LncRNA PlncRNA-1 accelerates the progression of prostate cancer by regulating PTEN/Akt axis

Clinical tissue samples and patients’ data

In total, 34 pairs of PCa tissue and matched normal tissues samples were collected from the Department of Urology, Shandong Provincial Hospital, between May 2014 and June 2020. Among them, 18 cases received endocrine therapy before operation, while 16 cases did not. All 34 pairs of PCa tissues and matched normal tissues were used for qPCR analysis. All patients signed informed consent form, and the study was approved by the Ethics Committee of Shandong Provincial Hospital (Jinan, China).

Culture of cell lines

Human PCa cell lines (DU145 and 22Rv1) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 22Rv1 and DU145 cells were cultured in RPMI-1640 (Gibco, CA, USA). All media were supplemented with 10% fetal bovine serum (Invitrogen, CA, USA) and 100 IU/ml penicillin (Sigma, MO, USA). Cell lines were maintained at 37°C and humidified atmosphere of 5% CO₂.

RNA isolation, reverse transcription and quantitative real-time PC

Total RNA was extracted from prepared tissues and cells using RNAiso Plus (Takara, Japan). The total RNA was reverse-transcribed into complementary DNA (cDNA) using the PrimeScript RT Reagent (Takara). PlncRNA-1 expression level was determined by qPCR reactions using the following primer sequences; forward, 5′- TGGCCAGGATCCTCGATAAGAC-3′, reverse 5′- CTTGTAGCCGCCAAGTTTCTGA-3′ using the SYBR Premix Ex Tap (Takara) on the LightCycler 480 II (Roche, Switzerland). Similarly, expression levels of PTEN was determined using primer sequences: forward, 5′-ACACCGCCAAATTTAATTGCAG-3′ and reverse, 5′-TGTCATCTTCACTTAGCCATTGGTC-3′. While, for Akt: forward 5′- AGCGACGTGGCTATTGTGAA-3′ and reverse 5′- CACGTTGGTCCACATCCTG-3′ primers were used. GAPDH mRNA served as the internal control. GAPDH primers used were: forward, 5′-ACACCGCCAAATTTAATTGCAG-3′ and reverse, 5′-ACACCGCCAAATTTAATTGCAG-3′. All experimental procedures were carried out in accordance to the manufacturer's protocol. Expression profiles obtained from qPCR results were analyzed using R program package ‘pcr’.

Cell transfection

siRNA and negative control siRNA vectors targeting PlncRNA-1 were purchased from Sangon Biotech (Shanghai, China). The sequences were: Control siRNA: 5′-UUC UCC GAA CGU GUC ACG UTT-3′; PlncRNA-1 siRNA: 5′-GGC GGC UAC AAG GAA UUA ATT-3′. The vectors were transfected into DU145 and 22Rv1 cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. All transfection experiments were carried out within a period of 48 h. The efficiency of transfection was tested using qPCR analysis.

Assessment of cell viability, migratory, invasion, apoptosis, and cell cycle

The Cell Counting Kit (CCK-8, Dojindo, Japan) assay was used to determine the proliferation of the prostate cancer cell lines. Cells were seeded in 96-well plates and they were then transfected siRNA. Cells were then cultured for 0, 24, 48, 72 and 96 h. At the end of the experiment, 10 μl CCK-8 solution was added into each well and the cells were incubated at 37°C in an incubator with 5% CO₂ for 1 h. Spectrophotometric absorbance values of each sample were recorded at 450 nm using Spectrophotometer Multiskan Go (Thermo Fisher Scientific, Finland). The wound healing assay was used to assess the migration of PCa cells. Briefly, transfected prostate cancer cells were grown to 90% confluence in a six-well plate. A wound was created with a 200 μl sterile pipette tip. The cells were incubated at 37°C with 5% CO₂ and imaged at 0 h, 24 h and 48 h. Transwell assays were performed to determine the migration and invasion of prostate cancer cell lines. After transfection, 3 × 10⁴ cells in serum-free medium were seeded on uncoated upper chambers (Costar, NY, USA) to measure cell migration ability. For the invasion assay, 5 × 10⁴ cells in serum-free medium were seeding on Matrigel-coated (BD Bioscience, CA, USA) upper chambers (Costar, NY, USA). A culture medium containing 10% FBS was added into the lower wells and incubated for further 24 h. Cells in three random fields were counted for the determination of cell migration and invasion. Flow cytometric analysis was performed to determine the cell apoptosis and cell cycle in the prostate cancer cell lines. After transfection with the siRNA or negative control in 6-well plates, the cells were cultured for 48 h. Next, FITC Annexin V Apoptosis Detection Kit (BD Biosciences) was used to analyze cell apoptosis according to the manufacturer's instructions, and DNA Content Quantitation Assay (Cell Cycle) (Solarbio, Beijing, China) was used to analyze cell cycle.

