The lncRNA NEAT1 Inhibits miRNA-216b and Promotes Colorectal Cancer Progression by Indirectly Activating YY1

Background Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) is commonly considered an oncogene in various cancers. The long noncoding RNA NEAT1 has been reported to be overexpressed in colorectal cancer (CRC). However, the exact role of NEAT1 in CRC remains unknown. Our research aimed to explore the function of NEAT1 in the tumorigenesis and the development of CRC. Methods Real-time quantitative PCR (qRT-PCR) was used to detect the NEAT1, miR-216b, and YIN-YANG-1 (YY1) mRNA levels in CRC tissues and cells, then immunohistochemistry (IHC) was used to detect the expression of YY1 in CRC tissues. Luciferase reporter, qPCR, western blot, and DNA pulldown assays were conducted to study the relationships between NEAT1, miR-216b, and YY1. Flow cytometry analysis was performed for cell cycle and apoptosis analyses, and a colony formation assay was performed to test cell proliferation. Transwell assays were performed to detect cell invasion and migration. Results The NEAT1 expression was significantly upregulated in CRC tissues compared with its expression in normal tissues, and downregulation of NEAT1 suppressed the proliferation, migration, and invasion of CRC cells. Moreover, we found NEAT1 decreased the miR-216b level directly, and the suppression of miR-216b could inhibit the function of downstream YY1. However, overexpression of YY1 accelerated CRC cell proliferation, migration, and invasion. Conclusion Our results indicated that NEAT1 acted as an oncogene in CRC and promoted the progression of CRC by directly sponging miR-216 b expression to activate the expression of YY1. The NEAT1/miR-216b/YY1 axis may be a novel therapeutic target for CRC.


Introduction
Colorectal cancer (CRC), including colon cancer and rectal cancer, is one of the most common malignancies in the world, which has threatened the global health [1]. Although the existing methods of diagnosis and treatment continue to improve, it also exhibits the low rate of 5-year overall survival and poor prognosis [2]. Due to its complex process of pathogenesis involving many factors, the clear mechanism of CRC still retain unknown, which motivate the clinical treatment to explore the CRC progression.
In our research, we detected the expression of NEAT1 in human CRC tissues and cells. Subsequently, functional experiments were prepared to verify the carcinogenic effect of NEAT1. In mechanism, the regulatory relationship between NEAT1 and micRNA-216b was further studied which has not reported previously. Notably, we provided evidence that NEAT1 directly sponged the expression of miR-216b, which consequently removed functional regulation of the downstream YY1. -is study provides new insights into clinical treatment and further intervention targets for CRC.

Patient Specimens.
All 57 paired CRC tissues and normal tissues stored in biobank were obtained from those diagnosed CRC patients who underwent no preoperative therapy prior to surgical resection at Zhejiang Cancer Hospital (Hangzhou, China) from 2013 to 2017. Tissue collection and manipulation were authorized by the Ethics Committee of Zhejiang Cancer Hospital (ethics number: IRB-2020-26). Informed consents were collected from all subjects.
-e specimens were frozen in liquid nitrogen after surgery and kept at −80°C. -e detailed clinical and pathological data from these 57 CRC specimens are shown in Table 1. -e overall survival time and time from diagnosis to death were recorded to detect the prognostic status. Conventional receiver operating characteristic (ROC) curve analysis was used to confirm the cut-off value of NEAT1 expression. Furthermore, Youden index, which consisted of specificity and sensitivity, was prepared to help calculate the cut-off of the expression level (specificity%+sensitivity%− 100%), the largest one of which was the cut-off value.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
Total RNA was isolated from the paired tissues using the Trizol reagent (number: 15596-018, Invitrogen, Carlsbad, USA) according to the standard protocol in the kit. After reverse transcription of total RNA by using a cDNA reverse transcriptase kit (Takara, code number: 6215A, purchased from ElifeBio, Hangzhou, China), we obtained the cDNA. -e expression levels of LncRNA NEAT1 and miR-216b were measured using Applied Biosystems 7500 Real Time PCR System with a Green One-Step qRT-PCR SuperMix Kit (Transgen, code number: AQ211-01, Beijing, China). -e reaction conditions were as follows: predenaturation at 95°C for 5 min, denaturation at 95°C for 10 s, and annealing at 60°C for 30 s for 35 cycles. -e results were normalized to the expression levels of GAPDH or U6 snRNA using the 2-ΔΔCT method for quantification. Primers were devised and produced by Sangon Biotech (Shanghai, China), and the sequences of PCR primers are shown in Table 2.

