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. 2022 Nov 21;27:101588. doi: 10.1016/j.tranon.2022.101588

LINC01311 exerts an inhibitory effect in thyroid cancer progression by targeting the miR-146b-5p/IMPA2 axis

Mengjiang Liu 1, Linghui Zhang 1, Juping Hu 1, Chong Yan 1, Yi Zhang 1, Zhaodan Yan 1,⁎
PMCID: PMC9685481  PMID: 36423383

Highlights

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    LINC01311 overexpression restrains TC tumor cell growth in vitro and in vivo.

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    LINC01311 targets miR-146b-5p.

  • •

    LINC01311 interacts with miR-146b-5p to counteract the tumor-suppressive role of LINC01311 in TC cells.

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    MiR-146b-5p recognizes IMPA2 and downregulates its expression.

  • •

    IMPA2 overexpression counteracts miR-146b-5p's oncogenic action in TC cells.

Keywords: Thyroid cancer, LINC01311, miR-146b-5p, IMPA2, Migration, Proliferation

Abstract

Background

A growing body of research suggests that long non-coding RNA (lncRNA) play an important role during the tumorigenesis and progression of cancers, including thyroid cancer (TC). Herein, we intended to uncover the role and mechanisms of LINC01311 in TC.

Methods

The relative LINC01311, miR-146b-5p, and IMPA2 expressions were quantified by subjecting TC cells and tissues to western blotting and RT-qPCR. CCK-8 and scratch-wound healing assays were carried out for the evaluation of the proliferation and migration of TC cells. The apoptosis was evaluated by flow cytometry assay and western blotting of Bax and Bcl-2 proteins. Xenograft tumor model was also used to study how LINC01311 functions during TC cell growth. Luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to ascertain miR-146b-5p's interactions with LINC01311 and IMPA2 3′UTR.

Results

The TC cells and tissues exhibited a downregulation of LINC01311 and IMPA2 and an upregulation of miR-146b-5p. LINC01311 overexpression retarded TC cell growth in vitro as well as in vivo. The luciferase reporter and RIP assays verified that miR-146b-5p recognizes LINC01311 and IMPA2 3′UTR by base pairing. LINC01311 overexpression could counteract the oncogenic effect of miR-146b-5p in vitro. Moreover, IMPA2 upregulation could offset the tumor-promoting effect of miR-146b-5p.

Conclusion

LINC01311-mediated inhibition of TC cell growth was achieved by targeting the miR-146b-5p/IMPA2 axis. These findings support that targeting the LINC01311/miR-146b-5p/IMPA2 axis may be a promising approach against TC progression.

Introduction

Thyroid cancer (TC) remains a predominant neoplasm in the endocrine system. TC has had a continuous increase in occurrence for decades, which has declined recently [1]. The progress in multimodal TC treatments has all achieved relatively low mortality rates [2]. In spite of that, TC still comprises about 3% of juvenile cancers [3]. Currently, over diagnosis might be the main reason for the high occurrence, creating a significant medical need for the exploration of possible therapeutics targets. Given these facts, it is crucial to investigate the mechanism behind TC progression.

Long non-coding RNA (lncRNA) is a subfamily of non-coding RNAs with over 200 nt [4]. LncRNA is highly abundant in the human genome, yet it was initially regarded as a transcription byproduct with no biofunction [5]. Recent evidence has underscored the biological importance of lncRNAs. This is due to the increasing exploration of their transcriptional and post-transcriptional regulatory functions, which are well-established mechanisms that may favor oncogenic drivers or suppressors [6]. During the malignant progression of TC, an increasing number of lncRNAs have been reported to act either as tumor-suppressors or tumor-promoters [7]. For instance, lncRNA XIST evokes the oncogenic PI3K-AKT signaling to facilitate TC progression [8]. LncRNA TUG1 benefits epithelial-mesenchymal transition, which has been linked to the metastatic and invasive properties of TC cells [9]. The oncogenic role of lncRNA RNAMALAT1 is triggered by increasing the expression of myelocytomatosis (MYC), which is a driver of TC cell proliferation [10]. LINC01311 is a novel lncRNA recently reported to be interfering with Alzheimer's disease via epigenetic regulation [11]. However, its role in TC remains obscure.