Nude mouse xenograft model

All animals experiments were approved by the Institutional Animal Care and Use Committee of Shandong Provincial Hospital. 1 × 10⁷ cells/0.1 ml DU145 single-cell suspension was injected subcutaneously into 4-week-old male BALB/c nude mice (Vital River, Beijing, China) for tumor xenotransplantation experiments. The length (L), width (W), and estimate of the height (H) of the subcutaneous xenograft tumor were measured every 3 days. The volume of each tumor was calculated according to the formula: V = π/6*(L*W*H), from which a tumor growth curve was drawn. After 4 weeks, nude mice were humanely sacrificed, and tumors were excised, weighed, and imaged.

Western blotting analysis

Protein samples were extracted from cells using RIPA Lysis Buffer (Beyotime, Shanghai, China). The proteins were separated on 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skimmed milk solution for 1 hour. The membrane was treated with the following primary antibodies: anti-PTEN (Cat. #9188, Cell Signaling Technology, Danvers, MA), anti-p-PTEN (Cat. #9549, Cell Signaling Technology), anti-Akt (ab8805, Abcam, MA, USA), anti-p-Akt (ab38449, Abcam), anti-E-cadherin (Cat. #3195, Cell Signaling Technology), anti-E-cadherin (Cat. #13116, Cell Signaling Technology) and anti-vimentin (ab92547, Abcam) and corresponding secondary antibodies conjugated with horseradish peroxidase. Protein bands were visualized using an enhanced chemiluminescence system (Millipore, Bedford, MA) and analyzed with Amersham image 800 (GE). The GAPDH protein (Cat. #5174, Cell Signaling Technology) served as the loading control.

RNA fluorescent in situ hybridization (FISH)

PlncRNA-1 FISH probes were designed and synthesized by the RiboBio Company (Guangzhou, China). The DU145 cells were collected after transfection for 48 h and mounted on glass coverslips. RNA FISH was performed using a fluorescent in situ hybridization kit (RiboBio) following the manufacturer’s instructions. Finally, a fluorescence scanning microscope (Leica, Germany) was used to measure fluorescence of cells.

Immunohistochemistry (IHC)

Tissue samples were paraffinized and analyzed using SP Link Detection kit (Rabbit Biotin-streptavidin HRP Detection Systems) as per the manufacturer’s instructions (ZSGB-Bio, Beijing, China). The sections were visualized under a fluorescent microscope (Vienna, Austria). The image pro plus software was calculated the integrated optical density (IOD) value of immunohistochemistry.

Statistical analysis

All statistical analyses and data visualization were performed using R 3.6.1. The R package ‘edgeR’ was used for differential analysis while R package‘ggplot’ was used to visualize histograms, box plots and line plots. Student’s t-test, ANOVA, Spearman’s rank correlation test and χ² test were used for statistical analysis. Data were presented as the mean ± SD of three independent experiments. A p < 0.05 was considered statistically significant.

