and cyclin-dependent kinase 1 (CDK1) [14,15]. The downregulated expression of miR-769-5p combined with let-7d-5p overexpression predicts poor prognosis of non-small cell lung cancer (NSCLC) [16]. Furthermore, miR-769-5p inhibits NSCLC cell proliferation, migration and invasion viaattenuating transforming growth factor beta receptor 1 (TGFBR1) [17]. miR-769-5p regulation of epidermal growth factor receptor (EGFR) mediates the oncogenic role of long noncoding RNA (lncRNA) LINC00460 in NSCLC [18]. However, miR-769-5p is highly expressed in melanoma and facilitates cell proliferation by repressing glycogen synthase kinase-3 beta (GSK3β) [19]. But, the expression level of miR-769-5p and its function in HCC are not reported yet. The current study verified the aberrant expression of miR-769-5p in HCC. Next, the clinical significance of miR-769-5p overexpression was analyzed. Additionally, the biological role of miR-769-5p and its potential mechanism in HCC progression were explored.
Table 1 Correlation between miR-769-5p expression and clinicopathologic characteristics of hepatocellular carcinoma. Variable
Age (y) Sex HBV infection Serum AFP level (ng/ mL) Tumor size (cm) No. of tumor nodules Cirrhosis Venous infiltration Edmondson-Steiner grading TNM tumor stage
n = 80
< 50 ≥50 Male Female Absent Present < 20 ≥20 <5 ≥5 1 ≥2 Absent Present Absent Present I + II III + IV I + II III + IV
33 47 64 16 26 54 22 58 25 55 64 16 32 48 41 39 43 37 62 18
miR-769-5p expression Low (n = 40)
High (n = 40)
16 24 31 9 16 24 14 26 17 23 35 5 19 21 26 14 25 15 35 5
17 23 33 7 10 30 8 32 8 32 29 11 13 27 15 25 18 22 27 13
P
0.820 0.576 0.152 0.133 0.030* 0. 094 0.171 0.014* 0.116 0.032*
HBV, hepatitis B virus; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis. * Statistically significant.
2. Materials and methods
2.4. Quantitative real-time PCR (qRT-PCR) assay
2.1. Clinical samples
Total RNAs were isolated from tissues and cells using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) and Trizol reagent (Invitrogen), and reversely transcribed with the miScript II RT kit (Qiagen, Hilden, Germany) and the reverse transcription kit (Invitrogen). qRT-PCR assay was performed with the SYBR® Premix Ex Taq™ II (Takara, Dalian, China) in a CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) following the instructions. The primers were presented in Supplementary Table 1.
Eighty pairs of HCC and adjacent noncancerous liver tissues were harvested from patients, who received hepatectomy at Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University. All tissue samples were pathologically confirmed. Patients who received any preoperative treatments, such as RFA, TACE and sorafenib, were excluded from this study. Clinical samples were collected after obtaining written informed consents and were maintained at -80 °C until being used. The clinical features of HCC patients were displayed in Table 1. This study was approved by the Ethnic Committee of The First Affiliated Hospital of Xi’an Jiaotong University.
2.5. Cell proliferation assay HCC cell viability was determined by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Dojindo, Japan) assay as previously described [23]. For colony formation assay, HCC cells (500 cells/well) were plated in a 6-well plate. After culturing 14 days, the colonies were fixed with methanol for 10 min and stained with 0.5% crystal violet solution for 30 min.
2.2. Cell culture and transfection The normal human hepatic cell line LO2 and HCC cell lines including HepG2, Hep3B, Huh7, MHCC97H and HCCLM3 were maintained in our laboratory and cultured under standard condition as previously mentioned [8]. The RING1 and YY1 binding protein (RYBP) expression vector (pcDNA3.1-RYBP) and the empty vector (pcDNA3.1) were obtained from Shanghai Genechem, China. miR-769-5p mimics, miR-769-5p inhibitors and matched negative controls were obtained from RiboBio (Guangzhou, China). Lentiviral vector-meidated miR769-5p inhibitors were purchased from GeneCopoeia (Guangzhou, China). The RYBP siRNA pool and control siRNA pool were purchased from GE Healthcare Dharmacon, Inc. (Lafayette, CO, USA). These plasmids and oligos were transfected into HCC cells using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
2.6. Transwell assay HCC cell migration and invasion ability were determined by transwell migration and invasion assays (Corning Costar, Shanghai, China) as previously described [24]. Briefly, 1 × 104 HCC cells were added into the upper chamber in serum-free culture medium. The lower chamber was filled with completed medium with 10% FBS. After 24 h incubation, the migrated or invaded cells were fixed in 4% formaldehyde and stained with 0.05% crystal violet in PBS for 30 min at room temperature, and counted under microscope in 5 different fields of each filter.
2.3. Public database analysis
2.7. Western blotting
The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGALIHC) data from starBase platform [20,21] and a GSE21362 dataset from Gene Expression Omnibus (GEO) database were used to compare the expression of miR-769-5p between HCC and non-tumor tissues. The prognostic significance of miR-769-5p was confirmed by analyzing TCGA-LIHC data from OncoLnc [22].
Transfected HCC cells were harvested and then lysed by RIPA buffer (Beyotime, Shanghai, China) for protein extraction. The protein concentration was confirmed by the BCA™ Protein Assay Kit (Beyotime). Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel 2
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Fig. 1. Elevated expression of miR-769-5p correlates with poor prognosis of HCC. (A) The expression levels of miR-769-5p in HCC (n = 80) and adjacent nontumor tissues (n = 80) were measured using qRT-PCR. (B) GEO data (GSE21362) indicated that miR-769-5p was highly expressed in HCC tissues (n = 73) compared to nontumor tissues (n = 73). (C) qRT-PCR was performed to detect the expression levels of miR-769-5p in the normal hepatic cell line LO2 and five HCC cell lines. (D) The overall survival of HCC patients between the low (n = 40) and high (n = 40) miR-769-5p expression group was compared. (E) TCGA data demonstrated that HCC patients with high miR-769-5p level had a prominent poorer survival compared to cases with low/medium miR-769-5p level. *P < 0.05.
