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miR-1307-3p promotes tumor growth and metastasis of hepatocellular carcinoma by repressing DAB2 interacting protein
Biomedicine & Pharmacotherapy 117 (2019) 109055
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miR-1307-3p promotes tumor growth and metastasis of hepatocellular carcinoma by repressing DAB2 interacting protein
T
Shuangjiang Chena,b,1, Liang Wanga,1, Bowen Yaoa, Qingguang Liua, , Cheng Guoa, ⁎
a b
⁎
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, 710061, China Department of General Surgery, Ankang People's Hospital, Ankang, Shaanxi Province, 725000, China
ARTICLE INFO
ABSTRACT
Keywords: HCC miR-1307-3p DAB2IP Tumor growth Tumor metastasis
Increasing studies provide evidence to support that microRNAs (miRNAs) play important roles in regulating hepatocellular carcinoma (HCC) initiation and progression. However, whether miR-1307-3p is aberrantly expressed in HCC and affects malignant behaviors of cancer cells remain unknown. In this study, we found that miR-1307-3p expression was obviously up-regulated in HCC compared to adjacent nontumor tissues. Moreover, miR-1307-3p expression was prominently higher in HCC cells compared with the normal hepatic cell line LO2. Patients with venous infiltration, tumor size ≥5 cm and advanced tumor stages (III + IV) had significant higher levels of miR-1307-3p in HCC tissues. Notably, the high level of miR-1307-3p predicted poor clinical outcomes of HCC patients. Functionally, miR-1307-3p knockdown inhibited the proliferation, migration and invasion of MHCC97H and HCCLM3 cells, and suppressed the in vivo growth and metastasis of HCCLM3 cells. Conversely, overexpression of miR-1307-3p facilitated Hep3B cell proliferation, migration and invasion. Mechanistically, DAB2 interacting protein (DAB2IP) was screened as a direct target of miR-1307-3p. The expression of DAB2IP mRNA was down-regulated and inversely correlated with miR-1307-3p level in HCC tissues. miR-1307-3p knockdown increased the level of DAB2IP in HCC cells. Luciferase reporter assay confirmed the direct interaction between miR-1307-3p and 3′UTR of DAB2IP. Importantly, DAB2IP overexpression significantly suppressed the proliferation, migration and invasion of HCCLM3 cells. DAB2IP knockdown rescued miR-1307-3p silencingattenuated HCC cell proliferation, migration and invasion. Taken together, our findings suggest that miR-13073p plays a driving role in HCC progression by targeting DAB2IP. Our study may provide new therapeutic targets for HCC treatment.
1. Introduction Hepatocellular carcinoma (HCC) is the most common primary liver cancer and a major cause of cancer-related death worldwide [1]. The long-term survival of HCC patients is still unsatisfactory due to lack of effective early diagnostic indicators, high aggressive features of tumor, and postoperative recurrence and metastasis [2,3]. Therefore, it is essential to investigate the molecular mechanism underlying HCC progression and explore new biomarkers for HCC diagnosis and treatment. MicroRNAs (miRNAs) are a group of small non-coding RNAs
involved in gene regulation by binding to the 3′-untranslated regions (3′-UTRs) of target mRNAs, and accordingly lead to mRNA degradation and/or translational repression [4]. Accumulating evidence supports that miRNAs exert crucial roles in physiological and pathological processes of human diseases including cancer [5,6]. Our group has revealed several aberrantly expressed miRNAs, which play critical roles in modulating cell proliferation, apoptosis, cell cycle progression, migration and invasion in HCC [7–14]. For instance, miR-1468 is highly expressed in HCC and contributes to tumor growth of HCC by activating peroxisome proliferator-activated receptor-γ (PPAR-γ)/AKT signaling
Abbreviations: HCC, hepatocellular carcinoma; miRNAs, microRNAs; 3′UTRs, 3’-untranslated regions; PPAR-γ, peroxisome proliferator-activated receptor-γ; EMT, epithelial-mesenchymal transition; UBE3C, ubiquitin protein ligase E3C; S100A4, S100 calcium binding protein A4; RCC, renal cell carcinoma; TKI, tyrosine kinase inhibitors; DAB2IP, DAB2 interacting protein; VKORC1L1, vitamin K epoxide reductase complex subunit 1 like 1; GATAD2B, GATA zinc finger domain containing 2B; EML2, EMAP like 2; KDM6B, lysine demethylase 6B; TMEM161A, transmembrane protein 161A; DNMTs, DNA methyltransferases; hnRNPK, heterogeneous nuclear ribonucleoprotein K; MMP2, matrix metalloproteinase 2 ⁎ 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. E-mail addresses: [email protected] (Q. Liu), [email protected] (C. Guo). 1 Contributed equally. https://doi.org/10.1016/j.biopha.2019.109055 Received 13 May 2019; Received in revised form 29 May 2019; Accepted 30 May 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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[8]. miR-542-3p functions as a tumor suppressor and a prognostic biomarker in HCC, and it suppresses epithelial-mesenchymal transition (EMT) and metastasis of HCC by repressing ubiquitin protein ligase E3C (UBE3C) [10]. Furthermore, miR-187-3p plays an anti-metastatic role in HCC and inversely regulates S100 calcium binding protein A4 (S100A4) abundance to restrain EMT of cancer cells [7]. miR-1307-3p is a newly identified cancer-related miRNA. miR1307-3p is a risk factor for metastatic renal cell carcinoma (RCC) progression under tyrosine kinase inhibitors (TKI) treatment [15]. Serum miR-1307-3p level is elevated in breast cancer and it is able to use for breast cancer early detection [16]. miR-1307-3p contributes to breast cancer cell proliferation in vitro and facilitates tumor formation in vivo [17]. Moreover, miR-1307-3p is implicated in capecitabine-based therapy in colon cancer [18]. However, the expression of miR-1307-3p and its biological role in HCC were still unclear. In the present study, we detected the expression of miR-1307-3p in HCC and adjacent nontumor tissues. Loss- and gain-of-function experiments were designed to investigate the biological function of miR1307-3p in HCC cell proliferation migration and invasion. Subsequently, the molecular mechanism involved in the role of miR1307-3p was disclosed. This study, for the first time, revealed that miR1307-3p was a tumor promoting factor via directly repressing DAB2 interacting protein (DAB2IP) in HCC.
