Elevated levels of hsa_circ_006100 in gastric cancer promote cell growth and metastasis via miR‐195/GPRC5A signalling

Abstract Objectives Circular RNAs (circRNAs) are non‐coding RNAs, some of which are thought to be involved in gastric cancer development. Here, we examined the functions of circRNA hsa_circ_006100 in gastric cancer cells and an animal model of gastric cancer. Materials and Methods The expression of hsa_circ_006100, miR‐195 and various functional genes was determined by quantitative RT‐PCR. Cell viability, clone formation, apoptosis and cell migration/invasion abilities were analysed by the CCK‐8 assay, crystal violet staining, Hoechst staining and Transwell assay, respectively. A tumour model was established by subcutaneously injecting tumour cells into nude mice. Levels of protein expression were analysed by Western blotting and immunohistochemistry. Results A bioinformatics analysis showed that miR‐195 was negatively co‐expressed with hsa_circ_006100. Patients with a high hsa_circ_006100 level or low miR‐195 level had tumours with a high TNM stage, poor cellular differentiation and lymph node metastasis. miR‐195 was targeted and inhibited by hsa_circ_006100. Overexpression of hsa_circ_006100 enhanced cellular viability and proliferation, while miR‐195 suppressed hsa_circ_006100‐enhanced cell growth and induced apoptosis in MGC‐803 and AGS cells. Forced hsa_circ_006100 expression promoted the migration and invasion of MGC‐803 and AGS cells, while those activities were inhibited by miR‐195. Mechanistically, GPRC5A was predicted as a target of miR‐195 and was upregulated in gastric cancer. A miR‐195 inhibitor restored cell viability, proliferation, migration and invasion, and repressed apoptosis via GPRC5A. In vivo studies showed that knockdown of hsa_circ_006100 delayed tumour growth, reduced PCNA expression and upregulated miR‐195 and BCL‐2 expression which was restored by miR‐195 inhibition due to GPRC5A/EGFR signalling, and changed the EMT phenotype in vivo. Conclusions Hsa_circ_006100 functions as an oncogene in gastric cancer and exerts its effects via miR‐195/GPRC5A signalling.


| INTRODUC TI ON
Gastric cancer (GC) is a common malignant tumour, and despite its declining incidence over the past century, it is a major cause of cancer-related death worldwide. In Eastern Asia, the GC mortality rate ranks third among all cancer types. 1 GC originates from superficial mucosal epithelial cells of glands in the stomach and usually manifests as adenocarcinoma. 2 Despite the great progress being made in cancer therapy, the survival rates of GC patients have remained dismal, largely because the disease is usually diagnosed at an advanced stage when extensive tissue invasion and distant metastasis have already occurred. In most areas of the world, the overall 5-year survival rate of GC patients is <25%. The development and occurrence of GC metastases are regulated by a variety of oncogenes and tumour suppressor genes, and this method of regulation might help facilitate the early diagnosis and treatment of gastric cancer. 3,4 Non-coding RNAs (ncRNAs) are distinguished by their sizes, which range from short RNAs (including microRNAs) to long RNAs that span up to hundreds of kbs (>200 nt), such as long non-coding RNA (LncRNAs) and circular RNAs (circRNAs). 5 Through their interactions with DNA, RNA, and proteins, ncRNAs influence protein stability and various gen regulatory activities, including chromatin remodelling, transcription and mRNA translation during many physiological and pathological processes, such as carcinogenesis. 6,7 We previously identified a series of ncRNAs involved in the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced transformation of GES-1-T human gastric epithelial cells. One of those ncRNAs (LncRNA LOC101927497) functions as a tumour suppressor by interacting with miR-574 to inhibit GC cell proliferation and migration, suggesting that ncRNAs might assist in regulating GC progression. 8 CircRNAs are newly discovered endogenous ncRNAs that are hundreds or even thousands of bases in length and share a covalently closed structure. CircRNAs exhibit tissue-and developmental stage-specific expression and play crucial roles in cancer development by serving as miRNA sponges that sequester miRNA molecules, and thereby affect the stability of target mRNAs and dynamically regulate mRNA translation. 9,10 The most investigated circRNAs include CDR1as, circ-Foxo3, cir-ITCH and circHIPK3, which are associated with hepatocellular carcinoma, lung cancer and osteosarcoma via their interactions with miR-7, FOXO3, miR-17 and miR-124 respectively. 11 Several other circRNAs have been reported to be associated with gastric cancer development.
For example, circPVRL3 expression was found to be correlated with GC incidence, a higher TNM stage and lower overall survival rates. 12 The levels of hsa_circ_000745 expression in GC tissues were found to be upregulated and correlate with tumour differentiation, while the levels of hsa_circ_000745 expression in plasma correlated with the stage of tumour-node-metastasis. 13 Recently, circRNA_100269 was found to be downregulated in gastric cancer and suppresses tumour cell growth by targeting miR-630. 14 However, the roles played by cir-cRNAs in GC progression require further investigation.
In this study, we screened the circRNAs-miRNA network that is reported to exist during the transformation of human gastric epithelial cells and found that circRNA hsa_circ_006100 was significantly upregulated in cancerous cells, while miR-195 expression was reduced. We subsequently investigated the roles of hsa_circ_006100 and miR-195 in gastric cancer tissues and corresponding adjacent non-cancerous tissues (n = 20). In addition, hsa_circ_006100 knockdown and overexpression studies were performed to determine the function of _hsa_circ_006100 in GC cell lines in vitro and in vivo.
We found that circRNA hsa_circ_006100 functions as an oncogene to promote GC cell proliferation and metastasis via regulation of miR-195 and its target gene GPRC5A.