Reportedly, lncRNA functions as a sponge of microRNA (miRNA), thereby releasing the target gene expressions from miRNA suppression [12]. LncRNA XIST sponges miR-34a and upregulates the expression of hepatocyte growth factor receptor (MRT), thus facilitating TC progression [8]. Furthermore, lncRNA-MIAT serves as miR-150–5p's ceRNA (competing endogenous RNA) to boost EZH2 expression, thereby favoring oncogenicity during TC progression. The ceRNA activity of LINC01311 to miR-146a-5p has also been described in Alzheimer's disease [11]. Hence, aside from evaluating the function of LINC01311 in vitro and in vivo, our subsequent mechanistic exploration was also conducted, which aimed to find the miRNA epigenetically silenced by LINC01311. Our bioinformatics analysis revealed that LINC01311 preferentially binds with miR-146b-5p and its tumorigenic role in TC piqued our curiosity [13], [14], [15], [16].

Therefore, we investigated the ceRNA activity of LINC01311 with miR-146a-5p during TC malignancy. We are hopeful that our present research may help identify new targets for the treatment of TC.

Methods

Tissue specimens

Twenty-eight [28] pairs of tumoral and noncancerous tissues were surgically obtained from individuals suffering from TC. Each patient submitted a written informed consent. None of the patients underwent neoadjuvant chemotherapy, chemoradiotherapy, or any therapy before the surgical resection. Specimens were sent for pathologic confirmation and were stored at −80 °C after snap-freezing for the next assays. Our hospital granted ethical approval to this study.

Cell culture

Normal human primary thyroid follicular epithelial cell line (Nythy-ori3–1) and one of the TC cell lines (TPC-1) were purchased from Millipore (Sigma, USA). Two other TC cell lines (KTC-1 and SW579) were from the Cell Bank of Typical Culture Preservation Committee of the Chinese Academy of Sciences (Shanghai, China). The fourth TC cell line (BCPAP) was from the DSMZ-German Collection of Microorganisms and Cell Cultures (GmbH Germany). BCPAP, KTC-1, TPC-1, and Nythy-ori3–1 were maintained in RPMI 1640 Medium (ThermoFisher, USA) while the SW579 cells were kept in DMEM (ThermoFisher, USA). All cells were grown in an incubator (95% air, 5% CO2; 37 °C). While the cells were being cultivated, 1% Penicillin-Streptomycin Solution (ThermoFisher, USA) and 10% FBS (ThermoFisher, USA) were added to the medium.

Cell transfection and infection

MiR-146b-5p mimic or mimic-NC, pcDNA-IMPA2 vectors (IMPA2-OE), and the empty vectors (Empty vector) were obtained from Gema, Shanghai, China. These products were introduced into KTC-1 and TPC-1 cells with the aid of Lipofectamine 2000 reagent (Invitrogen, USA). Forty-eight hours later, the transfection efficiency was ascertained by means of RT-qPCR.

For LINC01311 overexpression, empty plasmids and lentiviral particles harboring LINC01311 cDNA for LINC01311 ectopic expression were purchased from Guangzhou Yunzhou, Biological Technology Co. Ltd, China. The prepared lentiviral vectors were used to infect KTC-1 and TPC-1 cells before a 4-week puromycin maintenance. The survivals were propagated and subjected to RT-qPCR analysis.

RT-qPCR assay

A Total RNA Extraction Kit (Solarbio, China) was utilized to extract total RNA from cells and tissues. PrimeScript RT reagent Kit (Takara, Japan) and One step miRNA RT kit (Haigene, China) were employed for the reverse transcription of 1 μg RNA to cDNA. The cDNA was then amplified and quantified in a Bio-Rad iQ5 Real-Time System with the aid of a Fast SYBR Green Master Mix (Applied Biosystems, USA). The following thermal cycling conditions were used for qPCR: initial denaturation for 5 min at 95 °C, followed by 40 cycles of denaturation for 10 s at 95 °C and amplification/extension for 30 s at 60 °C, and one cycle of melt curve analysis according to the instrument's guidelines. GAPDH (for LINC01311 and IMPA2) or U6 (for miR-146b-5p) were adopted to normalize the data with 2−△△Ct. Table 1 presents all primers used.

Table 1.

The sequences of the primers in this study.

