LncRNA AC008972.1 as a Novel Therapeutic Target for Prostate Cancer

Background: Prostate cancer is the leading cause of disease and death in men. Long non-coding RNAs (lncRNAs), microRNA (miRNAs) and mRNAs networks mediate prostate cancer progression. Here, we aim to investigate functions of lncRNA AC008972.1/miR-143-3p/thousand-and-one-amino acid 2 kinase (TAOK2) in prostate cancer. Methods: The expression levels of lncRNA AC008972.1, miR-143-3p and TAOK2 are detected in prostate cancer tissues and cell lines by RT-qPCR. PC3 and LNCaP cells are used to establish lncRNA AC008972.1-knockdown, miR-143-3p-overexpressing, and TAOK2-down-regulated cells. Cell viability is examined by MTT and cell proliferation is detected by clone formation assay. Cell migration and invasion are tested by wound scratch assay and transwell chamber assay. The rate of apoptosis was analyzed by �ow cytometry. The protein expression is detected by western blot assay. The target is validated by RNA binding protein immunoprecipitation (RIP) assay and dual luciferase activity assay. A mouse xenograft model was conducted to investigate the oncogenic effect of lncRNA AC008972.1 on prostate cancer. Results: High expression of lncRNA AC008972.1 was associated with low overall survival in prostate cancer. Down-regulation of lncRNA AC008972.1 delayed prostate cancer process by inhibiting cell viability, proliferation, migration and invasion, as well as altering protein expression(cid:0)whereas cell apoptosis was markedly promoted. LncRNA AC008972.1 negatively regulated miR-143-3p expression and miR-143-3p overexpression promoted prostate cancer process in vitro. TAOK2 expression was decreased by miR-143-3p through the complementary targeting of TAOK2 mRNA. Down-regulation of lncRNA AC008972.1 mitigated prostate cancer process in vitro based on miR-143-3p/TAOK2 node. Furthmore, the data of xenograft model experiment showed that inhibition of lncRNA AC008972.1 suppressed tumor growth in vivo. Conclusions: Collectively, knockdown of lncRNA AC008972.1 inhibits prostate cancer cell growth based on down-regulation of TAOK2 induced by miR-143-3p. Here, we identify that lncRNA AC008972.1 exerts essential roles in the progression of prostate cancer and serves as a novel therapeutic target for prostate cancer.


Introduction
As the leading cause of disease and death in men, 1.6 million men are diagnosed with prostate cancer and 366,000 men die from prostate cancer every year [1]. Epidemiological studies of prostate cancer have disclosed lifestyle factors and individual biology in uence the survival rate of prostate cancer and the risk of developing prostate cancer in multiple ways [1,2]. Advances in prostate cancer diagnosis and treatment have ameliorated the capacity for stratifying patients by risk, thus allowing clinicians to recommend treatment according to cancer prognosis and patient preference [3,4]. Chemotherapy effectively improves survival rate compared with androgen deprivation therapy [3][4][5]. Additionally, drugs such as abiraterone and enzalutamide improve outcomes for men with metastatic prostate cancer that shows resistance to conventional hormone therapy [3,4].
As transcripts of more than 200 nucleotides, long non-coding RNAs (lncRNAs) are unable to code proteins. Their biological contributions have been associated with malignant tumors by organizing nuclear domains, mediating transcription in cis or trans, thus regulating mRNA processing and posttranscriptional management, and modulating protein activity [6][7][8]. Results from differential screens of the expression of genes show that 61 lncRNAs are differentially expressed in prostate cancer, such as lncRNA AC008972.1, lncRNA LINC00844 and lncRNA PCAT1 [9][10][11]. Some of these lncRNAs have been well acknowledged as diagnostic, therapeutic or prognostic molecules [12,13]. However, the biological roles of lncRNA AC008972.1 have not been precisely investigated. Furthermore, prior to clinical translation, it is signi cant to extensively understand the functional mechanism of lncRNAs.

