Clinical Tissues Collection
Eighty PCa patients at the Mindong Hospital Affiliated to Fujian Medical University, between Feb 2019 and Jan 2020, were enrolled into the study. The PCa tumor and adjacent normal tissues samples were collected. All patients were confirmed as PCa by histopathological analysis and without preoperative radiotherapy or chemotherapy. All fresh specimens were stored in liquid nitrogen immediately at −80 °C until use. The detailed criteria for the patients were that the pathological type is adenocarcinoma. The clinical parameters of the enrolled patients such as age, TNM stage and Gleason score et al were recorded and analyzed to correlate the in vitro findings with the clinical presentations (Table 1). All patients signed the relevant informed consent. The Ethics Committee of Mindong Hospital Affiliated to Fujian Medical University approved the study protocol. The batch number was  NingMin Medical Ethics No (0110-1). The investigation adhered to the principles outlined in the Declaration of Helsinki.
RNA extraction and qRT-PCR
Total RNA was extracted from PCa tissue and cells by NucleoZOL® (Gene Co., Ltd., Shanghai, China). Primers were resuspended by adding 250 µL of RNase-free water. Master mix was prepared for each mRNA-specific assay. Each single reaction included 10 µL of qPCR SYBR® Green Master Mix Universal (Takara Bio, Inc., Japan). About 10 µL of the primer was set for an individual miRNA. RNase free water (3 µL) and RT product (2 µL) were added to a single real-time PCR reaction tube as template.
Reverse transcription (RT) was performed using the RT System Kit (Takara Bio Inc., Tokyo, Japan). The synthesized cDNA was amplified by quantitative PCR by using HEAL FORCE (Xianggang, China). The reaction conditions included 42 °C for 60 min, followed by cooling to 4 °C. The resultant cDNA was used as template for subsequent PCR. Forty PCR amplification cycles were performed with initial incubation at 95 °C for 10 min and final extension at 72 °C for 5 min. Each cycle comprised denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The GAPDH mRNA was a control. The relative expression of candidate genes was calculated using the formula ΔΔCq = (Cq, target - Cq, reference) PCa -(Cq, target - Cq, reference)NC, and the estimated expression ratio was equal to 2-ΔΔCq . The estimated expression ratio was equal to 2−ΔΔCq. The primer set of candidate gens: LncRNA SNHG16 FORWARD primer was GTGCCTCAGGAAGTCTTGCC; REVERSE primer was ATCCAAACAAGTTATCAGCAGCAGCAC. TGFBR2 FORWARD primer was GTAGCTCTGATGAGTGCAATGAC; REVERSE primer was CAGATATGGCAACTCCCA GTG. GAPDH FORWARD primer was ATGGGGAAGTGAAGGTCG; REVERSE primer was TTACTCCTTGGAGCCATGTG.
Taqman RT- PCR
The RNA harvested from PCa tissue and cells was extracted by the miRNeasy mini kit (Qiagen, USA). Before RNA extraction, each sample was added with 100 ng exogenous and synthetic Caenorhabditis elegans miR-39 to normalize. Specifically, sample suspension (50 mg tissue) was mixed with Qiazol lysis reagent (1 mL), incubated or homogenate for 5 min in room temperature, and then mixed with 200 µL cholorform for 3 min. The RNA was separated at 12,000×g for 15 min at 4℃ and collected the aqueous phase. After centrifugal, the supernatant mixed with 100 % ethanol (600 µL). In order to purify RNA, 700 µL supernatant was added to miRNeasy mini spin column and centrifuged at 8,000 × g for 15s at room temperature. Then, miRNA was eluted by the addition of 30 µl RNase-free water. Concentration of RNA was measured by nanodrop.
To confirm hsa-mir-373-3p expression, the Taqman real-time PCR was used (Applied Biosystems, Foster City, CA, USA). 5 µL RNA was reversely transcribed into cDNA using the Taqman miRNA reverse transcription kit (Applied Biosystems, USA). The reaction conditions were 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Then, 10 µL of TaqMan® Universal PCR Master Mix (Applied Biosystems, USA), 1 µL of TaqMan Stem-loop miRNAassay, 7.5 µL of nuclease free water, and 1.5 µL of RT product were used for real-time PCR reaction. Forty cycles of PCR amplification were performed, with initial incubation at 95 °C for 10 min. Each cycle comprised at 95 °C for 15 s and annealing at 60 °C for 1 min. The stem-loop primers were 5 nM each: cel-miR-39: GTCGTATCCAGTGCAGGG TCCGAG-GTATTCGCACTGGATACACCAAGCT, hsa-miR-373-3p: GTCGT ATCCAGTG CAGGGTCCGAGGTATTCGCACTGGATACGACCTTCAC. The estimated expression ratio of hsa-miR-373-3p was equal to 2−ΔΔCq.
