Circ-RNF111 aggravates the malignancy of gastric cancer through miR-876-3p-dependent regulation of KLF12

Thirty-one GC patients at the First People’s Hospital of Xiaoshan were recruited in our research. After the study was permitted by the Ethics Committee of the First People’s Hospital of Xiaoshan and written informed consents were offered by the participants, the tumor tissues and adjacent non-tumor tissues were acquired and stored at – 80 °C before use.

GES-1 cells and AGS cells were obtained from Procell (Wuhan, China). GC cells (SNU-638) were acquired from Shanghai GuanDao Biological Engineering Co., Ltd. (Shanghai, China). The cells were cultivated at 37°C in DMEM (Sigma-Aldrich, St. Louis, MO, USA) added with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Sigma-Aldrich) in a humidified incubator.

After being extracted via RNAiso Plus (Takara, Dalian, China), the RNA was reversely transcribed into cDNAs with PrimeScript™ RT reagent Kit (Takara) or TaqMan miRNA assays (Applied Biosystems, Foster City, CA, USA). Then, qRT-PCR was conducted through the usage of SYBR Premix DimerEraser (Takara) and related primers (Sangon, Shanghai, China). The expression was estimated with the 2^−ΔΔCt way with normalization to GAPDH or U6. The primers included: circ-RNF111: (F: 5′-ACAATCCAGCTGTTCCCTCA-3′ and R: 5′-GGCTCTGGATGCAAAAGGAT-3′); miR-876-3p: (F: 5′-CTGTGGTGGTTTACAAAGTAATT-3′ and R: 5′-GTGCAGGGTCCGAGGT-3’); KLF12: (F: 5′-TGGCAAAGCACAAATGGAC-3′ and R: 5′-CTAAATGGTGAAATTGAACAAGG-3′); GAPDH: (F: 5′-GGAGTCCACTGGCGTCTTCA-3′ and R: 5′-GGTTCACACCCATGACGAAC-3′); U6: (F: 5′-ATTGGAACGATACAGAGAAGATT-3′ and R: 5′-GGAACGCTTCACGAATTTG-3′). To analyze the feature of circ-RNF111, total RNA was exposed to RNase R (3 U/μg; Epicentre, Madison, WI, USA) for 15 min at 37 °C and then circ-RNF111 and GAPDH levels were detected.

Circ-RNF111 siRNAs (si-circ-RNF111#1 and si-circ-RNF111#2), circ-RNF111 overexpression plasmid (circ-RNF111), miR-876-3p mimics, miR-876-3p inhibitors (anti-miR-876-3p), KLF12 overexpression plasmid (KLF12), circ-RNF111 shRNA (sh-circ-RNF111), and related controls (si-NC, Vector, miR-NC, anti-miR-NC, pcDNA, and sh-NC) were designed by GenePharma (Shanghai, China). GC cells were introduced with the compositions utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Firstly, 5 × 10³ cells were seeded into each well of 96-well plates. After 48 h of incubation, 10 μL CCK-8 (Sigma-Aldrich) was supplemented into the well and kept for an additional 2 h. At last, the absorption at 450 nm was measured.

Following indicated transfection, GC cells were seeded into 6-well plates for 14 days. When the colonies were visible, the culture was terminated. Next, the colonies were dyed with crystal violet (Sangon) and quantified utilizing a microscope (Olympus, Tokyo, Japan).

The apoptosis and cell cycle process were examined with Annexin V-FITC/PI Apoptosis Kit (Beyotime, Shanghai, China) based on the manufacturers’ instructions. To examine cell apoptosis, GC cells with various transfections were harvested, washed with PBS (Sangon), and then resuspended in binding buffer. Then, Annexin V-FITC and PI were adopted to dye the cells. For cell cycle process, after the transfected cells were washed, they were fixed with 70% ethanol and then kept with RNase (Solarbio, Beijing, China) in PBS (Sangon) for 1 h. Thereafter, the cells were interacted with PI. The apoptotic rate and cell cycle were assessed with a FACS flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

To determine the migration of GC cells, the transfected GC cells were added into 6-well plates and grown until 100% confluence. Then, the sterile pipette tip was utilized to make a scratch in the well. At 0 h and 24 h, the crosses were recorded.

