miR-1204 promotes hepatocellular carcinoma progression through activating MAPK and c-Jun/AP1 signaling by targeting ZNF418.

Emerging evidence has indicated that abnormal microRNAs (miRNAs) participated in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). Better understanding the association between miRNAs and HCC may contribute to discover novel therapeutic approaches for diagnosis and treatments. In the current study, we have shown that miR-1204 level was elevated in HCC tissues and cell lines, which was associated with malignant clinical features, including large tumor size and advanced TNM stage. Furthermore, gain-or loss-of function assays demonstrated that miR-1204 promoted cell proliferation in vitro and tumor growth in vivo as well as inhibited apoptosis in vitro. Luciferase reporter gene assays confirmed that ZNF418 was a direct downstream target of miR-1204. Recuse assays showed that ZNF418 mediates the biological function of miR-1204 on HCC cells through regulating MAPK and c-Jun signaling. In conclusion, our results suggest that miR-1204 functions as an oncogene to promote proliferation and inhibit apoptosis through regulating MAPK and c-Jun signaling by targeting ZNF418, and potentially serves as a novel prognostic biomarker and therapeutic target for HCC.

Introduction

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer (PLC), is one of the most common, aggressive malignancies and the third leading cause of cancer-related mortality worldwide 1-3. Although surgical resection and other comprehensive treatments have improved the prognosis of HCC patients, it is still unsatisfactory 4. Tumor recurrence and metastasis are the main causes for the poor survival rate of patients with advanced stage HCC. Therefore, identification of some novel therapeutic targets for HCC treatment is urgently needed 5.

MicroRNAs (miRNAs), a group of endogenous, small, single-strand, and non-coding short RNA with approximately 19-25 nucleotides in length, act as post-transcriptional modulator of gene expression in cancer progression by binding with complementary sequences within the 3'-untranslated region (UTR) of target mRNA to induce mRNA degradation or translational repression6-8. Accumulating evidence confirmed that aberrant miRNAs play critical roles in diverse biological progress including cellular proliferation, differentiation, apoptosis, migration and metastasis. miRNAs have been identified as promising therapeutic and prognostic biomarkers in HCC diagnosis and treatment9, 10. miR-1204, a novel cancer-related miRNA, was dysregulated in diverse cancers11-13. MiR-1204 sensitizes nasopharyngeal carcinoma cells to paclitaxel both in vitro and in vivo 14. miR-1204 targets VDR to promote epithelial-mesenchymal transition and metastasis in breast cancer15. However, ectopic expression of miR-1204 leads to increase p53 levels and causes cell death in a partially p53-dependent manner16. These data suggest that miR-1204 expression was cell type-dependent. Nevertheless, the functional importance of miR-1204 and its molecular mechanisms in HCC remain largely unknown.

Zinc-finger protein 418 (ZNF418), a novel Krüppel-associated box/Cys2His2 (KRAB/C2H2) zinc finger proteins, has been proved to be involved in tumor suppression, cell differentiation, proliferation and apoptosis 17, 18. In gastric cancer, ZNF418 overexpression protects against gastric carcinoma and prompts a good prognosis 19. ZNF418 overexpression protects against cardiac hypertrophy and fibrosis 20. However, its role in HCC human cancers including HCC remains unclear.

In this study, we attempted to explore the expression level, clinical importance, functions and potential mechanisms of miR-1204 in HCC. Gain- and loss-of-function analysis confirmed that the biological function of miR-1204 in vitro and in vivo on HCC progression. Notably, ZNF418 was identified as a direct target of miR-1204 and mediated the function of miR-1204 in HCC cells. Moreover, ZNF418 inhibits HCC progression through MAPK and c-Jun/AP1 pathway. Therefore, our results confirm that miR-1204 exerts a oncogenic role in HCC progression and represents a potential target for HCC diagnosis and treatment.

