Circ_0000144 acts as a miR-1178-3p decoy to promote cell malignancy and angiogenesis by increasing YWHAH expression in papillary thyroid cancer

Clinical specimens and ethics statement

33 pairs of PTC tissues and adjacent normal thyroid tissues were collected from patients who underwent surgical resection at the The First People’s Hospital of Fuyang District. All recruited PTC patients signed informed consents. All human-related procedures were conducted in accordance with the Declaration of Helsinki, and the utilization of human PTC tissues was approved by the Ethics Committee of the The First People’s Hospital of Fuyang District. The information on PTC patients was provided in Table 1.

Table 1 Association of circ_0000144 expression with clinicopathological factors in PTC patientsCell culture

Normal human primary thyroid follicular epithelial cells Nthy-ori 3-1, human umbilical vein endothelial cells (HUVECs), and PTC cells TPC-1 and IHH-4 were bought from COBIOER (Nanjing, China) and maintained in a humidified atmosphere at 37 °C with 5% CO2. Nthy-ori 3-1 and TPC-1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Thermo Fisher, Waltham, MA, USA) supplemented with 10% FBS (fetal bovine serum) (Sigma, St. Louis, MO, USA), while IHH-4 cells were cultured in a mixture (1:1) of RPMI-1640 (Thermo Fisher) and DMEM (Dulbecco’s modified Eagle’s medium) (Thermo Fisher) supplemented with 10% FBS (Sigma).

Oligonucleotides and plasmids

Small interference (si) RNA targeting circ_0000144 (si-circ_0000144), negative control (NC) for siRNA (si-NC), short hairpin (sh) RNA against circ_0000144 (sh-circ_0000144), NC for shRNA (sh-NC), miR-1178-3p inhibitor (anti-miR-1178-3p), NC for miR inhibitor (anti-miR-NC), miR-1178-3p mimic (miR-1178-3p), and NC for miR mimic (miR-NC) were synthesized by AoKe Biotech (Beijing, China). Transfection of PTC cells was performed with the Lipofectamine 3000 reagent (Thermo Fisher). The pcDNA-YWHAH (YWHAH) plasmid was constructed using the pcDNA vector (vector) (Addgene, Cambridge, MA, USA).

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Redwood, CA, USA). Total RNA (1 μg) was reversely transcribed using the Prime-Script RT reagent kit (TaKaRa, Dalian, China) or TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). RT-qPCR was performed using the SYBR Prime Script RT-PCR kit (TaKaRa). All primer sequences were synthesized by AoKe Biotech (Table 2). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and U6 were used as internal references. Relative expression was figured with the 2−ΔΔCt method. Each experiment was performed in triplicate.

Table 2 Primer sequences for RT-qPCRCell counting kit-8 (CCK-8) assay

PTC cells were transfected with specific plasmids and/or oligonucleotides, followed by culturing in 96-well plates at a density of 5 × 103 cells/well for 24, 48, or 72 h. Then, the CCK-8 reagent (10 μL, Dojindo, Kumamoto, Japan) was added and incubated for 1 h. The OD (optical density) value (450 nm) was evaluated using a microplate reader (Bio-TEK, Winooski, VT, USA).

Cell cycle analysis

The transfected PTC cells (1 × 104) were cultured for 48 h and then detached with 0.025% trypsin (Thermo Fisher). After fixation with 70% ethanol for overnight (4 °C), the cells (1 × 106) were stained with propidium iodide (PI) solution (400 μL), which contained 4 µg/mL PI (Sigma), 0.5 mg/mL RNase A (Thermo Fisher), and 1% Triton X-100 (Sigma). The cellular DNA content was evaluated with a FACS Verse flow cytometer (Becton Dickinson, San Jose, California, USA), followed by analyzing with the FlowJo software (FlowJo v7.6, LLC, Ashland, OR, USA).

Western blotting

Total protein was extracted using the RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher). Total protein (30 μg) was isolated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to the polyvinylidene fluoride (PVDF) membrane (Bio-Rad). After sealing with 5% non-fat milk, the membranes were incubated with primary antibodies cyclinD1 (sc-450, 1:200), p21 (sc-53870, 1:200), B-cell lymphoma-2 (Bcl-2) (sc-7382, 1:200), Bcl-2 associated X (Bax) (sc-23959, 1: 200), YWHAH (sc-293464, 1:200), and GAPDH (sc-47724, 1:200). Then, the membranes were incubated with the mouse IgGκ BP-HRP. All primary antibodies were bought from Santa Cruz (Santa Cruz, CA, USA). The blots were developed using the Western Blotting Luminol Reagent (Santa Cruz).

