LncRNA NORAD functions as a competing endogenous RNA to regulate KMT2D expression by targeting miR-204-5p in gastric cancer

Yan Zhou (  yan_zhou09@163.com ) Nanfang Hospital, Southern Medical University Zhenwei Deng Dongguan People's Hospital, Southern Medical University Ting Pan Dongguan People's Hospital, Southern Medical University Guohao Chen Dongguan People's Hospital, Southern Medical University Yongchang Cai Dongguan People's Hospital, Southern Medical University Yuxin Tang Dongguan People's Hospital, Southern Medical University Yijun Wang Dongguan People's Hospital, Southern Medical University Yucheng Wang Dongguan People's Hospital, Southern Medical University Ruiping Li Dongguan People's Hospital, Southern Medical University Libo Li Dongguan People's Hospital, Southern Medical University


Introduction
Gastric cancer (GC) is s one of the most common malignant tumors worldwide, constituting the sixth leading cause of cancer related-mortality (1,2). As most GC patients are at an advanced stage when diagnosed and the 5-year survival rates remain low, particularly for those with local and distant metastasis (3,4). Improved GC early diagnosis and survival outcomes is thus urgently needed.
Protein coding genes regulate many cell processes including differentiation, metabolism and tumorigenesis and have been well-studied in recent years (5,6). However, with the development of human genome sequencing, non-coding RNA (ncRNA) has gradually attracted increasing attention (7,8).
Emerging evidence suggests that lncRNAs are key players a multitude of cellular processes, especially resulting in aberrant expression of gene products link to the advance of diverse human cancers (9,10).
However, the role of lncRNAs and its molecular mechanism involved in GC is still largely unclear.
Non-coding RNA activated by DNA damage (NORAD), a newly discovered lncRNA that maps to 20q11.23 in human, has been reported as an oncogenic factor in numbers of human cancers (11)(12)(13)(14)(15)(16). For instance, a recent study revealed that NORAD was upregulated in GC tissues and promoted GC cell growth in vitro (17). Another report indicated that silencing of NORAD suppressed cell proliferation and invasion via positive modulation of RhoA/ROCK1 pathway in GC(18). Micro-RNAs (miRNAs) are a type of endogenous short-chain non-coding RNA molecules that involved in the development and progression of human cancers (19,20). Acting as competitive endogenous RNAs (ceRNAs) to sponge miRNAs is one of the most well-established biological roles of lncRNAs (21,22). Interestingly, NORAD also functioned as a ceRNA in many cancers (23)(24)(25)(26). MiR-204-5p is a miRNA implicated in the development and progression of human cancers, including GC (27)(28)(29)(30). Given that there are limited studies that reveals the relationship between NORAD and miR-204-5p in GC. In the present study, we aimed to comprehensively elucidate the functional roles of NORAD/miR-204-5p axis and their molecular mechanisms in GC, as well as their potential as therapeutic target for the disease.

Clinical samples
Our sample cohort contained a total of 60 pairs of matched normal and GC tissues, which were obtained from A liated Dongguan People's Hospital, Southern Medical University (Dongguan People's Hospital).
All the tissue samples after liquid nitrogen refrigeration were saved in the-80°C cryogenic refrigerator. All subjects have written informed consent. This study was approved by the Ethics Committee of Dongguan People's Hospital, Southern Medical University (KYKT-2020-026) and that this was conducted in accordance with the Declaration of Helsinki.
Quantitative qRT-PCR (qRT-PCR) assay Cellular RNAs were extracted using Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China) was used for total RNA reverse transcription. The expression of NORAD and KMT2D was detected by a LightCycler 480 Instrument (Roche, Basel, Switzerland), using SYBR Green PCR Kit (TaKaRa Biotechnology) and GAPDH was used as the internal control. Relative expression of miR-204-5p was measured using TaqMan MicroRNA Assays (Applied Biosystems) and U6 was treated as an internal control. All the primer sequences were listed in Table 1. Table 1 The sequences of the primers.

