Long noncoding RNA NEAT1 promotes tumorigenesis in H. pylori gastric cancer by sponging miR‐30a to regulate COX‐2/BCL9 pathway

Helicobacter pylori (H. pylori) is a carcinogenic factor for gastric cancer. Our previous study demonstrated that H. pylori decreased the expression of micro‐RNA (miRNA)‐30a to promote the tumorigenesis of gastric cancer. However, the upstream regulatory molecules of miR‐30a are not well elucidated. In this study, we found the long non‑coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) may sponge miR‐30a to regulate COX‐2/BCL9 pathway.


| BACKG ROU N D
Gastric cancer is one of the most frequently diagnosed digestive cancers, accounting for over million new cases globally in 2018.
Incidence rates of gastric cancer are markedly elevated in Asian countries, especially in China. 1 Compelling evidence supports that Helicobacter pylori (H. pylori) is a first class carcinogen leading to gastric adenocarcinoma 2 and is directly linked to the development of gastric cancer. 3 Early diagnosis of gastric cancer for radical cure largely improved the overall survival of patients 4 ; however, since early-stage gastric cancer is asymptomatic, or characterized by nonspecific symptoms, most gastric cancer patients are diagnosed at an advanced stage. 5 Thus, there is an urgent need to identify earlystage markers of gastric cancer.
Non-protein-coding RNAs (ncRNAs) were found to participate in genome encoded-transcripts 6 and almost all cellular processes. 7 ncRNAs are classified as long noncoding RNAs (lncRNAs) and small ncRNAs, such as microRNAs (miRNAs). 8 The interaction of lncRNAs, miRNAs, and mRNAs is critically implicated in various diseases, including cancer. 9 Multiple ncRNAs act as oncogenes or tumor suppressor genes during carcinogenesis. 9 Therefore, ncRNAs represent effective diagnostic biomarkers of cancer.
miR-30a has been identified to have tumor suppressor properties, and the double-stranded precursor generates two singlestranded miRNAs, including miR-30a-3p and miR-30a-5p, which regulate the proliferation and migration, respectively, of H. pyloriinfected gastric cancer cells. Specifically, miR-30a-3p was found to target the 3′UTR of COX-2 mRNA and regulate nuclear translocation of β-catenin, and miR-30a-5p was shown to target the 3'UTR of BCL9 mRNA to affect TCF/LEF promoter activity and regulate downstream gene expression of β-catenin. 10 LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) was reported to influence various diseases. NEAT1 is up-regulated in the Huntington's disease brain tissues to alleviate neuronal injury, 11 but repressed expression in the early stage of Alzheimer's disease. 12 NEAT1 also be induced by immune stimuli. 13,14 All these reports suggest that NEAT1 is an effector during stress and disease development. 15 Additionally, NEAT1 is aberrantly expressed in different types of cancer. High levels of NEAT1 may act as a biomarker of prostate cancer patients, 16 and NEAT1 promotes cervical cancer cell invasion and the progression of sarcoma metastasis. 17 NEAT1 is downregulated in acute promyelocytic leukemia. 18 We applied bioinformatics analysis, and it was predicted that NEAT1 sponges miR-30a. Hence, our study aimed at evaluating the function of NEAT1 in H. pylori gastric cancer, which might be valuable for the diagnosis and treatment of gastric cancer.  19 H. pylori was suspended in phosphate-buffered saline (PBS), and the suspension mixture was kept in an ice bath, then pulse sonicated for 5 min at 50% capacity. The suspension was centrifuged at 10,000 rpm for 15 min to remove bacterial debris, and the collected supernatant was sterilized by passing through a 0.22 μm cellulose acetate filter. Protein concentration was determined by targeted miR-30a (miR-30a-3p and miR-30a-5p). Because miR-30a (miR-30a-3p and miR-30a-5p) negatively regulates the expression of downstream COX-2 and BCL9, NEAT1 was identified to upregulate indirectly the expression of COX-2 and BCL9. IHC showed that the expression of COX-2 and BCL9 was increased in H. pylori gastric cancer tissues.

| Cell culture
Human MKN45 and SGC-7901 gastric cancer cell lines were commercially obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% streptomycin and penicillin, at 37℃, 5% CO 2 , and saturated humidity. MKN45 and SGC-7901 cells were treated with H. pylori filtrate at a concentration of 500 μg/ml.

| Cell transfection
Cell transfection was performed using the HilyMax kit (DOJINDO, Japan) according to the manufacturer's instructions. NEAT1-targeting siRNA (siNEAT1), miR-30a-3p mimics or inhibitor, miR-30a-5p mimics or inhibitor, were commercially provided by Genomeditech (Shanghai, China). Empty pcDNA3.1(+) vector, mimics control, and non-targeting siRNAs were used as negative controls. The miRNA mimics or inhibitors, and the siNEAT1 were introduced into cells at a final concentration of 50 nM. The empty pcDNA3.1(+) vector and p-NEAT1 vector were introduced into cells at a final concentration of 1 μg/ml. The sequences of siNEAT1 are listed in Table S6; mimics or inhibitor sequences of miR-30a-3p and miR-30a-5p are listed in Table S7.

