MicroRNA-1251-5p promotes tumor growth and metastasis of hepatocellular carcinoma by targeting AKAP12

Biomedicine & Pharmacotherapy 122 (2020) 109754

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MicroRNA-1251-5p promotes tumor growth and metastasis of hepatocellular carcinoma by targeting AKAP12

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Shaoshan Han1, Liang Wang1, Liankang Sun, Yufeng Wang, Bowen Yao, Tianxiang Chen, Runkun Liu, Qingguang Liu* Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province 710061, China

A R T I C LE I N FO

A B S T R A C T

Keywords: HCC MiR-1251-5p AKAP12 Growth Metastasis

MicroRNAs (miRNA) are small RNA molecules that have emerged as important regulators of gene expression in hepatocellular carcinoma (HCC). However, the expression, function and mechanism of miR-1251-5p in HCC remain poorly understood. In the present study, it was observed that miR-1251-5p expression was upregulated in HCC. Furthermore, higher miR-1251-5p level was correlated with poor prognosis, large tumor size, vascular invasion and high tumor-node-metastasis (TNM) stages of HCC patients. Functionally, miR-1251-5p drove HCC cell proliferation, migration and invasion in vitro, and promoted growth and metastasis of HCC cells in vivo. Akinase anchor protein 12 (AKAP12) was screened as a direct target of miR-1251-5p by using the starBase V3.0 online platform. The AKAP12 mRNA expression was downregulated and negatively correlated with miR-1251-5p level in HCC tissues. Furthermore, in vitro experiments confirmed that AKAP12 was targeted and negatively regulated by miR-1251-5p. Importantly, AKAP12 overexpression decreased HCC cell proliferation, migration and invasion, whereas inhibition of AKAP12 rescued the miR-1251-5p knockdown-attenuated HCC cell proliferation, migration and invasion. Overall, the present study indicates that miR-1251-5p plays an oncogenic role in HCC by targeting AKAP12, and may be a potential therapeutic target for HCC treatment.

1. Introduction Hepatocellular carcinoma (HCC) is the third-leading cause of cancer deaths and the fifth most common cancer worldwide [1]. The lack of early effective diagnostic indicators, and the high recurrence and metastasis rate after surgical operation leads to the poor prognosis of HCC patients [2,3]. Thus, there is an urgent need to identify the key criminal molecules involved in HCC progression. MicroRNAs, are small non-coding RNA molecules, regulating downstream target genes at the post-transcriptional and/or transcriptional level. Studies have reported that miRNAs play an important role in the progression of HCC [4–8]. For example, the study conducted by Ren W revealed that miRNA-196a/-196b regulated the progression of HCC by modulating the JAK/STAT pathway via targeting SOCS2 [9]. Wu H confirmed that miR-29c-3p regulated DNMT3B and LATS1 methylation to inhibit tumor progression in HCC [10].Our study group also identified several aberrantly expressed miRNAs in HCC. [11–17].

For instance, we found that miR-519c-3p forced the tumor growth and metastasis of HCC cells by targeting BTG3 [11]. MiR-1204 promoted HCC progression by activating MAPK and c-Jun/AP1 signaling through targeting ZNF418 [12]. Recently, the role of miR-1251-5p has been identified. The study conducted by Shao Y et al. revealed that miR1251-5p functioned as a tumor driver, which could promote cell proliferation and cell cycle progression in vitro and contribute to carcinogenesis and autophagy in vivo via targeting the tumor suppressor TBCC in ovarian cancer cells [18]. However, whether miR-1251-5p participates in the progression of HCC remains unclear. A-kinase anchor protein 12 (AKAP12) is a member of the A-kinase anchoring protein family, which functions as scaffold proteins in signal transduction, and exerts an anti-tumor role in various human cancers, including breast cancer [19], colorectal cancer [20,21], esophageal neoplastic progression [22], myeloid malignancies [23], gastric cancer [24], and HCC [25,26]. Previous studies have revealed that promoter hypermethylation led to the reduction of the tumor suppressor gene