(n = 4 each group) were subcutaneously injected into the right flank of nude mice. Tumor size was detected every week for 4 weeks using a caliper. The tumor volumes were calculated: V (mm3) = width2 (mm2) × length (mm)/2. Immunohistochemical staining of Ki-67 was carried out in the xenograft tumor tissues. For lung metastasis mouse model, 2 × 106 HCCLM3 cells with or without miR-769-5p knockdown (n = 4 each group) were injected into the nude mice viathe tail vein. After 10 weeks, the mice were sacrificed, and serial sections were made for every tissue block of the lung. The sections were stained with hematoxylin-eosin, and the total number of lung metastases was counted under the microscope. The animal studies were approved by the Institutional Animal Care and Use Committee of the Xi’an Jiaotong University, Xi’an, China.
electrophoresis (SDS-PAGE) at 200 V for 30–60 min and transferred onto polyvinylidene difluoride (PVDF) membranes at 300 mA for 2 h (Millipore, Bedford, MA, USA). Blots were incubated with primary antibodies at 4℃ overnight and secondary antibody at room temperature for 1 h. The rabbit-anti-human RYBP antibody (ab185971) and mouse-anti-human β-actin antibody (ab8226) were from Abcam (Cambridge, MA, USA). Proteins were visualized using ECL reagents (Millipore). 2.8. Luciferase reporter assay The wild type (wt) 3’UTR of RYBP containing the binding sites for miR-769-5p (wt) was inserted into the reporter vector (pMirGLO; Promega, Madison, WI, USA). The binding sites of miR-769-5p in the 3’UTR of RYBP was mutated by a QuickChange II Site-Mutagenesis kit (Agilent, San Jose, CA, USA). HCCLM3 cells were co-transfected pMirGLO-3’UTR-RYBP-wt/-mt plasmid and miR-769-5p mimics/inhibitors. Cells were harvested 48 h post-transfection and the Dual Luciferase Reporter Assay System (Promega) was performed to detect the fluorescence intensity of each group.
2.10. Statistical analysis The data was presented as mean ± standard deviation (SD) and analyzed by student’s t-test or analysis of variance (ANOVA) using GraphPad Prism 8.0 (GraphPad Inc., San Diego, CA, USA). The chisquare test was performed to confirm the correlations between clinical features and miR-769-5p expression. Kaplan–Meier method and logrank test were carried out for survival analysis. The correlation of miR769-5p with RYBP mRNA was analyzed by the Pearson correlation test. A P value less than 0.05 was statistically significant.
2.9. Animal experimental 4-week-old male BALB/C nude mice were obtained from Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China). These mice were maintained in our laboratory animal center for one week before receiving experimental operation. The HCCLM3 cells (5 × 106) with or without miR-769-5p knockdown 3
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Fig. 2. miR-769-5p knockdown suppresses the growth and invasion of HCC cells. (A) The viability of HCC cells was repressed by miR-769-5p knockdown as suggested by CCK-8 assay. (B) miR-769-5p silencing reduced the colonies formed by HCC cells. (C) miR-769-5p knockdown suppressed HCC cell migration and invasion potential in vitro. (D) The viability of Hep3B cells was promoted by miR-769-5p overexpression as suggested by CCK-8 assay. (E) miR-769-5p overexpression increased the colonies formed by Hep3B cells. (F) Ectopic expression of miR-769-5p facilitated Hep3B cell migration and invasion potential in vitro. *P < 0.05.
3. Results
3.3. miR-769-5p facilitates the malignant phenotype of HCC cells
3.1. miR-769-5p is highly expressed in HCC
Next, miR-769-5p was knocked down by synthetic inhibitors in HCCLM3 and MHCC97H cells (P < 0.05, Supplementary Fig. 2A). CCK-8 assay suggested that miR-769-5p knockdown markedly inhibited the proliferation of HCC cells (P < 0.05, Fig. 2A). As shown in Fig. 2B, the colonies formed by HCC cells were prominently reduced by miR769-5p silencing (P < 0.05). Furthermore, cell migration and invasion potential were significantly suppressed by miR-769-5p knockdown in both HCCLM3 and MHCC97H cells (P < 0.05, Fig. 2C). Conversely, force expression of miR-769-5p obviously promoted the proliferation, migration and invasion of Hep3B cells (P < 0.05, Supplementary Fig. 2B and D–F). Importantly, miR-769-5p knockdown remarkably restrained in vivo growth of HCCLM3 cells (P < 0.05, Fig. 3A). Subcutaneous xenograft tumor tissues in miR-769-5p knockdown group showed significant lower percentage of Ki-67 staining cells and miR769-5p expression level compared to those in control group (P < 0.05, Fig. 3B and C). Additionally, lung metastases formed by HCCLM3 cells were obviously reduced by miR-769-5p knockdown in mice (P < 0.05, Fig. 3D). Thus, miR-769-5p contributed to the aggressive phenotype of HCC cells.
The expression difference of miR-769-5p between HCC and adjacent noncancerous liver tissues was detected. We found that the expression of miR-769-5p in HCC tissues was prominently higher than that in corresponding noncancerous tissues (P = 0.0004, Fig. 1A). TCGA data indicated that miR-769-5p level in HCC was 1.28-fold higher than that in normal liver tissues (Supplementary Fig. 1). Additionally, GEO data (GSE21362) also revealed an up-regulated expression of miR-769-5p in HCC (P = 0.0006, Fig. 1B). Moreover, miR-769-5p level was markedly increased in HCC cells compared to LO2 cells (P < 0.05, Fig. 1C). Therefore, these findings indicated that miR-769-5p level was aberrantly up-regulated in HCC.
3.2. High level of miR-769-5p confers to poor prognosis of HCC The median expression level of miR-769-5p was recognized as a cutoff value, and HCC patients were categorized into low (< cutoff value) and high miR-769-5p group (≥cutoff value). Interestingly, HCC tissues from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages had significantly higher levels of miR-769-5p than those from control groups (P < 0.05, Table 1). Importantly, HCC patients highly expressing miR-769-5p had an obvious poorer survival rate compared to miR-769-5p low-expressing cases (P = 0.0119, Fig. 1D). TCGA data also confirmed that high level of miR-769-5p in tumor tissues predicted poor prognosis of HCC (P = 0.0240, Fig. 1E). Thus, the up-regulated expression of miR-769-5p potentially indicated poor clinical outcomes of HCC.
