Introduction
Thyroid carcinoma is the most frequent malignancy in different endocrine-related tumours. The occurrence rate of thyroid carcinoma has been increasing, and the rate takes 9th place in all cancers [1]. Most thyroid carcinomas are categorized as papillary thyroid carcinoma (PTC), which accounts for almost 80% of all cases [2]. Recent therapeutic regimens including surgery and iodine radiotherapy keep the overall 5-year survival rate over 95%. Nevertheless, some patients still develop progressive diseases due to the PTC metastasis and recurrence. Previous studies indicate that approximately 10–15% of PTC patients receiving regular regimens will develop metastasis [3]. Therefore, there is an urgent need to uncover the molecular mechanisms of PTC metastasis and provide possible ways to prevent its progression.
Long non-coding RNAs (lncRNAs) are composed of over 200 nucleotides, which lack protein coding ability [4]. Accumulative studies have shown that the occurrence and the progression of cancers are associated with dysregulated lncRNAs [5]. Our previous study showed that lncRNA ZFAS1 promotes the metastasis of PTC via regulation of the miR-373-3p/MMP3 axis. In addition, ZFAS1 is a target of transcription factor CREB3, which is also involved in the regulatory program in PTC progression [6]. Our previous results indicate that dysregulated lncRNA and its associated transcription factor may play an important role in the progression of PTC. Recently, numerous studies have suggested that lncRNA LINC00092 may serve as a tumour suppressor in different cancers, including breast cancer [7] and lung cancer [8], whereas the function of LINC00092 in PTC is still unclear. Therefore, it is intriguing to investigate the role of LINC00092 in PTC metastasis and progression.
The tumour promoter MYB is a transcription factor recognizing the binding motif t/cAACt/gG in the promoter of target genes [9]. In physiological conditions, MYB mainly stays in the high turnover tissues such as the haematopoietic system and intestinal epithelial tissues [10]. MYB can be stimulated by external signals via the extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signalling pathways [11, 12]. The dysregulation of MYB was validated to be correlated to the progression of different cancers including breast cancer, colorectal cancer, and leukaemia [13]. Here we set out to demonstrate that MYB functions as a transcription factor of LINC00092 in PTC.
Material and methods
Cell culture and patient samples
PTC cell line TPC-1 and a normal cell line Nthy-ori 3-1 were purchased from Sigma-Aldrich. Cells were cultured in RPMI 1640 medium with 10% foetal bovine serum (FBS, Gibco, USA), penicillin/streptomycin (2%), and amphotericin B (1%) at 37℃ and 5% carbon dioxide (CO2). Papillary thyroid carcinoma tissues were collected from 5 different patients who received treatments in our hospital from January 2019 to December 2021. The experiment on human samples was approved by the Ethics Committee (No. 2021011). The work associated with human samples was conducted in accordance with the Declaration of Helsinki (1964).
Vector cloning
Overexpression of LINC00092 and MYB was performed by transfected with the pcDNA3.1 system. The construction of ectopic vector containing the entire mRNA of LINC00092 and CDS region of MYB was accomplished and validated by Maokang Bio (Shanghai, China). For luciferase assay, 3’-UTR of LINC00092 was cloned into pGL3-control vector (Promega, USA). The seed match sequence of miR-4741 in the 3’-UTR of LINC00092 was mutated by using Mut Express II Fast Mutagenesis Kit V2 (Vazyme, China) according to the manufacturer’s instructions.
Transfection
For cell transfection, cells were seeded into 6-well plates at a density of 2 × 105 cells/well and cultivated overnight. When cell confluence reached 80%, the transfection process was performed based on the manufacturer’s instructions. Briefly, for transfection of vectors, Lipofectamine 2000 (Invitrogen, USA, 10 μL) was used and mixed with vectors (2.0 μg) in Opti-MEM media (Gibco, USA). The mixture was incubated at room temperature for 5 min, and then added to the 6-well plate dropwise for 48-hour incubation. For transfection of miRNAs, HiPerFect (Qiagen, Germany, 10 μL) was used and mixed with miRNAs (25 nM) in Opti-MEM media. The mixture was incubated at room temperature for 5 min and then added to the 6-well plate dropwise for 48-hour incubation.
Quantitative real-time polymerase chain reaction (qPCR)
qPCR was performed after different treatments. Total RNAs were isolated by using a High Pure RNA Isolation Kit (Roche, Switzerland) following the manufacturer’s instructions. A verso cDNA Synthesis Kit (Thermo Fisher, USA) was used to reverse transcribe RNA to cDNA. Gene expression was measured by qPCR with SYBR Green on the LightCycler 480 platform (Roche, Switzerland). The primers used in qPCR analysis were listed in Table 1 (5’–3’). The relative expression of genes was calculated by using the 2^(-DDCt) method.