2.4. Western blot Proteins were extracted from HCC cell lines using RIPA lysis buffer (Beyotime, Guangzhou, China) and the concentration was measured by the BCA protein assay kit (Pierce, Rockford, IL, USA). The protocols for western blot analysis were described in our previous study [20]. Rabbitanti-human DAB2IP primary antibody (ab87811) and mouse-antihuman DAB2IP primary antibody (ab8245) were purchase from Abcam (Cambridge, MA, USA). 2.5. Cell proliferation assay The cell proliferation was detected by the Cell Counting Kit-8 (CCK8; Dojindo, Kyushu, Japan) as previously described [20]. For the colony formation assay, HCC cells were seeded into a six-well plate (500 cells per well) and were cultured for two weeks. Then, the colonies were fixed with 4% methanol and stained with 0.1% crystal violet. Finally, the colonies were photographed and counted. 2.6. Cell migration and invasion assays The Transwell chemotaxis 24-well chamber (BD Biosciences, Franklin Lakes, NJ, USA) was used for cell migration assay. 50 μl Matrigel (BD Biosciences) was added into the basement membrane of filters for cell invasion assay. The detailed protocols for Transwell assay were described in our previous study [20].
2. Materials and methods 2.1. Patients and samples Eighty pairs of HCC and paracancerous tissues were obtained from patients who underwent hepatectomy in Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University. HCC patients that received preoperative treatments were excluded. All tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C until use. The experimental protocols were approved by the Ethics Committee of The First Affiliated Hospital of Xi’an Jiaotong University (No. 2018-G14, March 3, 2018) with the 1964 Helsinki declaration and its later amendments [19]. Informed consent was obtained from all patients.
2.7. Luciferase reporter assay The pEZX-MT06 vector containing the full length of DAB2IP 3′-UTR was purchased from GeneCopoeia Inc. The target sequence of miR1307-3p in the 3′UTR of DAB2IP was mutated by a QuickChange II SiteMutagenesis kit (Agilent, San Jose, CA, USA). Then, cells were cotransfected with luciferase reporter vector containing wild type (wt) or mutated (mt) 3′UTR of DAB2IP and miR-1307-3p mimics or inhibitors. After transfection for 48 h, the fluorescence intensity was measured by a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to renilla luciferase activity.
2.2. Cell lines and cell transfection Four human HCC cell lines Hep3B, Huh7, MHCC97H, HCCLM3 and normal hepatic cell line LO2 were maintained in our lab and cultured as previously described [12]. miR-1307-3p mimics and scrambled control were purchased from RioBio (Guangzhou, China). Lentivector-mediated miR-1307-3p inhibitors (anti-miR-1307-3p) and negative control (NC) were obtained from GeneCopoeia Inc. (Guangzhou, China). DAB2IP expression vector (pcDNA3.1-DAB2IP) was designed by GenePharma (Shanghai, China) and empty vector (pcDNA3.1) was used as negative control. A specific DAB2IP siRNA and a scrambled siRNA were synthesized by RioBio and sequences were shown in Supplementary Table 1. Vectors were transfected into HCC cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions.
2.8. Animal experimental All experiments involved in animal were approved by the Animal Care and Use Committee of Xi’an Jiaotong University. BALB/C nude mice (male, 4-week-old) were purchased from Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China). The in vivo tumor formation and pulmonary metastasis assays were conducted as previously described [20]. The xenograft tumor tissues were subjected to immunohistochemistry for Ki-67 staining and lung tissues were detected by H&E staining. 2.9. Statistical analysis
2.3. RNA extraction and real-time PCR analysis
All the statistical analysis of data was carried out using the GraphPad Prism Software (Version 8.0, GraphPad Software, La Jolla, CA, USA). Data were presented as mean ± standard deviation (SD) from three independent repeats. The difference between two independent groups was assessed using a two-tail Student’s t-test and multiple group comparisons were performed using ANOVA. Pearson correlation test was carried out to confirm the correlation between miR1307-3p and DAB2IP mRNA expression. Survival analysis was determined by Kaplan-Meier method and log-rank test. P < 0.05 was considered to be statistically significant.
Total RNA from tissues and cell lines was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) and Trizol reagent (Invitrogen) and reversely transcribed into cDNA using the miScript II RT kit and the reverse transcription kit (Invitrogen) following the manufacture’s protocol. Real-time PCR assay was performed using the SYBR® Premix Ex Taq™ II (Takara, Dalian, China) in a CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH and U6 were used as internal controls. The primers used are listed in Supplementary Table 1. 2
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Fig. 1. The expression and clinical significance of miR-1307-3p in HCC. (A) qRT-PCR analysis was performed to detect the expression difference of miR-1307-3p between HCC tissues (n = 80) and adjacent non-tumor tissues (n = 80). (B) The levels of miR-1307-3p in HCC cell lines (Hep3B, Huh7, MHCC97H and HCCLM3) and normal hepatic cell line LO2 were measured by qRT-PCR. (C) HCC patients with venous infiltration had a significant higher level of miR-1307-3p compared to cases without venous infiltration. (D) HCC patients with tumor size ≥5 cm had a significant higher level of miR-1307-3p compared to cases with tumor size < 5 cm. (E) HCC patients with advanced tumor stages (III + IV) had a significant higher level of miR-1307-3p compared to cases with early tumor stages (I + II). (F) HCC patients were divided into two subgroups (low/high miR-1307-3p group) according to the median level of miR-1307-3p in this cohort. HCC patients with high miR-1307-3p level (n = 40) showed a significant poorer overall survival compared to low miR-1307-3p group (n = 40). *P < 0.05.
E). Moreover, HCC patients with high miR-1307-3p level had an obvious poorer overall survival compared to miR-1307-3p low-expressing cases (P = 0.0099, Fig. 1F). These findings suggested that miR-1307-3p was a promising prognostic biomarker for HCC.
3. Results 3.1. miR-1307-3p expression is increased in HCC PCR analysis was performed to examine the expression difference of miR-1307-3p between HCC and adjacent nontumor tissues. We found that the expression of miR-1307-3p in HCC tissues was prominently higher than that in paracancerous tissues (P = 0.0038, Fig. 1A). Moreover, TCGA data from starBase V3.0 online platform [21,22] indicated that miR-1307-3p was significantly overexpressed in HCC tissues compared to normal liver tissues (P < 0.0001, Supplementary Fig. 1). Additionally, up-regulated expression of miR-1307-3p was detected in HCC cell lines (Hep3B, Huh7, MHCC97H, HCCLM3) compared with normal hepatic cell line LO2 (P < 0.05, Fig. 1B). These results indicated a putative tumor promoting role of miR-1307-3p in HCC.
