lncRNA HCG11 regulates cell progression by targeting miR-543 and regulating AKT/mTOR pathway in prostate cancer

Patients and tissue specimens

This experiment was approved by the Research Ethics committee of the Fourth Hospital of Hebei Medical University. Twenty pairs of PCa tissues (non-metastasis and metastasis tissues) and adjacent normal tissues were obtained from patients following informed written consent. All patients did not receive any pre-operation treatment.

Cell culture and transfection

Human prostate normal cell lines RWPE1 and human PCa cell lines LNCaP, PC-3, and C4-2B as well as HEK293T cell lines were obtained from RiboBio Co. (Guangzhou, China) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), penicillin and streptomycin at 37°C with 5% CO₂. Medium was changed every 2 days.

pcDNA3.1-miR-543 (MiR-17-5p mimics, 5′-AAACAUUCGCGGUGCACUUCUU-3′), pcDNA3.1-HCG11(pcDNA-HCG11) and their corresponding negative control (pcDNA3.1-NC, pcDNA3.1-vector) were synthesized and purchased from GenePharma (Shanghai, China). Small interfering RNA (siRNA) (Silencer R; Life Technologies, Shanghai, China) and siRNAs targeting miR-543 (anti-miR-543, 5′-AAGAAGUGCACCGCGAAUGUUU-3′) were constructed by GenePharma. Lipofectamine 2000 (Invitrogen, CA, USA) was applied to transfect all plasmid and oligos according to the manufacturer's instructions.

Quantitative real-time polymerase chain reaction

Total RNAs were extracted from tissues and cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA concentration was measured using spectrophotometry. Complementary DNA (cDNA) of miRNA was reverse transcribed using TaqMan miRNA Reverse-Transcription Kit (Applied Biosystems, Foster City, CA, USA). cDNA of messenger RNA (mRNA) was reverse transcribed using Reverse-Transcription Reagents (Applied Biosystems). Real-time qPCR was performed using SYBRH Select Master Mix for CFX (Invitrogen) following the manufacturer's instructions. The fluorescence was detected in an ABI 7300 System (Applied Biosystems). lncRNA and mRNA expression were normalized according to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). miRNA expression was normalized according to the expression of U6. method was used to calculate the relative expression of miRNA and mRNA. The primer sequences: lncRNA HCG11, forward, 5′-GCTCTATGCCATCCTGCTT-3′ and reverse, 5′-TCCCATCTCCATCAACCC-3′.

GAPDH, forward, 5′-TGTTCGTCATGGGTGTGAAC-3′ and reverse, 5′-ATGGCATGGACTGTGGTCAT-3′.

miR-543, forward, 5′-CCAGCTACACTGGGCAGCAGCAATTCATGTTT-3′ and reverse 5′-CTCAACTGGTGTCGTGGA-3′.

U6, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′.

Western blot

Total protein was extracted from cells lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, San Jose, CA, USA). Protein concentration was detected using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Then, the equivalent weights of protein samples (30 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. Then, the membrane was blocked in Tris-buffered saline (TBS) with 5% no-fat milk. Subsequently, the membrane was incubated with primary antibody against proliferating cell nuclear antigen (PCNA), cleavage-caspase 3 (cle-caspase 3), N-cadherin, E-cadherin, phosphorylation-AKT (p-AKT), AKT, p-mammalian target of rapamycin (mTOR), mTOR, and GAPDH (1:2,000; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After being washed with TBST buffer, the membrane was incubated with secondary antibodies goat anti-mouse immunoglobulin G (1:2,000 dilution; Cell Signaling Technology) at 37°C for 1 h. The western blot was detected using ECL detection kit (Thermo Fisher Scientific, Rockford, IL, USA), optical densities were measured using ChemiDoc XRS System (Bio-Rad).

Cell proliferation and apoptosis

Cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded into 96-well plates at a density of 2 × 10³ cells per well after transfection. Cultured 48 h later, 20 µL MTT solution (5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added into each well to stain all cells and the plate was incubated 37°C for 4 h. Then 200 µL of dimethyl sulfoxide (DMSO; Sigma-Aldrich) were added into each well. Cell proliferation was performed by detecting the absorbance at 490 nm using a Multiskan Ascent 354 microplate reader (Thermo Labsystems, Waltham, MA, USA).

Cell apoptosis was measured using flow cytometry. Cells were trypsinized and washed with phosphate-buffered saline. Then, each sample was stained with FITC-Annexin V and PI (BD Biosciences, San Jose, CA, USA) for 30 min. Cell apoptosis was determined by flow cytometry (BD Biosciences).

