LncRNA PCGEM1 promotes renal carcinoma progression by targeting miR-433-3p to regulate FGF2 expression

2.1 Tissue specimens

Human tissue samples used in current study were obtained from 47 renal carcinoma patients who underwent surgical resection in Taizhou Hospital of Zhejiang Province, Wenzhou Medical University. Patients received nothing therapy before surgery. After surgical resection, tissues were rapidly frozen in liquid nitrogen and then stored at - 80 ∘ C until RNA extraction. Study about tissues was approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province, Wenzhou Medical University. All patients enrolled in current study signed informed consent. This study was conducted according to the Declaration of Helsinki.

2.2 Cell lines and cell culture

All cell lines containing human renal tubular epithelial cells (HK-2) and renal cancer cells (OSRC-2, ACHN, A498 and 786O) were all acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). RPM11640 medium (Gibco) containing 10% fetal bovine serum (HyClone) was prepared for the culture of 786O and OSRC-2 cells and MEM medium (Gibco) containing 10% fetal bovine serum for the incubation of A498 and ACHN cells, in an incubator at 37 ∘ C with 5% CO 2 .

2.3 Cell transfection

For transfection, short hairpain RNAs (shRNAs) specifically targeting PCGEM1 (shPCGEM1#1/2/3), miR-433-3p mimics, miR-433-3p inhibitor and pcDNA3.1/FGF2 with relative negative controls (shNC, NC mimics, NC inhibitor and pcDNA3.1) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). 786-O and A498 cells (10 5 /well) were grown in a 6-pore culture plate prior to transfection. When cells reached 30–50% confluence, transfection of vectors was conducted by employing the Lipofectamine 2000 Transfection kit (Invitrogen, Thermo Fisher Scientific, Inc., USA) following the manufacturer’s directions.

2.4 Quantitative real-time polymerase chain reaction (qRT-PCR)

The extraction of total RNA from 786O and A498 cells was performed by use of TRIzol reagent (Invitrogen, USA) under the distributor’s instructions, followed by reverse transcription into cDNA with a First Strand cDNA Synthesis Kit (TOYOBO Life Science, Shanghai, China). A reaction mix of SYBR Green (Takara, China) was used to detect gene expression. The relative expression of PCGEM1 was normalized to that of GAPDH, and miR-433-3p to U6. The used primer sequences were as follows: 5’-TGCCTCAGC CTCCCAAGTAAC-3’ (sense) and 5’-GGCCAAAAT AAAACCAAACAT-3’ (antisense) for PCGEM1; 5’-C GATCATGATGGGCTCCTCG-3’ (sense) and 5’-GTG CAGGGTCCGAGGT-3’ (antisense) for miR-433-3p; 5’-ACTGGCTTCTAAATGTGTTACG-3’ (sense) and 5’-TTGGATCCAAGTTTATACTGCC-3’ (antisense) for FGF2; 5’-CTCGCTTCGGCAGCACA-3’ (sense) and 5’-AACGCTTCACGAATTTGCGT-3’ (antisense) for U6 and 5’-CACCCACTCCTCCACCTTTGA-3’ (sense) and 5’-ACCACCCTGTTGCTGTAGCCA-3’ (antisense) for GAPDH. The results were analyzed using the - Δ Ct method.

2.5 Microarray analysis

The microarray profiling was carried out using HK-2 cell and four kinds of renal cancer cells. RNA extraction and sequential microarray hybridization were performed by Biotechnology Company (Shanghai, China). Human lncRNA array V6.0 (4 × 180 K; Agilent Technologies, Inc., Santa Clara, CA, USA) was used for detection of the human genome transcripts. Raw data gained from microarrays were log2 transformed post quantile normalization. The absolute value of fold change (FC) $>$ 2 and the P -value 0.05 indicated that lncRNAs were differentially expressed in renal carci- noma cells. As for the screening of mRNAs affected by miR-433-3p, all experimented cells were divided into two groups: NC mimics and miR-433-3p mimics.

