General data

A total of 112 newly diagnosed prostate cancer patients and 80 healthy controls who received treatment in Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital from March 2020 to March 2022 were included in this study, and the whole process was conducted under the supervision of the Hospital Ethics Committee (No. 2019–229). Inclusion criteria: newly diagnosed patients with prostate cancer; patients who have not received antitumor therapy before; patients and their families agree to cooperate with this study. Exclusion requirements: patients with other malignant tumors; patients with secondary prostate cancer; patients with cardiovascular and cerebrovascular, diabetes or infectious diseases; patients who failed to sign informed consent. General information such as age, tumor size, and PSA level of the participating prostate cancers were collected and recorded. Meanwhile, lymph node metastasis refers to the process of prostate cancer cells spreading through the lymphatic system to other parts of the body. This is a sign of the aggressiveness of prostate cancer, which means that the cancer may have spread beyond the prostate itself to other parts of the body. We evaluated by imaging examination and histopathological examination, and PSMA PET/CT can be used as a means of auxiliary diagnosis.

Collection of blood specimens and tissue samples

Blood specimens were collected from all participants with the knowledge and consent of all participants, and serum was separated in time. The obtained serum samples were stored in a refrigerator at -80 °C for later use.

In addition, 54 prostate cancer tissue samples and paracancerous normal tissue samples were washed with PBS and similarly cryopreserved.

Purchase and culture of cells

Cell samples including prostate cancer cells (DU145, 22Rv1, LNCaP and PC3) and normal prostate cells RWPE-1 were purchased from ATCC in the United States, and they were inoculated in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% inactivated fetal bovine serum (FBS). All cells were incubated at 37 °C and maintained in an incubator containing 5% CO2.

Evaluation of PITPNA-AS1 and miR-129-5p expression

After predicting the PITPNA-AS1 and miR-129-5p contents in prostate cancer using the ENCORI database, their levels were determined in the serum samples collected in this study. Total RNA was extracted from serum samples with Trizol reagent. The concentration and purity of RNA were measured in time to determine the quality of RNA. Total RNA as template, cNDA was synthesized according to TIANGEN reverse transcription kit (Beijing, China). SYBR Green fluorescent polymerase chain reaction kit (Thermo Fisher, USA) was used to configure the reaction system, and RT-qPCR was performed with 7500 FAST PCR instrument (ABI, USA). Using GAPDH and U6 as internal parameters, the relative expression levels of PITPNA-AS1 and miR-129-5p in the samples to be tested were calculated through 2−ΔΔCT method.

Cell transfection and in vitro assay

The control group, silencing negative control (si-NC) and silencing PITPNA-AS1 (si-PITPNA-AS1) were transfected into prostate cancer cells LNCaP and PC3 in the presence of Lipofectamine 2000 reagent (Invitrogen, USA). Simultaneously, the transfection results were reflected by the relative expression of PITPNA-AS1.

Transfected cells (si-PITPNA-AS1-LNCaP and si-PITPNA-AS1-PC3) were cultured in 96-well plates under the same conditions as above. CCK-8 reagent was added at appropriate times and culture was continued, then OD value was detected 2 h later to understand the proliferation activity of cells. Transfected cells LNCaP and PC3 were inoculated in the upper chamber of the Transwell plate for cell metastasis assay, in which Matrigel-coated upper chambers were used for invasion level detection, and non-coated upper chambers were used for migration experiment. The cells were transferred to an upper chamber filled with RPMI-1640 medium, while the lower chamber was filled with RPMI-1640 medium supplemented with 10% FBS. The cells were incubated and calculated after crystal violet staining.

Western blot assay of cell markers

The expression of interstitial markers vimentin and N-cadherin and epithelial adhesion marker E-cadherin were detected by Western blot. Cells were cultured in 6-well plates for 48 h and then lysed by protein extraction kit (Biyuntian, China). Protein samples were added to SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were applied to antibody solutions for vimentin, N-cadherin, E-cadherin, and GAPDH, and peroxidase coupled secondary antibodies were added and incubated for 2 h. Protein bands were quantified and visualized using a luminescence image analyzer and ImageJ 1.42q software.

RIP assay

RIP assay was carried out with IgG antibody and Ago2-specific, and RNA in PC3 cells was extracted by RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). The interaction between PITPNA-AS1 and miR-129-5p was reflected according to the relative content of PITPNA-AS1.

Dual-luciferase reporter assay

The binding sites of PITPNA-AS1 and miR-129-5p were predicted by LncBook 2.0 database and then confirmed using luciferase activity assay. PC3 cells were cultured in 48-well plates to cell adherence, and then co-transfected with the control group, mimic NC, inhibitor NC, miR-129-5p mimic or miR-129-5p inhibitor and PITPNA-AS1-WT (wild-type) or PITPNA-AS1-MUT (mutant-type) with Lipofectamine 2000 reagent. Correlation analysis was performed with relative luciferase activity as reference.

Statistical analysis

After each experiment was repeated three times, data statistics were performed by SPSS 21.0 software. The enumeration data were tested by chi-square test, and the measurement data were expressed as mean ± standard deviation. The diagnostic value of PITPNA-AS1 was assessed by ROC curve, and the prediction accuracy is higher when AUC > 0.9.