3.3. miR-1307-3p promotes the growth and metastasis of HCC cells To determine the biological function of miR-1307-3p in HCC, MHCC97H and HCCLM3 cells were transfected with miR-1307-3p inhibitors and negative control (P < 0.05, Fig. 2A). CCK-8 assay showed that miR-1307-3p knockdown significantly suppressed the proliferation of HCC cells (P < 0.05, Fig. 2B). Furthermore, the colonies formed by HCC cells were obviously reduced by miR-1307-3p knockdown (P < 0.05, Fig. 2C). Additionally, miR-1307-3p silencing prominently repressed the in vivo growth of HCCLM3 cells (P < 0.05, Fig. 2D). The percentage of Ki-67 positive cells in xenograft tumor tissues from miR1307-3p group was markedly lower than that in control group (P < 0.05, Fig. 2E). Next, we found that knockdown of miR-1307-3p consistently inhibited the metastasis of HCC cells in vitro and in vivo (P < 0.05, Fig. 3). Conversely, miR1307-3p overexpression facilitated the proliferation, migration and invasion of Hep3B cells (P < 0.05, Supplementary Fig. 2). These results revealed the tumor promoting effects of miR-1307-3p in HCC.
3.2. Elevated expression of miR-1307-3p correlates with poor prognosis To disclose the clinical significance of miR-1307-3p, HCC patients were divided into two subgroups (low/high miR-1307-3p group) according to the median level of miR-1307-3p in this cohort. Interestingly, HCC patients with venous infiltration had a significant higher miR-1307-3p level compared to HCC patients without venous infiltration (P < 0.05, Fig. 1C). miR-1307-3p levels in HCC patents with tumor size ≥5 cm and advanced tumor stages (III + IV) were obviously higher than those in cases with tumor size < 5 cm and early tumor stages (I + II) (P = 0.0027 and 0.0001, respectively, Fig. 1D and
3.4. DAB2IP is a target for miR-1307-3p To study the molecular mechanism underlying the oncogenic role of 3
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Fig. 2. miR-1307-3p knockdown suppresses the growth of HCC cells. (A) miR-1307-3p inhibitors (anti-miR-1307-3p) or negative control (NC) were transfected into MHCC97H and HCCLM3 cells, and qRT-PCR was performed to detect miR-1307-3p expression. (B) CCK-8 assay revealed that miR-1307-3p knockdown suppressed the proliferation of HCC cells. (C) The colonies formed by HCC cells were reduced by miR-1307-3p knockdown. (D) HCCLM3 cells with or without miR-13073p knockdown (n = 4 each group) were implanted into nude mice via subcutaneous injection. Tumor growth curves indicated that miR-1370-3p knockdown restrained in vivo growth of HCC cells. (E) The percentage of Ki-67 positive staining cells in tumor tissues from miR-1307-3p group (n = 4) was significantly lower than that in control group (n = 4). *P < 0.05.
miR-1307-3p in HCC, we screened potential targets for miR-1307-3p based on starBase V3.0 online platform [21,22]. Six candidates including vitamin K epoxide reductase complex subunit 1 like 1 (VKORC1L1), GATA zinc finger domain containing 2B (GATAD2B), EMAP like 2 (EML2), DAB2IP, lysine demethylase 6B (KDM6B) and transmembrane protein 161A (TMEM161A) were predicted in three public databases. Notably, only DAB2IP is previously identified as a tumor suppressor in HCC [23] and its 3′UTR has complementary sequences for miR-1307-3p (Fig. 4A). We found that miR-1307-3p knockdown significantly increased the level of DAB2IP mRNA in HCCLM3 cells (P < 0.05, Supplementary Fig. 3). However, miR-13073p silencing did not change KDM6B mRNA expression (Supplementary Fig. 3). Moreover, the levels of DAB2IP protein in HCCLM3 and MHCC97H cells were up-regulated by miR-1307-3p knockdown (P < 0.05, Fig. 4B). The levels of DAB2IP mRNA in HCC tissues were prominently lower than those in adjacent nontumor tissues (P < 0.0001, Fig. 4C). And miR-1307-3p expression was inversely correlated with the level of DAB2IP mRNA in HCC tissues (r=-0.3835, P = 0.0004, Fig. 4D). Importantly, miR-1307-3p overexpression reduced, whereas miR-1307-3p knockdown enhanced the luciferase activities of vectors carrying wt 3′UTR of DAB2IP (P < 0.05, Fig. 4E). But, modulating miR-1307-3p level had no effect on luciferase activities
of vector containing mt 3′UTR of DAB2IP (Fig. 4E). Thus, our data recognized DAB2IP as a direct target of miR-1307-3p. 3.5. DAB2IP mediates the oncogenic role of miR-1307-3p in HCC cells To confirm whether DAB2IP is a downstream effector of miR-13073p in HCC, DAB2IP expression was restored by transfecting expression plasmid in HCCLM3 cells (P < 0.05, Fig. 5A). DAB2IP overexpression remarkably suppressed the proliferation, migration and invasion of HCCLM3 cells, which was consistent with the effects of miR-1307-3p knockdown (P < 0.05, Fig. 5B–D). Next, DAB2IP was knocked down by a specific siRNA in HCCLM3 cells with miR-1307-3p knockdown (P < 0.05, Fig. 6A). Interestingly, DAB2IP silencing rescued miR-13073p knockdown-attenuated HCCLM3 cell proliferation, migration and invasion (P < 0.05, Fig. 6B–D). Taken together, our results indicated that miR-1307-3p contributed to HCC cell proliferation, migration and invasion by targeting DAB2IP. 4. Discussion The aberrant expression of miRNAs has been identified as potential diagnostic and prognostic biomarker for human cancer [24,25]. For 4
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Fig. 3. Knockdown of miR-1307-3p represses the metastasis of HCC cells. (A) Transwell assays demonstrated that miR-1307-3p knockdown inhibited the migration and invasion of MHCC97H and HCCLM3 cells. (B) HCCLM3 cells with or without miR-1307-3p knockdown (n = 4 each group) were implanted into nude mice via tail vein injection. The metastases in lung tissues from miR-1307-3p knockdown group (n = 4) was obviously lower than those in control group (n = 4). *P < 0.05. Black arrows indicated lung metastases.
