SLCO4A1-AS1 Facilitates the Malignant Phenotype via miR-149-5p/STAT3 Axis in Gastric Cancer Cells

Solute carrier organic anion transporter family member 4A1 (SLCO4A1-AS1), a newly discovered lncRNA, may exert effects in tumors. Since its role in gastric cancer remains obscure, we sought to explore the mechanism of SLCO4A1-AS1 in gastric cancer. The relationship among SLCO4A1-AS1, miR-149-5p, and STAT3 was detected by bioinformatics, dual luciferase analysis, and Pearson's test, and the expressions of these genes were determined by quantitative real-time PCR and Western blot. Moreover, CCK-8, flow cytometry, wound healing assay, and Transwell analysis were performed to verify the function of SLCO4A1-AS1 in gastric cancer. Rescue experiments were used to detect the role of miR-149-5p. The expressions of SLCO4A1-AS1 and STAT3 were increased, while the expression of miR-149-5p was suppressed in gastric cancer tissues and cell lines. In addition, STAT3 expression was negatively correlated with miR-149-5p expression but was positively correlated with SLCO4A1-AS1 expression. Overexpression of SLCO4A1-AS1 promoted cell viability, migration, invasion, and STAT3 expression but suppressed apoptosis, while knockdown of SLCO4A1-AS1 had the opposite effect. SLCO4A1-AS1 bound to miR-149-5p and targeted STAT3. Moreover, miR-149-5p mimic inhibited the malignant development of gastric cancer cells and obviously reversed the function of SLCO4A1-AS1 overexpression. Our research reveals that abnormally increased SLCO4A1-AS1 expression may be an important molecular mechanism in the development of gastric cancer.


Introduction
Gastric cancer is a common cancer of the digestive tract [1]. Although the morbidity of gastric cancer has declined in recent years, its mortality rate is still among the highest in the world [2]. Diagnosis of the disease in the early stage together with certain treatment would help cure or prolong the survival of the patients; however, conventional treatment would be difficult or fail to treat patients when they are first diagnosed at advanced gastric cancer stage [3]. erefore, the study of new mechanisms would provide new ideas for the treatment of gastric cancer.
Noncoding RNA (ncRNA) has recently become a research hotspot in the molecular mechanisms of new diseases [4]. Studies have shown that the occurrence and development of gastric cancer are accompanied by a variety of ncRNA expression disorders, and abnormally expressed ncRNAs will eventually affect epigenetics and lead to the formation of tumor malignant phenotypes [5,6]. For example, the first studied microRNAs (miRNAs), such as miR-17-5p and miR-623, have shown significant regulatory effects on the proliferation, migration, and invasion of gastric cancer cells [7]. Circular RNA (circRNA) is a kind of ncRNA characterized by a closed covalent loop structure. Studies have found that circRNA has a more stable structure and can be detected through blood pathways (such as hsa_circ_0000745 and hsa_circ_0000419), which can guide the diagnosis and prognosis of gastric cancer to a certain extent [8,9]. Of course, long noncoding RNA (lncRNA) is also an important part of the progression of gastric cancer. Zhang et al. screened and analyzed lncRNA of tissue samples from gastric cancer patients and adjacent normal tissues and then found that the expressions of plasma lncRNAs (TINCR, CCAT2, AOC4P, BANCR, and LINC00857) were significantly upregulated in gastric cancer cell lines [10]. Wang et al. found that lncRNA UCA1 regulates the stability of GRK2 protein by promoting Cbl-c-mediated G proteincoupled receptor kinase 2 ubiquitination, thereby increasing the metastatic ability of gastric cancer cells [11]. is also reflects that lncRNAs may become potential markers and possible targets for gastric cancer diagnosis and tumor treatment.
Solute carrier organic anion transporter family member 4A1 (SLCO4A1) is a newly discovered lncRNA in recent years. It is located on the long arm of chromosome 20 and contains 1440 bp in full length [12]. Most of the research on SLCO4A1 focuses on colon cancer. Some studies have found that the sense chain of SLCO4A1-AS1, SLCO4A1, belongs to the superfamily of membrane transport systems, which may serve as a valuable marker for poor prognosis of colorectal cancer. It also plays an important role in colorectal cell proliferation, migration, invasion, and carcinogenesis [13,14]. In addition, SLCO4A1-AS1 also showed obvious cancer-promoting potential in bladder cancer and lung adenocarcinoma [15,16]. However, there is no research evidence about SLCO4A1-AS1 in gastric cancer.
More and more studies have shown that lncRNAs can regulate multiple pathways through miRNAs to regulate the progress of gastric cancer [17][18][19]. For example, CASC11 acts as a ceRNA by sponging miR-340-5p to regulate cell cycle signals and affects the growth and apoptosis of gastric cancer cells [20]. However, it is not clear whether SLCO4A1-AS1 can also be used as ceRNA, and binding miRNA plays a regulatory role in gastric cancer. erefore, we tried to explore the role of SLCO4A1-AS1 in gastric cancer and the possible regulatory pathways.