Cell culture and transfection
Human PCa DU-145 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI-1640 medium (Gibco, USA) and cultured in the media with 10% FBS (PAN biotech, Germany), 4 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2.
1×105 cells/well of DU-145 cell was inoculated into 24-well plates before transfection. Lipofectamine 2000 (Invitrogen, USA) was utilized for cell transfection. For SNHG16 gene knockdown experiment, shRNA was amplified by the following primers, then the product was digested with BamHI and EcoRI restriction enzymes (Fermentas, UK), and the fragment was inserted into the BamhI/EcoRI sites of the pLVX-shRNA2-Puro vector. The shRNA targeting SNHG16 was 5'- GATCCGGATGAGACTTAACTTAAATTCAAGAGATTTAAGTTAA GTCTCATCCTTTTT G-3', negative control shRNA was 5'-GATCCG TGTAGATGCGTTGTGATATTCAAGAGAT ATCACAACGCATCTACACTTTTTG-3' (Life Technologies, Carlsbad, CA, USA). For SNHG16 overexpression, the cells were transfected with the SNHG16 overexpression construct (pcDNA3.1-SNHG16). Specifically, the full-length of SNHG16 was obtained via PCR amplification using the primers: SNHG16-F: 5’-CGGGATCCCGGCGTTCTTTTCGAGGTCGGCCG-3’ and SNHG16-R: 5’-CCCTCGAGGGTGACGGTAGTTTCCCAAGTTTA-3’. Then, the amplification the product was digested with BamhI and XhoI restriction enzymes, and the fragment was inserted into the BamhI/XhoI sites of the PcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc., USA) to obtain a SNHG16 overexpression plasmid. Hsa-miR-373-3p mimics, inhibitors and negative control were purchased from GenePharma (Shanghai, China). The shRNA targeting TGFBR2 was 5'- GGTGGGAACTGCAAGATACATCUGUGCUGU CCATGTATCTTGCAGTTCCCACCTTTTT-3', the negative control shRNA was 5'-GATCCGAUCAAUACUAUUCAUCAATTCAAGAGAUUGAUGAAUAGUAUUGAUCTTTTTG-3'. The pLVX-shRNA2-Puro vector, EcoRI and BamhI were used to construct TGFBR2 knockdown and negative control plasmid. When cells grew to 30%–50% confluence, the plasmid (10 nM) was utilized to transfect. After 48 h of transfection, relevant tests were used for comparison.
CCK-8 assay for cell proliferation
The transfection cells were determined by Cell Counting Kit-8 assay (Dojindo, Japan). DU-145 cell suspensions (1×104/mL) were transferred to 96-well plates and incubated for 24, 48, 72, and 96 h. Each well was added with 10 μL of CCK-8 and incubated for 4 h. The values of each well were measured by a microplate reader (PERLONG, Beijing, Chia) at 450 nm. All experiments were repeated three times.
Flow cytometric analysis for cell apoptosis
The 1×105 cells/ well were cultured in 24-well plates and transfected until the confluence reached 60%–70%. After transfecting with plasmid for 48 h, the cells were washed two times with PBS and centrifuged for 5 min at 1000 rpm. According to the instruction, the cells were then incubated with 5 μL of Annexin V-FITC for 15 min and 10 μL of PI for 5min at room temperature and dark (BD Biosciences, USA). Finally, the cells were detected by flow cytometry (Becton Dickinson, USA). All experiments were repeated three times.
Wound healing assay for cell migration
Wound healing test was used to measure cell migration. After transfecting with plasmid for 48 h cells were reaped. A total of 3×105 cell/well were inoculated into 24-well plates, which each well has cell plug-in. Wound healing experiment was carried out when all the cells were adherent and just full. After incubation for 24 h, the cells were counted under the microscope. All experiments were repeated three times.
Transwell assay for cell invasion
Cell invasion was assayed using a Transwell chamber (8 μm pore size, Corning) deposited with Matrigel (BD Biosciences, USA). After transfecting with plasmid for 48 h, 1×105 cell/well was inoculated into Transwell’s upper chamber. The lower chamber was added with complete media. After 24 h, cells that remained in the upper membrane were removed. The migrated cells were incubated with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 15 min, and counted in the microscope. All experiments were repeated three times.