Fig. 4. DAB2IP is identified as a novel target of miR-1307-3p. (A) The 3′UTR of DAB2IP contained complementary sequences for miR-1307-3p. (B) miR-1307-3p inhibitors (anti-miR-1307-3p) or negative control (NC) were transfected into HCCLM3 and MHCC97H cells, and immunoblotting was performed to detect DAB2IP expression. (C) The expression of DAB2IP mRNA was detected by qRT-PCR between HCC tissues (n = 80) and adjacent non-tumor tissues (n = 80). (D) An inverse correlation between miR-1307-3p level and the expression of DAB2IP mRNA was found in HCC tissues (n = 80). (E) The vectors carrying wild type (wt) or mutated (mt) 3′UTR of DAB2IP and miR-1307-3p mimics or inhibitors were co-transfected into HCCLM3 cells and the relative fluorescence intensity was detected. *P < 0.05.
example, the aberrantly expressed serum miR-106b-3p, miR-101-3p and miR-1246 may be useful biomarkers for HCC diagnosis [26]. Circulating miR-103 level is increased in HCC patients and acts as a promising marker for tumor metastasis [27]. Our previous studies have
revealed the differentially expressed miRNAs, such as miR-187-5p, miR1468 and miR-23c, as prognostic biomarkers for HCC [7–9]. Here, we found that miR-1307-5p was highly expressed in HCC tissues and cell lines. HCC patients with venous infiltration, tumor size ≥5 cm and 5
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Fig. 5. DAB2IP overexpression represses the proliferation, migration and invasion of HCCLM3 cells. (A) pcDNA3.1-DAB2IP or empty vector (EV) was transfected into HCCLM3 cells and immunoblotting was performed to detect the expression of DAB2IP protein. (B) CCK-8, (C) colony formation, and (D) Transwell assays were performed to determine the proliferation, migration and invasion of HCCLM3 cells. *P < 0.05.
Fig. 6. DAB2IP silencing rescues miR-1307-3p knockdown-attenuated HCCLM3 cell proliferation, migration and invasion. (A) DAB2IP was knocked down by a specific siRNA in HCCLM3 cells with miR-1307-3p knockdown and immunoblotting was performed to detect DAB2IP protein. (B) CCK-8, (C) colony formation, and (D) Transwell assays were performed to determine the proliferation, migration and invasion of HCCLM3 cells transfected with corresponding vectors. *P < 0.05. 6
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advanced tumor stages (III + IV) showed higher levels of miR-1307-3p. Furthermore, the high miR-1307-3p expression was strongly correlated with poor overall survival of HCC patients. These data indicated a potential prognostic value of miR-1307-3p in HCC. Previous study reports that the high level of serum miR-1307-3p is a promising indicator for early breast cancer [16]. Thus, it is worth to further detect the serum miR-1307-3p level in HCC patients. Previous studies have disclosed several mechanisms involved in deregulation of miRNAs in HCC. For instance, miR-122 is epigenetically suppressed by via DNA methyltransferases (DNMTs)-mediated DNA methylation [28]. LncRNA MCM3AP-AS1 functions as a molecular sponge to inversely regulate miR-194-5p abundance in HCC cells [29]. Moreover, miR-671-5p and miR-204 are hypoxia-responsive miRNAs and mediate hypoxia-induced HCC progression [12,13]. It will be interesting to investigate the molecular mechanism underlying the aberrant expression of miR-1307-3p in HCC. More and more evidences have solidly demonstrated that miRNAs are critical drivers or suppressors in the tumorigenesis and progression of HCC [30]. miR-1307-3p has previously reported to be implicated in breast cancer, RCC and colon cancer [15–18]. Here, we for the first time demonstrated that miR-1307-3p contributed to tumor growth and metastasis of HCC via gain- and loss-of-function experiments. Moreover, DAB2IP expression was inversely regulated by miR-1307-3p in HCC cells and it was accordingly identified as a direct target for miR-13073p as suggested by luciferase reporter assay. DAB2IP is a common tumor suppressor in human cancer [31]. Mutant p53 enhances the tumor promoting effects of insulin by repressing DAB2IP in breast and prostate cancer [32]. Moreover, DAB2IP underexpression contributes to invasion and metastasis of colorectal cancer by targeting heterogeneous nuclear ribonucleoprotein K/matrix metalloproteinase 2 (hnRNPK/ MMP2) axis [33]. Previous study finds that DAB2IP expression is suppressed in HCC [34]. Additionally, DAB2IP is confirmed to be a potential prognostic marker in HCC and inhibits cancer cell proliferation and invasion [23], which is consistent with our results in this study. miR-149-3p, miR-367 and miR-889 have been proved to be negative post-transcriptional regulators of DAB2IP [35–37]. This study provided a novel upstream regulatory mechanism for DAB2IP inactivation in HCC. Importantly, DAB2IP silencing rescued miR-1307-3p knockdownattenuated HCC cell proliferation, migration and invasion. Collectively, miR-1307-3p exerted an oncogenic role by targeting tumor suppressor DAB2IP in HCC. In conclusion, our findings indicated that overexpression of miR1307-3p conferred to poor clinical outcomes and promoted the proliferation, migration and invasion of cancer cells by repressing tumor suppressor DAB2IP in HCC. This study might provide a potential biomarker and promising therapeutic target for HCC treatment.
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Zhou, Y.Q. Chen, L.H. Qu, starBase: a database for exploring microRNA-mRNA interaction maps from Argonaute CLIP-Seq and Degradome-Seq data, Nucleic Acids Res. 39 (Database issue) (2011) D202–9. [22] J.H. Li, S. Liu, H. Zhou, L.H. Qu, J.H. Yang, starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data, Nucleic Acids Res. 42 (Database issue) (2014) D92–7. [23] X. Zhang, N. Li, X. Li, W. Zhao, Y. Qiao, L. Liang, Y. Ding, Low expression of DAB2IP contributes to malignant development and poor prognosis in hepatocellular carcinoma, J. Gastroenterol. Hepatol. 27 (6) (2012) 1117–1125.
