Long intergenic noncoding RNA00265 promotes proliferation of gastric cancer via the microRNA-144-3p/Chromobox 4 axis

ABSTRACT The expression and biological function of long intergenic noncoding RNA00265 (LINC00265) in gastric cancer (GC) have not yet been explored. This study aimed to detect LINC00265 expression in GC tissues and cell lines, investigate its roles in the proliferation of GC cells in vitro, and elucidate the regulatory mechanisms of LINC00265 action. It was found that LINC00265 expression was significantly upregulated in GC tissue samples and cell lines compared with their normal counterparts. Additionally, LINC00265 knockdown could inhibit GC cell proliferation in vitro. Further investigation revealed that LINC00265 acted as a competing endogenous RNA for microRNA-144-3p (miR-144-3p) and inhibition of miR-144-3p markedly counteracted LINC00265 knockdown-meditated suppression on GC cell proliferation. Additionally, Chromobox 4 (CBX4) was upregulated in GC and silencing CBX4 could reduce GC cell proliferation. Then, CBX4 mRNA was demonstrated to be a direct target of miR-144-3p in GC cells and LINC00265/miR-144-3p axis could regulate CBX4 expression. Taken together, LINC00265 may promote GC cell proliferation via the miR-144-3p/CBX4 axis.


Introduction
Gastric cancer is one of the most common malignant cancer in the world and ranks the third most common causes of cancer mortality worldwide [1] . The incidence rate has been dramatically increasing in Eastern Asia during the past decades [2,3]. Because GC exhibits no specific symptoms at early stage and sensitive biomarkers for GC are not available currently, GC patients are always diagnosed at advanced stage, especially in China [4,5]. Even worse, current treatment options for this devastating disease are extremely limited [6]. As result, GC patients at advanced stage have rather poor long-term survival outcome. Therefore, it is urgent to further reveal the molecular mechanisms underlying the occurrence and development of gastric cancer [7], in order to develop novel therapeutic methods.
There are many risk factors contributing to the incidence and development of GC, such as Helicobac ter pylori (H.pylori) infection, dietary habits, tobacco use, and obesity [8]. Helicobacter pylori is a wellknown risk factor for GC and played an important pro-tumor role in GC. The mechanisms underlying the pro-tumor role of in H.pylori are rather complex. For example, gastritis caused by H.pylori infection could change the immune environment to induce carc inogenesis, and H.pylori could also directly act on epithelial cells to induce gene mutation, which aberrantly activates tumor-associated signaling pathways, ultimately resulting GC progression [9,10]. Interestin gly, growing evidence suggests that H.pylori infection could contribute to GC tumorigenesis by regulating lncRNAs. Han et al. suggested that H.pylori infection could upregulate lncRNA SNHG17 in GC cells. Fur thermore, they uncovered that lncRNA SNHG17/ NONO and SNHG17/miR-3909/RING1/Rad51 pathways could accelerate GC tumorigenesis through disturbing the DNA repair system [11]. In addition, a re cent study indicated that H.pylori could promote GC cell growth and migration by upregulating THAP9-AS1 [12].
LncRNAs are a class of non-coding RNAs wi th longer than 200 nucleotides and their dysregulations can promote cancer cell proliferation, metastatic cascades, and chemoradiotherapy res istance via post-transcription regulatory mecha nisms, such as acting as miRNA sponge [13][14][15].
Dysregulation of lncRNA occurs frequently in GC and closely correlates with dise ase progression [16]. For example, lncRNA KCNQ1OT1 was found to be downregulated in GC and it could function as a competing end ogenous RNA for microRNA-9 (miR-9) to regulate LMX1A expression, consequently repressing GC cell proliferation [17]. Besides, a recent study indicated that lncRNA PVT1 was highly expressed in GC tissues and can promote tumor progression by interacting with FoxM1, and PVT1 [18]. Furthermore, Xiao et al. reported that lncRNA TRPM2-AS exerted a pro-tumor effect by sponging miR-612 in GC cells [19]. In addition, there are many other dysregulated lncRNAs involved in GC progression, such as LINC00152, PANDA, MAGI2-AS3, PWRN1 and TUBA4B [20]. With the development and application of high throughput sequence technology, more and more novel lncRNAs dysregulated in cancers are identified. Recently, LIN C00265 was shown to be upregulated in colorectal cancer (CRC) and it could enhance cell viability and glycolysis through negatively regulating miR-216b-5p [21]. Additionally, it has been demonstrated that LINC00265 was highly expressed in bone marrow and serum of acute myeloid leukemia (AML) patients, and it could promote AML cell proliferation and migration by activating PI3K/AKT signaling pathway [22]. However, no information regarding the role of LINC00265 in GC is not available currently.
MicroRNAs (miRNAs) are noncoding RNAs 20-24 nt in length, which have the ability to modulate the stability and translational efficiency of targeted mRNAs [23]. Solid evidence has demonstrated that miRNAs participate in various biological processes, such as cell proliferation, different iation, apoptosis and metabolism [24]. Notably, numerous studies indicated that a multiple of miRNAs are dysregulated in many cancer types, including GC [25]. Particularly, Faheim M. et al revealed that miR-144 was downregulated in GC cells, and miR-144 could inhibit GC cell proliferation and invasion by directly targeting activating enhancer-binding protein 4 (AP4) [26]. Yao. et al. showed that miR-144 could negatively regulate the expression of cyclooxygenase-2 (COX-2) to repress GC cell viability [27]. However, the mechanisms responsible for low miR-144 expression and protumorigenic mechanisms in GC are not completely deciphered currently.
Chromobox family, a subgroup of proteins in the PcG family, has eight members including CBX1-8. These proteins display distinct biological functions in different tissues. Chromobox 4 (CBX4), also known as polycomb 2 (Pc2), is a sp ecial chromobox protein, since it is not only a tr anscriptional repressor but also a SUMO E3 lig ase [28]. Increasing evidence suggested that CB X4 might be an oncogene and could be exploited as a therapeutic target in several cancers, including lung cancer, cervical cancer, breast cancer and osteosarcoma [29][30][31]. For example, CBX4 could promote the proliferation and metastasis of lung cancer cells in vitro through enhancing BMI-1-mediated expression and activation of P53, CDK2, Cyclin E, MMP2, MMP9 and CXC R4 [32]. However, CBX4 was found to play a tu mor suppressor in colorectal carcinoma via recru itment of HDAC3 to the runx2 Promoter [33]. Therefore, whether CBX4 functions as an oncogene or tumor suppressor may depend on the genetic context of cancer. To date, the role of CBX4 in gastric cancer has never been studied.
In this study, we initially evaluated the expression status of LINC00265 in GC tissues and cell lines, and explored its effect on GC cell proliferation and the corresponding mechanisms, so as to find the potential therapeutic targets and prognostic biomarkers. Our data suggest that LINC00265 is overexpressed in GC and exerts oncogenic activities via the miR-144-5p/CBX4 signaling pathway.

Ethics statement
Investigation was conducted in compliance with the principles of the Declaration of Helsinki and according to national and international guidelines. Moreover, this study protocol was approved by the Ethics Committee of The Second Affiliated Hospi tal of Nanchang University. Informed consent has been obtained from all patients.

RNA isolation and reverse-transcription quantitative PCR
The TRIzol® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from tissue samples or cells. The miScript Reverse Transcri ption Kit was purchased from Qiagen GmbH (Hilden, Germany), and then used for the synthesis of cDNA from the total RNA. After reverse transcription, reverse-transcription quantitative PCR (RT-PCR) was performed using the miScript SYBR Green PCR Kit (Qiagen GmbH) to determine miR-144-3p expression. The expression level was determined in relation to U6 small nuclear RNA. To quantitate LINC00265 and CBX4 expression, the synthesis of cDNA was performed using the PrimeScript RT-Reagent Kit (Takara Bio, Kusatsu, Japan), followed by RT-PCR with the SYBR Premix Ex Taq™ Kit (Takara Bio). Expression levels of LINC00265 and CBX4 were normalized to GAPDH. All the data were analyzed by the 2 −ΔΔCq method.