Primer Sequences
miR-146b-5p Forward: 5′-TGACCCATCCTGGGCCTCAA-3′
Reverse: 5′-CCAGTGGGCAAGATGTGGGCC-3′
IMPA2 Forward: 5′-TAAGCAGCTAGCATGAAGCCGAGCGGC-3′
Reverse: 5′- TGTTTAGAATTCTCACTTCTCATCATCCCGC-3′
GAPDH Forward: 5′-CTGGGCTACACTGAGCACC-3′
Reverse: 5′-AAGTGG TCGTTGAGGGCAATG-3′
U6 Forward: 5′-TGCGGGTGCTCGCTTCGGCAGC-3′
Reverse: 5′-GGATCCGACGGCTGGGTCTTCTCAGA-3′
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Western blot

The cells were harvested 48 h after transfection and lysed with RIPA buffer (ThermoFisher, USA). After centrifugation (10,000 g at 4 °C for 10 min), the concentration of the protein in the resulting supernatant was determined using a BCA Protein Assay Reagent kit (Pierce, USA). Afterward, 20 µg protein samples were resolved on a 10% SDS-PAGE gel before being transferred to PVDF membranes. Each membrane was subsequently blocked with 5% skim milk powder at room temperature for 30 min. Next, the membranes were exposed to diluted specific primary antibodies at 4 °C overnight. Thereafter, horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (Sigma-Aldrich, USA) were added to the membranes at room temperature for 3 h. Finally, a Pierce ECL Western Blotting Substrate was utilized for the visualization of the bands and the same were photographed on a GelDoc XR+ system (Bio-Rad, USA). The antibodies used are as follows: anti-Bcl-2 antibodies (Cat#: 13–8800; 1:10,000; ThermoFisher, USA), anti-Bax antibodies (Cat#: MA5–14,003; 1:10,000; ThermoFisher, USA), anti-IMPA2 antibodies (Cat#: ab256410;1:10,000; Abcam, USA), anti-GAPDH antibodies (Cat#: ab99252; 1:10,000; Abcam, USA), anti-mouse HRP secondary antibody (Cat#:31,430,1:1000, Invitrogen, USA), and anti-goat HRP secondary antibody (Cat#:31,402,1:1000, Invitrogen, USA).

Flow cytometry

Apoptosis was evaluated by flow cytometry assay with the help of an FITC Annexin V Apoptosis Kit (abcam, USA). TC cells (5 × 105), with or without LINC01311-OE transfection, were incubated at room temperature with 100 μl of 1 × binding buffer supplemented with 5 μl of propidium iodide solution and 5 μl of FITC Annexin V. Finally, a BD Biosciences flow cytometer was utilized in analyzing the cells.

In vitro proliferation assay

The CCK-8 kit (Sigma, USA) was used to assess TC cell proliferation in vitro. In brief, transfected TC cells (2 × 103 cells/well) were seeded into 96-well culture plates and subjected to 24-h, 48-h, and 72-h incubation. Next, 10 μL of CCK-8 reagent was added to each well and incubated for 3 h. Finally, each well was measured for absorbance (OD) at 450 nm with a microplate reader (Promega, USA).

In the vitro migration assay

A scratch wound healing assay was carried out to evaluate cellular migration. TC cells (5 × 103 cells/well) were inoculated into 6-well plates. Upon reaching 95% confluence, linear scratch wounds were created on the cell monolayers using 200 μL pipette tips. The scratched cells were removed, and the plates were maintained in a serum-free culture medium for 24 h. Photographs of the plates were taken at 0 h and 24 h.

Luciferase reporter assay

The putative binding sequences of LINC01311 and IMPA2 3′UTR were amplified and inserted into psi-CHECK2TM vectors (Promega, USA) to construct the LINC01311 wild-type (WT) and IMPA2 3′UTR WT luciferase vectors. The corresponding mutant (MUT) vectors were also produced. KTC-1 and TPC-1 cells were transfected with a combination of one of the developed vectors and either a miR-NC or miR-146b-5p mimic. Forty-eight hours post-transfection, the luciferase activities was measured. Normalized activity = Firefly Luciferase activity ÷ Renilla Luciferase activity.

RNA immunoprecipitation (RIP) assay

RNA Immunoprecipitation Kit (Millipore Sigma, USA) was utilized in the detection of LINC01311’s interaction with miR-146b-5p. KTC-1 and TPC-1 (4 × 104 cells) were lysed in a 200 µL harsh lysis buffer. The collected cell lysate was incubated with Protein A Magnetic Beads pre-conjugated with rat anti-IgG or Monoclonal anti-Ago2 antibodies overnight. After that, the magnetic beads were isolated, washed, and digested in a buffer containing Proteinase K. Finally, the RNA was extracted from the immunoprecipitates, and RT-qPCR was applied to analyze the enrichment level of LINC01311 and miR-146b-5p.

