Upregulation of lncRNA DQ679794 contributes to preventing the progression of hepatocellular carcinoma both in vitro and in vivo

Background Hepatocellular carcinoma (HCC) is an aggressive tumor with a poor prognosis. Long noncoding RNAs (lncRNAs) are key regulators of tumor development. However, lncRNA profiles in HCC remain largely unknown. In previous studies, we found that lncRNA DQ786243 plays an important role in the pathogenesis of HCC and promotes the development of HCC. In this study, we investigated the role of lncRNA DQ679794 in the pathogenesis of HCC. Methods and Results We first used quantitative real-time PCR among 64 paired HCC tissues, and the level of lncRNA DQ679794 was found to be significantly lower in tumors than in normal tissues. In addition, the effects of lncRNA DQ679794 were assessed by overexpression in vitro and in vivo . We found that the level of apoptosis was increased and that cell proliferation was weakened in HepG2 cells overexpressing DQ679794. Finally, the transplanted tumor experiment confirmed that after the overexpression of lncRNA DQ679794, the growth of transplanted tumors formed by liver cancer cells was inhibited. Conclusion This study suggests that lncRNA DQ679794 is an oncogene that inhibits tumor progression, and we believe that lncRNAs may be a key regulatory center in HCC progression.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common and malignant tumors in the world and causes approximately 600,000 deaths each year [1]. HCC is the sixth most common cancer worldwide and the third leading cause of cancer-related death [2]. Chronic hepatitis B/C virus infections are significant development factors of HCC [3]. Frequent intrahepatic and extrahepatic metastases lead to low respectability, poor prognosis and high recurrence rate in the early stage [4]. Therefore, it is important to find effective methods for the early diagnosis and treatment of HCC.
Long noncoding RNAs (lncRNAs), a novel class of noncoding RNAs, are commonly defined as RNA molecules longer than 200 nucleotides in length [5]. Unlike smaller noncoding miRNAs, the functions of most lncRNAs are not fully understood. However, with the continuous improvement of transcriptome spectroscopy techniques, emerging research has indicated that lncRNAs play a vital role in human tumorigenesis and progression by serving as tumor oncogenes or suppressors [6,7].
Abnormal expression of lncRNAs has been found in a variety of tumors, including HCC, and is associated with the proliferation, growth, apoptosis, invasion and metastasis of tumor cells [8,9]. lncRNAs that have been widely reported in HCC include MALAT1, H19, HULC and MEG3. For example, the levels of MALAT1 and H19 in normal liver cells were very low, and their expression significantly increased when liver cancer occurred [10]. The expression of H19 in most primary liver cancers is higher than alpha-fetoprotein, and its combined detection with alpha-fetoprotein can improve the diagnosis rate of early liver cancer [11]. HULC can be used as a marker for liver metastasis of colorectal tumors [12]. CUDR can inhibit cysteine aspartic proteinase 3-induced drug resistance [13].
Overexpression of MEG3 could inhibit the growth of HCC cells by inducing the apoptosis of HCC cells [14,15].
However, most of the lncRNA profiles in HCC remain largely unknown. In previous studies, we found that lncRNA DQ786243 plays an important role in the pathogenesis of HCC and promotes the development of HCC [16]. In the present study, we focused on lncRNA DQ679794 to identify the key lncRNA associated with HCC and to study its biological functions in HCC progression. Overexpression of DQ679794 inhibited the proliferation of HCC cells, suggesting its potential utility as a prognostic marker or a therapeutic target.

Patients and tissue specimens
The present study was approved by the Ethics Committee of Peking University Shenzhen Hospital

