Down-regulation of miR-146b-5p by long noncoding RNA MALAT1 in hepatocellular carcinoma promotes cancer growth and metastasis

MicroRNAs play an important role in liver cancer genesis and progression. In this study, we identified down-regulation of miR-146b-5p associated with tumor growth, metastasis and poor survival in hepatocellular carcinoma (HCC) patients. miR-146b-5p could suppress proliferation, migration, and invasion and induced apoptosis in vitro and in vivo. Remarkably, TNF receptor associated factor 6 (TRAF6) was confirmed as a direct target of miR-146b-5p in HCC and miR-146b-5p exerted the tumor suppression roles through inhibiting the phosphorylation of Akt mediated by TRAF6. Furthermore, we identified long non-coding RNA MALAT1 as a molecular sponge of miR-146b-5p to down-regulate its expression in HCC. In general, our results indicate that miR-146b-5p inhibits tumor growth and metastasis of HCC by targeting TRAF6 mediated Akt phosphorylation.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common digestive cancers in China [1]. MicroRNAs represent as a class of endogenous short single-chain RNAs that regulate various cellular processes through binding to the 3' untranslated region (3'-UTR) of target mRNAs [2]. Plenty of reports have well established their roles in metabolism [3], growth [4], senescence [5], angiogenesis [6] and metastasis [7].
Data in mounting numbers revealed that miR-146b-5p was involved in cancer genesis and progression [8]. However, miR-146b-5p is a friend or foe that depends on the cancer types. Up-regulation of miR-146b-5p was found in osteosarcoma tissues [9], and it promoted proliferation, migration and invasion through inhibiting ZNRF3 in osteosarcoma cells. Nevertheless, miR-146b-5p inhibited glioma cells' migration and invasion through silencing MMP-16 expression [10]; it also attenuated stemness and radioresistance through restraining HuR/ lincRNA-p21/β-catenin pathway [11]. In papillary thyroid carcinoma (PTC), the role of miR-146b-5p was more equivocal. An elevated expression of miR-146b-5p was observed in PTC tissues [12,13]. Over-expression of miR-146b-5p enhanced growth and metastasis of PTC cells [14,15]. However, recent study discovered that exosomes derived from TPC-1 cells were rich in miR-146b-5p, but these exosomes served as a negative regulator for cell proliferation to both TPC-1 and normal thyroid follicular cells [16]. Even the role of miR-146b-5p has been uncovered in many cancers, the expression and functions of miR-146b-5p in HCC are still unclear.
In this study, we disclosed that miR-146b-5p was down-regulated in HCC tissues and correlated with poor prognosis. In vitro and in vivo experiments showed that miR-146b-5p could suppress proliferation, migration, and invasion and induce apoptosis through inhibiting TRAF6/ p-Akt signaling pathway.

miR-146b-5p inhibits growth and metastasis in HCC cells
We detected the expression levels of miR-146b-5p in four HCC cell lines (MHCC97-H, SMMC-7721, Hep3B and HepG2) and a human immortal liver cell line (LO2). As shown in Figure 2A, compared with the expression of miR-146b-5p in LO2, MHCC97-H and Hep3B had the lowest and highest expression levels of miR-146b-5p in these four cell lines, repectively (P<0.001, respectively). So we selected these two cell lines for further experiments. First, we stably over-expressed miR-146b-5p in MHCC97-H cells and knocked down miR-146b-5p expression in Hep3B cells (P<0.001, respectively, Figure 2B). Next, we concluded from CCK-8 assays and plate clone formation assays that miR-146b-5p over-expression suppressed cell viability and proliferation in MHCC97-H cells; on the contrary, inhibition of miR-146b-5p depressed cell viability and proliferation in Hep3B cells (P<0.01, respectively, Figure  2C and 2D). Additionally, determined by flow cytometry and caspase 3/7 activity assays, up-regulation of miR-146b-5p induced apoptosis in MHCC97-H cells and downregulation of miR-146b-5p reduced apoptosis in Hep3B Figure 1: Low expression of miR-146b-5p in HCC tissues relates to poor prognosis. A. The expression levels of miR-146b-5p in HCC tissues and matched tumor-adjacent tissues. *** P<0.001. B. Down-regulation of miR-146b-5p was detected in 73.33% (44/60) of HCC tissues. C and D. Low expression of miR-146b-5p was significantly associated with the poorer 5-year overall survival (C, log-rank=5.606, P=0.018) and disease-free survival (D, log-rank=7.692, P=0.005) of HCC patients. Patients were divided into high and low expression subgroups according to the mean value of miR-146b-5p. cells (P<0.01, respectively, Figure 2E and 2F). We further determined cell migration and invasion through transwell chambers. As shown in Figure 2G, up-regulation of miR-146b-5p significantly decreased the migration and invasion in MHCC97-H cells (P<0.01, respectively). On contrary, down-regulation of miR-146b-5p increased the migration and invasion in Hep3B cells (P<0.01, respectively).

