Long non-coding RNA colon cancer–associated transcript 1 functions as a competing endogenous RNA to regulate cyclin-dependent kinase 1 expression by sponging miR-490-3p in hepatocellular carcinoma progression

Abstract

Hepatocellular carcinoma is an aggressive neoplasm and is one of the most common human cancers. Recently, long non-coding RNAs have been demonstrated to participate in pathogenesis of many diseases including the progression in several cancers. In this study, we found that the long non-coding RNA colon cancer–associated transcript 1 was upregulated in hepatocellular carcinoma tissues (p < 0.05), and high colon cancer–associated transcript 1 expression level was positively associated with tumor volume (p < 0.05) and American Joint Committee on Cancer stage (p < 0.05) in hepatocellular carcinoma patients. Luciferase reporter assays and RNA-pulldown assays showed that colon cancer–associated transcript 1 is a target of miR-490-3p. Real-time quantitative polymerase chain reaction and Western blot analysis indicated that colon cancer–associated transcript 1 regulated cyclin-dependent kinase 1 expression as a competing endogenous RNA by sponging miR-490-3p in hepatocellular carcinoma cells. Furthermore, colon cancer–associated transcript 1 silencing decreased hepatocellular carcinoma cells proliferation and invasion and overexpression promoted cell proliferation and invasion in vitro. These data demonstrated that the colon cancer–associated transcript 1/miR-490-3p/cyclin-dependent kinase 1 regulatory pathway promotes the progression of hepatocellular carcinoma. Inhibition of colon cancer–associated transcript 1 expression may be a novel therapeutic strategy for hepatocellular carcinoma.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common human cancers in the world with high mortality and ranks as the fifth most common malignancy.¹ Although surgical treatment measures have been improved in recent years including the most effective treatment approaches for HCC, such as surgical resection or transplantation, the 5-year survival rate of HCC patients is still only approximately 5%.² Therefore, it is important to explore the molecular and biological functions underlying HCC progression, which could help to identify novel cancer biomarker and therapeutic targets.

Long non-coding RNAs (lncRNAs) compose a group of non-protein-coding RNAs that are more than 200 nucleotides in length. Recent studies have shown that lncRNAs play important roles in regulating gene expression and can reprogram the transcriptome to modulate cellular processes involved in cellular growth and differentiation and thereby contribute to tumorigenesis.^3,4

Colon cancer–associated transcript 1 (CCAT1), a ~2-kb lncRNA located at chromosome 8q24.21, is first found to be upregulated in colon cancer.⁵ Recently, some reports have found that CCAT1 is involved in tumor progression and tumorigenesis in various types of cancers including colon cancer,⁶ gastric cancer,⁷ and colorectal cancer.⁸ The competitive endogenous RNA (ceRNA) mechanism for CCAT1 and microRNAs involving in tumor progression has been reported in some studies, for example, Zhou et al.⁹ reported the lncRNA CCAT1/miR-490 axis regulated gastric cancer cell migration by targeting hnRNPA1. Ma et al.¹⁰ reported that lncRNA CCAT1 promoted gallbladder cancer development via negative modulation of miRNA-218-5p. In HCC, CCAT1 promoted HCC progression by functioning as let-7 sponge.¹¹ But the role of CCAT1 functioned as a competing endogenous RNA (ceRNA) in HCC progression still needs to be further explored.

In this study, we found that CCAT1 expression was upregulated in HCC tissues and knockdown of CCAT1 inhibited HCC cells proliferation and invasion. The expression of CCAT1 was negatively correlated with miR-490-3p and functioned as a ceRNA to regulate cyclin-dependent kinase 1 (CDK1) expression by sponging miR-490-3p. The CCAT1/miR-490-3p/CDK1 regulatory axis may be a novel therapeutic target for HCC.

Materials and methods

Patients and samples

The 40 cases of HCC tissues and their pair-matched adjacent normal hepatic tissues utilized in this study were obtained from patients with HCC who had undergone radical resection between January 2014 and December 2015 at the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Chinese PLA General Hospital. All cases were confirmed as HCC. The patients were staged according to the tumor node metastasis (TNM) staging system (the 7th edition) of the American Joint Committee on Cancer (AJCC). Patients recruited in this study received no other treatments prior to surgery. Specimens were obtained immediately after surgical resection and stored at −80°C for further analysis. The study was approved by the Human Ethics Committee of The First Affiliated Hospital of Chinese PLA General Hospital. Written consent for publication had been obtained from all patients. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki.