Wound-healing assay
Cells were seeded into a 6-well plate at the density of 5 × 105 cells/well. After 24–48 hours, and when the cell confluence reached 90%, the cells were scratched with a 20 μL tip. The debris was removed by washing with Hank’s balanced salt solution (HBSS) twice. After one-hour incubation, the image of the scratch was taken as the initial stage of wound healing (d0, µm). After 48 hours, the image of the scratch was taken as the end stage of wound healing (dt, µm). The migration rate (%) was determined by the following formula: (dt-d0)/d0 × 100%.
Chromatin immunoprecipitation qPCR (ChIP-qPCR)
Q-ChIP was performed based on the protocol of Auto iDeal ChIP-qPCR Kit (Diagenode, USA). MYB was overexpressed in TPC-1 cells for 24 hours, and then cells were lysed to isolate chromatin. Obtained chromatin was sonicated for 30 seconds and 30 cycles to obtain DNA fragments of appropriate size (less than 400 bp). The enrichment of LINC00092 was evaluated by q-PCR.
Bioinformatics analysis
The data of the patient cohorts were obtained from Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/). The microRNA target prediction was performed through miRDB (http://mirdb.org/custom.html). The transcription factor screening was performed using Cistrome DB (http://cistrome.org/db/#/) and JASPAR (https://jaspar.genereg.net/).
Statistics
All data are presented as mean ± standard deviation (SD) (at least 3 independent experiments were included). Analyses were achieved by 2-way analysis of variance and t-test. All analyses were performed using GraphPad Prism8 software. P-values of < 0.05 or < 0.01 were considered statistically significantly.
Results
LINC00092 is downregulated in PTC
To determine the expression level of LINC00092 in thyroid carcinoma, we analysed TCGA-THCA patient cohorts. In the unpaired and paired comparisons, the expression of LINC00092 was significantly repressed in thyroid carcinoma tissues (Fig. 1A and 1B). In addition, the expression of LINC00092 was negatively correlated to the advanced TNM stages of thyroid carcinoma compared to normal groups (Fig 1C–E). Therefore, the downregulation of LINC00092 suggests promoted progression of thyroid carcinoma. To validate the findings of bioinformatics analysis, we performed q-PCR analysis on LINC00092 expression in 5 paired tissues of PTC patients. Indeed, the expression of LINC00092 was downregulated in PTC tissues compared to paired adjacent normal tissues (Fig. 1F). Furthermore, the expression of LINC00092 was also repressed in the TPC-1 cell, a cell line derived from a papillary thyroid carcinoma of a female patient (Fig. 1G). Collectively, LINC00092 is downregulated in PTC, which indicates that LINC00092 may play a tumour suppressive role in PTC.
LINC00092 represses EMT of PTC
Because the results of bioinformatics analysis indicated that LINC00092 may function as a tumour suppressor, here we evaluated the function of LINC00092 in the migration of PTC. Ectopic expression of LINC00092 repressed the migration ability of TPC-1 cells, whereas silencing LINC00092 promoted the migration ability of TPC-1 cells (Fig. 2A). In addition, ectopic LINC00092 induced E-cadherin and repressed vimentin (VIM) expression in TPC-1 cells (Fig. 2B), indicating that LINC00092 represses migration of PTC via regulation of the EMT process. Furthermore, MTT assay results showed that ectopic LINC00092 significantly repressed the proliferation of TPC-1 cells (Fig. 2C). Conversely, silencing LINC00092 promoted the proliferation of TPC-1 cells (Fig. 2D). Taken together, our findings suggest that LINC00092 functions as a tumour suppressor via repressing the EMT of PTC.
LINC00092 is a direct target of MYB
Next, we set out to clarify the potential regulator of LINC00092 in PTC. By screening with the JASPR online tool, MYB appeared to be a transcription factor of LINC00092 (Fig. 3A). Indeed, the chromatin immunoprecipitation-sequencing (ChIP-seq) data of previous studies obtained from the Cistrome DB online tool showed that MYB was enriched in the promoter region of LINC00092, represented by significant ChIP peaks (Fig. 3B). Ectopic expression of MYB repressed the expression of LINC00092 (Fig. 3C). In addition, q-ChIP analysis validated that LINC00092 is a direct target of MYB (Fig. 3D). Furthermore, the newly synthesized RNA of LINC00092 was suppressed by ectopic MYB in TPC-1 cells (Fig. 3E). Therefore, LINC00092 is a direct target of MYB, and MYB directly represses the expression of LINC00092.