Nucleocytoplasmic fractionation assay
The cytoplasmic and nuclear components were separates using PARIS Kit (Life Technologies, MA, USA). The nuclear and cytoplasmic fractions was detected by qRT-PCR assay to determine NORAD distribution using GAPDH and U6 as the cytoplasmic reference and nuclear reference, respectively.
RNA immunoprecipitation (RIP) assay RIP was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. An anti-Argonaute-2 (Ago-2) or anti-IgG antibodies was also used. Enrichment of NORAD and miR-204-5p was determined by qRT-PCR assay.
The membrane was blocked in 5% nonfat skim milk and incubated with primary antibodies and

Statistical analysis
All statistical analysis were performed using SPSS 21.0 (SPSS, Chicago, IL, USA) or GraphPad Prism (GraphPad Prism, Inc., La Jolla, CA, USA). Each experiment was performed in three independent replicates and the data are presented as the mean ± standard deviation (SD). Student's t-test or one-way ANOVA was used to compare the means of two or three groups. P<0.05 was considered statistically signi cant.

NORAD was highly expressed in GC tissues and cell lines
A cohort containing 60 pairs of fresh frozen GC tissues and paired adjacent normal tissues was involved in our study. qRT-PCR assay was performed to determine different expression levels of NORAD. As shown in Fig. 1A, NORAD was obviously upregulated in the GC tissues compared with the matched adjacent normal tissues (P<0.001). We also examined the expression of NORAD in different cell lines. The results showed that the expression of NORAD was signi cantly higher in several GC cell lines (MKN-45, SGC-7901, AGS, BGC-823, MGC-803) than in the gastric epithelium cell line (GES-1) (Fig. 1B). Among these cell lines, MKN-45 and SGC-7901 cells exhibited the highest level and were employed in the following experiments. Collectively, these results indicate that NORAD is highly expressed in GC.

NORAD silencing hampered GC cell growth in vitro and in vivo
To evaluate the biological roles of NORAD in the behaviors of GC cells, two shRNAs speci cally targeting NORAD (shNORAD-1 and shNORAD-2) were designed and constructed. They were transfected into MKN-45 and SGC-7901 cells, respectively. qRT-PCR assay showed that shNORAD-1 and shNORAD-2 could signi cantly reduce the expression levels of NORAD in MKN-45 and SGC-7901 cells ( Fig. 2A, B), which indicated a satisfactory interfering e ciency for further loss-function experiments.
Sustained proliferative signaling is one of the hallmarks of cancer. The role of NORAD silencing on cell growth in GC were rstly investigated. The results of CCK-8 demonstrated that cell proliferation was obviously suppressed in shNORAD group compared with shControl group (Fig. 2C, D). We then evaluated the effect of NORAD on GC growth in mouse models. As shown in Fig. 2E, F, the size and weight of tumors in shNORAD silencing group were signi cantly lower than those in shControl group at 18 days after inoculation.
NORAD silencing leads to cell cycle arrest but did not affect cell migration and invasion in GC  Fig. 3A, B).
Activating invasion and metastasis is another malignant characteristic of cancer cells. The ability of cell invasion and migration were assessed by Transwell assay. As shown in Fig. S1A, transfection with shNORAD did not affect GC cell invasion in contrast with transfection with shControl. The changes of cell migratory ability had the same trend with cell invasion (Fig. S1B). These results implicated that NORAD silencing arrested cell cycle, but not changed cell invasion or migration in GC.