3.4. miR-769-5p directly targets RYBP for post-transcriptional regulation The online platform starBase V3.0 [20,21] was used to screen the candidate targets of miR-769-5p and twenty-two genes were predicted by at least four databases cross-validation. As miR-769-5p was identified as a tumor promoting factor in HCC, we focused on tumor suppressors in HCC among these genes. RYBP, bone morphogenetic protein receptor type 2 (BMPR2) and insulin like growth factor 2 receptor (IGF2R) have been identified as tumor suppressors in HCC [25–27]. Notably, miR-769-6p knockdown markedly increased the expression of RYBP mRNA (P < 0.05, Supplementary Fig. 3), while did not affect the 4
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Fig. 3. miR-769-5p silencing restrains the growth and metastasis of HCCLM3 cells in vivo. (A) Lenti-viral vector-mediated miR-769-5p inhibitors (anti-miR-7695p) and corresponding negative control (NC) were respectively transfected into HCCLM3 cells, which were implanted into the frank of nude mice viasubcutaneous injection. Tumor volume in miR-769-5p knockdown group (n = 4) was obviously lower than that in control group (n = 4). Scale bar: 5 mm. (B) The percentage of Ki67 staining cells in tumor tissues from miR-769-5p knockdown group (n = 4) was obviously lower than that in control group (n = 4). Original magnification 400 × . (C) The expression of miR-769-5p in tumor tissues from miR-769-5p knockdown group (n = 4) was obviously lower than that in control group (n = 4). (D) Lung metastases formed by HCCLM3 cells with miR-769-5p knockdown (n = 4) were significantly less than those formed by control cells (n = 4). Original magnification 100 × . *P < 0.05.
yet. The dysregulation of miRNAs has been recognized as a crucial driver in HCC development and progression [28]. The expression of miR-769-5p is down-regulated in NSCLC, OSCC and CRC [13–15,17]. Whereas, miR-769-5p is highly expressed in melanoma [19]. But, the expression level of miR-769-5p is yet unknown in HCC. Here, we found that miR-769-5p level was obviously up-regulated in HCC tissues. TCGA and GEO data consistently supported the elevated expression of miR769-5p in HCC. Furthermore, miR-769-5p was also highly expressed in HCC cells compared to the normal hepatic cell line. Previous studies report that Bcl-xL knockdown induces miR-769-5p down-regulation and hypoxia leads to increased expression of miR-769-5p in A549 cells [29,30]. Our prior studies demonstrate that hypoxia contributes to the tumor growth and metastasis of HCC viamodulating miRNAs [7,8,11]. Therefore, it is interesting to further confirm whether miR-769-5p mediates hypoxia-induced HCC progression. More and more studies reveal that miRNAs have been identified as promising tools for diagnosis and prognosis prediction of HCC [31]. The aberrant level of miR-769-5p is strongly linked to diagnosis of lung cancer and OSCC [13,16]. Moreover, down-regulated expression of miR-769-5p in tumor tissues predicts poor clinical outcomes of NSCLC and CRC [14,17]. In this study, we found that miR-769-5p expression was significantly up-regulated in HCC tissues arising from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages. Additionally, both our data and TCGA data demonstrated that the high level of miR-769-5p indicated a prominent worse overall survival of HCC patients. Our previous studies have identified miR-1296, miR-187-3p and miR-876-5p as tumor suppressors, whereas miR-1468, miR-519a and miR-92a as drivers for HCC progression [9–12,32,33]. Currently, we demonstrated that miR-769-5p silencing suppressed HCC cell proliferation, migration and invasion, and repressed in vivo growth and
levels of BMPR2 and IGF2R mRNA in HCCLM3 cells (Supplementary Fig. 3). The 3’UTR of RYBP contained complementary sequences for miR-769-5p (Fig. 4A). miR-769-5p silencing prominently up-regulated RYBP protein expression in both HCCLM3 and MHCC97H cells (P < 0.05, Fig. 4B). Importantly, ectopic expression of miR-769-5p significantly reduced, but miR-769-5p knockdown remarkably enhanced the luciferase activity of vectors carrying wt 3’UTR of RYBP rather than mt 3’UTR of RYBP (P < 0.05, Fig. 4C). The expression of RYBP mRNA was down-regulated in HCC tissues (P < 0.0001, Fig. 4D) and negatively correlated with miR-769-5p level (r=-0.7465, P < 0.0001, Fig. 4E). Moreover, the expression of RYBP protein in HCC tissues with high miR-769-5p was obviously lower than that in HCC tissues with low miR-769-5p (P < 0.05, Fig. 4F). Herein, RYBP was a direct target of miR-769-5p. 3.5. RYBP mediated the tumor promoting role of miR-769-5p in HCC RYBP overexpression was performed in HCCLM3 cells viaplasmid transfection (P < 0.05, Fig. 5A). Functional experiments demonstrated that HCCLM3 cell proliferation, migration and invasion potential were strongly suppressed by RYBP overexpression (P < 0.05, Fig. 5B–D). Next, a specific siRNA was used to inhibit RYBP expression in HCCLM3 cells with miR-769-5p knockdown (P < 0.05, Fig. 6A). Interestingly, RYBP knockdown partially abolished miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells (P < 0.05, Fig. 6B–D). In summary, our data supported that miR-769-5p exerted an oncogenic role in HCC possibly by attenuating RYBP. 4. Discussion The molecular mechanism of HCC pathogenesis remains unclear 5
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Fig. 4. RYBP is a direct target of miR-769-5p. (A) The complementary sequences between 3’UTR of RYBP and miR-769-5p. (B) Synthetic miR-769-5p inhibitors (anti-miR-769-5p) and corresponding negative control (NC) were transduced into HCCLM3 and MHCC97H cells, and western blotting was used to detect RYBP expression. (C) The reporter vectors carrying wild type (wt) or mutated (mt) 3’UTR of RYBP and miR-769-5p mimics or inhibitors were co-transfected into HCCLM3 cells and the fluorescence intensity was detected. (D) The expression levels of RYBP mRNA in HCC (n = 80) and adjacent nontumor tissues (n = 80) were measured using qRT-PCR. (E) The correlation of miR-769-5p level with the expression of RYBP mRNA was confirmed in HCC tissues (n = 80). (F) The expression of RYBP protein in HCC tissues with high miR-769-5p (n = 8) was significantly lower than that in HCC tissues with low miR-769-5p (n = 8). *P < 0.05.
