LINC00922 acts as a novel oncogene in gastric cancer

Background Long non-coding RNAs (lncRNAs) have been discovered to participate in various cancer developments. However, the biological function of lncRNAs associated with gastric cancer (GC) has not been fully elucidated. Methods Quantitative RT-PCR (qRT-PCR) assay was performed to measure lncRNAs, microRNAs (miRNAs) and message RNA (mRNA) expression. Cell Counter Kit-8 (CCK-8), clone formation, wound healing, and transwell assays were performed to investigate cell proliferation, migration, invasion, and apoptosis. Fluorescence in situ hybridization (FISH) assay was used to analyze LINC00922 in either the cytoplasm or nucleus. The potential binding among lncRNA, miRNA, and mRNA was evidenced by bioinformatics, luciferase reporter assay. Mouse-xenograft experiments were used to explore the tumorigenesis in vivo. Results LINC00922 was upregulated in GC, and high LINC00922 expression was associated with poor prognosis. Inhibition of LINC00922 suppressed GC cell proliferation, migration, invasion, and activated cell apoptosis in vitro and inhibited tumorigenesis in vivo. Besides, LINC00922 was markedly located in the cytoplasm. The mechanistic analysis demonstrated that LINC00922 acted as a sponge of miR-204-5p, thereby inhibiting the expression of the target gene-High Mobility Group AT-hook 2 (HMGA2). Conclusion LINC00922 accelerated the progression of GC by miR-204-5p/HMGA2 axis. These findings support LINC00922 may be a promising option for the diagnosis and therapy of GC. Supplementary Information The online version contains supplementary material available at 10.1186/s12957-022-02569-3.


Introduction
Gastric cancer (GC) is the third leading cause of cancer death worldwide [1]. Although advances in chemotherapy, radiotherapy and targeted therapy have partly improved clinical outcomes, the prognosis of advanced GC remains poor [2,3]. Early GC has no specific symptoms, leading to late diagnosis and limited clinical treatment [4]. Hence, identifying molecules of GC is vital for the development of diagnostic markers and new effective treatment methods in GC.
Long non-coding RNAs (lncRNAs) are a kind of functional transcript with a length of more than 200 nucleotides [5]. The disorder of lncRNAs expression is deeply implicated in the occurrence and development of numerous human tumor diseases including GC [6][7][8][9][10][11][12]. For instance, LncRNA SNHG7 is considered a vital oncogene in GC [8,9]. Besides, high expression of LINC00483 promotes cell viability, migration and invasion [12,13]. Therefore, lncRNAs are potential molecular markers and therapeutic targets for tumor diagnosis and prognosis [14]. Unfortunately, the roles of a large number of lncRNAs associated with GC have not been elucidated. Although LINC00922 has been proven to modulate osteosarcoma [15], lung cancer [16], breast cancer [17], and its biological function in GC remain unclear.
Here, we purposed to shed light on the molecular mechanism of LINC00922 in GC. We identified LINC00922 contributed to the progression of GC by acting as a ceRNA of miR-204-5p, thereby inhibiting the level of target gene-High Mobility Group AT-hook 2 (HMGA2).

Tissues collection
Forty pairs of GC tissues and the corresponding nontumor tissues were collected from The First Affiliated Hospital of Soochow University between April 2018 and September 2018. The characteristics of participants were listed in Supplementary Table 1. The consent agreement was signed and approved by the Ethics Committee of The First Affiliated Hospital of Soochow University.

Quantitative RT-PCR (qRT-PCR) assay
Total RNA was isolated using Trizol reagent (Invitrogen, USA) and evaluated by NanoDrop (Thermo, USA). TaqMan miRNA probes and SYBR Green dye (Ambion, USA) were used to quantify miRNA and mRNA. qRT-PCR was performed using SYBR Premix Ex TaqII (Takara, Japan). Fold-changes were calculated by 2 −ΔΔCT method with U6 and GAPDH as internal reference. Primers were listed in Supplemental Table 3.

Clone formation assay
Cells (5 × 10 3 ) were seeded into 6-well plates for 2 weeks. Then the cells were fixed in methanol (Sigma, USA) for 15 min and stained with crystal violet (Sigma, USA) for 20 min. Cell colonies were observed under a light microscope (Olympus, Japan).