Cell invasion and migration

Cell invasion was performed using Transwell chamber with Matrigel (BD Biosciences) and cell migration was displayed using Transwell chamber without Matrigel. 1 × 10⁵ cells were seeded into the upper chamber and DMEM with 10% FBS was added into the lower chamber. After the plate was incubated at 37°C for 24 h, the cells on the upper chamber were wiped away. Then, the membrane was fixed with 90% ethyl alcohol at 37°C for 10 min and was stained with 0.2% crystal violet for 30 min. The cell migration and invasion were counted using an inverted microscope.

Dual-luciferase reporter assay

3′-Untranslated region (3′-UTR) of lncRNA HCG11 was amplified using PCR and inserted into pMIR-REPORT™ (Thermo Fisher Scientific) to construct XISY wild-type reporter vector (HCG11-wt). The HCG11 mutant-type (HCG11-mut) was made with GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific). Then, HCG11-WT or HCG11-MUT and miR-NC or miR-543 were transfected into HEK293T cells. Otherwise, HCG11-wt or HCG11-mut and anti-NC or anti-miR-543 were transfected into HEK293T cells. After transfection for 48 h, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) following the protocols. The Renilla luciferase activity was normalized to calculate firefly luciferase activity.

Animal experiment

HCG11 and negative control (vector) were stably transfected into C4-2B cell lines. Then the transfected cell lines were injected subcutaneously to the right side of the posterior flank of female BALB/c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd. China) at 4–5-week-old. Tumor volumes were measured every 5 days. After injected for 30 days, all mice were sacrificed. Tumor xenografts were collected and weighted. The animal experiment was approved by the Institutional Animal Care and Use Committee of the Fourth Hospital of Hebei Medical University.

Statistical analysis

The analysis of all data was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). All comparisons were analyzed using a Student's t test to assess the significance of the results unless otherwise specified. Pearson's correlation analysis was used to analyze the correlation between HCG11 expression and miR-543 expression. P < 0.05 was considered to be statistically significant.

LncRNA HCG11 was downregulated in PCa tissues and cells

To confirm the expression of HCG11 in PCa tissues and cells, qRT-PCR assay was used in this experiment. As shown in Figure 1A, HCG11 has lower expression in non-metastasis and metastasis tissues compared with that in normal tissues. This result indicated that lncRNA HCG11 was downregulated in PCa tissues and was associated with metastasis in PCa. Then, we obtained the normal cell line (RWPE1) and PCa cell lines (LNCaP, PC-3, and C4-2B) for the following experiments, the results showed that HCG11 expression was obviously decreased in prostatic carcinoma cells compared with that in normal cell lines (RWPE1) and was lowest in C4-2B cell line (Figure 1B).

Upregulated lncRNA HCG11 expression inhibited cell growth and induced apoptosis in PCa

To further investigate the function of HCG11 in PC, we constructed the cell lines of transfection with HCG11, which significantly increased HCG11 expression (Figure 2A). Then the cell viability of HCG11 was measured using the MTT assay. The result showed that overexpressed HCG11 was significantly lower than that of vector and control cell lines (Figure 2B). Meanwhile, flow cytometry showed that the apoptosis cells with overexpressed HCG11 were sharply increased compared with that of vector and control cells (Figure 2C). Additionally, high HCG11 expression significantly inhibited the PCNA protein level and induced cle-caspase 3 protein expression (Figure 2D).

More than that, we also investigated the effects of HCG11 on tumor metastasis and invasion. The transwell assay displayed that the metastasis and invasion of PC cells lines was decreased by HCG11 overexpression (Figures 2E and 2F). Moreover, the protein level of N-cadherin was decreased and the protein level of E-cadherin was increased by upregulating HCG11 (Figure 2G). Therefore, these results showed that upregulated HCG11 expression could inhibit cell proliferation, metastasis, and invasion as well as induced cell apoptosis in PCa.

miR-543 was a target miRNA of lncRNA HCG11

On the basis of the bioinformatics analysis, we found that miR-543 had complementary sequences with HCG11 3′-UTR (Figure 3A). The results of luciferase reporter assay showed that miR-543 mimic decreased the luciferase activity by binding with HCG11-3′-UTR (wt), while HCG11-mut has no effect with it (Figure 3B). Moreover, anti-miR-543 increased the luciferase activity by binding with HCG11-3′-UTR (wt) rather than HCG11-mut (Figure 3C). In addition, overexpression of HCG11 greatly decreased the expression of miR-543, whereas downregulation of HCG11 remarkably increased the miR-543 expression (Figure 3D).