regulated in HCC and might be a useful prognostic biomarker. Additionally, miR-769-5p contributed to tumor growth and metastasis of HCC possibly by suppressing RYBP. These results suggest that miR796-5p/RYBP axis may serve as potential novel therapeutic targets for HCC.
metastasis of HCCLM3 cells. Whereas, ectopic expression of miR-769-5p enhanced the aggressive phenotype of Hep3B cells. These results suggested a tumor promoting role of miR-769-5p in HCC, which was consistent with the oncogenic role of miR-769-5p in melanoma [19]. But, miR-769-5p is previously identified to be a cancer-suppressing factor in CRC and NSCLC [14,15,17,18]. Downstream regulatory targets determine the function of miR-769-5p in tumors, which may be the mechanism underlying its opposite role in different tumors. Accordingly, the candidate targets for miR-769-5p were predicted using the starBase V3.0 online platform. After screening, RYBP, a reported tumor suppressor in HCC [25,34], caught our attention. Previous studies report that RYBP under-expression is frequently detected in HCC tissues and correlates with poor clinical outcomes [25,34]. RYBP overexpression suppresses cell growth and invasion, reduces chemoresistance and induces apoptosis of HCC cells [25]. Kruppel like factor 4 (KLF4) and SP1 have been identified as upstream regulator of RYBP in HCC and regulate HCC cell growth viamodulating RYBP [34]. Furthermore, miR-125b, miR-29 and miR-9 are post-transcriptional regulator of RYBP [35–37]. Here, RYBP was demonstrated as a novel downstream target of miR-769-5p. miR-769-5p inversely modulated RYBP expression in HCC. Moreover, we further confirmed that RYBP was under-expressed in HCC tissues and inhibited cancer cell growth and invasion. Importantly, RYBP knockdown partially abrogated miR769-5p silencing-induced tumor suppressive effectors on HCCLM3 cells. Collectively, this study provided evidence to support that miR-769-5p promoted HCC progression possibly by attenuating RYBP. To conclude, this study disclosed that miR-769-5p level was up-
5. Conclusions In this study, we describe that miR-769-5p expression is dramatically increased in HCC tissues, and inversely correlated with the prognosis of patients. In vitro and in vivo approaches verify that miR769-5p promotes the growth and metastasis of HCC cells. Mechanistically, RYBP is identified as a novel target of miR-769-5p and partially mediates the oncogenic role of miR-769-5p in HCC. These data provide further insights into the complex regulatory network of HCC pathogenesis. Declaration of Competing Interest All authors declare no conflicts of interest. Acknowledgments This work was supported by grants from Natural Science Basic Research Plan in Shaanxi Province of China (2019JM-133), Innovation Capacity Support Plan in Shaanxi Province of China (2018KJXX-045) and International Science and Technology Cooperation Program in 6
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Fig. 5. RYBP overexpression inhibits HCCLM3 cell proliferation, migration and invasion. (A) HCCLM3 cells were transfected with empty vector (EV) and pcDNA3.1-RYBP, respectively, and detected by western blotting for RYBP expression. (B) The viability of HCCLM3 cells was decreased by RYBP overexpression. (C) Ectopic expression of RYBP knockdown reduced the colonies formed by HCCLM3 cells. (D) Overexpression of RYBP repressed the migration and invasion of HCCLM3 cells. *P < 0.05.
Fig. 6. RYBP knockdown abolishes miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells. (A) RYBP was knocked down by a specific siRNA against RYBP (siRYBP) in HCCLM3 cells with miR-769-5p knockdown and immunoblotting was performed to detect RYBP protein. (B–D) CCK-8, colony formation, and transwell assays were performed to detect the proliferation, migration and invasion of HCCLM3 cells transfected with different vectors. *P < 0.05. 7
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Shaanxi Province of China (2018KW-062).
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Original article
MicroRNA-769-5p contributes to the proliferation, migration and invasion of hepatocellular carcinoma cells by attenuating RYBP ⁎⁎
Yao Xiana, Liang Wangb, Bowen Yaob, Wei Yangb, Huanye Mob, Lei Zhangc, , Kangsheng Tub,
T
⁎
a
Department of Nutrition, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province 710061, China Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province 710061, China c Department of Geriatric Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi Province 710061, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma miR-769-5p RYBP Proliferation Tumor metastasis
Hepatocellular carcinoma (HCC) is the commonest primary liver cancer with highly aggressive features. MicroRNAs (miRNAs) are demonstrated to play important roles in the tumorigenesis and progression of HCC. miR-769-5p is a recently identified cancer-associated miRNA. But, the expression level of miR-769-5p and its function in HCC are unexplored. In this study, we found that miR-769-5p expression was obviously increased in HCC samples compared to adjacent noncancerous liver tissues. Additionally, we revealed that miR-769-5p was over-expressed in HCC cells as compared with LO2 cells. Notably, HCC tissues from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages showed higher levels of miR-769-5p compared to those from corresponding controls. Interestingly, our data indicated that HCC patients highly expressing miR-769-5p had significant shorter survivals. Next, functional experiments verified that miR-769-5p knockdown markedly suppressed HCC cell proliferation, migration and invasion. Conversely, ectopic expression of miR-769-5p promoted these biological behaviors of Hep3B cells. Furthermore, depletion of miR-769-5p repressed the growth and metastasis of HCCLM3 cells in vivo. Importantly, miR-769-5p inversely modulated RING1 and YY1 binding protein (RYBP) by directly binding to 3’ untranslated region (UTR) in HCC cells. The expression of RYBP mRNA was down-regulated in HCC tissues and negatively correlated with miR-769-5p level. RYBP overexpression remarkably inhibited the proliferation, migration and invasion of HCCLM3 cells. Accordingly, knockdown of RYBP partially abolished miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells. In summary, our study revealed the up-regulated expression of miR-769-5p, which contributed to HCC progression possibly by targeting RYBP.
