Long Noncoding RNA MIR100HG Knockdown Attenuates Hepatocellular Carcinoma Progression by Regulating MicroRNA-146b-5p/Chromobox 6

Purpose Hepatocellular carcinoma (HCC) accounts for approximately ninety percent of primary liver cancer. This study attempted to investigate the effects of the long noncoding RNA MIR100HG (MIR100HG) in HCC and the underlying molecular mechanism. Materials and Methods qRT-PCR was implemented to analyze the expression of MIR100HG, microRNA-146b-5p (miR-146b-5p), and Chromobox 6 (CBX6). The correlation between MIR100HG and clinicopathological features of HCC patients was assessed. Additionally, the effects of MIR100HG knockdown on HCC cell viability, migration, and invasion were explored. The interactions among MIR100HG, miR-146b-5p, and CBX6 were confirmed. Furthermore, rescue experiments were conducted to investigate whether MIR100HG knockdown modulates HCC cell behaviors through modulating the miR-146b-5p/CBX6 axis. Results The expression of MIR100HG and CBX6 was enhanced, while miR-146b-5p was inhibited in HCC cells. High MIR100HG expression was positively associated with the TNM tumor stage and Edmondson-Steiner grading in HCC patients. MIR100HG knockdown considerably reduced the HCC cell viability, migration, and invasion. In addition, MIR100HG directly targeted miR-146b-5p, and miR-146b-5p directly targeted CBX6 in HCC cells. Moreover, miR-146b-5p suppression or CBX6 elevation evidently rescued the suppressed viability, migration, and invasion of HCC cells caused by MIR100HG knockdown. Conclusions Knockdown of MIR100HG inhibited the viability, migration, and invasion of HCC cells by targeting the miR-146b-5p/CBX6 axis, offering a potential therapeutic target for HCC therapy.


Introduction
Primary liver cancer is the fourth most common reason of cancer mortality [1]. Hepatocellular carcinoma (HCC) is a frequently malignant tumor of primary liver cancer [2]. HCC is usually accompanied by cirrhosis and chronic viral hepatitis B or C [3]. Despite advances in the clinical treatment of HCC, the prognosis for HCC patients is still poor [4]. Thus, it is crucial to discover new therapeutic targets for HCC treatment.
Emerging evidences reveal that long noncoding RNAs (lncRNAs) are involved in the regulation of cell proliferation, migration, and invasion in several types of human cancers, such as osteosarcoma [5], breast cancer [6], and lung cancer [7]. Surely, some lncRNAs acting as oncogenes are also reported to participate in HCC tumorigenesis. lncRNA FAL1 inhibits miR-1236 expression to promote HCC cell proliferation and metastasis [8]. lncRNA KTN1-S1 silencing reduces viability and triggers apoptosis of HCC cells [9]. lncRNA MALAT1 contributes to HCC progression via mTOR activation and SRSF1 elevation [10]. It has been documented that lncRNA MIR100HG (MIR100HG) serves as an oncogene to take part in solid tumors. MIR100HG elevation accelerates the metastasis of colorectal cancer and is related to poor outcome [11]. MIR100HG overexpression accelerates the laryngeal squamous cell carcinoma progression by inhibiting miR-204-5p [12]. Elimination of MIR100HG attenuates the cell proliferation in gastric cancer [13]. However, the specific regulatory function of MIR100HG in HCC remains unknown.
Chromobox 6 (CBX6), one of the polycomb paralogs, functions in cancer progression [20]. Certain CBX family members exert a tumor-promoting role in HCC. CBX8 interacts with YBX1 to accelerate HCC cell proliferation [21]. CBX4 silencing impairs cell cycle progression and hampers cell proliferation in HCC [22]. Importantly, CBX6 overexpression is an independent prognostic factor for shorter overall survival of HCC patients [23]. However, the regulatory relationship between MIR100HG and CBX6 in HCC remains to be elucidated.

Quantitative Real-Time Polymerase
Chain Reaction (qRT-PCR) and Western Blot. qRT-PCR and western blot were performed as previously described [24]. The primers are depicted in Table 1. MIR100HG, miR-146b-5p, and CBX6 expression was normalized to GAPDH, U6, and β-actin expression. The antibodies for western blot analysis including anti-CBX6 Gastroenterology Research and Practice viability was measured via the MTT cell viability assay kit (Sigma) by the manufacturer.
2.6. Wound Healing Assay. Hep3B and SK-HEP1 cells (1 × 10 6 /well) were incubated in 6-well plates. The cell monolayer was then wounded with a 10 μl pipette tip and cultured in serum-free medium. Cell migration images were captured at 0 and 24 h.

