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Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family Zai-Bo Li a, Hong-Tao Chu b, Min Jia b, Lin Li b, * a b
Department of Hepatology, Zaozhuang Municipal Hospital, Zaozhuang, Sandong, China Department of Cardiovascular Medicine, Zaozhuang Municipal Hospital, Zaozhuang, Shandong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 January 2020 Accepted 17 February 2020 Available online xxx
Long non-coding RNAs (lncRNAs) have obtained growing attention due to their potential effects as novel regulators in various tumors. This study aimed to investigate the expression and roles of lncRNA LINC01139 (LINC01139) in the progression of hepatocellular carcinoma (HCC). We found that LINC01139 was over-expressed in HCC specimens and cell lines, and its upregulation was observed to be associated with advanced TNM stage, lymph node metastasis and poor clinical prognosis of HCC patients. Multivariate analyses confirmed that LINC01139 expression was an independent poor prognostic factor for HCC patients. Functionally, the knockdown of LINC01139 suppressed cell proliferation, clone formation and metastasis of HCC cells. Moreover, luciferase assays and rescue experiments revealed that LINC01139/miR-30/MYBL2 established the ceRNA network involved in the modulation of cell proliferation and metastasis of HCC cells. Overall, LINC01139 may exhibit an oncogenic function in HCC via acting as a sponge for miR-30 to upregulate MYBL2, and may serve as a potential therapeutic target and a prognostic biomarker for HCC patients. © 2020 Elsevier Inc. All rights reserved.
Keywords: LncRNA LINC01139 miR-30 family MYBL2 Hepatocellular carcinoma Biomarker
1. Introduction Hepatocellular carcinoma (HCC), accounting for approximately 85% of primary liver neoplasms, is the 3rd most common cause of tumor-associated mortality worldwide [1,2]. There are >750,000 new cases of HCC and estimated 55,000e85000 deaths due to this neoplasm annually [3]. Though considerable advancements have been made in the clinical treatments for HCC, the clinical outcomes of HCC patients remain unsatisfactory [4]. One of the biggest challenges for the clinical therapeutics is their diagnosis at an advanced stage and frequent metastasis of HCC cells, which results in their short survivals, especially in older patients [5]. Elucidation of the mechanisms involved in tumor metastasis and progression may display great help in the development of effective diagnosis and treatments, which will further improve the clinical outcome of HCC patients. Long non-coding RNAs (lncRNAs) represent a subgroup of
* Corresponding author. Department of Cardiovascular Medicine, Zaozhuang Municipal Hospital, Zaozhuang, 277100, Shandong, China. E-mail address: [email protected] (L. Li).
noncoding RNAs characterized by limited protein coding potential and long nucleotide sequences [6]. Growing evidences have shown that lncRNAs exhibit extensive regulatory functions in several biological processes, such as DNA damage and repair, vasculogenesis, miRNAs silencing, sex-hormone-driven diseases and apoptosis [7]. Besides, it is also confirmed that lncRNAs acted as imperative modulators in the development of inflammation, immune cell and cancer metastasis, highlighting the potential possibility of lncRNAs used as therapeutic targets and novel biomarkers for tumor patients [8,9]. In the past few years, a lot of lncRNAs have been reported to be tumor promotors or anti-oncogenes [10,11]. However, a large number of lncRNAs remained to be functionally identified in HCC. In this study, we identified a novel HCC-related lncRNA, lncRNA LINC01139 (LINC01139) which was also known as LINK-A and located in 1q43. The roles of LINC01139 in tumor progression were rarely reported. Previously, the distinct upregulation of LINC01139 and its oncogenic roles in several tumors have been reported, such as breast cancer and ovarian carcinoma [12,13]. However, whether LINC01139 exhibited dysregulated expression in HCC and its potential functions involved in tumor behaviors remained unknown.
https://doi.org/10.1016/j.bbrc.2020.02.116 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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2. Materials and methods
2.10. Subcellular fractionation assays
2.1. Patients and tissue specimens
The details are described in the Supplemental Materials and Methods.
HCC tissues were obtained from one hundred and nine patients with HCC between January 2011 and December 2014 at Zaozhuang Municipal Hospital. Before surgical resection, the patients did not receive anti-cancer therapy. The written informed consents were obtained from all patients. All specimens were immediately frozen using liquid nitrogen for the following experiments. The protocols were approved by the Ethics Committee of Zaozhuang Municipal Hospital. 2.2. Cell transfection The details are described in the Supplemental Materials and Methods. 2.3. Real-time PCR The details are described in the Supplemental Materials and Methods. The primers were listed in Table S1. 2.4. CCK-8 assays About 2 103 LINC01139-siRNAs-transfected cells (per well) were placed into 96-well plates. Afterwards, CCK-8 reagents (10 ml per well; Qianshang, Wuxi, Jiangsu, China) were placed into the plates for 24, 48, 72 and 96 h, respectively. The 450 nm absorbance of the plates was determined by a microplate reader.
2.11. RNA pull-down The details are described in the Supplemental Materials and Methods. 2.12. Dual-luciferase reporter assays The details are described in the Supplemental Materials and Methods. 2.13. Statistical analysis Statistical analysis was carried out by SPSS 19.0 (Chicago, IL, USA). Two-tailed Student’s t-tests was used for statistical comparisons. Associations between clinicopathologic parameters and LINC01139 expression were analyzed by the application of c2-tests. The Kaplan-Meier methods with the log-rank tests were used for analyzing the overall survivals. The variables were further analyzed by multivariate models to identify the independent prognostic values. P value less than 0.05 was considered statistically significant. 3. Results 3.1. Up-regulation of LINC01139 in HCC was correlated with poor prognosis
2.5. Clonogenic assays Trypsin was used for digesting the HCC cells which were transfected with si-LINC01139#1 or si-LINC01139#2. Then, the cells (500 cells/well) were placed into 6-well plates. Subsequently, the plates were put into an incubator with 5% CO2 at 37 C. The cultivation was terminated after 2 weeks. Then, methanol and crystal violet were utilized for treating the colonies. After washing with PBS, a camera was used for imaging the pictures of the colonies. 2.6. Cell apoptosis determination The cells after transfection with si-LINC01139#1 or siLINC01139#2 were collected into flow tubes. Subsequently, the cells were washed with PBS and added with 350 ml binding buffer. Then, Annexin V-FITC (5 ml; Yunfeng, Kunming, Yunnan, China)) reagents and 5 ml PI solution were placed into the flow tubes. After incubation for 15 min without light, the cells were washed using PBS and subjected to flow cytometry analysis. 2.7. Western blot assays The details are described in the Supplemental Materials and Methods. 2.8. Wound-healing assays The details are described in the Supplemental Materials and Methods. 2.9. Transwell assays The details are described in the Supplemental Materials and Methods.