In vivo animal experiment

Four to five weeks old BALB/C nude mice (∼ 20 g,) were bought from the Center for Animal Experiment/Animal Biosafety Level III laboratory (ABSL-III lab) of Wuhan University (Wuhan, Hubei, China). The in vivo tumor models (n = 5/group) were constructed by subcutaneously injecting each mouse with 2 × 103 KTC-1 cells infected with lentiviral particles carrying LINC01311 or empty plasmids. Tumor sizes were monitored once every four days. After 28 days passed, the mice were euthanized by CO2 inhalation. The xenograft tumors were subsequently excised and weighed. The Institutional Animal Care and Ethics Committee of our hospital granted approval to this animal experiment.

Statistical analysis

Data analysis was performed in GraphPad Prism 9.0 (GraphPad Prism, USA). The data were indicated as mean ± SD. Data from the two groups were compared by applying a paired student t-test. Meanwhile, the differences among multiple groups were tested by One-way ANOVA and Tukey multiple-comparison test. Pearson correlation coefficient was applied to ascertain the correlation of miR-146b-5p with LINC01311 and IMPA2. Lastly, a P < 0.05 was indicative of significance.

Results

LINC01311 overexpression suppresses TC cell growth in vitro and in vivo

We first assessed LINC01311 expression in various TC cell lines. TC cells (KTC-1, BCPAP, TPC-1, and SW579), particularly KTC-1 and TPC-1 cells, expressed lower LINC01311 compared to Nythy-ori3–1 cells (Fig. 1A). We subsequently analyzed the expression of LINC01311 in 28 paired TC and normal tissues and observed a similar downregulation of LINC01311 in TC tissues as well (Fig. 1B). To further characterize the function of LINC01311 in TC cells, we determined its localization within the KTC-1 and TPC-1 cells. The outcomes showed that it is dominantly located in the cytoplasm (Fig. 1C), suggesting its ceRNA activity. To assess the function of LINC01311 in TC cells, we enhanced its expression in the TPC-1 and KTC-1 cells by introducing pCDNA-LINC01311 (LINC01311-OE) into both the cell lines. The results of RT-qPCR analysis revealed that LINC01311 expression was enhanced after the transfection (Fig. 1D). Next, we investigated how LINC01311 overexpression altered TC cell malignant phenotypes such as proliferation, apoptosis, and migration. We found that the proliferative potential of both the TC cell lines was significantly decreased when LINC01311 was overexpressed (Fig. 1E). Furthermore, in KTC-1 and TPC-1 cells overexpressing LINC01311, the relative protein levels of Bax were increased while those of Bcl-2 were decreased, implying that LINC01311 overexpression induces apoptosis (Fig. 1F). Similar results were obtained through flow cytometry, which revealed that overexpression of LINC01311 promoted apoptosis in these cell lines (Fig. 1G). The migratory abilities of both the TC cell lines overexpressing LINC01311 were also reduced as compared to the control cell line (Fig. 1H).

Fig. 1.

Fig. 1

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LINC01311 overexpression suppresses TC cell growth in vitro. (A) RT-qPCR analysis of LINC01311 in TC cells (KTC-1, BCPAP, TPC-1, and SW579) and normal cells (Nythy-ori3–1). [**P < 0.001 vs. Nythy-ori3–1; n = 3/group; Dunnett's post hoc test]. (B) RT-qPCR analysis of LINC01311 in TC tissues and normal tissues [**P<0.0001 vs. normal tissues; n = 28/group; Welch's t-test]. (C) LINC01311 localization within the nucleus and the cytoplasm of TC cells was tested by RT-qPCR. (D) RT-qPCR analysis of LINC01311 expression in KTC-1 and TPC-1 cells transfected with Empty vectors or LINC01311-overexpressing vectors (LINC01311-OE). (E) CCK-8 assessment of TC cells transfected with Empty vectors or LINC01311-OE. (F) Western blotting analysis of the expressions of Bcl-2 and Bax in TC cells carrying Empty vectors or LINC01311-OE. (G) Cell apoptosis of TC cells transfected with Empty vectors or LINC01311-OE was examined by Annexin V-FITC/PI-labeled flow cytometry. (H) Scratch wound-healing assay transfected TC cells carrying Empty vectors or LINC01311-OE. [D to H: **P < 0.001 vs. Empty vector; n = 3/group; Student's t-test].

