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

Long non-coding RNAs are key regulators of tumor development and progression, with the potential to be biomarkers of tumors. This study aimed to explore the role of PlncRNA-1 in the progression of prostate cancer (PCa). We found that PlncRNA-1 was up-regulated in 85.29% of PCa tissues and could predict the T stage of PCa patients to a certain extent. Results showed that inhibition of PlncRNA-1 expression potentially promoted cell apoptosis, suppressed the proliferation, migration, and invasion of cells, and triggered G2/M cycle arrest in vitro and in vivo. PlncRNA-1 was mainly localized in the nucleus and PlncRNA-1 expression and phosphatase and tensin homologue (PTEN) expression were negatively correlated. Mechanistically, knockdown of PlncRNA-1 increased expression levels of PTEN protein and phosphorylated PTEN protein, and decreased expression levels of Akt protein and phosphorylated Akt protein. Rescue experiments demonstrated that PTEN inhibitors abolished the changes in PTEN/Akt pathway caused by PlncRNA-1 interference. PlncRNA-1 can promote the occurrence and development of PCa via the PTEN/Akt pathway. PlncRNA-1 may, therefore, be a new candidate target for the treatment of PCa.


INTRODUCTION
Prostate cancer is the second most common cancer and malignant tumor of the male genitourinary system. In 2020, the American Cancer Society reported an estimated 191,930 new cases of PCa, accounting for 21% of all male tumors and its incidence is the highest of all male tumors [1]. It results in an estimated 33,330 deaths in 2020, making it the second leading cause of cancer-related mortality in men [1]. Nowadays, radical prostatectomy and radiotherapy are the standard treatments for patients with localized PCa, while androgen suppression therapy is the main treatment for recurrent disease and advanced PCa [2]. Although androgen suppression therapy is initially effective, nearly all PCa patients eventually develop metastatic castration-resistant PCa [3]. The average overall survival of patients with metastatic castration-resistant PCa varies between 13 to 32 months, and the 5-year survival rate is less than 15% [4]. Despite the wide spread application of prostate-specific antigen in clinical screening, its low specificity leads to poor diagnosis and treatment [5]. Therefore, more sensitive biomarkers should be developed to improve early detection and diagnosis of PCa.
PTEN is a tumor suppressor gene that is frequently destroyed in a variety of cancers. PTEN, as the most important negative regulator of phosphatidylinositol 3 kinase (PI3K) signaling pathway, has been studied in various research fields due to its ability to regulate diverse physiological functions. Broadly, PTEN inhibits cell proliferation, cell survival, regulates genomic stability, cell migration, energy metabolism, cell structure, stem cell self-renewal, and tumor microenvironment [6,7]. The expression and function of PTEN are altered in cancer [8]. In PCa, deletion of PTEN and alterations of PTEN-PI3K pathway activity have been reported in many advanced PCa [9,10]. Loss of PTEN is strongly associated with poor prognosis of PCa [11]. Therefore, a comprehensive understanding of the pathological role of PTEN will undoubtedly lead to the rational design of new PCa therapies.
In this study, we characterized the biological functions of PlncRNA-1/PTEN in PCa. Expression of PlncRNA-1 in PCa tissues and its correlation with PTEN was evaluated using quantitative real-time polymerase chain reaction (qPCR). Further, we explored how PlncRNA-1 regulates the proliferation, migration, invasion, apoptosis and cell cycle. Finally, the effect of PlncRNA-1 on PTEN was examined using qPCR and western blot (WB) techniques. Our results reveal that PlncRNA-1 maybe a potential therapeutic target in PCa.

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.
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 × 10 4 cells in serum-free medium were seeded on uncoated upper chambers (Costar, NY, USA) to measure cell migration ability. For the invasion assay, 5 × 10 4 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 × 10 7 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 AGING

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 χ 2 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. AGING 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.

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.

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.

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.

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) 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).

AGING
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 AGING 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 AGING 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 AGING 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.

DISCUSSION
Prostate cancer is a common urological tumor and a leading cause of cancer-related deaths in men [34].
Most PCa are androgen-dependent, and hence androgen deprivation therapy is considered the standard first-line treatment for advanced PCa. This is achieved through surgical castration, medical castration, anti-androgen and androgen biosynthesis inhibitors. These therapies effectively relieve symptoms, reduce tumor burden, and prolong patient survival. However, it is unfortunate that hormone deprivation therapy rarely cures cancer itself AGING because most PCa cases recur, leading to deadly castration-resistant PCa [35]. This calls for further studies to determine the mechanism of PCa occurrence and development.
Previous studies have shown that several lncRNAs are dysregulated in tumors, and these affects tumorigenesis and tumor progression [36,37]. The level of lncRNAs can reflect the stage of tumor development in PCa patients [38]. Our study found that the expression level of PlncRNA-1 was significantly higher in 85.29% PCa tissues. Analysis of clinical information of PCa patients showed that PlncRNA-1 was related to the T stage of PCa patients. Consequently, PlncRNA-1 was found to  Several studies have shown that lncRNA-1 can regulate various biological processes in PCa. For instance, lncRNA DSCAM-AS1 and LINC00675 promote the progression of castration-resistant PCa [39], lncRNA SNHG17 regulates the proliferation, invasion, migration, epithelial-mesenchymal transition and apoptosis of PCa cells [8], and lncRNA PCAT7 promotes bone metastasis of PCa [16]. In the present study, we found that in vitro silencing of PlncRNA-1 expression significantly inhibited the proliferation, migration and invasion of PCa cells, promoted cell apoptosis, and caused G2/M cycle arrest. In vivo experiments confirmed that PlncRNA-1 expression significantly decreased the weight and volume of the implanted tumor as well as decreased expression of Ki-67, indicating that in vivo silencing of PlncRNA-1 significantly reduced proliferation ability of PCa cells.
Furthermore, this study shows that the expression level of PTEN was lower in 67.65% PCa tissues than in normal tissues. Correlation analysis revealed an inverse relationship between the expression levels of PlncRNA-1 and PTEN. Results of RNA FISH assay showed that PlncRNA-1 was mainly localized in the nucleus, and a partly in the cytoplasm. Hence, the distribution of PTEN and PlncRNA-1 was nearly similar. Based on the above results, we postulate that PlncRNA-1 and PTEN may interact directly or indirectly. Subsequently, we silenced PlncRNA-1 expression in PCa cells which increased expression of PTEN protein and phosphorylated PTEN protein, and decreased expression levels of Akt protein and phosphorylated Akt protein. It was further observed that treatment with PTEN inhibitors alleviated the changes in the PTEN/Akt pathway caused by PlncRNA-1 silencing.

CONCLUSIONS
These findings demonstrate that PlncRNA-1 is upregulated in PCa tissues and it can predict T stage of PCa patients. In addition, silencing PlncRNA-1 inhibits the proliferation, migration and invasion of PCa cells, promotes apoptosis, and causes G2/M cycle arrest in vitro and in vivo. Mechanistic studies showed that the effects of PlncRNA-1 in prostate cancer were mediated by the PTEN/Akt axis. Therefore, this study reveals that PlncRNA-1 has a significant predictive, diagnostic or therapeutic value in PCa.

AUTHOR CONTRIBUTIONS
ZC, QW, YD and WK performed experiments. HW, MW and JW analyzed the data. HG, DZ and XJ designed the study. NY, PS and TQ wrote the