CCK-8 assay and colony formation assay
After 24 h transfection, cells were maintained in 96-well plates in 10% FBS-supplemented DMEM for 0, 24, 48, or 72 h. At every time point, 10 µl of the CCK-8 solution (Dojindo Laboratories, Kuma moto, Japan) was added into each well, and the cells were incubated further at 37°C for 2 h. Optical density was measured at 450 nm wavelength on a Sunrise™ microplate reader (Tecan Gro up, Ltd., Mannedorf, Switzerland). As for colony formation assay, GC cells were gathered and maintained in in 6-well plates after 24 h transfection. Then, the cells were cultured with complete medium at 37°C with 5% CO2 for half a month. On day 15, GC cells were fixed in 4% paraformaldehyde and stained using 0.5% crystal violet. At the last step, the colonies were counted under micr oscope.

Nuclear/cytoplasmic fractionation
Briefly, cells were washed twice with cold PBS, and lysed in cell lysis buffer (20 mmol/L Tris-HCl pH 8, 0.5% NP40, 10 mmol/L NaCl, 3 mmol/L MgCl2 supplemented with protease inhibitors) for 30 min on ice with vigorous pipetting every 5 min. Nuclei were sedimented by centrifugation at 5000 × g for 5 min at 4°C and the supernatant (cytoplasmic fraction) was collected. The nuclear pellet was was hed 3 times in cell lysis buffer to eliminate cytoplasmic traces and the nuclei were lysed in nuclear lysis buffer (50 mmol/L Tris-HCl, 1% SDS) on ice for 20 min followed by brief sonication to ensure nuclei rupture. Nuclear debri was collected by centrifugation at 12,500 × g for 10 min at 4°C and the supernatant (nuclear fraction) was gather ed. Nuclear and cytoplasmic fractions were stored at −80°C prior to analysis by SDS-PAGE and western blotting.

Bioinformatics prediction and luciferase reporter assay
A target prediction tool, starBase 3.0 (http://star base.sysu.edu.cn/), was used to predict potential miRNAs targeted by LINC00265. The potential targeted mRNAs of miR-144-3p were predicted usi ng two miRNA target prediction databases: starBa se 3.0 and TargetScan (http://www.targetscan.org/ ). The CBX4 3 -UTRs containing either the wildtype (wt) binding sequence or the mutant (mut) binding sequence for miR-144-3p were synthesized by GenePharma Co., Ltd., and inserted into the pmirGLO luciferase reporter vector (Promega, Madison, WI, USA). The resultant plasmids are referred to as CBX4-wt and CBX4-mut, respectively. The LINC00265-wt and LINC00265-mut reporter plasmids were produced by similar experi mental procedures. Either agomiR-144-3p or agomir-NC was co-transfected into cells with either a 'wt' or 'mut' reporter plasmid using the Lipofec tamine® 2000 reagent. Luciferase activity was determined 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega, Madi son, WI, USA). Renilla luciferase activity was normalized to that of firefly luciferase.

Western blotting analysis
Total protein was isolated by means of the RIPA buffer (Beyotime Institute of Biotechnology, Shang hai, China). The Bicinchoninic Acid Protein Assay Kit (Beyotime Institute of Biotechnology, Shangh ai, China) was used to determine the protein concentration. The protein samples were resolved by SDS-PAGE on a 10% gel and transferred onto polyvinylidene difluoride membranes, followed by 2 h blocking with 5% fat-free milk diluted in Trisbuffered saline containing 0.1% of Tween 20 (TBST). After incubation with a primary antibody against CBX4 (cat. 18,544-1-AP; dilution 1:500; proteintech) or against GAPDH (cat. 60,004-1-Ig; dilution 1:5000; proteintech), the membranes were washed thrice with TBST, probed with a goat antirabbit immunoglobulin G antibody conjugated with horseradish peroxidase (cat. No. ab205718; dilution 1:5,000; Abcam) (secondary antibody) and, then, treated with the Pierce™ ECL Western Bl otting Substrate (Pierce; Thermo Fisher Scientific, Inc.) for visualization of the protein signals.

Statistical analysis
Data were analyzed using SPSS version 13.0 software (SPSS; Chicago, USA). Data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. The comparisons between two groups were performed by Student's t test; oneway analysis of variance, followed by the Student-Newman-Keuls test, was applied to evaluate the differences among multiple groups. Data with P < 0.05 were considered statistically significant.