5. Conclusions To conclude, this study reveals that miR-1307-3p overexpression is a frequent event and confers poor clinical outcomes in HCC. Functionally, miR-1307-3p promotes cell proliferation, migration and invasion in vitro, and enhances tumor growth and metastasis of HCC in vivo. Tumor suppressor DAB2IP is recognized as a novel direct target of miR-1307-3p and participates in miR-1307-3p-induced HCC progression. These observations support the notion that miR-1307-3p may be a novel potential target for HCC therapy. Conflict of interests The authors declare that they have no conflict of interests. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81874069, 81572847) and the Key 7
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carcinoma, J. Cell. Physiol. 234 (8) (2019) 12188–12225. [31] A. Bellazzo, G. Di Minin, L. Collavin, Block one, unleash a hundred. Mechanisms of DAB2IP inactivation in cancer, Cell Death Differ. 24 (1) (2017) 15–25. [32] E. Valentino, A. Bellazzo, G. Di Minin, D. Sicari, M. Apollonio, G. Scognamiglio, M. Di Bonito, G. Botti, G. Del Sal, L. Collavin, Mutant p53 potentiates the oncogenic effects of insulin by inhibiting the tumor suppressor DAB2IP, Proc. Natl. Acad. Sci. U. S. A. 114 (29) (2017) 7623–7628. [33] X.H. Zhu, J.M. Wang, S.S. Yang, F.F. Wang, J.L. Hu, S.N. Xin, H. Men, G.F. Lu, X.L. Lan, D. Zhang, X.Y. Wang, W.T. Liao, Y.Q. Ding, L. Liang, Down-regulation of DAB2IP promotes colorectal cancer invasion and metastasis by translocating hnRNPK into nucleus to enhance the transcription of MMP2, Int. J. Cancer 141 (1) (2017) 172–183. [34] G.H. Qiu, H. Xie, N. Wheelhouse, D. Harrison, G.G. Chen, M. Salto-Tellez, P. Lai, J.A. Ross, S.C. Hooi, Differential expression of hDAB2IPA and hDAB2IPB in normal tissues and promoter methylation of hDAB2IPA in hepatocellular carcinoma, J. Hepatol. 46 (4) (2007) 655–663. [35] A. Bellazzo, G. Di Minin, E. Valentino, D. Sicari, D. Torre, L. Marchionni, F. Serpi, M.B. Stadler, D. Taverna, G. Zuccolotto, I.M. Montagner, A. Rosato, F. Tonon, C. Zennaro, C. Agostinis, R. Bulla, M. Mano, G. Del Sal, L. Collavin, Cell-autonomous and cell non-autonomous downregulation of tumor suppressor DAB2IP by microRNA-149-3p promotes aggressiveness of cancer cells, Cell Death Differ. 25 (7) (2018) 1224–1238. [36] W. Cai, H. Jiang, Y. Yu, Y. Xu, W. Zuo, S. Wang, Z. Su, miR-367 regulation of DOC2/DAB2 interactive protein promotes proliferation, migration and invasion of osteosarcoma cells, Biomed. Pharmacother. 95 (2017) 120–128. [37] Y. Xu, J. He, Y. Wang, X. Zhu, Q. Pan, Q. Xie, F. Sun, miR-889 promotes proliferation of esophageal squamous cell carcinomas through DAB2IP, FEBS Lett. 589 (10) (2015) 1127–1135.
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miR-1307-3p promotes tumor growth and metastasis of hepatocellular carcinoma by repressing DAB2 interacting protein
T
Shuangjiang Chena,b,1, Liang Wanga,1, Bowen Yaoa, Qingguang Liua, , Cheng Guoa, ⁎
a b
⁎
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, 710061, China Department of General Surgery, Ankang People's Hospital, Ankang, Shaanxi Province, 725000, China
ARTICLE INFO
ABSTRACT
Keywords: HCC miR-1307-3p DAB2IP Tumor growth Tumor metastasis
Increasing studies provide evidence to support that microRNAs (miRNAs) play important roles in regulating hepatocellular carcinoma (HCC) initiation and progression. However, whether miR-1307-3p is aberrantly expressed in HCC and affects malignant behaviors of cancer cells remain unknown. In this study, we found that miR-1307-3p expression was obviously up-regulated in HCC compared to adjacent nontumor tissues. Moreover, miR-1307-3p expression was prominently higher in HCC cells compared with the normal hepatic cell line LO2. Patients with venous infiltration, tumor size ≥5 cm and advanced tumor stages (III + IV) had significant higher levels of miR-1307-3p in HCC tissues. Notably, the high level of miR-1307-3p predicted poor clinical outcomes of HCC patients. Functionally, miR-1307-3p knockdown inhibited the proliferation, migration and invasion of MHCC97H and HCCLM3 cells, and suppressed the in vivo growth and metastasis of HCCLM3 cells. Conversely, overexpression of miR-1307-3p facilitated Hep3B cell proliferation, migration and invasion. Mechanistically, DAB2 interacting protein (DAB2IP) was screened as a direct target of miR-1307-3p. The expression of DAB2IP mRNA was down-regulated and inversely correlated with miR-1307-3p level in HCC tissues. miR-1307-3p knockdown increased the level of DAB2IP in HCC cells. Luciferase reporter assay confirmed the direct interaction between miR-1307-3p and 3′UTR of DAB2IP. Importantly, DAB2IP overexpression significantly suppressed the proliferation, migration and invasion of HCCLM3 cells. DAB2IP knockdown rescued miR-1307-3p silencingattenuated HCC cell proliferation, migration and invasion. Taken together, our findings suggest that miR-13073p plays a driving role in HCC progression by targeting DAB2IP. Our study may provide new therapeutic targets for HCC treatment.
1. Introduction Hepatocellular carcinoma (HCC) is the most common primary liver cancer and a major cause of cancer-related death worldwide [1]. The long-term survival of HCC patients is still unsatisfactory due to lack of effective early diagnostic indicators, high aggressive features of tumor, and postoperative recurrence and metastasis [2,3]. Therefore, it is essential to investigate the molecular mechanism underlying HCC progression and explore new biomarkers for HCC diagnosis and treatment. MicroRNAs (miRNAs) are a group of small non-coding RNAs
involved in gene regulation by binding to the 3′-untranslated regions (3′-UTRs) of target mRNAs, and accordingly lead to mRNA degradation and/or translational repression [4]. Accumulating evidence supports that miRNAs exert crucial roles in physiological and pathological processes of human diseases including cancer [5,6]. Our group has revealed several aberrantly expressed miRNAs, which play critical roles in modulating cell proliferation, apoptosis, cell cycle progression, migration and invasion in HCC [7–14]. For instance, miR-1468 is highly expressed in HCC and contributes to tumor growth of HCC by activating peroxisome proliferator-activated receptor-γ (PPAR-γ)/AKT signaling
Abbreviations: HCC, hepatocellular carcinoma; miRNAs, microRNAs; 3′UTRs, 3’-untranslated regions; PPAR-γ, peroxisome proliferator-activated receptor-γ; EMT, epithelial-mesenchymal transition; UBE3C, ubiquitin protein ligase E3C; S100A4, S100 calcium binding protein A4; RCC, renal cell carcinoma; TKI, tyrosine kinase inhibitors; DAB2IP, DAB2 interacting protein; VKORC1L1, vitamin K epoxide reductase complex subunit 1 like 1; GATAD2B, GATA zinc finger domain containing 2B; EML2, EMAP like 2; KDM6B, lysine demethylase 6B; TMEM161A, transmembrane protein 161A; DNMTs, DNA methyltransferases; hnRNPK, heterogeneous nuclear ribonucleoprotein K; MMP2, matrix metalloproteinase 2 ⁎ 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. E-mail addresses: [email protected] (Q. Liu), [email protected] (C. Guo). 1 Contributed equally. https://doi.org/10.1016/j.biopha.2019.109055 Received 13 May 2019; Received in revised form 29 May 2019; Accepted 30 May 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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[8]. miR-542-3p functions as a tumor suppressor and a prognostic biomarker in HCC, and it suppresses epithelial-mesenchymal transition (EMT) and metastasis of HCC by repressing ubiquitin protein ligase E3C (UBE3C) [10]. Furthermore, miR-187-3p plays an anti-metastatic role in HCC and inversely regulates S100 calcium binding protein A4 (S100A4) abundance to restrain EMT of cancer cells [7]. miR-1307-3p is a newly identified cancer-related miRNA. miR1307-3p is a risk factor for metastatic renal cell carcinoma (RCC) progression under tyrosine kinase inhibitors (TKI) treatment [15]. Serum miR-1307-3p level is elevated in breast cancer and it is able to use for breast cancer early detection [16]. miR-1307-3p contributes to breast cancer cell proliferation in vitro and facilitates tumor formation in vivo [17]. Moreover, miR-1307-3p is implicated in capecitabine-based therapy in colon cancer [18]. However, the expression of miR-1307-3p and its biological role in HCC were still unclear. In the present study, we detected the expression of miR-1307-3p in HCC and adjacent nontumor tissues. Loss- and gain-of-function experiments were designed to investigate the biological function of miR1307-3p in HCC cell proliferation migration and invasion. Subsequently, the molecular mechanism involved in the role of miR1307-3p was disclosed. This study, for the first time, revealed that miR1307-3p was a tumor promoting factor via directly repressing DAB2 interacting protein (DAB2IP) in HCC.