Cell culture

The liver normal cell lines LO2 and four human HCC cell lines MHCC97H, MHCC97L, Hep3B, and SMCC-7721 were obtained from the Chinese Academy of Sciences Cell Bank. Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY, USA) including 10% fetal bovine serum (FBS, HyClone, Invitrogen, Camarillo, CA, USA), 100 µg/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were incubated at 37°C with 5% CO₂.

RNA preparation, reverse transcription, and quantitative polymerase chain reaction

Total RNA was prepared from HCC tissues and cells using TRIzol (TaKaRa, Dalian, China) according to the manufacturer’s protocol. All samples were synthesized from 1 µg of total RNA by Prime Script RT Master Mix kit (TaKaRa). The reactions were incubated at 95°C for 60 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. Real-time polymerase chain reaction (PCR) was performed using a SYBR Green PCR kit (TaKaRa), and real-time reverse transcription polymerase chain reaction (RT-PCR) reactions were performed on an ABI 7500 system (Applied Biosystems, Carlsbad, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal controls for lncRNAs and U6 was used as internal controls for microRNAs, respectively. The primer sequences used were as follows: GAPDH (forward): 5′-GTCAACGGATTTGGTCTGTATT-3′, GAPDH (reverse): 5′-AGTCTTCTGGGTGGCAGTGAT-3′; CCAT1 (forward): 5′-CATTGGGAAAGGTGCCGAGA-3′, CCAT1 (reverse): 5′-ACGCTTAGCCATACAGAGCC-3′; CDK1 (forward): 5′-CGG ACA TTA CTG GCC TGT TC-3′, CDK1 (reverse): 5′-TAG GGC AGC TTG AAG GAA ACC-3′; MiR-490-3p (forward): AACATGCCATGGGGGCCGGAGCGGAGT. The relative expression fold change of messenger RNAs (mRNAs) was calculated by the 2^−ΔΔCt method. All experiments were performed in triplicate.

Cell proliferation assays

The Cell Counting Kit-8 (CCK-8) proliferation assay was performed with MHCC97H and MHCC97L cells (Dojindo, Tokyo, Japan) according to the manufacturer’s protocol. Furthermore, si-NC, si-CCAT1, miR-490-3p inhibitor, and si-CCAT1+miR-490-3p inhibitor were transfected into MHCC97H or MHCC97L cells, respectively. A total of approximately 3 × 10³ cells were plated in 96-well plates. CCK-8 assay solution (10 µL) was added to each well after incubation for 24, 48, 72, and 96 h. Then, after incubation for another 2 h, optical density (OD) at 450 nm was measured with an enzyme immunoassay analyzer (Thermo Fisher Scientific, Inc., USA) to estimate cell proliferation among different groups.

Western blot analysis

Whole cells were washed in phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Sigma, Shanghai, China) followed by incubation on ice for 20 min. The supernatant (45 µg of protein) was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Beijing, China). The following primary antibodies were used according to the manufacturer’s instructions and antibodies against CDK1 (1:1000, CST, USA) and GAPDH (1:1000, CST). Blots were incubated with anti-rabbit secondary antibody (1:2000, CST) and visualized using enhanced chemiluminescence (ECL, Thermo Scientific, Beijing, China). All experiments were performed in triplicate.

RNAi and transfection

Two CCAT1-siRNAs were purchased from Genepharm (Shanghai, China). A negative control siRNA was also provided by Genepharm. The siRNA sequences are as follows: si-CCAT1-1, 5′-CGGCAGGCATTAGAGATGAACAGCA-3′; si-CCAT1-2, 5′-CCATTCCATTCATTTCTCTTTCCTA-3′; negative control, sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. The concentrations of relative siRNAs, mimics and inhibitors were 20 µM, and the working concentration was 20 nM. Short interfering RNA plasmids were transfected into cells in 6-well plates using Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The miR-490-3p mimic and inhibitor were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) The CCAT1 sequence was synthesized and subcloned into the pCDNA3.1 vector (Invitrogen, Beijing, China). Ectopic expression of CCAT1 was achieved through pcDNA-CCAT1 transfection, with an empty pcDNA vector used as a control.