1. Introduction Hepatocellular carcinoma (HCC) is the commonest primary liver cancer with high mortality and one of the leading cause of cancer-related deaths worldwide [1]. In the last decades, surgical resection, liver transplantation, radiofrequency ablation (RFA), transarterial chemoembolization (TACE) and sorafenib effectively improved the prognosis of HCC patients [2]. However, high recurrence rate in patients with HCC within two years after surgery causes an unsatisfied long-
term survival [3]. Thus, investigating the mechanisms involved in HCC progression may be essential to improve the clinical outcomes. MicroRNAs (miRNAs) are crucial regulators in various biological and pathological processes viadirectly binding to 3’ untranslated region (UTR) of mRNAs to induce degradation and/or translation inhibition [4,5]. A large number of studies suggest that miRNAs are widely deregulated in HCC and regulate tumor growth and metastasis [6–12]. Our study reports that miR-187-3p functions as an anti-metastatic factor by repressing S100 calcium binding protein A4 (S100A4) in HCC
Abbreviations: HCC, hepatocellular carcinoma; RFA, radiofrequency ablation; TACE, transarterial chemoembolization; miRNAs, microRNAs; 3’UTR, 3’-untranslated region; S100A4, S100 calcium binding protein A4; SRPK1, SRSF protein kinase 1; PI3K, phosphatidylinositol 3-kinase; OSCC, oral squamous cell carcinoma; CRC, colorectal cancer; HEY1, hes related family bHLH transcription factor with YRPW motif 1; CDK1, cyclin-dependent kinase 1; NSCLC, non-small cell lung cancer; TGFBR1, transforming growth factor beta receptor 1; EGFR, epidermal growth factor receptor; lncRNA, long noncoding RNA; RYBP, RING1 and YY1 binding protein; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; KLF4, Kruppel like factor 4 ⁎ Corresponding author at: Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi Province 710061, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (L. Zhang), [email protected] (K. Tu). https://doi.org/10.1016/j.biopha.2019.109343 Received 15 June 2019; Received in revised form 1 August 2019; Accepted 7 August 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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[12]. miR-1296 is frequently under-expressed in HCC, and suppresses tumor metastasis by inhibiting SRSF protein kinase 1 (SRPK1) and inactivating phosphatidylinositol 3-kinase (PI3K)/AKT pathway [11]. Additionally, miR-671-5p and miR-204 are deregulated under hypoxia condition and participate in regulating the growth and metastasis of HCC [7,8]. Recently, miR-769-5p is identified as a cancer-associated miRNA. miR-769-5p expression is down-regulated in oral squamous cell carcinoma (OSCC) tissues and has a promising diagnosis potential [13]. miR-769-5p suppresses colorectal cancer (CRC) progression viadirectly targeting hes related family bHLH transcription factor with YRPW motif 1 (HEY1) and cyclin-dependent kinase 1 (CDK1) [14,15]. The downregulated expression of miR-769-5p combined with let-7d-5p overexpression predicts poor prognosis of non-small cell lung cancer (NSCLC) [16]. Furthermore, miR-769-5p inhibits NSCLC cell proliferation, migration and invasion viaattenuating transforming growth factor beta receptor 1 (TGFBR1) [17]. miR-769-5p regulation of epidermal growth factor receptor (EGFR) mediates the oncogenic role of long noncoding RNA (lncRNA) LINC00460 in NSCLC [18]. However, miR-769-5p is highly expressed in melanoma and facilitates cell proliferation by repressing glycogen synthase kinase-3 beta (GSK3β) [19]. But, the expression level of miR-769-5p and its function in HCC are not reported yet. The current study verified the aberrant expression of miR-769-5p in HCC. Next, the clinical significance of miR-769-5p overexpression was analyzed. Additionally, the biological role of miR-769-5p and its potential mechanism in HCC progression were explored.
Table 1 Correlation between miR-769-5p expression and clinicopathologic characteristics of hepatocellular carcinoma. Variable
Age (y) Sex HBV infection Serum AFP level (ng/ mL) Tumor size (cm) No. of tumor nodules Cirrhosis Venous infiltration Edmondson-Steiner grading TNM tumor stage
n = 80
< 50 ≥50 Male Female Absent Present < 20 ≥20 <5 ≥5 1 ≥2 Absent Present Absent Present I + II III + IV I + II III + IV
33 47 64 16 26 54 22 58 25 55 64 16 32 48 41 39 43 37 62 18
miR-769-5p expression Low (n = 40)
High (n = 40)
16 24 31 9 16 24 14 26 17 23 35 5 19 21 26 14 25 15 35 5
17 23 33 7 10 30 8 32 8 32 29 11 13 27 15 25 18 22 27 13
P
0.820 0.576 0.152 0.133 0.030* 0. 094 0.171 0.014* 0.116 0.032*
HBV, hepatitis B virus; AFP, alpha-fetoprotein; TNM, tumor-node-metastasis. * Statistically significant.
2. Materials and methods
2.4. Quantitative real-time PCR (qRT-PCR) assay
2.1. Clinical samples
Total RNAs were isolated from tissues and cells using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) and Trizol reagent (Invitrogen), and reversely transcribed with the miScript II RT kit (Qiagen, Hilden, Germany) and the reverse transcription kit (Invitrogen). qRT-PCR assay was performed with the SYBR® Premix Ex Taq™ II (Takara, Dalian, China) in a CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) following the instructions. The primers were presented in Supplementary Table 1.
Eighty pairs of HCC and adjacent noncancerous liver tissues were harvested from patients, who received hepatectomy at Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University. All tissue samples were pathologically confirmed. Patients who received any preoperative treatments, such as RFA, TACE and sorafenib, were excluded from this study. Clinical samples were collected after obtaining written informed consents and were maintained at -80 °C until being used. The clinical features of HCC patients were displayed in Table 1. This study was approved by the Ethnic Committee of The First Affiliated Hospital of Xi’an Jiaotong University.
2.5. Cell proliferation assay HCC cell viability was determined by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Dojindo, Japan) assay as previously described [23]. For colony formation assay, HCC cells (500 cells/well) were plated in a 6-well plate. After culturing 14 days, the colonies were fixed with methanol for 10 min and stained with 0.5% crystal violet solution for 30 min.
