LncRNA PFAR facilitates the proliferation and migration of papillary thyroid carcinoma by competitively binding to miR-15a

Papillary thyroid carcinoma (PTC) is type of aggressive tumor, with a markedly declined survival rate when distant metastasis occurs. It is of great significance to develop potential biomarkers to evaluate the progression of PTC. LncRNAs are recently widely claimed with biomarker value in malignant tumors. Herein, the role of LncRNA PFAR in PTC was investigated to explore potential prognostic marker for PTC. Compared to NTHY-ORI 3-1 cells, LncRNA PFAR was found markedly upregulated in PTC cell lines. In LncRNA PFAR knockdown TPC-1 cells, markedly declined cell viability, increased apoptotic rate, enhancive number of migrated cells, and elevated migration distance were observed, accompanied by a suppressed activity of the RET/AKT/mTOR signaling. In LncRNA PFAR overexpressed BCPAP cells, signally increased cell viability, declined apoptotic rate, reduced number of migrated cells, decreased migration distance, and increased tumor volume and tumor weight in nude mice xenograft model were observed, accompanied by an activation of the RET/AKT/mTOR signaling. The binding site between LncRNA PFAR and miR-15a, as well as miR-15a and RET, was confirmed by the dual luciferase reporter assay. The FISH study revealed that LncRNA PFAR was mainly located in the cytoplasm. Furthermore, the impact of the siRNA targeting LncRNA PFAR against the growth and migration of PTC cells was abolished by the inhibitor of miR-15a or SC79, an activator of AKT/mTOR signaling. Collectively, LncRNA PFAR facilitated the proliferation and migration of PTC cells by mediating the miR-15a/RET axis. Supplementary Information The online version contains supplementary material available at 10.1007/s00210-023-02779-w.


Introduction
Thyroid carcinoma (TC) is one of the most common endocrine diseases in women, which is influenced by multiple factors, such as genetics, environment, immunosuppression, and unscientific diet.TC is a malignant tumor with the fastest increasing incidence in the past two decades, and also one of the main causes of death (Hu et al. 2021;Haddad et al. 2022).TC is divided into four main histological types, which were PTC, follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), and anaplastic thyroid carcinoma (ATC) (Asa 2019).Among them, PTC accounts for about 80.85% of all TC cases, occupying an absolute dominant position (Zhang et al. 2020;Xu et al. 2020).Although PTC is a low-grade tumor, and the overall 5-year survival rate of patients with PTC after effective treatment is generally higher than 95% (Tong et al. 2020), PTC will be developed into aggressive cancer in the presence of distant metastasis, with a reduced survival to about 50% (Pinto et al. 2011).Therefore, it is important to investigate the migration process and molecular mechanism of PTC cells to further improve the survival rate of PTC patients.
With the rapid development of high-throughput sequencing technology, researchers have discovered a class of long non-coding RNAs (LncRNAs) with a length of more than 200 nucleotides, which are localized in the nucleus or cytoplasm.By mediating the gene expression at the level of chromatin transcription, modification, and post-transcriptional expression (Park et al. 2022;Lin et al. 2020), LncRNAs are involved in the regulation of cell development, migration, pluripotency, cycle, apoptosis, and invasion (Lin et al. 2020).It is confirmed that LncRNAs play a critical role in the development of human diseases, especially malignant tumors (Yang et al. 2022;Bridges et al. 2021).LncRNAs are abnormally expressed in a variety of tumors and closely related to the changes of tumor biological behavior, which are involved in tumor invasion, metastasis, autophagy, differentiation and other biological processes (Entezari et al. 2022).In PTC, LncR-NAs are also reported to act as "oncogenes" or "tumor suppressor genes" (Mahmoudian-Sani et al. 2019;Sedaghati and Kebebew 2019;Zheng et al. 2016).MicroR-NAs (miRNAs) were first discovered in Caenorhabditis elegans, and later similar non-coding endogenous small RNAs were found in different organisms (Gierlikowski and Gierlikowska 2022;Pozniak et al. 2022).MiRNAs are highly conserved, with 18-22 nucleotides length (Komatsu et al. 2023), and play a role in RNA silencing and posttranscriptional regulation of gene expression by binding to the 3 'Untranslated regions (UTR) of target genes, leading to degradation or inhibition of transcription of target genes (Jonas and Izaurralde 2015).MiRNA regulates a variety of cellular processes, including proliferation, apoptosis, differentiation, and metastasis (Zhao et al. 2022).Studies have found that the dysregulation of miRNA is related to a variety of human malignant tumors.Similar to LncRNAs, miRNAs also act as "oncogenes" or "cancer suppressor genes" (Zhang et al. 2007).By affecting tumor invasion and migration of PTC, miRNAs may be used as prognostic biomarkers for PTC (Wei et al. 2015;Zhang et al. 2014;Wang et al. 2013).Recently, it is claimed that miR-15a is downregulated in PTC tissues compared to normal tissues and inhibits the PTC cell proliferation and invasion (Jin et al. 2019).LncRNA PFAR is reported to target miR-15a and regulates its expression (Sun et al. 2019).However, the function of LncRNA PFAR in PTC remains uncertain.Herein, the role of LncRNA PFAR-miR-15a axis in PTC was investigated to explore potential prognostic markers for PTC.
Transfection To knockdown LncRNA PFAR in TPC-1 cells, cells were transfected with siRNAs (siR-PFAR-1, siR-PFAR-2, and siR-PFAR-3) targeting PFAR under the assistance of lipofectamine 3000 (Invitrogen, USA) for 48 h, followed by evaluating the transfection efficacy using RT-PCR to screen the optimized siRNA.SiR-NC was taken as the negative control.To obtain the LncRNA PFAR-overexpressed BCPAP cells, the overexpression-vector of LncRNA PFAR (pcDNA3.1-PFAR) was imported into lentiviral particles, which were transfected into BCPAP cells for 48 h, followed by evaluating the transfection efficacy using RT-PCR assay.pcDNA3.1-NCwas taken as the negative control.
RT-PCR assay Total RNAs were obtained from cells using the TRIzol reagent and were quantified with the Nanodrop one (Thermo Scientific, USA).A cDNA synthesis kit (CW2569, CWBIO, China) was utilized for conducting the transcription from RNAs to cDNAs, followed by performing the PCR reaction utilizing a SYBR Green kit (11201ES08, YEASEN, China).The 2 −ΔΔCt method was used for the calculation of gene levels.Sequences of primers were shown in Table 1.For the detection of LncRNA PFAR level, GAPDH or RNU6 snRNA was taken as the negative control.For the detection of miR-15a level, U6 was taken as the negative control.