The invasion of GC cells was evaluated with the transwell insert chambers (BD Biosciences) pre-covered Matrigel (BD Biosciences). In short, the transfected AGS and SNU-638 cells in DMEM (Sigma-Aldrich) with serum-free were added into the upper chamber. The bottom chamber was filled with DMEM (Sigma-Aldrich) including 10% FBS (Sigma-Aldrich). Twenty-four hours later, the invaded cells were dyed with crystal violet (Sangon) and quantified utilizing a microscope (Olympus; × 100 magnification).

The lactate assay kit (Sigma-Aldrich), glucose assay kit (Sigma-Aldrich), and ATP assay kit (Sigma-Aldrich) were employed to test glucose uptake, lactate production, and ATP production levels according to the manufacturers’ protocols.

The protein was contained by lysing tissues and cells into RIPA buffer (Beyotime), electrophoresed on SDS-PAGE (Solarbio, Beijing, China) and then blotted onto PVDF membranes (Millipore, Billerica, MA, USA). After blockage in 5% skim milk for 1 h, the proteins were interacted overnight with primary antibodies and secondary antibody (bs-0295M-HRP; Bioss, Beijing, China) for 1.5 h. The bands were visualized with ECL reagent (Beyotime) and band intensity was examined with ImageJ (NIH, Bethesda, MD, USA). The primary antibodies including β-actin (bs-0061R; Bioss), hexokinase2 (HK-2; bs-3993R; Bioss), CyclinD1 (bs-0623R; Bioss), MMP9 (bs-4593R; Bioss), or KLF12 (bs-16783R; Bioss)

The fragments of wild-type (WT) circ-RNF111, or KLF12 3′UTR including miR-876-3p binding sites or mutant (MUT) circ-RNF111 or KLF12 3′UTR lacking miR-876-3p binding sequences were introduced into pmirGLO (Promega, Fitchburg, WI, USA) to generate WT-circ-RNF111, MUT-circ-RNF111, WT-KLF12 3′UTR, and MUT-KLF12 3′UTR, respectively. Next, the generated vectors and miR-NC/miR-876-3p were administrated into GC cells. The luciferase activity was measured utilizing Dual-Luciferase Reporter Assay System (Promega).

In brief, after AGS and SNU-638 cells were lysed in RIP buffer, the cell lysates were maintained with protein A/G sepharose beads conjugated with antibody IgG or Ago2. Next, total RNA in immunoprecipitates was subjected to qRT-PCR for the abundance of circ-RNF111, miR-876-3p, and KLF12.

The BALB/c nude mice from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were assigned into 2 groups (n = 5/group). Sh-NC or sh-circ-RNF111 transfected AGS cells (2 × 10⁵) were suspended in 0.2 mL PBS (Sangon) and then subcutaneously introduced into the flank of the mice. After 7 days, tumor size was examined every 5 days and estimated via the formula: (LengthWidth²) × 0.5. At 32 days, the mice were sacrificed and xenograft tumors were weighted. The in vivo study obtained permission from the Ethics Committee of Animal Research of the First People’s Hospital of Xiaoshan.

The expression of KLF12 and ki67 in the xenograft tumor tissues was examined by IHC assay, as previously described [24]. The antibodies against KLF12 (bs-16783R) and ki67 (bs-23103R) were provided by Bioss.

The results from three independent experiments were analyzed using GraphPad Prism 7 and exhibited as mean ± SD. The relationship between miR-876-3p level and circ-RNF111 level or KLF12 level in GC tissues was evaluated by Spearman’s correlation coefficient. Student’s t test or one-way analysis of variance was utilized for different analysis. P < 0.05 was thought to be significant.

To explore the effect of circ-RNF111 in GC development, the expression level of circ-RNF111 in GC tissues and normal tissues was determined. The results exhibited that circ-RNF111 was highly expressed in GC tissues compared to adjacent normal tissues (Fig. 1A). Moreover, we found that circ-RNF111 was conspicuously increased in AGS and SNU-638 cells relative to GES-1 cells (Fig. 1B). Besides, RNase R assay showed that GAPDH was digested by RNase R treatment, but circ-RNF111 was resistant to RNase R, indicating that circ-RNF111 was stable (Fig. 1C).

