Inhibition of long noncoding RNA cancer susceptibility candidate 7 attenuates hepatocellular carcinoma development by targeting microRNA-30a-5p

ABSTRACT Long non-coding RNA (lncRNA) cancer susceptibility candidate 7 (CASC7) was reported to be participated in tumor development. This study was carried out to investigate the functions of CASC7 in hepatocellular carcinoma (HCC) progression. The expression of CASC7 and microRNA-30a-5p (miR-30a-5p) in HCC tissues and cells were detected by quantitative Real-time PCR (qRT-PCR). The expression of Krueppel-like factor 10 (KLF10), transforming growth factor-β (TGF-β), and SMAD3 were detected by Western Blot analysis. Transwell assay, flow cytometry, Cell Counting Kit-8 (CCK-8) assay and colony formation assay were performed to evaluate the effects of CASC7, KLF10 and miR-30a-5p on cell function. The relationship among CASC7, KLF10 and miR-30a-5p was evaluated by luciferase reporter assay and bioinformatics analyses. Tumor growth was detected in nude mice. The expression levels of CASC7 were increased and the expression levels of miR-30a-5p were reduced in HCC cells and tissues. Knockdown of CASC7 and overexpression of miR-30a-5p reduced tumor growth as well as HCC cell proliferation, invasion and migration. In HCC tumor tissues, the expression of miR-30a-5p was negatively correlated with the expression of CASC7. Moreover, as a target of miR-30a-5p, KLF10 was regulated by CASC7 and miR-30a-5p, and CASC7 regulated the KLF10/TGF-β/SMAD3 pathway via binding to miR-30a-5p, thereby promoting HCC cell progression.


Introduction
HCC has become the sixth most common malignant tumor in the world [1,2], and most of them are diagnosed in late stages with multiple complications and the surgical resection rate is extremely low [3]. At present, the treatment for liver cancer is mainly surgical resection, combined with radiotherapy, chemotherapy and liver transplantation [4]. Most patients often suffer from metastasis and recurrence after surgery [5]. Recent studies have shown that HCC is closely related to abnormal expression of long non-coding RNAs (lncRNAs) and cell signal transductions [6].
LncRNAs are expressed in different cells and tissues and can interact with other macromolecules such as DNAs, miRNAs and proteins [7,8].
With the advance in high-throughput sequencing technologies, various lncRNAs have been identified [9]. Moreover, lncRNAs might serve as the biomarkers for tumor progression and recurrence in HCC patients [10,11]. For example, it has been reported that lncRNA MIAT promotes HCC cell invasion and proliferation via sponging miR-214 [12]. LncRNA cancer susceptibility candidate 7 (CASC7) was revealed to be involved in glioma [13], non-small cell lung cancer [14], and colon cancer [15]. However, the functions of CASC7 in HCC remain unclear.
MicroRNAs (miRNAs) widely exist in many organisms and are involved in the regulation of gene expression [16]. Studies have found that lncRNAs can regulate the expression of miRNAs and tumorigenesis [17]. Moreover, miRNAs are also involved in liver cancer [18] and function as oncogenes or tumor suppressive genes [19]. Meanwhile, miRNAs are related to the clinicopathological characteristics of liver cancer such as pathological type and the degree of malignancy [20]. As a newly discovered miRNA, miR-30a-5p has been reported to be involved in invasion and migration of a variety of cancers [21]. Moreover, studies have found that miR-30a-5p is significantly downregulated and suppresses HCC tumor growth and cell invasion and proliferation in HCC tissues [22]. We therefore speculated that miR-30a-5p may mediate the function of CASC7 in HCC. The TGF-β/SMAD3 signaling participates in HCC migration and metastasis [15,23]. In addition, it has been reported that miRNA-491 is involved in anti-angiogenesis in HCC induced by arsenic trioxide by inhibiting the TGF-β/SMAD3 signaling [14].
Furthermore, the TGF-β/SMAD3 pathway mediated the anti-oncogene of miR-133a/FOSL2 in HCC. These studies suggest that the TGF-β/ SMAD3 signaling can be regulated by miRNAs in HCC. We therefore hypothesized that CASC7 might regulate the KLF10/TGF-β/SMAD3 pathway through miR-30a-5p in HCC development. This study was therefore carried out to investigate the role and underlying mechanism of CASC7 in HCC.

