Blockage of MDM2‐mediated p53 ubiquitination by yuanhuacine restrains the carcinogenesis of prostate carcinoma cells by suppressing LncRNA LINC00665

Prostate cancer (PCa) is a challenging issue for men's health worldwide due to its uncontrolled proliferation and high metastatic potential. Increasing evidence has supported plant extracts and natural plant derivatives as promising antitumor therapy with less toxic side effects. Yuanhuacine is an active component isolated from Daphne genkwa and can effectively suppress the tumorigenesis of several cancers. However, its role in PCa remains unclear. In this study, yuanhuacine dose‐dependently inhibited the proliferation and induced apoptosis of PCa cells. Moreover, yuanhuacine also restrained the invasion and migration of PCa cells. Mechanically, yuanhuacine decreased the ubiquitination and degradation of p53 protein, and ultimately increased p53 levels, which was regulated by inhibiting the phosphorylation and total protein levels of mouse double minute 2 (MDM2). Moreover, elevation of MDM2 reversed the suppressive efficacy of yuanhuacine in PCa cell viability, invasion, and migration. The network pharmacologic and bioinformatics analysis confirmed that MDM2 might be a common target of D. genkwa and LINC00665. Furthermore, yuanhuacine inhibited LINC00665 expression. Upregulation of LINC00665 reversed yuanhuacine‐mediated inhibition in MDM2 protein expression and suppressed p53 levels by enhancing its ubiquitination in yuanhuacine‐treated cells. Importantly, the inhibitory effects of yuanhuacine on cell viability and metastatic potential were offset after LINC00665 elevation. Together, the current findings highlight that yuanhuacine may possess tumor‐suppressive efficacy by inhibiting LINC00665‐mediated MDM2/p53 ubiquitination signaling. Therefore, this study indicates that yuanhuacine may be a promising candidate for the treatment of PCa.


| INTRODUCTION
Prostate cancer (PCa) constitutes to be a major public health problem for men worldwide and is the second leading cause of cancer-related death. Epidemiologic statistical assay reveals that PCa accounts for 54% of all cancer incident cases in men. [1] Currently, the incidence of PCa remains stable from 2014 through 2018, whilst the proportion of PCs diagnosed at a distant stage continues to increase from 3.9% to 8.2% over the past decade. [2] Approximately 268,000 new cases and 34,500 new deaths of PCa are predicted in the United States in 2020. [2] In China, the incidence and burden of PCa is increasing rapidly. [3] Although recent advances in the diagnosis and treatment of PCa, the prognosis for men with advanced PCa is poor with less than one third surviving 5 years after diagnosis. [4] Therefore, a better understanding of the molecular events underlining the progression of PCa is urgently needed for developing an effective therapeutic strategy.
Increasing research has supported the notion that the wide utility of natural agents opens a novel avenue for cancer therapy through their ability to target multiple cancer-related pathways. [5] Yuanhuacine is a daphnane diterpenoid ( Figure 1A) traditional herbal medicine from the flowers of Daphne genkwa (Thymelaeaceae) and has a range of biological activities, including abortifacient, antivirus, and anti-inflammation. [6,7] In recent years, increasing interest has focused on the pharmacological activities of yuanhuacine, especially its antitumor activity, [8] including lung cancer, [9] colon cancer, [10] and breast cancer. [11] For instance, yuanhuacine inhibits human lung cancer cell proliferation and exhibits little cytotoxicity to normal lung epithelial cells. [9] Moreover, administration with yuanhuacine suppresses tumor growth in nude mouse models of non-small cell lung cancer. [12] Furthermore, yuanhuacine also exerts potential antitumor efficacy in triple-negative breast cancer. [11] However, its role in PCa remains unclear.
P53 is a proverbial tumor suppressor by regulating diverse signaling pathways involved in cell growth, invasion, migration and chemoresistance. [13,14] Notably, the levels and activity of p53 are usually controlled by the ubiquitin protein ligase mouse double minute 2 (MDM2). Convincingly, targeting the MDM2/p53 axis is a promising challenge for successful clinical therapy for cancers. [13,15] In the current study, we predicted MDM2 as a potential target of yuanhuacine using the HERB database. Therefore, we hypothesized that yuanhuacine might inhibit the carcinogenesis of PCa by regulating the MDM2/p53 axis. Here, we sought to explore the roles of yuanhuacine in cancer cell growth, invasion and migration in PCa. Moreover, the involvement of the MDM2/p53 axis was also investigated in the above processes. The current study will provide a new promising therapeutic agent for PCa.

| Cell culture and treatment
The human normal prostate stromal immortalized cell line (WPMY-1) and two PCa cell lines (LNCaP and PC-3) were purchased from the American Type Culture Collection (ATCC).

| Construction of recombinant MDM2 plasmids
The cDNA encoding MDM2 was prepared by PCR amplification using a cDNA template that was synthesized via a SuperScript II First-Strand Synthesis System (Invitrogen). After treatment with restriction enzymes, the MDM2 cDNA was subcloned into the pcDNA3.1 (Thermo Fisher Scientific), and subsequently named pcDNA-MDM2.