Ethics Statement and Tissue.
e collection of clinical tissues was approved by the Ethics Committee of the ird Affiliated Hospital of Soochow University (ZC201903024), and the informed consent signed by the patients was obtained. e 46 pairs of tissue samples were all from pathologically and histologically confirmed gastric cancer patients who received treatment in our hospital from Apr 2019 to May 2020.

Cell Counting
en the AGS or SNU-16 cells were continuously cultivated for 2 h. Lastly, Microplate Absorbance Reader (E0228) from Beyotime (China) was employed to detect the absorbance (450 nm).

Flow Cytometry.
e changes of cell apoptosis were detected by flow cytometry according to Annexin V-FITC Apoptosis Detection Kit (CA1020, Solarbio, China). After digestion with trypsin, AGS or SNU-16 cells were collected, followed by suspending the cells with 1 ml of 1 × binding buffer to bring the cell density to 1 × 10 6 cells/ml. 100 μl of cells (1 × 10 5 cells) was added to each tube, and 5 μl of Annexin V-FITC was added to the tube (room temperature, protected from light, and gently mixed for 10 min). Lastly, the cell mixture was performed by flow cytometer (Cyto-FLEX, Backman Coulter, USA) within 1 h to detect cell apoptosis.

Wound Healing
Assay. AGS or SNU-16 cells (3 × 10 5 cells/ml) were seeded into 6-well plates and cultured to 100% cell fusion. 100 μl pipette tip was used to draw a line wound through the layer of fused cells. After washing with PBS, the culture plate was cultured in serum-free medium for 24 h. At last, the wound closure was photographed and measured by the BX53M microscope from Olympus (Japan) (magnification ×100).

Transwell.
Transwell chamber (3422, Corning, USA) precoated with Matrigel (354230, BD, USA) was inserted into a 24-well culture plate. e AGS or SNU-16 cell suspension and the medium without FBS were mixed and added to the upper chamber and then cultured for 24 h. Subsequently, after being fixed with methanol, the cells on the membrane were stained with 0.1% crystal violet solution (C8470, Solarbio, China). e cells on the membrane were photographed under a BX53M microscope (magnification ×200).

Western Blot.
As mentioned in the literature previously [22], the total protein of AGS or SNU-16 cells was lysed and extracted by RIPA buffer containing 1% PMSF (R0010, Solarbio, China), and then the protein concentration was detected in the BCA kit (PC0020, Solarbio). After electrophoretic separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a PVDF membrane (ISEQ00010) produced by Millipore (USA). Antibodies of STAT3 (ab119352, 88 kDa, 1/1000, Abcam, UK) or GAPDH (ab8245, 36 kDa, 1/1000) were added to the membranes and incubated at 4°C for 16 h. Afterwards, the membranes were incubated with the Goat Anti-Mouse (ab205719, 1/5000) for 2 h, followed by exposure with ultrasensitive ECL chemiluminescent substrate (SW2040, Solarbio, China). Lastly, LabWorks Image Acquisition and Analysis Software (UVP, Upland, USA) was used to analyze the data.
2.11. Statistical Analysis. By GraphPad Prism 8.0, independent-sample t-test or one-way ANOVA was performed to analyze the difference between two or among more groups. e correlation between target genes (SLCO4A1-AS1, miR-149-5p, STAT3) was evaluated by Pearson correlation coefficient. P < 0.05 was considered as statistically significant.