Wound healing assay
In brief, cells (5 × 10 5 ) were seeded into 6-well plates. When the cells have grown and fused, a sterile tube was used to scratch the cells. 0 h and 24 h after scratches, the width of the scratch was calculated by taking photos with a light microscope (Olympus, Japan).

Transwell assay
For cell migration, cells (5 × 10 4 ) were seeded into the upper transwell chamber with serum-free medium (Invitrogen, USA). For cell invasion, cells were seeded into the upper transwell chamber and pre-treated with Matrigel (BD biosciences, USA). The culture medium with 20% FBS was added into the lower transwell chamber. After 24 h, the migration cells were fixed in methanol (Sigma, USA) for 15 min and crystal violet (Sigma, USA) for 20 min. The number of cell migration and invasion was observed under a light microscope (Olympus, Japan).

Flow cytometry assay
Cells were harvested and resuspended in Binding Buffer. Then, 5 μL Annexin V-FITC and 10 μL PI (Beyotime, China) were added to 500 μL cells for 10 min. The incubated cells were protected from light. The apoptotic rate was detected immediately by a flow cytometer (BD Biosciences, USA).

Fluorescence in situ hybridization (FISH) assay
The probe signal was detected using FISH kit (Gene-Script, China). In brief, cells were fixed in 4% paraformaldehyde for 15 min and hybridized with LINC00922 probe for 30 min. Then, the nuclei were stained with DAPI. The images were observed by a fluorescence microscope (Nikon, Japan). The sequence for LINC00922 probe was 5′-CCT CCA CCA CCT CCT CTT CTG-3′.

RNA immunoprecipitation (RIP) assay
RIP analysis was performed using RIP kit (Millipore, USA). In short, the collected cells were centrifuged at 2500×g for 15 min to pellet the nuclei. The nuclear pellet was then resuspended in RIP buffer for 30 min. The cell extracts were incubated with protein A/G sepharose beads coupled with antibodies against Ago2 for 2 h. Total RNA from whole cell lysates was analyzed by qRT-PCR.

Western blot assay
Total protein was isolated with RIPA and quantified by BCA assay kit (Beyotime, China). The same amount of protein was placed by SDS-PAGE, and then shifted it to the PVDF membrane. Then, the membrane was incubated with the primary antibody (anti-HMGA2, Ab207301,1:1000, Abcam, USA; anti-GAPDH, ab9485, 1:5000, Abcam, USA) at 4 °C for 12 h, and the secondary antibody for 1 h. Finally, ECL kit (ThermoFisher, USA) was used to visualize the membrane.

Tumor xenograft model
Animal care and euthanasia were carried out with the approval by the Ethics Committee of The First Affiliated Hospital of Soochow University. 4-6-week-old nude mice were divided into two groups: sh-NC group (injection of HGC-27 cells with sh-NC), sh-LINC00922 group (injection of HGC-27 cells with sh-NC). In brief, 200 μL HGC-27 cell suspension (1 × 10 7 cells) was subcutaneously injected into the mice. After four weeks, mice were sacrificed with 100 mg/kg sodium pentobarbital. The gastric tumors were dissected, photographed, and weighted.

Hematoxylin and eosin (HE) staining
The gastric tumors were fixed in 4% paraformaldehyde (Sigma, USA) 48 h, and embedded in paraffin. Thereafter, tissues were sliced and the sections were dyed with hematoxylin for 2 min and eosin for 1 min. Images were viewed by a light microscope (Nikon, Japan).

Immunohistochemistry (IHC) staining
The sections of gastric tumors were incubated with anti-Ki67 (ab16667, 1:200, Abcam, USA) at 4 °C for 12 h and secondary antibody at room temperature for 30 min. At last, the sections were visualized with DAB (Abcam, USA) for 1 min. Images were viewed by a light microscope (Nikon, Japan).

TUNEL staining
TUNEL staining was performed using TUNEL detection kit (Beyotime, China). The sections of gastric tumors were incubated with proteinase K for 15 min and 3% H2O2 for 20 min. Then incubated with 50 μl Streptavidin-HRP for 30 min and immersed by DAB. Images were viewed by a light microscope (Nikon, Japan).

Statistical analysis
All experiments were independently repeated three times. The data were presented as the mean ± standard deviation (SD). SPSS (18.0 version) and GraphPad (6.0 version) were used to determine each group statistic data. Moreover, Student's t test and one-way ANOVA were used to compare two or multiple groups.

