Circ-MAP4K3 promotes papillary thyroid cancer progression by sponging miR-758-3p to enhance RRM2 expression

Accumulation studies confirmed that circRNAs play an important role in regulating malignant TPC progression [ 26 , 27 ]. A large number of differential circRNAs were found between PTC and paracarcinoma tissues in the present study (Fig. 1 A). FISH assay results showed that circ-MAP4K3 expression was increased in TPC tissues and that circ-MAP4K3 was localized in the cytoplasm (Fig. 1 B). RT-qPCR results also revealed that circ-MAP4K3 expression was increased in TPC tissues (Fig. 1 C) and in TPC cells when compared to that in Nthy-ori 3 − 1 cells (Fig. 1 D). Both TPC-1 and B-CPAP cells demonstrated a higher circ-MAP4K3 expression level and were thus selected for use in further investigation. Bioinformatics analysis determined that hsa_circ_0054181 is derived from 13 cyclizing exons in the MAP4K3 gene located at chr2:39476421–39,515,421. MAP4K3 is 39,000 bp in length, while the spliced mature circRNA is 2,708 bp long (Fig. 1 E). Thus, hsa_circ_0054181 was named circ-MAP4K3. RNase R was used to treat MAP4K3 and circ-MAP4K3 mRNAs in TPC-1 and B-CPAP cells, showing that circ-MAP4K3 was resistant to RNase R. The MAP4K3 mRNA levels were almost unaffected by the RNase R treatment (Fig. 1 F and G). Furthermore, circ-MAP4K3 was abundant in the cytoplasm as determined by the FISH assays (Fig. 1 H). Fig. 1 Circ-MAP4K3 validation and expression in PTC tissues and cells. ( A ) Heatmap of all differentially expressed circRNAs between PTC and paracarcinoma tissues. T, tunor; N, normal. ( B ) RNA FISH revealed that circ-MAP4K3 expression was increased in PTC and paracarcinoma tissues. ( C ) RT-qPCR analysis results showing circ-MAP4K3 expression ( C ) between PTC and paracarcinoma tissues (data are expressed as mean ± SD; *** p  < 0.001 vs. Adjacent) and ( D ) in PTC cell lines compared to that in Nthy-ori 3 − 1 cell line. Data are expressed as mean ± SD. * p  < 0.05, ** p  < 0.01, and *** p  < 0.001 vs. Nthy-ori 3 − 1. ( E ) Genomic loci of MAP4K3 gene and circ-MAP4K3. ( F and G ) circ-MAP4K3 and MAP4K3 mRNA-level expression was analyzed using RT-qPCR after RNase R treatment and compared to controls. Data are expressed as mean ± SD. * p  < 0.05, ** p  < 0.01, and *** p  < 0.001 vs. NC. (H) RNA FISH analysis revealed that circ-MAP4K3 was localized in the cytoplasm of PTC cells. circ-MAP4K3 probes were labeled with FITC and the nuclei were stained with DAPI Circ-MAP4K3 validation and expression in PTC tissues and cells. ( A ) Heatmap of all differentially expressed circRNAs between PTC and paracarcinoma tissues. T, tunor; N, normal. ( B ) RNA FISH revealed that circ-MAP4K3 expression was increased in PTC and paracarcinoma tissues. ( C ) RT-qPCR analysis results showing circ-MAP4K3 expression ( C ) between PTC and paracarcinoma tissues (data are expressed as mean ± SD; *** p  < 0.001 vs. Adjacent) and ( D ) in PTC cell lines compared to that in Nthy-ori 3 − 1 cell line. Data are expressed as mean ± SD. * p  < 0.05, ** p  < 0.01, and *** p  < 0.001 vs. Nthy-ori 3 − 1. ( E ) Genomic loci of MAP4K3 gene and circ-MAP4K3. ( F and G ) circ-MAP4K3 and MAP4K3 mRNA-level expression was analyzed using RT-qPCR after RNase R treatment and compared to controls. Data are expressed as mean ± SD. * p  < 0.05, ** p  < 0.01, and *** p  < 0.001 vs. NC. (H) RNA FISH analysis revealed that circ-MAP4K3 was localized in the cytoplasm of PTC cells. circ-MAP4K3 probes were labeled with FITC and the nuclei were stained with DAPI To explore the involvement of circ-MAP4K3 in PTC cell functions, circ-MAP4K3 small hairpin RNAs and circ-MAP4K3 overexpression plasmids were designed and transfected into PTC cells to establish stable circ-MAP4K3-knockdown or -overexpressing cells. The transfection efficiency of circ-MAP4K3 and subsequent MAP4K3 expression levels were examined using RT-qPCR. The results showed that knockdown or overexpression of circ-MAP4K3 did not influence the MAP4K3 mRNA levels (Fig. 2 A and B). CCK-8 (Fig. 2 C and D) and EdU (Fig.  2 E and G) assays demonstrated that circ-MAP4K3 downregulation inhibited TPC-1 and B-CPAP cell