Invasion Assay.
A Transwell chamber (8 mm, Corning Incorporated, Corning, NY, USA) was used to measure cell invasion. Briefly, Hep3B and SK-HEP1 cells (2:5 × 10 5 /well) in serum-free medium were added into the upper chamber. Medium containing 10% FBS was added to the lower chamber and acted as a chemoattractant. After 24 h, cells that had invaded the pore were fixed with methanol and stained with 0.05% crystal purple. The invading cells were counted.

Gastroenterology Research and Practice
presented as the mean ± SD. The differences between two groups or among multiple groups were assessed by Student's t-test or one-way ANOVA followed by Tukey's post hoc test. The significance of the correlations was determined by Pearson's correlation analysis. P values < 0.05 were considered statistically significant. Figure 1(a) exhibits that MIR100HG expression was considerably increased in tumor tissues of HCC patients (P < 0:001). Besides, MIR100HG expression was clearly enhanced in tumors at   Gastroenterology Research and Practice TNM III/IV (P < 0:01, Figure 1(b)). As depicted in Table 2, increased MIR100HG expression was related to the TNM tumor stage and Edmondson-Steiner grading in HCC patients (P < 0:05). Moreover, MIR100HG expression was dramatically upregulated in Hep3B, HepG2, SK-HEP1, and Huh7 cells compared to that in LO2 cells (P < 0:001, Figure 1(c)).