To uncover the aberrantly expressing lncRNAs in HCC, we carried out bioinformatics analysis using TCGA. The heatmap and volcano map was presented in Fig. 1A and B. Among these aberrantly expressing lncRNAs, LINC01139 which highly expressed in TCGA HCC tumor specimens attracted our attention (Fig. 1C). In addition, using lnCAR program, we also found that LINC01139 was also up-regulated in the HCC tumor samples involved in GSE36736, GSE55092 and GSE76427 microarray data (Fig. 1D). Moreover, the co-expression work of LINC01139 was also generated by lnCAR program (Fig. 1E). Additionally, KEGG pathway analysis using lnCAR program revealed that LINC01139 was associated with metabolic pathways and pathways in cancer (Fig. 1F). Then, we performed RTPCR to determine the dysregulated expression of LINC01139 in our cohort (109 HCC patients), finding that the expression of LINC01139 in HCC specimens was distinctly high than that in matched normal specimens (Fig. 1G). In addition, cellular experiments also revealed that LINC01139 was overexpressed in seven HCC cell lines compared to LO2 cells (Fig. 1H). Overall, our findings provided robust evidence that LINC01139 was an overexpressed lncRNA in HCC. Then, to explore the clinical significance of LINC01139 in HCC patients, the LINC01139 levels were classified as high or low in relation to the median value. As shown in Table S2, the results of chi-square test revealed that high LINC01139 expression was associated with advanced TNM stage (p ¼ 0.015) and lymph node metastasis (p ¼ 0.031). Then, Kaplan-Meier assays indicated that the patients with high LINC01139 expression displayed dramatically poorer overall survival rates than those with low LINC01139 expression (p ¼ 0.0011, Fig. 1I). To further confirm the prognostic value of overexpressed LINC01139 in HCC patients, our group performed multivariate assays, confirming that high LINC01139 expression (HR ¼ 2.721, 95% CI: 1.289e4.183, p ¼ 0.019) was an independent prognostic marker for HCC patients (Table S3).
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 1. Bioinformatics analysis revealed that LINC01139 was up-regulated in HCC cancer tissues. (A) Heatmap of differentially expressed lncRNAs in TCGA data. (B) Volcano map. (C) The expression of LINC01139 in TCGA HCC specimens. (D) LINC01139 was up-regulated in the HCC tumor samples involved in GSE36736, GSE55092 and GSE76427. The data was analyzed by lnCAR program and the diagrams were modified. (E) The co-expression work of LINC01139. (F) KEGG pathway analysis. (G) qPCR detected the LINC01139 levels in 109 HCC samples. (H) qPCR measured the LINC01139 levels in various HCC cell lines. (I) Overall survival. *p < 0.05, **p < 0.01.
3.2. LINC01139 depletion suppressed malignant behaviors in HCC The above findings that LINC01139 was remarkable upregulation in HCC tumor tissues and cell lines suggested that LINC01139 might act as a tumor promoter in HCC. Therefore, we next sought to investigate whether LINC01139 was capable to influence the malignant behaviors of HCC cells. First, we synthesized siRNAs targeting LINC01139 (si-LINC01139#1, siLINC01139#2) and the knockdown efficiency of these siRNAs in HepG2 cells was determined by qPCR (Supplementary Fig. 1A). Afterwards, CCK-8 assays revealed that LINC01139 depletion led to notable suppression of HepG2 cell proliferation (Supplementary Fig. 1B). Then, cell colony assays also demonstrated that LINC01139 deficiency caused markedly decreased colony formation abilities of HepG2 cells (Supplementary Fig. 1C). Besides, flow cytometry was utilized for determining the effects of LINC01139 depletion on HepG2 cell apoptosis. The data suggested that the apoptotic rates of HepG2 cells were significantly increased after their LINC01139 was knocked down (Supplementary Fig. 1D). Subsequently, we conducted the above
experiments using another HCC cells (SMMC-7721 cells), and similar results were also observed (Supplementary Fig. 1E-H). Hence, these data validated that LINC01139 served as crucial regulatory functions in HCC cell proliferation and apoptosis. 3.3. LINC01139 knockdown impaired metastatic properties of HCC cells The effects of LINC01139 on HCC cell metastatic potentials were then investigated. Data from wound-healing assays revealed that silencing the expression of LINC01139 notably inhibited the wound closures of HCC cells, which indicated that LINC01139 could regulate HCC cell migration (Fig. 2A and B). Afterwards, the influence of LINC01139 on HCC cell invasion was also evaluated using transwell assays. The results demonstrated that transfection with LINC01139 siRNAs caused dramatically decreased invasion cell number, which indicated that knockdown of LINC01139 was capable to impair the invasive capacities of HCC cells (Fig. 2C and D). Collectively, these results demonstrated that LINC01139 was able to modulate the mobility of HCC cells.
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 2. LINC01139 facilitated HCC cell migration and invasion. (A and B) The migratory abilities of HCC cells were determined by wound-healing assays. (C and D) Transwell assays assessed the invasive capabilities of HCC cells. *p < 0.05, **p < 0.01.
