Specimens collection
60 PTC tissues and adjacent normal thyroid tissues were obtained from PTC patients who underwent surgery at The First Affiliated Hospital of Guangxi Medical University and frozen at -80 °C until use. These patients did not receive any treatment prior to this surgical procedure.
Cell Culture
Human thyroid cell line Nthy-ori3-1 was purchased from BNBIO (Beijing, China), PTC cell lines TPC-1, K-1 and SW1736 were bought from Shanghai Institute of Cell Biology (Shanghai, China) and PTC cell line SW579 was obtained from Procell (Wuhan, China). All cells were kept in a humidified incubator (95% air/5% CO2) at 37 °C in RPMI1640 (Invitrogen, Carlsbad, CA, USA) containing 1% penicillin-streptomycin (Solarbio, Beijing, China) and 10% fetal bovine serum (FBS; Solarbio).
Quantitative Real-time Polymerase Chain Reaction (qrt-pcr)
Total RNA was isolated using TRIzol reagent (Beyotime, Shanghai, China) and determined on a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription experiment was conducted using PrimeScript™ RT reagent Kit (Takara, Dalian, China) or TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Next, the BeyoFast™ SYBR Green qPCR Mix (Beyotime) was utilized to conduct qRT-PCR. The expression was estimated using the 2−ΔΔCt method with U6 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference. The sequences of primers were: circ_LDLR: (F: 5’-GTGAGGGCTCTGTCCATTGT-3’ and R: 5’-GGTGGTCCTCTCACACCAGT-3’); LDLR: (F: 5’-CAATGTCTCACCAAGCTCTG-3’ and R: 5’-TCTGTCTCGAGGGGTAGCTG-3’); miR-195-5p: (F: 5’-GGGGTAGCAGCACAGAAAT-3’ and R: 5’-TCCAGTGCGTGTCGTGGA-3’); LIPH: (F: 5’-CTGATGCTCTACACAAGGA-3’ and R: 5’-ATGGACAATGAAGGTGGTT-3’); GALNT7: (F: 5’-GGTACCATGGCCTCATGTTG-3’ and R: 5’-GCCACCACACTGCCATATCT-3’); PSD3: (F: 5’-GCTCTGTACAACTCAATCAAGAATG-3’ and R: 5’-CCAATACGACTGATGGTCTTTG-3’); ITGA2: (F: 5’-CCTACAATGTTGGTCTCCCAGA-3’ and R: 5’-AGTAACCAGTTGCCTTTTGGATT-3’); GAPDH: (F: 5’-CTGGGCTACACTGAGCACC-3’ and R: 5’-AAGTGGTCGTTGAGGGCAATG-3’); U6: (F: 5’-TTATGGGTCCTAGCCTGAC-3’ and R: 5’-CACTATTGCGGGTCTGC-3’).
Rnase R Digestion Assay
10 µg total RNA was incubated with RNase R (20 mg/mL; Solarbio) for 20 min at 37 °C. Total RNA treated without RNase was used as control. Then qRT-PCR was carried out to measure the levels of circ_LDLR and LDLR mRNA.
Actinomycin D Assay
After TPC-1 and SW579 cells were sowed into 24-well plates and incubated overnight, 2 µg/mL Actinomycin D (Abcam, Cambridge, MA, USA) was added to block transcription at indicated time points. QRT-PCR was employed to examine the expression of circ_LDLR and LDLR mRNA.
Cell Transfection
The small interfering RNA targeting circ_LDLR (si-circ_LDLR), mimics of miR-195-5p (miR-195-5p), inhibitors of miR-195-5p (anti-miR-195-5p), the overexpression vector of LIPH (LIPH), short hairpin RNA against circ_LDLR (sh-circ_LDLR) and their controls were bought from Sangon (Shanghai, China). Lipofectamine 2000 (Invitrogen) was adopted for cell transfection.