2.4. Western blot Proteins were extracted from HCC cell lines using RIPA lysis buffer (Beyotime, Guangzhou, China) and the concentration was measured by the BCA protein assay kit (Pierce, Rockford, IL, USA). The protocols for western blot analysis were described in our previous study [20]. Rabbitanti-human DAB2IP primary antibody (ab87811) and mouse-antihuman DAB2IP primary antibody (ab8245) were purchase from Abcam (Cambridge, MA, USA). 2.5. Cell proliferation assay The cell proliferation was detected by the Cell Counting Kit-8 (CCK8; Dojindo, Kyushu, Japan) as previously described [20]. For the colony formation assay, HCC cells were seeded into a six-well plate (500 cells per well) and were cultured for two weeks. Then, the colonies were fixed with 4% methanol and stained with 0.1% crystal violet. Finally, the colonies were photographed and counted. 2.6. Cell migration and invasion assays The Transwell chemotaxis 24-well chamber (BD Biosciences, Franklin Lakes, NJ, USA) was used for cell migration assay. 50 μl Matrigel (BD Biosciences) was added into the basement membrane of filters for cell invasion assay. The detailed protocols for Transwell assay were described in our previous study [20].
2. Materials and methods 2.1. Patients and samples Eighty pairs of HCC and paracancerous tissues were obtained from patients who underwent hepatectomy in Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University. HCC patients that received preoperative treatments were excluded. All tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C until use. The experimental protocols were approved by the Ethics Committee of The First Affiliated Hospital of Xi’an Jiaotong University (No. 2018-G14, March 3, 2018) with the 1964 Helsinki declaration and its later amendments [19]. Informed consent was obtained from all patients.
2.7. Luciferase reporter assay The pEZX-MT06 vector containing the full length of DAB2IP 3′-UTR was purchased from GeneCopoeia Inc. The target sequence of miR1307-3p in the 3′UTR of DAB2IP was mutated by a QuickChange II SiteMutagenesis kit (Agilent, San Jose, CA, USA). Then, cells were cotransfected with luciferase reporter vector containing wild type (wt) or mutated (mt) 3′UTR of DAB2IP and miR-1307-3p mimics or inhibitors. After transfection for 48 h, the fluorescence intensity was measured by a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to renilla luciferase activity.
2.2. Cell lines and cell transfection Four human HCC cell lines Hep3B, Huh7, MHCC97H, HCCLM3 and normal hepatic cell line LO2 were maintained in our lab and cultured as previously described [12]. miR-1307-3p mimics and scrambled control were purchased from RioBio (Guangzhou, China). Lentivector-mediated miR-1307-3p inhibitors (anti-miR-1307-3p) and negative control (NC) were obtained from GeneCopoeia Inc. (Guangzhou, China). DAB2IP expression vector (pcDNA3.1-DAB2IP) was designed by GenePharma (Shanghai, China) and empty vector (pcDNA3.1) was used as negative control. A specific DAB2IP siRNA and a scrambled siRNA were synthesized by RioBio and sequences were shown in Supplementary Table 1. Vectors were transfected into HCC cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions.
2.8. Animal experimental All experiments involved in animal were approved by the Animal Care and Use Committee of Xi’an Jiaotong University. BALB/C nude mice (male, 4-week-old) were purchased from Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China). The in vivo tumor formation and pulmonary metastasis assays were conducted as previously described [20]. The xenograft tumor tissues were subjected to immunohistochemistry for Ki-67 staining and lung tissues were detected by H&E staining. 2.9. Statistical analysis
2.3. RNA extraction and real-time PCR analysis
All the statistical analysis of data was carried out using the GraphPad Prism Software (Version 8.0, GraphPad Software, La Jolla, CA, USA). Data were presented as mean ± standard deviation (SD) from three independent repeats. The difference between two independent groups was assessed using a two-tail Student’s t-test and multiple group comparisons were performed using ANOVA. Pearson correlation test was carried out to confirm the correlation between miR1307-3p and DAB2IP mRNA expression. Survival analysis was determined by Kaplan-Meier method and log-rank test. P < 0.05 was considered to be statistically significant.
Total RNA from tissues and cell lines was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) and Trizol reagent (Invitrogen) and reversely transcribed into cDNA using the miScript II RT kit and the reverse transcription kit (Invitrogen) following the manufacture’s protocol. Real-time PCR assay was performed using the SYBR® Premix Ex Taq™ II (Takara, Dalian, China) in a CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH and U6 were used as internal controls. The primers used are listed in Supplementary Table 1. 2
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Fig. 1. The expression and clinical significance of miR-1307-3p in HCC. (A) qRT-PCR analysis was performed to detect the expression difference of miR-1307-3p between HCC tissues (n = 80) and adjacent non-tumor tissues (n = 80). (B) The levels of miR-1307-3p in HCC cell lines (Hep3B, Huh7, MHCC97H and HCCLM3) and normal hepatic cell line LO2 were measured by qRT-PCR. (C) HCC patients with venous infiltration had a significant higher level of miR-1307-3p compared to cases without venous infiltration. (D) HCC patients with tumor size ≥5 cm had a significant higher level of miR-1307-3p compared to cases with tumor size < 5 cm. (E) HCC patients with advanced tumor stages (III + IV) had a significant higher level of miR-1307-3p compared to cases with early tumor stages (I + II). (F) HCC patients were divided into two subgroups (low/high miR-1307-3p group) according to the median level of miR-1307-3p in this cohort. HCC patients with high miR-1307-3p level (n = 40) showed a significant poorer overall survival compared to low miR-1307-3p group (n = 40). *P < 0.05.