Discussion
The lncRNA elevation often facilitates HCC progression [25]. The expression of lncRNAs, such as MALAT1 [10], AB019562 [24], and CARLo-5 [26], is enhanced in HCC patients. In the present research, MIR100HG expression was elevated in HCC, indicating that MIR100HG may be an oncogenic lncRNA in HCC. Besides, MIR100HG expression was found to be correlated with the TNM tumor stage and Edmondson-Steiner grading in HCC patients. Some lncRNAs have the same function in HCC. lncRNA CARLo-5 is upregulated in HCC patients with a higher Edmondson-Steiner grade [26]. High-level lncRNA LINC00470 is markedly correlated with an advanced TNM stage in HCC patients [27]. Above all, MIR100HG expression was enhanced in HCC and related to the HCC progression. Numerous researches have demonstrated that MIR100HG partakes in tumor behavior of different cancers [11,12]. Knockdown of MIR100HG has an inhibitory effect on the tumorigenesis of cancers. For instances, MIR100HG suppression impairs the cell viability, thus attenuating the tumorigenesis of acute megakaryoblastic leukemia [28]. MIR100HG deficiency impedes cell viability while accelerating cell apoptosis to suppress HCC progression [29]. MIR100HG silencing upregulates miR-5590-3p and downregulates OTX1, resulting in the decrease in cell viability and invasion in breast cancer [30]. In this study, MIR100HG knockdown decreases the HCC cell viability, invasion, and migration, suggesting that MIR100HG acts as a therapeutic target for HCC treatment. In addition, sorafenib, an oral kinase inhibitor, has been confirmed to be a gold quality standard to enhance survival in patients affected by advanced HCC in the first-line treatment [31]. Interestingly, numerous lncRNAs are overexpressed in HCC tissues and are closely associated with sorafenib resistance, such as SNHG1 [32], SNHG3 [33], HEIH [34], and NEAT1 [35]. We speculated that the high expression level of MIR100HG may also promote sorafenib resistance to affect the progression of HCC when sorafenib is administered in clinical practice. Further researches on the interaction of MIR100HG with sorafenib are urgently needed. lncRNAs often function as a molecular sponge or competing endogenous RNA in regulating miRNA in HCC. For instances, lncRNA n335586 facilitates CKMT1A expression through competitively binding with miR-924 to enhance HCC cell migration and invasion [36]. lncRNA SNHG15 serves as a molecular sponge of miR-141-3p to exert its oncogenic effect in HCC [37]. Notably, lncRNA MALAT1 accelerates HCC progression via sponging miR-146b-5p [19]. Here, miR-146b-5p was a target of MIR100HG and negatively related to MIR100HG expression, indicating that MIR100HG may be involved in HCC by regulating miR-146b-5p. miR-146b-5p is often inhibited in solid tumors, such as glioma [38] and gallbladder cancer [39]. In this study, miR-146b-5p was suppressed in HCC, suggesting that miR-146b-5p may be an anti-oncomiR in HCC. It has been documented that miR-146b-5p serves as a mediator in tumor progression. miR-146b-5p upregulation leads to MMP16 knockdown, thus hindering glioma cell growth [38]. miR-146b-5p overexpression retards cell proliferation and facilitates cell apoptosis in nasopharyngeal carcinoma by regulating HNRNPA2B1 [40]. Importantly, miR-146b-5p is inhibited in HCC cells and is a biomarker for the gene therapy of HCC [41]. Here, we determined that miR-146b-5p restrained tumorigenesis of HCC cells, and miR-146b-5p deficiency weakened the antitumor function of MIR100HG suppression in HCC cells. Taken together, MIR100HG silencing may inhibit tumorigenesis of HCC cells through upregulating miR-146b-5p.
CBX family members are frequently elevated in various tumors, such as CBX2 in ovarian cancer [42], CBX4 in breast cancer [43],, and CBX8 in muscle invasive bladder cancer [44]. Similarly, CBX6 expression was increased in HCC in this study, indicating that CBX6 may be a participant in HCC. CBX family numbers are involved in HCC progression. CBX1 interacts with HMGA2 to accelerate HCC cell proliferation and migration [45]. CBX2 suppression attenuates proliferation and induces apoptosis in HCC cells by modulating YAP [46]. Notably, CBX6 elevation promotes HCC cell growth and is predictive of a poor outcome in HCC [47]. Additionally, the CBX family member can be involved in HCC progression as a target gene for miRNAs. For example, miR-195 overexpression restrains proliferative and invasive capacities of the HCC cells by inhibiting CBX4 [48]. In the present study, CBX6 was demonstrated to be a target of miR-146b-5p, which was inversely modulated through miR-146b-5p. We assumed that miR-146b-5p may suppress HCC progression through inhibiting CBX6. Furthermore, we discovered that CBX6 expression was positively related to MIR100HG in HCC. Considering the MIR100HG/miR-146b-5p axis, we speculated that MIR100HG may influence CBX6 via modulating miR-146b-5p. Importantly, rescue experiment exhibited that CBX6 overexpression reversed the suppressed HCC cell behaviors caused by MIR100HG silencing. To sum up, MIR100HG silencing may attenuate the tumorigenesis of HCC cells by regulating the miR-146b-5p/CBX6 axis. Additionally, the expression of MIR100HG/-miR-146b-5p/CBX6 in HCC tissues was based on hepatectomy in this study. In many cases, however, biomarkers are not uniformly present in all cancer cells, and such heterogeneity might hinder the therapeutic efficacy of tailored therapies [49]. In recent years, there has been an increasing development of liquid biopsy to replace tissue biopsy [50]. Bloodbased liquid biopsy consists in the isolation and analysis of tumor-derived or tumor-associated components that circulate in the bloodstream, including circulating tumor cells (CTCs), circulating leukocytes, and tumor-derived circulating nucleic acids, such as cell-free circulating tumor DNA (ctDNA), miRNA, and lncRNAs [51,52]. In fact, these components are deeply involved in the epithelial-to-mesenchymal transition (EMT) process of HCC, which greatly affect cell invasion and migration [53][54][55]. We speculated that if so, the measurement of MIR100HG/miR-146b-5p/CBX6 expression by liquid biopsy may be a more efficient and rapid method to determine whether the metastasis occurs in HCC patients. This may be also a limitation of this study, and we will consider this in future studies. Even so, our findings for the first time uncover new insight for the molecular mechanism of HCC and provide a potential target for HCC treatment.

Data Availability
All data are available through the responsible corresponding author.

Ethical Approval
This study was conducted after obtaining local ethical committee approval of Wei Fang Yi Du Central Hospital (no. 2017-002). We confirm that all experiments were performed following relevant named institutional and national guidelines and regulations.

Conflicts of Interest
The authors declare that they have no conflict of interest.