2.6 Cell Counting Kit-8 (CCK-8) assay

For the CCK-8 assay, we firstly inoculated cells (0.2 × 10 3 cells/well) into a 96-pore culture plate, and secondly supplemented CCK-8 solution (10 μ L) into each pore for extra incubation at 37 ∘ C. Finally, a microplate reader (BioTek, Winooski, VT, USA) was used for detection of the absorbance value at 450 nm.

2.7 5’-Ethynyl-2’-deoxyuridine (EdU) incorporation assay

An EdU assay kit (Guangzhou Ribobio Co., Ltd., Guanzhou, China) was manipulated following the supplier’s protocol. In brief, cells were cultivated in culture media complemented with 10% FBS with 5 μ M EdU for two hours at 37 ∘ C. Lastly, the DNA contents were dyed using DAPI at 37 ∘ C and imaged under a fluorescent microscope (magnification × 100; Nikon Corporation, Tokyo, Japan).

2.8 Flow cytometry

Cell apoptosis was determined using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (R&D Systems Europe, Ltd., Abingdon, UK) and analyzed by FACSCalibur flow cytometer, following the quantification with CellQuest 6.0 software (BD Biosciences, Franklin Lakes, NJ, USA).

2.9 Caspase-3 activity assay

Caspase-3 assay kit (Beyotime, Haimen, China) was applied to assess the caspase-3 activity following the protocols by the supplier. Stably transfected cells (5 × 10 6 ) were grown in 50- μ l cold Cell Lysis Buffer, followed by the separation of cell supernatant and analysis of protein density. After that, protein (50- μ l) was cultured utilizing 50- μ l 2 × Reaction Buffer and 5- μ l 4 mM DEVD-p-NA substrate (final concentration at 200 μ M) for about 2 h. The evaluation of absorbance value at 405 nm was implemented with a microplate reader (Tecan, Switzerland).

2.10 Transwell assay

The migratory capacity was evaluated through the migration number crossing Transwell inserts. Routinely, cell (3 × 10 5 cells) was cultured in 24-well plate-sized inserts (8-micron chamber; Corning Life Sciences, USA). Non-serum medium is proper for the upper chamber and the medium plus 10% fetal bovine serum for the lower chamber. Twenty-four hours later, cells which had not migrated across the pores were intensively removed by a wet cotton swab. All cells that had migrated from the top to the bottom part of the filter were fastened with 4% paraformaldehyde and subsequently stained with 1% crystal violet. They were then calculated and visualized via a light microscope (Olympus Corporation, Tokyo, Japan).

2.11 Fluorescence in situ hybridization (FISH)

Fluorescence-conjugated PCGEM1 probes were utilized for RNA FISH experiment. DNA probe sets (Ribobio, Guangzhou, China) were performed for the hybridization, and an NA1.4 inverted Leica DMI6000 microscope (Leica, Heidelberg, Germany) for the observation according to the distributor’s instructions. The images were visualized under a Hamamatsu ORCA-R2 camera (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed by LAS AF software (Leica).

2.12 Subcellular fractionation assay

As the supplier’s recommendation directed, subcellular fractionation assay was conducted using PARIS TM Kit (Invitrogen). Renal carcinoma cells were placed into cell fractionation buffer for centrifugation. The supernatant was collected and the remaining lysates were washed by cell fractionation buffer. Cell nuclei were lysed via cell disruption buffer. Next, the lysate was grown with the supernatant, ethanol and 2 × lysis/binding solution. Eventually, qRT-PCR detection was utilized to determine the cytoplasmic and nuclear RNAs.

2.13 Luciferase reporter assay

PCGEM1 full length which included the wild-type or mutant binding site for miR-433-3p were inserted into the pmirGLO vector (Promega, Madison, WI, USA), forming the reporter plasmids PCGEM1-WT and PCGEM1-Mut. FGF2 3’UTR containing miR-433-3p-binding sites or mutated miR-433-3p-binding sites were also inserted into pmirGLO vector (Promega, Madison, WI, USA) to construct FGF2-WT or FGF2-Mut reporter. A total of 2 × 10 4 cells were plated into 96-well culture plates and co-transfected with PCGEM1-WT or PCGEM1-Mut reporter plasmid and miR-433-3p mimics or NC mimics with the assistance of Lipofectamine 2000 (Invitrogen). For the relationship between FGF2 and miR-433-3p, cells were co-transfected with FGF2-WT or FGF2-Mut reporter plasmid and miR-433-3p mimics or NC mimics. Luciferase activities were examined 48 h post transfection by Dual-Luciferase Assay (Promega) obeying the manufacturer’s manual.