Western Blot
Assays. -e details of western blot assays were performed as described in our previous published research [10]. Fresh tumor tissues and normal tissues treated with liquid nitrogen were crushed and then lysed in RIPA lysis buffer supplemented with proteinase inhibitor cocktail (Roche, number: 5892970001). In brief, lysed proteins were isolated by SDS-PAGE, transferred to PVDF membranes, and incubated with the following primary antibodies: anti-GAPDH antibody (1 : 3000 dilution, rabbit, Abcam, ab9485) andanti-YY1 antibody (1 : 500 dilution, rabbit, Abcam, ab109228). Following incubation with the appropriate HRP-conjugated secondary antibodies, the bands were visualized using Pierce ™ ECL western blotting substrate (-ermo Scientific, number: 32106, USA). -e signal intensity of the protein was further determined by ImageJ software (Madison, WI, USA).

Immunohistochemistry (IHC)
. -e detail of the assay referred to the previous study [16]. After cut into 4-µm sections all the tissues were deparaffinized and treated with EDTA (pH 9.0) to antigen retrieval in a microwave for 20 min. -en, Autostainer Link 48 machine (Dako, Denmark A/S, Denmark) was performed for staining. Subsequently, primary anti-YY1 antibody (1 : 100, Abcam,

Cell Invasion and Migration Assays.
Cell invasion and migration were tested by a transwell chamber with Matrigel (8-μm pore size; BD Biosciences, USA) according to the manufacturer's protocol. -e detail referred to the previous study [12].

Flow Cytometry Cell Cycle and Apoptosis Analysis.
Flow cytometry assay for cell cycle and apoptosis was prepared referring to a previous article [17].

Pull-Down Assay with Biotinylated NEAT1 DNA Probe.
-e DNA fragment containing the full-length NEAT1 sequence or negative control sequence was PCR amplified by a T7-containing primer and then bound to GV394 (Invitrogen, Shanghai, China). -e obtained plasmid DNAs were digested to a linear form by the restriction enzyme XhoI. Biotin-labeled RNAs were conversely transcribed, and then qRT-PCR was used to analyze target the RNA expression according to the method described previously [18].

Statistical Analysis.
All experimental data were analyzed by GraphPad Prism 8.0, and the results are shown as the mean ± SD (standard deviation). Chi-square tests were performed to detect the correlation between the NEAT1 expression and clinicopathological factors. Student's t-test was performed to compare the differences between the two groups. Comparisons among multiple groups were made by one-way ANOVA. Survival analysis was performed via the Kaplan-Meier method accompanied by Cox regression analysis for univariate and multivariate analyses. P < 0.05 was considered significant.

Detecting NEAT1 from TCGA in Cancers by Online Tools.
According to the public, database "UALCAN" (http:// ualcan.path.uab.edu) from the Cancer Genome Atlas (TCGA) in cancers, we found NEAT1 overexpressed in  patients with metastasis especially nodal metastasis and liver metastasis exhibited a higher expression level of NEAT1 than nonmetastasis ones (Figures 2(e)-2(g), p < 0.01). In order to identify the association of NEAT1 in prognosis, we explored the cut-off value of NEAT1 in our CRC specimen, the result of which showed 2.888 was the threshold for NEAT1 (compared with GAPDH). Exactly mRNA levels of NEAT1 >2.888 was considered as "high" and those ≤2.888 as "low" (Figure 2(j), AUC � 0.9070). In our result, higher expression level of NEAT1 displayed the lower overall survival time ( Figure 2(h), p � 0.0012) and progression-free survival time ( Figure 2(i), p � 0.0064). We also found high expression of NEAT1 mRNA significantly correlated with distant metastasis (p < 0.01), lymph node metastasis (P � 0.0001), and tumor grade (P � 0.011) ( Table 1).