Luciferase reporter assay

The MHCC97H cells (2.0 × 10⁴) grown in a 96-well plate were co-transfected with 150 ng of empty pmir-GLO-NC, pmir-GLO-CCAT1-Wt, or pmir-GLO-CCAT1-Mut (Sangon Biotech, China) and 2 ng of pRL-TK (Promega, Madison, WI, USA) with a miR-490-3p mimic or miR-NC into cells using Lipofectamine 2000 (Invitrogen, USA). The relative luciferase activity was normalized to Renilla luciferase activity 48 h after transfection. The transfection was independently repeated three times.

Biotin-labeled miRNA-pulldown assays

Biotinylated-NC, biotinylated miR-490-3p, and biotinylated miR-490-3p-mut were transfected into MHCC97H cells. RNA-pulldown assays were performed as described previously and had a small revision.¹² MHCC97H cells were collected after transfection at 48 h. The cells lysates were incubated with M-280 streptavidin magnetic beads (Invitrogen, San Diego, CA, USA). To prevent non-specific binding of RNA and protein complexes, the beads were coated with RNase-free bovine serum albumin (BSA) and yeast transfer RNA (tRNA; both from Sigma). The beads were incubated at 4°C for 3 h and washed three times with ice-cold lysis buffer and once with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0 and 500 mM NaCl). The bound RNAs were purified using TRIzol for the analysis.

Statistical analysis

All data are reported as the mean ± standard deviation (SD). Pearson’s correlation coefficient (r) was used to measure linear correlations between two variables. The differences between groups were analyzed using Student’s t-test. The difference was statistically significant at p < 0.05.

Results

CCAT1 is upregulated in HCC tissues and cells

To define the role of CCAT1 in human HCC, we analyzed 40 pairs of human HCC tissues and matched adjacent normal tissues using quantitative real-time PCR (qRT-PCR) analysis. The results found that CCAT1 expression levels were significantly upregulated in HCC tissue samples compared to adjacent normal tissues (Figure 1(a), p < 0.05). The CCAT1 expression rate was 27/40 (67.5%). Moreover, the high-CCAT1 group (n = 25: CCAT1 expression ratio ⩾ median ratio) and low-CCAT1 group (n = 15; CCAT1 expression ratio ⩽ median ratio) were classified according to median ratio of relative CCAT1 mRNA expression (2.95) in 40 cases of tumor tissues. Higher CCAT1 expression level in HCC was positively associated with tumor volume and AJCC stage (Figure 1(b) and (c), Table 1, p < 0.05). To explore the biological functions of CCAT1 in HCC, we compared CCAT1 expression in four HCC cell lines (MHCC97H and MHCC97L, SMCC-7721 and Hep3B cells) with a human normal hepatocellular cell LO2 (Figure 1(d)). We found that expression level of CCAT1 was significantly upregulated in the tumor cell lines. Furthermore, two different siRNAs oligos against CCAT1 were transfected into MHCC97H cells. Both of two siRNAs could efficiently knock down the endogenous CCAT1 (Figure 1(e)). The siRNA-CCAT1-2 was used in the later experiment for efficiently silencing CCAT1 and MHCC97L cell was used for overexpression of CCAT1 (Figure 1(f)).

Figure 1.

CCAT1 was upregulated in 40 cases of hepatocellular carcinoma tissues. (a) CCAT1 expression levels in HCC tissues and adjacent normal tissues were detected by qRT-PCR (n = 40). The relative expression fold change of mRNAs was calculated by the 2^−ΔΔCt method. The expression of CCAT1 was normalized to GAPDH. (b) CCAT1 expression was significantly higher in larger tumors (tumor size > 5 cm). (c) CCAT1 expression was significantly higher in patients with an advanced clinical stage than in those with an early clinical stage. (d) The CCAT1 expression levels in four HCC cell lines (MHCC97H, MHCC97L, Hep3B, and SMCC-7721) and the human normal hepatocellular LO2 cell were detected by qRT-PCR. The expression of CCAT1 was normalized to that in LO2. (e) MHCC97H was transfected with two different siRNAs against CCAT1. (f) MHCC97L was transfected with pcDNA3.1-CCAT1.

Error bars represent the mean ± SD of triplicate experiments. **p < 0.05.

[Figure omitted. See PDF]

Table 1.

The correction between expression of CCAT1 and clinical characteristics in 40 hepatocellular carcinoma patients.