NORAD could bind to miR-204-5p
Cytoplasmic lncRNAs are more often involved in post-transcriptional regulation, such as playing the role of miRNA sponges. In the present study, we determined the cytoplasmic localization of NORAD in GC cells (Fig. 4A, B). As NORAD was mainly colocalized in cytoplasm, we further analyzed whether it could post-transcriptionally regulate gene expression by acting as a ceRNA. Firstly, miR-204-5p was predicted as a target of NORAD and their putative binding sites were obtained (Fig. 4C). Moreover, to determine the interaction between NORAD and miR-204-5p, a dual-luciferase reporter gene assay was performed in MKN-45 and SGC-7901 cells. The results showed that overexpression of miR-204-5p signi cantly reduced the luciferase activity of wild-type NORAD (NORAD WT) vector but did not change the luciferase activity of wild-type NORAD (NORAD WT) vector indicating the interaction between NORAD and miR-204-5p. RIP assay further con rmed that NORAD can bind to miR-204-5p, as higher levels of NORAD and miR-204-5p in the Ago2 antibody group than in the IgG control group (Fig. 4F, G). Subsequently, qRT-qPCR assay showed that NORAD silencing increased miR-204-5p expression levels (Fig. 4H, I). miR-204-5p expression was found to be lower in GC tissues (Fig. 4J). Furthermore, a negative correlation between NORAD and miR-204-5p expression in GC tissues was determined by Pearson correlation analysis (Fig. 4K). In all, our data suggested that NORAD could directly bind to miR-204-5p.
KMT2D is a target gene of miR-204-5p in GC Based on bioinformatic prediction, miR-204-5p could target KMT2D (Fig. 5A). Their interaction was then con rmed by a dual-luciferase reporter gene assay. The results showed that the luciferase activity of MKN-45 and SGC-7901 cells with KMT2D WT vector decreased upon overexpression of miR-204-5p, whereas no signi cant change was observed in MKN-45 and SGC-7901 cells with KMT2D MUT vector (Fig.5B, C). Moreover, overexpression of miR-204-5p dramatically reduced KMT2D mRNA and protein expression in MKN-45 and SGC-7901 cells (Fig. 5D, E). We also con rmed a statistical negative correlation between KMT2D mRNA and protein expression in GC tissues (r=-0.3674, P<0.001; Fig. 5F). Further qRT-PCR assay and Western blot uncovered that the mRNA and protein levels of KMT2D were suppressed in MKN-45 and SGC-7901 cells with NORAD knockdown (Fig. 5G, H). Finally, Pearson correlation analysis validated a positive correlation between NORAD and KMT2D mRNA expression in the same sample cohort (Fig. 5I). Taken together, NORAD might function as a ceRNA against miR-204-5p to regulate KMT2D.
The inhibitory effects of NORAD silencing on GC cell growth were mediated by miR-204-5p/KMT2D axis Based on the above assays, we speculated that the NORAD/miR-204-5p/KMT2D axis played an important role in GC growth, therefore rescue assays were then performed. As shown in Fig. 6A-D, miR-204-5p antangomir reversed the anti-proliferation effects on MKN-45 and SGC-7901 cells induced by NORAD silencing. In addition, NORAD silencing resulted in an increase of G2/M phase arrest while miR-204-5p antangomir rescued the increase. However, such rescuing effects could be partially abolished by the co-transfection of siKMT2D. Based on the above results, we highly proved that NORAD/miR-204-5p/KMT2D axis exerts potential regulatory effects on GC.
NORAD/miR-204-5p/KMT2D axis regulated PTEN/PI3K/AKT signaling pathway in GC Increasing information has suggested that PTEN/PI3K/AKT signaling pathway is a biological participator in tumor progression. In the present study, we investigated whether NORAD led to the aberrant activation of PTEN/PI3K/AKT signaling pathway in GC cells. We measured the protein levels of PTEN/PI3K/AKT pathway factors and found that NORAD silencing signi cantly increased the expression of PTEN, but reduced the expression of PI3K and phosphorylated AKT (pAKT), indicating the potential regulatory effect of NORAD on PTEN/PI3K/AKT signaling pathway. Such regulatory effect was also mediated by miR-204-5p/KMT2D axis (Fig. 7).