Patients
This study obtained the approval from the Ethics Committee of Jiangsu Province Hospital of Chinese medicine (NO. NL-129-02). We obtained the informed written consents from all patients for utilizing the pathologic tissue specimens. The clinical characterization and tissues of patients with prostate cancer were collected from A liated Hospital of Nanjing University of Chinese Medicine (Nanjing, China). Tumor grades were classi ed according to the World Health Organization's Classi cation. All men in the cohort were followed for 100 months. All experimental procedures were implemented in accordance with the Declaration of Helsinki of 1975 (revised in 2000). The clinicopathologic ndings were shown in Table   1. LncRNA AC008972.1 expression in tissues was examined by reverse transcription-quantitative PCR (RT-qPCR).

Animal study
All animal experiments were performed with the approval of the Animal Ethics Committee of Jiangsu Health Vocational College (No. JHVC-IACUC-2020-B002). 8-week-old male nude mice were randomly divided into two groups: sh-NC or sh-lncRNA AC008972.1 group (3 in each group). The PC3 cells transfected with sh-NC vector or sh-lncRNA AC008972.1 vector were injected subcutaneously into mice.
The tumor size was monitored every weak. After four weeks, the mice were sacri ced. The tumor volumes were calculated by using the following formula: Tumor volume = (width2 × length)/2. Tumor tissues were xed for H&E (Solarbio, China), IHC (Solarbio, China) and Tunel assay (Promega) following the manufacture's instruction.

Cell lines
Cell lines RWPE-1, PC3 and DU145 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). P4E6 cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), and LNCaP from the Cell Lines Service (CLS, Eppelheim, Germany).

Cell transfection
To arti cially modulate lncRNA AC008972.1, miR-143-3p, or TAOK2 expression, transfection was carried out in PC3 and LNCaP cells. In short, PC3 and LNCaP cells were seeded in a 6-well plate with a density of 10 5 cells/mL and pre-incubated for 24 h. Transfection was carried out using Lipofectamine RNAiMAX reagent (Thermo Fisher Scienti c, Somerset, NJ, USA) according to the manufacturer's instructions. The cells were transfected with 10 pmol/mL of lncRNA AC008972.1 short-hairpin RNA (sh-LncRNA AC008972.1), or negative control (sh-NC) (Sigma-Aldrich). To up-regulate or down-regulate miR-143-3p, the cells were transfected with miR-143-3p mimic (NC mimic as a negative control) or miR-143-3p inhibitor (NC inhibitor as a negative control) (Sigma-Aldrich). To down-regulate TAOK2 expression, shRNA for TAOK2 was transfected into PC3 and LNCaP cells, with sh-NC as a negative control (Sigma-Aldrich).

Cell viability
All cells were collected 48 h after transfection. Cell viability was assayed by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method. The cells were incubated with 200 μL of 0.5 mg/mL MTT (Sigma-Aldrich) prepared in phosphate-buffered saline at 37 o C. After 4 h, the supernatant was removed, and the formazan crystals were dissolved in 100 μL of dimethyl sulfoxide. The absorbance of each well was detected at 540 nm using a microplate reader (BioTek Instruments, Winooski, VA, USA).
Colony formation PC3 and LNCaP cancer cells were seeded in 6-well plates at a density of 100 cells each well. The transfected cells were kept at 37 o C in 5% CO 2 for 14 days, and the medium was changed every 2 days.
The formed colonies were xed using 70% ethanol, followed by staining with 0.5% crystal violet. The colony consisting of 50 cells were counted using Image J software (NIH, Bethesda, MD, USA). The experiment was carried out in triplicate.

Scratch assay
After transfection, PC3 and LNCaP cells were cultured in 6-well plates for 10 h. Then, two parallel wounds were created with a 200-μL pipette tip. The debris was cleaned with phosphate buffer saline (PBS). The cells were continually cultured for 48 h. Wound closure was monitored using a microscope (Olympus, Tokyo, Japan) and imaged with ImageJ software.

Flow cytometry assay
To detect apoptosis, cells were harvested, following by staining with annexin V-FITC/PI in the dark. The rate of apoptosis was analyzed by ow cytometry. All determinations were performed independently in triplicate.