Clinical characteristics	Case number	CCAT1 expression		p-value
		Low	High
Gender				0.285
Female	12	6	6
Male	28	9	19
Age				0.505
⩽60	24	8	16
>60	16	7	9
Tumor size				0.010**
<5 cm	14	9	5
>5 cm	26	6	20
Histological grade				0.870
Well and moderately	22	8	14
Poorly and others	18	7	11
Lymph node metastasis
Yes	23	7	16	0.283
No	17	8	9
AFP level (ng/mL)				0.412
⩽400	18	8	10
>400	22	7	15
AJCC stage				0.023**
I–II	15	9	6
III–IV	25	6	19

CCAT1: colon cancer–associated transcript 1; AFP: alpha-fetoprotein; AJCC: American Joint Committee on Cancer.

**

p < 0.05.

CCAT1 directly targeted miR-490-3p and inhibits its expression in HCC cells

A previous study had reported that CCAT1 may be an oncogenic lncRNA and promoted HCC cell proliferation and invasion.¹¹ Some studies have demonstrated that LncRNA could function as a ceRNA in modulating the expression and biological functions of miRNA.¹³ We performed a bioinformatics analysis using Starbase 2.0 (http://starbase.sysu.edu.cn) and then found that miR-490-3p contains a binding site for CCAT1 (Figure 2(a)). The expression levels of miR-490-3p in normal tissues and tumor specimens were analyzed by quantitative real-time PCR; we found that miR-490-3p was downregulated in the HCC tissue specimens (Figure 2(b)), but CCAT1 was upregulated in the HCC tissues (Figure 1(a)), these results indicated a significant negative correlation between CCAT1 and miR-490-3p (R = −0.389, p < 0.05, Figure 2(c)). We then performed knockdown experiments to explore whether knockdown of CCAT1 affects miR-490-3p in MHCC97H or SMCC-7721 cells. MiR-490-3p was significantly upregulated after silencing of CCAT1 (Figure 2(d) and (e), p < 0.05). MiR-490-3p was significantly downregulated after overexpression of CCAT1 in MHCC97L cells (Figure 2(f), p < 0.05). Furthermore, we demonstrated that the expression level of CCAT1 was decreased after transfection with a miR-490-3p mimic and increased after transfecting with a miR-490-3p inhibitor in MHCC97H cells (Figure 2(g), p < 0.05). Moreover, we also detected the miR-490-3p expression in four HCC cell lines (MHCC97H and MHCC97L, SMCC-7721 and Hep3B cells) with a human normal hepatocellular cell LO2, and the results demonstrated that miR-490-3p was downregulated in HCC cells (Figure 2(h), p < 0.05).

Figure 2.

Identification of miR-490-3p was a target of CCAT1. (a) Alignment of potential CCAT1 sequences with miR-490-3p as identified by starbase v2.0 (http://starbase.sysu.edu.cn/mirLncRNA.php). CCAT1 mutated at the putative binding site. (b) MiR-490-3p was downregulated in HCC tissues compared to adjacent normal tissues as determined by qRT-PCR assays. The expression of miR-490-3p was normalized to U6. (c) The correlation between CCAT1 and miR-490-3p expression level was measured in 40 HCC tissues (R = −0.389, p < 0.05). The ΔCt values were subjected to Pearson’s correlation analysis. (d) and (e) The expression of miR-490-3p was upregulated after CCAT1 silencing in MHCC97H or SMCC-7721 cells. (f) The expression of miR-490-3p was downregulated after overexpression of CCAT1 in MHCC97L cells. (g) The expression level of CCAT1 was upregulated by transfecting the miR-490-3p mimic and was downregulated by transfecting the miR-490-3p inhibitor into MHCC97H cells. (h) MiR-490-3p is downregulated in four HCC cells compared to the level in LO2 cell as determined by qRT-PCR assays. The expression of miR-490-3p was normalized to U6.

Error bars represent the mean ± SD of triplicate experiments. **p < 0.05.

[Figure omitted. See PDF]

To explore the underlying biological function between the CCAT1 and miR-490-3p, the dual-luciferase reporter assay was performed with MHCC97H cells. The results demonstrated that the luciferase activity was significantly decreased by the co-transfection of miR-490-3p mimic + pmiR-Glo-CCAT1-wt, but no significant difference in the relative luciferase activity between the co-transfection of miR-490-3p mimic + miR-NC and miR-490-3p mimic + pmiR-Glo-CCAT1-mut in MHCC97H cells (Figure 3(a)). Above results demonstrated that CCAT1 is a target of miR-490-3p.