Clinical specimens
The tumor tissues and adjacent non-tumor tissues were obtained from 38  between clinicopathologic characteristics and the expression of CASC7 was analyzed in HCC (Table 1).

RNA pull-down assay
Cells were transfected with biotin-labeled mutant (Mut)-and the wild-type (WT)-bio-miR-30a-5p (50 nM) for 48 h. After washing with PBS, a specific lysis buffer was added to incubate the cells for 10 min. After pre-coating with BSA, M-280 streptavidin magnetic beads were used to incubate the lysate at 4°C for 3 h. Finally, The binding RNA was purified and its concentration was measured [25].

Apoptosis assay
Cells (5 x 10 5 cells/well) were plated and grown to the logarithmic growth phase. Cells were then collected, counted and separated by centrifugation and regenerated by adding 195 μl annexin V-FITC. Propidium iodide (10 μl) and Annexin V-FITC (5 μl) staining solutions were then added. Cells were incubated in darkness for 10-20 min and then placed in an ice bath [26]. Flow cytometry results were then collected (Jiyuan, Guangzhou, China).

Luciferase reporter assay
Luciferase reporter assay was carried out as previously described [27]. The WT and MUT primers were used to amplify CASC7. pGL3-Bashc luciferase reporter vector carrying the CASC7 fragment was named as CASC7-WT-Luc. The mutant plasmid CASC7-Mut-Luc was constructed by mutagenizing the binding region in CASC7 and miR-30a-5p. pRLTK and miR-30a-5p mimic were co-transfected into cells with CASC7-Mut-Luc and CASC7-WT-Luc. Cells were collected after 6 h of transfection. Luciferase activity was determined using a Luciferase Assay Kit.

Colony formation assay
Approximately 1,000 cells were plated and cultured in DMEM medium for 7 d. Then, cells were stained with Wright's stain, followed by staining with Sorensen phosphomolybdate buffer solution and Giemsa dye solution (9:1) for 10 min. Colonies were then counted under a microscope [28].

Transwell assay
HCC cells were transfected with shCASC7, pcCASC7, miR-30a-5p inhibitor and mimic or NC. Briefly, 3 × 10 5 HCC cells were treated with 2.5 μg/mL mitomycin C to inhibit cell division for 24 h. After culturing, appropriate amount of trypsin digestion was added, cells were washed with PBS buffer and resuspend in serum-free medium. Then, cells were cultured in the basement with complete medium for 24 h. The superfluous medium was discarded. Cells were then fixed with polyformaldehyde (4%) for 30 min, followed by staining with crystal violet (0.1%) for 10 min. Ten visual fields were randomly selected, cells were observed, photographed, and counted [29].

IHC staining
Liver cancer tissues were deparaffinized and heated after hydration. The sections were blocked with normal goat serum (10%) and then incubated with anti-Ki-67. The Envision™ ABC kit (Beinuo, Shanghai, China) was used for immunological detection. Finally, the Leica DM4000B/M microscope was used for observation [31].

Tumor xenograft models in nude mice
Male athymic nude mice were kept under standard conditions. Approximately 5 × 10 6 HCC cells transfected with CASC7 lentiviral vector or miR-30a-5p inhibitor were separately subcutaneously inoculated into nude mice. All mice were divided to different groups. The tumor size was determined every week for 4 weeks. Mice were sacrificed and tumors were weighted.

Statistical methods
Data were presented as the mean ± stand deviation (SD). All statistical analyses were performed using the SPSS 13.0 software. Student's t-test was used for comparisons. p value < 0.05 was considered as significant difference.

Results
In this study, we aimed to investigate whether and how CASC7 was involved in HCC progression. We found that CASC7 regulated the KLF10/TGFβ/SMAD3 axis via binding to miR-30a-5p, thereby promoting HCC cell progression.