Cell apoptosis analysis

After culturing for 48 h, the cells were collected, detached, and re-suspended in 1 × binding buffer (1 mL). Subsequently, the cells were stained with the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Solarbio, Beijing, China) in accordance with the manufacturer’s instructions. The apoptotic rate was determined by the FACS Verse flow cytometer (Becton Dickinson).

Wound-healing assay

The transfected PTC cells (1 × 105 cells/well) were cultured overnight and then the wounds were made with a sterile pipette tip (200 μL) in the cell monolayer. The wounded gaps were captured with a microscope (Nikon Eclipse E600, Nikon Instruments, Melville, NY, USA) at 0 and 24 h, followed by analyzing with the Image J software (NIH, Bethesda, MD, USA).

Transwell migration and invasion assays

Transwell chambers with Matrigel (#354,480, Costar, Cambridge, MA, USA) or without Matrigel (#3422, Costar) were used to assess the invasion and migration of transfected PTC cells. The transfected PTC cells (1 × 105) were re-suspended in cell culture medium lacking FBS and then placed on the apical chamber. The cell culture medium encompassing 10% FBS (Sigma) was added to the basolateral chamber. After culturing for 24 h, the migrating and invading cells were fixed with 4% paraformaldehyde (Sigma) and stained with 0.1% crystal violet (Thermo Fisher). The migrating or invading cells in five random fields were counted under the microscope (Nikon Eclipse E600, Nikon Instruments).

Tube formation assay

HUVECs were placed on 96-well plates, which were pre-cooled, coated with Matrigel (70 μL, Becton Dickinson), and then placed at 37 °C for 30 min. The cells were incubated with the supernatant of PTC cells. The tubule branches were imaged with a microscope (Nikon Eclipse E600, Nikon Instruments), followed by the calculation of the number of tubule branches using the Image J software (NIH).

Xenograft assay

To obtain TPC-1 cells stable knockdown of circ_0000144, the sequence of sh-circ_0000144 was inserting into the pLKO.1 vector (Thermo Fisher), and sh-NC was utilized as an NC. Then, these plasmids were transfected into HEK293T cells (COBIOER) together with the lentiviral packaging plasmids (Thermo Fisher). Subsequently, TPC-1 cells were infected with lentiviral particles produced by HEK293T cells, followed by selection with puromycin (2 μg/mL) (Sigma).

For the xenograft assay, TPC-1 cells carrying sh-circ_0000144 or sh-NC were subcutaneously injected into the flank of each BALB/c nude mouse (4–6 weeks old, 15–20 g). Tumor volumes were measured once a week. After injection for 4 weeks, the tumor tissues of the euthanized mice were stripped for tumor weight evaluation and subsequent analysis. 10 BALB/c nude mice (Vital River Laboratory, Beijing, China) were divided into 2 groups by random number table. Tumor volume was calculated based on the following equation: Volume = (length × width2)/2. The animal experiments were approved by the Animal Ethics Committee of The First People’s Hospital of Fuyang District.

Immunohistochemistry (IHC) staining

IHC was conducted to detect the proliferative ability of PTC cells in vivo. IHC staining was performed using the Vectastain Universal Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) based on the manufacturer’s instructions. Paraffin-embedded xenograft tissue Sects. (4 μm thick) were incubated with anti-Ki67 antibody (sc-23900, 1:200, Santa Cruz) at 4 °C for 12 h.

Dual-luciferase reporter assay

The putative binding sites between circ_0000144 or YWHAH and miR-1178-3p were predicted by the circular RNA interactome or targetscan database. The wild-type (WT) and mutant (MUT) sequences of circ_0000144 and YWHAH 3’ untranslated region (UTR) were synthesized and then inserted into the psiCHECK2 vector (Promega, Madison, WI, USA), respectively. The luciferase activity in PTC cells co-transfected with miR-1178-3p mimic or miR-NC and the luciferase plasmids carrying WT-circ_0000144, MUT-circ_0000144, WT-YWHAH 3’UTR, or MUT-YWHAH 3’UTR was evaluated using a dual-luciferase reporter assay kit (BioVision, Milpitas, CA, USA).

Statistical analysis

At least three biological repeats were conducted for each experiment, and all data were presented as mean ± standard deviation. GraphPad Prism 7 software (GraphPad, La Jolla, CA, USA) was utilized for statistical analyses. Differences between two or more groups were evaluated using Student’s t-test or analysis of variance. All statistical tests were considered significant when P < 0.05.

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