Figure 3.

The underlying mechanism of the regulation of miR-490-3p and CCAT1. (a) Luciferase reporter activity in MHCC97H cells was detected after co-transfection with miR-490-3p mimic and luciferase reporters containing nothing, wild CCAT1 or mutant transcripts. Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (b) Amount of CCAT1 bound to miR-490-3p was detected by qRT-PCR after RNA-pulldown assays in MHCC97H cells. (c) CDK1 expression levels in HCC tissues and adjacent normal tissues were detected by qRT-PCR (n = 40). The expression of CCAT1 was normalized to GAPDH. (d) and (e) The mRNA or protein levels of CDK1 in MHCC97H cells transfected with si-NC, si-CCAT1, si-CCAT1+ miR-490-3p inhibitor, and miR-490-3p inhibitor. (f) and (g) The mRNA or protein levels of CDK1 in MHCC97L cells transfected with pcDNA3.1, pcDNA3.1-CCAT1, pcDNA3.1-CCAT1+ miR-490-3p mimic, and miR-490-3p mimic.

Error bars represent the mean ± SD of triplicate experiments. **p < 0.05, n.s., not statistically significant.

[Figure omitted. See PDF]

In addition, we performed RNA-pulldown assays to detect whether miR-490-3p could directly bind to CCAT1. MHCC97H cells were transfected with biotinylated miR-490-3p and then harvested for biotin-based pulldown assays. As shown by qRT-PCR assays, CCAT1 was pulled down by biotin-labeled miR-490-3p oligos, but not the mutated oligos (binding sites were mutated to the complementary sequences) that disrupted base pairing between CCAT1 and miR-490-3p (Figure 3(b)). Thus, our results indicated that miR-490-3p directly bound to CCAT1.

Knockdown of CCAT1 inhibits CDK1, a target of miR-490-3p

Overexpression of CDK1 promoted tumor proliferation and was reported to be a target of miR-490-3p in ovarian epithelial carcinoma and miR-490-3p suppressed tumorigenesis and progression via inhibition of CDK1.¹⁴ Our results detected that CDK1 mRNA expression was upregulated in HCC tissues compared with adjacent normal tissues (Figure 3(c)).The qRT-PCR and Western blot assays were used to measure the mRNA and protein level of CDK1, and we found that knockdown of CCAT1 in MHCC97H cells led to a significant decrease in endogenous CDK1 mRNA and protein expression. The miRNA-490-3p inhibitor reversed these effects, which indicated that CCAT1 partially modulated CDK1 by competing with miRNA-490-3p (Figure 3(d) and (e)). Overexpression of CCAT1 in MHCC97L cells led to a significant increase in endogenous CDK1 mRNA and protein expression, but the miRNA-490-3p mimic reversed the effect (Figure 3(f) and (g)). Together, the above results suggested that CCAT1 acts as a ceRNA by sponging to CDK1, modulating the CDK1 expression by binding miR-490-3p.

CCAT1 promotes HCC cell proliferation and invasion by competing with miRNA-490-3p in vitro

To detect the effect of CCAT1 on cell progression in HCC, a siRNA-CCAT1 oligo was stably transfected into MHCC97H and pcDNA3.1-CCAT1 was transfected into MHCC97L cells. The growth curves detected by CCK-8 assays showed that CCAT1 silencing significantly inhibited HCC cell proliferation. Whereas, this was reversed by co-transfection of si-CCAT1 and a miR-490-3p inhibitor in MHCC97H cells (Figure 4(a)). The overexpression of CCAT1 significantly promoted HCC cell proliferation, but was reversed by co-transfection of pcDNA3.1-CCAT1 and miR-490-3p mimic in MHCC97L cell (Figure 4(b)). Cell invasion assays demonstrated that the invasive cell number was significantly reduced in the CCAT1 silencing group compared with the si-NC group, but was reversed by co-transfection of si-CCAT1 and the miR-490-3p inhibitor in MHCC97H cells (Figure 4(c) and (d)). Overexpression of CCAT1 significantly promoted HCC cell invasion, but was reversed by co-transfection of pcDNA3.1-CCAT1 and miR-490-3p mimic in MHCC97L cells (Figure 4(e) and (f)).Thus, these results indicated that CCAT1 promoted HCC cell proliferation and invasion by competing with miRNA-490-3p in vitro.