2.2. Cell culture and transfection The normal human hepatic cell line LO2 and HCC cell lines including HepG2, Hep3B, Huh7, MHCC97H and HCCLM3 were maintained in our laboratory and cultured under standard condition as previously mentioned [8]. The RING1 and YY1 binding protein (RYBP) expression vector (pcDNA3.1-RYBP) and the empty vector (pcDNA3.1) were obtained from Shanghai Genechem, China. miR-769-5p mimics, miR-769-5p inhibitors and matched negative controls were obtained from RiboBio (Guangzhou, China). Lentiviral vector-meidated miR769-5p inhibitors were purchased from GeneCopoeia (Guangzhou, China). The RYBP siRNA pool and control siRNA pool were purchased from GE Healthcare Dharmacon, Inc. (Lafayette, CO, USA). These plasmids and oligos were transfected into HCC cells using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
2.6. Transwell assay HCC cell migration and invasion ability were determined by transwell migration and invasion assays (Corning Costar, Shanghai, China) as previously described [24]. Briefly, 1 × 104 HCC cells were added into the upper chamber in serum-free culture medium. The lower chamber was filled with completed medium with 10% FBS. After 24 h incubation, the migrated or invaded cells were fixed in 4% formaldehyde and stained with 0.05% crystal violet in PBS for 30 min at room temperature, and counted under microscope in 5 different fields of each filter.
2.3. Public database analysis
2.7. Western blotting
The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGALIHC) data from starBase platform [20,21] and a GSE21362 dataset from Gene Expression Omnibus (GEO) database were used to compare the expression of miR-769-5p between HCC and non-tumor tissues. The prognostic significance of miR-769-5p was confirmed by analyzing TCGA-LIHC data from OncoLnc [22].
Transfected HCC cells were harvested and then lysed by RIPA buffer (Beyotime, Shanghai, China) for protein extraction. The protein concentration was confirmed by the BCA™ Protein Assay Kit (Beyotime). Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel 2
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Fig. 1. Elevated expression of miR-769-5p correlates with poor prognosis of HCC. (A) The expression levels of miR-769-5p in HCC (n = 80) and adjacent nontumor tissues (n = 80) were measured using qRT-PCR. (B) GEO data (GSE21362) indicated that miR-769-5p was highly expressed in HCC tissues (n = 73) compared to nontumor tissues (n = 73). (C) qRT-PCR was performed to detect the expression levels of miR-769-5p in the normal hepatic cell line LO2 and five HCC cell lines. (D) The overall survival of HCC patients between the low (n = 40) and high (n = 40) miR-769-5p expression group was compared. (E) TCGA data demonstrated that HCC patients with high miR-769-5p level had a prominent poorer survival compared to cases with low/medium miR-769-5p level. *P < 0.05.
(n = 4 each group) were subcutaneously injected into the right flank of nude mice. Tumor size was detected every week for 4 weeks using a caliper. The tumor volumes were calculated: V (mm3) = width2 (mm2) × length (mm)/2. Immunohistochemical staining of Ki-67 was carried out in the xenograft tumor tissues. For lung metastasis mouse model, 2 × 106 HCCLM3 cells with or without miR-769-5p knockdown (n = 4 each group) were injected into the nude mice viathe tail vein. After 10 weeks, the mice were sacrificed, and serial sections were made for every tissue block of the lung. The sections were stained with hematoxylin-eosin, and the total number of lung metastases was counted under the microscope. The animal studies were approved by the Institutional Animal Care and Use Committee of the Xi’an Jiaotong University, Xi’an, China.
electrophoresis (SDS-PAGE) at 200 V for 30–60 min and transferred onto polyvinylidene difluoride (PVDF) membranes at 300 mA for 2 h (Millipore, Bedford, MA, USA). Blots were incubated with primary antibodies at 4℃ overnight and secondary antibody at room temperature for 1 h. The rabbit-anti-human RYBP antibody (ab185971) and mouse-anti-human β-actin antibody (ab8226) were from Abcam (Cambridge, MA, USA). Proteins were visualized using ECL reagents (Millipore). 2.8. Luciferase reporter assay The wild type (wt) 3’UTR of RYBP containing the binding sites for miR-769-5p (wt) was inserted into the reporter vector (pMirGLO; Promega, Madison, WI, USA). The binding sites of miR-769-5p in the 3’UTR of RYBP was mutated by a QuickChange II Site-Mutagenesis kit (Agilent, San Jose, CA, USA). HCCLM3 cells were co-transfected pMirGLO-3’UTR-RYBP-wt/-mt plasmid and miR-769-5p mimics/inhibitors. Cells were harvested 48 h post-transfection and the Dual Luciferase Reporter Assay System (Promega) was performed to detect the fluorescence intensity of each group.
2.10. Statistical analysis The data was presented as mean ± standard deviation (SD) and analyzed by student’s t-test or analysis of variance (ANOVA) using GraphPad Prism 8.0 (GraphPad Inc., San Diego, CA, USA). The chisquare test was performed to confirm the correlations between clinical features and miR-769-5p expression. Kaplan–Meier method and logrank test were carried out for survival analysis. The correlation of miR769-5p with RYBP mRNA was analyzed by the Pearson correlation test. A P value less than 0.05 was statistically significant.
2.9. Animal experimental 4-week-old male BALB/C nude mice were obtained from Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China). These mice were maintained in our laboratory animal center for one week before receiving experimental operation. The HCCLM3 cells (5 × 106) with or without miR-769-5p knockdown 3
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Fig. 2. miR-769-5p knockdown suppresses the growth and invasion of HCC cells. (A) The viability of HCC cells was repressed by miR-769-5p knockdown as suggested by CCK-8 assay. (B) miR-769-5p silencing reduced the colonies formed by HCC cells. (C) miR-769-5p knockdown suppressed HCC cell migration and invasion potential in vitro. (D) The viability of Hep3B cells was promoted by miR-769-5p overexpression as suggested by CCK-8 assay. (E) miR-769-5p overexpression increased the colonies formed by Hep3B cells. (F) Ectopic expression of miR-769-5p facilitated Hep3B cell migration and invasion potential in vitro. *P < 0.05.