miR-146b-5p inhibits tumor growth and metastasis in vivo
In order to further confirm our in vitro results, we subcutaneously injected the recombinant MHCC97-H and Hep3B cells into nude mice, measured the tumor volume each week for 4 weeks and drew the tumor growth curves. As shown in Figure 3A, miR-146b-5p over-expression significantly repressed xenograft tumor growth, whereas miR-146b-5p knockdown promoted tumor growth (P<0.01, respectively, Figure 3A). Determined by Ki-67 IHC staining and TUNEL assays, miR-146b-5p was demonstrated to inhibit proliferation (P<0.01, respectively, Figure 3B) and induce apoptosis (P<0.01, respectively, Figure 3C). Moreover, we established the lung metastasis mice model through the tail vein injection. Lung tissue sections were stained with hematoxylin and eosin. Compared with control groups, miR-146b-5p overexpression decreased the lung metastasis but miR-146b-5p knockdown increased the lung metastasis (P<0.01, respectively, Figure 3D).

miR-146b-5p attenuates TRAF6/p-Akt signal pathway in HCC
TRAF6 is thought to achieve its functions partly through contributing the phosphorylation of Akt [17]. In order to explore whether miR-146b-5p performs its anti-cancer role though inhibiting the TRAF6-mediated phosphorylation of Akt, we detected the expression of total Akt (t-Akt) and phosphorylated Akt (p-Akt) in transfected cells. As shown in Figure 5, miR-146b-5p over-expression decreased the expression of p-Akt in MHCC97-H cells, whereas miR-146b-5p knockdown increased the expression of p-Akt in Hep3B cells (P<0.05, respectively, 1 st row). However, no significant expression changes of total Akt (t-Akt) were detected in these two cell lines (P>0.05, respectively, 2 nd row). Subsequently, we detected a series of downstream genes of the TRAF6/p-Akt signaling pathway, such as Bcl-2, Mcl-1 and MMP-9. Western blot results showed that the proteinlevels of these three molecules were down-regulated in MHCC97-H cells and up-regulated in Hep3B cells (P<0.05, respectively, 3 rd to 5 th rows).

Long non-coding RNA MALAT1 serves as an endogenous sponge of miR-146b-5p in HCC
Studies have shown that long noncoding RNAs (lncRNAs) act as an endogenous sponge of microRNAs. We used Starbase v.2.0 to predict that a long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), had a complementary sequence of miR-146b-5p. We found that the expression of MALAT1 was higher in HCC tissues than that in tumor-adjacent tissues (P<0.01, Figure 7A). Furthermore, a negative relationship was identified between these two non-coding RNAs, the correlation index was -0.483 (P=0.007, Figure  7B). MHCC97-H and SMMC-7721 cells which had high original MALAT1 expression levels (P<0.01, respectively, Figure 7C) were selected to silence MALAT1 expression by siRNA (P<0.001, respectively, Figure 7D). Real-time PCR results showed that MALAT1 knockdown rescued miR-146b-5p expression in MHCC97-H and SMMC-7721 Figure 5: miR-146b-5p exerts its functions through inhibiting the TRAF6/p-Akt signaling pathway. In MHCC97-H cells with miR-146b-5p over-expression, the phosphorylation of Akt (p-Akt) was impaired and the expression levels of Bcl-2, Mcl-1 and MMP-9 were decreased. In Hep3B cells with miR-146b-5p knockdown, enhanced the phosphorylated Akt induced the expression of Bcl-2, Mcl-1 and MMP-9. Neither over-expression nor knockdown of miR-146b-5p could find any significant changes of total Akt (t-Akt) expression. * P<0.05. Immunoblotting was performed at least in triplicate and the data are presented as the (mean ± SD). www.impactjournals.com/oncotarget cells (P<0.001, respectively, Figure 7E). These findings might explain why miR-146b-5p expression was lower in HCC.