E). Moreover, HCC patients with high miR-1307-3p level had an obvious poorer overall survival compared to miR-1307-3p low-expressing cases (P = 0.0099, Fig. 1F). These findings suggested that miR-1307-3p was a promising prognostic biomarker for HCC.
3. Results 3.1. miR-1307-3p expression is increased in HCC PCR analysis was performed to examine the expression difference of miR-1307-3p between HCC and adjacent nontumor tissues. We found that the expression of miR-1307-3p in HCC tissues was prominently higher than that in paracancerous tissues (P = 0.0038, Fig. 1A). Moreover, TCGA data from starBase V3.0 online platform [21,22] indicated that miR-1307-3p was significantly overexpressed in HCC tissues compared to normal liver tissues (P < 0.0001, Supplementary Fig. 1). Additionally, up-regulated expression of miR-1307-3p was detected in HCC cell lines (Hep3B, Huh7, MHCC97H, HCCLM3) compared with normal hepatic cell line LO2 (P < 0.05, Fig. 1B). These results indicated a putative tumor promoting role of miR-1307-3p in HCC.
3.3. miR-1307-3p promotes the growth and metastasis of HCC cells To determine the biological function of miR-1307-3p in HCC, MHCC97H and HCCLM3 cells were transfected with miR-1307-3p inhibitors and negative control (P < 0.05, Fig. 2A). CCK-8 assay showed that miR-1307-3p knockdown significantly suppressed the proliferation of HCC cells (P < 0.05, Fig. 2B). Furthermore, the colonies formed by HCC cells were obviously reduced by miR-1307-3p knockdown (P < 0.05, Fig. 2C). Additionally, miR-1307-3p silencing prominently repressed the in vivo growth of HCCLM3 cells (P < 0.05, Fig. 2D). The percentage of Ki-67 positive cells in xenograft tumor tissues from miR1307-3p group was markedly lower than that in control group (P < 0.05, Fig. 2E). Next, we found that knockdown of miR-1307-3p consistently inhibited the metastasis of HCC cells in vitro and in vivo (P < 0.05, Fig. 3). Conversely, miR1307-3p overexpression facilitated the proliferation, migration and invasion of Hep3B cells (P < 0.05, Supplementary Fig. 2). These results revealed the tumor promoting effects of miR-1307-3p in HCC.
3.2. Elevated expression of miR-1307-3p correlates with poor prognosis To disclose the clinical significance of miR-1307-3p, HCC patients were divided into two subgroups (low/high miR-1307-3p group) according to the median level of miR-1307-3p in this cohort. Interestingly, HCC patients with venous infiltration had a significant higher miR-1307-3p level compared to HCC patients without venous infiltration (P < 0.05, Fig. 1C). miR-1307-3p levels in HCC patents with tumor size ≥5 cm and advanced tumor stages (III + IV) were obviously higher than those in cases with tumor size < 5 cm and early tumor stages (I + II) (P = 0.0027 and 0.0001, respectively, Fig. 1D and
3.4. DAB2IP is a target for miR-1307-3p To study the molecular mechanism underlying the oncogenic role of 3
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Fig. 2. miR-1307-3p knockdown suppresses the growth of HCC cells. (A) miR-1307-3p inhibitors (anti-miR-1307-3p) or negative control (NC) were transfected into MHCC97H and HCCLM3 cells, and qRT-PCR was performed to detect miR-1307-3p expression. (B) CCK-8 assay revealed that miR-1307-3p knockdown suppressed the proliferation of HCC cells. (C) The colonies formed by HCC cells were reduced by miR-1307-3p knockdown. (D) HCCLM3 cells with or without miR-13073p knockdown (n = 4 each group) were implanted into nude mice via subcutaneous injection. Tumor growth curves indicated that miR-1370-3p knockdown restrained in vivo growth of HCC cells. (E) The percentage of Ki-67 positive staining cells in tumor tissues from miR-1307-3p group (n = 4) was significantly lower than that in control group (n = 4). *P < 0.05.
miR-1307-3p in HCC, we screened potential targets for miR-1307-3p based on starBase V3.0 online platform [21,22]. Six candidates including vitamin K epoxide reductase complex subunit 1 like 1 (VKORC1L1), GATA zinc finger domain containing 2B (GATAD2B), EMAP like 2 (EML2), DAB2IP, lysine demethylase 6B (KDM6B) and transmembrane protein 161A (TMEM161A) were predicted in three public databases. Notably, only DAB2IP is previously identified as a tumor suppressor in HCC [23] and its 3′UTR has complementary sequences for miR-1307-3p (Fig. 4A). We found that miR-1307-3p knockdown significantly increased the level of DAB2IP mRNA in HCCLM3 cells (P < 0.05, Supplementary Fig. 3). However, miR-13073p silencing did not change KDM6B mRNA expression (Supplementary Fig. 3). Moreover, the levels of DAB2IP protein in HCCLM3 and MHCC97H cells were up-regulated by miR-1307-3p knockdown (P < 0.05, Fig. 4B). The levels of DAB2IP mRNA in HCC tissues were prominently lower than those in adjacent nontumor tissues (P < 0.0001, Fig. 4C). And miR-1307-3p expression was inversely correlated with the level of DAB2IP mRNA in HCC tissues (r=-0.3835, P = 0.0004, Fig. 4D). Importantly, miR-1307-3p overexpression reduced, whereas miR-1307-3p knockdown enhanced the luciferase activities of vectors carrying wt 3′UTR of DAB2IP (P < 0.05, Fig. 4E). But, modulating miR-1307-3p level had no effect on luciferase activities
of vector containing mt 3′UTR of DAB2IP (Fig. 4E). Thus, our data recognized DAB2IP as a direct target of miR-1307-3p. 3.5. DAB2IP mediates the oncogenic role of miR-1307-3p in HCC cells To confirm whether DAB2IP is a downstream effector of miR-13073p in HCC, DAB2IP expression was restored by transfecting expression plasmid in HCCLM3 cells (P < 0.05, Fig. 5A). DAB2IP overexpression remarkably suppressed the proliferation, migration and invasion of HCCLM3 cells, which was consistent with the effects of miR-1307-3p knockdown (P < 0.05, Fig. 5B–D). Next, DAB2IP was knocked down by a specific siRNA in HCCLM3 cells with miR-1307-3p knockdown (P < 0.05, Fig. 6A). Interestingly, DAB2IP silencing rescued miR-13073p knockdown-attenuated HCCLM3 cell proliferation, migration and invasion (P < 0.05, Fig. 6B–D). Taken together, our results indicated that miR-1307-3p contributed to HCC cell proliferation, migration and invasion by targeting DAB2IP. 4. Discussion The aberrant expression of miRNAs has been identified as potential diagnostic and prognostic biomarker for human cancer [24,25]. For 4
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Fig. 3. Knockdown of miR-1307-3p represses the metastasis of HCC cells. (A) Transwell assays demonstrated that miR-1307-3p knockdown inhibited the migration and invasion of MHCC97H and HCCLM3 cells. (B) HCCLM3 cells with or without miR-1307-3p knockdown (n = 4 each group) were implanted into nude mice via tail vein injection. The metastases in lung tissues from miR-1307-3p knockdown group (n = 4) was obviously lower than those in control group (n = 4). *P < 0.05. Black arrows indicated lung metastases.
