lncRNA CASC2 Enhances 131I Sensitivity in Papillary Thyroid Cancer by Sponging miR-155

Long noncoding RNA cancer susceptibility candidate 2 (CASC2) has been reported to play an anticancer role in papillary thyroid cancer (PTC). Radioiodine (131I) is a common option for the treatment of PTC. However, the role and mechanism of CASC2 in 131I sensitivity remain unclear. In this study, 131I-resistant cells were constructed through continuous treatment of 131I. The expression levels of CASC2 and miR-155 were measured by qRT-PCR. The IC50 of 131I was analyzed by cell viability using MTT assay. Flow cytometry was conducted to determine cell apoptosis induced by 131I. The association between CASC2 and miR-155 was evaluated by luciferase assay and RNA immunoprecipitation. A mouse xenograft model was built to explore the effect of CASC2 on the growth of 131I-resistant PTC cells in vivo. Results showed that CASC2 expression was decreased in PTC tissues and cells, and low expression of CASC2 was associated with poor outcome of patients. CASC2 level was reduced in 131I-resistant cells. Knockdown of CASC2 inhibited 131I sensitivity in thyroid cancer cells. Overexpression of CASC2 enhanced 131I sensitivity in constructed resistant PTC cells. CASC2 was a decoy of miR-155, and CASC2-mediated promotion of 131I sensitivity was weakened by decreasing miR-155. Abundance of CASC2 inhibited the growth of 131I-resistant cells in vivo. As a conclusion, CASC2 increases 131I sensitivity in PTC by sponging miR-155, providing a novel target for the treatment of thyroid cancer patients with 131I resistance.


Introduction
Papillary thyroid cancer (PTC) is regarded as a low-risk thyroid cancer with high morbidity [1]. In recent years, great development has been gained on the diagnosis and treatment of thyroid cancer [2]. Radioiodine ( 131 I) exposure is the standard adjuvant treatment for patients with thyroid cancer [3]. However, some patients would fail to respond to 131 I therapy with an overall survival less than 50% due to the local recurrence and distant metastasis [4]. Noncoding RNAs are associated with PTC development and can enhance 131 I therapeutic function and improve the survival of patients [5][6][7].
Noncoding RNAs, including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), play as important biomarkers by regulating cancer development and treatment in endocrine-related cancers, including thyroid cancer [8]. lncRNAs with >200 nucleotides in length and absent ability of coding proteins have been reported to play important roles in the diagnosis, prognosis, and therapeutics of thyroid cancer [9]. Moreover, lncRNA can drive radioresponse and regulate the outcomes of patients after radiotherapy [10]. For example, Liu et al. report that lncRNA maternally expressed gene 3 (MEG3) can increase 131 I sensitivity by sponging miR-182 in thyroid cancer [6]. Moreover, Xiang et al. suggest that lncRNA solute carrier family 6 member 9 (SLC6A9) sensitizes PTC cells to 131 I treatment [7]. Long noncoding RNA cancer susceptibility candidate 2 (CASC2) has been demonstrated as a tumor suppressor by regulating cell proliferation, apoptosis, migration, and chemoresistance in human cancers, including hepatocellular carcinoma, gastric cancer, and prostate cancer [11][12][13]. Furthermore, emerging evidences suggest that CASC2 is lowly expressed and inhibits cell proliferation in PTC [14,15]. However, little is known about the role of CASC2 in 131 I sensitivity to PTC.
In the present study, we first constructed the 131 I-resistant cells. Moreover, we investigated the effect of CASC2 on 131 I sensitivity to PTC cells by detecting cell viability and apoptosis. In addition, we explored the target association between CASC2 and miR-155 to elucidate the regulatory mechanism of CASC2.

Patients and Tissues.
A total of 46 paired cancer tissues and surrounding normal samples were collected from patients with PTC during surgical resection at Xinyang Central Hospital and then stored at -80°C in the Key Laboratory of Geriatric Diseases of Xinyang until used. The clinical features of patients are displayed in Table 1. Furthermore, another 50 PTC patients (23 males and 27 females; age: 45-60 years old; 39 with lymph node metastasis and 11 without) who have received 131 I treatment at least 1 year (200 mCi for patients with lymph node metastasis and 100 mCi for those without lymph node metastasis) prior to the study were recruited, and a follow-up study was performed for analysis of the overall survival of patients. All participants have provided the written informed consent, and this study was approved by the ethics committee of Xinyang Central Hospital.