Discussion
GC results from a multistep process through the accumulation of numerous genetic and epigenetic alterations in oncogenes and tumor suppressor genes (31,32). LncRNAs were once considered to be simply cloning artifacts or transcriptional noise. (33); however, in recent years, multiple studies have highlighted the potential roles of lncRNAs in GC pathogenesis (34). The current study delineated that NORAD expression was elevated in GC tissues and cell lines, and silencing of NORAD suppressed the GC cells growth via induction of cell cycle arrestment, suggesting the potential role of this lncRNA in GC progression.
Our study found that NORAD post-transcriptionally regulated the progression of GC through the ceRNA mechanism. The ceRNA network hypothesis state that lncRNA can absorb miRNAs by binding to them and subsequently exhibiting a miRNA sponge function, nally constructing a framework to systematically functionalize miRNA-response elements (MRE)-harboring noncoding RNAs and integrate them with the protein-coding RNA dimension (21,22). As reported previously, long intergenic nonprotein coding RNA 00184 (LINC00184) encourages proliferation and invasion of GC cells by targeting miR-145/ANGPT2 pathway (35). Chen et al. reported that lncRNA HCP5 inhibits GC progression via functioning as a ceRNA for miR-106b-5p to affect p21 expression(36). In line with these reports, our study found that NORAD interacted with miR-204-5p, which has been also reported in Parkinson's disease (37). The present data showed that KMT2D was validated as a downstream target of miR-204-5p. In our previous study, we found that KMT2D was overexpressed in GC tissues(38). Moreover, we showed that depletion of KMT2D suppressed cell proliferation in vitro and in vivo. In present study, we found that KMT2D had a negative correlation with miR-204-5p but a positive correlation with NORAD in GC tissues. Based on the luciferase reporter assay, we eventually veri ed a direct interaction between KMT2D and miR-204-5p in GC cells. Tus, we hypothesized that NORAD sponged miR-204-5p to regulate KMT2D in GC cells.
Phosphoinositide 3-kinases (PI3Ks), consisted in PI3K/AKT signaling pathway, a family of signaling enzymes which regulate diverse cellular processes including apoptosis, metabolism, cell proliferation and cell growth (39). The tumor suppressor PTEN, decreased or absent in many tumors, can block the biological processes to inhibit the development of tumors through inactivating PI3K/AKT signaling pathway (40). It has also been fully-studied that PTEN/PI3K/AKT signaling pathway is able to confer tumorigenic properties GC. In our previous study, KMT2D depletion triggered the changes in the PTEN/PI3K/AKT pathway(38). Another study reported by Lv et al. found that KMT2D sustained carcinogenesis by activating PI3K/AKT pathway (41). In the present study, NORAD, which mediated the expression of KMT2D by targeting miR-204-5p, was veri ed as a positive regulator of cell proliferation and cell cycle via PTEN/PI3K/AKT pathway in GC cells. Most importantly, rescue experiments further con rmed that NORAD regulated the malignant features by targeting miR-204-5p/KMT2D axis in GC cells. These data unveiled a novel role for NORAD as a therapeutic target for GC.

Conclusions
Our study identi ed NORAD as a promising oncogene in GC. NORAD contributed to GC growth through modulation of KMT2D expression via acting as a ceRNA for miR-204-5p. These ndings enhance our knowledge of the role of NORAD and reveal new therapeutic strategies for GC.

Acknowledgements
Not applicable.
Authors' contributions RL, LL and YZ designed and supervised the study. ZD and TP performed the experiments and acquired result data. GC, YC and YT helped to perform the partial experiments. YW helped to review the statistical analysis. ZD drafted the manuscript. ZD and TP critically revised the manuscript. All authors read and approved the nal version of the manuscript.

Funding
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Dongguan People's Hospital, Southern Medical University. Signed written informed consents were obtained from the patients.

Consent for publication
Not applicable.

Supplementary Files
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