DISCUSSION
miR-146b-5p showed paradoxical roles in human cancers. It was reduced in gallbladder cancer tissues and correlated with large tumor size and inferior cell differentiation [18]. A prognostic value of miR-146b-5p was also embodied in ER-negative breast cancer patients [19], large B-cell lymphoma patients [20] and gastric cancer [21]. Interestingly, Yoon et.al reported that high expression of miR-146b-5p in the proximal resection margin tissues of gastric cancer was a strong risk factor for recurrence and poor survival [22]. In the present study, for the first time, we discovered that the expression of miR-146b-5p was significantly decreased in HCC tissues. Down-regulation of miR-146b-5p was correlated with malignant clinical features and poor survival in HCC patients. All above results indicate that decreased expression of miR-146b-5p is an unfavorable factor and may lead to the disorder of cell growth and invasion in HCC.   To confirm this hypothesis, a series of in vitro and in vivo assays were designed to investigate the biological functions of miR-146b-5p. We revealed that up-regulation of miR-146b-5p reduced proliferation, migration and invasion, promoted apoptosis and caspase 3/7 activity in vitro, and arrested tumor growth and lung metastasis in vivo. Even though miR-146b-5p promoted the proliferation and invasion of osteosarcoma cells [23], our results were still consistent with the previous reports in prostate and pancreatic cancers [24].
Functionally, TRAF6 exhibits an E3 ubiquitin ligase activity. Unlike other E3 ubiquitin ligases catalyze the synthesis of polyubiquitin chains linked through lysine-48 (K48), the chains catalyzed by TRAF6 was linked through the point of lysine-63 (K63) [25]. These unique chains can lead to the activation of TAK1 [26], MAPK [27] and many other kinases' activity. We used dual-luciferase reporter system to confirm that only wildtype miR-146b-5p changed the luciferase activity of 3'-UTR of TRAF6 mRNA. miR-146b-5p could decrease the TRAF6 expression in vitro and in vivo. Finally, we provided a negative correlation between TRAF6 and miR-146b-5p expression in a cohort of HCC patients. Our results also showed that miR-146b-5p only imapired the phosphorylation of Akt through inhibiting the expression of TRAF6 but not influenced the total Akt expression. Because the K48 linked ubiquitination usually caused degradation of target proteins, we could partly confirm that K63 might be the catalytic site of TRAF6 for Akt ubiquitination. We also detected three downstream genes of Akt signaling pathway which could enhance cell growth and metastasis. First, the expression levels of Bcl-2 and Mcl-1 were down-regulated through inhibiting TRAF6/ p-Akt activation by miR-146b-5p. Mcl-1 served as a substrate of caspase-3 [28], its down-regulation could be explained by the enhancement of caspase 3/7 activity after the miR-146b-5p over-expression. Next, our study also found that miR-146b-5p inhibited the expression of MMP-9, and that might explain why miR-146b-5p could inhibit cell invasion and lung metastasis. Furthermore, we rescued the expression of TRAF6 in MHCC97-H miR-146b-5p cells and silenced TRAF6 expression in Hep3B anti-miR-146b-5p cells. Our results showed that TRAF6 partly abrogated the effects of miR-146b-5p on HCC cell viability, proliferation, apoptosis, migration, invasion through reactivated Akt signaling pathway.
Previous studies showed that deregulation of lncRNAs profoundly influenced the expression of microRNAs [29]. For example, lncRNA HOTAIR contained a conserved binding site for miR-326. Silencing HOTAIR by siRNA could rescue the expression of miR-326 in human glioma cells [30]. Inspired by these studies, we identified a negative correlation between lncRNA MALAT1 and miR-146b-5p expression in HCC tissues. Down-regulation of lncRNA MALAT1 expression significantly increased the expression of miR-146b-5p in HCC cells.
In conclusion, this study demonstrated that downregulation of miR-146b-5p in HCC tissues was related to malignant clinical features and poor prognosis. Using in vitro and in vivo studies, miR-146b-5p was demonstrated as a novel inhibitor for tumor growth and metastasis in HCC. The multiple anti-cancer functions of miR-146b-5p were due to the inhibition of the TRAF6/p-Akt pathway ( Figure 8). Together, our findings suggested that miR-146b-5p could become a novel prognostic bio-marker and potential therapeutic target in HCC.  Table 1. Informed consent was obtained in accordance with a protocol approved by the Ethics Committee of Xi'an Jiaotong University.