Results
Growing evidence has demonstrated that many lncRNAs are dysregulated in GC and play important roles in promoting disease progression [33], suggesting these lncRNAs may be potential diagnostic biomarkers and therapeutic targets for GC. Previous studies reported that LINC00265 was highly expressed in CRC and AML, and performed oncogenic functions [21,22]. However, LINC00265 expression and its role in GC have never been explored yet. In this study, we thus detected LINC 00265 expression in GC, clarified its effect on GC cell proliferation in vitro and clarified the underlying mechanisms, in order to determine whether LINC00265 could be exploited as the potential therapeutic target and prognostic biomarker for GC.

LINC00265 is overexpressed in GC tissue samples and cell lines
To determine the role of LINC00265 in GC, we first examined its expression profile in 32 pairs of GC tissue samples and adjacent normal tissue sam ples using RT-PCR. LINC00265 was found to be overexpressed in the GC tissue samples relative to the adjacent normal gastric tissues (Figure 1a, P < 0.05). Moreover, it was also found that LINC 00265 expression was significantly higher in a panel of GC cell lines (KATO-III, SGC-7901, BGC-823, HGC-27, AGS, NCI-N87, SNU-1, and SNU-16) compared with that in normal gastric cells (GES cells) (Figure 1b, P < 0.05). These resu lts implied that LINC00265 is upregulated in GC.

LINC00265 knockdown inhibits GC cell growth in vitro
Considering the aberrant upregulation of LINC00265 in GC, we next attempted to determine the functions of LINC00265 in GC progression. NCI-N87 and KATO III cells were chosen for subsequent experiments and were transfected with either a small interfering RNA (siRNA) against LINC00265 (si-LINC 00265) or a negative control siRNA (si-NC). LINC0 0265 was successfully knocked down in NCI-N87 and KATO III cells after transfection of si-LINC00265 (Figure 2a, P < 0.05). The Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the influence of si-LINC00265 on GC cell proliferation. The si-LINC00265 transfection obviously reduced the proliferative ability of NCI-N87 and KATO III cells compared with si-NC transfection (Figure 2b, P < 0.05). Then, colony formation assay was conducted to further confirm whether LINC00265 knockdown inhibit GC cell growth in vitro. As expected, the colo ny number of NCI-N87 and KATO III cells was signi ficantly reduced after transfection with si-LINC00265 (Figure 2c, P < 0.05). In general, these findings suggested that LINC00265 knockdown could inhibit GC cell growth in vitro.

LINC00265 promotes GC cell growth by inhibiting miR-144-3p as a competing endogenous RNA (ceRNA)
To investigate the molecular events involved in LINC00265-mediated GC proliferation, a nuclear/ cytoplasmic fractionation assay was first conducted to determine the distribution of LINC00265 in GC cells. The data indicated that LINC00265 was mainly located in the cytoplasm of GC cells (Figure 3a), whi ch suggested that this lncRNA may serve as a ceRNA for some miRNA(s) [34]. Then, starBase 3.0. was employed to predict the candidate miRNAs probably inactivated by LINC00265. The result showed that LINC00265 contained one conserved binding site for miR-144-3p (Figure 3b). Luciferase reporter was performed to further characterize the relation between LINC00265 and miR-144-3p in GC cells. MiR-144-3p agomir (agomiR-144-3p) transfectionmediated upregulation of miR-144-3p (Figure 3c, P < 0.05) noticeably decreased the luciferase activity of the LINC00265-WT plasmid (the plasmid expressing LINC00265 containing the wild-type binding site for miR-144-3p; P < 0.05) in both NCI-N87 and KATO III cells; however, the luciferase activity of LINC00265-MUT (the plasmid expressing LINC 00265 containing a mutant binding site for miR-144-3p) was unaffected when miR-144-3p was overexpressed, as evidenced by the luciferase reporter assay (Figure 3d). These results suggested that miR-144-3p was a target of LINC00265 in GC cells. Next, we detected miR-144-3p expression in the 40 pairs of GC tissue samples and adjacent normal gastric tissues by RT-qPCR. MiR-144-3p was found to be significantly downregulated in the GC tissue samples (Figure 3e, P < 0.05). Furthermore, the expression of miR-144-3p was evaluated in LINC00265-deficient NCI-N87 and KATO III cells. The LINC00265 knockdown remarkably upregulated miR-144-3p in NCI-N87 and KATO III cells (figure 3f, P < 0.05). Strikingly, previous studies suggested that miR-144-3p functioned as a tumor suppressor in GC [35,36]. Consistently, in this study it was found that transfection with agomiR-144-3p appreciably repressed proliferation (Figure 4a&B, P < 0.05) of NCI-N87 and KATO III cells. In general, these results indicated that LINC00265 promoted GC cell proliferation by inhibiting miR-144-3p as a ceRNA.