Materials and Methods

Tissues samples

30 pairs HCC and corresponding adjacent non-tumor tissues were collected from the First Affiliated Hospital of Xi'an Jiaotong University. None of them received any therapies before surgery. All HCC and normal tissues were collected and restored in liquid nitrogen. All the patients were provided written informed consent. Approval was obtained from the Ethics Committee of Xi'an Jiaotong University on the basis of the Declaration of Helsinki.

Cell culture, transfection and reagent

The human HCC cell lines (Hep3B, HCCLM3, Huh7, MHCC-97H and SMMC-7721) and the normal immortalized human hepatocyte LO2 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM medium (Gibco-BRL, Grand Island, NY, USA) containing with 10% fetal bovine serum (FBS) (Gibco-BRL) in humidified atmosphere with 5% CO2 at 37°C.

miR-1204 overexpression vectors (HmiR0708) and miRNA inhibitors (HmiR-AN0049), and their corresponding control vectors (CmiR0001-MR04 and CmiR-AN0001-AM02) were purchased from GeneCopoeia Inc. (Guangzhou, China). For ZNF418 overexpression, a full-length human ZNF418 cDNA was amplified by PCR and subcloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA). Small interfering RNA (siRNA) used for ZNF418 silencing and nontargeting (NT) siRNA were purchased from GenePharma (Shanghai, China). Cells transfection was performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. HCC cells were harvested for further analysis 48h after transfection.

Cell viability, proliferation, colony formation and flow cytometry of apoptosis assay

CCK8, colony formation, Edu, and apoptosis were performed as described previously 21-23.

RNA extraction and quantitative real-time PCR (qPCR)

Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract RNA from tissues and cells following as the manufacture procedure. TakaRa PrimeScript™ RT kit (Takara, Dalian, China) was used to perform reverse transcription. The level of miR-1204 was dectected by TaqMan miRNA assays (Applied Biosystems, Foster City, CA, USA). qPCR of ZNF418 mRNA was carried out using SYBR Green Premix PCR Master Mix (Roche, Mannheim, Germany) in a Step One Plus real-time PCR system (Applied Biosystems). The relative level of miR-1204 and ZNF418 mRNA was normalized to U6 and GAPDH, respectively.

Western blot

Western blotting was carried out according to the protocol described previously 21, 24, 25.

Immunohistochemical staining (IHC)

IHC was performed according to the protocol described previously 21, 24, 25.

Tumor Xenograft model

BALB/c nude mice (male, 4-week-old) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and randomly divided into two groups. Hep3B cells (1×106 per injection) transfected with control and miR-1204 were implanted into the right flank of the mice via subcutaneous injection. Tumor volumes were measured every 4 days after being apparently observed and calculated with the following formula: Volume = (length × width2)/2. After 3 weeks, all mice were sacrificed under anesthesia. The animal experiments were approved by the Animal Care and Use Committee of Xi'an Jiaotong University.

Luciferase reporter assay

The 3'-UTR of ZNF418 containing predicted miR-1204 binding sites (wild type and mutant) were subcloned into the pmirGLO vector (Promega, Madison, WI, USA). For the luciferase reporter assay, HCC cells were transfected with different combinations of miR-1204 mimics, control mimics and pGL3- ZNF418 3'-UTR wild type or mutant. The relative luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized to Renilla activity.

Statistical analysis

Data were shown as Mean ± SD performed at least three independent replicates. SPSS software, 24.0 (SPSS Inc., Chicago, IL, USA) and Graphpad Prism 6.0 (San Diego, CA, USA) were used for one-way ANOVA (multiple groups), a two-tailed Student t-test (2 groups). Kaplan-Meier method and Log-rank test were used for survival analysis. Spearman Pearson correlation analysis was performed to analyse the correlation between miR-1204 and ZNF418 mRNA expression. Differences were defined as statistically significant if P<0.05.