Data analysis
Data were analyzed using GraphPad Prism software. Statistical analysis was performed using unpaired t-test measures ANOVA followed by the Bonferroni post hoc test, which were specifically stated in the Results. All results are shown as the mean ± S.E.M. P < 0.05 was considered statistically significant.
Result lncRNA DQ679794 expression in patients with HCC and cell lines 6 The expression levels of lncRNA DQ679794 in patients with HCC and paired nontumor tissues were determined using qRT-PCR. The results showed that lncRNA DQ679794 expression was significantly lower in HCC tumors than in corresponding nontumors (Fig. 1A, **p < 0.01). Moreover, we detected several different HCC cell lines, and the results showed that the level of lncRNA DQ679794 in the HepG2 group and Hep3B group was significantly lower than that in the L02 group (Fig. 1B, **p < 0.01). These data suggested that abnormal lncRNA DQ679794 expression may be related to HCC progression.
Effect of lncRNA DQ679794 on HCC cell proliferation and apoptosis in vitro In addition, we determined whether apoptosis contributes to cell growth inhibition by lncRNA DQ679794 overexpression. We detected the effect of lncRNA DQ679794 on apoptosis by flow cytometry, and the results showed that the apoptosis level of the DQ-OE group increased significantly compared to that of the DQ-veh group (Fig. 2C, 2D, *p < 0.05). These results indicate that the overexpression of DQ679794 may promote apoptosis in HCC cells.
Moreover, we examined the expression of certain proteins involved in apoptosis regulation by western blot. A significant elevation in proapoptotic protein (Caspase-3 and Caspase-9) levels was observed in the DQ-OE group, and the level of Bax was unchanged compared to the DQ-veh group (Fig. 2E, 2F, *p < 0.05, **p < 0.01). The expression of the antiapoptotic protein (Bcl-2) was significantly decreased in the DQ-OE group compared to that in the DQ-veh group (Fig. 2E, 2F, **p < 0.01). These results suggest that the dysregulation of certain cell apoptosis-related proteins explains the lncRNA DQ679794-dependent effects on HCC cell proliferation. lncRNA DQ679794 is involved in cell cycle progression 7 Since the induction of cell cycle arrest is an important antiproliferation mechanism, we investigated whether the growth inhibitory activity of lncRNA DQ679794 overexpression was involved in the control of cell cycle progression. To detect the expression of intracellular proteins that regulate cell cycle progression in the G2/M-phase, we measured the protein levels of Cyclin B1, CDC2 and p-CDC2 (Tyr-15). Our results showed that the level of Cyclin B1 in the DQ-OE group was decreased dramatically, and the level of CDC2 was also significantly decreased compared to that in the DQ-veh group (Fig. 3A, 3B, **p < 0.01). The levels of the inhibitory p-CDC2 (phosphorylated at Tyr-15) in the DQ-OE group were increased dramatically compared to those in the DQ-veh group (Fig. 3A, 3B, *p < 0.05). These results suggest that the dysregulation of certain cell cycle-related proteins explains the lncRNA DQ679794-dependent effects on HCC cell proliferation.

Overexpression of lncRNA DQ679794 suppressed the tumorigenicity of HCC cells in vivo
To further investigate the role of lncRNA DQ679794 in the progression of HCC in vivo and to determine the therapeutic potential of targeting lncRNA DQ679794 in HCC, we established xenograft tumor models using Hep3B and HepG2 cells, which were stably transfected with targeting lncRNA DQ679794 or empty vector and injected subcutaneously into the right dorsal tissues of nude mice.
After 30 days, the tumors were harvested as shown in Fig. 4A. We measured the size and weight of the tumor tissue, and the results showed that the tumor size in the overexpressed lncRNA DQ679794 (HepG2-DQ) group was significantly lower than that of the HepG2-veh group (Fig. 4B, **p < 0.01). In addition, the weight of tumors in the HepG2-DQ group was also significantly lower than that in the HepG2-veh group (Fig. 4C, **p < 0.01). These results suggest that upregulating lncRNA DQ679794 inhibits tumor growth in vivo.