3.4. LINC01139 acted as a sponge of miR-30 family in HCC cells Accumulating evidences had validated that lncRNAs, particularly existing in cytoplasm, modulated their downstream targets via serving as ceRNAs [14]. Hence, we first used lncLocator program to determine the subcellular localization of LINC01139 in cells. The predicting results showed that LINC01139 mainly expressed in cytoplasm (Fig. 3A). Subsequently, the subcellular fractionation assays were carried out in HepG2 cells and the similar results were also observed (Fig. 3B). Hence, LINC01139 was possibly a sponge for specific miRNAs. We next generated the ceRNA network using lnCAR program and found that the miR-30 family members were included in that network (Fig. 3C). Therefore, we employed starbase program to predict the potential targeting miRNAs of LINC01139. Interestingly, miR-30 family members (miR-30a-e) were all the possible targets of LINC01139 (Fig. 3D). Afterwards, the expressing analysis using TCGA data by starbase program revealed that four members (miR-30a, b, c and e) of miR-30 family were significantly down-regulated in HCC tumor samples (Fig. 3E). Then, TCGA data analysis using starbase program indicated that the levels of LINC01139 were notably negatively correlated with miR-30a, miR30b and miR-30d in HCC tumor samples (Fig. 3F). Therefore, we next attempted to verify whether miR-30 family members were the exact targets of LINC01139. To achieve that, RNA-pull down was conducted. The results showed that biotinylated-LINC01139 (Biotin-LINC01139) was able to directly precipitate miR-30 family members, while biotinylated-LINC01139 with mutant predicting binding sites (Biotin-LINC01139 mut) could not precipitate miR-30 family members (Fig. 3G). To further verify the bindings, luciferase reporter assays were carried out. The data validated that cotransfection of wild-type LINC01139 (LINC01139 wt) luciferase reporters with indicated miR-30 family mimics dramatically reduced the luciferase activities in 293T cells, while the luciferase activities were affected when the cells were co-transfected with mutant-type LINC01139 (LINC01139 mut) luciferase reporters and indicated miR30 family mimics (Fig. 3H). In addition, qPCR analysis confirmed that overexpressing LINC01139 (ov-LINC01139) resulted in significantly decreased expression of miR-30 family membranes, while enhancing the mutant-type LINC01139 (ov-LINC01139-mut)
expression had no influence on the levels of miR-30 family membranes (Fig. 3I). On the contrary, knocking down LINC01139 led to remarkably increased levels of miR-30 family membranes (Fig. 3J). Overall, the above findings validated that LINC01139 directly targeted miR-30 family membranes in HCC cells. 3.5. MYBL2 was a target of miR-30 family members in HCC cells Next, we sought to investigate the downstream targets of miR30 family members. First, we used miRDB program to predict the common targeting genes of the five miR-30 family members, and then the common targeting genes were intersected with the upregulated genes in HCC tumor samples, and 10 genes were found (Fig. 4A). Subsequently, we analyzed the expressing correlation between LINC01139 and the 10 genes using GEPIA program. The results showed that among these 10 genes, only MYBL2 and SNX8 were notably positively correlated with LINC01139 expression in HCC (Fig. 4B). In addition, analyzing the overall survival using GEPIA program revealed that, among MYBL2 and SNX8, only the high expression of MYBL2 was significantly associated with lower overall survivals of HCC patients (Fig. 4C). Hence, we next sought to clarify whether MYBL2 was a target of miR-30 family members. The predicting binding sites between the 30 UTR of MYBL2 and miR-30 family members were presented in Fig. 4D. Furthermore, using starbase program, we found that the expression of MYBL2 in HCC tumor samples of TCGA data was negatively correlated with miR30a and miR-30e (Fig. 4E). Moreover, through analyzing TCGA data using UALCAN program, we discovered that MYBL2 was upregulated in most cancer types (Fig. 4F). Additionally, KEGG analysis revealed that MYBL2-positively correlated genes were relevant with cell cycle, which indicated that MYBL2 might play critical roles in regulating HCC malignancies (Fig. 4G). Next, we thereby performed luciferase reporter assays to verify the binding between 30 UTR of MYBL2 and miR-30 family members. The 3’ UTR of MYBL2 mRNA including the predicting miR-30 family binding site (wildtype: wt) or the mutated sequence (mutant-type: mut) was constructed into pGL3 vectors to form MYBL2 wt or MYBL2 mut luciferase reporters. It was observed that the five miR-30 family members remarkably reduced the luciferase activities in 293T cells
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 3. MiR-30 family members was directly interacted with LINC01139 in HCC cells. (A) lncLocator program predicted the subcellular localization of LINC01139. (B) Subcellular fractionation assays. (C) The ceRNA network of LINC01139 was generated by lnCAR program. (D) The predicting binding sites between LINC01139 and miR-30 family members were generated by starbase program. (E) The expression of miR-30 family members was conducted by starbase program. (F) The correlation between LINC01139 and miR-30 family members was analyzed by starbase program. (G) RNA pull-down assays. (H) Luciferase reporter assays. (I) qPCR detected the levels of miR-30 family members in HepG2 cells after transfection with ov-LINC01139 or ov-LINC01139-mut plasmids. (J) qPCR evaluated the levels of miR-30 family members in HepG2 cells after transfection with LINC01139 siRNAs. *p < 0.05, **p < 0.01.
transfected with MYBL2 wt reporters, instead of the mutant-type reporters (Fig. 4H). Subsequently, we synthesized siRNAs targeting MYBL2 (si-MYBL2#1, si-MYBL2#2, si-MYBL2#3) and qPCR analysis indicated that si-MYBL2#2 had the highest knockdown efficiency (Fig. 4I). Afterwards, si-MYBL2#2 was transfected into HepG2 cells and the relative expression of miR-30 family members was assessed by qPCR. The data confirmed that the relative levels of miR-30 family members were dramatically increased in HCC cells with MYBL2 deficiency (Fig. 4J). Therefore, these results validated that miR-30 family members targeted MYBL2 in HCC cells.
3.6. LINC01139 modulated MYBL2 expression trough sponging miR30 family members in HCC cells To elucidate the detail molecular mechanisms by which LINC01139 served as an essential regulator in HCC malignancies, we next respectively transfected the mimics of miR-30 family members into HepG2 cells. The data from qPCR analysis validated that enhancing the expression of miR-30 family members contributed to remarkably decreased levels of LINC01139 and MYBL2 (Supplementary Fig. 2A). On the contrary, the depression of miR-30
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 4. MiR-30 family members targeted MYBL2 in HCC cells. (A) Venn diagram of miRDB program predicting the target genes of miR-30 family members (left). The intersection of the common targeting genes was intersected with the up-regulated genes (from TCGA data using GEPIA analysis) in HCC tumor samples (right). (B) The expressing correlation between LINC01139 and the 10 genes using GEPIA program. (C) The overall survival was analyzed by GEPIA program. (D) The predicting binding sites between 30 UTR of MYBL2 and miR-30 family members were presented. (E) The expressing correlation between MYBL2 and miR-30 family members was analyzed by starbase program. (F) UALCAN program analyzed the expression of MYBL2 across diverse cancer types. (G) KEGG pathway analysis of MYBL2-positively-correlated genes in HCC. (H) Luciferase reporter assays. (I) qPCR determined the expression of MYBL2 in HepG2 cells after transfection with MYBL2 siRNAs. (J) qPCR evaluated the expression of miR-30 family members. *p < 0.05, **p < 0.01.
family members by transfecting corresponding inhibitors resulted in markedly increased levels of LINC01139 and MYBL2 (Supplementary Fig. 2B). Then, we depleted LINC01139 in HepG2 cells by transfecting LINC01139 siRNAs and utilized qPCR to evaluate the expression of MYBL2. We found that the levels of MYBL2 were dramatically suppressed by LINC01139 depletion (Supplementary Fig. 2C). Similarly, Western blot assays also revealed that LINC01139 deficiency resulted in markedly decreased protein levels of MYBL2 (Supplementary Fig. 2D). Next, we mixed the miRNA mimics of miR-30 family members (miR-30 mix) and
performed qPCR to assess the expressing changes of MYBL2 in HepG2 cells after transfection with LINC01139 overexpressing plasmids (ov-LINC01139) or miR-30 mix. The results demonstrated that ectopic expression of LINC01139 was able to increase MYBL2 expression, and transfection of miR-30 mix led to a remarkable decline of MYBL2 levels (Supplementary Fig. 2E). However, forced expression of LINC01139 could significantly restore the levels of MYBL2 which were impeded by transfection of miR-30 mix (Supplementary Fig. 2E). Similar results were also observed by utilizing Western blot assays (Supplementary Fig. 2F).