Abbreviations: HCC, hepatocellular carcinoma; MiRNA, microRNAs; 3′UTR, 3′-untranslated regions; AKAP12, A-kinase anchor protein 12; TPD52, Tumor protein D52 ⁎ Corresponding author at: Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi Province 710061, China. E-mail address: [email protected] (Q. Liu). 1 Contributed equally. https://doi.org/10.1016/j.biopha.2019.109754 Received 21 September 2019; Received in revised form 22 November 2019; Accepted 27 November 2019 0753-3322/ © 2019 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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AKAP12 in human hepatocarcinogenesis [25,27]. In addition, Xia W et al. reported that MiR-103 regulates HCC growth by targeting AKAP12 [26]. In the present study, the level of miR-1251-5p in HCC tissues and cells was tested. In vitro experiments were conducted to explore the function of miR-1251-5p in HCC cell proliferation, migration and invasion. Subsequently, the downstream target mediating the role of miR1251-5p was identified. Together, we confirmed that miR-1251-5p plays an oncogenic role in HCC cells by targeting AKAP12.

Table 2 Primers used in this study.

2. Methods and materials 2.1. Clinical specimens and data

Primers name

Primer sequence

AKAP12 forward AKAP12 reverse β-actin forward β-actin reverse miR-1251-5p forward miR-1251-5p reverse U6 forward U6 reverse

5′-CCTCTCTATGGCAGGAAGACATTC-3′ 5′-ATGGAATCGCAACTGTGATGGC-3′ 5′-TGACGTGGACATCCGCAAAG-3′ 5′-CTGGAAGGTGGACAGCGAGG-3′ 5′-ACTCTAGCTGCCAAAGGCG-3′ 5′-GAACATGTCTGCGTATCTC-3′ 5′-CTCGCTTCGGCAGCACA-3′ 5′-AACGCTTCACGAATTTGCGT-3

(miR-control, miR1N0000001-1-5), miR-1251-5p mimics (miR-12515p, miR10005903-1-5), inhibitors control (anti-miR-NC, miR2N0000001-1-5) and miR-1251-5p inhibitors (anti-miR-1251-5p, miR20005903-1-5) were purchased from RioBio (Guangzhou, China). Lentivector-mediated miR-1251-5p inhibitors (Lv-anti-miR-1251-5p, HmiR-AN0083) and the negative control (Lv-anti-miR-NC) were obtained from GeneCopoeia Inc. (Guangzhou, China). AKAP12 expression plasmid (pCMV6-AKAP12, RC214439), specific siRNA against AKAP12 (si-AKAP12, SR306381), and the corresponding negative control (EV and si-control) were constructed and purchased from RioBio (Guangzhou, China). Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) was used to perform the cell transfection, according to manufacturer’s instructions.

Fresh tumor samples and matched tumor-adjacent tissues were randomly collected from 57 HCC patients who underwent curative resection at the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China). Clinical specimens were used after obtaining informed consent from each patient. None of the patients had received any prior radiotherapy or chemotherapy. The demographic and clinicopathological data of these patients were obtained through review of medical records, and are listed in Table 1. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University, according to the Declaration of Helsinki. 2.2. Cell culture and transfection

2.3. Real-time qPCR

Five human liver cancer cell lines (Hep3B, PLC/PRF/5, Huh7, MHCC97-L, and MHCC97-H), and the human immortalized normal hepatocyte cell line (L02) were obtained from the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in our laboratory and cultured as previously described [11]. The mimics control

Trizol (Invitrogen, Carlsbad, CA, USA) and a miRVana miRNA Isolation Kit (Ambio, Austin, TX, USA) were used to extract the RNA from tissues and cells, according to manufacturer’s instructions. The reverse transcription was performed using TIANScript RT Kit (Tiangen Biotech, Beijing, China). The qPCR were performed using a TaqMan Human MiRNA Assay Kit (Genecopoeia, Guangzhou, China) and a SYBR Premix Ex Taq™ Kit (TaKaRa, Shiga, Japan). The primers for AKAP12 and β-actin were purchased from Realgene (Nanjing, China). The primers for miR-1251-5p and U6 were obtained from RiboBio (Guangzhou, China). The 2−ΔΔCt method was used to quantify the expression levels. All primers are listed in Table 2.