| Dual-luciferase reporter assay
Dual-luciferase assays were used to verify the predicted relationships between NEAT1 and miR-30a-3p or miR-30a-5p. The constructed luciferase reporters were co-transfected into MKN45 and SGC-7901 cells with Renilla reporters. For luciferase assays, cells in 24-well plates were co-transfected with miR-30a (miR-30a-3p or miR-30a-5p) mimics or inhibitor and 200 ng/well luciferase reporter constructs, and SV-Renilla luciferase plasmid was applied at 5 ng/ well as the internal control. After 24 h transfection, the luciferase activity was detected using the Dual Luciferase Reporter Assay kit (Promega). Firefly luciferase activities were normalized to Renilla luciferase activity.

| Reverse transcription and quantitative realtime PCR
Total RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Vazyme, Nanjing, China) and a miRNA qPCR Quantitation Kit (Tiangen) to synthesize cDNA. Quantification of NEAT1 expression was normalized to GAPDH expression, and quantification of miR-30a-3p and miR-30a-5p expression was normalized to U6 expression using the 2 −ΔΔCt method. RT-qPCR was performed using the following primers:

| Fluorescence in situ hybridization
The fluorescence in situ hybridization (FISH) assay was used to analyze the location and expression of NEAT1. were labeled with Spectrum-Red.

| Immunohistochemistry
Immunohistochemistry (IHC) was carried out to analyze the loca-

| Expression of NEAT1 and miR-30a in gastric cancer tissues
Our previous study proved that miR-30a affected the H. pyloriinduced gastric cancer, and bioinformatics analysis by RNAhybrid and starBase v.2.0 revealed that lncRNA NEAT1 could effectively bind to the two strands of miR-30a, including miR-30a-3p and miR-30a-5p.
Hence, we detected the expressions of NEAT1, miR-30a-3p, and miR-30a-5p in gastric cancer tissues to determine whether these genes

MKN45 and SGC-7901 cells, the dual-luciferase assay validated that
miR-30a-3p mimics and miR-30a-5p mimics suppressed the expression of a reporter plasmid carrying the wild-type gene sequence, but not the mutant sequence, of NEAT1 in MKN45 and SGC-7901 cells.
In addition, either the miR-30a-3p inhibitor or the miR-30a-5p inhibitor slightly increased the expression of a reporter plasmid carrying the wild-type gene sequence of NEAT1, though the difference was not statistically significant ( Figure 3C). Overexpression of NEAT1 obviously decreased the expression of miR-30a-3p and miR-30a-5p in MKN45 cells and SGC-7901cells, whereas silencing NEAT1 significantly increased miR-30a-3p or miR-30a-5p expression in MKN45 and SGC-7901 cells ( Figure 3D,E).

| Immunohistochemical detection of COX-2/ BCL9 protein in gastric cancer tissues
Using the gastric cancer tissues with or without H. pylori infection, as well as the adjacent tumor tissues, we evaluated the expression of COX-2 and BCL9 protein by IHC staining. Positive COX-2/BCL9 immunostaining was mainly localized in the cytoplasm of gastric cancer tissue cells. According to the positive expression area, compared with non-H. pylori infection gastric cancer tissue, expression of both COX-2 and BCL9 proteins was increased in the H. pylori-infected gastric cancer tissues ( Figure 6A,B). All of the above findings suggest that NEAT1 may accelerate tumorigenesis in H. pylori gastric cancer, by sponging miR-30a (miR-30a-3p, miR-30a-5p) to regulate COX-2/ BCL9 pathway ( Figure 6C).  lncRNAs participate in transcriptional regulation processes, and miRNAs are an important link between lncRNAs and mRNA. ln-cRNAs compete with miRNAs, which is known as "miRNA sponging", 27 miRNAs negatively regulate mRNA expression by targeting mRNA for directing translational inhibition, 28 and each miRNA is able to target multiple genes. This reciprocal regulatory mechanism is involved in many biological processes, including cancer development. [29][30][31] Previous study showed that miR-30a inhibited H. pylori-induced gastric cancer. 10  ing miR-342-3p in macrophages. 37 Silencing NEAT1 reduced the expression of COX-2 in epilepsy cell by sponging miR-129-5p 38 ; NEAT1 was also reported to regulate expression of COX2 via its role in competing with endogenous RNAs. 39 Our previous study revealed that miR-30a regulates the COX-2/BCL9 pathway, and this current study verified that NEAT1 indirectly regulates the protein expression of COX-2 and BCL9 in a miR-30a-dependent manner.
Additionally, the IHC staining positive expression area showed that the protein expression of COX-2 and BCL9 in H. pylori-infected gastric cancer tissues was increased compared with non-H. pyloriinfected gastric cancer tissues. NEAT1 regulates various biological processes, and we found that it promotes tumorigenesis in H. pylori gastric cancer by sponging miR-30a to regulate COX-2/BCL9 pathway.

| CON CLUS ION
Our results indicate that lncRNA NEAT1 is elevated in gastric cancer tissues, especially in H. pylori-infected gastric cancer tissues.
Aberrant expression of NEAT1 in H. pylori-infected gastric cancer cells affects the proliferation, migration, and invasion of gastric cancer cells. The tumor-promoting activity of NEAT1 is achieved mainly by sponging miR-30a and subsequently upregulating the expression of COX-2 and BCL9.

ACK N OWLED G EM ENTS
Not applicable.

CO N FLI C T O F I NTE R E S T
All other authors declare no conflicts of interest.

AUTH O R S' CO NTR I B UTI O N S
QL, QJ, XL, CH, and HJG supervised the project. XWR and NNL performed the experiments. RJ, YYF, and ZZZ analyzed the experiments. GH, NLS, RC, and ZYW provided clinical samples. LHZ wrote the manuscript. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All datasets supporting the conclusion for this study are included in the article.