proliferation, while circ-MAP4K3 overexpression promoted it. Wound healing experiments (Fig. 2 H–K) and Transwell migration (Fig. 2 L–N) analysis showed that circ-MAP4K3 downregulation inhibited TPC-1 and B-CPAP cell migration, while circ-MAP4K3 overexpression promoted it. Fig. 2 Circ-MAP4K3 plays an important role in regulation of PTC cell migration and proliferation abilities. ( A ) RT-qPCR analysis results showing circ-MAP4K3 and MAP4K3 mRNA expression in both TPC-1 and B-CPAP cells after transfection with short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( B ) RT-qPCR analysis results showing circ-MAP4K3 and MAP4K3 mRNA expression levels in both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( C and D ) CCK-8 assay results showing the proliferation ability in both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. ** p  < 0.01, *** p  < 0.001. ( E – G ) EdU results showing the proliferation ability of both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. si-circ-MAP4K3. ( H – K ) Wound healing experiment results showing the migration ability of both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ## p  < 0.01, ### p  < 0.001 vs. si-circ-MAP4K3. ( L – N ) Transwell assay results showing the migration ability of both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. si-circ-MAP4K3 Circ-MAP4K3 plays an important role in regulation of PTC cell migration and proliferation abilities. ( A ) RT-qPCR analysis results showing circ-MAP4K3 and MAP4K3 mRNA expression in both TPC-1 and B-CPAP cells after transfection with short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( B ) RT-qPCR analysis results showing circ-MAP4K3 and MAP4K3 mRNA expression levels in both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( C and D ) CCK-8 assay results showing the proliferation ability in both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. ** p  < 0.01, *** p  < 0.001. ( E – G ) EdU results showing the proliferation ability of both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. si-circ-MAP4K3. ( H – K ) Wound healing experiment results showing the migration ability of both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ## p  < 0.01, ### p  < 0.001 vs. si-circ-MAP4K3. ( L – N ) Transwell assay results showing the migration ability of both TPC-1 and B-CPAP cells after transfection with MAP4K3 overexpression vector or short hairpin RNAs. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. si-circ-MAP4K3 In vivo experiments confirmed that circ-MAP4K3 downregulation inhibited tumor growth based on both tumor volume and weight (Fig. 3 A–C). Immunohistochemistry analysis of Ki67 expression showed that circ-MAP4K3 silencing inhibited Ki67 expression in tumor tissues (Fig. 3D and E). Live imaging revealed that circ-MAP4K3 silencing led to a decrease in pulmonary metastasis of TPC-1 cells as indicated by a decreasing number of metastatic foci in HE-stained lung tissue (Fig.  3 F and H). Fig. 3 Downregulation of circ-MAP4K3 inhibits growth and migration. ( A – C ) In vitro xenograft mouse assay results for tumor volume and weight in nude mice injected with TPC-1 cells transfected with sh-circ-MAP4K3 or sh-NC. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( D and E ) Immunohistochemical analysis results for Ki67 staining showing tumor growth capacity. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( F ) Live imaging results showing pulmonary metastasis in TPC-1 cells. ( G and H ) Numbers of metastatic foci in lung tissues calculated based on HE staining results. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC Downregulation of circ-MAP4K3 inhibits growth and migration. ( A – C ) In vitro xenograft mouse assay results for tumor volume and weight in nude mice injected with TPC-1 cells transfected with sh-circ-MAP4K3 or sh-NC. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( D and E ) Immunohistochemical analysis results for Ki67 staining showing tumor growth capacity. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC. ( F ) Live imaging results showing pulmonary metastasis in TPC-1 cells. ( G and H ) Numbers of metastatic foci in lung tissues calculated based on HE staining results. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC Bioinformatics analysis determined that circ-MAP4K3 interacted with a series of miRNAs, including miR-3164, miR-758-3p, miR-338-3p, miR-324-5p, miR-493-5p, miR-511-3p, miR-98-5p, miR-196a-5p, and miR-196b-5p. A luciferase reporter analysis confirmed that miR-758-3p inhibited luciferase activity in wild-type cells but not in mutated cell lines (Fig. 4 A–C), suggesting that miR-758-3p was the circ-MAP4K3 target. EdU assays demonstrated that miR-758-3p inhibition rescued the proliferation ability of TPC-1 cells after circ-MAP4K3 silencing (Fig.  4 D and E). Transwell assay showed that miR-758-3p inhibition rescued the migration ability of TPC-1 cells after circ-MAP4K3 silencing (Fig. 4 F and G). Fig. 4 Circ-MAP4K3 regulation of PTC cell progression using target miR-758-3p. ( A ) Luciferase reporter assay showing relative luciferase activity 48 h after transfection of TPC-1 cells with different miRNA mimics and luciferase reporting vector. ( B ) Bioinformatics analysis predicting binding sites for miR-758-3p in circ-MAP4K3. Mutant version of circ-MAP4K3 is shown. ( C ) Relative luciferase activity determined 48 h after transfection of TPC-1 cells with miR-758-3p mimic/NC or circ-MAP4K3 wild-type/Mut. Data are expressed as mean ± SD. *** p  < 0.001. ( D and E ) EdU assay results showing the proliferation ability of TPC-1 cells. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. ( F and G ) Transwell assay results showing TPC-1 cell migration ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. mimic, miR-758-3p overexpression Circ-MAP4K3 regulation of PTC cell progression using target miR-758-3p. ( A ) Luciferase reporter assay showing relative luciferase activity 48 h after transfection of TPC-1 cells with different miRNA mimics and luciferase reporting vector. ( B ) Bioinformatics analysis predicting binding sites for miR-758-3p in circ-MAP4K3. Mutant version of circ-MAP4K3 is shown. ( C ) Relative luciferase activity determined 48 h after transfection of TPC-1 cells with miR-758-3p mimic/NC or circ-MAP4K3 wild-type/Mut. Data are expressed as mean ± SD. *** p  < 0.001. ( D and E ) EdU assay results showing the proliferation ability of TPC-1 cells. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. ( F and G ) Transwell assay results showing TPC-1 cell migration ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. mimic, miR-758-3p overexpression Bioinformatics analysis found that RRM2 was the downstream target of miR-758-3p. To further confirm the relationship between miR-758-3p and RRM2, wild-type or mutated 3’UTR-RRM2 sequences containing the miR-758-3p binding sequence were incorporated into a luciferase reporter vector (Fig. 5 A). The luciferase reporter vector was then transfected into TPC-1 cells with or without the miR-758-3p mimic. Luciferase reporter analysis determined that miR-758-3p inhibited luciferase activity in wild-type cells but not in mutated cell lines (Fig. 5 B), suggesting that RRM2 was the miR-758-3p target. RT-qPCR results demonstrated that circ-MAP4K3 silencing inhibited RRM2 expression at the mRNA level. However, RRM2 expression increased significantly after transfection with RRM2 overexpression vector (Fig. 5 C). EdU assay results revealed that RRM2 overexpression rescued the proliferation ability of TPC-1 cells after circ-MAP4K3 silencing (Fig.  5 D and E). Transwell assays showed that RRM2 overexpression rescued the migration ability of TPC-1 cells after circ-MAP4K3 silencing (Fig. 5 F and G). Fig. 5 Circ-MAP4K3 regulation of PTC cell progression via RRM2 regulation. ( A ) Bioinformatics analysis predicting binding sites for miR-758-3p in RRM2. Mutant version of RRM2 is shown. ( B ) Relative luciferase activity determined 48 h after transfection of TPC-1 cells with miR-758-3p mimic/NC or RRM2 wild-type/Mut. Data are expressed as mean ± SD. *** p  < 0.001. ( C ) RT-qPCR assay results showing RRM2 expression. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. ( D and E ) EdU assay results showing the proliferation ability of TPC-1 cells. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. ( F and G ) Transwell assay results showing TPC-1 cell migration ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3 Circ-MAP4K3 regulation of PTC cell progression via RRM2 regulation. ( A ) Bioinformatics analysis predicting binding sites for miR-758-3p in RRM2. Mutant version of RRM2 is shown. ( B ) Relative luciferase activity determined 48 h after transfection of TPC-1 cells with miR-758-3p mimic/NC or RRM2 wild-type/Mut. Data are expressed as mean ± SD. *** p  < 0.001. ( C ) RT-qPCR assay results showing RRM2 expression. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. ( D and E ) EdU assay results showing the proliferation ability of TPC-1 cells. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3. ( F and G ) Transwell assay results showing TPC-1 cell migration ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. sh-circ-MAP4K3 RT-qPCR analysis showed that miR-758-3p overexpression inhibited RRM2 expression. However, RRM2 overexpression did not influence miR-758-3p expression (Fig. 6 A and B). EdU assays demonstrated that RRM2 overexpression rescued the proliferation ability of TPC-1 cells after miR-758-3p overexpression (Fig. 6 C and D). Transwell assays revealed that RRM2 overexpression rescued the migration ability of TPC-1 cells after miR-758-3p overexpression (Fig.  6 E and F). Fig. 6 RRM2 overexpression rescued the migration and proliferation abilities after miR-758-3p overexpression. ( A and B ) RT-qPCR analysis results showing miR-758-3p and RRM2 expression. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. mimic. ( C and D ) EdU assay results showing TPC-1 proliferation ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. mimic. ( E and F ) Transwell assay results showing TPC-1 migration ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. mimic RRM2 overexpression rescued the migration and proliferation abilities after miR-758-3p overexpression. ( A and B ) RT-qPCR analysis results showing miR-758-3p and RRM2 expression. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. mimic. ( C and D ) EdU assay results showing TPC-1 proliferation ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. mimic. ( E and F ) Transwell assay results showing TPC-1 migration ability. Data are expressed as mean ± SD. *** p  < 0.001 vs. NC, ### p  < 0.001 vs. mimic

BALB/c mice aged 4–6 weeks and weighing 15–20 g were acquired from Shanghai SLAC Laboratory Animals Co. Ltd., Shanghai, China. The animal ethics committee at Shanghai Gongli Hospital approved this research. The animal experiments followed the ARRIVE guidelines ( https://arriveguidelines.org/arrive-guidelines ). First, 2.5 µg of total RNA was extracted from TPC-1 and B-CPAP cells and then incubated for 15 min at 37 °C with or without 10 U RNase R (Geneseed, Guangzhou, China). The expression levels of circTIAM1 and TIAM1 were then determined using RT-qPCR and the results were normalized to GAPDH . Human PTC cell lines TPC-1, B-CPAP, KTC-1, and IHH-4 and human thyroid follicular epithelial cell line Nthy-ori 3 − 1 were kindly provided by Stem Cell Bank, Chinese Academy of Sciences or ATCC (Manassas, VA, USA) and confirmed by STR profiling analysis. No mycoplasma contamination was detected. PTC and human thyroid follicular epithelial cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). B-CPAP cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and MEM non-essential amino acid solution (100X; Invitrogen, 11140050). Cells were cultured under proper conditions at 37 °C with 5% CO 2 . The siRNA was transfected against circ-MAP4K3 (si-circ-MAP4K3), circ-MAP4K3 overexpression vector (OE-circ-MAP4K3), RRM2 overexpression vector (RRM2), miR-758-3p inhibitor (5ʹ-GGUUAGUGGACCAGGUCACAAA-3ʹ), and miR-758-3p mimic (5ʹ-UUUGUGACCUGGUCCACUAACC-3ʹ; GenePharma, Shanghai, China) in TPC-1 cells using Lipofectamine 2000 transfection reagent (Invitrogen) for two days prior to further analyses. PTC samples were procured from Shanghai Gongli Hospital. The informed consent was obtained from all patients participating in the study. Both normal and PTC tumor samples were extracted. The tumor tissue was not treated with any chemotherapy drugs before the surgery. Ethics