Upregulation of NEAT1 in CRC Tissues and Cells.
After transfection, the expression of NEAT1 significantly decreased in the siRNA NEAT1 group compared with the We also detected NEAT1 knockdown inhibited cell proliferation by the colony formation assay (Figure 3(f )). Suppression of NEAT1 restrain much more cells than the negative control group in G0/G1 which inhibited cell proliferation (Figure 3(c), P < 0.05). Loss function of NEAT1 elevated the apoptotic rate in SW620 and HCT-116 by the flow cytometry apoptosis assay (Figure 3(d), P < 0.05). Afterwards, the transwell assay further indicated the silenced of NEAT1 decreased invasive and migratory cell numbers, which revealed NEAT1 led to CRC invasion and migration (Figure 3(e), P < 0.05).

Suppression of NEAT1 in Repressing Cell Proliferation and
Invasion of CRC. We identified miR-216 b as a most potential candidate by bioinformatics database "Diana Tools" (Diana, http:// diana.imis.athena-innovation.gr/ DianaTools) (Figure 4(a)). Since then, we constructed plasmids of NEAT1 to detect whether the decreased expression of NEAT1 could lead to the increased expression of miR-216 b as well as designed the luciferase reporter containing exact or mutant miR-216 b binding sites to determine the binding effect between NEAT1 and miR-216 b. We found NEAT1 knockdown resulted in the increased expression of miR-216 b by qPCR (Figure 4(b)). Furthermore, we found the decreased miR-216 b expression in the CRC tissue (Figure 4(c), P < 0.05). -e luciferase reporter showed miR-216 b mimic significantly reduced luciferase activity of the wild-type NEAT1 plasmid, however, miR-216b inhibitor significantly increased it in HCT-116 ell line (Figure 4(d), P < 0.05). Moreover, miR-216b inhibitor significantly upregulated the expression of NEAT1 (Figure 4(e), P < 0.05). In contrast, knockdown of NEAT1 also induced the upregulation of miR-216b (Figure 4(f ), P < 0.05). Subsequently, the biotin-labeled pulldown system was applied to further confirm whether NEAT1 could pulldown miR-216 b. We observed miR-216 b in CRC cells pulled down by   Journal of Oncology biotinylated NEAT1 which indicated miR-216b could directly bind to NEAT1 at the microRNA recognition site (Figure 4(g), P < 0.05). -e transwell assay was prepared to explore the function of NEAT1 and miR-216b in migration and invasion, the result from which identified SW620 and HCT-116 transfected with NEAT1-silenced and cotransfected with miR-216b mimic significantly repressed the cell invasion and migration but failed when cotransfected with the miR-206 inhibitor (Figure 4(h), p < 0.01). -e SW620 and HCT-116 cell lines with NEAT1 silence showed poor ability in proliferation and it achieved better when cotransfected with miR-216b mimic. However, when cell lines with NEAT1-silenced and cotransfected with miR-216b inhibitor displayed better ability in proliferation than that with NEAT1-silenced and cotransfected with miR-216b mimic (Figure 4(i), p < 0.01).

NEAT1 Sponged miR-216b to Activate YY1 to Regulate the Progression of CRC Cells.
To explore candidate target of miR-216b, bioinformatics online tools (miRDB, http:// mirdb.org/) were prepared to predict the suitable one that finding miR-216b possessed the matched binding site with YY1 ( Figure 5(a)), which overexpressed in the CRC tissues not normal tissues ( Figure 5(b), p < 0.01). In addition, the IHC staining indicated the strong staining of YY1 in the CRC tissues and liver metastatic tissues compared with the almost no staining in the CRC situ tissues especially in  ( Figure 5(g), p < 0.01). Taken together, NEAT1 sponged miR-216b to activate YY1 to accelerate the ability of cell viability, apoptosis, and invasion on colorectal cancer cells.