Transwell chamber models
The 8 μm pore-sized transwell inserts (Nalge Nunc, Penfield, NY, USA) were used to detect cell migration, or coated with matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at 1 mg/mL on the inner layer to detect cell invasion. Cells were re-suspended with reduced serum DMEM medium (Gibco, Carlsbad, CA, USA) and added into upper-chamber, and a 750 μL completed DMEM medium was added into the lower-chamber, then incubating for 24 hours. Cells were fixed in 4% paraformaldehyde for 2 min and stained with 0.3% crystal violet. Migrated or invaded cells on the under-surface were counted under a light microscope. 4 weeks BALB/c nude mice were purchased from the center of laboratory animals of Xi'an Jiaotong University. 5×10 6 cells were suspended in 100 μL PBS and injected into the subcutis. Width and length of the neoplasm were measured with calipers every week. Tumor volume was calculated with the formula: π×length×width2/6. 4 weeks later, mice were excused. 100 mg tissue was collected from every sample to isolate total RNA. Neoplasms were fixed with 4% paraformaldehyde, embedded in paraffin and made into 4 μm slices. Ki-67 antibodies (#9027, CST, Danvers, MA, USA) were used to determine cell proliferation by immunohistochemical (IHC) staining as we described previously [33]. In situ cell death detection kit, POD (Roche, Mannheim, Germany) was used to detected cell apoptosis according to the manufacturer's instruction. The scores of TUNEL positive staining cells were expressed as the following grades: 0, <5%; 1, 6%-25%; 2, 26%-50%; 3, 51%-75%; and 4, >75%.

In vivo tumor growth and metastasis assays
1×10 5 cells with 100 μL PBS were injected into mouse systemic circulation through the tail vein. Lungs were obtained after four weeks feeding and fixed with 4% paraformaldehyde. 4 μm consecutive sections were made and stained with hematoxylin and eosin. The number of metastatic clusters was calculated to analyze the effects of different treatments.

Luciferase reporter assay
The 3'-UTR region of TRAF6 was inserted into pmiR-RB-Report ™ vector (RiboBio, Guangzhou, China). miR-146b-5p and recombinant vectors were co-transfected into MHCC97-H cells with Lipofectamine ® 2000. Ranilla luciferase activity was detected using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) to reflect the binding between miR-146b-5p and the 3'-UTR region of TRAF6. Firefly luciferase activity was served as the internal control.

Statistical analysis
Measurement data is presented as Mean ± SD and processed by the SPSS statistical package for Window Version 21 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 software (GraphPad Inc., San Diego, CA, USA). Student's t test or one-way ANOVA were used to analyze the difference among/between sample groups. Patients' survival was reflected by Kaplan-Meier and analyzed by log-rank test. Person correlation analysis was used to evaluate the relationship between miR-146b-5p and MALAT1. P<0.05 was considered to be statistically significant.