LncRNA FOXN3-AS2 modulated the growth of papillary thyroid carcinoma via miR-34a/SOX4 axis

Expression of miR-34a in PTC tissues and cell lines

To evaluate the effects of miR-34a in PTC, we firstly detected miR-34a expression in PTC clinical tissues and cell lines. As shown in Figure 1A, the mRNA level of miR-34a was decreased in PTC tissues comparing with adjacent normal tissues. In addition, we examined miR-34a expression in PTC cell lines. Compared with normal thyroid gland cells Nthy-ori 3-1, miR-34a expressions in PTC cell lines HIM-1 and TCP-1 were remarkably reduced (Figure 1B). Then, we analyzed the 60-months survival rate of PTC patients. According to the average expression of miR-34a in 1A, the 40 PTC patients were divided into low and high miR-34a expression groups. The patients with low miR-34a expression had a lower survival rate (Figure 1C). These data suggested a potential regulatory role of miR-34a in the pathological process of PTC.

Figure 1. LncRNA FGD5-AS1 is increased in OC tissue and cells. (A) The expression of miR-34a in PTC tissues (n = 40) and adjacent normal tissues (n = 40) determined by qRT-PCR (^*p < 0.05). (B) qRT-PCR assay analyzed the expression of miR-34a in normal human thyroid cells (Nthy-ori 3-1) and PTC cell lines (TCP-1 and HIM-1) (^*p < 0.05). (C) The 60-months survival rate of PTC patients with high and low miR-34a expression (^*p < 0.05).

Overexpression of miR-34a suppresses proliferation, migration and invasion of PTC cells

To further explore the role of miR-34a in PTC, miR-34a mimics or its negative control (NC) was transfected into TCP-1 cells. The transfection of miR-34a mimics significantly increased miR-34a expression (Figure 2A). Next, we used CCK8 and colony formation assay to determine cell proliferation. The overexpression of miR-34a inhibited the proliferated capacity of TCP-1 cells (Figure 2B and 2C). Migration and invasion are critical to the growth of cancer cells, and we then detected the migration and invasion of TCP-1 cells upon miR-34a mimics or NC transfection. The wound healing results showed miR-34a significantly suppressed cell migration (Figure 2D). Meanwhile, transwell assay uncovered a lower invasive ability in miR-34a mimics transfected TCP-1 cells (Figure 2E).

Figure 2. Upregulation of miR-34a inhibits proliferation, migration and invasion in TCP-1 cells. miR-34a mimics and its negative control (NC) was transfected into TCP-1 cells. (A) The expression of miR-34a was determined by qRT-PCR (^*p < 0.05). (B) CKK-8 assay was used to examine the cell growth at 0, 24, 48 and 72 h (^*p < 0.05). (C) Colony formation assay was used to calculated cell proliferation (^*p < 0.05). (D) Wound healing assay was used to detect cell migration (^*p < 0.05). (E) Transwell assay was performed to check cell invasive ability (^*p < 0.05).

MiR-34a suppresses PTC progression by targeting SOX4 in PTC cells

SOX4 is abnormally expressed in a variety of tumor tissues, and is closely related to the occurrence, development and metastasis of tumors. In our study, we surprisedly found that there were complementary base pairs between SOX4 and miR-34a using multiple databases (Targetscan, miRanda and TarBase) (Figure 3A), which revealed SOX4 might be a potential target of miR-34a in regulating PTC progression. To test our hypothesis, we performed luciferase assay and found that miR-34a mimics significantly decreased luciferase activity of WT 3′UTR of SOX4 rather than mutant SOX4 (Figure 3B). Furthermore, miR-34a mimics inhibited both mRNA and protein level of SOX4, while miR-34a inhibitor increased SOX4 expression (Figure 3C and 3D). Moreover, we found an obvious increase of SOX4 protein in PTC tissues and cells (Figure 3E and 3F). Besides, we analyzed the correlation of miR-34a and SOX4 expression in PTC tissues, which showed that miR-34a expression was negatively related to SOX4 level (Figure 3G).