Results

1. Expression and clinical significance of miR-1204 in HCC

Real-time quantitative PCR (qPCR) was performed to detect miR-1204 level in 30 pairs HCC tissues and the adjacent non‑tumor tissues. Results showed that miR-1204 was prominently upregulated in HCC compared to adjacent non-tumor tissues (P<0.05, Fig. 1A). In accordance, higher miR-1204 level was observed in HCC cell lines (Hep3B, HCCLM3, Huh7, MHCC-97H, SMMC-7721) compared to the immortalized normal liver cell line (LO2) (P<0.05, Fig. 1B). To unveil the clinical role of miR-1204 in HCC, patients were divided into different subgroups according to the median value. As shown in Table 1, high miR-1204 was associated with large tumor size (P=0.018) and TNM stage (P=0.024). Results from the Kaplan‑Meier analysis revealed that high miR-1204 patients had a worse overall survival than the patients with low miR-1204 level (P=0.0012, Fig. 1C). Taken together, our data indicated that miR-1204 is a potential prognostic biomarker for HCC patients.

Figure 1

miR-1204 is increased in HCC and predicted poor prognosis. (A) The expression level of miR-1204 was measured in 30 pairs HCC tissues and adjacent normal tissues. (B) miR-1204 expression was up-regulated in HCC cell lines compared to normal hepatic cell line LO2. (C) Kaplan-Meier analyses of the overall survival in HCC patients with high or low levels of miR-2014. *P<0.05, **P<0.01.

Table 1

Clinical correlation of miR-1204 expression in HCC.

Clinical parameters	Cases(n = 97)	Expression level		P-value
		miR-1204high(n=49)	miR-1204low(n=48)
Age				0.770
< 50 years	41	20	21
≥50 years	56	29	27
Gender				0.812
Male	82	41	41
Female	15	8	7
Tumor size				0.018*
<5cm	80	36	44
≥5cm	17	13	4
Tumor number				0.393
solitary	84	41	43
multiple	13	8	5
Edmondson				0.446
Ⅰ+Ⅱ	61	29	32
Ⅲ+Ⅳ	36	20	16
TNM stage				0.024*
Ⅰ+Ⅱ	78	35	43
Ⅲ+Ⅳ	19	14	5
Capsular infiltration				0.537
Present	70	34	36
Absent	27	15	12
Venous infiltration				0.563
Present	12	7	5
Absent	85	42	43
AFP				0.509
<400ng/ml	23	13	10
≥400ng/ml	74	36	38
Liver cirrhosis				0.325
Absent	33	18	15
Present	64	31	33
HBsAg				0.976
positive	89	45	44
negative	8	4	4

* p<0.05

2. miR-1204 facilitates HCC cell proliferation and inhibits apoptosis in vitro

To determine the effects of miR-1204 on the proliferation and apoptosis of HCC cells in vitro, we constructed the stably overexpressed miR-1204 in Hep3B cell with lentivirus and knocked down miR-1204 in HCCLM3 cell with miRNA inhibitors (P<0.05, Fig. 2A). The results of CCK8 assays, EdU and colony formation assays revealed that miR-1204 overexpression promoted Hep3B cell viability and proliferation (P<0.05, Fig. 2B, C, D). The results of flow cytometry assay revealed that miR-1204 overexpression suppressed apoptosis of Hep3B cell (P<0.05, Fig. 2E). While miR-1204 knockdown showed opposite effects on HCCLM3 cells (P<0.05, Fig. 2B-E). Moreover, results of western blot showed that miR-1204 regulated Bcl-2 and Bax level (P < 0.05, Fig. 2F). These results suggested that miR-1204 inhibits HCC cell apoptosis and promotes proliferation in vitro.

Figure 2

miR-1204 promotes cell viability, proliferation, colony formation and inhibits apoptosis of HCC cells. (A) Hep3B and HCCLM3 cells that were transfected with different vectors were subjected to qRT-PCR for miR-1204 expression. Overexpression of miR-1204 promoted cell viability (B), cell proliferation (C), colony formation (D) and inhibited apoptosis (E) in Hep3B cells, while down-regulation of miR-1204 inhibited cell viability (B), cell proliferation (C), cell colony formation (D) and promoted apoptosis (E) in HCCLM3 cells. (F) Western blot checked apoptosis-related protein Bcl2/Bax expression in the ectopic-expression of miR-1204. *P<0.05.

