Abstract
Circular RNA has been reported to be a new noncoding RNA which plays important roles in tumor progression. One of the most common functions of circular RNA is to regulate microRNA expression by acting as a microRNA sponge. However, the circular RNA expression profile and function remain mostly unclear in gastric cancer. In the study, we explored the expression and function of circCOL1A1 (hsa_circ_0044556) in gastric cancer. We performed RT-PCR with divergent primers, mRNA stability assay, and RNase R digestion assay to characterize circCOL1A1 in gastric cancer cell lines. qRT-PCR was applied to detect the level of circCOL1A1 in both gastric cancer cell lines and tissues. Gain- and loss-of-function studies were carried out to detect the influence of circCOL1A1 on gastric cancer cells by performing CCK8, migration, and invasion assays. The regulation of the downstream genes was identified by qRT-PCR, western blot assay, dual luciferase assay, and RNA pull-down assay. The results showed that circCOL1A1 was highly expressed in both gastric cancer cells and tissues. Silence of circCOL1A1 inhibited the proliferation, migration, and invasion of gastric cancer cells. circCOL1A1 regulated the expression of miR-145 by acting as a microRNA sponge, and the influence of circCOL1A1 could be abrogated by miR-145 mimics. Our research shows that miR-145 plays its functions through targeting and regulating RABL3. Inhibition of circCOL1A1/miR-145/RABL3 could effectively suppress gastric cancer cell proliferation, migration, and invasion. circCOL1A1 also promote the transformation of M1 into M2 macrophage. Our study identified circCOL1A1 as a novel oncogenic circRNA and will provide more information for gastric cancer therapy.
Highlights
The results showed that silencing of circCOL1A1 weakened the proliferation of AGS (Figure 2(e)) and BGC-823 (Figure 2(f)), and overexpression of circCOL1A1 enhanced the proliferation of MKN-45 (Figure 2(g)) and SGC-7901 (Figure 2(h))
We identified the level of miR-145 in gastric cancer cell lines and clinical gastric cancer tissues by Quantitative real-time PCR (qRT-PCR)
The results show that miR-145 acts as a regulator of RABL3 in gastric cancer. miR-145/RABL3 might be a possible target for gastric cancer
Summary
MKN-45, SGC-7901, AGS, MGC-803, and BGC-823 gastric cancer cell lines were obtained from ATCC (American Type Culture Collection). Gastric cancer cell lines were cultured in RPMI 1640 with 10% FBS. 20 gastric cancer and adjacent tissues were obtained from Huai’an Second People’s Hospital. We applied Transwell chambers (Corning, NY, USA) to detect cancer cell migration. We applied Transwell chambers with 50 μl Matrigel (Corning, NY, USA) to detect cancer cell invasion. CircCOL1A1 siRNA, circCOL1A1 overexpression plasmids, miRNA inhibitor, and miRNA mimics were brought from GenePharma (Shanghai, China) and transfected into gastric cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) under the direction of the manufactory. Cells were cultured in 24-well plates and transfected with the indicated plasmids. QRT-PCR was applied to measure the RNA level. QRT-PCR was carried out to detect cytoplasmic and nuclear circCOL1A1 levels.
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