Differential expression of PlncRNA-1 in prostate cancer

A total of 34 pairs of PCa and matched normal tissues were collected from the Department of Urology, Shandong Provincial Hospital from March 2014 to June 2020. As shown in Table 1, 18 patients received endocrine therapy before operation while 16 patients did not. The average age of patients was 68.147 ± 6.977 years old with a preoperative prostate-Specific Antigen (PSA) level of 25.335 ± 29.737. From the results, 20 patients recorded a Gleason score of 6–7, while 14 patients had Gleason score of 8–9. In 19 patients, tumors were localized in the prostate, whereas in 15 patients, tumors extended beyond the prostate. Additionally, 6 cases of PCa patients with lymph node metastasis and 28 cases without lymph node metastasis were found.

Compared with normal tissues, PlncRNA-1 mRNA level was significantly elevated in 85.29% (29/34) PCa patients (p < 6.9e-06) (Figure 1A–1B). Subgroup analysis showed that the expression level of PlncRNA-1 in PCa patients with T3-T4 stage was significantly higher than in those with T2 stage (p < 0.02) (Figure 1C). However, the expression level of PlncRNA-1 was not significantly related with age, total preoperative PSA level, preoperative treatment, Gleason score, tumor size, and lymph node metastasis of PCa patients (Figure 1D–1I). PCa patients were divided into high and low expression levels of PlncRNA-1 based on the median value of 2.696. As noted from Table 1, the proportion of patients with T3-T4 PCa was 64.71% (11/16) in the high PlncRNA-1 expression group, whereas the proportion of patients with T3-T4 PCa was 23.53% (4/17) in the low PlncRNA-1 expression group, and the difference was significant (p < 0.038). In other words, if a patient's expression level of PlncRNA-1 exceeded 2.696, the probability of the patient being diagnosed with T3-T4 staging was 64.71%. Conversely, if a patient's expression level of PlncRNA-1 was less than 2.696, the probability of the patient being diagnosed with T3-T4 staging was 23.53%. PlncRNA-1 showed the potential to predict the T stage of PCa patients to a certain extent. In the high and low PlncRNA-1 expression groups, age, preoperative treatment, preoperative PSA level, Gleason score, and lymph node metastasis were not correlated with PlncRNA-1 expression. Hence, the expression level of PlncRNA-1 may be utilized as a predictor of the clinical stage of PCa.

Figure 1. The level of PlncRNA-1 expression in PCa tissues. (A–B) The expression level of PlncRNA-1 in 34 pairs of PCa and normal matched tissues. (C–I) The correlation between the expression level of PlncRNA-1 and T stage, age, preoperative tPSA, preoperative treatment, Gleason score, tumor size, and N stage.

PlncRNA-1 promotes the proliferation, migration and invasion of PCa cells in vitro

As an oncogene, PlncRNA-1 is highly expressed in PCa tissues. Therefore, siRNA was synthesized to interfere with the expression of PlncRNA-1 in PCa DU145 cells and 22Rv1 cell lines. Compared with the control group, the expression level of PlncRNA-1 in the siPlncRNA-1 group was significantly lower indicating that siRNA effectively reduced the expression of PlncRNA-1 in PCa DU145 cells and 22Rv1 cell lines (Figure 2A). Moreover, results of the CCK-8 showed that PlncRNA-1 significantly reduced the proliferation ability of DU145 cells and 22Rv1 cells in vitro (Figure 2B–2C). The wound healing assays demonstrated that the migration ability of DU145 cells and 22Rv1 cells began to decrease at 24 hours after silencing PlncRNA-1 expression (Figure 2D–2G). After 48 hours of transfection, the migration ability of PCa DU145 cells and 22Rv1 cells was significantly reduced. The transwell experiment revealed that the migration and invasion of PCa DU145 cells and 22Rv1 cells were inhibited following PlncRNA-1 silencing (Figure 2H–2J). These findings indicate that inhibition of PlncRNA-1 in vitro can significantly inhibit the proliferation, migration and invasion of PCa cells.