Table 1 Correlations of miR-1251-5p with clinicopathological features of HCC. Characteristics

Number (n = 57)

Age(years) ≥60 38 < 60 19 Gender Male 41 Female 16 HBV infection Negative 10 Positive 47 Liver cirrhosis Absent 8 Present 49 AFP(ng/ml) ≤20 13 > 20 44 Tumor size ≤5cm 27 > 5cm 30 Tumor multiplicity Single 35 Multiple 22 Vascular invasion No 36 Yes 21 Edmondson grade Ⅰ+Ⅱ 32 Ⅲ+Ⅳ 25 TNM stage Ⅰ+Ⅱ 40 Ⅲ+Ⅳ 17

miR-1251-5p levels High (n = 29)

Low (n = 28)

20 9

18 10

23 6

18 10

P-value

0.708

2.4. MTT assay

0.207

MTT (Sigma, St. Louis, MO, USA) assay was used to assess the cell viability. The protocols for MTT were described in the previous study of the investigators [11]. A microplate reader (Bio-Rad, USA) was used to read the absorbance.

0.682 4 25

6 22

3 26

5 23

0.412

2.5. Ethynyl deoxyuridine (EdU) incorporation assay 0.308

5 24

8 20

An EdU kit (C10310-1, RioBio, Guangzhou, China) was used to assess cell proliferations. The results were acquired using a Zeiss fluorescence photomicroscope (Carl Zeiss, Oberkochen, Germany).

0.047* 10 19

17 11

17 12

18 10

14 15

22 6

14 15

18 10

16 13

24 4

0.661

2.6. Transwell migration and invasion assays Transwell chambers (Millipore, USA) were used for the cell migration and invasion assays. The protocols for MTT were described in the previous study of the investigators [11]. The results were acquired using the camera under the microscope, and quantified by counting at least 10 random fields. Each experiment was carried out in triplicate wells and repeated at least 3 times.

0.018*

0.223

0.012*

2.7. In vivo experiments

HBV hepatitis B virus, AFP alpha-fetoprotein, TNM tumor-node-metastasis. * P < 0.05, statistically significant difference.

Four-week-old male BALB/c athymic nude mice (obtained from 2

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Fig. 1. The expression and prognostic value of miR-1251-5p in HCC. (A) The levels of miR1251-5p in 57 paired human HCC and adjacent normal tissues. P < 0.0001 by Student’s t-test. (B) The level of miR-1251-5p in HCC tissues was obviously higher than that in normal liver tissues in the TCGA database obtained from the starBase V3.0 platform. P = 7 × 10−8 by Student’s t-test. (C) The levels of miR-1251-5p in HCC cell lines (Hep3B, PLC/PRF/5, Huh7, MHCC97-L, and MHCC97-H) and L02 cells. *P < 0.05, by Student’s t-test vs. L02; n = 3. (D) The Kaplan-Meier survival analysis revealed that HCC patients with a higher expression of miR-1251-5p (n = 29) have poorer overall survival (P = 0.0116, by Log-rank test), when compared to those with low miR-12515p expression (n = 28).

Animal Experiment Center of Xian Jiaotong University) were kept under pathogen-free conditions on a standard diet. The Xenograft model and pulmonary metastasis model were developedas previously described [28]. The tumors and lung tissues were harvested and embedded in paraffin for routine pathological examination and immunohistochemistry for Ki-67. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University.

between groups was carried out using the Student's t-test or one way ANOVA. Correlation of miR-1251-5p with clinical and pathologic variables was analyzed using Pearson correlation tests. Survival curves were plotted using Kaplan-Meier’s method and compared between groups by the log-rank test. A value of P < 0.05 was considered to be statistically significant.

2.8. Western blot

3.1. MiR-1251-5p was significantly upregulated in HCC and associated with poor prognosis of HCC patients

3. Results

RIPA Buffer was used to extract the proteins from HCC cells and tissues, and the concentration of proteins was measured using a BCA Kit. The protocols for the western blot analysis were described in the previous study [11]. AKAP12 (1:1,000, ab49849; Abcam, Cambridge, MA, UK), TPD52 (1:1,000, ab182578; Abcam, Cambridge, MA, UK), and β-actin (1:1,000, sc-47778, SANTA CRUZ, Dallas, Texas, USA) antibodies were used in the present study.

