Silencing of Long Non-coding RNA ENST00000606790.1 Inhibits the Malignant Behaviors of Papillary Thyroid Carcinoma through the PI3K/AKT Pathway
Zhihua Zuo , Ling Liu , Bin Song , Juan Tan , Dafa Ding & Yibing Lu
To cite this article: Zhihua Zuo , Ling Liu , Bin Song , Juan Tan , Dafa Ding & Yibing Lu (2020):
Silencing of Long Non-coding RNA ENST00000606790.1 Inhibits the Malignant Behaviors of Papillary Thyroid Carcinoma through the PI3K/AKT Pathway, Endocrine Research, DOI: 10.1080/07435800.2020.1804928
To link to this article: https://doi.org/10.1080/07435800.2020.1804928
Published online: 13 Aug 2020.
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KEYWORDS:Long non-coding RNA; thyroid carcinoma; microarray analysis; siRNA; Pi3k/AKT pathway
Introduction
Thyroid carcinoma (TC) is one of the most common types of malignant endocrine tumors, and is more pre- valent in women than in men. Papillary thyroid carci- noma (PTC) accounts for approximately 85–90% of all cases of TC.1 In recent decades, the global incidence of thyroid cancer has gradually increased.2 In general, patients with PTC are considered to recover favorably and the prognosis is good. However, patients with cer- tain clinicopathological factors at diagnosis may have a poor prognosis, including advanced age, a primary tumor larger than 3 cm, extrathyroidal extension, lymph node metastasis, distant metastasis, and advanced TNM stage.3,4 However, the current methods of diag- nosis and treatment options remain limited. Therefore, it is of great importance to find an effective biomarker to improve the early diagnosis of PTC, particularly highly invasive PTC.
Long-noncoding RNAs (lncRNAs), a subset of 200- nucleotide-long non-coding RNAs, are involved in epige- netic and post-transcriptional regulation.5–7 Accumulating evidence has demonstrated the role of lncRNAs in promot- ing cancer development, including bladder cancer, gastric cancer, pancreatic cancer, breast cancer, and colorectal cancer.8–16 Similar to protein-coding genes, lncRNAs also act as oncogenes, and have been demonstrated to serve as potential biomarkers and/or therapeutic targets for tumor diagnosis and treatment. A number of lncRNAs (such as BRAF, UCA1, HOTAIR, H19, PVT1, and NEAT1) are currently being applied to clinical diagnosis or prognostic determination.8,13,15-19 In addition, the BRAF-activated lnc RNA (BANCR) and lncRNA PTC susceptibility candidate 3 (PTCSC3) have been demonstrated to play an important role in PTC development and progression.20,21 However, the specificity and mechanism of these markers remain largely unknown. New findings are still needed.
Our previous microarray analysis of lncRNA expression profiles (unpublished data) revealed that lncRNA ENST00000606790.1 (ENST) was signifi- cantly upregulated in PTC tissues compared to nor- mal para-cancerous thyroid (NPTC) tissues. Therefore, we hypothesized that ENST may play an important role in promoting the development of PTC. To test this hypothesis, first we determined the expression of ENST in human tumor and non- tumor-tissues or -cell lines. Second, the correlation of ENST expression with clinical outcomes was ana- lyzed. Third, we evaluated the effect of silencing of ENTS on tumor cell development in vitro.
The P13K/AKT pathway has been shown to play a central role in promoting tumor growth, proliferation, invasion, and blood vessel growth, and to be involved in the malignant transformation or metastasis of TC.22,23 Therefore, we investigated whether ENST regulates the development of PTC through modification of PI3K/ AKT signaling. This study may provide a novel insight into the molecular mechanisms underlying carcinogen- esis of PTC, and provide a new molecular marker for diagnosis and treatment of PTC.