LINC00922 is up-regulated in GC tissues and cell lines
To identify the level of LINC00922 in GC, qRT-PCR assay was firstly employed in GC samples. The abundance of LINC00922 in GC tissues was higher (mean ratio of 3.59-fold) than adjacent tissues (Fig. 1A). Besides, LINC00922 was associated with poor prognosis and survival time in patients with GC (Fig. 1B). Consistent with clinical samples, LINC00922 level was markedly increased in MKN-45, HGC-27, AGS, NCI-N87 compared with GES-1 (Fig. 1C). As LINC00922 expression in MKN-45, HGC-27 had a higher ratio (2.04-fold and 2.27-fold, respectively), MKN-45 and HGC-27 were chosen for the next experiments (Fig. 1C). Above all, LINC00922 expression is increased in GC, which indicates that upregulation of LINC00922 may exert a function in GC progression.

Inhibition of LINC00922 suppresses GC progression in vitro
To verify the exact roles of LINC00922 in gastric progression, sh-LINC00922 sequences were transfected to knock down LINC00922 in MKN-45 and HGC-27. The transfection effect of sh-LINC00922 was showed in Fig. 2A. As sh-LINC00922-1 group had a lower ratio of LINC00922 (0.24-fold and 0.35-fold, respectively) in MKN-45, HGC-27, sh-LINC00922-1 was selected for the further experiment ( Fig. 2A). Inhibition of   (Fig. 2B, C). Consistently, wounding healing and transwell assays clarified that inhibition of LINC00922 reduced cell migration and invasion (Fig. 2D-E). Furthermore, flow cytometry assay displayed that cell apoptotic rate was markedly elevated by knockdown of LINC00922 (Fig. 2F). These results demonstrate that suppression of LINC00922 reduces cell proliferation, migration, invasion, and activates cell apoptosis in GC.

MiR-204-5p targets HMGA2 in GC
To find the putative targets of miR-204-5p, we performed three online databases (Targetscan 5.0, miRDB, and miR-Walk 2.0) to predict common targets (Fig. 5A). Besides, miRDB screening conditions were energy ≤ − 25, and miRDB screening conditions were Targetscore ≥ 50. After screening, we finally found 11 intersecting genes of miR-204-5p (Fig. 5A). Additionally, we checked the expression of 11 intersecting target genes in GC cell lines. The result showed that HMGA2 expression had a higher ratio in MKN-45, HGC-27 (4.52-fold and 4.76-fold, respectively) (Fig. 5B). Therefore, HMGA2 was chosen as a candidate gene for further study. We further constructed a luciferase reporter gene that mutated the putative miR-204-5p binding site in the HMGA2 sequence (Fig. 5C). We found that miR-204-5p was specifically bound to HMGA2 in GC (Fig. 5D). Besides, miR-204-5p negatively regulated HMGA2 level as indicated by qRT-PCR and western blot assays (Fig. 5E, F). We also found HMGA2 level was increased in the GC tissues (Fig. 5G,  H). In short, these results demonstrate that HMGA2 is a target of miR-204-5p.