committee of the Shanghai Gongli Hospital approved the research study. Total RNA samples from TPC tissues were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, 3 µg of total RNA was used to remove ribosomal RNA and retain non-coding RNAs employing VAHTS Total RNA-Seq (H/M/R) Library Prep kits for Illumina (Vazyme Biotech Co., Ltd, Nanjing, China). RNA was treated with 40 U of RNase R (Epicenter) at 37 °C for 3 h followed by TRIzol purification. RNA-Seq libraries were prepared via KAPA Stranded RNA-Seq Library Prep kits (Roche, Basel, Switzerland) and utilized for deep sequencing (Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China). DNA synthesis and cell proliferation were analyzed using EdU assay kit (RiboBio, Guangzhou, China). Then, 1 × 10 4 TPC-1 and B-CPAP cells were seeded into 96-well plates and incubated overnight. On the second day, EdU solution (25 µM) was added to the plates and incubated for 24 h. Next, 4% formalin was used to fix the cells for 2 h at room temperature before 0.5% Triton X-100 permeabilization treatment for 10 min. Finally, Apollo reaction solution (200 µL) was added prior to EdU and DAPI (200 µL) staining of the nuclei for 30 min. DNA synthesis and cell proliferation were measured using a Nikon microscope (Nikon, Tokyo, Japan). Cells were incubated in 10% CCK-8 solution diluted in normal culture medium at 37 ℃ until visible color change occurred. Proliferation rates were determined 24, 48, and 72 h after transfection. The absorbance values of samples at 450 nm were acquired via a microplate reader (Bio-Rad Laboratories; Hercules, CA, USA). Cell proliferation index was calculated as the relative value of sample absorbance to a non-treatment control. The Transwell migration assay was performed using a Transwell chamber according to the manufacturer’s protocols (BD Biosciences, Bedford, MA, USA). Briefly, 200 µL of serum-free medium containing 5 × 10 4 treated cells was added to the upper chambers. Then, 600 µL of complete medium was added to the lower chambers, and the cells were incubated for 24 h. Representative cell images were obtained under an inverted light microscope (Zeiss, Primovert). Migrated cells were quantified by counting the number of migrated cells in at least three randomly selected fields of view. Both TPC-1 and B-CPAP cells were incubated until they formed confluent monolayers in six-well plates, which were then scored with a 200-µL pipette tip. The cells were then incubated for three days with oridonin after rinsing twice with phosphate-buffered saline. Light microscope images of the wounds were obtained. Total RNA samples were extracted using TRIzol Reagent (Invitrogen). Then, cDNA synthesis was performed utilizing the TransScript All-in-One First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China). RT-qPCR was carried out in a Bio-Rad PCR machine (Bio-Rad, Hercules, CA, USA) with 2 × Taq PCR Master Mix (Solarbio, Beijing, China) according to the manufacturer’s instructions. Relative fold changes were determined using the mean values of the relative quantifications. The 2 −ΔΔCT method was used to determine the relative fold differences in expression. U6 and GAPDH served as the internal references. Primers employed to assay circ-MAP4K3 expression were as follows (forward/reverse): 5′-CATATTGTAAAATAAACATG-3′ / 5′-CATTTCCTGTGGTATGAAG-3′; miR-758-3p : 5′-ACACTCCAGCTGGGTTTGTGACCTGGTCCA-3′ / 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGTTAGTG-3′; RRM2 : 5′-AGTGGAAGGCATTTTCTTTTCC-3′ / 5′-GCAAAATCACAGTGTAAACCCT-3′; U6: 5′-CTCGCTTCGGCAGCACATATACTA-3′ / 5′-ACGAATTTGCGTGTCATCCTTGCG-3′; GAPDH: 5′-TGACTTCAACAGCGACACCCA-3′ / 5′-CACCCTGTTGCTGTAGCCAAA-3′; and β-actin: 5′-CATGTACGTTGCTATCCAG-3′ / 5′-CTCCTTAATGTCACGCAC-3′. The interaction relationship among circRNA, miRNA, and mRNA was predicted using the starBase database ( http://starbase.sysu.edu.cn/ ). Circ-MAP4K3 and RRM2 3′-UTRs containing the predicted binding sequence for miR-758-3p were amplified and inserted into several cloning sites of the pMIR-REPORT luciferase reporter vector. Next, TPC-1 cells were co-transfected with 0.1 µg of luciferase reporter vectors comprising wild-type (WT) or