Materials and Methods
Patients
From January 2016 to October 2017, a total of 62 PTC patients were recruited for this study at the Second Affiliated Hospital of Nanjing Medical University and the Jiangning Hospital affiliated to Nanjing Medical University. All surgical PTC and NPTC specimens of patients were stored immediately in liquid nitrogen after resection. A hospital pathol- ogist confirmed the diagnosis of PTC in all cases. None of the patients included in this study received preoperative cervical irradiation or radiotherapy/che- motherapy. Two hospital institutional ethics review boards approved the study and all patients provided written informed consent.
RNA Extraction
RNA was extracted from PTC and NPTC tissues using Trizol reagent (cat no.1596018, Thermo Fisher, Waltham, USA), according to the manufacturer instruc- tions. The ratio of absorbance at 260 nm to the absor- bance at 280 nm (A260/A280) was used to estimate the purity of extracted RNA; the extracted RNA samples with the value of A260/A280 ranging from 1.8 to 2.0 were used for further analysis.
Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR)
Reverse transcription was performed using a commercial kit (cat. no. K1622, Thermo Fisher, Waltham, USA). The obtained cDNA was stored at −20°C. The cDNA was used as the template for qPCR to detect the expression of ENST in 62 PTC and NPTC tissues. All primers were synthesized by TaKaRa (Nanjing, China). Sequences of primers were listed as follows: ENST: 5ʹ-GCCAATGTCACCATGCA CTC-3ʹ (forward) and5ʹ-GCTGAGAGAACGTCAGCT CC-3ʹ (reverse), and GAPDH:5ʹ-CAGGAGGCATTGCTGATGAT-3ʹ (forward) and 5ʹ-GAAGGCTGGGGCTC ATTT-3ʹ (reverse). The qPCR reaction mix (10 μL) con- sisted of SYBR Green Master Mix (5ul), forward and reverse primers (0.2 uL of each), RNase-free ddH2 O (3.6 μL), and diluted cDNA (1 μL). A total of 40 cycles were performed for the cyclic reaction: pre-denaturation at 95°C for 5 min, denaturation at 95°C for15 s, and annealing at 60°C for 1 min. The lncRNA expression level was calculated according to the cyclic threshold (Ct value) using the 2−ΔΔCt method.
Cell Culture and siRNA Transfection
The human PTC cell lines (IHH4 and TPC-1), and normal thyroid cells (Nthy-ori-3-1) were kindly donated by the Endocrine Laboratory of Integrative Medicine Hospital of Jiangsu Province. IHH4 and TPC-1cells were maintained in a mixture medium (RPMI1640 and Dulbecco’s modified Eagle at a ratio of 1:1; Gibco, Thermo Fisher, Waltham, USA) supplemented with 10% fetal bovine serum (FBS). All cells were maintained at 37°C in a humidified atmosphere of 5% CO2. ENST- specific small interfering RNA (siRNA) and negative control (NC) were designed and synthesized by Ribo Technology (Ribo, Nanjing, China). When IHH4 cells reached a confluence of 35%-45%, they were transfected with siRNA or NC (150 nM/sample) using lipofecta- mine 2000 reagent (Invitrogen, Shanghai, China).
Cell Viability Assay
A Cell Counting Kit-8 (CCK-8, Beyotime, Nanjing, China) was used to measure cell viability. CCK-8 solution (10 uL) was added to each well. Then, the cells were incubated with the solution at 37°C for 3 h. The absorbance was measured at 450 nm to calculate the number of viable cells.
Cell Cycle Analysis
The cultured cells were digested to a single cell suspen- sion and fixed with 75% cold ethanol at 4°C overnight.Following removal of the ethanol, cells were incubated with propidium iodide (PI; Thermo Fisher, Waltham, USA) for 30 min at room temperature in dark. All samples were analyzed by flow cytometery.
Transwell Assay
Cell migration was detected by a transwell invasion method. In brief, matrix (25 g) gel was used to coat an 8-μm-pore polycarbonate membrane (BD Biological Science, New York, USA). The cells (5 × 104) were seeded in medium containing 0.5–1% FBS in the upper chamber, and the culture medium in the lower chamber containing 5–10% FBS. The cells that migrated through the filter were immobilized with methanol and stained with 0.5% crystal violet. Five random fields for each sample were recorded by a microscope for calculating the mean cells per field. Each experiment was conducted in triplicate.