| Flow cytometry
Cell apoptosis was evaluated using Annexin V-FITC/propidium iodide (PI) staining kit (Nanjing Jiancheng Bioengineering Institute). Briefly, cells under LINC00665, pcDNA-MDM2 and yuanhuacine conditions were collected and centrifuged at 1000 g for 5 min. After resuspending in 500 μl binding buffer, 10 μl of Annexin V-FITC and 5 μl of PI were added for further incubation at room temperature of 10 min under the dark. All specimens were then subjected to a flow cytometer (CytoFlex, Beckman Coulter).

| Transwell invasion assay
The Transwell chambers (8-μm pore filters; Corning) were used to assess cell invasion ability. PCa cells under various treatments were adjusted to 1 × 10 5 cells/ml, and then were seeded into the upper chamber. The lower chamber was then filled with 400 μl complete medium supplemented with 10% FCS that was used as a chemoattractant. After a 48-h incubation and removal of cells on the bottom surface of the filter, 4% paraformaldehyde was adopted for fixing the invaded cells, following staining with 0.1% crystal violet solution.
Finally, a microscope was used to count cell invasion ability at 200× magnification over five random fields.

| Wound healing analysis
Cell migration was analyzed using a conventional scratch test. Posttreatment cells were placed in six-well plates at a concentration of 1 × 10 5 per well and allowed to grow to 80%-90% confluence. Then, a cross-shape was scratched in the middle of each well with sterile 200 μl plastic pipette tips. After incubation in an air atmosphere of 37°C for 24 h, non-adherent cells were gently rinsed and photograph images were taken over time using an inverted microscope (IX71; Olympus). The migration area was calculated as (%) = ((original gap area − gap area at X h)/original gap area) × 100%.

| Yuanhuacine facilities p53 ubiquitination and degradation by interfering with MDM2 to exert the antitumor potential in PCa cells
Abundant evidence has supported that p53 often acts as a well-known tumor suppressor to participate in cell apoptosis, epithelial-mesenchymal transition, and metastatic potential. [14] Herein, treatment with yuanhuacine increased p53 protein levels by decreasing p53 ubiquitination ( Figure 3A). Noticeably, p53 levels and activity are majorly controlled by the ubiquitin proteinligase MDM2. [13] We therefore further elaborated on the correlation between yuanhuacine and MDM2 and found that yuanhuacine decreased the mRNA levels of MDM2 in LNCaP cells and PC-3 cells relative to the control groups ( Figure 3B).
Moreover, yuanhuacine treatment dramatically reduced the phosphorylation of MDM2 and total MDM2 protein levels ( Figure 3C,D), which in turn resulted in the decrease of p53 degradation. Thus, these results indicate that yuanhuacine may regulate MDM2 expression to mediate the ubiquitination and degradation of p53. To further investigate the involvement of MDM2 in yuanhuacine-mediated anti-tumorigenesis potential, we overexpressed MDM2 protein levels ( Figure 3E). Moreover, upregulation of MDM2 reversed yuanhuacine-induced inhibition of cell viability ( Figure 3F) and apoptosis ( Figure 3F). Concomitantly, the suppressive effects of yuanhuacine on LNCaP cell invasion ( Figure 3G) and migration ( Figure 3H) were also offset after MDM2 overexpression.

F I G U R E 2 Treatment with yuanhuacine inhibits PCa cell invasion and migration. (A) LNCaP (A) and PC-3 cells (B)
were treated with 20 µM yuanhuacine. A Transwell assay was then performed to detect cell invasion. (C, D) Cell migration ability was evaluated by wound healing analysis. PCa, prostate cancer. *p < 0.05 versus control group.

| Yuanhuacine affects MDM2-mediated ubiquitination of p53 by blocking LINC00665
Our previous study confirmed high expression of LINC00665 in PCa tissues that could act as an oncogene to facilitate the progression of PCa. [16] To further decipher the mechanism underlying yuanhuacinemediated MDM2 downregulation in the progression of PCa, we explored the involvement of LINC00665 during this process. As shown in Figure 4A, network pharmacology analysis had identified 112 target genes of Flos Genkwa using the HERB database. Moreover, we also identified 937 target genes of LINC00665 by AnnoLnc2 database and corroborated that there was a common target gene MDM2 between Flos Genkwa and LINC00665 ( Figure 4B). qRT-PCR assay found that yuanhuacine treatment decreased LIN0065 expression in PCa cells ( Figure 4C). Transfection with recombinant LINC00665 plasmids enhanced LINC00665 expression ( Figure 4D). Bioinformatics tool GIPA2 substantiated there was no significant correlation between LINC00665 and MDM2 mRNA ( Figure 4E). Consistent with the above prediction, LINC00665 overexpression did not affect the expression of MDM2 mRNA in yuanhuacine-treated PCa cells ( Figure 4F). Intriguingly, upregulation of LINC00665 antagonized the inhibitory effects of yuanhuacine on MDM2 protein expression and phosphorylation ( Figure 4G,H). Furthermore, LINC00665 enhancement inhibited p53 protein levels by increased p53 ubiquitination relative to yuanhuacinetreated groups ( Figure 4I).