To address whether LINC01311 impacts TC tumorigenesis in vivo, we constructed a mouse xenograft tumor model by subcutaneously injecting LINC01311-overexpressing vectors into KTC-1 cells. The results demonstrated that LINC01311 overexpression impeded tumor growth in vivo by markedly reducing the size, volume, and weight of the tumors (Fig. 2A-C). Collectively, LINC01311 is an anti-oncogenic lncRNA that suppresses the malignant behavior of TC cells, both in vivo and in vitro.

Fig. 2.

Fig. 2

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LINC01311 overexpression restrains TC tumor cell growth in vivo. Four to five-weeks-old BALB/C nude mice were subcutaneously injected with KTC-1 cells infected with lentiviral particles carrying LINC01311 or empty plasmids. Tumor sizes were monitored once every four days. Twenty-eight days later, the mice were euthanized by CO2 inhalation, and the tumor weight was recorded upon the excision of the xenograft tumors. (A) Representative photograph of xenograft tumors in LINC01311-OE and empty vector groups. (B) Tumor size was measured every four days in empty vectors and LINC01311-OE groups. (C) Tumor weight was determined after tumor isolation in LINC01311-OE and empty vector groups. [B and C: **P < 0.001 vs. Empty vector; n = 5/group; Student's t-test].

LINC01311 targets miR-146b-5p

Next, we investigated for the target miRNA of LINC01311 in order to understand its ceRNA activity in influencing the malignant behaviors of TC cells. Through starBase analysis, we found that LINC01311 preferentially binds with miR-146b-5p (Fig. 3A). Furthermore, the miR-146b-5p mimic decreased the luciferase activity of KTC-1 and TPC-1 cells transfected with LINC01311-WT but did not affect their luciferase activity when transfected with LINC01311-MUT (Fig. 3B). The results of the RIP assay demonstrated that the enrichment of LINC01311 and miR-146b-5p was considerably higher in the Anti-Ago2 group compared to the Anti-IgG group in the KTC-1 and TPC-1 cells, indicating that LINC01311 recognized miR-146b-5p by base pairing (Fig. 3C). Contrary to the poor expression of LINC01311 in TC cells and tissues, miR-146b-5p expression was enhanced in TC cells and tissues (Fig. 3D and E). Furthermore, we observed a negative correlation between the expressions of LINC01311 and miR-146b-5p (Fig. 3F).

Fig. 3.

Fig. 3

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LINC01311 targets miR-146b-5p. (A) StarBase predicted that miR-146b-5p might bind with LINC01311. (B) Luciferase activity in extracts from KTC-1 and TPC-1 cells co-transfected with LINC01311-WT/MUT luciferase vectors and miR-146b-5p mimic or miR-NC. [**P < 0.001 vs. miR-NC; n = 3/group; Student's t-test]. (C) Ago2 RNA immunoprecipitation (RIP) assay was performed to detect miR-146b-5p and LINC01311 levels in KTC-1 and TPC-1 cells with Ago2. [**P < 0.001 vs. Anti-Ago2; n = 3/group; Student's t-test]. (D) RT-qPCR analysis of miR-146b-5p in TC cells (KTC-1 and TPC-1) and normal cells (Nythy-ori3–1). [**P < 0.001 vs. Nythy-ori3–1; n = 3/group; Dunnett's post hoc test]. (E) RT-qPCR analysis of miR-146b-5p in normal and TC tissues. [**P<0.0001 vs. normal tissues; n = 28/group; Welch's t-test]. (F) Pearson correlation coefficient indicating the correlation of LINC01311 expression with that of miR-146b-5p in TC tissues. (G) RT-qPCR analysis of LINC0311 and miR146b-5p expression in xenograft tumors. [**P < 0.001 vs. Empty vector; n = 5/group; Student's t-test]. (H) LINC01311 overexpressing vectors (LINC01311-OE), Empty vectors, miR-146b-5p mimic (mimic), mimic-NC, and LINC01311-OE (OE)+mimic were transfected into KTC-1 and TPC-1 cells. Relative miR-146b-5p levels in the transfected TC cells were quantified via RT-qPCR analysis. [⁎⁎P < 0.001 vs. Empty vector; ##P < 0.001 vs. mimic-NC; ^^P < 0.001 vs. LINC01311-OE (OE)+mimic; n = 3/group; Dunnett's post hoc test].