Colony Formation Assay
The transfected IHH4 cells at the logarithmic phase of growth were digested with a 0.25% trypsin solution, and then 100 cells were seeded into a 6-well culture plate. At day 14 of culture, the colonies became visible to the naked eye, and the cell culture was terminated. The cells were fixed with methanol for 20 min and stained with 0.5% crystal violet. The number of colonies was counted under a microscope in five randomly selected fields. The colony formation rate (CFR) was calculated according to the following formula: CFR = (clone count/seed cell count) × 100%. The experiment was conducted in triplicate.
Western Blot Analysis
The transfected cells were washed in phosphate buffered saline (PBS) and the protein lysate was obtained using a protein analysis kit (Roche, Shanghai, China). Total cell proteins were extracted, and the protein concentration was measured using a colorimetric method. Proteins were separated by 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred (semi- dry) to a nitrocellulose membrane. After being soaked in 5% skimmed milk at room temperature for 1 h, the membranes were incubated with the following primary antibodies overnight at 4°C: anti-AKT antibody (1:1000, ab82538, Abcam, Shanghai, China), anti-P-AKT (1:1000, Cell Signaling Technology, Shanghai, China), anti-GAPDH (1:5000, Santa Cruz Biotechnology, Shanghai, China), anti-CHK1 (1:800 ab137400; Abcam, Shanghai, China), and anti-CDC25C (1:1000 ab208035; Abcam, Shanghai, China). Then, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (1:200, Abcam, Shanghai, China) for 1 h at room temperature. The protein bands were visualized by staining with chemilu- minescent HRP substrate, and photos were taken by a digital gel image analysis system (Tanon-4500; Tanon Science and Technology, Shanghai, China).
Statistical Analysis
All statistical analyses were performed with SPSS 20.0 software (IBM SPSS, Chicago, ILA, USA). The rank-sum test was used to compare the lncRNA expression between PTC and NPTC tissues; the correlation of lncRNA expression and clinico- pathological characteristics was evaluated using the chi-square test; Student’s t-test was used for com- parisons between two groups; one-way ANOVA was used to compare data from three groups, followed by Tukey’s test for multiple comparisons. P < .05 was regarded as statistically significant. Data are represented as mean ±SEM. Results Expression of IncRNA ENST in PTC Tissues and Cell Lines The expression level of ENST in 62 PTC and NPTC tissues was determined by RT-qPCR. Results showed that the expression level of ENST was significantly higher in PTC than in NPTC tissues (Figure 1a). Consistently, the expression level of ENST was signifi- cantly higher in the cancer cell lines IHH4 and TPC-1 than in normal cells, Nthy-ori-3-1 (Figure 1b). Correlation of IncRNA ENST Expression with the Clinicopathological Features of PTC We retrospectively analyzed the clinical data of 62 patients with PTC, including 46 women and 16 men, aged 11–73 years (42.3 ± 14.43 mean). Of the 62 patients with PTC, 48 patients had tumors <2 cm, 18 had thyroid exter- nal extension, and 41 had lymph node metastasis, but there were no cases of distant metastasis. As shown in Table 1, high expression of ENST was significantly correlated to lymph node metastasis and tumor size, but not with other factors. Figure 1. Expression of ENST in PTC tissues and cell lines. The expression levels of ENST in (a) PTC and NPTC tissues, (b) normal thyroid cell line and PTC cell lines. NPTC tissue vs. PTC tissues, or normal thyroid cell line vs. PTC cell lines. ***P < .001. The cell experiment was conducted in triplicate. Effect of Silencing of ENST on PTC Cell Proliferation, Colony Formation and Invasion To determine whether ENST plays a critical role in pro- moting the development of PTC, we silenced the expres- sion of ENST in a cultured tumor cell line IHH4. The results from RT-qPCR showed that our constructed siRNA could significantly inhibit the expression of ENST (Figure 2a). Silencing of ENST could significantly inhibit cell growth and tumor cell colony