LINC00265 sponges miR-144-3p to upregulate CBX4 expression in GC cells
We next continued to ask how LINC00265/miR-144-3p axis regulated GC cell growth in vitro. To this end, we first employed starBase 3.0 and TargetScan to predict the potential targets of miR-144-3p. CBX4 was predicted as a potential target gene of miR-144-3p (Figure 5a). To validate this prediction, a luciferase reporter assay was  performed on NCI-N87 and KATO III cells after cotransfection with either agomiR-144-3p or agom ir-NC and either plasmid CBX4-WT (a plasmid expressing luciferase mRNA containing the CBX4 3 -UTR harboring a wild-type binding site for miR-144-3p) or plasmid CBX4-MUT (a plasmid expressing luciferase mRNA containing the CBX4 3 -UTR harboring a mutated binding site for miR-144-3p). The ectopic expression of miR-144-3p significantly reduced the luciferase activity of CBX4-WT in NCI-N87 and KATO III cells (P < 0. 05). By contrast, mutation of the binding site abrogated this phenomenon (Figure 5b). Next, the expression levels of CBX4 in miR-144-3p-over expressing NCI-N87 and KATO III cells were determined to investigate whether miR-144-3p could target CBX4 in GC cells. As expected, the protein levels (Figure 5c, P < 0.05) and mRNA (Figure 5d, P < 0.05) of CBX4 in NCI-N87 and KATO III cells diminished in response to the agomiR-144-3p transfection. Collectively, these res ults identified CBX4 mRNA as a direct target of miR-144-3p in GC cells.

CBX4 is upregulated in GC and exerts a promotive effect on GC cell growth in vitro
To further verify LINC00265 knockdown inhibited GC cell proliferation through regulating miR-144-3p/CBX4 axis, we explored the role of CBX4 in GC. For this purpose, we first examined CBX4 expression in 40 pairs of GS tissue samples and adjacent normal tissue samples, as well as GC cell lines and GES cells. CBX4 was found to be overexpressed in GC tissue samples (RT-qPCR; Figure 6a, P < 0.05) and GC cell lines (Figure 6b, P < 0.05). Next, we assessed the effect of CBX4 on GC cell proliferation by silencing CBX4. As shown in Figure 7 A&B, CBX4 was successfully knocked down in NCI-N87 and KATO III  showed CBX4 exerted a promotive effect on GC cell growth in vitro, further confirming that LINC 00265 knockdown inhibited GC cell proliferation through regulating miR-144-3p/CBX4 axis.