2.14 RNA immunoprecipitation (RIP)

RNA immunoprecipitation was carried out by employing RIP kit (Millipore, USA, 17-700) under the franchiser’s instructions. Briefly, the cell lysate by RIP lysis buffer was co-cultured with magnetic beads conjugated with human antibody against Argonaute2 (Ago2) or a negative control mouse Immunoglobulin G (IgG). Antibodies Ago2 and IgG were acquired from Abcam. qRT-PCR was adopted to detect the levels of PCGEM1, miR-433-3p and FGF2 in the RISC complex and also the levels of predicted miRNAs.

2.15 Tumor xenograft analysis

Male nude mice were obtained from Shanghai Institute of Material Medical (Chinese Academy of Science). With the approval of Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, the animal experiments were conducted as previously documented [15]. Immunohistochemical staining was conducted based on previous report for Ki-67 expression [16].

2.16 Statistical analysis

Data are demonstrated as the mean ± standard deviation (SD). All statistics were subjected to SPSS software (version 19.0; Inc., Chicago, IL, USA). Student’s t -test was utilized for comparing the differences between two groups while differences for more than two groups were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. All experiments were conducted thrice. P value below 0.05 indicated a statistically significant difference.

3.1 High level of PCGEM1 was a potential prognostic factor in patients with renal carcinoma

According to the results from the microarray, we focused on the top five overexpressed lncRNAs (LINC00958, LINC00899, SBF2-AS1, NR2F2-AS1, PCGEM1) in renal cancer cells (Fig. 1A). Besides, we probed their expression in human renal cancer tissues and normal control via qRT-PCR. No significant alterations were discovered in LINC00958, LINC00899, SBF2-AS1, and NR2F2-AS1 expressions (Fig. 1B–F). Of note, PCGEM1 was obviously overexpressed in renal carcinoma tissues. The cellular expression of PCGEM1 was also probed and results suggested its abundance in renal cancer cells, particularly in A498 and 786O cells (Fig. 1G). Meanwhile, highly expressed PCGEM1 predicted poorer survival in renal carcinoma patients (Fig. 1H). More importantly, we disclosed the close correlation of PCGEM1 intensity with TNM stage, tumor size and distant metastasis (Table 1).

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To inquire whether PCGEM1 took part in the initiation and development of renal carcinoma, we attempted to knock down PCGEM1 by transfecting shPCGEM1#1/2/3 into 786O and A498 cells for loss-of-function assays. PCGEM1 expression was decreased after transfection, among which shPCGEM1#1/2 exhibited higher transfection efficacy than shPCGEM1#3 (Fig. 2A). It was revealed by CCK-8 and EdU assays that PCGEM1 silencing restrained cell proliferation (Fig. 2B and C). Flow cytometry analysis exhi- bited that more apoptotic cells were produced when PCGEM1 was silenced (Fig. 2D). Caspase-3 activity assay uncovered that silencing PCGEM1 promoted cell apoptosis (Fig. 2E). And tranwell assay demonstrated that cell migration was inhibited when PCGEM1 was silenced (Fig. 2F). Besides, in vivo animal experiments were conducted. Tumors developed from shPCGEM1#2 cells were smaller and lighter than those from shNC cells (Fig. 2G–I). Besides, qRT-PCR assay also demonstrated the suppressed level of PCGEM1 in shPCGEM1#2-induced tumors in comparison to shNC-induced tumors (Fig. 2J). Immumohistochemical staining showed an inhibited Ki-67 level in shPCGEM1#2-induced tumors (Fig. 2K). To sum up, silencing of PCGEM1 repressed the growth and migration of renal carcinoma cells.