CCK-8 assay
Transfected cells were digested, resuspended, counted, and plated at a cell density of 6 × 10 3 cells/well.After adhesion, 10 μL of CCK8 reagent was added to each well and incubated in the incubator for 2 h.The absorbance value of each well was measured at a wavelength of 450 nm by a microplate reader (CMaxPlus, MD, USA).
Apoptosis detection using the flow cytometry Cells of each group were placed in a constant temperature incubator for 48 h, followed by resuspended and adding 10 μL Annexin V reagent and 5 μL PI reagent.Cells were incubated at room temperature for 10 min, and the appropriate amount of cell suspension was sucked and mixed with PBS in flow assay tubes, followed by loading onto the flow cytometry (Novo-Cyte, Agilent, USA) for apoptosis detection.Transwell study for migration detection 500 μL of complete medium was added to the lower chamber.The cell suspension was added to the upper chamber and incubated for 12 h at 37℃ and 5% CO2 in the medium without serum.The upper chamber medium was discarded, and the Transwell chambers were washed twice with PBS at room temperature, followed by transferred to the well of a 24-well plate preadded with 700 μl 4% formaldehyde.After fixing for 20 min, 700 μl of 0.1% crystal violet was added and washed.The images were taken under an inverted microscope (AE2000, Motic, China), with five fields counted.
Wound healing assay Cells (2 × 10 5 cells /mL) were seeded in 6-well plates.After reaching 80%-90% density, cells were scraped with a yellow pipet, and the scratch area was washed twice with PBS to remove floating cells.The scratch width was recorded at 0 h.After continuous incubation for 48 h in medium containing 5% serum, the wound was photographed again and the scratch width and wound healing rate were calculated.
Western blotting assay Total proteins were extracted from cells and quantified with the BCA method, which were separated using the 12% SDS-PAGE, followed by transferred to the PVDF membrane.Following blocking, primary antibodies against RET (1:1000, AF6120, Affinity, USA), p-AKT (1:1000, AF0016, Affinity, USA), AKT (1:1000, AF6259, Affinity, USA), mTOR (1:1000, AF6308, Affinity, USA), p-mTOR (1:1000, AF3308, Affinity, USA), and GAPDH (1:10000, 1094-1-AP, Proteintech, USA) were added to be cultured for 12 h at 4 ℃, followed by incubation with the secondary antibody (1:6000, 7074, CST, USA) and incubated for 60 min.Lastly, the ECL solution was added for exposure and the expression level was quantified with the Image J software.Fluorescence in situ hybridization (FISH) Cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then permeabilized in precooled PBS containing 0.5% Triton X-100 for 5 min.After washed three times for 10 min each in PBS and rinsed once in 2 × SSC buffer, hybridization was performed with probes for LncRNA PFAR, U6, and 18S for 12 to 16 h at 37 ℃.Cells were then washed with 4 × SSC buffer, 2 × SSC buffer, and 1 × SSC buffer, respectively.Finally, cells were counterstained with DAPI for 10 min and visualized by laser confocal microscopy (LSM880, ZEISS, Germany).Statistical analysis Data were expressed using mean ± SD and analyzed with the one-way ANOVA method followed by Tukey's test using the GraphPad prism software 6.0.P < 0.05 was a significant difference.