3. miR-1204 promotes HCC growth in vivo

We constructed subcutaneous tumor model to investigate the role of miR-1204 on tumorigenicity in vivo. Results showed that miR-1204 overexpression dramatically promoted the Hep3B cells growth in nude mice (P<0.05, Fig. 3A). Results of IHC for Ki67 and TUNEL staining in the xenografted tissues revealed that miR-1204 elevated the positive rate of Ki67 and degraded the number of apoptotic cells for TUNEL positive (P<0.05, Fig. 3B, C). These results showed that miR-1204 contributed to HCC growth in vivo.

Figure 3

miR-1204 promotes tumor growth (A) Tumor growth curve and tumor weight revealed that miR-1204 overexpression significantly promoted tumor growth in vivo. (B) Tumor nodules were subjected to immunohistochemical staining for Ki-67 assays and (C) TUNEL and quantitative analysis. *P < 0.05.

4. ZNF418 is a direct target of miR-1204 in HCC

TargetScan was used to predict the candidate target of miR-1204 and and the results showed that 3'-UTR of ZNF418 had the complementary sequence of miR-1204 (Fig.4A). The results of luciferase reporter assay showed that overexpression miR-1204 suppressed, while silencing miR-1204 enhanced luciferase activity of wt-3'UTR of ZNF418 but not of the mut-3'UTR (P<0.05, Fig. 4B). Moreover, miR-1204 overexpression significantly suppressed the mRNA and protein expression of ZNF418 in Hep3B cells (P<0.05, respectively, Fig. 4C, D). By contrast, silencing miR-1204 increased the mRNA and protein of ZNF418 in HCCLM3 cells (P<0.05, respectively, Fig. 4C, D). These data indicated that ZNF418 was a direct target of miR-1204 in HCC.

Figure 4

ZNF418 is a direct target of miR-1204 in HCC cells. (A) miR-1204 and its putative binding sequences in the 3'-UTR of ZNF418. The mutant binding site was generated in the complementary site for the seed region of miR-1204. (B) miR-1204 overexpression significantly suppressed the luciferase activity that carried wild-type (wt) but not mutant (mt) 3'-UTR of ZNF418. miR-1204 knockdown showed the opposite effects. miR-1204 overexpression reduced the expression of ZNF418 mRNA (C) and protein (D) in Hep3B cells and miR-1204 knockdown increased the level of ZNF418 mRNA (C) and protein (D) in HCCLM3 cells. *P<0.05.

5. miR-1204 negatively correlates with ZNF418 in HCC

Next, we confirmed the relation between ZNF418 and miR-1204 in HCC tissues. The results showed that ZNF418 mRNA and protein levels were prominently lower in high miR-1204 subgroup than that in low miR-1204 subgroup in HCC tissues (P<0.05, Fig. 5A, B). Furthermore, the mRNA level of ZNF418 in the HCC tissues was negatively correlated with miR-1204 expression ((r=-0.7812, P<0.0001, Fig. 5C). In addition, ZNF418 staining in miR-1204 high-expression tumors was remarkedly lower than miR-1204 low-expression tumors (P<0.05, Fig. 5D). Moreover, miR-1204 ectopic-expression showed a weak ZNF418 staining (P<0.05, Fig. 5E). Collectively, these results demonstrated that ZNF418 level was negatively correlated with miR-1204 in HCC.