Figure 3. SOX4 was a direct target of miR-34a. (A) Multiple databases (Targetscan, miRanda and TarBase) identified that the SOX4 had complementary base pairs with miR-34a. (B) WT and mutant SOX4 luciferase plasmids were transfected into HEK293 cells with miR-34a mimics or NC. The luciferase activity was measured by dual-luciferase reporter assay system. (^*p < 0.05). miR-34a or miR-34a inhibitor or its NC was transfected into TCP-1 cells. (C) The mRNA level of SOX4 was analyzed by qRT-PCR, (D) and the protein level of SOX4 was tested by Western blot (^*p < 0.05). The protein expression of SOX4 in PTC tissues (E) and cells (F) was detected by Western blot (^*p < 0.05). (G) The correlation analysis of miR-34a and SOX4 expression in PTC tissues.

To verify whether miR-34a regulated PTC by targeting SOX4, we forced expression of SOX4 in TCP-1 cell (Figure 4A and 4B), and cotransfected miR-34a mimics. As shown in Figure 4C and 4D, upregulation of SOX4 reversed the inhibitory effect of miR-34a on TCP-1 cells’ proliferation. SOX4 also restored the migrative and invasive ability (Figure 4E and 4F). These data indicated that miR-34a inhibited the malignancy of PTC cells via inhibiting SOX4.

Figure 4. MiR-34a inhibits PTC development by downregulating SOX4. SOX4 or NC was transfected into TCP-1 cells. (A) The mRNA expression of SOX4 was determined by qRT-PCR, (B) and the protein level of SOX4 was tested by Western blot(^*p < 0.05). (C) CKK-8 assay was used to examine the cell growth at 0, 24, 48 and 72 h (^*p < 0.05). (D) Colony formation assay was used to calculated cell proliferation (^*p < 0.05). (E) Wound healing assay was used to detect cell migration (^*p < 0.05). (F) Transwell assay was performed to check cell invasive ability (^*p < 0.05).

FOXN3-AS2 acts as a sponge of miR-34a

Evidence is mounting that lncRNAs competitively bind to miRNAs and further inhibit its activity [16]. Thus, we used Starbase, LncBook and DIANA databases to identify the lncRNAs with miR-34a biding sites. Our data revealed that lncRNA FOXN3-AS2 could bind with miR-34a (Figure 5A). Following luciferase assay showed that overexpression of miR-34a increased luciferase activity of WT FOXN3-AS2 but not Mut one (Figure 5B). In addition, FOXN3-AS2 or si-FOXN3-AS2 was transfected into TCP-1 cells. FOXN3-AS2 plasmid promoted FOXN3-AS2 level, and si-FOXN3-AS2 inhibited FOXN3-AS2 expression (Figure 5C). Meanwhile, FOXN3-AS2 suppressed miR-34a and increased SOX4 expression, while silencing of FOXN3-AS2 had the opposite effect (Figure 5C). Then, FOXN3-AS2 plasmid and miR-34a were cotransfected into TCP-1 cells, which showed that miR-34a abolished the promoting role of FOXN3-AS2 on SOX4 expression (Figure 5D). These data suggested FOXN3-AS2 promoted SOX4 expression via inhibiting miR-34a. What’s more, FOXN3-AS2 was highly expressed in PTC tissues and cells (Figure 5E and 5F). In addition, the expression of FOXN3-AS2 expression in PTC tissues was negatively related to miR-34a expression (Figure 5G). Together, FOXN3-AS2 promoted SOX4 expression via inhibiting miR-34a.

Figure 5. FOXN3-AS2 directly regulates miR-34a and inhibits its expression in PTC. (A) Starbase, LncBook and DIANA databases used to identify the lncRNAs with miR-34a biding sites. (B) WT and mutant miR-34a luciferase plasmids were transfected into HEK293 cells with pcDNA3.1 or FOXN3-AS2. The luciferase activity was measured by dual-luciferase reporter assay system (^*p < 0.05). (C) FOXN3-AS2 or si-FOXN3-AS2 or its NC was transfected into TCP-1 cells. The mRNA level of FOXN3-AS2, miR-34a and SOX4 were analyzed by qRT-PCR (^*p < 0.05). (D) FOXN3-AS2 and miR-34a were cotransfected into TCP-1 cells, the mRNA level of SOX4 were tested (^*p < 0.05). The protein level of miR-34a in PTC tissues (E) and cells (F) was detected by qRT-PCR (^*p < 0.05). (G) The correlation analysis of miR-34a and FOXN3-AS2 expression in PTC tissues.