Fig. 4. DAB2IP is identified as a novel target of miR-1307-3p. (A) The 3′UTR of DAB2IP contained complementary sequences for miR-1307-3p. (B) miR-1307-3p inhibitors (anti-miR-1307-3p) or negative control (NC) were transfected into HCCLM3 and MHCC97H cells, and immunoblotting was performed to detect DAB2IP expression. (C) The expression of DAB2IP mRNA was detected by qRT-PCR between HCC tissues (n = 80) and adjacent non-tumor tissues (n = 80). (D) An inverse correlation between miR-1307-3p level and the expression of DAB2IP mRNA was found in HCC tissues (n = 80). (E) The vectors carrying wild type (wt) or mutated (mt) 3′UTR of DAB2IP and miR-1307-3p mimics or inhibitors were co-transfected into HCCLM3 cells and the relative fluorescence intensity was detected. *P < 0.05.
example, the aberrantly expressed serum miR-106b-3p, miR-101-3p and miR-1246 may be useful biomarkers for HCC diagnosis [26]. Circulating miR-103 level is increased in HCC patients and acts as a promising marker for tumor metastasis [27]. Our previous studies have
revealed the differentially expressed miRNAs, such as miR-187-5p, miR1468 and miR-23c, as prognostic biomarkers for HCC [7–9]. Here, we found that miR-1307-5p was highly expressed in HCC tissues and cell lines. HCC patients with venous infiltration, tumor size ≥5 cm and 5
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Fig. 5. DAB2IP overexpression represses the proliferation, migration and invasion of HCCLM3 cells. (A) pcDNA3.1-DAB2IP or empty vector (EV) was transfected into HCCLM3 cells and immunoblotting was performed to detect the expression of DAB2IP protein. (B) CCK-8, (C) colony formation, and (D) Transwell assays were performed to determine the proliferation, migration and invasion of HCCLM3 cells. *P < 0.05.
Fig. 6. DAB2IP silencing rescues miR-1307-3p knockdown-attenuated HCCLM3 cell proliferation, migration and invasion. (A) DAB2IP was knocked down by a specific siRNA in HCCLM3 cells with miR-1307-3p knockdown and immunoblotting was performed to detect DAB2IP protein. (B) CCK-8, (C) colony formation, and (D) Transwell assays were performed to determine the proliferation, migration and invasion of HCCLM3 cells transfected with corresponding vectors. *P < 0.05. 6
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advanced tumor stages (III + IV) showed higher levels of miR-1307-3p. Furthermore, the high miR-1307-3p expression was strongly correlated with poor overall survival of HCC patients. These data indicated a potential prognostic value of miR-1307-3p in HCC. Previous study reports that the high level of serum miR-1307-3p is a promising indicator for early breast cancer [16]. Thus, it is worth to further detect the serum miR-1307-3p level in HCC patients. Previous studies have disclosed several mechanisms involved in deregulation of miRNAs in HCC. For instance, miR-122 is epigenetically suppressed by via DNA methyltransferases (DNMTs)-mediated DNA methylation [28]. LncRNA MCM3AP-AS1 functions as a molecular sponge to inversely regulate miR-194-5p abundance in HCC cells [29]. Moreover, miR-671-5p and miR-204 are hypoxia-responsive miRNAs and mediate hypoxia-induced HCC progression [12,13]. It will be interesting to investigate the molecular mechanism underlying the aberrant expression of miR-1307-3p in HCC. More and more evidences have solidly demonstrated that miRNAs are critical drivers or suppressors in the tumorigenesis and progression of HCC [30]. miR-1307-3p has previously reported to be implicated in breast cancer, RCC and colon cancer [15–18]. Here, we for the first time demonstrated that miR-1307-3p contributed to tumor growth and metastasis of HCC via gain- and loss-of-function experiments. Moreover, DAB2IP expression was inversely regulated by miR-1307-3p in HCC cells and it was accordingly identified as a direct target for miR-13073p as suggested by luciferase reporter assay. DAB2IP is a common tumor suppressor in human cancer [31]. Mutant p53 enhances the tumor promoting effects of insulin by repressing DAB2IP in breast and prostate cancer [32]. Moreover, DAB2IP underexpression contributes to invasion and metastasis of colorectal cancer by targeting heterogeneous nuclear ribonucleoprotein K/matrix metalloproteinase 2 (hnRNPK/ MMP2) axis [33]. Previous study finds that DAB2IP expression is suppressed in HCC [34]. Additionally, DAB2IP is confirmed to be a potential prognostic marker in HCC and inhibits cancer cell proliferation and invasion [23], which is consistent with our results in this study. miR-149-3p, miR-367 and miR-889 have been proved to be negative post-transcriptional regulators of DAB2IP [35–37]. This study provided a novel upstream regulatory mechanism for DAB2IP inactivation in HCC. Importantly, DAB2IP silencing rescued miR-1307-3p knockdownattenuated HCC cell proliferation, migration and invasion. Collectively, miR-1307-3p exerted an oncogenic role by targeting tumor suppressor DAB2IP in HCC. In conclusion, our findings indicated that overexpression of miR1307-3p conferred to poor clinical outcomes and promoted the proliferation, migration and invasion of cancer cells by repressing tumor suppressor DAB2IP in HCC. This study might provide a potential biomarker and promising therapeutic target for HCC treatment.
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5. Conclusions To conclude, this study reveals that miR-1307-3p overexpression is a frequent event and confers poor clinical outcomes in HCC. Functionally, miR-1307-3p promotes cell proliferation, migration and invasion in vitro, and enhances tumor growth and metastasis of HCC in vivo. Tumor suppressor DAB2IP is recognized as a novel direct target of miR-1307-3p and participates in miR-1307-3p-induced HCC progression. These observations support the notion that miR-1307-3p may be a novel potential target for HCC therapy. Conflict of interests The authors declare that they have no conflict of interests. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81874069, 81572847) and the Key 7
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