| The bioinformatics procedures and algorithms
The EBseq algorithm was applied to filter the differentially expressed genes, and gene co-expression networks were used to assess the relationships among different miRNAs and ncRNAs according to the normalized expression values of genes selected from genes associated with significant GO terms and pathways. Core regulatory factors were determined by the degree of differences between two class samples.

| RNA isolation and qRT-PCR
Total RNA was extracted using Trizol reagent (Invitrogen) according to a standard RNA isolation protocol. The synthesis of cDNA from 2 μg of total RNA was performed using a High-Capacity RNA-to-cDNA™ Kit (Thermo Fisher Scientific) according to the manufacturer's The primers used for qRT-PCR are listed in Table 1.

| CCK-8 assay
MGC-803 and AGS cells were seeded into the wells of a 96-well plate

| Luciferase reporter assay
MGC-803 and AGS cells were co-transfected with vectors containing the negative control construct, pcDNA3.1-hsa_circ_006100, psiCHECK2-GPRC5A and miR-195 mimics. Luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Briefly, firefly luciferase acted as a reporter gene for the normalized control.

| Transwell assay
The migration and invasion capabilities of MGC-803 and AGS cells were analysed using a Transwell culture system. For assessing cell migration, 3 × 10 4 MGC-803 or AGS cells that had been cultured in serum-free medium were added to the upper chamber, and culture medium supplemented with 10% FBS was added to the lower chamber.

| Western blot studies
To determine the expression of BCL-2, caspase-3 and EMT markers, total proteins were isolated from tissues using RIPA buffer. A sample of total proteins (~30 µg) extracted from each tissue specimen was

| Immunohistochemistry
PCNA expression in tumour tissues was analysed via IHC staining performed as previously described. 15

| Statistical analysis
All results were analysed using SPSS for Windows, version 16.0 (SPSS Inc) and Prism, version 5.0 software (GraphPad Software Inc). The unpaired t test or the Mann-Whitney U test was used for comparisons between two groups, and one-way ANOVA was used for multiple group comparisons. A P-value <0.05 was considered statistically significant. All experiments were repeated at least three times.

| Hsa_circ_006100 was upregulated and miR-195 was downregulated in malignantly transformed gastric cells
We  Figure 1A,B). Simultaneously, a circRNA-miRNA co-expression network was created and analysed. It showed that cancer-related miRNA-195 was negatively co-expressed with hsa_circ_006100 ( Figure 1C). A bioinformatics analysis of possible binding targets indicated that hsa_circ_006100 harbours a target site for miR-195, suggesting that hsa_circ_006100 might interact with miR-195 during the malignant transformation of human gastric epithelial cells ( Figure 1D).  Tables 2 and 3. Our results showed that GC tissues had higher levels of hsa_circ_006100 expression and lower levels of miR-195 expression when compared with the non-cancerous tissues (Figure 2A,B).