formation (Figure 2b,f, g). Cell cycle analysis revealed that silencing of ENST arrested the cell cycle in G2/M phase (Figure 2c). Further analysis revealed that CDC25Cwas significantly down- regulated while CHK1 was significantly up-regulated in ENST-silenced tumor cells (Figure 2d,e). Furthermore,tumor cell migrative capacity was significantly inhibited by silencing of ENST (Figure 2h,i). Silencing the Expression of ENST Down-regulated the Expression of Molecules Involved in the PI3K/ AKT Pathway As the PI3K/AKT signaling pathway is crucial for the development of PTC, we determined whether silencing of ENST in tumor cells would alter the expression of molecules in the PI3K/AKT pathway. Results showed the levels of PI3K, p-PI3K, AKT and p-AKT were sig- nificantly down-regulated in ENST-silenced IHH4 cells (Figure 3a,b). Re-activating PI3K/AKT Signaling Partially Abolished the Effect of Silencing of ENST on PTC Cells To determine whether PI3K/AKT signaling mediated the inhibitory effect of silencing of ENST on tumor cell viability and migration, we treated ENST-silenced cells with an activator of PI3K, 740-Y-P. 740-Y-P, the cell- permeable phosphopeptide activator of PI3K, has been widely used in scientific research to enhance the biolo- gical activity of PI3K.24–26 Results showed that 740- Y-P treatment significantly restored cell growth and cell colony formation capacity, and promoted cell migration of ENST-silenced cells (Figure 4a–e). Discussion The incidence of PTC is increasing globally. However, the exact mechanisms driving PTC development and progres- sion remain to be elucidated. Recent studies have high- lighted the role of lncRNAs in regulating the biological processes of various cancers, including TC.27–29 LncRNAs are emerging as novel molecular biomarkers for the diagnosis or prognosis of cancers.30–33 In the present study, we found that lncRNA ENST was significantly up- regulated in human PTC tissues and PTC cell lines. More importantly, the expression of ENST was strongly correlated to lymph node metastasis and tumor size at diagnosis in patients with PTC, suggesting that ENST may potentially serve as a marker in reflecting the devel- opmental stage of PTC. Figure 2. Effect of silencing ENST on PTC cell proliferation, colony formation and migration.IHH4 cells were transfected with NC or ENST siRNA. Twenty-four hours after transfection, (a) ENST expression was determined by RT- qPCR, (b) cell viability was detected by CCK8, (c) the cell cycle was analyzed by staining with PI, (d) CHK1 and CDC25 were analyzed by western blot, (e) the relative expression of CHK1 and CDC25 was quantified by densitometric analysis. Twelve days after transfection, (f) colony formation was assessed and (g) the number of colonies was quantified. Twenty-four hours after transfection, (h) cell migrative ability was assessed and (i) the number of cells was quantified. siRNA vs. NC, **P < .01, ***P < .001. The experiment was conducted in triplicate. Figure 3. Silencing of ENST downregulated the expression of molecules involved in the PI3K/AKT pathway.IHH4 cells were transfected with NC or ENST siRNA. Twenty-four hours after transfection, (a) western blot analysis of p-AKT, AKT, PI3K, and p-PI3K and (b) the relative expression of proteins was quantified. siRNA vs. NC, *P < .05, **P < .01, ***P < .001. The experiment was conducted in triplicate. Figure 4. Re-activating PI3K/AKT signaling partially abolished the effect of silencing of ENST on PTC cells.IHH4 cells were pre-treated with 740-Y-P (25ug/mL), then transfected with ENST siRNA, (a) cell viability was detected by CCK8. (b) Twelve days after transfection, colony formation was assessed, and (c) the number of colonies was quantified. (i) Forty-eight hours after transfection, cell migrative ability was assessed, and (j) the number of migrative cells was quantified. siRNA vs. NC, *P < .05, **P < .01,***P < .001; siRNA+740-Y-p vs. siRNA, ##P < .01, ###P < .001. The experiment was conducted in triplicate. Targeting cell cycle progression is one of the most common strategies for cancer treatment. It is known that cell cycle