Results
LncRNA PFAR was upregulated in PTC cells and the transfection in TPC-1 cells and BCPAP cells was identified Firstly, the level of LncRNA PFAR in human thyroid normal cells and human PTC cells were compared.Compared to NTHY-ORI 3-1 cells, the LncRNA PFAR was found markedly increased in BHP5-16, TPC-1, and K1 cells, and slightly elevated in BCPAP cells with no significant difference (Fig. 1, Fig. S1A).To explore the potential function of LncRNA PFAR in PTC, LncRNA PFAR was knocked down in TPC-1 cells, in which highest level of LncRNA PFAR was observed.Furthermore, LncRNA PFAR was overexpressed in BCPAP cells.The knockdown efficacy was evaluated by RT-PCR and siR-PFAR-2 with the highest transfection efficacy was chosen for subsequent assays.Furthermore, LncRNA PFAR was found markedly upregulated in pcDNA3.1-PFARtransfected BCPAP cells (Fig. 2, Fig. S1B-C).

LncRNA PFAR facilitated the in vivo growth of PTC cells
To confirm the function of LncRNA PFAR, Nude mice were subcutaneous implanted with BCPAP cells transfected with pcDNA3.1-NCand pcDNA3.1-PFAR,respectively, with nude mice implanted with normal BCPAP cells as the control group.Images of tumors were shown in Fig. 6A.Compared to the pcDNA3.1-NCgroup, the tumor volume (Fig. 6B) and tumor weight (Fig. 6C) were markedly increased in the pcDNA3.1-PFARgroup.