FOXN3-AS2 promotes proliferation, migration and invasion via miR-34a/SOX4 axis in PTC cells

To assess the role of FOXN3-AS2, we transfected FOXN3-AS2 plasmid into TCP-1 cells, and qRT-PCR assay showed an increase of FOXN3-AS2 level (Figure 6A). Next, we cotransfected miR-34a mimics or si-SOX4 with FOXN3-AS2 plasmid into TCP-1 cells. Followed functional experiments showed FOXN3-AS2 promoted proliferation, migration and invasion of TCP-1 cells, while this facilitated effect was removed by miR-34a mimics or si-SOX4 (Figure 6B–6E). Taken together, FOXN3-AS2 regulated the progression of PTC via miR-34a/SOX4 axis.

Figure 6. LncRNA FOXN3-AS2 promotes OC progression via miR-34a/SOX4 axis. (A) FOXN3-AS2 plasmid was transfected into TCP-1 cells, and qRT-PCR analysis was used to detect the transfection efficiency (^*p < 0.05). miR-34a or si-SOX4 was cotransfected into TCP-1 cells with FOXN3-AS2 plasmid. CCK8 assay (B) and colony formation assay (C) was used to calculate cell proliferation (^*p < 0.05 vs. pcDNA3.1, ^#p < 0.05 vs. FOXN3-AS2). (D) Wound healing assay was used to detect cell migration (^*p < 0.05 vs. pcDNA3.1, ^#p < 0.05 vs. FOXN3-AS2). (E) Transwell assay was performed to check cell invasive ability (^*p < 0.05 vs. pcDNA3.1, ^#p < 0.05 vs. FOXN3-AS2).

FOXN3-AS2 promotes in vivo tumor growth in the nude mice

For further explore the function of FOXN3-AS2 in PTC tumor, we set up xenograft nude mice model. 30 mice were divided into two group randomly, TCP-1 cells stably expressing FOXN3-AS2 or pcDNA3.1 were subcutaneously injected in right lower limb of the nude mice. The mice with FOXN3-AS2 transfected cells showed a bigger tumor volume, and tumors grew faster (Figure 7A). The tumors were isolated at 30 days after injection, FOXN3-AS2 significantly increased tumors weight than that pcDNA3.1 (Figure 7B). In addition, we isolated these tumor tissues and found that the expression of FOXN3-AS2 was increased in FOXN3-AS2 overexpression mice (Figure 7C). Moreover, FOXN3-AS2 decreased the mRNA level miR-34a, but increased SOX4 expression in tumor tissues (Figure 7D and 7E). These data indicated that FOXN3-AS2 accelerated PTC tumorigenesis via modulating miR-34a/SOX4 axis.

Figure 7. LncRNA FOXN3-AS2 promotes in vivo tumor growth in the nude mice. TCP-1 cells line stably expressing FOXN3-AS2 or pcDNA3.1 were subcutaneously injected in right lower limb of the nude mice. (A) The tumor volume was assessed in the nude mice every 5 days (^*p < 0.05). (B) Tumor weight was determined in the isolated tumors from the nude mice (^*p < 0.05). (C) The relative expression of FOXN3-AS2 was determined by qRT-PCR in the isolated tumor tissues (^**p < 0.01). qRT-PCR was performed to detect the relative mRNA expression of miR-34a (D) and SOX4 (E) (^*p < 0.05).

Clinical samples

Fresh cancer tissue samples and adjacent normal tissue samples were taken from 40 PTC patients undergoing surgical procedures at Jilin Cancer Hospital. All patients did not receive radiotherapy, chemotherapy, and immunization before cancer tissue collection, and all tissues were confirmed by pathological examination. All of the patients or their guardians provided written consent, and our experiments were supported by the Ethics Committee and Institutional Review Board of Jilin Cancer Hospital.

Cell culture

The cell lines (Nthy-ori 3-1, HIM-1 and TCP-1) were purchased from the Science Cell Laboratory. Cells were cultured in RPMI 1640 (GIBCO, USA) supplemented with 10% fetal bovine serum (Cromwell, USA) and 100 μL/mL penicillin and streptomycin (Sigma-Aldrich, USA) and placed at 37°C with 5% CO₂.