RNA extraction and RT-qPCR
Total RNA from tissues and cells were extracted using QIAzol Lysis reagent (QIAGEN, Hilden, Germany) and miRNeasy kit (QIAGEN). DNase digestion was performed. Cell fractionation was carried out using the NE-PER nuclear extraction kit (Thermo Fisher Scienti c) to obtain cytoplasmic RNA and nuclear RNA.
RNA binding protein immunoprecipitation (RIP) assay RIP assay was carried out using EZMagna RIP kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. PC3 and LNCaP cells at 80%-90% con uency were collected and lysed in RIPA buffer. Cell extract was incubated with RIP buffer containing magnetic beads linked to anti-Ago2 antibody (Millipore) or normal mouse IgG (Millipore). To remove the protein, the extract was incubated with proteinase K at 55 o C for 30 min. RNA content was detected with a NanoDrop (Thermo Fisher Scienti c). The immunoprecipitated lncRNA AC008972.1 and miR-143-3p were quanti ed by qRT-PCR.

Statistical analysis
Statistical analysis was done with GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). Student's t-test was used to analyze the statistical signi cance between two groups. One-way ANOVA followed by Tukey test was used to perform comparison between multiple groups. Data were presented as mean ± standard deviation (SD). p-values less than 0.05 were considered to be signi cant.

Results
High expression of lncRNA AC008972.1 is associated with low overall survival in prostate cancer To investigate lncRNA AC008972.1 expression in prostate cancer, prostate cancer tissues and paracancerous tissues were exposed to RT-qPCR. We con rmed that lncRNA AC008972.1 was enriched in prostate cancer tissues compared with para-cancerous tissues (p < 0.01) ( Figure 1A). Besides, patients with grade III-IV prostate cancer showed higher expression of lncRNA AC008972.1 relative to patients with grade I-II prostate cancer (p < 0.01) ( Figure 1B). Overall survival was signi cantly lower in patients with high expression of lncRNA AC008972.1 compared with those with low expression of lncRNA AC008972.1 (p < 0.001) ( Figure 1C). Compared with human prostate epithelial RWPE-1 cell line, the expression of lncRNA AC008972.1 was apparently higher than those in prostate cancer cells P4E6, PC3, LNCaP and DU145 cells (p < 0.01 or p < 0.001) ( Figure 1D), among which the PC3 and LNCaP cells exhibited the highest lncRNA AC008972.1 expression. Hence, the PC3 and LNCaP cell lines were selected for the subsequent experiment. In conclusion, high expression of lncRNA AC008972.1 was associated with prostate cancer process.

MiR-143-3p decreases TAOK2 expression by complementary targeting of TAOK2 mRNA
Similarly, TAOK2 mRNA shared the complementary elements with miR-143-3p, as predicted by TargetScan ( Figure 5A). Experimental con rmation was carried out by dual luciferase activity assay.

Down-regulation of lncRNA AC008972.1 mitigates prostate cancer process in vivo
To investigate the oncogenic effect of lncRNA AC008972.1 on prostate cancer, an in vivo study was conducted. PC3 cell lines were transfected with sh-lncRNA AC008972.1 vector or sh-NC vector, and subsequently implanted into nude mice to construct mouse xenograft model. The tumors volume (p < 0.01) and weight (p < 0.001) of sh-lncRNA AC008972.1 group were signi cantly down-regulated compared with sh-NC group ( Figure 7A-7C). Furthermore, the Hematoxylin and Eosin (HE) staining and immunohistochemistry assay (IHC) results illustrate that the tumors developed from sh-lncRNA AC008972.1 cells displayed alterations in shape and the reduction of Ki-67 compared with tumors formed from sh-NC vector-transfected cells (p < 0.001). While the Tunel assay result shows that the percentage of apoptotic bodies was increased incredibly in the tumors from sh-lncRNA AC008972.1 cells (p < 0.001) ( Figure 7D). Finally, we veri ed the lncRNA AC008972.1/miR-143-3p/TAOK2 regulatory axis in vivo. The qRT-PCR was conducted to detect the RNA relative expression level. The expression of lncRNA AC008972.1 was shut down in tumors derived from sh-lncRNA AC008972.1 cells (p < 0.001). On the contrary, miR-143-3p was up-regulated due to lncRNA AC008972.1 knocking down (p < 0.001), which led to the reduction of TAOK2 (p < 0.001) ( Figure 7E). Taken together, Knockdown of lncRNA AC008972.1 regulates TAOK2 by reducing the sponge effect on miR-143-3p to inhibit tumor development and promote tumor cell apoptosis.