3. Results
3.3. miR-769-5p facilitates the malignant phenotype of HCC cells
3.1. miR-769-5p is highly expressed in HCC
Next, miR-769-5p was knocked down by synthetic inhibitors in HCCLM3 and MHCC97H cells (P < 0.05, Supplementary Fig. 2A). CCK-8 assay suggested that miR-769-5p knockdown markedly inhibited the proliferation of HCC cells (P < 0.05, Fig. 2A). As shown in Fig. 2B, the colonies formed by HCC cells were prominently reduced by miR769-5p silencing (P < 0.05). Furthermore, cell migration and invasion potential were significantly suppressed by miR-769-5p knockdown in both HCCLM3 and MHCC97H cells (P < 0.05, Fig. 2C). Conversely, force expression of miR-769-5p obviously promoted the proliferation, migration and invasion of Hep3B cells (P < 0.05, Supplementary Fig. 2B and D–F). Importantly, miR-769-5p knockdown remarkably restrained in vivo growth of HCCLM3 cells (P < 0.05, Fig. 3A). Subcutaneous xenograft tumor tissues in miR-769-5p knockdown group showed significant lower percentage of Ki-67 staining cells and miR769-5p expression level compared to those in control group (P < 0.05, Fig. 3B and C). Additionally, lung metastases formed by HCCLM3 cells were obviously reduced by miR-769-5p knockdown in mice (P < 0.05, Fig. 3D). Thus, miR-769-5p contributed to the aggressive phenotype of HCC cells.
The expression difference of miR-769-5p between HCC and adjacent noncancerous liver tissues was detected. We found that the expression of miR-769-5p in HCC tissues was prominently higher than that in corresponding noncancerous tissues (P = 0.0004, Fig. 1A). TCGA data indicated that miR-769-5p level in HCC was 1.28-fold higher than that in normal liver tissues (Supplementary Fig. 1). Additionally, GEO data (GSE21362) also revealed an up-regulated expression of miR-769-5p in HCC (P = 0.0006, Fig. 1B). Moreover, miR-769-5p level was markedly increased in HCC cells compared to LO2 cells (P < 0.05, Fig. 1C). Therefore, these findings indicated that miR-769-5p level was aberrantly up-regulated in HCC.
3.2. High level of miR-769-5p confers to poor prognosis of HCC The median expression level of miR-769-5p was recognized as a cutoff value, and HCC patients were categorized into low (< cutoff value) and high miR-769-5p group (≥cutoff value). Interestingly, HCC tissues from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages had significantly higher levels of miR-769-5p than those from control groups (P < 0.05, Table 1). Importantly, HCC patients highly expressing miR-769-5p had an obvious poorer survival rate compared to miR-769-5p low-expressing cases (P = 0.0119, Fig. 1D). TCGA data also confirmed that high level of miR-769-5p in tumor tissues predicted poor prognosis of HCC (P = 0.0240, Fig. 1E). Thus, the up-regulated expression of miR-769-5p potentially indicated poor clinical outcomes of HCC.
3.4. miR-769-5p directly targets RYBP for post-transcriptional regulation The online platform starBase V3.0 [20,21] was used to screen the candidate targets of miR-769-5p and twenty-two genes were predicted by at least four databases cross-validation. As miR-769-5p was identified as a tumor promoting factor in HCC, we focused on tumor suppressors in HCC among these genes. RYBP, bone morphogenetic protein receptor type 2 (BMPR2) and insulin like growth factor 2 receptor (IGF2R) have been identified as tumor suppressors in HCC [25–27]. Notably, miR-769-6p knockdown markedly increased the expression of RYBP mRNA (P < 0.05, Supplementary Fig. 3), while did not affect the 4
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Fig. 3. miR-769-5p silencing restrains the growth and metastasis of HCCLM3 cells in vivo. (A) Lenti-viral vector-mediated miR-769-5p inhibitors (anti-miR-7695p) and corresponding negative control (NC) were respectively transfected into HCCLM3 cells, which were implanted into the frank of nude mice viasubcutaneous injection. Tumor volume in miR-769-5p knockdown group (n = 4) was obviously lower than that in control group (n = 4). Scale bar: 5 mm. (B) The percentage of Ki67 staining cells in tumor tissues from miR-769-5p knockdown group (n = 4) was obviously lower than that in control group (n = 4). Original magnification 400 × . (C) The expression of miR-769-5p in tumor tissues from miR-769-5p knockdown group (n = 4) was obviously lower than that in control group (n = 4). (D) Lung metastases formed by HCCLM3 cells with miR-769-5p knockdown (n = 4) were significantly less than those formed by control cells (n = 4). Original magnification 100 × . *P < 0.05.
yet. The dysregulation of miRNAs has been recognized as a crucial driver in HCC development and progression [28]. The expression of miR-769-5p is down-regulated in NSCLC, OSCC and CRC [13–15,17]. Whereas, miR-769-5p is highly expressed in melanoma [19]. But, the expression level of miR-769-5p is yet unknown in HCC. Here, we found that miR-769-5p level was obviously up-regulated in HCC tissues. TCGA and GEO data consistently supported the elevated expression of miR769-5p in HCC. Furthermore, miR-769-5p was also highly expressed in HCC cells compared to the normal hepatic cell line. Previous studies report that Bcl-xL knockdown induces miR-769-5p down-regulation and hypoxia leads to increased expression of miR-769-5p in A549 cells [29,30]. Our prior studies demonstrate that hypoxia contributes to the tumor growth and metastasis of HCC viamodulating miRNAs [7,8,11]. Therefore, it is interesting to further confirm whether miR-769-5p mediates hypoxia-induced HCC progression. More and more studies reveal that miRNAs have been identified as promising tools for diagnosis and prognosis prediction of HCC [31]. The aberrant level of miR-769-5p is strongly linked to diagnosis of lung cancer and OSCC [13,16]. Moreover, down-regulated expression of miR-769-5p in tumor tissues predicts poor clinical outcomes of NSCLC and CRC [14,17]. In this study, we found that miR-769-5p expression was significantly up-regulated in HCC tissues arising from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages. Additionally, both our data and TCGA data demonstrated that the high level of miR-769-5p indicated a prominent worse overall survival of HCC patients. Our previous studies have identified miR-1296, miR-187-3p and miR-876-5p as tumor suppressors, whereas miR-1468, miR-519a and miR-92a as drivers for HCC progression [9–12,32,33]. Currently, we demonstrated that miR-769-5p silencing suppressed HCC cell proliferation, migration and invasion, and repressed in vivo growth and