Real-time qPCR was performed to detect the level of miR-1251-5p in 57 pairs HCC tissues and adjacent non‑tumor tissues, and it was found that miR-1251-5p expression was upregulated in HCC (P < 0.0001, Fig. 1A). Consistently, TCGA data obtained from the starBase V3.0 online platform revealed that miR-1251-5p significantly increased in HCC tissues, when compared to normal liver tissues (P = 7 × 10−8, Fig. 1B). Furthermore, a higher miR-1251-5p level was detected in HCC cell lines (Hep3B, PLC/PRF/5, Huh7, MHCC97-L, and MHCC97-H), when compared to the immortalized normal liver cell line (L02) (Fig. 1C). In order to explore the clinical significance of miR-1251-5p, HCC patients were divided into two groups based on the median value of miR-1251-5p. The results revealed that the high levels of miR-12515p were significantly associated with larger tumor size (P = 0.047), vascular invasion (P = 0.018), and advanced tumor-node‑metastasis (TNM) stage (P = 0.012) (Table1). The Kaplan‑Meier analysis showed that patients with higher miR-1251-5p level was associated with worse overall survival (OS) (P = 0.0116, Fig. 1D). Taken together, miR-12515p is upregulated in HCC, and associated with poor prognosis of HCC patients.

2.9. Luciferase reporter assay The wild-type (WT) andthe mutant (MT) 3′UTR of AKAP12 mRNA were synthesized and inserted into the downstream of the pEZX-MT06 vector (GeneCopoeia, Guangzhou, China). Cells transfected with the miR-1251-5p mimics or inhibitors, or the corresponding control vectors were transfected with AKAP12-3′UTR-wt and AKAP12-3′UTR-mut. Cells were collected after 48 h, and the luciferase activity was quantified using a Luc-Pair™ Duo-Luciferase Assay Kit (GeneCopoeia, Guangzhou, China). The protocols for the Luciferase reporter assay were described in our previous study [11]. 2.10. Statistical analysis

3.2. MiR-1251-5p contributes to the proliferation, migration and invasion of HCC cells in vitro

For continuous variables, data are shown as the means ± SD. The SPSS V24.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (San Diego, CA, USA) were used used to analyze data. Comparison

In order to investigate the biological functions of miR-1251-5p in 3

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Fig. 2. MiR-1251-5p promotes HCC cell proliferation and motility in vitro. (A) The MTT assay revealed that miR-1251-5p mimics increased the viability of Hep3B. *P < 0.05, by ANOVA; n = 3. (B) The miR-1251-5p mimics enhanced the proliferation of Hep3B, as detected by the EdU assay. *P < 0.05, by Student’s ttest; n = 3. (C) MiR-1251-5p inhibitors restrained the viability of MHCC97-H, as detected by the MTT assay. *P < 0.05, by ANOVA; n = 3. (D) The EdU assay revealed that miR-1251-5p inhibitors suppressed the proliferation of MHCC97-H. *P < 0.05, by Student’s t-test; n = 3. (E) The Transwell migration and invasion assay revealed that miR-1251-5p mimics enhanced the motility of Hep3B, when compared to the miR-control group. *P < 0.05, by Student’s t-test; n = 3. (F) The Transwell migration and invasion assay revealed that miR-1251-5p inhibitors impaired the motility of MHCC97-H, when compared to the control group. *P < 0.05, by Student’s t-test; n = 3.