Inhibition of LINC00922 suppresses GC progression in vivo
To further evaluate the therapeutic potential of LINC00922 in vivo, we implemented the xenograft model in mice. As similar to the results of the in vitro study, knockdown of LINC00922 repressed the tumorigenicity of HGC-27 cells, and repressed the tumor growth and weight (Fig. 7A-C). In addition, HE staining suggested knockdown of LINC00922 inhibited tumor infiltration (Fig. 7D). IHC and TUNEL staining showed knockdown of LINC00922 suppressed Fig. 3 LINC00922 targets miR-204-5p in GC. A FISH analysis was performed to analyze LINC00922 in either the cytoplasm or nucleus. B The online databases RegRNA 2.0 and LncBase v.2 were used to predict possible miRNAs. C The expression of miRNAs was measured by qRT-PCR assay in MKN-45 and HGC-27, GES-1. *P < 0.05, **P < 0.01 vs. GES-1 group. D The potential binding sites of miR-204-5p with 3′UTR of LINC00922. E Luciferase reporter assay and F RNA pull-down assays were used to confirm the interaction between LINC00922 and miR-204-5p. **P < 0.01 vs. NC mimic group, ## P < 0.01 vs. NC inhibitor group. G miR-204-5p level in 40 pairs of GC tissues and adjacent normal tissues was verified by qRT-PCR assay. **P < 0.01 vs. Normal group. H Kaplan-Meier analysis assessed the survival curves in GC patients based on miR-204-5p expression the expression of the proliferating protein Ki67, and led to cell apoptosis (Fig. 7E). Besides, knockdown of LINC00922 promoted miR-204-5p level, and decreased HMGA2 level (Fig. 7F, G). In conclusion, these findings unravel that LINC00922 accelerated the deterioration of GC.
Emerging studies have declared that lncRNAs participated in the pathogenesis of GC [29]. Here, we identified the upregulated LINC00922 in GC. Aberrant expression of LINC00922 has been revealed to be associated with cancers. For instance, previous studies suggested that upregulation of LINC00922 promoted lung cancer cell proliferation [16]. Besides, abnormal expression of LINC00922 promoted epithelial-mesenchymal transformation and the occurrence of breast cancer by Wnt pathway [17]. Additionally, Gu Z et al. suggested that LINC00922 regulated the progression of doxorubicin-resistant osteosarcoma [15]. Here, the functional role of LINC00922 was investigated in GC cells and xenograft model. Our data revealed that inhibition of suppressed GC progression in vitro and in vivo, indicating LINC00922 could be an oncogene in GC.
Intracellular localization of lncRNA is the basis of its function [30]. LncRNAs located in the nucleus participate in epigenetic modification or transcriptional regulation [31,32]. However, lncRNAs located in cytoplasm are involved in post-transcriptional regulation mainly by affecting the stability of mRNA molecules. Our data revealed that LINC00922 was markedly located in the cytoplasm, indicating LINC00922 might serve as a ceRNA in GC. Besides, we identified miR-204-5p was a target of LINC00922 in GC. In the present study, we found miR-204-5p negatively correlated with gastric cell growth. These results were consistent with Chen X [24], Liang Y [33]. Liang Y et al. suggested that miR-204-5p contributed to the activation of GC through inhibiting organic cation transporter 1 (OCT1) expression level [33]. In addition, miR-204-5p has been proved to have  5). B Tumor volume and C tumor weight of the xenograft were measured. D HE staining was conducted to observe tumor infiltration. E IHC and TUNEL staining were used to assess Ki67 expression and cell apoptosis. F qRT-PCR was used to assess miR-204-5p and HMGA2 mRNA levels in GC tissues. G Western blot assays were used to assess HMGA2 protein level in GC tissues. *P < 0.05, **P < 0.01 vs. sh-NC group important biological functions in many diseases, such as osteoarthritis [34], prostate cancer [35], and cervical cancer [36].
It is accepted that miRNAs bind to the 3′-UTR complementary nucleotide sequence of target gene, thereby inhibiting the translation or degradation of mRNA and realizing the post-transcriptional regulation of genes [37]. In the present study, we found that miR-204-5p negatively regulated the expressions of HMGA2 by directly targeting its 3′-UTR. HMGA2 is considered as an oncogene and is involved in many carcinogenic signaling networks as p53, and Wnt/β-catenin signaling pathways [38][39][40]. Here, HMGA2 level was upregulated in GC. Moreover, the rescue experiment found that down-regulation of HMGA2 partially reversed the oncogenic role caused by miR-204-5p inhibitor.
Liang T et al. revealed that miR-204 reversed the promotive effects of LINC00922 on the cellular behaviors of lung cancer, and provided a link between LINC00922 and miR-204 in lung cancer [16]. However, the roles of LINC00922/miR-204-5p axis on GC progression remain unknown. In the present study, the online databases RegRNA 2.0 and LncBase v.2 showed that miR-204-5p might be bound to LINC00922, and miR-204-5p was downregulated in GC. The Functional experiments indicated that LINC00922 promoted GC progression via targeting miR-204-5p. Given the feature that lncRNA binds to multiple miRNAs to participate in cancers. Therefore, it is worthwhile to add considerable effort to reveal additional miRNAs that are potentially bound to LINC00922 during GC progression.

Conclusions
We firstly identified the participation of LINC00922 in GC and in-depth study the mechanism of LINC00922. LINC00922 functions as GC-promoting ceRNA via targeting miR-204-5p/HMGA2 axis. The LINC00922/miR-204-5p/HMGA2 axis might provide novel insight for GC-targeted therapy.