CircKIAA0907 Retards Cell Growth, Cell Cycle, and Autophagy of Gastric Cancer In Vitro and Inhibits Tumorigenesis In Vivo via the miR-452-5p/KAT6B Axis

Abstract

BACKGROUND The significant roles of circular ribonucleic acids (RNAs) in cancers have been discussed in many studies. This report aimed to investigate the biological functions of circKIAA0907 and its action mechanism in gastric cancer (GC). MATERIAL AND METHODS Relative RNA expression levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The examination of cell proliferation was performed via 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide assay. Flow cytometry was used to analyze the apoptosis rate and cell cycle. Protein levels were quantified using western blot. Biotinylated RNA pull-down assay was used to find the microRNA target of circKIAA0907; target binding was validated through dual-luciferase reporter assay. The assay in vivo was executed via a xenograft tumor model to explore the role of circKIAA0907 in GC. RESULTS CircKIAA0907 was downregulated in GC and had higher stability than its linear isoform. Functionally, circKIAA0907 upregulation resulted in the repression of proliferation, cell cycle, and autophagy and promotion of apoptosis in GC cells. Mechanistically, circKIAA0907 bound to miR-452-5p as a specific sponge for it; lysine acetyltransferase 6B (KAT6B) was a target gene of miR-452-5p, so circKIAA0907 elevated KAT6B levels via sponging miR-452-5p. Reversion assays indicated that circKIAA0907 served as a tumor inhibitor by inhibiting miR-452-5p and increasing KAT6B; miR-452-5p inhibition impeded GC development by upregulating KAT6B. The miR-452-5p/KAT6B axis was also accountable for circKIAA0907-induced tumorigenesis suppression in vivo. CONCLUSIONS This work demonstrated that circKIAA0907 has diagnostic and therapeutic value in GC by acting as an oncogenic molecule via the miR-452-5p/KAT6B axis.

Figure 1

CircKIAA0907 expression was lessened in gastric cancer (GC) tissues and cells. (A) Genomic location of KIAA0907 gene and circKIAA0907. (B, C) Level of circKIAA0907 was assayed using quantitative real-time polymerase chain reaction (qRT-PCR) in GC tissues (B) and cells (C). (D–G) qRT-PCR analysis of circKIAA0907 and KIAA0907 was implemented in HGC27 and AGS cells after treatment with actinomycin D (D, E) and RNase R (F, G). (H, I) CircKIAA0907 localization was analyzed through comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 via qRT-PCR detection. * P<0.05.

Figure 2

Overexpression of circKIAA0907 triggered proliferation and autophagy inhibition, cell cycle arrest, and apoptosis promotion of gastric cancer (GC) cells in vitro. (A, B) Evaluation of circKIAA0907 overexpression efficiency was conducted via quantitative real-time polymerase chain reaction (qRT-PCR) after transfection of vector or circKIAA0907. (C, D) Proliferation analysis in transfected GC cells was performed by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay. (E, F) Apoptosis rate of transfected cells was determined using flow cytometry. (G, H) Apoptosis-related proteins were assayed using western blot. (I, J) Cell cycle was analyzed via flow cytometry. (K, L) Western blot was used for detecting the autophagy-associated markers. * P<0.05.

Figure 3

CircKIAA0907 could sponge miR-452-5p in gastric cancer (GC) cells. (A, B) Expression level of circKIAA0907 was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) after biotinylated-circKIAA0907 pull-down assay in HGC27 and AGS cells transfected with vector or circKIAA0907. (C, D) qRT-PCR was used to quantify the relative expression of candidate micro(mi)RNAs in HGC27 and AGS cells after administration of biotinylated-circKIAA0907 pull-down. (E) Binding sites of circKIAA0907 and miR-452-5p presented after analysis of circBank. (F, G) Pull-down assay was executed to prove the capture of circKIAA0907 by miR-452-5p. (H, I) Luciferase activities of transfected HGC27 and AGS cells were determined using the dual-luciferase reporter system. (J, K) MiR-452-5p level was examined using qRT-PCR after actinomycin D treatment at 0, 12, and 24 h in GC cells transfected with vector or circKIAA0907. (L, M) miR-452-5p was measured in GC tissues (L) and cells (M) by qRT-PCR. (N, O) MiR-452-5p level was determined through qRT-PCR after GC cells were transfected with vector or circKIAA0907. * P<0.05.

Figure 4

Increase of miR-452-5p reversed the inhibition of circKIAA0907 on the progression of gastric cancer (GC) cells. (A, B) After transfection of vector, circKIAA0907, circKIAA0907+miR-NC, and circKIAA0907+miR-452-5p in HGC27 and AGS cells, the expression of miR-452-5p was assayed via quantitative real-time polymerase chain reaction (qRT-PCR). (C, D) 3-(4, 5-Dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used for measuring proliferation ability in transfected cells. (E–H) Cell apoptosis was assessed with flow cytometry and western blot. (I, J) The percentage of each phase in transfected GC cells was examined by cell cycle detection. (K, L) Evaluation of autophagy was carried out by detecting associated proteins after western blot. * P<0.05.

Figure 5

CircKIAA0907 sponged miR-452-5p to elevate expression of its target KAT6B. (A) The starbase v2.0 was utilized for analyzing the binding sites of KAT6B 3′-UTR and miR-452-5p. (B, C) The dual-luciferase reporter assay was used to determine whether miR-452-5p interacted with KAT6B. (D) The impact of anti-miR-452-5p on KAT6B was assayed using western blot. (E, F) Analysis of KAT6B level in gastric cancer (GC) tissues (E) and cells (F) via western blot. (G) After the respective transfection of circKIAA0907, circKIAA0907+miR-452-5p, or relative controls in HGC27 and AGS cells, the protein expression of KAT6B was determined via western blot. * P<0.05.

Figure 6

CircKIAA0907 worked as a tumor repressor in gastric cancer (GC) progression via the miR-452-5p-mediated KAT6B upregulation. (A, B) KAT6B was measured using western blot in HGC27 and AGS cells transfected with anti-miR-NC, anti-miR-452-5p, anti-miR-452-5p+si-NC, or anti-miR-452-5p+si-KAT6B. (C, D) Examination of proliferation ability in transfected cells implemented through 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay. (E–H) Flow cytometry and western blot to evaluate cell apoptosis. (I, J) Cell cycle analysis using flow cytometry. (K, L) Cellular autophagy assessed utilizing western blot. * P<0.05.

Figure 7

CircKIAA0907 inhibited tumorigenesis of gastric cancer (GC) in vivo via the miR-452-5p/KAT6B axis. (A) Tumor volume was recorded weekly after injecting HGC27 and AGS cells transfected with circKIAA0907 or vector. (B, C) Tumors were weighed (B) after excision from mice (C). (D) Quantitative real-time polymerase chain reaction (qRT-PCR) was used for examining expression of circKIAA0907, miR-452-5p, and KAT6B mRNA. (E) KAT6B protein level detected using western blot. * P<0.05.