3.3. MiR-1251-5p silencing inhibits the growth and metastasis of HCC cells in vivo

HCC, the expression level of miR-1251-5p in Hep3B and MHCC97-H was manipulated with miR-1251-5p mimics and inhibitors. In addition, qPCR was performed to confirm the transfection efficiency (Supplementary Fig. 1A and B). MTT and EdU assays were conducted to assess the influence of miR-1251-5p on the proliferation ability of HCC cells in vitro. The MTT assay revealed that the viability of Hep3B was significantly enhanced by miR-1251-5p mimics (P < 0.05, Fig. 2A), while miR-1251-5p inhibitors prominently inhibited the MHCC97-H viability (P < 0.05, Fig. 2C). Consistently, the EdU assay revealed that miR-1251-5p mimics obviously elevated the percentage of EdU positive cells in Hep3B (P < 0.05, Fig. 2B), while the percentage of EdU positive cells was remarkably reduced in MHCC97-H transfected with miR1251-5p inhibitors (P < 0.05, Fig. 2D). Next, Transwell assays were preformed to assess the influence of miR-1251-5p on the motility of HCC cells in vitro, and the results indicated that miR-1251-5p mimics notably increased the number of Hep3B cells passing through the membrane (P < 0.05, respectively; Fig. 2E). On the contrary, the number of MHCC97-H cells passing through the membrane was remarkably decreased by miR-1251-5p inhibitors (P < 0.05, respectively; Fig. 2F). Based on these above results, it can be confirmed that miR-1251-5p promotes the proliferation and motility of HCC cells in vitro.

To investigate whether silencing of of miR-1251-5p expression in HCC cells impedes their ability to grow and metastasize in vivo, we knocked down endogenous miR-1251-5p levels in highly invasive HCC cells. Briefly, MHCC97-H cells were infected with lentiviral vectors to establish two stable lines that either expressed antisense RNA against miR-1251-5p (Lv-anti-miR-1251-5p) or contained a control vector (Lvanti-miR-NC). Cellswere implanted into nude mice via subcutaneous injection and tail vein injection. The results of the tumor growth curves suggested that miR-1251-5p silencing significantly impaired the tumor growth in nude mice (P < 0.05, Figs. 3A and B). Subsequently, tumor tissues were harvested to detected the content of miR-1251-5p by qPCR. It was observed that the level of miR-1251-5p in tumor tissues that developed by injectingLv-anti-miR-1251-5p cells was significantly lower than that in the control group (P < 0.05, Fig. 3C). Furthermore, the results of the Ki-67 immunostaining revealed that the percentage of Ki-67 positive cells in tumor tissues from the Lv-anti-miR-1251-5p group was remarkably lower than that in the Lv-anti-miR-NC group (P < 0.05, Fig. 3D). In addition, the H&E staining of lung tissues revealed that the number of metastatic lung nodules in the Lv-anti-miR4

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Fig. 3. MiR-1251-5p silencing restrains the growth and metastasis of HCC cells in vivo. (A) MHCC97-H cells with miR-1251-5p silencing (n = 4) or controls (n = 4) were implanted into nude mice via subcutaneous injection. The tumor growth curves indicate that miR-1251-5p silencing suppressed the growth of HCC cells in mice. *P < 0.05, by ANOVA. (B) The subcutaneous tumors were harvested and weighted at the 21st day after implantation. *P < 0.05, by Student’s t-test. (C) The tumor tissues were subjected to qPCR to determine the expression of miR-1251-5p. (D) The immunostaining of Ki-67 xenograft tissues from the miR-1251-5p silencing group and control group is shown. *P < 0.05, by Student’s t-test. (E) The H&E staining of metastatic lung nodules in the miR-1251-5p silencing group and control group. *P < 0.05, by Student’s t-test.

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Fig. 4. AKAP12 is a direct target of miR-1251-5p in HCC. (A) The putative miR-1251-5p binding site in the 3′UTR of AKAP12 was predicted by the starBase V3.0 online platform. The mutant binging site was generated in the complementary site for the seed region of miR-1251-5p. (B) The mRNA levels of AKAP12 in 57 paired HCC and adjacent normal tissues are shown. P < 0.001, by Student’s t-test. (C) The Pearson’s correlation analysis revealed that there was a significant inverse correlation between the mRNA of AKAP12 and miR-1251-5p in HCC tissues. (D) The levels of AKAP12 protein in 4-paired human HCC and adjacent normal tissues are shown. *P < 0.05, by Student’s t-test. (E) The western blot revealed that miR-1251-5p could significantly and negatively regulate AKAP12 expression in HCC cells. *P < 0.05, by Student’s t-test; n = 3. (F) The luciferase reporter gene assay revealed that miR-1251-5p overexpression was suppressed, while miR-1251-5p knockdown enhanced the luciferase activity of the vector carrying the wt-3′UTR of AKAP12, but not the mut-3′UTR, in HEK-293 T cells. *P < 0.05, by Student’s ttest; n = 3. Fig. 5. AKAP12 overexpression suppresses the proliferation, migration and invasion of MHCC97-H cells. (A) The pCMV6-AKAP12 or empty vector (EV) was transfected into MHCC97-H cells, and the transfection efficiency was confirmed by western blot. *P < 0.05, by Student’s t-test; n = 3. (B) MTT, (C) EdU and (D) Transwell assays were performed to assess the proliferation, migration and invasion abilities of MHCC97-H cells. *P < 0.05, by Student’s t-test; n = 3.