Discussion
NEAT1 participates in many critical biological processes and acts as a potential predictor or target for prognosis in CRC, lung cancer, gastric cancer, and breast cancer [9,12,19,20]. However, further research studies are required to elucidate how NEAT1 function. Herein, we discovered high expression of NEAT1 in the CRC tissue, and associated with histological differentiation, overall survival, distant metastasis, and nodal metastasis, acting as an independent prognostic factor for overall survival in patients with CRC, indicating the poor prognosis. In the present study, we also observed that NEAT1 knockdown or miR-216b overexpression remarkably attenuated the ability of cell viability, apoptosis, and invasion on colorectal cancer cells. Mechanismly, our results disclosed that NEAT1 sponged the miR-216b to facilitate the function of the downstream regulators YY1, thus to promote CRC proliferation, invasion, and migration. Accumulating evidence has indicated that LncRNA could regulate the expression of microRNA as ceRNA [3,4,[21][22][23]. NEAT1 displayed multiple function in regulating microRNA that it not only sponged miR-133b to promote migration and invasion of breast cancer cells [24], but also inactivated miR-101 to play potential oncogene in breast cancer [25]. Moreover, in lung cancer, NEAT1 competed against let-7a to contribute to nonsmall-cell lung cancer proliferation and metastasis [26], and another research in lung cancer also revealed downregulation of NEAT1 led to cell invasion in NSCLC via sponging miR-153-3p [27]. In CRC, it was illustrated that the NEAT1 expression level was an independent prognostic factor for disease-free survival and overall survival which mechanismly promoted colorectal cancer progression by competitively binding miR-34a [12] resulting in activation of Wnt/beta-catenin signaling pathway. What's more, NEAT1 also could facilitate the sensitivity of 5-FU in CRC cells via miR-150-5p [14]. Although a little study displayed the anti-cancer role of NEAT1 in CRC, a flood of investigations have still been verified its oncogenic role especially acting as ceRNA to regulate microRNAs [20,27]. However, Xiong's team revealed that the NEAT1 expression in the CRC tissues were not significantly different compared with the normal tissues [28]. -erefore, the function of NEAT1 in CRC needs to be further explored.
Like previous studies, our research also identified NEAT1 acting as oncogene in CRC and considered as an independent prognostic factor for disease-free survival and overall survival. With the help of online bioinformatics tools "DIANA" and "MIRDB", we have explored microRNA-216b that NEAT1 may sponge to discover the deep mechanism. MicroRNA-216 b from our result was first reported molecular which can be regulated by NEAT1. MicroRNA-216 b was considered as the tumor suppressor in lung cancer, gastric cancer, and pancreatic cancer which could regulate cell proliferation, apoptosis, and invasion [29,30]. Here, we found that miR-216b, acting as a connecting carrier, targets both NEAT1 and the 3′-UTR of YY1 by luciferase reporter assays. YY1 showed the oncogenic role in CRC, especially playing the epigenetic regulative role on cancer stem cell transcription factors to accelerate tumor metastasis [31,32]. -is constitutes the identification of a competitive endogenous RNA network in colorectal cancer. Although we verified NEAT1 sponged the miR-216b to facilitate the function of the downstream regulators YY1, our limitations should not be ignored in this study: our results were from experiments in vitro not in vivo, which made us to finish the further studies as well as the epigenetic regulation of YY1 in this axis.

Conclusion
Our results illustrate that NEAT1 functions as an oncogenic lncRNA to facilitate the carcinogenesis and progression of CRC by competitively sponging miR-216b to activate YY1. -e present results elucidate a potential mechanism underlying the tumor-oncogenic role of NEAT1 in colorectal cancer and indicate that NEAT1 could serve as a useful marker and potential therapeutic target in CRC.

Data Availability
-e data that support the findings of this study are available from the corresponding authors upon reasonable request.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Research Ethics Committee of Zhejiang Cancer Hospital.

Conflicts of Interest
-e authors declare that they have no conflicts of interest.