levels of BMPR2 and IGF2R mRNA in HCCLM3 cells (Supplementary Fig. 3). The 3’UTR of RYBP contained complementary sequences for miR-769-5p (Fig. 4A). miR-769-5p silencing prominently up-regulated RYBP protein expression in both HCCLM3 and MHCC97H cells (P < 0.05, Fig. 4B). Importantly, ectopic expression of miR-769-5p significantly reduced, but miR-769-5p knockdown remarkably enhanced the luciferase activity of vectors carrying wt 3’UTR of RYBP rather than mt 3’UTR of RYBP (P < 0.05, Fig. 4C). The expression of RYBP mRNA was down-regulated in HCC tissues (P < 0.0001, Fig. 4D) and negatively correlated with miR-769-5p level (r=-0.7465, P < 0.0001, Fig. 4E). Moreover, the expression of RYBP protein in HCC tissues with high miR-769-5p was obviously lower than that in HCC tissues with low miR-769-5p (P < 0.05, Fig. 4F). Herein, RYBP was a direct target of miR-769-5p. 3.5. RYBP mediated the tumor promoting role of miR-769-5p in HCC RYBP overexpression was performed in HCCLM3 cells viaplasmid transfection (P < 0.05, Fig. 5A). Functional experiments demonstrated that HCCLM3 cell proliferation, migration and invasion potential were strongly suppressed by RYBP overexpression (P < 0.05, Fig. 5B–D). Next, a specific siRNA was used to inhibit RYBP expression in HCCLM3 cells with miR-769-5p knockdown (P < 0.05, Fig. 6A). Interestingly, RYBP knockdown partially abolished miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells (P < 0.05, Fig. 6B–D). In summary, our data supported that miR-769-5p exerted an oncogenic role in HCC possibly by attenuating RYBP. 4. Discussion The molecular mechanism of HCC pathogenesis remains unclear 5
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Fig. 4. RYBP is a direct target of miR-769-5p. (A) The complementary sequences between 3’UTR of RYBP and miR-769-5p. (B) Synthetic miR-769-5p inhibitors (anti-miR-769-5p) and corresponding negative control (NC) were transduced into HCCLM3 and MHCC97H cells, and western blotting was used to detect RYBP expression. (C) The reporter vectors carrying wild type (wt) or mutated (mt) 3’UTR of RYBP and miR-769-5p mimics or inhibitors were co-transfected into HCCLM3 cells and the fluorescence intensity was detected. (D) The expression levels of RYBP mRNA in HCC (n = 80) and adjacent nontumor tissues (n = 80) were measured using qRT-PCR. (E) The correlation of miR-769-5p level with the expression of RYBP mRNA was confirmed in HCC tissues (n = 80). (F) The expression of RYBP protein in HCC tissues with high miR-769-5p (n = 8) was significantly lower than that in HCC tissues with low miR-769-5p (n = 8). *P < 0.05.
regulated in HCC and might be a useful prognostic biomarker. Additionally, miR-769-5p contributed to tumor growth and metastasis of HCC possibly by suppressing RYBP. These results suggest that miR796-5p/RYBP axis may serve as potential novel therapeutic targets for HCC.
metastasis of HCCLM3 cells. Whereas, ectopic expression of miR-769-5p enhanced the aggressive phenotype of Hep3B cells. These results suggested a tumor promoting role of miR-769-5p in HCC, which was consistent with the oncogenic role of miR-769-5p in melanoma [19]. But, miR-769-5p is previously identified to be a cancer-suppressing factor in CRC and NSCLC [14,15,17,18]. Downstream regulatory targets determine the function of miR-769-5p in tumors, which may be the mechanism underlying its opposite role in different tumors. Accordingly, the candidate targets for miR-769-5p were predicted using the starBase V3.0 online platform. After screening, RYBP, a reported tumor suppressor in HCC [25,34], caught our attention. Previous studies report that RYBP under-expression is frequently detected in HCC tissues and correlates with poor clinical outcomes [25,34]. RYBP overexpression suppresses cell growth and invasion, reduces chemoresistance and induces apoptosis of HCC cells [25]. Kruppel like factor 4 (KLF4) and SP1 have been identified as upstream regulator of RYBP in HCC and regulate HCC cell growth viamodulating RYBP [34]. Furthermore, miR-125b, miR-29 and miR-9 are post-transcriptional regulator of RYBP [35–37]. Here, RYBP was demonstrated as a novel downstream target of miR-769-5p. miR-769-5p inversely modulated RYBP expression in HCC. Moreover, we further confirmed that RYBP was under-expressed in HCC tissues and inhibited cancer cell growth and invasion. Importantly, RYBP knockdown partially abrogated miR769-5p silencing-induced tumor suppressive effectors on HCCLM3 cells. Collectively, this study provided evidence to support that miR-769-5p promoted HCC progression possibly by attenuating RYBP. To conclude, this study disclosed that miR-769-5p level was up-
5. Conclusions In this study, we describe that miR-769-5p expression is dramatically increased in HCC tissues, and inversely correlated with the prognosis of patients. In vitro and in vivo approaches verify that miR769-5p promotes the growth and metastasis of HCC cells. Mechanistically, RYBP is identified as a novel target of miR-769-5p and partially mediates the oncogenic role of miR-769-5p in HCC. These data provide further insights into the complex regulatory network of HCC pathogenesis. Declaration of Competing Interest All authors declare no conflicts of interest. Acknowledgments This work was supported by grants from Natural Science Basic Research Plan in Shaanxi Province of China (2019JM-133), Innovation Capacity Support Plan in Shaanxi Province of China (2018KJXX-045) and International Science and Technology Cooperation Program in 6
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Fig. 5. RYBP overexpression inhibits HCCLM3 cell proliferation, migration and invasion. (A) HCCLM3 cells were transfected with empty vector (EV) and pcDNA3.1-RYBP, respectively, and detected by western blotting for RYBP expression. (B) The viability of HCCLM3 cells was decreased by RYBP overexpression. (C) Ectopic expression of RYBP knockdown reduced the colonies formed by HCCLM3 cells. (D) Overexpression of RYBP repressed the migration and invasion of HCCLM3 cells. *P < 0.05.
Fig. 6. RYBP knockdown abolishes miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells. (A) RYBP was knocked down by a specific siRNA against RYBP (siRYBP) in HCCLM3 cells with miR-769-5p knockdown and immunoblotting was performed to detect RYBP protein. (B–D) CCK-8, colony formation, and transwell assays were performed to detect the proliferation, migration and invasion of HCCLM3 cells transfected with different vectors. *P < 0.05. 7
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Shaanxi Province of China (2018KW-062).
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