We then examined the expression of LINC0311 and miR146b-5p in xenograft tumors to confirm the negative connection between the two. The results demonstrated that LINC01311 expression was elevated whereas miR-146b-5p expression was downregulated in tumors overexpressing LINC01311, confirming the negative correlation (Fig. 3G). Notably, overexpression of LINC01311 mitigated the elevated miR-146b-5p levels caused by the miR-146b-5p mimic in KTC-1 and TPC-1 cells (Fig. 3H). The aforementioned findings suggest that LINC01311 sponges miR-146b-5p to downregulate miR-146b-5p expression.

LINC01311 interacts with miR-146b-5p to antagonize the anti-oncogenic role of LINC01311 in TC

To investigate the functionality of the LINC01311–miR-146b-5p interaction, we studied the alternation of phenotypes in TC cells co-transfected with LINC01311 overexpressing vectors and miR-146b-5p mimic. Intriguingly, a significant rise in cell proliferation was observed in KTC-1 and TPC-1 cells that overexpressed miR-146b-5p. This increase in proliferation was nearly negated upon the additional transfection of LINC01311 overexpressing vector in both the TC cell lines (Fig. 4A). Conversely, the overexpression of miR-146b-5p induced by the transfection of the miR-146b-5p mimic constrained TC cell apoptosis. This was demonstrated by elevated Bcl-2 and reduced Bax protein levels in KTC-1 and TPC-1 cells (Fig. 4B). However, this suppression of apoptosis was offset by the overexpression of LINC01311. In scratch wound-healing assays, miR-146b-5p-overexpressing TC cells manifested increased cell migration. Meanwhile, the co-introduction of LINC01311 overexpressing vectors and miR-146b-5p mimic overturned this increase (Fig. 4C). These findings indicate that LINC01311 may directly interact with miR-146b-5p to constrain the malignant behaviors of TC cells.

Fig. 4.

Fig. 4

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LINC01311 interacts with miR-146b-5p to counteract the tumor-suppressive role of LINC01311 in TC cells. KTC-1 and TPC-1 cells were transfected with LINC01311 overexpressing vectors (LINC01311-OE), Empty vectors, miR-146b-5p mimic (mimic), mimic-NC, and LINC01311-OE (OE)+mimic. after 48 h. (A) Cell viability in different groups was tested by CCK-8. (B) Western blot analysis of the different groups using anti-Bax and anti-Bcl-2 antibodies. (C) Analysis of tumor cells migration by scratch wound-healing assay in different groups. [**P < 0.001 when compared to the Empty vectors; ##P < 0.001 when compared to mimic-NC; ^^P < 0.001 vs. LINC01311-OE (OE)+mimic; n = 3/group; Dunnett's post hoc test].

MiR-146b-5p recognizes IMPA2 and downregulates its expression

Next, we attempted to understand the mechanism by which miR-146b-5p promoted cellular proliferation and migration by exploring possible transcription silencing in TC cells with miR-146b-5p overexpression. The starBase prediction showed that miR-146b-5p might target IMPA2 (Fig. 5A). To substantiate this, we performed a lucifearse reporter assay and observed that miR-146b-5p mimic could evoke a considerable reduction in the luciferase activity of TC cells transfected with IMPA2-WT while having no effect on the luciferase activity of TC cells transfected with IMPA2-MUT (Fig. 5B). The result indicated that miR-146b-5p could directly target the 3′ UTR of IMPA2. A notable downregulation of IMPA2 was also detected in TC cells and tissues (Fig. 5C and D). In addition, IMPA2 expression was found to be negatively correlated with miR-146b-5p expression TC tissues (Fig. 5E). More importantly, miR-146b-5p mimic offset the overexpression of IMPA2 in TC cells, which was engendered by the transfection of IMPA2 overexpressing vectors (Fig. 5F). Based on the findings presented above, it is reasonable to conclude that miR-146b-5p interferes with IMPA2 expression by binding with its 3′UTR.

Fig. 5.