mutant (MUT) RRM2 3′-UTR and miR-758-3p mimic or miR-control using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The relative luciferase activity was calculated by normalizing the firefly luminescence to Renilla luminescence using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions two days post-transfection. To construct a subcutaneous TPC model, 2 × 10 6 viable Wt or sh-circ-MAP4K3 TPC-1 cells were injected into the right flanks of nude mice. Tumor volumes were measured every five days for 30 consecutive days using a Vernier caliper. The tumor volume was calculated as 4/3π (diameter1/2 × diameter2/2 × diameter 3/2). For tumor metastasis analysis, luminescence-labeled TPC-1 cells were stably transfected with NC or sh-circ-MAP4K3. The TPC-1 cells were then resuspended in sterile phosphate-buffered saline and injected into the tail vein of each nude mouse. Lung metastasis was evaluated after four weeks via an in vivo bioluminescence imaging system. The numbers of metastatic foci in lung tissues were calculated based on the HE staining. Tumor tissue samples were fixed in 10% formalin solution, embedded in paraffin, and sliced into 5-µm sections. Ki67 staining was used to show Ki67 expression in tumor tissues. HE staining was utilized to determine the number of lesions in the lung tissues. Axiophot light microscope (Zeiss, Oberkochen, Germany) was employed to photograph the lesions with a digital camera. Ki67-positive cells were quantified using 6 field. Statistical analysis was conducted using GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA). Overall survival analysis was performed using Kaplan–Meier curves and log-rank tests. Two-tailed Student’s t tests were used to explore the significance between two groups, and one-way analysis of variance with the post hoc Bonferroni test was used for 3 groups. Correlations were determined using the Pearson correlation test. Data are expressed as the mean ± standard deviation. A P value of < 0.05 indicated a statistically significant difference.

Previous studies have confirmed that circRNA play an important role in progress of PTC including hsa_circ_102002, hsa_circ_0004851, hsa_circ_0092278, hsa_circ_0007694 [ 28 – 31 ]. The present study found that hsa_circ_0054181 (circ-MAP4K3) expression was increased in both PTC tissues and cell lines. Hsa_circ_0054181 was derived from 13 cyclizing exons in the MAP4K3 gene located at chr2:39476421–39,515,421. Accumulation studies confirmed that an increase in mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) level was observed in tumor tissues, including lung cancer, glioblastoma, and hepatocellular carcinoma, compared to that in normal tissues [ 32 – 34 ]. The present study found that circ-MAP4K3 expression was increased and was abundant in the cytoplasm. Downregulation of circ-MAP4K3 inhibited the proliferation, migration, tumor growth and pulmonary metastasis of PTC in both in vivo and in vitro experiments, suggesting that circ-MAP4K3 plays a role in PTC progression regulation. Bioinformatics analysis confirmed that miR-758-3p was the downstream target of circ-MAP4K3. The circ-MAP4K3 silencing promoted the expression of miR-758-3p. The present study also found that circRNAs can function as miRNA sponges to regulate target gene expression, forming a regulatory circRNA-miRNA-mRNA network [ 35 ]. In addition, miR-758 has been determined to serve as a tumor-suppressing miRNA in cervical, lung, breast, bladder, and esophageal cancers [ 36 – 39 ]. In the present study, miR-758-3p inhibition rescued the migration ability of TPC-1 cells after circ-MAP4K3 silencing. Bioinformatics analysis and fluorescent luciferase reporter assay also confirmed that ribonucleotide reductase regulatory subunit M2 (RRM2) was the downstream circ-MAP4K3 target. Silencing of circ-MAP4K3 inhibited the RRM2 expression. The differential RRM2 expression is closely related to the occurrence, development, and prognosis of pancreatic cancer, gastric cancer, non-small cell lung cancer, liver cancer, malignant endometriosis, and other tumors [ 40 – 43 ]. RRM2 showed elevated levels in liver cancer and inhibited ferroptosis by stimulating GSH