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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3.7. LINC01139 exerted the oncogenic functions via sponging miR30 family members in HCC cells To certify whether LINC01139 exerted the oncogenic functions via sponging miR-30 family members in HCC cells, we conducted a series of functional studies. First, CCK-8 assays revealed that the forced expression of LINC01139 obviously promoted the proliferation of HCC cells, while concurrent overexpression of miR-30 family members (miR-30 mix) abolished the proliferation of HCC cell. However, re-introduction of LINC01139 (ov-LINC01139) markedly reversed the impeded influence of miR-30 mix on HCC cellular growth (Supplementary Fig. 3A). Similar results were also observed using clonogenic assays (Supplementary Fig. 3B). Besides, the results of wound-healing assays showed that enhancing the expression of LINC01139 caused markedly elevated migratory abilities of HCC cells (Supplementary Fig. 3C and D). Although overexpressing miR-30 family members led to notable inhibition of HCC cell migration, the re-overexpressing LINC01139 markedly reversed the cellular migratory abilities (Supplementary Fig. 3C and D). Taken together, these data demonstrated that LINC01139 promoted malignant behaviors of HCC in a miR-30 family dependent manner. 4. Discussion The identification of sensitive biomarkers for prognosis and diagnosis and novel therapeutic targets has a very essential clinical significance for the improvement of long-term survival of HCC patients [15]. In recent years, convincing evidence has confirmed the essential roles of lncRNAs in tumor progression and metastasis. In this study, we performed bioinformatics analysis and identified a novel overexpressed lncRNA LINC01139 in HCC patients. Then, the results of RT-PCR confirmed LINC01139 levels were distinctly upregulated in both HCC specimens from 109 patients and cell lines. Then, we explored the clinical significance of LINC01139 in HCC patients, finding that the upregulation of LINC01139 was positively associated with advanced TNM stage, lymph node metastasis and shorter overall survival, suggesting the clinical value of LINC01139 used as a prognostic biomarker. On the other hand, we performed loss-of-function assays, confirming that knockdown of LINC01139 suppressed the proliferation, migration and invasion. Overall, our findings revealed that LINC01139 could serve as a novel prognostic biomarker and therapeutic target in HCC. The competing endogenous RNA (ceRNA) hypothesis was proposed to describe the function of many lncRNAs as ceRNA to protect the genuine targets of miRNAs from silencing or translational suppression [14]. Recently, several studies on HCC studies have reported the mechanisms involved in lncRNA-miRNA-mRNA axis. Although the expression and function of LINC01139 have been reported in previous studies in several tumors, the potential mechanisms involved LINC01139 mediated ceRNA have not been investigated. Interestingly, we found that LINC01139 was expressed both in the nucleus and cytoplasm. Then, by the use of bioinformatics analysis and several online tools, our attention focused on miR-30 family which may interact with LINC01139. Previously, miR-30 family acting as a tumor suppressor had been well studied in many tumors, including HCC [16,17]. These finding supported the possibility of LINC01139 exhibiting its effects via suppressing miR30 family. Moreover, luciferase activity assays and RNA pull-down assays demonstrated the direct binding relationship between lncRNA LINC01139 and miR-30 family. Overall, our finding firstly provided evidence that LINC01139 served as a tumor promoter in HCC via directly binding to miR-30 family members. MYB proto-oncogene like 2 (MYBL2), located on chromosome 20q13, is a highly conserved member of the MYB family of transcription factors [18]. Previously, MYBL2 has been reported to be
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dysregulated in several tumors [19]. Functionally, overexpression of MYBL2 was shown to mediate resistance to chemotherapeutic agents and have a positive influence in the proliferation, metastasis and EMT progress in several tumors [20]. In HCC, overexpression of MYBL2 exhibited a positive role in the expression of several tumorrelated genes, resulting in an increased ability of HCC cells in the proliferation and metastasis [21]. In this study, we also provided evidence that MYBL2 was highly expressed in HCC and predicted a poor prognosis, which was consistent with the above results. In addition, we performed comprehensive assays, finding that MYBL2 may be a target of miR-30 family members. Moreover, the results of luciferase reporter assays confirmed that MYBL2 gene was one of the direct targets of miR-30 family members. To further explored the association among LINC01139, miR-30 family and MYBL2, we silenced or overexpressed the levels of miR-30 family members, finding that knockdown of miR-30 family members distinctly suppressed the expression of LINC01139 and MYBL2, while their suppression exhibited an opposite result. Rescue experiments indicated that overexpression of LINC01139 could increase the mRNA and protein levels of MYBL2 which was decreased by miR-30 family suppressors. More importantly, functional assays revealed that the transfection of ov-LINC01139 could promote the proliferation, colony formation and migration ability attenuated by the upregulation of miR-30 family members in HCC cells. Overall, findings indicated that LINC01139 served as a tumor promoter via modulating miR-30/MYBL2 axis. 5. Conclusions Our findings firstly suggested that LINC01139 act as an oncogenic lncRNA which promoted HCC progression via the miR-30/ MYBL2 axis. Overall, our results revealed that LINC01139 could act as a potential therapeutic target and a prognostic biomarker for HCC patients. Declaration of competing interest The Authors declare that they have no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.116. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.116. References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Canc. 136 (2015) E359eE386. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA A Cancer J. Clin. 68 (2018) 7e30. [3] L. Kulik, H.B. El-Serag, Epidemiology and management of hepatocellular carcinoma, Gastroenterology 156 (2019) 477e491, e471. [4] T. Clark, S. Maximin, J. Meier, S. Pokharel, P. Bhargava, Hepatocellular carcinoma: review of epidemiology, screening, imaging diagnosis, response assessment, and treatment, Curr. Probl. Diagn. Radiol. 44 (2015) 479e486. [5] S.D. Colquhoun, Hepatocellular carcinoma: the current role of surgical intervention, Crit. Rev. Oncog. 21 (2016) 93e103. [6] J. Jarroux, A. Morillon, M. Pinskaya, History, discovery, and classification of lncRNAs, Adv. Exp. Med. Biol. 1008 (2017) 1e46. [7] S.U. Schmitz, P. Grote, B.G. Herrmann, Mechanisms of long noncoding RNA function in development and disease, Cell. Mol. Life Sci. 73 (2016) 2491e2509. [8] F. Kopp, J.T. Mendell, Functional classification and experimental dissection of
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Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family Zai-Bo Li a, Hong-Tao Chu b, Min Jia b, Lin Li b, * a b
Department of Hepatology, Zaozhuang Municipal Hospital, Zaozhuang, Sandong, China Department of Cardiovascular Medicine, Zaozhuang Municipal Hospital, Zaozhuang, Shandong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 January 2020 Accepted 17 February 2020 Available online xxx
Long non-coding RNAs (lncRNAs) have obtained growing attention due to their potential effects as novel regulators in various tumors. This study aimed to investigate the expression and roles of lncRNA LINC01139 (LINC01139) in the progression of hepatocellular carcinoma (HCC). We found that LINC01139 was over-expressed in HCC specimens and cell lines, and its upregulation was observed to be associated with advanced TNM stage, lymph node metastasis and poor clinical prognosis of HCC patients. Multivariate analyses confirmed that LINC01139 expression was an independent poor prognostic factor for HCC patients. Functionally, the knockdown of LINC01139 suppressed cell proliferation, clone formation and metastasis of HCC cells. Moreover, luciferase assays and rescue experiments revealed that LINC01139/miR-30/MYBL2 established the ceRNA network involved in the modulation of cell proliferation and metastasis of HCC cells. Overall, LINC01139 may exhibit an oncogenic function in HCC via acting as a sponge for miR-30 to upregulate MYBL2, and may serve as a potential therapeutic target and a prognostic biomarker for HCC patients. © 2020 Elsevier Inc. All rights reserved.