Discussion
HCC is a leading cause of cancer-related death worldwide, and the recurrence and metastasis of tumors are the main factors leading to poor prognosis of patients with HCC [17]. As a new class of ncRNAs, thousands of lncRNAs have been found to be important regulators of human gene expression. Although most lncRNAs have been functionally described, the vast majority of these RNAs have not been fully described [18]. In addition, recent studies have greatly improved our 8 understanding of the important role of lncRNAs in HCC. In this study, we found differences in lncRNA DQ679794 between HCC tissues and adjacent normal tissues. Our functional test results showed that DQ679794 could effectively inhibit cell proliferation both in vivo and in vitro, suggesting that it plays an important role in inhibiting HCC proliferation.
In the past decade, there have been many reports on lncRNAs and HCC. For example, Wang et al [19]. demonstrated that the expression levels of lncRNA HOXA-AS2 were significantly upregulated in HCC tissues and 4 cell lines. In addition, knocking down HOXA-AS2 significantly inhibited proliferation, colony formation, migration and invasion of HCC cells and obviously induced cancer cell apoptosis [20]. Xiao et al [21]. identified that lncRNA Ftx was upregulated in human HCC tissues and cell lines, especially in relation to invasive clinicopathological features. Overexpression of lncRNA Ftx promoted the proliferation, invasion and migration of HCC cells, while knockdown of lncRNA Ftx had the opposite effect [22,23]. In our previous studies, we also found that lncRNA DQ786243 was elevated in both HCC tissue samples and serum, which was associated with a low survival rate and poor clinicopathological characteristics [16]. In addition, multivariate analysis showed that lncRNA DQ786243 expression was an independent prognostic factor for HCC. Knockdown of DQ786243 induces inhibition of cell proliferation, migration, and invasion in vivo and in vitro [21].
In contrast to our study, the above lncRNA levels were highly expressed in HCC tissues, while the DQ679794 we studied in this paper showed a low expression level in HCC tissues. This suggests that the deletion of DQ679794 may be an important factor affecting the accelerated HCC process. There are also many lncRNAs with low expression in HCC tissues, such as uc.134 and RGMB-AS1 [24]. Wen et al [25]. indicated that the expression of a novel lncRNA uc.134 was repressed in HCC specimens and that the expression of uc.134 was significantly correlated with the overall survival of patients.
Moreover, the overexpression of uc.134 suppressed HCC cell proliferation, invasion, and metastasis in vitro and in vivo [26]. Nan et al [27]. demonstrated that lncRNA RGMB-AS1 expression was decreased in hepatocellular carcinoma tissues and cell lines, and low lncRNA RGMB-AS1 expression correlated with malignant status and poor prognosis of patients with hepatocellular carcinoma. Overexpression of lncRNA RGMB-AS1 obviously suppressed hepatocellular carcinoma cell proliferation, migration, and invasion and promoted cell apoptosis [28]. This suggests that lncRNA DQ679794 is a general target for antitumor therapy. Moreover, to investigate the possible mechanism responsible for the proliferation enhancement effect of DQ679794, we detected proteins associated with the cell cycle and found that overexpression of DQ794697 can block the cell cycle and promote apoptosis in G2/M phase, suggesting that DQ786243mediated HCC cell proliferation may be associated with regulation of the cell cycle and apoptosis. To further elucidate the regulatory mechanism of DQ794697 in the cell cycle and apoptosis, we analyzed the proteins involved in the cell cycle and apoptosis by western blot. Our results showed that overexpression of DQ794697 significantly reduced the expression of Cyclin B1 and increased the phosphorylation of CDC2. It is widely accepted that the Cyclin B1-CDC2 complex is necessary for the transition from G2 phase to M phase [29]. We also observed that DQ794697 gene overexpression in HepG2 cells decreased the expression of the antiapoptotic protein Bcl-2 and increased the expression of the proapoptotic proteins Caspase-9 and Caspase-3. These results may extend our current understanding of the downstream genes of DQ794697 to include these cell cycle-and apoptosisrelated proteins.
Although lncRNAs have been reported to be involved in the progression and development of tumors, the underlying molecular mechanisms are unclear [30]. In this study, we found that the expression of lncRNA DQ794697 was correlated with HCC tumor and cell lines. Of course, as the number of HCC cell lines available is limited, these cultured cell lines cannot represent all subtypes of colorectal tumors, and more systematic studies using clinical HCC samples are required, however, it provides a possible overexpression of lncRNA DQ794697 that may be associated with colorectal neoplasia.

Conclusion
In conclusion, this study suggests that lncRNA DQ679794 is an oncogene that inhibits tumor progression, and we believe that lncRNAs may be a key regulatory center in HCC progression.     Overexpression of lncRNA DQ679794 suppressed the tumorigenicity of HCC cells in vivo. A.
Nude mice were injected with Hep3B and HepG2 cells transfected with DQ679794 (HepG2-DQ group) or vector (HepG2-veh group) via peritoneal injection. After 30 days, the mice were sacrificed, and the tumors were harvested for analysis. B. The tumor volume was measured. C. The tumor weight was measured. Values are expressed as the mean ± SEM (error bars). n=8-11, *p < 0.05, **p < 0.01 versus the Hep3B group or HepG2-veh group.