Down-regulation of CASC7 restrained HCC progression in vivo
It was found that inhibition of miR-30a-5p enhanced the tumor volume and weight, and knockdown of CASC7-2 reduced the tumor volume and weight (Figure 6a-6c, p < 0.05), which was attenuated by inhibition of miR-30a-5p. Immunohistochemical results showed that Ki67 positive cell number was increased in miR-30a-5p inhibitor group and decreased in shCASC7-2 group, which was attenuated by miR-30a-5p inhibitor (Figure 6d). ShCASC7-2 reduced the expression levels of KLF10, TGF-β and p-SMAD3 (P < 0.05), which was attenuated by miR-30a-5p inhibitor (P < 0.05, Figure 6e). Compared with the Control, inhibitor-NC and sh-NC group, shCASC7-2 significantly reduced the expression levels of KLF10, p-SMAD3 and TGFβ, which was attenuated by miR-30a-5p inhibition (supplementary Figure 1, P < 0.05). Altogether, these results suggested that knockdown of CASC7 inhibited HCC progression in vivo.

Discussion
Even with encouraging progress that has been made in the understanding of the molecular mechanisms of liver cancer development, in advanced liver cancer patients, the prognosis remains unfavorable [32]. It is known that altered expression of lncRNAs are closely related to the occurrence and development of liver cancer [33,34]. Here, we showed that the expression levels of CASC7 were increased in HCC tissues and cells. Importantly, CASC7 promoted HCC tumor malignant processes and tumor growth in vivo, suggesting that CASC7 plays an oncogenic role in HCC.
LncRNAs were shown to be involved in pathological processes of human disease [35]. In tumor development process, lncRNAs exert their functions as pro-tumor factors or suppressors in tumors through various mechanisms, such as regulating the expression of neighboring genes or affecting the expression of related genes [36]. Studies have identified many abnormally expressed lncRNAs in HCC, which are involved in HCC prognosis, metastasis and occurrence [37]. For example, the highly upregulated expression of ANRIL is closely related to HCC poor prognosis [38] and induces HCC cell invasion and proliferation [39]. In a variety of malignancies, CASC7 was found to be abnormally expressed. For example, the expression levels of CASC7 in colorectal cancer are significantly decreased, while overexpression of CASC7 can inhibit the survival of colorectal cancer cells [40]. In glioma tissues and cell lines, CASC7 is down-regulated and involved in glioma cell proliferation and apoptosis [13]. Moreover, it was reported that CASC7 suppresses malignant behaviors of breast cancer by regulating the miR-21-5p/ FASLG axis [27]. Here, we demonstrated that the expression levels of CASC7 were significantly increased in HCC. Moreover, knockdown of CASC7 inhibited HCC cell invasion, migration, and proliferation and induced HCC cell apoptosis. These suggested that CASC7 acts as an oncogene in the occurrence of HCC. Our findings further enriched our understanding of CASC7 in different types of tumor.
Studies have shown that lncRNAs have sponge adsorption on miRNAs, and thus participate in tumorigenesis [41]. For example, lncRNA DLEU2 sponges miR-30a-5p to accelerate non-small cell lung tumorigenesis and invasion [42]. LncRNA MALAT1 sponges miR-30a-5p to regulate vimentin expression in HCC [43]. Here, miR-30a-5p was found to be a target gene of CASC7. The expression levels of miR-30a-5p were reduced in HCC and reversely correlated with the expression levels of CASC7. In addition, overexpression of miR-30a-5p reduced HCC cell migration, invasion and proliferation and induced apoptosis of HCC cells, and it could reverse the effects of shCASC7 on HCC tumor growth and cell apoptosis, invasion, migration, proliferation. These suggest that miR-29a-3p mediated the function of CASC7 in HCC.
KLF10 is a transcription factor containing a zinc finger protein domain, and TGF-β is an early  responsive gene [44]. It has been reported that the TGF-β/SMAD3 axis is closely related to HCC occurrence [45]. Here, KLF10 was a target gene of miR-30a-5p. The expression levels of KLF10, p-SMAD3 and TGF-β were reduced after overexpression of miR-30a-5p. ShCASC71 could reduce the expression levels of KLF10, p-SMAD3 and TGF-β, which was reversed by miR-30a-5p inhibitor. These indicate that CASC7 regulates KLF10/TGF-β/ SMAD3 through miR-30a-5p to promote HCC proliferation. In future, we will further investigate the roles of SMAD2 and SMAD4 in HCC proliferation.

Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.