Figure 4.

CCAT1 promoted hepatocellular carcinoma cell proliferation and invasion by competing with miRNA-490-3p in vitro. (a) Cell proliferation was evaluated in MHCC97H cells transfected with si-NC, si-CCAT1, si-CCAT1+ miR-490-3p inhibitor, and miR-490-3p inhibitor and (b) was evaluated in MHCC97L cells transfected with pcDNA3.1, pcDNA3.1-CCAT1, pcDNA3.1-CCAT1+ miR-490-3p mimic, and miR-490-3p mimic by CCK-8 assays after cell transfection for 24, 48, 72, and 96 h. Absorbance at 450 nm is shown. (c) and (d) The cell invasion ability and invasive number was evaluated in MHCC97H cells transfected with si-NC, si-CCAT1, si-CCAT1+ miR-490-3p inhibitor, and miR-490-3p inhibitor. (e) and (f) The cell invasion ability and invasive number was evaluated in MHCC97L cells transfected with pcDNA3.1, pcDNA3.1-CCAT1, pcDNA3.1-CCAT1+ miR-490-3p mimic, and miR-490-3p mimic.

Error bars represent the mean ± SD of triplicate experiments. **p < 0.05, n.s., not statistically significant.

[Figure omitted. See PDF]

Discussion

Recent studies have demonstrated that lncRNA CCAT1 expression was increased in many types of cancers and was involved in various cellular processes related to carcinogenesis. In the field of HCC research, aberrant expression of CCAT1 has recently been reported functionally to be involved in tumorigenesis and silencing of CCAT1 in human HCC cells suppressed cell proliferation and invasion.¹¹ Our present data indicated that the expression level of CCAT1 was significantly overexpression in HCC samples compared with adjacent normal tissues and silencing of CCAT1 inhibited HCC cell proliferation and invasion. These results demonstrated that CCAT1 may function as an oncogene, and its overexpression could contribute to HCC progression.

Recently, a growing number of reports have suggested that lncRNAs act as “sponges” to bind specific miRNAs and regulate their function. For example, Gao et al.¹⁵ demonstrated that LincRNA ROR functioned as a ceRNA to regulate Nanog expression by sponging to miR-145 and predicted poor prognosis in pancreatic cancer. In HCC, highly upregulated in liver cancer (HULC) functioned as a ceRNA by sequestering the miR-200a-3p to mediate epithelial to mesenchymal transition (EMT) via upregulating ZEB1 to facilitate HCC metastasis.¹⁶ lncRNA RP11-838N2.4 enhanced the cytotoxic effects of temozolomide by inhibiting the functions of miR-10a in glioblastoma cell lines.¹⁷ Hypoxia-induced lncRNA-NUTF2P3-001 contributed to tumorigenesis of pancreatic cancer by derepressing the miR-3923/KRAS pathway.¹⁸ Similarly, the lncRNA CCAT1/miR-490 axis regulated gastric cancer cell migration by targeting hnRNPA1.¹⁹

However, the ceRNA mechanisms for CCAT1 deregulation in HCC have not been thoroughly elucidated. Here, by bioinformatics analysis and luciferase reporter assays, we demonstrated that CCAT1 was a target of miRNA-490-3p and further demonstrated that CCAT1 was pull down by miRNA-490-3p, and these experiments demonstrated that CCAT1 acted as a ceRNA sponging miR-490-3p. Furthermore, qRT-PCR and Western blot analysis showed that knockdown of CCAT1 resulted in a significant decrease in endogenous CDK1 mRNA and protein expression by binding to miR-490-3p in MHCC97H cells and overexpression of CCAT1 increased the endogenous CDK1 mRNA and protein expression by binding to miR-490-3p. Moreover, we also demonstrated that knockdown of CCAT1 inhibited cell proliferation and decreased the cell invasion by miR-490-3p. These results indicated that CCAT1 influenced tumor progression via directly binding to miR-490-3p and regulated the CDK1 expression.

In conclusion, this study demonstrated that CCAT1 influenced tumor progression through directly binding to the miR-490-3p and regulated the CDK1. Targeting the ceRNA regulatory network involving in CCAT1 may be a novel therapeutic strategy for HCC.

The authors thank the Department of Hepatobiliary Surgery, the First Affiliated Hospital of Chinese PLA General Hospital, for its generous help.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.