Discussion
Increasing evidence has suggested that many lncRN As were dysregulated in GC and played important roles in promoting tumorigenesis and metastasis [33]. Therefore, these lncRNAs may be potential diagnostic biomarkers and therapeutic targets for GC. In this study, we initially tested whether LINC00265 was dysregulated in GC, clarified its effect on GC cell proliferation in vitro and clarified the underlying mechanisms. Our data showed that LINC00265 expre ssion was significantly upregulated in GC tissue samples and cell lines compared to their normal counterparts. Besides, LINC00265 knockdown could inhibit GC cell proliferation in vitro. Further investigation uncovered that LINC00265 functioned as a competi ng endogenous RNA for miR-144-3p and Chromo box 4 (CBX4) mRNA was as a direct target of miR-144-3p in GC cells. Moreover, inhibition of miR-144-3p could markedly counteract LINC00265 knockdown-meditated suppression on GC cell proliferation.
It has been reported that LINC00265 was upregulated in CRC and played a key role in disease progression. For instance, Ge et al. suggested that increased LINC00265 expression closely correlated with lymph node metastases and advanced pathological stage in CRC, and LINC00265 could promote HT29 cell proliferation and invasion by upregulating the expression of EGFR [37]. Sun et al. also demonstrated that LINC00265 was upregulated in CRC and intimately correlated with poorer patient prognosis. Moreover, they uncovered that LINC00265 could positively regulated CRC cell viability, glucose uptake, pyruvate production and lactate production via the miR-216b-5p/TRIM44 axis [38]. Another study further revealed that LINC00265 was able to regulate ZMIZ2 and USP7-mediated stabilization of β-Catenin, thereby facilitating colorectal tumorigen esis [39]. Interestingly, it was reported that H. pylori can upregulate the expression of EGFR [40] and reduce the expression and activity of intracellular ubiquitinase USP7 in GC cells [41]. Therefore, we boldly hypothesized that H.pylori may induce LINC00265 expression in GC and LIN C00265 may contribute to GC progression. Here in, we only focused on the latter due to our financial limitation. As expected, our data showed that LINC00265 was upregulated in GC tissues and cell lines. Additionally, it was observed that LINC002 65 knockdown significantly restricted GC cell proliferation in vitro, similar to the previous studies in CRC [37], suggesting that LINC00265 may serve as an oncogene in GC.
We next elucidated the molecular mechanisms underlying the oncogenic action of LINC00265 in GC. Solid evidence has revealed that many lncR NAs can perform biological functions by acting as ceRNAs or molecular sponges to inactivate miRN As. MiR-144-3p has been reported to be downregulated in cervical cancer [42], ovarian cancer [43], CRC [44] and GC [45], indicating that miR-144-3p may exert tumor suppressive effects in the se cancers. For example, miR-144-3p can inhibit GC cell migration by negatively regulating met proto-oncogene (MET) [46] and proliferation [27], and induce cell apoptosis and cell cycle arrest through downregulating COX-2 expression [27]. Strikingly, herein our data showed that LINC002 65 served as a ceRNA for miR-144-3p. Moreover, inhibition of miR-144-3p markedly counteracted LINC00265 knockdown-meditated suppression on GC cell proliferation, suggesting that LINC00265 may promote GC cell proliferation by repressing miR-144-3p. Several studies have suggested that CBX4 played dual roles in cancer progression. For example, CBX4 was found to be highly expres sed in lung cancer and could promote tumor cell proliferation and metastasis by regulating BMI-1 expression [32,47]. In colorectal cancer, CBX4 can interact with histone deacetylase 3 (HDAC3) to maintain recruited HDAC3 to the Runx2 promoter, which maintains a deacetylated histone H3K27 state to inhibit Runx2 expression, consequently repressing tumor metastasis [48]. With respect to GC, Ying Luo et al reported that CBX4 rs77447679 polymorphism was positively associated with GC, and individuals with CC genotype had lower risk of GC [49,50]. In the present study, we for the first time demonstrated that CBX4 was upregulated in GC tissues and cell lines and CBX4 could promoted GC cell proliferation, indicating it may play a pro-tumor role in GC. Of interest, our fur ther exploration showed that CBX4 was a direct target of miR-144-3p and LINC00265/miR-144-3p axis could regulate CBX4 expression in GC cells.

Conclusion
In summary, LINC00265 was highly expressed in GC tissues and cell lines. Additionally, knockdown of LINC00265 or CBX4, and miR-144-3p overexpression could inhibit GC cell growth in vitro. Furthermore, LINC00265 could act as a sponge for miR-144-3p to up-regulate CBX4 expression in GC cells. Therefore, LINC00265/miR-144-3p/ CBX4 axis may serve as potential promising therapeutic targets for GC [50].

Disclosure statement
No potential conflict of interest was reported by the author(s).

Highlights:
LINC00265 is highly expressed in GC.
LINC00265 acts as a competing endogenous RNA for miR-144-3p in GC cells.