Discussion
Here we detected that lncRNA AC008972.1 high expressed in prostate cancer was associated with the low overall survival rate. LncRNA AC008972.1 knockdown mitigated prostate cancer cell growth. Further, we documented a critical function of miR-143-3p together with its downstream targeting gene TAOK2.
Whole genome transcriptomic analysis has revealed that lncRNA transcripts are implicated in physiological and pathological processes such as prostate cancer [26]. Accumulating studies have evidenced that the pathology and progression of prostate is generally associated with the aberrant expression of lncRNAs [13,27,28]. Currently, we rstly detected the up-regulation of lncRNA AC008972.1 in prostate cancer, and this high expression was more signi cant in grade III-IV prostate cancer. Besides, the high expression of lncRNA AC008972.1 presented a low overall survival rate. Consequently, lncRNA AC008972.1 showed the promise as a predictive, diagnostic and prognostic biomarker in prostate cancer. However, the functional role of lncRNA AC008972.1 remained partially understood. Here, we proved that knockdown of lncRNA AC008972.1 decreased prostate cancer cell viability, restrained colony formation, inhibited wound closure, and abated migration and invasion. Further, knockdown of lncRNA AC008972.1 increased E-cadherin expression. E-cadherin, as an epithelial-mesenchymal transition transcription factor, plays roles in metastasis and drug resistance of prostate cancer [29]. Of note, sh-lncRNA AC008972.1 decreases the expression of vimentin that is associated with a higher likelihood of metastasis.
It was noticed that miR-143-3p was down-regulated in prostate cancer tissues compared to that in nontumor tissues, which was consistently reported by previous studies [30,31]. What's more, the fact that lncRNA AC008972.1 shared the complementary sequence with miR-143-3p suggested the links between lncRNA AC008972.1 and miR-143-3p expression. Here, we demonstrated that lncRNA AC008972.1 negatively modulated miR-143-3p generation by targeting miR-143-3p. In addition, prostate cancer cell proliferation and migration was abrogated by miR-143-3p [30][31][32], which is consistently con rmed in vitro in our study. It has been reported that the modulatory networks consisting of lncRNAs and miRNAs are implicated in the molecular mechanisms and tumourigenesis in prostate cancer [9]. It was worth noting that miR-143-3p up-regulation mediated by lncRNA AC008972.1 knockdown restrained prostate cancer cell growth, implying that miR-143-3p was a critical mediator for knockdown of lncRNA AC008972.1 to inhibit prostate cancer.
TAOK2 has been characterized as MAPKKKs activating downstream MKK3 and MKK6, which exhibits speci city for stress-induced activation of p38 MAPK in prostate cancer [19][20][21][22]. Currently, we observed signi cant up-regulation of TAOK2 in prostate cancer tissues, which had not been reported previously. It was also veri ed that miR-143-3p mimic lessened TAOK2 at mRNA and protein levels. Of note, TAOK2 mRNA is predicted to possess the complementary sequence with miR-143-3p [33], which is further experimentally veri ed. Studies have provided evidences miR-143-3p inhibits cancer progression through targeting its downstream genes and regulating protein expression [18,34,35]. However, whether miR-143-3p inhibited prostate cancer cell growth via mediating TAOK2 expression lacked the exact evidences. Here, we con rmed that knockdown of lncRNA AC008972.1 inhibited prostate cancer process depending on miR-143-3p-mediated down-regulation of TAOK2.

Conclusions
Summarily, lncRNA AC008972.1 is highly expressed in prostate cancer, which indicates low overall survival rates. Knockdown of lncRNA AC008972.1 inhibits prostate cancer cell growth based on miR-143-3p-induced down-regulation of TAOK2. Our research provides a potential therapeutic target for prostate cancer, despite more detailed mechanisms need further research.

Consent for publication
All authors agree with the content of the manuscript.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.   Positve 6 11 Table 2 The sequence of speci c primers for RT-qPCR