| LINC00665 accounts for the antitumor efficacy of yuanhuacine in PCa cells
The above results indicated that yuanhuacine could induce MDM2/p53 axis by inhibiting LINC00665. We next elucidated  Figure 5A). Moreover, the proapoptotic efficacy of yuanhuacine was reversed after LINC00665 upregulation ( Figure 5B). Additionally, overexpression of LINC00665 enhanced PCa cell invasion ( Figure 5C) and migration ( Figure 5D) relative to yuanhuacinetreated groups. These data suggest that yuanhuacine may exert the suppressive efficacy in the carcinogenesis of PCa by blocking LINC00665.

| DISCUSSION
PCa is a challenging global issue in men's health and its increasing burden necessitates seeking novel and alternative therapies. In recent years, increasing evidence has supported plant extracts and natural plantderivatives as promising antitumor therapeutics as their less toxic side effects. [5,17] Yuanhuacine is an active component isolated from D. genkwa and has been proven to exert inhibitory efficacy in cell growth in several cancers, including lung cancer, breast cancer and colon carcinoma. [8] Nevertheless, its role in PCa remains elusive. The present study confirmed that yuanhuacine dose-dependently suppressed PCa cell viability and induced cell apoptosis. Moreover, yuanhuacine exhibited little cytotoxicity to normal prostate cells.
Analogously, a previous study also corroborated the low toxicity of yuanhuacine to normal lung epithelial cells. [9] The exception to the uncontrolled proliferation, most cancers share a common basic characteristic, the potential to be highly metastatic. It is generally believed that high invasion and migration usually lead to high metastasis that is correlated with treatment failure and poor prognosis for cancer patients at advanced stages, including PCa. [18,19] Currently, though therapeutic advances in cancer treatment, metastasis remains the principal cause of cancer-related death, which makes blocking cancer cell metastatic potential to be a promising strategy to fight cancer by refs. [20,21]. Therefore, we next examined the role of yuanhuacine in PCa cell metastatic potential and found that treatment with yuanhuacine restrained cancer cell invasion and migration, suggesting its potential to inhibit metastasis in PCa.
The p53 protein, a tumor suppressor, is a central safeguard that protects cells against genome instability and malignant transformation. [13,15] Given that p53 protein stability is mainly controlled by ubiquitin-dependent degradation pathway. Inducing protein expression of p53 by decreasing its ubiquitination regulates the carcinogenesis, including cancer cell growth, chemoresistance, invasion, migration and metastasis. [13] Generally, E3 ubiquitin ligase MDM2 can act as a suppressor to reduce intracellular p53 levels by inducing p53 ubiquitination and subsequent degradation. [15] To further analyze the mechanism underlying the inhibitory efficacy of yuanhuacine in the carcinogenesis of PCa, we investigated the correlation between yuanhuacine and p53 signaling. As excepted, yuanhuacine treatment inhibited p53 ubiquitination, degradation and ultimately increased p53 levels. More importantly, this process was regulated by blocking MDM2 phosphorylation. Notably, overexpression of MDM2 reversed the suppressive efficacy of yuanhuacine in cell growth and metastatic potential in PCa cells. Thus, yuanhuacine may act as an antitumor agent by regulating the MDM2/p53 axis.
F I G U R E 5 LINC00665 involves in Yuanhuacine-mediated suppression of carcinogenesis in PCa cells. Cells transfected with LINC00665 vectors were exposed to yuanhuacine. Then, cell viability (A), apoptosis (B), invasion (C), and migration (D) were analyzed by CCK-8, flow cytometry, Transwell and wound healing analysis, respectively. PCa, prostate cancer. *p < 0.05 versus control group. # p < 0.05 versus YC-treated group.
Long noncoding RNAs are responsible for the majority of human genome transcriptions and their aberrant expression involves in various pathological processes including cancer development. [22] Among them, LINC00665 is located at chromosome 19q13.12 and is abnormally upregulated in several cancers. [23][24][25] Our previous finding confirmed that LINC00665 could serve as an oncogene in the progression of PCa. [16] Intriguingly, evidence from the bioinformatics database predicted MDM2 as a common target of LINC00665 and D.
genkwa. Moreover, yuanhuacine also inhibited LIN0065 expression in PCa cells. We therefore further investigated the involvement of LINC00665 in yuanhuacine-mediated antitumor progression in PCa.
Intriguingly, the elevation of LINC00665 expression overturned yuanhuacine-mediated inhibition of MDM2 expression at posttranscriptional levels to regulate p53 ubiquitination and degradation.
Additionally, overexpression of LINC00665 offset yuanhuacinemediated suppression in PCa cell viability, invasion and migration.

| CONCLUSIONS
Collectively, the current findings first revealed that yuanhuacine could act as an effective suppressor to reduce PCa cell viability and metastatic potential by blocking LINC00665-mediated MDM2/p53 signaling. Therefore, this study highlights that yuanhuacine may act as a therapeutic agent against PCa. However, the current findings

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article.