Figure 5

miR-1204 inversely correlated with ZNF418 expression in HCC. (A) The expression of ZNF418 mRNA in miR-1204 high-expressing tumors was significantly lower than that in miR-1204 low-expressing tumors. (B) The expression of ZNF418 protein in miR-1204 high-expressing tumors was significantly lower than that in miR-1204 low-expressing tumors. (C) A significant inverse correlation between the mRNA levels of ZNF418 and miR-1204 was observed in HCC tissues. (D) Representative immunohistochemical staining showed a weak staining of ZNF418 in miR-1204 high-expressing HCC tissue and strong staining of ZNF418 in the miR-1204 low-expressing tumor. (E) Xenografts tissues immunohistochemical staining of ZNF418 in miR-1204 overexpression subcutaneous tumor nodules. *P<0.05.

6. ZNF418 is downregulated in HCC and inhibits tumor cell progression in HCC

Results of western blot showed that ZNF418 was downregulated in HCC compared to adjacent non-tumor tissues (P<0.05, Fig. 6A). In accordance, ZNF418 was decreased in HCC cell lines compared to LO2 (P<0.05, Fig. 6B). To confirm the effects of ZNF418, we overexpressed or knockdown ZNF418 in HCC cells (P<0.05, Fig. 6C). Our data showed that ZNF418 overexpression remarkably inhibited cell viability, proliferation, colony formation and promoted apoptosis in HCCLM3 cells (P<0.05, Fig. 6D-G), while ZNF418 knockdown showed opposite function on Hep3B cells (P<0.05, Fig.6D-G). Furthermore, ZNF418 also regulated Bcl-2 and Bax expression (P < 0.05, Fig. 6H). To conclude, the results suggested that ZNF418 was involved in HCC progression and its downregulation mimicked the function of miR-1204 on HCC cells.

Figure 6

ZNF418 inhibits cell viability, proliferation, colony formation and promotes apoptosis in HCC cells (A) The expression level of ZNF418 was measured in tumor tissues and adjacent normal tissues of HCC patients. (B) ZNF418 expression was down-regulated in HCC cell lines compared to normal hepatic cell line LO2. (C) HCCLM3 and Hep3B cells that were transfected with corresponding vectors were subjected to western blot for ZNF418 expression. Overexpression of ZNF418 suppressed cell viability (D), proliferation (E), colony formation (F) and promoted apoptosis (G) in HCCLM3 cells, while down-regulation of ZNF418 enhanced viability (D), proliferation (E), cell colony formation (F) and inhibited apoptosis (G) in Hep3B cells. (H) Western blot analysis of apoptosis-related protein Bcl2/Bax expression in the ZNF418 overexpression or knockdown. *P<0.05.

7. ZNF418 mediates the functional effects of miR-1204 in HCC cells

To investigate whether ZNF418 mediated the effects of miR-1204 on HCC cells, we reversed the expression of ZNF418 in Hep3B-miR-1204 cells or HCCLM3-anti-miR-1204 cells (P<0.05, Fig.7A). ZNF418 restoration significantly abolished the effects of miR-1204 on cell viability, proliferation, colony formation and apoptosis of Hep3B cells (P<0.05, Fig. 7B-E). Furthermore, the effects of miR-1204 knockdown on HCCLM3 cells were abrogated by co-transfection with ZNF418 siRNA (P < 0.05, Fig. 7B-E). In addition, ZNF418 alternation also reversed the effects of miR-1204 on Bcl-2 and Bax expression (P<0.05, Fig. 7F). These data indicated that ZNF418 is partly mediated the effects of miR-1204 on HCC cells.

Figure 7

Alteration of ZNF418 partially abolishes miR-1204 mediated biological function in HCC. (A) miR-1204-overexpressing Hep3B cells that were transfected with empty vector (EV) or ZNF418 overexpression vector were subjected to western blot for ZNF418. miR-1204-suppressive HCCLM3 cells that were transfected with control siRNA or ZNF418 siRNA were subjected to western blot for ZNF418. ZNF418 restoration abrogated the effects of miR-1204 overexpression on cell viability (B), proliferation (C), cell colony formation (D) and apoptosis (E) of Hep3B cells. ZNF418 knockdown reversed the effects of miR-1204 knockdown in HCCLM3 cells (B-E). (F) Western blot analysis of apoptosis-related protein Bcl2/Bax expression in the ZNF418 alteration. *P<0.05.