Then, we further investigated the association between HCG11 and miR-543 using qRT-PCR. As shown in Figures 3E and 3F, miR-543 was upregulated in PCa tissues and cells compared with normal tissues and cells (Figures 3E and 3F). Moreover, there was a negative correlation between HCG11 and miR-543 expression in PC tissues (Figure 3G). These results showed that the suppressive effect of HCG11 occurred via inhibiting miR-543 expression.

miR-543 was involved in HCG11-mediated regulation of cell proliferation, apoptosis, migration, and invasion in PCa cells

Next, we investigated the underlying mechanism of HCG11 in PCa cells and validated the function of miR-543 in HCG11-mediated PCa process. The control, vector and HCG11 were transfected into PCa cells, respectively. Meanwhile, HCG11 was co-transfected with miR-543 or miR-NC into PCa cells, respectively. HCG11 transfection significantly decreased the miR-543 expression (Figure 4A). In addition, compared with HCG11 + miR-NC group, the expression of miR-543 was obviously increased in HCG11 + miR-543 group (Figure 4A). Thus, miR-543 transfection improved miR-543 expression. According to the results of MTT assay and flow cytometry, we found that the overexpression of HCG11 inhibited cell proliferation and induced cell apoptosis in PCa cells, while the overexpression of miR-543 significantly blocked the effects of high HCG11 expression on PCa cell proliferation and apoptosis (Figures 4B and 4C). Meanwhile, Figure 4D revealed that PCNA protein expression was inhibited but cle-caspase 3 protein expression was induced by overexpression of HCG11, while these effects were impaired by upregulating miR-543.

Furthermore, transwell migration and invasion analyses performed that HCG11 transfection significantly decreased PCa cell invasion and migration, which were restored by miR-543 overexpression (Figures 4E and 4F). More than that, overexpression of HCG11 inhibited N-cadherin protein expression, whereas enhanced E-cadherin protein expression, upregulation of miR-543 reversed the effects of HCG11 transfection on the protein level of N-cadherin and E-cadherin (Figure 4G). These findings demonstrated that miR-543 was a functional target miRNA of HCG11 and that upregulation of miR-543 could reverse the effects of high HCG11 expression on PCa cell proliferation, apoptosis, invasion, and migration.

LncRNA HCG11 inhibited phosphoinositide-3 kinase/protein kinase B (PI3K/AKT) pathway to suppress PCa progression

PI3K/AKT pathway is an important signaling pathway in the regulation of cell cycle, which is associated with oncogenesis and development (Skeen et al., 2006; Iżycka-Świeszewska et al., 2010). P-AKT, AKT, p-mTOR, and mTOR were crucial proteins of PI3K/AKT pathway, which belonged to a part of the PI3K/PKB/AKT/mTOR pathway. P-AKT and p-mTOR protein expressions were significantly inhibited or induced, while AKT and mTOR protein expressions showed no significant changes, suggesting that PI3K/AKT pathway was inhibited or upregulated, so we measured their protein levels with western blot. The results showed that upregulation of HCG11 significantly reduced p-AKT and P-mTOR protein expression, whereas overexpression of miR-543 largely reversed the effects of upregulation of HCG11 on P-AKT and p-mTOR protein expression in PCa cells. However, AKT and mTOR protein expression has no significant change in all groups (control, vector, HCG11, HCG11 + miR-NC, and HCG11 + miR-543 groups) (Figure 5). These findings showed that lncRNA HCG11 affected PCa cell progression by inhibiting PI3K/AKT pathway.

LncRNA HCG11 regulated tumor growth by inhibiting miR-543 in vivo

To further verify the role of lncRNA HCG11 in tumor growth in vivo, C4-2B cells transfected with vector or HCG11 were injected into the flank of nude mice, respectively. As shown in Figure 6A, after injection for 25 days to 30 days, tumor volume of HCG11 was significantly lower than that of vector. The mice underwent surgery and the tumor weight was measured after 30 days of injection, tumor weight was notably reduced in HCG11 group compared with that in vector group (Figure 6A). Additionally, HCG11 transfection improved HCG11 expression but reduced miR-543 expression (Figures 6B and 6C). These findings demonstrated that HCG11 inhibited tumor growth via downregulating miR-543 in PCa.