Colony Formation Assay
After relevant transfection, PTC cells were seeded into 6-well plates (300 cells/well). The culture medium was changed every 3 days. 10 days later, the colonies were fixed with 4% paraformaldehyde (Sangon) and stained with 0.2% crystal violet (Solarbio). Finally, the colonies were counted and photographed.
3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium Bromide (mtt) Assay
The proliferation of PTC cells was assessed through MTT assay. Transfected PTC cells were collected and plated into 96-well plates and kept overnight. Then, 20 µL MTT (5 mg/mL; Solarbio) was added into the well at indicated time points and cultured for another 4 h. Afterward, the formazan crystals were dissolved through adding 150 µL dimethyl sulfoxide (DMSO; Solarbio) followed by determination of OD value at 490 nm with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Western Blot Assay
Total protein was isolated with RIPA buffer (Beyotime) and determined with a BCA protein assay kit (Beyotime). Then the extracts were separated by sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Next, the membranes were maintained in 5% slim milk for 1 h and incubated overnight at 4 °C with primary antibodies: Ki67 (ab16667; Abcam), Twist1 (ab49254; Abcam), E-cadherin (ab231303; Abcam), LIPH (ab192615; Abcam), GALNT7 (ab113743; Abcam), PSD3 (ab62194; Abcam), ITGA2 (ab133557; Abcam) or GAPDH (ab181602; Abcam). Afterward, the samples were subjected to secondary antibody (ab205719; Abcam) for 2 h at room temperature. At last, the protein bands were determined using ECL western blot kit (Beyotime).
Flow Cytometry Analysis
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Beyotime) was adopted for cell apoptosis. Briefly, transfected PTC cells were harvested and suspended. Then Annexin-FITC and PI were added to stain cells in the dark. 15 min later, the apoptotic cells were analyzed by FACScan® flow cytometry (BD Biosciences, San Jose, CA, USA).
Transwell Assay
Transwell insert chambers (Corning, Corning, NY, USA) coated with (for cell invasion assay) or without (for cell migration assay) Matrigel (Corning) were adopted to evaluate the invasion and migration of PTC cells. In brief, about 1 × 106 transfected cells suspended in 300 µL serum-free RPMI1640 (Invitrogen) were added in the upper chamber and 500 µL complete medium was added into the bottom chamber. After 24 h, migrated/invaded cells were stained with crystal violet (Solarbio) and observed with an inverted microscope (Olympus, Tokyo, Japan).
Dual-luciferase Reporter Assay
The fragments of circ_LDLR and LIPH 3’UTR containing the predicted binding sites of wild-type or mutant miR-195-5p were inserted into pmirGLO plasmid (Promega, Madison, WI, USA) and luciferase reporter vectors circ_LDLR-wt, circ_LDLR-mut, LIPH-wt and LIPH-mut were constructed. Then PTC cells were seeded into 24-well plates and transfected with miR-NC or miR-195-5p together with corresponding luciferase reporter vector. After 48 h, Dual-Luciferase Reporter Assay Kit (Promega) was applied to measure the luciferase activity.
Murine Xenograft Model
Lentivirus-mediated sh-circ_LDLR or sh-NC was transfected into TPC-1 cells. Then transfected cells were injected into the BALB/c nude mice (Shanghai SLAC Laboratory Animals Co., Ltd, Shanghai, China). Tumor volume was measured weekly and calculated using the formula: (length × width2)/2. After 4 weeks, the mice were euthanized and tumors were weighted. The collected tumor samples were preserved at -80 °C.
Statistical analysis
The experiments were repeated at least three times. The obtained data were analyzed by GraphPad Prism 7 (GraphPad Inc., La Jolla, CA, USA) and exhibited as mean ± standard deviation. Differences were estimated by Student’s t-test or one-way analysis of variance (ANOVA). The correlations between the expression of circ_LDLR and miR-195-5p, as well as miR-195-5p and LIPH mRNA were analyzed by Spearman’s correlation coefficient analysis. It was deemed as statistically significant if P < 0.05.