Results
e expression of SLCO4A1-AS1 was increased in gastric cancer and played a role as an oncogene. e analysis of the starBase database showed that the expression of SLCO4A1-AS1 in stomach adenocarcinoma (STAD) patients (n � 375) was higher than that of normal samples (n � 32) (P � 1.2e − 7, Figure 1(a)). We tested 46 gastric cancer tissues and matched paracancerous tissues and found that SLCO4A1-AS1 expression was upregulated in gastric cancer tissues (P < 0.01, Figure 1(b)). As shown in Figure 1(b), the expression of SLCO4A1-AS1 in AGS, SNU-16, NCI-N87, SNU-5, and RF-48 cell lines was significantly higher than that in GES-1 cell line (P < 0.01). Among them, the highest expression of SLCO4A1-AS1 was observed in AGS cells and the smallest expression of that is in SNU-16 cells (P < 0.01, Figure 1(c)). In order to investigate whether SLCO4A1-AS1 has an effect on the malignant phenotype of gastric cancer cells, SLCO4A1-AS1 was successfully induced to increase or suppress in gastric cancer cells (P < 0.01, Figures 1(d) and 1(e)). en we found that overexpression of SLCO4A1-AS1 enhanced cell viability, inhibited apoptosis, and significantly accelerated cell migration and invasion in gastric cancer cells, while knockdown of SLCO4A1-AS1 had the opposite effect (P < 0.05, Figures 1(f ), 1(g), and 2(a)-2(l)). SLCO4A1-AS1 targeted and negatively regulated miR-149-5p, which was downregulated in gastric cancer tissues.
As shown in Figure 6(a), STAT3 was targeted by miR-149-5p. e luciferase activity of miR-149-5p mimic was obviously inhibited in STAT3-wt group, while there was no significant difference of that in STAT3-mut group (P < 0.01, Figures 6(b) and 6(c)). We found that the expression of STAT3 in cancer tissues was significantly abnormally upregulated (P < 0.01, Figure 6(d)).
en we tested the correlation between STAT3 and SLCO4A1-AS1 or miR-149-5p. e results showed that the expressions of STAT3 and SLCO4A1-AS1 were positively correlated in gastric cancer tissues (r � 0.142, P < 0.05, Figure 6(e)), but STAT3 was negatively correlated with miR-149-5p (r � -0.622, P < 0.001, Figure 6(f )). In addition, we further examined the protein content of STAT3 and found that STAT3 expression was decreased in the mimic group but was increased in the pc-SLCO4A1-AS1 group, and the mimic + pc-SLCO4A1-AS1 group significantly counteracted the effect of mimic group or pc-SLCO4A1-AS1 group on STAT3 expression (P < 0.05, Supplementary Figures 1(a)-1(c) and Supplementary   Figure 2(a)). Conversely, STAT3 expression was increased in the inhibitor group but was decreased in the si-SLCO4A1-AS1 group, and the inhibitor + si-SLCO4A1-AS1 group significantly reversed the inhibitory effect of the inhibitor group or si-SLCO4A1-AS1 group on STAT3 expression (P < 0.01, Supplementary Figures 1(d)-1(f ) and Supplementary Figure 2(b)).