Cell Culture, 131 I-Resistant Cell Construction, and
Transfection. Thyroid cancer cell lines (FTC-133, TPC-1, BCPAP, and IHH-4) and human thyroid follicular cell line Nthy-ori 3-1 cells were purchased from BeNa Culture Collection (Beijing, China). The cells were maintained at 37°C and 5% CO 2 . Cell culture medium was RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum and changed every three days. TPC-1 and IHH-4 cells with the lowest level of CASC2 were chosen to construct 131 Iresistant cells (res-TPC-1 and res-IHH-4). In brief, TPC-1 and IHH-4 cells were exposed to a median-lethal dose of 131 I for continuous passaging. After treatment for 12 h, IC50 of 131 I radioactivity was analyzed by cell viability using MTT assay. The resistant cells were obtained after passaging for 8 generations (G) and identified using flow cytometry by avoiding apoptosis under 131 I exposure. TPC-1 and res-TPC-1 cells were exposed to 1 mCi 131 I for 12 h, and IHH-4 and res-IHH-4 cells were exposed to 0.5 mCi 131 I for 12 h, followed by further study.

Quantitative Real-Time Polymerase Chain Reaction
(qRT-PCR). For detection of the abundances of CASC2 and miR-155 in PTC tissues and cells, Trizol reagent (Thermo Fisher) was used for total RNA isolation. Then, 1 μg RNA was reversely transcribed to cDNA using TaqMan micro-RNA reverse transcription kit or High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher).The cDNA was mixed with SYBR Green Mix (Thermo Fisher) and specific primers and used for qRT-PCR on a 7900HT Fast RT-PCR. The amplification procedure was as follows: 95°C for 5 min  were used. The level of CASC2 enriched in complex by RIP was detected by qRT-PCR.

In Vivo Tumor Growth
Assay. BALB/c nude mice (six weeks old, male, 18-22 g) were obtained from Vital River Laboratory Animal Technology (Beijing, China) and housed under standard conditions. For tumor growth assay, 1 × 10 6 res-TPC-1 cells (empty group) or res-TPC-1 cells transfected with a control lentivirus (lenti-NC group) or a recombinant lentivirus-expressing CASC2 (lenti-CASC2 group) were subcutaneously injected into the back of mice. After inoculation, tumor volume was monitored and calculated every week for a total of five weeks according to the formula ðlength × width 2 Þ/2. At five weeks after injection, mice were euthanized by intraperitoneal phenobarbital injection (150 mg/kg; Guangdong Bangmin Pharmaceutical Co., Ltd., Guangdong, China), and tumors were removed for weight and qRT-PCR assay. The animal work was performed at Xinyang Vocational and Technical College and with the approval of Animal Ethics Committee of our college.
2.9. Statistical Analysis. Data of three independent experiments in graph were expressed as mean ± standard deviation (S.D.) and investigated by Student's t test or ANOVA followed by Tukey's post hoc test, supported by GraphPad Prism 7 software (La Jolla, CA, USA). Overall survival of patients with PTC after 131 I therapy was analyzed by Kaplan-Meier plot and log-rank test. The association between CASC2 level and clinical features of PTC patients was analyzed by the χ 2 test. P < 0:05 was considered statistically significant ( * P < 0:05, * * P < 0:01, and * * * P < 0:001).

The Expression of CASC2 Is Reduced in PTC.
To explore the role of CASC2 in PTC, we first measured its expression in 46 cancer tissues. As shown in Figure 1(a), the level of CASC2 was significantly decreased in cancer tissues in comparison to that in the matched normal tissues. Moreover, the patients were divided into high (n = 21) or low (n = 25) CASC2 level group according to its mean abundance in cancer tissues. Table 1 summarizes that low expression of CASC2 was associated with pathological stage, tumor size, and lymph node metastasis (P < 0:05) but not with age and gender (P > 0:05 ) of patients. Furthermore, CASC2 was obviously lowly expressed in thyroid cancer cells (FTC-133, TPC-1, and IHH-4) when compared with the control group (Nthy-ori 3-1) (Figure 1(b)). In addition, another 50 patients with treatment of 131 I were divided into two groups according to the median value of CASC2, and the patients with low expression of CASC2 (n = 25) exhibited poorer overall survival than those with high expression (n = 25) (hazard ratio ðHRÞ = 0:3828; 95% confidence interval (CI) (0.1507-0.9723); P = 0:0441) (Figure 1(c)).  Figures 3(a) and 3(b), the expression of CASC2 was greatly reduced in res-TPC-1 and res-IHH-4 cells compared with the corresponding TPC-1 and IHH-4 cells. To investigate the effect of CASC2 on 131 I sensitivity in sensitive PTC cells, si-CASC2 was used to knock down the abundance of CASC2 in TPC-1 and IHH-4 cells, which was confirmed in Figures 3(c) and 3(d). Furthermore, silencing CASC2 promoted cell viability induced by 131 I in the two cell lines compared with treatment of si-NC (Figures 3(e) and 3(f)). In addition, knockdown of CASC2 inhibited 131 Icaused apoptosis in TPC-1 and IHH-4 cells (Figure 3(g)). 3.5. miR-155 Is a Target of CASC2. lncRNA could serve as miRNA sponge in cancer progression. The analysis of star-Base online predicted the binding sites of miR-155 and CASC2 at chr10: 119813301-119813323 ( Figure 5(a)). In order to validate the target association between CASC2 and miR-155, luciferase assay and RIP assay were performed in res-TPC-1 and res-IHH-4 cells. As described in Figures 5(b) and 5(c), overexpression of miR-155 resulted in more than 55% reduction of luciferase activity in res-TPC-1 and res-IHH-4 cells, respectively, in the CASC2-WT group, while the activity was not changed in the CASC2-MUT group. Moreover, overexpression of miR-155 induced 5.1-fold and 7.7-fold elevation in enrichment level of CASC2 by Ago2 RIP, but IgG failed to display the enrichment (Figures 5(d) and 5(e)).  BioMed Research International 1D). Moreover, by phylogenetic assay using the UCSC Genome Browser (http://genome.ucsc.edu/), miR-155 binding site across species is highly conserved among mammals (Supplementary Figure 1E).

