Overexpression of miR-623 suppresses progression of hepatocellular carcinoma via regulating the PI3K/Akt signaling pathway by targeting XRCC5

2.1 Patients and specimens

The study was approved by the Research Ethics Committee of Ningbo No.2 Hospital. A total of 53 pairs of human HCC tissues and their corresponding adjacent liver tissues were collected from patients (male/female: 38/15, age ≤50/>50 years: 29/24) undergoing liver resection surgery at the Ningbo No.2 Hospital between February 2012 and January 2018. Written informed consent was obtained from all the patients. Clinicopathologic histories for these patients are shown in Table 1. Patients were subcategorized in either the miR-623 downregulated group (n = 39) or the intact group (n = 14). We did this by comparing the median value of miR-623 expression among the HCC tissue samples that were analyzed.

2.2 Cells culture

Liver cancer cell lines Hep3B, 7721, Huh7, HepG2, and Bel-7402 cells were seeded in Roswell Park Memorial Institute-1640 (HyClone) medium enriched with 10% fetal bovine serum (FBS). 7721, Huh7, and Bel-7402 cells were transiently transfected with either the pCMV-MIR-miR-623 plasmid (2.5 µg) when they grew to 80% fusion, and pCMV-MIR was performed as negative control (NC).

For XRCC5 transfection, cells (2 × 10⁴) were grown in a six-well plate and cultured for 24 hours and transfected with siRNA-XRCC5 (50 nM; Ribobio) using Lipofectamine 2000 to block the expression of XRCC5, and scrambled siRNA was used as negative control. The expressing vector (pcDNA3.1-XRCC5; Ribobio) was conducted and cells were cotransfected with miR-623 and XRCC5 using Lipofectamine 2000, and the blank vector was used as negative control. The siRNA-XRCC5 sequence was 5′-GGATGTGATTCAACATGAA-3′.

2.3 Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA from HCC cells were extracted by means of the Ultrapure RNA Kit (Cat #CW057; CWBIO). Complementary DNA was reverse-transcribed from RNA by use of the miRNA cDNA Synthesis Kit (Cat #CW2141; CWBIO). The related expression of miR-623 was then measured by the miRNA qPCR Assay Kit (Cat #CW2142S; CWBIO). The primer used in this study was synthesized from (Ribobio) miR-623: 5′-AUCCCUUGCAGGGGCUGU-3′; XRCC5: 5′-CCATGAGCTTGGCAAAGAAAG-3′, 5′-GTGCAGCAGACACTGAAATAATC-3′; β-actin: 5′-CCCGAGCCGTGTTTCCT-3′, 5′-GTCCCAGTTGGTGACGATGC-3′.

2.4 Western blot assay

After being transfected for 48 hours, 20 μg of samples extracted from cells were then loaded to each lane in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and were electrotransferred to the polyvinylidene fluoride membrane, which was then blocked with 5% nonfat milk followed by incubation with primary antibodies and with secondary antibodies for 1 hour. The ECL substrate was applied to visualize the protein bands. Glyceraldehyde 3-phosphate dehydrogenase was used as the loading control and the relative expression of proteins was normalized to NC group.

2.5 CCK-8 assay

After being transfected for 24 hours, cells were grown in a 96-well plate and then incubated with cell counting kit-8 (CCK-8) reagent (10 μL/well; CWBIO) for 1.5 hour. The optical density value was measured every 24 hours at 450 nm.

2.6 Colony formation assay

Cells were grown into a 6-cm plate and incubated with 5% CO₂ for 10 days. After washing with phosphate-buffered saline, colonies were fixed with 4% paraformaldehyde followed by staining with 500 μL of GIEMSA (Cat #AR-0752; DingGuo Bio, Shanghai, China) for 10 minutes.

2.7 Transwell assay

Transwell chambers coated with Matrigel were performed for assessment of cell invasion. About 1 × 10⁴ of HCC cells were seeded in the upper compartment, and the lower compartment was filled with 600 µL of medium supplemented with FBS. After incubation for 24 hours, the invaded cells were treated with 4% paraformaldehyde, with 0.1% crystal violet and then stained for 5 minutes. The stained cells were captured (magnification, ×100). Transwell chamber did not require Matrigel in the migration assay.

2.8 Apoptosis assay

The Annexin V-FITC- PI Apoptosis Detection Kit (4A Biotech, Beijing, China) was used to measure the rate of apoptosis in HCC cells. The rate of apoptosis was calculated by using the BD FACSDiva Software (BD FACSC anto II, BD Biosciences, Bedford MA).

2.9 Dual-luciferase reporter assay

A total of 5 × 10⁵ HCC cells were inoculated into culture plates. After incubation for 24 hours, luciferase reporter plasmids, pmirGLO-XRCC5-3′UTR-wild type (wt) or pmirGLO- XRCC5-3′UTR-mutant type (mut), were cotransfected with either pCMV-pMIR-miR-623 (miR-623) or pCMV-MIR (NC) into the HCC cells. The luciferase activity of cells was measured when cells were cultured for 48 hours.

2.10 Statistical analysis

The data were shown as means ± SD and statistically analyzed by the SPAA 18.0 saftware (SPSS Inc., Chicago, IL). The differences between groups were analyzed using the Student t test or one-way analysis of variance. The association between two variables was assessed by the χ² test. Pearson's correlation coefficient was used for correlation analysis.

3.1 Downregulation of miR-623 is correlated with poor clinical outcomes of HCC

Our data illustrated that miR-623 was downregulated in 73.6% (39/53) HCC tissues (P < .01; Figure 1A) when compared with the corresponding normal liver tissues. Moreover, the expression of miR-623 was correlated with HBV-DNA load (P = .010), tumor size (P = .0278), vascular invasion (P = .0136), and tumor stages (P = .0328) (Table 1). Collectively, miR-623 may play a role in the progression of HCC.