Discussion
ere are a large number of regulatory interaction sites in lncRNAs, which provides a broader platform for the development of new structure-based anticancer drugs [23]. In addition, in view of their involvement in multiple cell signaling pathways and tissue-specific expression, lncRNAs can also be used in new strategies for the diagnosis and targeting of specific cancer subtypes [24].
is study found that SLCO4A1-AS1 has a cancer-promoting effect in gastric cancer cells, which may regulate the biological characteristics of gastric cancer cells by targeting STAT3 with miR-149-5p, suggesting that SLCO4A1-AS1 may be used for the treatment of gastric cancer.
Gastric cancer, as a malignant tumor of the digestive tract with a high mortality rate, has already attracted the attention of scholars at home and abroad [25]. It is necessary to study the molecular mechanism of gastric cancer, hoping to provide effective assistance for the treatment of gastric cancer. For example, LncRNA-PCTA6 participates in the endogenous competition of miR-30 by targeting the Makorin RING finger protein 3 to promote the malignant transformation of gastric cancer [26]. Wang et al. revealed that the expression of lncRNA AB007962 is downregulated in gastric cancer tissues, and the expression level is negatively correlated with tumor size; furthermore, it indicates that the expression of AB007962 is significantly correlated with poor prognosis [27]. Our research revealed that SLCO4A1-AS1 is elevated in gastric cancer, which is similar to the expression trend of LncRNA-PCTA6 [26], so we speculate that SLCO4A1-AS1 may also have a role in  Journal of Oncology 7 promoting malignant transformation of gastric cancer. SLCO4A1-AS1 has different roles in different cancers, but the cell experiments in this study proved our conjecture, showing that pc-SLCO4A1-AS1 promotes cell viability, migration, and invasion and inhibits apoptosis, while si-SLCO4A1-AS1 inhibits cell growth and migration and promotes apoptosis. It suggests that SLCO4A1-AS1 may be involved in the growth and metastasis of gastric cancer tumors, and our results are consistent with the research of Ouyang et al. [28]. Compared with the sudden rise of lncRNA research, miRNA has always been the focus of cancer research [29]. erefore, the target miRNA based on bioinformatics is essential to more fully reveal the tumorrelated pathways involved in lncRNA. We experimentally verified that SLCO4A1-AS1 can bind to miR-149-5p.
ere have also been numerous reports of miRNA research in gastric cancer. Recently, the expression of miR-582 has been found to increase in gastric cancer, and it may activate PI3K/AKT/Snail axis through FOXO3 to regulate the proliferation and metastasis of gastric cancer cells [30]. miR-149-5p is one of the two active chains produced by miR-149 precursors, which can act on their respective target genes to exert biological effects. e role of miR-149-5p in a variety of diseases has been reported, including various cancers [31,32], atherosclerosis [33], and diabetes [34]. e regulation is mostly related to the sponge mechanism of lncRNA-miRNA. Consistent with the report of Zhang et al. [35], our results confirmed the downregulation of miR-149-5p in gastric cancer and the inhibition of cancer cell function.
To the best of our knowledge, miRNAs can regulate the upregulation or downregulation of mRNA by targeting mRNA and then activate related pathways to play a role in cancer cells [36]. Mechanically, we explained that STAT3 is a downstream target gene of miR-149-5p and that the two have a negative regulatory relationship. Many studies have shown that STAT3 plays an important role in JAK/STAT, promoting cell proliferation, migration, and invasion [37]. Wu et al. explored that cancer-associated fibroblasts can facilitate gastric cancer migration and EMT in the tumor microenvironment by IL-6/JAK2/STAT3 signaling [38]. Although we demonstrated that SLCO4A1-AS1 positively regulates STAT3 through sponging miR-149-5p in gastric cancer cells, the downstream regulatory mechanism of miRNA : 3′ cccucacuucugugccUCGGUCu 5′ STAT3-mut : 5′ agggaauaugguucuuAUGAGu 3′ STAT3-wt : 5′ agggaauaugguucuuAGCCAGu 3′   Figure 6: e regulatory relationship and expression of SLCO4A1-AS1-miR-149-5p-STAT3 in gastric cancer. (a-c) STAT3 was the target gene of miR-149-5p, which was predicted by starBase database (https://starbase.sysu.edu.cn/) and verified by dual luciferase reporter gene. (d) STAT3 was elevated in gastric cancer tissues, detected by RT-qPCR (n � 46). Experiment was repeated three times independently. (e, f ) Pearson correlation coefficient was used to analyze the relationship between STAT3 and SLCO4A1-AS1 (r � 0.142 and P < 0.05), as well as STAT3 and miR-149-5p (r � −0.622 and P < 0.001). Each experiment was repeated three times independently. * * P < 0.01 versus blank; ## P < 0.01 versus adjacent tissue. STAT3, signal transducer and activator of transcription 3.
STAT3 in gastric cancer still needs to be verified. Our next work may focus on in vivo functional verification and more regulatory networks.

Conclusion
To sum up, our research reveals that SLCO4A1-AS1 may explain part of the progression of gastric cancer and may target STAT3 through competitive binding of miR-149-5p to affect the growth and metastasis of gastric cancer tumors.
Data Availability e analyzed data sets generated during the study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare no conflicts of interest.