CASC2 Decreases 131 I-Resistant PTC Cell Growth In Vivo.
To explore whether CASC2 affected the growth of 131 I-resistant cells in vivo, res-TPC-1 cells or res-TPC-1 cells stably transfected with lenti-NC or lenti-CASC2 were injected into the mice. Tumor volumes were examined every week, and xenografts were removed at the fifth week. As presented in Figure 7(a), tumor volumes kept increasing after injection. Compared with the empty and lenti-NC groups, overexpression of CASC2 decreased tumor volumes at each time point.
Moreover, tumor weight was also significantly reduced in the CASC2-expressing group relative to the empty and lenti-NC groups (Figure 7(b)). The results of qRT-PCR assay revealed that stable transfection of lenti-CASC2 markedly increased the level of CASC2, but decreased miR-155 level in tumor tissues (Figures 7(c) and 7(d)).

Discussion
In this study, we found that CASC2 was lowly expressed in PTC tissues and cells and indicated poor prognosis of patients, which is also in agreement with former efforts [14,15,18]. 131 I therapy is a common strategy for the treatment of thyroid cancer. However, the interaction between CASC2 This validated the successful construction of 131 I-resistant cells. Then, the qRT-PCR results of reduced CASC2 in resistant cells revealed that low expression of CASC2 might be associated with 131 I resistance. A former work suggested the antiproliferation and proapoptosis role of CASC2 in PTC cells [15]. Similarly, by loss-of-function and gain-offunction experiments, we also found that CASC2 silence increased cell viability but decreased apoptosis and DNA  BioMed Research International damage in PTC cells exposed by 131 I, while CASC2 addition played an opposite effect. In vivo experiments also indicated that abundance of CASC2 inhibited the growth in 131 I-resistant cell-formed tumor, which indicated that CASC2 could act as a therapeutic target for 131 I therapy of PTC.
Previous studies indicated that CASC2 could serve as a competing endogenous RNA (ceRNA) or miRNA sponge in the development of cancers [12,19]. Accruing reports demonstrated that miR-155 is an oncogenic miRNA involved in drug resistance and radioresistance in human cancers [20][21][22]. Furthermore, it is suggested that miR-155 expression was increased in papillary thyroid carcinoma and its overexpression promoted cell proliferation by regulating adenomatous polyposis coli and Wnt/β-catenin signaling [23][24][25]. In this study, we first confirmed the association between CASC2 and miR-155 in PTC cells by luciferase assay and RIP. Moreover, we found that the expression of miR-155 was higher in resistant cells than in sensitive cells, uncovering that upregulation of miR-155 might contribute to 131 I resistance. By res-cue experiments, results showed that miR-155 reversed CASC2-mediated promotion of 131 I sensitivity, reflecting that CASC2 could regulate 131 I sensitivity by sponging miR-155 in PTC. In order to better understand that ceRNA mechanism allows CASC2 in 131 I sensitivity, the target of miR-155 might be helpful. We used DIANA tools to predict 10 targets of miR-155, which were reported to be relevant to radioresistance [21,[26][27][28][29][30][31][32][33][34]. As one of the predicted targets, forkhead box O3 (FOXO3) was involved in miR-155mediated regulation of radio-or chemoresistance in thyrocytes [35,36]. This study also confirmed that FOXO3 was targeted and negatively regulated via miR-155 in res-TPC-1 and res-IHH-4 cells. Hence, we hypothesized that FOXO3 might be responsible for CASC2 in 131 I sensitivity to PTC by the crosstalk of miR-155, which would be further confirmed in the future.
In conclusion, low expression of CASC2 was showed in PTC and indicated poor outcomes of patients. CASC2 knockdown reduced 131 I sensitivity in PTC cells, while its overexpression increased the sensitivity, possibly by regulation of miR-155. This study indicates a new mechanism for the development of 131 I resistance and provides a novel target for the treatment of PTC.

Data Availability
The data displayed in this manuscript is available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declare that there are no competing interests associated with the manuscript.