Discussion
Several researches have claimed the important role of LncR-NAs in PTC.Lan (Lan et al. 2015) performed gene microarray analysis of 62 pairs of human PTC tissues and adjacent tissues, and found that 1192 LncRNAs were upregulated and 2307 LncRNAs were downregulated.GO analysis and signaling pathway analysis showed that the differentially expressed LncRNAs and miRNAs were related to cancerrelated signaling pathways such as p53, MAPK, and PPAR, In thyroid cancer tissues, the study by He and Pallante showed that miR-146b-5p, miR-221-3p and miR-222-3p were upregulated in PTC (He et al. 2005;Pallante et al. 2006).Zhang (Zhang et al. 2013) used gene chip technology to find that 10 miRNAs were upregulated and 5 miRNAs were downregulated in thyroid cancer, among which miR-146b-5p was the most significantly upregulated and miR-335 was the most significantly downregulated miRNA.The target genes of miRNA predicted by bioinformatics tools were involved in cell proliferation, differentiation, apoptosis, cell cycle, and signal transduction.These researches highlighted the important function of miRNAs in PTC.Herein, miR-15a was found to be a sponging miRNA of LncRNA PFAR, which is in line with data presented by Sun (Sun et al. 2019).Furthermore, the suppressive function of siR-PFAR-2 against the proliferation and migration of PTC cells was abolished by the inhibitor of miR-15a, implying a tumor suppressor function of miR-15a.In fact, miR-15a is previously found to be markedly downregulated in PTC (Hu et al. 2017).Moreover, miR-15a is reported to exert an inhibitory effect on PTC progression by regulating expressions of several proteins, including HOXA3 (Jiang et al. 2019) and AXIN2 (Zhao et al. 2015).These researches on the function of miR-15a in PTC were in accordance with data obtained in the present study.
RET is located on human chromosome 10 and can be cleaved into different forms of receptor tyrosine kinases such as RET9, RET43, and RET51, which constitute the single transmembrane tyrosine kinase receptor protein RET family (Li et al. 2019).Thyroid cancer is found closely related to the activation of RET.Studies have shown that RET tyrosine kinase inhibitors (vandetanib and cabozantinib) exert a significant effect on PTC (Paziewska et al. 2011;Carr et al. 2010;Pao and Girard 2011).Herein, RET was found to be targeted by miR-15a, which is in line with the research by Jin (Jin et al. 2019).RET is reported to participate in the development of malignant tumors by activating the AKT/mTOR signaling (Couto et al. 2012).Herein, LncRNA PFAR was found to signally activate the RET/AKT/mTOR signaling.
Considering the interaction between LncRNA PFAR and miR-15a, as well as miR-15a and RET, we suspected that LncRNA PFAR sponged miR-15a to upregulate RET, which contributed to the activation of the AKT/mTOR signaling.Furthermore, the influence of siR-PFAR-2 on the proliferation and apoptosis of PTC cells was abolished by the inhibitor of AKT/mTOR signaling, which confirmed that LncRNA PFAR facilitated the progression of PTC by activating the AKT/mTOR signaling.In the future work, the involvement of RET in the regulatory function of LncRNA PFAR against PTC will be further confirmed by treating RET-knockdown PTC cells with the pcDNA3.1-PFAR.In addition, the prognosis value of LncRNA PFAR in patients with PTC will be analyzed in our future work to further confirm the biofunction of LncRNA PFAR.Furthermore, there are limitations in the present study.Compared to BCPAP cells, LncRNA PFAR was not knocked down in the Nthy-ori3-1 cell line, and the effect of LncRNA PFAR downregulation on proliferation, apoptosis, migration, invasion in normal thyroid gland epithelial cells was not observed, which should be further studied in our future work.In addition, RET mutation is previously reported in TPC-1 cells (Gilbert-Sirieix et al. 2010).Considering the negative control (siR-NC and Fig. 9 The impact of LncRNA PFAR on the RET/AKT/mTOR signaling was abolished by miR-15a inhibitor or SC79.The expression level of RET, p-AKT, AKT, p-mTOR, and mTOR was detected by Western blotting assay (**p < 0.01 vs. siR-NC+ inhibitor NC, ## p < 0.01 vs. siR-PFAR-2, n = 3) inhibitor NC) utilized in the present study, the influence of RET mutation on the miR-15a/RET axis in TPC-1 cells has been narrowed down.Moreover, it is reported that RET mutation has not been observed in BCPAP cells (Meireles et al. 2007).In BCPAP cells the influence of RET mutation on the miR-15a/RET axis was excluded.
Collectively, LncRNA PFAR facilitated the proliferation and migration of PTC cells by mediating the miR-15a/ RET axis.
Xenograft model 18 female nude mice were divided into 3 groups: Control, pcDNA3.1-NC,and pcDNA3.1-PFAR.Nude mice were subcutaneous implanted with BCPAP cells (1 × 10 7 /mice) transfected with pcDNA3.1-NCand pcDNA3.1-PFAR,respectively, with nude mice implanted with normal BCPAP cells as the control group.The tumor volume was measured every 3 days (7 times in total), and the tumor tissue was weighed on the 21 st day post implantation.Dual luciferase reporter assay Cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well.The next day, 200 ng of pMir-reporter fluorescent vector containing the 3' UTR region of LncRNA PFAR or RET and wild-type miR-15a, 200 ng of pMir-reporter fluorescent vector containing the 3' UTR of LncRNA PFAR or RET and mutant miR-15a, 200 ng miR-15a, and 200 ng negative control oligonucleotide (miR-Ctrl) were transfected into 293 T cells (ATCC, USA) using lipofectamine 3000 (Invitrogen, USA).After

Fig. 3 Fig. 4
Fig. 3 The proliferation was facilitated and the apoptosis was inhibited by LncRNA PFAR in PTC cells.A The cell viability of TPC-1 cells and BCPAP cells was measured by CCK-8 assay (n = 6).B The

Fig. 6
Fig. 6 LncRNA PFAR facilitated the in vivo growth of BCPAP cells in the xenograft model of nude mice.A Images of tumors isolated from nude mice (n = 6).B The volume of tumor tissues was recorded (n = 6).C The tumor weight was recorded (**p < 0.01 vs. pcDNA3.1-NC,n = 6)

Fig. 7
Fig. 7 LncRNA PFAR upregulated the RET level by sponging miR-15a.A The binding site between LncRNA PFAR and miR-15a was predicted.B The binding between LncRNA PFAR and miR-15a was confirmed by the dual luciferase reporter assay (n = 3).C The binding site between the 3'UTR region of RET and miR-15a was predicted.

Fig. 8
Fig.8The influence of LncRNA PFAR on the proliferation and migration of PTC cells was abolished by miR-15a inhibitor or SC79.A The level of LncRNA PFAR was determined by the RT-PCR assay (n = 3).B The level of miR-15a was determined by the RT-PCR assay