miR-4741 is a direct target of MYB, and miR-4741 represses LINC00092
Because previous studies have shown that lncRNAs are widely regulated by miRNAs via the 3’-UTR target, herein we screened the potential target miRNAs of LINC00092. In the screened miRNA candidates, miR-4741 was upregulated in PTC tissues and TPC-1 cells (Fig. 4A and 4B). Luciferase assay validated that LINC00092 is a target of miR-4741 (Fig. 4C). Ectopic expression of miR-4741 largely repressed the expression of LINC00092 in TPC-1 cells (Fig. 4D). In addition, ectopic expression of miR-4741 largely abrogated the effect of LINC00092 on EMT markers of TPC-1 cells (Figs. 4E and 4F). Interestingly, the ChIP-seq data from a previous study, obtained from the Cistrome DB online tool, indicated that MYB is enriched in the promoter of MIR4741 gene (Fig. 4G), thereby miR-4741 may serve as a direct target of MYB. Next, we performed qChIP analysis and validated that MYB is a transcription factor of miR-4741 (Fig. 4H). In addition, ectopic MYB induced the expression of miR-4741 and the nascent mRNA of MIR4741 (pri-miR-4741) in TPC-1 cells (Figs. 4I and 4J). Furthermore, inhibition of miR-4741 largely abrogated the effect of MYB on repressing LINC00092 in TPC cells (Fig. 4K). Collectively, MYB could repress LINC00092 by inducing the expression of miR-4741.
MYB promotes the EMT of PTC by repressing LINC00092
Because we had identified that MYB directly or indirectly represses LINC00092, we would like to ascertain whether MYB promotes EMT of PTC via regulation of LINC00092. Ectopic expression of MYB induced VIM expression and repressed E-cadherin expression in TPC-1 cells, whereas ectopic LINC00092 largely abrogated the effect of MYB (Figs. 5A and 5B). The above results indicate that MYB promotes EMT via repression of LINC00092. The proliferation of TPC-1 cells was promoted by ectopic MYB (Fig. 5C). Furthermore, this effect was reversed by ectopic LINC00092 (Fig. 5C). Therefore, MYB promotes the progression of PTC via repression of LINC00092.
Discussion
In this study, we demonstrated that LINC00092 functions as a tumour suppressor in PTC. In addition, the tumour promoter MYB can directly or indirectly repress LINC00092, as summarized in Fig. 5D. Our previous study shows that lncRNA ZFAS1 plays an oncogenic role in PTC via induction of MMP3 by repressing miR-373-3p [6]. The transcription factor CREB3 directly induces the expression of ZFAS1, thereby promoting the progression of PTC. Herein, we validated that LINC00092, a downregulated lncRNA in PTC, inhibits the EMT of PTC. Similarly, the expression of LINC00092 is also controlled by a specific transcription factor, MYB. Therefore, the regulatory pattern of transcription factor/lncRNA/miRNA could be critical in cancer progression. Of note, the function of LINC00092 is contradictory in different cancers. LINC00092 is highly expressed in ovarian cancer [14]. LINC00092 drives glycolysis and progression of ovarian cancer. However, the lower expression of LINC00092 indicates a poorer prognosis in lung adenocarcinoma and colon cancer [8, 15]. In addition, LINC00092 suppresses the malignant progression of breast cancer by inducing SFRP1 through the miR-1827 target. Our findings suggest that LINC00092 plays a tumour suppressive role in PTC.
The transcription factor MYB is highly conserved, ranging from plant to vertebrates [16], suggesting that MYB has an essential function in different cells and organisms. Previous studies have identified that MYB targets genes at a global level: over 10,000 promoters can be targeted by MYB in MCF-7 cells [17]. The validated targets of MYB include MYC, CCNB1, JUN, and KLF4. In line with previous findings, herein we show that MYB plays an oncogenic role in PTC, inducing EMT and proliferation. Interestingly, MYB repressed the expression of LINC00092 by acting as a transcription factor. Our results imply that reversing the expression of genes repressed by MYB could counteract the effect of MYB.
Interestingly, LINC00092 was also regulated by MYB indirectly via miR-4741. Herein, we confirmed that MIR4741 gene is a direct target of MYB; hence, ectopic MYB induces the expression of miR-4741. Furthermore, LINC00092 was identified as a target of miR-4741, which enables miR-4741 to mediate the function of MYB by repressing downstream LINC00092. Our findings reveal a robust regulation between MYB and LINC00092, which confers the progression of PTC. MYB, LINC00092, and miR-4741 form a coherent feed forward loop. Because MYB regulates downstream genes in a global level, the validation and demonstration of more potential target miRNAs are warranted. In addition, the function of LINC00092 and the correlated regulatory axis require in vivo evidence.
Conclusion
Our study validated that LINC00092 is a tumour suppressor in PTC. LINC00092 is regulated by MYB directly through promoter binding or indirectly through the miR-4741 target. The axis of MYB-LINC00092 promotes progression of PTC.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Ethics statement
The experiment on human samples was approved by the Ethics Committee of Southwest Medical University (No. 2021011).
Author contributions
L.C. and X.D. designed the study. L.C. performed experiments and analysed the results. Y.L. performed bioinformatics analysis. L.C. and X.D. wrote the paper. All authors read and approved the paper.
Conflict of interest
The authors declare that there is no conflict of interest for this study.
Funding
Not applicable.
Acknowledgments
None.