An analysis of the correlation between hsa_circ_006100 and miR-195
showed that hsa_circ_006100 was negatively correlated with miR-195 expression in the GC tissues ( Figure 2C). In addition, patients with high levels of hsa_circ_006100 expression or low levels of expression of miR-195 expression had worse clinical outcomes, were at an advanced TNM stage, showed poor cell differentiation and had lymph node metastases ( Figure 2 D-H). These findings indicated that hsa_circ_006100 participates in the progression of GC, while miR-195 might function as a tumour suppressor in GC.

| Oncogene GPRC5A was a target gene of miR-195
Previous studies indicated that the G protein-coupled receptor, class C, group 5, member A (GPRC5A) plays important pathogenic roles, and its dysregulation can promote the development of several different types of cancer via its effect on EGFR signalling. 16 Our bioinformatics study showed that GPRC5A was a target gene of miR-195 in GC cells; we also found that GPRC5A expression was upregulated in GC tissues ( Figure 4A

| GPRC5A contributes to the tumour suppressor role of miR-195 in gastric cancer
The function of miR-195/GPRC5A signalling in GC cells was analysed. The viability ( Figure 5A,B) and proliferation rates ( Figure 5C) of MGC-803 and AGS cells were enhanced when GPRC5A was overexpressed, but this effect was repressed by the miR-195 mimics.
Apoptosis showed similar trends in both two cell lines ( Figure 5D,E).
The migration and invasion abilities of MGC-803 and AGS cells were also increased by the overexpression of GPRC5A, but were impaired by the `miR-195 mimics ( Figure 5 F,G). These in vitro results suggested that miR-195 suppresses GC cell growth via its effect on GPRC5A.

| Knockdown of hsa_circ_006100 inhibited tumour growth in vivo via miR-195 signalling
The tumorigenic function of hsa_circ_006100 in GC was analysed restored the tumorigenic activity of hsa_circ_006100 ( Figure 6A,B).
The expression of a proliferation indicator (PCNA, Figure 6C) and  expression. 20 The levels of circ_002059 expression in the plasma of postoperative gastric cancer patients were found to be significantly higher than those in the plasma of preoperative gastric cancer patients and were also associated with distant metastasis, TNM stage, patient gender and age. 21 In this study, we found that hsa_ and also function as molecular decoys or sponges of miRNAs. 22 CircRNA Cdr1as is massively bound by miR-7 and miR-671 in human and mouse brain tissues. 23 In osteoarthritis (OA), circRNA-CER was reported to function as a sponge by competitively binding miR-136 and could also target and regulate MMP13 via TGFβ, JNK and ERK pathways. 24 Here, we found that miR-195 levels were reduced in

| CON CLUS IONS
In summary, we found that hsa_circ_006100 induces the malignant transformation of gastric cells and exerts this effect by inhibiting siRNA-hsa_circ_006100 and/or miR-195 inhibitor/mimics, as determined by IHC assays. E, Levels of the anti-apoptosis proteins BCL-2 and caspase-3, as well as EGFR and EMT proteins, in tumours derived from MGC-803 cells treated with lentivirus-siRNAhsa_ circ_006100 and/or miR-195 inhibitor/mimics, as determined by Western blotting. GAPDH was used as the internal standard. Mice harbouring tumours were sacrificed four weeks after model establishment. Bcl-2, B-cell lymphoma-2; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPRC5A, G protein-coupled receptor, family C, group 5, member A; NC, negative control; data are expressed as the mean ± SD * P < 0.05, ** P < 0.01 expression of the tumour suppressor, miR-195. Inhibition of miR-195 expression induced GPRC5A to increase cell viability and promote the proliferation and metastasis of GC cells.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
No competing financial interests exist.