synthesis via GSS [ 44 ]. The high RRM2 expression was associated with a worse prognosis in patients with breast cancer [ 45 ]. In the present study, RRM2 overexpression rescued the migration ability of TPC-1 cells after circ-MAP4K3 silencing. Luciferase reporter analysis confirmed that RRM2 was the downstream target of miR-758-3p. RRM2 overexpression rescued the proliferation and migration abilities after miR-758-3p overexpression. This regulatory network needs to be topological analysis in the future [ 46 ]. In summary, the present study found that circ-MAP4K3 levels were increased in PTC tissues and cells. Additionally, circ-MAP4K3 absence inhibited PTC cell proliferation, migration, and migration via the miR-758-3p/RRM2 axis. This regulatory network needs to be verified using clinical assays in the future. The study data may provide innovative ideas for PTC management.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, accounting for approximately 90% of all thyroid cancers [ 1 ]. Although there have been some advances in detection and treatment methods and prognosis has improved, metastatic tumors usually involve cervical lymph nodes and lungs [ 2 ]. The incidence of lung metastases is 7%–30% in children and adolescents ≤ 20 years of age and only ~ 4% in adults [ 3 , 4 ]. Moreover, multiple organ metastasis is associated with a higher mortality rate (50.4%) than single organ involvement (36.7%) [ 5 ]. The accumulated research has confirmed that tumor microenvironment can influencing therapeutic response [ 6 , 7 ]. Metabolic reprogramming is associated with the malignant progression of tumors [ 8 ]. The study also found that immune checkpoint genes play an important role in regulation the progress of PTC [ 9 ]. Therefore, exploring the molecular mechanisms involved in the pathogenesis of PTC metastasis may provide alternative intervention targets and improve therapeutic strategies. Accumulation studies confirmed that circular RNAs (circRNAs) play an important role in PTC progression regulation [ 10 ]. CircRNAs have been identified as endogenous non-coding tissue-specific RNAs that are widely present in eukaryotes [ 11 – 13 ]. It has been reported that circRNAs act as molecular sponges of microRNA (miRNA) by competing for the rich miRNA-binding sites and interfering its expression with target messenger RNAs (mRNAs) [ 14 ]. They are characterized by a covalently closed continuous ring, which can resist the cutting of RNA extrac or bonucnase and thus become a regulator of miRNA binding, protein interaction, and regulatory splicing [ 15 – 17 ]. Previous studies have confirmed that circ-AGTPBP1, circ-EIF3I, circ-PTPRM, and circ-NDST1 play an important role in regulating malignancy progression in PTC [ 18 – 21 ]. The dysregulation of circRNA can affect cell proliferation, apoptosis, epithelial-mesenchymal transition, migration, tumor immune escape, and gene mutation and modification and ultimately promote tumor progression and invasive metastasis, becoming a potential tumor biological marker [ 21 , 22 ]. However, the role of circRNA remains largely unclear. Research also indicates that in a hypoxic microenvironment, tumor cells consume more glucose and produce more lactic acid compared to normal conditions [ 5 ]. More studies have found that aerobic glycolysis in solid tumors promotes malignant progression, including papillary thyroid carcinoma (PTC) [ 23 ]. RRM2 play an improtant role in regulation the progress of aerobic glycolysis [ 24 ]. Aerobic glycolysis is a promising therapeutic intervention for cancer because it provides sufficient biomass energy for cancer cells, which is the building material for cell growth and proliferation, and also helps cancer cells adapt to a high oxidative microenvironment, thereby enhancing their survival [ 25 ]. Whether circular RNAs can regulate the progression of PTC by influencing aerobic glycolysis remains unclear. The present study determined that circ-MAP4K3 plays an important role in regulation of the migration and proliferation abilities of PTC cells. The mechanism study confirmed that circ-MAP4K3 promotes PTC progression via the miR-758-3p/RRM2 signaling pathway.