3.3 PCGEM1, located in the cytoplasm, interacted with miR-433-3p

LncRNAs located in the cytoplasm have been widely recognized to serve as ceRNAs. It was viewed that PCGEM1 was mainly situated in the cytoplasm, as suggested in FISH assay and subcellular fractionation assay (Fig. 3A and B). We searched on starBase and obtained the predicted miRNAs as target of PCGEM1. Ago2-RIP assay was performed to examine the enrichment of all these miRNAs and indicated the highest enrichment of miR-433-3p in RNA induced silencing complex (RISC) (Fig. 3C). The wild-type and mutant binding sites of PCGEM1 and miR-433-3p sequence were presented in Fig. 3D. Then miR-433-3p was overexpressed by miR-433-3p mimics for follow-up luciferase reporter experiment (Fig. 3E). In luciferase reporter experiment, only the luciferase activity of PCGEM1-WT was reduced by miR-433-3p mimics while PCGEM1-MUT had no change (Fig. 3F). In RIP experiment, the enrichment of PCGEM1 or miR-433-3p was found in Ago2 group (Fig. 3G). In addition, miR-433-3p expression was significantly low in renal carcinoma cells, and could be increased by shPCGEM1#2 (Fig. 3H and I). Collectively, PCGEM1 was located in the cytoplasm and targeted miR-433-3p.

3.4 PCGEM1 functioned as a ceRNA to upregulate FGF2 via sponging miR-433-3p

To further probe the regulation mechanism underlying PCGEM1, we continued to browse starBase v3.0 and gained 498 shared mRNAs from miRmap and PITA datasets (Fig. 4A). Heatmap illustrated that FGF2, a noted oncoprotein, exhibited the highest down-regulation in response to miR-433-3p promotion (Fig. 4B). The feasible wild-type and mutated binding sites between miR-433-3p and FGF2 were presented in Fig. 4C. As expected, the luciferase activity of FGF2-WT was dramatically suppressed by miR-433-3p overexpression, and both miR-433-3p and FGF2 were abundant in the mixture precipitated by anti-Ago2 (Fig. 4D and E). Besides, FGF2 expression in mRNA and protein levels were overtly elevated in renal carcinoma cells and obviously lessened by miR-433-3p upregulation (Fig. 4F and G). In order to explore the co-regulation of miR-433-3p and PCGEM1 on FGF2, miR-433-3p expression was abated by transfecting miR-433-3p inhibitor (Fig. 4H). FGF2 mRNA and protein expression was decreased by PCGEM1 knockdown but recovered by miR-433-3p inhibition (Fig. 4I). Taken together, PCGEM1 functioning as a ceRNA sponged miR-433-3p to regulate FGF2.

3.5 PCGEM1 sponged miR-433-3p to promote renal cancer cell growth and migration via upregulating FGF2

Finally, to certify whether miR-433-3p or FGF2 could function in PCGEM1-regulated renal cancer growth and migration, we performed a series of cell biological experiments. Cell proliferation, apoptosis and migration regulated by silenced PCGEM1 were rescued by the inhibition of miR-433-3p (Fig. 5A–E). Similarly, miR-433-3p induced inhibition on cell proliferation and migration as well as the promotion on cell apoptosis were rescued after co-transfection with FGF2 expression vector (Fig. 5F–J). Further, we employed rescue assays to verify the mechanism of PCGEM1 in renal carcinoma. FGF2 mRNA and protein expression were promoted by pcDNA3.1/FGF2 in 786O cells (Fig. 6A). As examined by CCK-8 and EdU experiments, cell proliferation was repressed by PCGEM1 downregulation, whereas regained by FGF2 overexpression, as shown in the promotion of cell viability and increase of EdU positive cells in three groups (Fig. 6B and C). As for cell apoptosis, flow cytometry revealed that apoptotic cells were firstly increased with PCGEM1 silencing and then restrained with FGF2 enhancement (Fig. 6D). Further, in caspase-3 activity assay, the PCGEM1 silence-activated cell apoptosis was abolished by FGF2 promotion (Fig. 6E). It was unveiled by transwell assay that cell migration retarded by shPCGEM1#2 was relieved by co-transfection of pcDNA3.1/FGF2 (Fig. 6F). In conclusion, PCGEM1 modulated cell progression in renal carcinoma by FGF2.