3.2 Overexpression of miR-623 suppresses the proliferation and viability of HCC cells

The expression of miR-623 in different liver cancer cell lines were detected, and we observed that the level of miR-623 in 7721, Huh7, and Bel-7402 cells was much low (Figure 1B; P < .0001). The pCMV-MIR-miR-623 plasmid was transfected into 7721, Huh7, and Bel-7402 cells to investigate the effects of miR-623 on HCC cells (Figure 1C). As shown in Figure 1 D, overexpression of miR-623 caused a significant reduction in the viability of 7721 cells. Similar inhibition caused by upregulation of miR-623 was also observed in Huh7 and Bel-7402 cells (Figure 1E, F). Moreover, our results showed that the colony count was markedly decreased in miR-623 overexpressed cells (Figure 1G,H). Collectively, these results suggested an antigrowth role of miR-623 in HCC cells.

3.3 Upregulation of miR-623 inhibits cell migration and invasion in HCC cells in vitro

To investigate the effect of miR-623 on HCC cell mobility, a Transwell assay was performed. Our data confirmed that upregulation of miR-623 reduced the migration abilities of 7721, Huh7, and Bel-7402 cells (Figure 2A-D). In addition, cell invasion abilities were obviously suppressed by upregulation of miR-623 (Figure 2E-H).

3.4 Overexpression of miR-623 induces apoptosis

We found that rate of cell apoptosis was significantly promoted by upregulation of miR-623 in HCC cells (Figure 3A,B). Moreover, we observed that the expression of Bcl-2 was downregulated in miR-623 overexpressed cells, and the level of both total casapse3 and total caspase9 was decreased, while the expression of Bax, cleaved caspase3 and cleaved caspase9 was significantly increased (Figure 3C,D). These data suggested that upregulation of miR-623 induced apoptosis in HCC cells might be done through the mitochondrial apoptotic pathway.

3.5 Upregulation of miR-623 suppresses the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways

The PI3K/Akt pathway is indispensable in cell growth and survival as it regulates cell cycle–related proteins and Bcl-2-related proteins, both of which have been confirmed to be involved in HCC progression.22, 25 As shown in Figure 4A,B, we found that overexpression of miR-623 did not affect the total level of Akt and mammalian target of rapamycin (mTOR), whereas phosphorylated form p-Akt and p-mTOR was significantly inhibited in HCC cells. In addition, overexpression of miR-623 also downregulated the expressions of cyclin D1 and p70S6K in HCC cells (Figure 4A,B). Collectively, our data suggested that upregulation of miR-623 inhibited the PI3K/Akt signaling pathway.

Moreover, we found that the expression of Wnt3 and β-catenin were decreased in miR-623 overexpressed cells (Figure 4C,D). In addition, we observed that the expression levels of downstream protein E-cad were different in the three cell lines, which was due to the diversity of cell lines. However, the expression of E-cad was upregulated by miR-623 in 7721, Huh7, and Bel-7402 cells (Figure 4C,D). Together, these data suggested that the Wnt/β-catenin pathway was also inhibited by the overexpression of miR-623 in HCC cells in vitro. In addition, upregulation of miR-623 decreased the phosphorylated forms of ERK1/2 and JNK (Figure 4E,F), suggesting that upregulation of miR-623 could inhibit the ERK/JNK pathway in HCC cells.

3.6 miR-623 directly targets XRCC5 3ʹUTR in HCC

As shown in Figure 5A, we found that XRCC5 3′UTR had a putative binding site for miR-623 from the TargetScan database. To validate it, cells were cotransfected with miR-623 and pmirGLO-XRCC5 3′UTR-wt or pmirGLO-XRCC5 3′UTR-mut. We observed that miR-623 clearly inhibited the luciferase activity of XRCC5-3′UTR in HCC cells (Figure 5B), whereas the mutant counterpart blocked the same decrease caused by miR-623, indicating that XRCC5 was a target of miR-623. Western blot analysis revealed that the protein expression of XRCC5 was significantly downregulated by miR-623 (Figure 5C). Collectively, these results indicated that miR-623 directly bound to XRCC5 and downregulated its expression in HCC cells.

3.7 Upregulation of XRCC5 reversed the miR-623-induced suppression for HCC

We predicted that the suppressive effect of miR-623 on growth and metastasis of HCC might be mediated by regulating XRCC5. To validate this prediction, HCC cells were cotransfected with XRCC5 over-expressing vector (lacking its 3′UTR) and pCMV-MIR-miR-623; and siRNA targeting XRCC5 was transfected into HCC cells to block XRCC5 (Figure S1A and S1B). As shown in Figure 5D, silencing XRCC5 significantly inhibited the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways by downregulating p-Akt, Wnt3, and p-ERK1/2, similar to that seen in HCC cells transfected with miR-623. Moreover, the inhibitory effect on p-Akt, Wnt3, and p-ERK1/2 caused by overexpression of miR-623 was significantly reversed by upregulation of XRCC5 (Figure 5D). Furthermore, as shown in Figure 6A-C, upregulation of XRCC5 (Figure S1C and S1D) reversed the suppression of cell proliferation induced by overexpression of miR-623 in HCC cells. In addition, the decreased migration and invasion capabilities induced by overexpression of miR-623 in HCC cells were significantly restored by the upregulation of XRCC5 (Figure 6D,E). Therefore, miR-623 may inhibit cell growth and invasion abilities of HCC cells through targeting XRCC5, and the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways may be involved.