Figure 2. Effect of PlncRNA-1 knockdown on the proliferation, migration, and invasion of PCa cells. (A) qPCR analysis for the transfection efficiency of PlncRNA-1-siRNA in 22Rv1 and DU145 PCa cells. (B–C) CCK-8 assay for the proliferation of 22Rv1 and DU145 PCa cells after PlncRNA-1 silencing. (D–G) Wound healing assays for the migration of 22Rv1 and DU145 PCa cells after PlncRNA-1 silencing (Scale bar, 200 μm). (H) Transwell migration and invasion assays for the migration and invasion of 22Rv1 and DU145 PCa cells after PlncRNA-1 silencing (Scale bar, 100 μm). (I–J) Quantitative analysis transwell migration (I) and invasion (J) assays of 22Rv1 and DU145 PCa cells after PlncRNA-1 silencing.

PlncRNA-1 regulates cell apoptosis and cell cycle in vitro

Flow cytometry was used to detect the effect of PlncRNA-1 on cell apoptosis. The number of apoptotic 22Rv1 cells increased from 6.05% to 17.88% following PlncRNA-1 silencing compared with the control group and the difference was statistically significant (P < 0.031) (Figure 3A and 3B). Similarly, the number of apoptotic DU145 cells increased from 8.00% to 15.71% (p < 0.014) after silencing PlncRNA-1 expression (Figure 3A and 3B). This indicated that silencing PlncRNA-1 expression promoted apoptosis of PCa cells. Further analysis showed that PlncRNA-1 blocked the cell cycle of 22Rv1 cells at G2/M cycle. Notably, the number of cells in the G2/M cycle increased from 11.14% to 23.66% following PlncRNA-1 silencing (Figure 3C and 3D). Similarly, the number of DU145 cells in the G2/M cycle increased from 9.37% to 20.58% following PlncRNA-1 silencing (Figure 3C and 3D). These results show that silencing PlncRNA-1 expression can cause G2/M cycle arrest in PCa cells.

Figure 3. Flow cytometry detects the changes in apoptosis (A–B) and cell cycle (C–D) in 22Rv1 and DU145 PCa cells after interference with PlncRNA-1.

Silencing PlncRNA-1 expression inhibits the tumorigenicity of PCa cells in vivo

DU145 cells in which PlncRNA-1 was silenced were subcutaneously injected into nude mice. This resulted in inhibition of tumor growth after 4 weeks compared with the control group (Figure 4A–4B). Silencing PlncRNA-1 expression decreased the volume (Figure 4C) and weight (Figure 4D) of the implanted tumor. IHC analysis of the implanted tumor showed that silencing PlncRNA-1 expression inhibited Ki-67 expression (Figure 4E–4F). These results proved that inhibiting PlncRNA-1 expression in vivo significantly inhibited the tumorigenicity of PCa cells.

Figure 4. Xenograft tumor formation of DU145 cells in PlncRNA-1-silenced nude mice. (A) Photograph of nude mice with Xenografted tumors on day 28. (B) Pictorials of subcutaneous tumors on day 28. (C) Growth curves for xenografted tumors. (D) The tumor weights in different groups. (E) Immunohistochemistry assay for the expression of Ki-67 in the tumor xenografts (Scale bar, 100 μm). (F) Quantitative analysis immunohistochemistry assay for the expression of Ki-67 in the tumor xenografts.

Correlation between PlncRNA-1 and PTEN expression

In vivo and in vitro experiments showed that PlncRNA-1 regulated the proliferation, migration, invasion, apoptosis and cell cycle of PCa. However, it was not clear whether PlncRNA-1 could down-regulate PTEN. Thus, we assessed the relationship between PlncRNA-1 and PTEN in 34 pairs of PCa tissues. The mRNA expression level of PTEN in 67.65% (23/34) of PCa tissues was significantly lower than that of normal tissue (p < 0.0032), (Figure 5A–5B). Similarly, the expression level of PTEN protein in PCa tissue was lower than in normal tissue as revealed by IHC (Figure 5C–5D). A negative relationship was found between PlncRNA-1 and PTEN expression (R = –0.28, p < 0.021) in PCa tissues (Figure 5E)