To explore the functions of circ-RNF111 in GC, AGS and SNU-638 cells with circ-RNF111 silencing were constructed by transfecting si-circ-RNF111#1 or si-circ-RNF111#2 into AGS and SNU-638 cells. As a result, the transfection of si-circ-RNF111#1 or si-circ-RNF111#2 led to a distinct suppression in circ-RNF111 expression in AGS and SNU-638 cells compared to si-NC control groups (Fig. 2A). The results of CCK-8 assay showed that circ-RNF111 knockdown repressed the viability of AGS and SNU-638 cells relative to control groups (Fig. 2B). Colony formation assay indicated that the colony formation ability of AGS and SNU-638 cells was inhibited by the downregulation of circ-RNF111 in comparison with si-NC control groups (Fig. 2C). Flow cytometry analysis indicated that circ-RNF111 silencing facilitated the apoptosis of AGS and SNU-638 cells in comparison with control groups (Fig. 2D). As illustrated by wound-healing assay and transwell assay, the migration and invasion of AGS and SNU-638 cells were restrained by circ-RNF111 knockdown relative to control groups (Fig. 2E, F). Moreover, circ-RNF111 silencing arrested cell cycle in G0/G1 phase, as analyzed by flow cytometry analysis (Fig. 2G, H). Circ-RNF111 knockdown reduced the levels of cell cycle-related protein CyclinD1 and cell metastasis-related protein MMP9 (Fig. 2I, J). Besides, the impact of circ-RNF111 deficiency on glycolysis was explored. It was found that circ-RNF111 deficiency reduced the levels of glucose consumption, lactate production, ATP production and HK-2 protein in AGS and SNU-638 cells compared to control groups, suggesting the repression of glycolysis in AGS and SNU-638 cells after circ-RNF111 knockdown (Fig. 2K–N). Taken together, circ-RNF111 knockdown suppressed GC cell growth, metastasis and glycolysis, promoted apoptosis, and arrested cell cycle.

Through analyzing circular RNA interactome, miR-876-3p was found to share the binding sites with circ-RNF111 (Fig. 3A). Dual-luciferase reporter assay was then performed to estimate the relationship between circ-RNF111 and miR-876-3p. The results exhibited that the luciferase activity of WT-circ-RNF111 in AGS and SNU-638 cells was restrained by miR-876-3p transfection, while the luciferase activity of MUT-circ-RNF111 was not affected (Fig. 3B). Thereafter, RIP assay further demonstrated the interaction between circ-RNF111 and miR-876-3p for the abundance of circ-RNF111 and miR-876-3p was elevated in Ago2 pellets relative to IgG control groups (Fig. 3C). Indeed, miR-876-3p level was decreased in GC tissues and cells compared to normal tissues and cells (Fig. 3D, E). Moreover, there was an inverse correlation between miR-876-3p level and circ-RNF111 level in GC tissues (Fig. 3F). Besides, si-circ-RNF111 or circ-RNF111 was successfully transfected into AGS and SNU-638 cells to reduce or elevate circ-RNF111 expression, which was demonstrated by qRT-PCR assay (Fig. 3G). Of note, our results exhibited that circ-RNF111 silencing increased miR-876-3p expression in AGS and SNU-638 cells, while circ-RNF111 overexpression showed the opposite results (Fig. 3H). To summarize, circ-RNF111 directly targeted miR-876-3p to regulate miR-876-3p expression.

Based on the above results, we further investigated whether circ-RNF111 could alter GC cell progression by sponging miR-876-3p through transfecting si-NC, si-circ-RNF111, si-circ-RNF111 + anti-miR-NC, or si-circ-RNF111 + anti-miR-876-3p into AGS and SNU-638 cells. The results showed that anti-miR-876-3p transfection restored si-circ-RNF111-caused upregulation of miR-876-3p level in both AGS and SNU-638 cells compared to anti-miR-NC control groups (Fig. 4A). Furthermore, we found that circ-RNF111 knockdown-mediated inhibitory effects on GC cell viability, colony formation ability, migration, invasion and cell cycle process, and promotional effect on GC cell apoptosis were ameliorated by reducing miR-876-3p (Fig. 4B–G). The effects of circ-RNF111 on CyclinD1 and MMP9 in AGS and SNU-638 cells were also reversed by decreasing miR-876-3p (Fig. 4H, I). Additionally, our results showed that miR-876-3p inhibition reversed the impacts of circ-RNF111 deficiency on glucose consumption, lactate production, ATP production, and HK-2 protein levels in both AGS and SNU-638 cells (Fig. 4J–N). Thus, we concluded that circ-RNF111 silencing repressed GC cell growth, metastasis, and glycolysis and promoted cell apoptosis and cell cycle arrest by targeting miR-876-3p.