Cell transfection

About 5 × 10⁵ cells per well were seeded in 6-well plates, 2000 ng small interference RNA (siRNA) or negative control of siRNA (si-NC), 2000 ng plasmid or empty vector (pcDNA3.1), 20 nmol miRNA mimics or negative control (NC) of miRNA mimics, and 20 nmol miRNA inhibitor or negative control of miRNA inhibitor (NC inhibitor) were transfected into the cells for 48 h using Lipo2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s recommendations.

qRT-PCR

The RNA Isolation Kit (Vazyme, China) was used to extract total RNA from tissues or cells, and the concentration and quality of the purified RNA were measured using Nanodrop 2000/2000C spectrophotometer. RNA was reverse transcribed into cDNA template at the following reaction temperatures: 37°C 15 min, 50°C 5 min, 98°C 5 min, 4°C hold. The cDNA was stored at −20°C.Then the primers synthesized by Shanghai Jikegein Company were used for quantitative PCR. The reaction system of 20 μL was prepared. The reaction was carried out according to the following steps: pre-denaturation at 95°C for 15 min, denaturation at 95°C for 15s and annealing extension at 60°C for 1min for 35–40 cycles. LncRNA FOXN3-AS2 and SOX4 were standardized by GAPDH, and miR-34a was standardized by U6. The expression l of FOXN3-AS2 and SOX4 and miR-34a was analyzed by 2^−ΔΔCT.

Western blot

The tissues or cells in each group were collected and treated with RIPA lysate. Then, the protein concentration was evaluated by BCA kit (Beyotime, China). And the proteins in loading buffer were separated by SDS-PAGE and transferred to PVDF membrane. The membrane carrying proteins was sealed with blocking fluid, and was incubated with SOX4 (1: 500) and GAPDH (1: 1000) overnight at 4°C. The next day, HRP-labeled secondary antibody (1: 5000) was added and incubated at room temperature for 2 h. The membrane was photographed by ECL exposure and analyzed the gray value of the target bands. The primary antibodies were purchased from Abcam, SOX4 (ab80261), and GAPDH (ab8245) was used as an internal control.

CCK8 assay

The logarithmic growth phase cells were digested by trypsin and resuspended in complete medium and counted. Cells were inoculated in a 96-well plate at a density of 2500 cells/well and cultured in a cell incubator. From the second day to the fifth day after planking, 10 μl CCK-8 reagent (Vazyme, China) was added 2–4 h before culture termination every day. After 4 h, the 96-well plate was placed on the oscillator and oscillated for 5 min. The OD value was detected at 450 nm with a microplate analyzer. The final values of each group were plotted and the results were analyzed.

Wound healing assay

5 × 10⁵ cells were planted in a 6-well plate, and when the cells grew to fuse, two vertical parallel lines were drawn with 10 μL suction heads against the ruler. The floating cells were washed with PBS and cultured in serum-free medium for 24 h. Images were taken at 0 and 24 h of cell culture, respectively.

Transwell assay

Cells in logarithmic growth phase were adjusted to 2 × 10⁵ cells/well of medium (without serum) and plated into the upper chamber insert pre-coated with 1 μg/μl Matrigel. Lower chamber was added with 500 μL of medium (with 10% FBS), and then incubate the chamber at 37°C for 48 h. Then the invading cells were visualized by the crystal violet and inverted microscope.

Colony formation assay

Cells were seeded into 6-well plates at a density of 100 cells/well and cultured for 14 days. Cells clone were fixed with 4% paraformaldehyde and stained with 0.1% Crystal Violet (Sigma-Aldrich. MO, USA) at room temperature for 15 min. Then cells were rinsed with distilled water, and the colonies were visualized by inverted microscope.

Dual luciferase reporter assay

psiCHECK-2 luciferase reporter plasmid was inserted with the wildtype (WT) 3′UTR of SOX4/FOXN3-AS2 or mutant (Mut) 3′UTR SOX4/FOXN3-AS2 sequences that contain the putative binding sites of miR-34a. NC or miR-34a mimics were transfected with reporter vectors into HEK293 cells. The cells were collected after 48 h post-transfection and lysed to detect the luciferase activity (Promega).

In vivo tumor growth assay

Nude mice were purchased from Beijing Charles river. The TCP-1 cell line stably expressing FOXN3-AS2 or pcDNA3.1 were constructed by Cyagen Biosciences (China). The TCP-1 cells (5 × 10⁶) were subcutaneously injected in right lower limb of the nude mice. Tumor size was measured every five days. After 30 days of cells injection, mice were killed and the tumors were removed for follow-up study. The animal experimental procedures were approved by the animal experimental ethics committee of Jilin Cancer Hospital, and the experiments were carried out following the Guide for the Care and Use of Experimental Animals.

Statistical analysis

Significant differences were calculated using two-tailed t-test through Graphpad 8.0. The P values less than 0.05 were considered significance.