Keywords: LncRNA LINC01139 miR-30 family MYBL2 Hepatocellular carcinoma Biomarker
1. Introduction Hepatocellular carcinoma (HCC), accounting for approximately 85% of primary liver neoplasms, is the 3rd most common cause of tumor-associated mortality worldwide [1,2]. There are >750,000 new cases of HCC and estimated 55,000e85000 deaths due to this neoplasm annually [3]. Though considerable advancements have been made in the clinical treatments for HCC, the clinical outcomes of HCC patients remain unsatisfactory [4]. One of the biggest challenges for the clinical therapeutics is their diagnosis at an advanced stage and frequent metastasis of HCC cells, which results in their short survivals, especially in older patients [5]. Elucidation of the mechanisms involved in tumor metastasis and progression may display great help in the development of effective diagnosis and treatments, which will further improve the clinical outcome of HCC patients. Long non-coding RNAs (lncRNAs) represent a subgroup of
* Corresponding author. Department of Cardiovascular Medicine, Zaozhuang Municipal Hospital, Zaozhuang, 277100, Shandong, China. E-mail address: [email protected] (L. Li).
noncoding RNAs characterized by limited protein coding potential and long nucleotide sequences [6]. Growing evidences have shown that lncRNAs exhibit extensive regulatory functions in several biological processes, such as DNA damage and repair, vasculogenesis, miRNAs silencing, sex-hormone-driven diseases and apoptosis [7]. Besides, it is also confirmed that lncRNAs acted as imperative modulators in the development of inflammation, immune cell and cancer metastasis, highlighting the potential possibility of lncRNAs used as therapeutic targets and novel biomarkers for tumor patients [8,9]. In the past few years, a lot of lncRNAs have been reported to be tumor promotors or anti-oncogenes [10,11]. However, a large number of lncRNAs remained to be functionally identified in HCC. In this study, we identified a novel HCC-related lncRNA, lncRNA LINC01139 (LINC01139) which was also known as LINK-A and located in 1q43. The roles of LINC01139 in tumor progression were rarely reported. Previously, the distinct upregulation of LINC01139 and its oncogenic roles in several tumors have been reported, such as breast cancer and ovarian carcinoma [12,13]. However, whether LINC01139 exhibited dysregulated expression in HCC and its potential functions involved in tumor behaviors remained unknown.
https://doi.org/10.1016/j.bbrc.2020.02.116 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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2. Materials and methods
2.10. Subcellular fractionation assays
2.1. Patients and tissue specimens
The details are described in the Supplemental Materials and Methods.
HCC tissues were obtained from one hundred and nine patients with HCC between January 2011 and December 2014 at Zaozhuang Municipal Hospital. Before surgical resection, the patients did not receive anti-cancer therapy. The written informed consents were obtained from all patients. All specimens were immediately frozen using liquid nitrogen for the following experiments. The protocols were approved by the Ethics Committee of Zaozhuang Municipal Hospital. 2.2. Cell transfection The details are described in the Supplemental Materials and Methods. 2.3. Real-time PCR The details are described in the Supplemental Materials and Methods. The primers were listed in Table S1. 2.4. CCK-8 assays About 2 103 LINC01139-siRNAs-transfected cells (per well) were placed into 96-well plates. Afterwards, CCK-8 reagents (10 ml per well; Qianshang, Wuxi, Jiangsu, China) were placed into the plates for 24, 48, 72 and 96 h, respectively. The 450 nm absorbance of the plates was determined by a microplate reader.
2.11. RNA pull-down The details are described in the Supplemental Materials and Methods. 2.12. Dual-luciferase reporter assays The details are described in the Supplemental Materials and Methods. 2.13. Statistical analysis Statistical analysis was carried out by SPSS 19.0 (Chicago, IL, USA). Two-tailed Student’s t-tests was used for statistical comparisons. Associations between clinicopathologic parameters and LINC01139 expression were analyzed by the application of c2-tests. The Kaplan-Meier methods with the log-rank tests were used for analyzing the overall survivals. The variables were further analyzed by multivariate models to identify the independent prognostic values. P value less than 0.05 was considered statistically significant. 3. Results 3.1. Up-regulation of LINC01139 in HCC was correlated with poor prognosis
2.5. Clonogenic assays Trypsin was used for digesting the HCC cells which were transfected with si-LINC01139#1 or si-LINC01139#2. Then, the cells (500 cells/well) were placed into 6-well plates. Subsequently, the plates were put into an incubator with 5% CO2 at 37 C. The cultivation was terminated after 2 weeks. Then, methanol and crystal violet were utilized for treating the colonies. After washing with PBS, a camera was used for imaging the pictures of the colonies. 2.6. Cell apoptosis determination The cells after transfection with si-LINC01139#1 or siLINC01139#2 were collected into flow tubes. Subsequently, the cells were washed with PBS and added with 350 ml binding buffer. Then, Annexin V-FITC (5 ml; Yunfeng, Kunming, Yunnan, China)) reagents and 5 ml PI solution were placed into the flow tubes. After incubation for 15 min without light, the cells were washed using PBS and subjected to flow cytometry analysis. 2.7. Western blot assays The details are described in the Supplemental Materials and Methods. 2.8. Wound-healing assays The details are described in the Supplemental Materials and Methods. 2.9. Transwell assays The details are described in the Supplemental Materials and Methods.