Figure 5. The relationship between PTEN and PlncRNA-1. (A–B) The expression level of PTEN in 34 pairs of PCa and normal matched tissues. (C) Immunohistochemistry assay for the expression of PTEN in the PCa and normal matched tissues (Scale bar, 100 μm). (D) Quantitative analysis immunohistochemistry assay for the expression of PTEN in the PCa and normal matched tissues. (E) The correlations between level of PlncRNA-1 expression and PTEN in PCa tissues. (F) RNA-FISH images for subcellular localization of PlncRNA-1 in PCa cells (Scale bar, 50 μm).

RNA Fish was used to locate the position of PlncRNA-1 in the cell. We choose U6 and 18S as internal reference genes. U6, exhibiting a red Cy3 fluorescence was distributed in the nucleus, and 18S with a red Cy3 fluorescence was distributed in the cytoplasm. We found that PlncRNA-1 was mainly distributed in the nucleus and partly distributed in the cytoplasm (Figure 5F). PTEN is normally distributed in the nucleus and cytoplasm, almost similar to PlncRNA-1. Based on the similar subcellular localization and negative relationship between PlncRNA-1 and PTEN, we speculate that they may be mutually regulated.

PlncRNA-1 regulates PTEN/Akt pathway in prostate cancer cells

To determine the regulatory mechanism between PlncRNA-1 and PTEN, we silenced PlncRNA-1 expression in DU145 and 22Rv1 cells to measure the expression levels of PTEN and Akt with qPCR and WB. Notably, expression level of PTEN mRNA increased, whereas the expression level of Akt mRNA decreased after PlncRNA-1 silencing (Figure 6A). The protein level of PTEN and phosphorylated PTEN were increased after PlncRNA-1 silencing in the PCa DU145 and 22Rv1 cells (Figure 6B–6C). However, the expression levels of Akt protein and phosphorylated Akt protein were decreased after PlncRNA-1 silencing in PCa DU145 and 22Rv1 cells (Figure 6B–6C).

Figure 6. Regulation of PTEN expression by PlncRNA-1. (A) qPCR analysis for the expression level of PTEN and Akt in PlncRNA-1 silent PCa cells. (B) Western blot for the expression level of PTEN and Akt in siPlncRNA-1 group. (C) The histogram shows the statistical analysis of Western blot in Figure 6B. (D–E) The relative expression levels of PTEN and Akt protein were detected in difference group by Western blot. (F) The histogram shows the statistical analysis of Western blot in Figure 6D–6E.

To further explore whether the function of PlncRNA-1 was mediated via the PTEN/Akt axis, we conducted a rescue experiment in the PCa DU145 and 22Rv1 cells. The experiment was performed in four groups: control group, siPlncRNA-1 group, PTEN inhibitor (SF1670) group and the siPlncRNA-1+SF1670 group. In order to verify the efficiency of SF1640 in inhibiting PTEN/ Akt pathway, compared with the control group, the expression levels of PTEN protein and phosphorylated PTEN protein decreased, whereas the expression levels of phosphorylated Akt protein increased in SF1670 group (Figure 6D–6F), which providing evidence that PTEN inhibitors could effectively inhibit PTEN/ Akt pathway. Compared with the control group, silencing PlncRNA-1 expression with PCa DU145 and 22Rv1 cells increased PTEN protein and phosphorylated PTEN protein levels but decreased the expression levels of Akt protein and phosphorylated Akt protein in siPlncRNA-1 group (Figure 6D–6F). Compared with siPlncRNA-1 group, treatment with PTEN inhibitor in siPlncRNA-1 group significantly inhibited the upregulation of PTEN and phosphorylated PTEN induced by PlncRNA-1 interference and promoted the downregulation of Akt and phosphorylated Akt induced by PlncRNA-1 interference (Figure 6D–6F). Collectively, these results showed that PlncRNA-1 regulated PTEN/Akt axis in PCa cell lines.