Fig. 5

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MiR-146b-5p recognizes IMPA2 and downregulates its expression. (A) The binding sites shared by miR-146b-5p and IMPA2 3′UTR, as predicted by starBase. (B) Dual-luciferase reporter experiment verified the binding of MPA2 3′UTR with miR-146b-5p. [**P < 0.001 vs. miR-NC; n = 3/group; Student's t-test]. (C) RT-qPCR analysis of IMPA2 expression in TC tissues and normal tissues. [**P<0.0001 vs. normal tissues; n = 28/group; Welch's t-test]. (D) RT-qPCR analysis of IMPA2 expression in TC cells (KTC-1 and TPC-1) and normal cells (Nythy-ori3–1). [⁎⁎P < 0.001 vs. Nythy-ori3–1; n = 3/group; Dunnett's post hoc test]. (E) Pearson correlation coefficient revealed the association of IMPA2 expression with that of miR-146b-5p in TC tissues. (F) KTC-1 and TPC-1 cells were transfected with miR-146b-5p mimic (mimic), mimic-NC, Empty vector, IMPA2 overexpressing vectors (IMPA2-OE), and IMPA2-OE (OE)+mimic. Western blotting was performed to estimate IMPA2 expression in the different groups. [⁎⁎P < 0.001 when compared to Empty vector; ##P < 0.001 when compared to mimic-NC; ^^P < 0.001 when compared to IMPA2-OE (OE)+mimic; n = 3/group; Dunnett's post hoc test].

IMPA2 overexpression overturns miR-146b-5p's oncogenic activity in TC cells

After determining the direct interaction of miR-146b-5p with the IMPA2 3′UTR, we further investigated whether IMPA2 downregulation is a crucial step for the oncogenic activity of miR-146b-5p. As a result, the ectopic expression of IMPA2 impaired the proliferative capacity of TC cells. This anti-proliferative potential of IMPA2, however, was reversed upon the additional introduction of the miR-146b-5p mimic (Fig. 6A). The outcomes of the western blot experiment performed using anti-Bcl-2 and anti-Bax antibodies revealed that IMPA2-overexpressing TC cells manifested elevated Bax and reduced Bcl-2 protein expression levels, indicating enhanced apoptosis. This outcome, however, was mitigated by the addition of the miR-146b-5p mimic (Fig. 6B). In addition, we observed that IMPA2 overexpression reduced the migration of TC cells. This anti-migratory action, again, was counteracted by the miR-146b-5p mimic (Fig. 6C). These results suggest that the oncogenic role of miR-146b-5p may be reliant, at least in part, on the downregulation of IMPA2.

Fig. 6.

Fig. 6

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IMPA2 overexpression counteracts miR-146b-5p's oncogenic action in TC cells. KTC-1 and TPC-1 cells were transfected with miR-146b-5p mimic (mimic), mimic-NC, Empty vector, IMPA2 overexpressing vectors (IMPA2-OE), and IMPA2-OE (OE)+mimic. After 48 h. (A) Cell viability in different groups was tested by CCK-8. (B) Western blot analysis of the different groups using anti-Bax and anti-Bcl-2 antibodies. (C) Analysis of tumor cell migration in the different groups by means of the scratch wound-healing assay. [⁎⁎P < 0.001 vs. Empty vector; #P < 0.05, ##P < 0.001 vs. mimic-NC; ^^P < 0.001 vs. IMPA2-OE (OE)+mimic; n = 3/group; Dunnett's post hoc test].

Discussion

In the present work, we found poor expression of LINC01311 in TC cells and tissues, implying that LINC01311 may play a role in TC progression. Furthermore, we found that ectopic expression of LINC01311 reduced the proliferative and migratory potentials of TC cells in vitro and triggered apoptosis. This inhibitory effect was also manifested in vivo. To the best of our knowledge, our results are the first to demonstrate the tumor-suppressive activity of LINC01311 in cancer. Mechanistically, we further characterized the novel ceRNA activity of LINC01311 wherein it sponged miR-146b-5p. This reversed the miR-146b-5p-mediated IMPA2 silencing, thereby suppressing the malignant behaviors of the TC cells. This newly discovered ceRNA regulatory network of LINC01311/miR-146b-5p/IMPA2 establishes the foundation for targeted therapies against TC.