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Fig. 6. AKAP12 knockdown rescues the miR-1251-5p knockdown-attenuated HCC cell proliferation, migration and invasion. (A) AKAP12 was knocked down by the siRNA in MHCC97-H cells with miR-1251-5p knockdown, and the transfection efficiency was confirmed by western blot assay. *P < 0.05, by Student’s t-test; n = 3. (B) MTT, (C) EdU and (D) Transwell assays were performed to assess the proliferation, migration and invasion abilities of MHCC97-H cells transfected with the corresponding vectors. *P < 0.05, by Student’s t-test; n = 3.

transfecting pCMV6-AKAP12 plasmid in MHCC97-H cells (P < 0.05, Fig. 5A). The functional experiments confirmed that AKAP12 overexpression significantly inhibited the proliferation and motility of MHCC97-H cells (P < 0.05, Figs. 5B–D). Subsequently, AKAP12 expression was blocked with a specific siRNA in MHCC97-H cells with miR-1251-5p knockdown (P < 0.05, Fig. 6A). Importantly, the functional experiments showed that silencing AKAP12 expression could reverse the inhibitory effect of the miR-1251-5p inhibitor on HCC cell proliferation, migration and invasion. (P < 0.05, Figs. 6B-D). In conclusion, these results indicated that AKAP12 was a functional mediator of miR-1251-5p on the proliferation, migration and invasion of HCC cells.

1251-5p group was significantly lesser than that in the Lv-anti-miR-NC group (P < 0.05, Fig. 3E). These results suggest that miR-1251-5p silencing inhibits the growth and metastasis of HCC cells in vivo. 3.4. AKAP12 is a direct functiontarget of miR-1251-5p The starBase V3.0 online platform was used to predict the potential targets of miR-1251-5p. Two tumor suppressor genes, AKAP12 [27] and TPD52 [29] were screened among the results. However, merely AKAP12 could be significantly regulated by miR-1251-5p at both the mRNA and protein level (P < 0.05; Supplementary Figs. 2B and C, 4E). The complementary sequence between the miR-1251-5p and 3′UTR of AKAP12 is presented in Fig. 4A. The western blot and qPCR results revealed an obviously lower expression of AKAP12 in HCC tissues, when compared to adjacent normal tissues (P < 0.0001 and P < 0.05, respectively; Figs. 4B and D). Consistently, the TCGA data indicated that AKAP12 was prominently decreased in HCC tissues, when compared to normal liver tissues (P = 0.0062, Supplementary Fig. 2A). In addition, miR-1251-5p expression was inversely correlated with the mRNA level of AKAP12 in HCC tissues (r=-0.6328, P < 0.0001; Fig. 4C). To test whether AKAP12 is a direct target of miR-1251-5p, the wild type and mutant 3′UTR of AKAP12 gene were cloned downstream of firefly luciferase. The luciferase reporter assay results revealed that miR-1251-5p overexpression suppressed, while miR-1251-5p knockdown enhanced the luciferase activity of the vector carrying the wild type 3′UTR of AKAP12, but not of the mutant -3′UTR in HEK-293 T cells (P < 0.05, Fig. 4F). Therefore, miR-1251-5p directly targets AKAP12 through the identified binding sites in the 3′UTR.