progression is monitored by checkpoints in G1, G2, and metaphase.34 When cells encounter damage, such as DNA damage, these checkpoints will be activated to arrest cell cycle progression, thereby allowing for damage repair. It has been demonstrated that most tumors show a defective G1 checkpoint but an intact G2 checkpoint.35 Therefore, targeting G2 check- point has emerged as a new strategy for tumor treatment.35 It has been reported that the tumor inhibi- tory effect of several chemicals was associated with blocking G2/M transition.36,37 In the present study, we found that silencing the expression of ENST signifi- cantly decreased cell viability and increased cells in the G2/M phase, demonstrating a role of ENST in regulating the process of G2/M transition.G2/M transition is regulated partly by CHK1- CDC25C pathway.38 CHK1 can directly phosphorylate CDC25C on serine-216, which leads to inhibition of Cdc25C’s ability to dephosphorylate and activate CDC2.38 It is well established that CDC2-cyclin B complex is crucial for meiotic G2/M transition.39 Therefore, up-regulation of CHK1 decreases the level of unphosphorylated CDC25C, resulting in the reduced formation of CDC2-cyclin B complex and a blockage of G2/M transition. In the present study, we found that silencing of ENST up-regulated the expression of CHK1 and down-regulated the expression of CDC25C, indicat- ing that the role of ENST in regulating G2/M transition may go through modification of CHK1-CDC25C path- way. Besides, we demonstrated that ENST had a role in promoting tumor cell migration. Based on these find- ings, we conclude that ENST plays a critical role in promoting the development of PTC. The Pl3K/AKT pathway is an important intracellular signal transduction pathway that regulates cell proliferation, metabolism, and tumorigenesis.22 It has been widely reported that the PI3K/AKT pathway plays a critical role in promoting the development of various tumors, including TC.23,40 Several studies have shown that lncRNA regulated the development of cancers through modifications of PI3K/ AKT signaling.41,42 Therefore, we investigated whether the tumor-promoting effect of ENST was mediated by modification of PI3K/AKT signaling. In the present study, we showed that the expression of PI3K, p-PI3K, AKT, and p-AKT was downregulated by silencing of ENST, and a re- activated PI3K/AKT pathway largely abolished the effect of silencing of ENST on tumor cells. Therefore, we demon- strated that the PI3K/AKT pathway, at least partly, mediated the effect of ENST on cell viability and migration. Previous studies showed that PI3K/AKT signaling was associated with G2/M transition and cell migration of tumor cells. Liu et al. reported that inhibition of the PI3K/AKT signaling pathway led to G2/M cell cycle arrest in hepato- cellular carcinoma cells.43 Similar results were reported in human bladder cancer cells.44 Numerous studies have indi- cated that PI3K/AKT signaling may play a central role in promoting cell migration of PTC.45–47 Basing on previous studies and our results in the present study, we concluded that ENST promoted G2/M transition and cell migration of PTC perhaps through activating PI3K/AKT signaling. Some limitations of our study should be mentioned. First, we included only a small number of cases. Owing to time and funding limitations, we were not able to carry out a larger and longer-term follow-up study. Second, how ENST modifies the PI3K/AKT signaling remains unknown. Third, other mechanisms of action underlying the regulatory role of ENST in PTC were not explored, which will be investigated in future research. Despite these limitations, our preliminary results strongly suggest that ENST can serve as a potential novel biomarker for the diagnosis or prognosis of PTC. Disclosure Of Interest No potential conflicts of interest relevant to this article were reported. The authors alone are responsible for the content and writing of the paper. Funding This work was supported by the Key Research and Development Project of Jiangsu Province under [grant BE2015723]. 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