8. miR-1204 affects the MAPK and c-Jun/AP1 signaling pathways in HCC

ZNF418 is involved in the regulation of the MAPK and AP1 signaling pathway. To determine whether miR-1204 affects the MAPK and c-Jun/AP1 signaling pathways in HCC. Western blot was used to measure the levels of MAPK and c-Jun/AP1 in HCC cells co-transfection with miR-1204 and ZNF418. And the results showed that miR-1204 overexpression increased p-P38/MAPK and p-c-Jun expression, while ZNF418 restoration reversed the effects of miR-1204 in Hep3B cells (Figure 8). The opposite effects were shown in HCCLM3 cells. These results suggest that miR-1204 promotes the activation of the MAPK and c-Jun/AP1 signaling pathways in HCC by regulation of ZNF418.

Figure 8

miR-1204 affects the MAPK and c-Jun/AP1 signaling pathways in HCC. miR-1204 overexpression suppresses the MAPK and c-Jun signaling pathways in HCC. ZNF418 overexpression reversed the promotive effects. Western blot analysis was conducted to measure the protein expression. *P<0.05.

Discussion

Accumulating studies demonstrated that aberrant miRNAs play a critical role in the development and progression of HCC 26-28. Recently, some miRNAs have been identified as diagnostic, therapeutic and prognostic markers. miR-1204, a novel cancer-related miRNA, was up-regulated in HCC tissues and cell lines. Moreover, up-regulated miR-1204 was significantly correlated with malignant clinicopathological features, including large tumor size and advanced TNM stage. Kaplan-Meier survival cure showed that miR-1204 was a novel prognostic factor in predicting survival of HCC patients. Functional experiments demonstrated that miR-1204 promoted cell viability, proliferation, colony formation and inhibited apoptosis by gain- and loss-of-function experiment on HCC cells. Moreover, in vivo data, the results showed that miR-1204 promoted tumor growth. These data indicated that miR-1204 functions as an oncogene in HCC progression and maybe a promising biomarker for HCC therapy.

ZNF418, also named zinc-finger protein 418, which spans 13.5kb on chromosome 19q13.43 encompassing six exons, and transcribes a 3.7 kb mRNA that encodes a protein with 676 amino acid residues 18. ZNF418 was significantly downregulated in advanced stage GC patients. Moreover, a decreased ZNF418 expression was associated with the presence of GC and also with higher TNM stage and lower OS rates compared with non-tumor tissues 19. These data showed that ZNF418 performed an important role in cancer development. Here, we demonstrated that miR-1204 inhibited ZNF418 expression by directly binding to its 3'UTR. Furthermore, miR-1204 was negatively correlated with ZNF418 expression in clinical tissues. ZNF418 was decreased in HCC tissues and inhibited cell viability, proliferation, colony formation and increased apoptosis. Moreover, ZNF418 restoration reversed the function of miR-1204 on HCC cells. Moreover, we confirmed that miR-1204 regulated the MAPK and c-Jun signaling pathways in HCC. MAPK and c-Jun signaling pathways were involved in multiple biological function, including proliferation, apoptosis and autophagy et al in HCC. Taken together, miR-1204 serves as an oncogene in HCC by directly targeting ZNF418 and regulating the MAPK and c-Jun/AP1 signaling pathways.

In conclusion, our data confirmed that miR-1204 was upregulated in HCC tissues and cell lines. Its ectopic-expression predicted a poorer survival of patients. Moreover, miR-1204 promoted HCC cell viability, proliferation, colony formation and inhibited apoptosis through binding to the 3'UTR of ZNF418 and regulating its expression. ZNF418 inhibited HCC progression through MAPK and c-Jun signaling pathways and mediated the effects of miR-1204. Taken together, our results indicated that miR-1204 functions as an oncogene in HCC and may be a promising therapeutic target for HCC treatment.