After transfecting 48 h, DU-145 cells were collected. RIPA buffer (Takara Bio Inc., Tokyo Japan) and protease inhibitor (Roche Diagnostics) were used for cell lysis. BCA assay kit was used to detect protein concentration (Epizyme, China). The protein samples were separated using SDS–PAGE and shifted to PVDF. The PVDF membranes were saturated with blocking buffer (5% skim milk in 20 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature and incubated with primary antibodies (1:1000) at 4 °C overnight. The antibodies of TGFBR2, c-MYC, E2F4, SMAD2, p-SMAD2, SMAD3, p-SMAD3, and β-actin (Cell Signaling Technology, Beverly, MA, USA) were used. After washing two times with PBS, the cells were incubated with secondary-HRP-antibodies (Protein-Tech, USA) for 2 h at room temperature. The membranes were added with ECL Western blotting reagents (GE Healthcare, USA) to detect protein levels. The bands were imaged by Tanon 5200 Biotanon. Image J software was used to calculate the gray value.
Double-luciferase reporter assay
SNHG16-3′-UTR-wild-type (WT) sequence was gag tttcagagag taatgcttaa ccccagttac acaggatgcc gtcttgtgtt tcctcttgtt tagttaccca ctacagtgat tttgtgatct gctaatgggt tgccacccac aaccattgct ttaACGCTTt tacttcaaat caatgaagga ttgataaaag ttctcctggt gtctccgcag agtgccttcc aggaacagat ctttgcatag aatatcagtg gtttcctttt ttgtttcaaa tagtggtcaga. SNHG16-3′-UTR-mutant (MUT) sequence was gag tttcagagag taatgcttaa ccccagttac acaggatgcc gtcttgtgtt tcctcttgtt tagttaccca ctacagtgat tttgtgatct gctaatgggt tgccacccac aaccattgct ttagcacttt tacttcaaat caatgaagga ttgataaaag ttctcctggt gtctccgcag agtgccttcc aggaacagat ctttgcatag aatatcagtg gtttcctttt ttgtttcaaa tagtggtcaga. TGFBR2 3′-UTR -WT sequence was ggaataaca ttctgtagtt cctaaaaata ctgacttttt tcactactat acataaaggg aaagttttat tcttttatgg aacacttcag ctgtactcat gtattaaaat aggaatgtga atgctatata ctctttttat atcaaaagtc tcaagcactt atttttattc tatgcattgt ttgtctttta cataaataaa atgtttatta gattgaataa agcaaaatac tcaggtgagc atcctgcctc ctgttcccat tcctagtagct. Then, THBS1 3′-UTR- MUT sequence was ggaataaca ttctgtagtt cctaaaaata ctgacttttt tcactactat acataaaggg aaagttttat tcttttatgg aacacttcag ctgtactcat gtattaaaat aggaatgtga atgctatata ctctttttat atcaaaagtc tcaCAGAtCt atttttattc tatgcattgt ttgtctttta cataaataaa atgtttatta gattgaataa agcaaaatac tcaggtgagc atcctgcctc ctgttcccat tcctagtagct. These sequences were synthesized (GenePharma, Shanghai, China) and digested with XhoI and NotI restriction enzymes (Fermentas, UK). Then, the 3′-UTR fragment was inserted into the XhoI/NotI sites of the psiCHECK-2 vector (Promega, USA) to obtain luciferase reporter plasmid.
DU-145 cell suspensions (1×105 cell/well) were transferred to 24-well plates and incubated for 24 h. In order to verify the targeting effect of SNHG16 and hsa-miR-373-3p, the psiCHECK-2-SNHG16-WT or psiCHECK-2-SNHG16-MUT reporter plasmids (20 ng) were cotransfected with hsa-miR-373-3p mimic or mimic negative control (10 nM) into cells by using Lipofectamine 2000 (Invitrogen, USA). When verifying the targeting effect of TGFBR2 and hsa-miR-373-3p, the psiCHECK-2-TGFBR2-WT or psiCHECK-2-TGFBR2-MUT reporter plasmids (20 ng) were cotransfected with hsa-miR-373-3p mimic or mimic negative control (10 nM) into cells by using Lipofectamine 2000. After 36 h of transfection, cell lysates were prepared by using lysis buffer (Promega, Madison, USA). The firefly and Renilla luciferase activities were detected according to dual luciferase reporter assay (Promega, Madison, USA). The Renilla luciferase values were then divided by the firefly luciferase activity values to normalize the difference in transfection efficiency. All experiments were repeated three times.
All experiments were analyzed by the IBM SPSS Statistics 20.0 software (IBM Corp., Armonk, New York, USA). If data followed normal distribution, the one-way ANOVA analysis with LSD post-hoc test was performed. If the data did not follow normal distribution, Mann–Whitney U test was used. Spearman correlation was used to analyze the correlation between SNHG16 and hsa-mir-373-3p, hsa-mir-373-3p and TGFBR2. Chi-square test was used to descriptive analysis. Datas were presented as mean ± standard deviation, and P < 0.05 indicated significant difference.