To uncover the aberrantly expressing lncRNAs in HCC, we carried out bioinformatics analysis using TCGA. The heatmap and volcano map was presented in Fig. 1A and B. Among these aberrantly expressing lncRNAs, LINC01139 which highly expressed in TCGA HCC tumor specimens attracted our attention (Fig. 1C). In addition, using lnCAR program, we also found that LINC01139 was also up-regulated in the HCC tumor samples involved in GSE36736, GSE55092 and GSE76427 microarray data (Fig. 1D). Moreover, the co-expression work of LINC01139 was also generated by lnCAR program (Fig. 1E). Additionally, KEGG pathway analysis using lnCAR program revealed that LINC01139 was associated with metabolic pathways and pathways in cancer (Fig. 1F). Then, we performed RTPCR to determine the dysregulated expression of LINC01139 in our cohort (109 HCC patients), finding that the expression of LINC01139 in HCC specimens was distinctly high than that in matched normal specimens (Fig. 1G). In addition, cellular experiments also revealed that LINC01139 was overexpressed in seven HCC cell lines compared to LO2 cells (Fig. 1H). Overall, our findings provided robust evidence that LINC01139 was an overexpressed lncRNA in HCC. Then, to explore the clinical significance of LINC01139 in HCC patients, the LINC01139 levels were classified as high or low in relation to the median value. As shown in Table S2, the results of chi-square test revealed that high LINC01139 expression was associated with advanced TNM stage (p ¼ 0.015) and lymph node metastasis (p ¼ 0.031). Then, Kaplan-Meier assays indicated that the patients with high LINC01139 expression displayed dramatically poorer overall survival rates than those with low LINC01139 expression (p ¼ 0.0011, Fig. 1I). To further confirm the prognostic value of overexpressed LINC01139 in HCC patients, our group performed multivariate assays, confirming that high LINC01139 expression (HR ¼ 2.721, 95% CI: 1.289e4.183, p ¼ 0.019) was an independent prognostic marker for HCC patients (Table S3).
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 1. Bioinformatics analysis revealed that LINC01139 was up-regulated in HCC cancer tissues. (A) Heatmap of differentially expressed lncRNAs in TCGA data. (B) Volcano map. (C) The expression of LINC01139 in TCGA HCC specimens. (D) LINC01139 was up-regulated in the HCC tumor samples involved in GSE36736, GSE55092 and GSE76427. The data was analyzed by lnCAR program and the diagrams were modified. (E) The co-expression work of LINC01139. (F) KEGG pathway analysis. (G) qPCR detected the LINC01139 levels in 109 HCC samples. (H) qPCR measured the LINC01139 levels in various HCC cell lines. (I) Overall survival. *p < 0.05, **p < 0.01.
3.2. LINC01139 depletion suppressed malignant behaviors in HCC The above findings that LINC01139 was remarkable upregulation in HCC tumor tissues and cell lines suggested that LINC01139 might act as a tumor promoter in HCC. Therefore, we next sought to investigate whether LINC01139 was capable to influence the malignant behaviors of HCC cells. First, we synthesized siRNAs targeting LINC01139 (si-LINC01139#1, siLINC01139#2) and the knockdown efficiency of these siRNAs in HepG2 cells was determined by qPCR (Supplementary Fig. 1A). Afterwards, CCK-8 assays revealed that LINC01139 depletion led to notable suppression of HepG2 cell proliferation (Supplementary Fig. 1B). Then, cell colony assays also demonstrated that LINC01139 deficiency caused markedly decreased colony formation abilities of HepG2 cells (Supplementary Fig. 1C). Besides, flow cytometry was utilized for determining the effects of LINC01139 depletion on HepG2 cell apoptosis. The data suggested that the apoptotic rates of HepG2 cells were significantly increased after their LINC01139 was knocked down (Supplementary Fig. 1D). Subsequently, we conducted the above
experiments using another HCC cells (SMMC-7721 cells), and similar results were also observed (Supplementary Fig. 1E-H). Hence, these data validated that LINC01139 served as crucial regulatory functions in HCC cell proliferation and apoptosis. 3.3. LINC01139 knockdown impaired metastatic properties of HCC cells The effects of LINC01139 on HCC cell metastatic potentials were then investigated. Data from wound-healing assays revealed that silencing the expression of LINC01139 notably inhibited the wound closures of HCC cells, which indicated that LINC01139 could regulate HCC cell migration (Fig. 2A and B). Afterwards, the influence of LINC01139 on HCC cell invasion was also evaluated using transwell assays. The results demonstrated that transfection with LINC01139 siRNAs caused dramatically decreased invasion cell number, which indicated that knockdown of LINC01139 was capable to impair the invasive capacities of HCC cells (Fig. 2C and D). Collectively, these results demonstrated that LINC01139 was able to modulate the mobility of HCC cells.
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 2. LINC01139 facilitated HCC cell migration and invasion. (A and B) The migratory abilities of HCC cells were determined by wound-healing assays. (C and D) Transwell assays assessed the invasive capabilities of HCC cells. *p < 0.05, **p < 0.01.