To further explore the related regulatory mechanism of circ-RNF111/miR-876-3p in GC development, TargetScanHuman 7.2 was utilized to analyze the potential target gene of miR-876-3p. It was found that KLF12 was the target gene of miR-876-3p (Fig. 5A). Dual-luciferase reporter assay showed that the overexpression of miR-876-3p inhibited luciferase activity of WT-KLF12 3′UTR rather than MUT-KLF12 3′UTR in both AGS and SNU-638 cells (Fig. 5B). RIP assay showed that miR-876-3p and KLF12 were enriched by Ago2 RIP compared to IgG RIP, further verifying the combination between miR-876-3p and KLF12 (Fig. 5C). As expected, KLF12 mRNA level was increased in GC tissues and cells in comparison with normal tissues and cells (Fig. 5D, E). Moreover, KLF12 mRNA level was negatively correlated with miR-876-3p level in GC tissues (Fig. 5F). Western blot assay presented that the protein level of KLF12 was elevated in GC tissues and cells relative to normal tissues and cells (Fig. 5G, H). As shown in Fig. 5I, miR-876-3p transfection increased miR-876-3p expression, while anti-miR-876-3p transfection decreased miR-876-3p expression in AGS and SNU-638 cells. Besides, miR-876-3p overexpression reduced KLF12 protein level and miR-876-3p inhibition elevated KLF12 protein level in both AGS and SNU-638 cells (Fig. 5J, K). Collectively, miR-876-3p directly interacted with KLF12.

To investigate the relationship between miR-876-3p and KLF12 in regulating GC progression, rescue experiments were conducted. As shown in Fig. 6A, B, miR-876-3p overexpression reduced the mRNA and protein levels of KLF12 in AGS and SNU-638 cells, while KLF12 overexpression vector transfection rescued the impacts. CCK-8 assay and colony formation assay indicated that miR-876-3p overexpression suppressed the viability and colony formation of AGS and SNU-638 cells, with KLF12 elevation reversed the impacts (Fig. 6C, D). As demonstrated by flow cytometry analysis, miR-876-3p overexpression facilitated AGS and SNU-638 cell apoptosis, while the effect was abrogated by elevating KLF12 (Fig. 6E). The results of wound-healing assay and transwell assay, miR-876-3p upregulation suppressed the capacities of AGS and SNU-638 cells to migrate and invade, whereas these effects were ameliorated by KLF12 overexpression (Fig. 6F, G). The cell cycle process of AGS and SNU-638 cells was arrested by miR-876-3p overexpression, with KLF12 elevation abrogated the effect (Fig. 6H). Western blot assay showed that miR-876-3p overexpression reduced CyclinD1 and MMP9 protein levels in AGS and SNU-638 cells, with KLF12 elevation abrogated the effects (Fig. 6I, J). Besides, overexpression of miR-876-3p reduced the levels of glucose uptake, lactate production, ATP production, as well as HK-2 protein in AGS and SNU-638 cells, while KLF2 elevation abated the impacts (Fig. 6K–N). Taken together, miR-876-3p overexpression suppressed the malignancy of GC cells by targeting KLF12.

As exhibited in Fig. 7A, B, circ-RNF111 silencing led to an apparent suppression in KLF12 protein level in both AGS and SNU-638 cells, while miR-876-3p inhibition restored the impact. The findings indicated that circ-RNF111 positively regulated KLF12 expression in GC cells by adsorbing miR-876-3p.

At last, the functional role of circ-RNF111 in tumor progression in vivo was explored. Our results presented that tumor size and tumor weight were inhibited after circ-RNF111 knockdown compared to sh-NC control groups (Fig. 8A–C). Moreover, the levels of circ-RNF111, KLF12 mRNA, and KLF12 protein were declined and the level of miR-876-3p was enhanced in the tumors in sh-circ-RNF111 groups compared to control groups (Fig. 8D, E). IHC assay also showed that reduced KLF12 and Ki-67 levels in tumors in sh-circ-RNF111 groups compared to sh-NC groups (Fig. 8F). Collectively, circ-RNF111 contributed to tumorigenesis in vivo.