Previously, miR-146b-5p has been reported to act as either a tumor promoter or suppressor in different malignancies [17], [18], [19]. For example, miR-146b-5p has been shown to be an oncogenic miRNA in colorectal and papillary thyroid carcinoma in vitro and in vivo [20,21]. On the contrary, it has been reported to function as a tumor suppressor miRNA in non-small cell lung cancer and T-cell acute lymphoblastic leukemia [19]. We observed elevated miR-146b-5p expression in TC cells and tissues and our functional assays demonstrated that the high miR-146b-5p levels promoted TC cell migration and proliferation and impeded apoptosis. Hence, our results suggests that miR-146b-5p act as an oncogenic miRNA in TC. Our findings are consistent with previous reports that showed miR-146b-5p overexpression increased TC cell migration and proliferation [13,14], validating miR-146b-5p's oncogenic function in TC progression. Interestingly, via luciferase reporter and RIP assays, we discovered that miR-146b-5p is a target of LINC01311’s ceRNA activity. LINC01311 overexpression suppressed the expression of miR-146b-5p as well as its oncogenic role, therefore inhibiting the progression of TC. Although other lncRNAs have been reported to target miR-146b-5p [19,[22], [23], [24]], our study is the first to reveal the interaction of miR-146b-5p with LINC01311, which suggests a novel regulatory mechanism for miR-146b-5p modulation.

LncRNAs sequester miRNAs by sponging them and thereby attenuate the repressive effect of miRNAs on mRNAs [25]. Here, we found that miR-146b-5p targeted IMPA2 3′ UTR by our bioinformatics analysis as well as luciferase reporter assay. The IMPA2 gene is located on chromosome 18p11.21 and consists of 9 exons. This gene encodes an inositol monophosphatase which catalyzes the dephosphorylation of inositol monophosphate and executes a significant role in phosphatidylinositol signaling [26]. In cervical cancer, IMPA2 expression is reportedly increased in the tumor and the short hairpin RNA (shRNA)-mediated IMPA2 silencing suppressed the malignant potential of tumor cells by attenuating ERK phosphorylation [26]. In clear cell renal cell carcinoma, IMPA2 downregulation is a novel biomarker of dismal prognosis of the tumor, and its silencing has been reported to drive the proliferation of tumor cells [27,28]. In TC, we found reduced expression of IMPA2 in tumors and overexpressing IMPA2 hampered the migration and proliferation of TC cells while boosting apoptosis. We are first to report the tumor-suppressive role of IMPA2 in TC. Furthermore, rescue assays also demonstrated that the miR-146b-5p mimic could offset the IMPA2-overexpression-induced suppression of migration and proliferation as well as the stimulation of apoptosis in TC cells. Our findings provide novel insights on the epigenetic modification of miR-146b-5p during TC progression.

This study has some limitations, and future research is needed to address them. First, the ceRNA regulatory network constituted by LINC01311, miR-146b-5p, and IMPA2 may be more complex due to the different possible targets among them. For example, miR-146b-5p regulates lncRNA MALAT1 expression and drives the progression of TC [14]. Our future research will concentrate on these complex regulatory networks. Second, some regulatory factors have been reported to play anti-tumor roles in cancers such as granulocyte colony-stimulating factor [29], bruton's tyrosine kinase [30], shikonin [31]. Therefore, we will explore the key anti-tumor factors associated with LINC01311/miR-146b-5p/IMPA2 axis in TC in the future. In addition, the clinical outcome requires additional confirmation with a large sample size, and the downstream effector of IMPA2 needs to be investigated further.

Conclusion

We identified LINC01311 as a tumor suppressor, both in vitro and in vivo. LINC01311 mitigates TC cell migration and proliferation and promotes apoptosis via sponging miR-146b-5p to upregulate IMPA2 expression. Overall, our research establishes the groundwork for TC intervention.

Declarations

Funding

This study did not receive any funding in any form.

Ethics approval

The present study was approved by the Ethics Committee of Hubei NO.3 People's Hospital of Jianghan University (Wuhan, China). The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. All patients signed written informed consent.

This animal experiment was conducted in accordance with the ARRIVE guidelines and was authorized by the Institutional Animal Care and Ethics Committee of Hubei NO.3 People's Hospital of Jianghan University.

Consent to participate

All patients signed a written informed consent.

Consent for publication

Consent for publication was obtained from the participants.

Availability of data and material

All data generated or analyzed during this study are included in this article.

Authors' contributions

MJL and LHZ conducted the experiments and data analysis. JPH and CY conceived and designed the study. YZ and ZDY acquired the data. ZDY and MJL carried out the analysis and interpretation of data. All authors have read and approved this manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

None.

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Data Availability Statement

All data generated or analyzed during this study are included in this article.

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