3.4. LINC01139 acted as a sponge of miR-30 family in HCC cells Accumulating evidences had validated that lncRNAs, particularly existing in cytoplasm, modulated their downstream targets via serving as ceRNAs [14]. Hence, we first used lncLocator program to determine the subcellular localization of LINC01139 in cells. The predicting results showed that LINC01139 mainly expressed in cytoplasm (Fig. 3A). Subsequently, the subcellular fractionation assays were carried out in HepG2 cells and the similar results were also observed (Fig. 3B). Hence, LINC01139 was possibly a sponge for specific miRNAs. We next generated the ceRNA network using lnCAR program and found that the miR-30 family members were included in that network (Fig. 3C). Therefore, we employed starbase program to predict the potential targeting miRNAs of LINC01139. Interestingly, miR-30 family members (miR-30a-e) were all the possible targets of LINC01139 (Fig. 3D). Afterwards, the expressing analysis using TCGA data by starbase program revealed that four members (miR-30a, b, c and e) of miR-30 family were significantly down-regulated in HCC tumor samples (Fig. 3E). Then, TCGA data analysis using starbase program indicated that the levels of LINC01139 were notably negatively correlated with miR-30a, miR30b and miR-30d in HCC tumor samples (Fig. 3F). Therefore, we next attempted to verify whether miR-30 family members were the exact targets of LINC01139. To achieve that, RNA-pull down was conducted. The results showed that biotinylated-LINC01139 (Biotin-LINC01139) was able to directly precipitate miR-30 family members, while biotinylated-LINC01139 with mutant predicting binding sites (Biotin-LINC01139 mut) could not precipitate miR-30 family members (Fig. 3G). To further verify the bindings, luciferase reporter assays were carried out. The data validated that cotransfection of wild-type LINC01139 (LINC01139 wt) luciferase reporters with indicated miR-30 family mimics dramatically reduced the luciferase activities in 293T cells, while the luciferase activities were affected when the cells were co-transfected with mutant-type LINC01139 (LINC01139 mut) luciferase reporters and indicated miR30 family mimics (Fig. 3H). In addition, qPCR analysis confirmed that overexpressing LINC01139 (ov-LINC01139) resulted in significantly decreased expression of miR-30 family membranes, while enhancing the mutant-type LINC01139 (ov-LINC01139-mut)
expression had no influence on the levels of miR-30 family membranes (Fig. 3I). On the contrary, knocking down LINC01139 led to remarkably increased levels of miR-30 family membranes (Fig. 3J). Overall, the above findings validated that LINC01139 directly targeted miR-30 family membranes in HCC cells. 3.5. MYBL2 was a target of miR-30 family members in HCC cells Next, we sought to investigate the downstream targets of miR30 family members. First, we used miRDB program to predict the common targeting genes of the five miR-30 family members, and then the common targeting genes were intersected with the upregulated genes in HCC tumor samples, and 10 genes were found (Fig. 4A). Subsequently, we analyzed the expressing correlation between LINC01139 and the 10 genes using GEPIA program. The results showed that among these 10 genes, only MYBL2 and SNX8 were notably positively correlated with LINC01139 expression in HCC (Fig. 4B). In addition, analyzing the overall survival using GEPIA program revealed that, among MYBL2 and SNX8, only the high expression of MYBL2 was significantly associated with lower overall survivals of HCC patients (Fig. 4C). Hence, we next sought to clarify whether MYBL2 was a target of miR-30 family members. The predicting binding sites between the 30 UTR of MYBL2 and miR-30 family members were presented in Fig. 4D. Furthermore, using starbase program, we found that the expression of MYBL2 in HCC tumor samples of TCGA data was negatively correlated with miR30a and miR-30e (Fig. 4E). Moreover, through analyzing TCGA data using UALCAN program, we discovered that MYBL2 was upregulated in most cancer types (Fig. 4F). Additionally, KEGG analysis revealed that MYBL2-positively correlated genes were relevant with cell cycle, which indicated that MYBL2 might play critical roles in regulating HCC malignancies (Fig. 4G). Next, we thereby performed luciferase reporter assays to verify the binding between 30 UTR of MYBL2 and miR-30 family members. The 3’ UTR of MYBL2 mRNA including the predicting miR-30 family binding site (wildtype: wt) or the mutated sequence (mutant-type: mut) was constructed into pGL3 vectors to form MYBL2 wt or MYBL2 mut luciferase reporters. It was observed that the five miR-30 family members remarkably reduced the luciferase activities in 293T cells
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 3. MiR-30 family members was directly interacted with LINC01139 in HCC cells. (A) lncLocator program predicted the subcellular localization of LINC01139. (B) Subcellular fractionation assays. (C) The ceRNA network of LINC01139 was generated by lnCAR program. (D) The predicting binding sites between LINC01139 and miR-30 family members were generated by starbase program. (E) The expression of miR-30 family members was conducted by starbase program. (F) The correlation between LINC01139 and miR-30 family members was analyzed by starbase program. (G) RNA pull-down assays. (H) Luciferase reporter assays. (I) qPCR detected the levels of miR-30 family members in HepG2 cells after transfection with ov-LINC01139 or ov-LINC01139-mut plasmids. (J) qPCR evaluated the levels of miR-30 family members in HepG2 cells after transfection with LINC01139 siRNAs. *p < 0.05, **p < 0.01.
transfected with MYBL2 wt reporters, instead of the mutant-type reporters (Fig. 4H). Subsequently, we synthesized siRNAs targeting MYBL2 (si-MYBL2#1, si-MYBL2#2, si-MYBL2#3) and qPCR analysis indicated that si-MYBL2#2 had the highest knockdown efficiency (Fig. 4I). Afterwards, si-MYBL2#2 was transfected into HepG2 cells and the relative expression of miR-30 family members was assessed by qPCR. The data confirmed that the relative levels of miR-30 family members were dramatically increased in HCC cells with MYBL2 deficiency (Fig. 4J). Therefore, these results validated that miR-30 family members targeted MYBL2 in HCC cells.
3.6. LINC01139 modulated MYBL2 expression trough sponging miR30 family members in HCC cells To elucidate the detail molecular mechanisms by which LINC01139 served as an essential regulator in HCC malignancies, we next respectively transfected the mimics of miR-30 family members into HepG2 cells. The data from qPCR analysis validated that enhancing the expression of miR-30 family members contributed to remarkably decreased levels of LINC01139 and MYBL2 (Supplementary Fig. 2A). On the contrary, the depression of miR-30
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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Fig. 4. MiR-30 family members targeted MYBL2 in HCC cells. (A) Venn diagram of miRDB program predicting the target genes of miR-30 family members (left). The intersection of the common targeting genes was intersected with the up-regulated genes (from TCGA data using GEPIA analysis) in HCC tumor samples (right). (B) The expressing correlation between LINC01139 and the 10 genes using GEPIA program. (C) The overall survival was analyzed by GEPIA program. (D) The predicting binding sites between 30 UTR of MYBL2 and miR-30 family members were presented. (E) The expressing correlation between MYBL2 and miR-30 family members was analyzed by starbase program. (F) UALCAN program analyzed the expression of MYBL2 across diverse cancer types. (G) KEGG pathway analysis of MYBL2-positively-correlated genes in HCC. (H) Luciferase reporter assays. (I) qPCR determined the expression of MYBL2 in HepG2 cells after transfection with MYBL2 siRNAs. (J) qPCR evaluated the expression of miR-30 family members. *p < 0.05, **p < 0.01.
family members by transfecting corresponding inhibitors resulted in markedly increased levels of LINC01139 and MYBL2 (Supplementary Fig. 2B). Then, we depleted LINC01139 in HepG2 cells by transfecting LINC01139 siRNAs and utilized qPCR to evaluate the expression of MYBL2. We found that the levels of MYBL2 were dramatically suppressed by LINC01139 depletion (Supplementary Fig. 2C). Similarly, Western blot assays also revealed that LINC01139 deficiency resulted in markedly decreased protein levels of MYBL2 (Supplementary Fig. 2D). Next, we mixed the miRNA mimics of miR-30 family members (miR-30 mix) and
performed qPCR to assess the expressing changes of MYBL2 in HepG2 cells after transfection with LINC01139 overexpressing plasmids (ov-LINC01139) or miR-30 mix. The results demonstrated that ectopic expression of LINC01139 was able to increase MYBL2 expression, and transfection of miR-30 mix led to a remarkable decline of MYBL2 levels (Supplementary Fig. 2E). However, forced expression of LINC01139 could significantly restore the levels of MYBL2 which were impeded by transfection of miR-30 mix (Supplementary Fig. 2E). Similar results were also observed by utilizing Western blot assays (Supplementary Fig. 2F).
Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116
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3.7. LINC01139 exerted the oncogenic functions via sponging miR30 family members in HCC cells To certify whether LINC01139 exerted the oncogenic functions via sponging miR-30 family members in HCC cells, we conducted a series of functional studies. First, CCK-8 assays revealed that the forced expression of LINC01139 obviously promoted the proliferation of HCC cells, while concurrent overexpression of miR-30 family members (miR-30 mix) abolished the proliferation of HCC cell. However, re-introduction of LINC01139 (ov-LINC01139) markedly reversed the impeded influence of miR-30 mix on HCC cellular growth (Supplementary Fig. 3A). Similar results were also observed using clonogenic assays (Supplementary Fig. 3B). Besides, the results of wound-healing assays showed that enhancing the expression of LINC01139 caused markedly elevated migratory abilities of HCC cells (Supplementary Fig. 3C and D). Although overexpressing miR-30 family members led to notable inhibition of HCC cell migration, the re-overexpressing LINC01139 markedly reversed the cellular migratory abilities (Supplementary Fig. 3C and D). Taken together, these data demonstrated that LINC01139 promoted malignant behaviors of HCC in a miR-30 family dependent manner. 4. Discussion The identification of sensitive biomarkers for prognosis and diagnosis and novel therapeutic targets has a very essential clinical significance for the improvement of long-term survival of HCC patients [15]. In recent years, convincing evidence has confirmed the essential roles of lncRNAs in tumor progression and metastasis. In this study, we performed bioinformatics analysis and identified a novel overexpressed lncRNA LINC01139 in HCC patients. Then, the results of RT-PCR confirmed LINC01139 levels were distinctly upregulated in both HCC specimens from 109 patients and cell lines. Then, we explored the clinical significance of LINC01139 in HCC patients, finding that the upregulation of LINC01139 was positively associated with advanced TNM stage, lymph node metastasis and shorter overall survival, suggesting the clinical value of LINC01139 used as a prognostic biomarker. On the other hand, we performed loss-of-function assays, confirming that knockdown of LINC01139 suppressed the proliferation, migration and invasion. Overall, our findings revealed that LINC01139 could serve as a novel prognostic biomarker and therapeutic target in HCC. The competing endogenous RNA (ceRNA) hypothesis was proposed to describe the function of many lncRNAs as ceRNA to protect the genuine targets of miRNAs from silencing or translational suppression [14]. Recently, several studies on HCC studies have reported the mechanisms involved in lncRNA-miRNA-mRNA axis. Although the expression and function of LINC01139 have been reported in previous studies in several tumors, the potential mechanisms involved LINC01139 mediated ceRNA have not been investigated. Interestingly, we found that LINC01139 was expressed both in the nucleus and cytoplasm. Then, by the use of bioinformatics analysis and several online tools, our attention focused on miR-30 family which may interact with LINC01139. Previously, miR-30 family acting as a tumor suppressor had been well studied in many tumors, including HCC [16,17]. These finding supported the possibility of LINC01139 exhibiting its effects via suppressing miR30 family. Moreover, luciferase activity assays and RNA pull-down assays demonstrated the direct binding relationship between lncRNA LINC01139 and miR-30 family. Overall, our finding firstly provided evidence that LINC01139 served as a tumor promoter in HCC via directly binding to miR-30 family members. MYB proto-oncogene like 2 (MYBL2), located on chromosome 20q13, is a highly conserved member of the MYB family of transcription factors [18]. Previously, MYBL2 has been reported to be
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dysregulated in several tumors [19]. Functionally, overexpression of MYBL2 was shown to mediate resistance to chemotherapeutic agents and have a positive influence in the proliferation, metastasis and EMT progress in several tumors [20]. In HCC, overexpression of MYBL2 exhibited a positive role in the expression of several tumorrelated genes, resulting in an increased ability of HCC cells in the proliferation and metastasis [21]. In this study, we also provided evidence that MYBL2 was highly expressed in HCC and predicted a poor prognosis, which was consistent with the above results. In addition, we performed comprehensive assays, finding that MYBL2 may be a target of miR-30 family members. Moreover, the results of luciferase reporter assays confirmed that MYBL2 gene was one of the direct targets of miR-30 family members. To further explored the association among LINC01139, miR-30 family and MYBL2, we silenced or overexpressed the levels of miR-30 family members, finding that knockdown of miR-30 family members distinctly suppressed the expression of LINC01139 and MYBL2, while their suppression exhibited an opposite result. Rescue experiments indicated that overexpression of LINC01139 could increase the mRNA and protein levels of MYBL2 which was decreased by miR-30 family suppressors. More importantly, functional assays revealed that the transfection of ov-LINC01139 could promote the proliferation, colony formation and migration ability attenuated by the upregulation of miR-30 family members in HCC cells. Overall, findings indicated that LINC01139 served as a tumor promoter via modulating miR-30/MYBL2 axis. 5. Conclusions Our findings firstly suggested that LINC01139 act as an oncogenic lncRNA which promoted HCC progression via the miR-30/ MYBL2 axis. Overall, our results revealed that LINC01139 could act as a potential therapeutic target and a prognostic biomarker for HCC patients. Declaration of competing interest The Authors declare that they have no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.116. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.116. References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Canc. 136 (2015) E359eE386. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA A Cancer J. Clin. 68 (2018) 7e30. [3] L. Kulik, H.B. El-Serag, Epidemiology and management of hepatocellular carcinoma, Gastroenterology 156 (2019) 477e491, e471. [4] T. Clark, S. Maximin, J. Meier, S. Pokharel, P. Bhargava, Hepatocellular carcinoma: review of epidemiology, screening, imaging diagnosis, response assessment, and treatment, Curr. Probl. Diagn. Radiol. 44 (2015) 479e486. [5] S.D. Colquhoun, Hepatocellular carcinoma: the current role of surgical intervention, Crit. Rev. Oncog. 21 (2016) 93e103. [6] J. Jarroux, A. Morillon, M. Pinskaya, History, discovery, and classification of lncRNAs, Adv. Exp. Med. Biol. 1008 (2017) 1e46. [7] S.U. Schmitz, P. Grote, B.G. Herrmann, Mechanisms of long noncoding RNA function in development and disease, Cell. Mol. Life Sci. 73 (2016) 2491e2509. [8] F. Kopp, J.T. Mendell, Functional classification and experimental dissection of
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Please cite this article as: Z.-B. Li et al., Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.02.116