4. Discussion There are ample studies which have proven that miRNAs play crucial roles in HCC. Hence, it is very helpful for the treatment of HCC identifying the novel miRNAs which take part in the progression of HCC. The key finding of the current study is that miR-1251-5p is remarkably elevated in HCC cells and tissues, compared with normal hepatocytes and liver tissues. The TCGA data also showed that miR1251-5p expression was elevated in HCC tissues, which was accordance with the present data. Furthermore, the clinical association analysis suggested that higher miR-1251-5p level was correlated with poor prognosis, large tumor size, vascular invasion and high tumor-nodemetastasis (TNM) stages of HCC patients. Mechanistically, previous studies have illustrated that the hypoxic microenvironment enhances the expression of miR-182 and miR-210 [30,31]. In addition, the previous studies conducted by the our team demonstrated that long noncoding RNA AGAP2-AS1, which act as a competitive endogenous RNA, regulated the miR-16-5p expression [32]. According to these above, we wonder whether the expression of miR-1251-5p is regulated by hypoxia or lncRNA in HCC, so the accurate mechanisms involved in the regulation of miR-1251-5p expression would be explored later.

3.5. AKAP12 is essential for miR-1251-5p-enhanced cell proliferation and motility in HCC In order to validate whether AKAP12 is the downstream effector of miR-1251-5p in HCC, AKAP12 expression was upregulated by 7

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References

The function of miR-1251-5p has been identified recently. A study conducted by Shao Y et al. revealed that miR-1251-5p promoted carcinogenesis and autophagy via targeting the tumor suppressor TBCC in ovarian cancer cells [18]. However, it remains unclear whether miR1251-5p is involved in the regulation of HCC progression. In this study, a series of functional experiments were preformed, whose results showed that miR-1251-5p facilitated HCC cell proliferation, migration and invasion in vitro, and promoted the growth and metastasis of HCC cells in vivo. Overall, these present results suggest that miR-1251-5p works as an oncogene in HCC. There are many hard evidences which have revealed that AKAP12 acts as a tumor suppressor in various human cancers. For instance, a study conducted by Soh RYZ et al. revealed that AKAP12 inhibited cell migration in breast cancer [19]. He P et al. reported that the upregulation of AKAP12 with HDAC3 depletion suppressed the progression and migration of colorectal cancer [20]. In addition, Xia W et al. reported that overexpressed AKAP12 suppressed the cell proliferation and induced apoptosis in HCC [26]. In the present study, sufficient evidence was provided to confirm that AKAP12 is the direct functional target of miR-1251-5p in HCC. Firstly, miR-1251-5p was inversely correlated with the mRNA level of AKAP12 in HCC tissues. Secondly, miR-1251-5p significantly affected the luciferase activity of vectors carrying the wt-3′UTR, but not the mut-3′UTR, of AKAP12. Thirdly, miR-1251-5p negatively regulated the AKAP12 expression in HCC cells at both the mRNA and protein level. More importantly, the restoration of AKAP12 expression could reverse the oncogenic function of miR-1251-5p in HCC cells. Results above indicate that AKAP12 mediate the role of miR-1251-5p in HCC. In the present study, it was observed that miR-1251-5p was significantly upregulated in HCC tissues and cell lines, which was associated with poor prognosis. Importantly, miR-1251-5p contributed to the proliferation, migration and invasion of HCC cells by targeting AKAP12 in vitro and in vivo. The present study indicates that miR-12515p may be a potential therapeutic target for HCC treatment.

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5. Conclusion In conclusion, we explored the function of miR-1251-5p in HCC. We observed that miR-1251-5p was upregulated in both HCC tissues and cell lines, which was significantly associated with poor prognosis and clinicopathological features. Functionally, miR-1251-5p drove HCC cell proliferation, migration and invasion in vitro, and facilitated the growth and metastasis of HCC cells in vivo. Mechanistically, AKAP12 which mediated the function of miR-1251-5p on HCC cell proliferation, migration and invasion was screened and verified as the target of miR1251-5p. These results indicate that miR-1251-5p exerts as an oncogene in HCC and may serve as a potential therapeutic target for HCC treatment.

Declaration of Competing Interest The authors declare no conflict of interest.

Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (81602566 and 81874069), and the Natural Science Basic Research Plan in Shaanxi Province of China (2016JQ8029).

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109754. 8

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