Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.

Identification of expression profiles and prognostic value of RFCs in colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, with its incidence closely tied to DNA damage. The Replication Factor C (RFC) complexes comprises five protein subunits: RFC1, RFC2, RFC3, RFC4, and RFC5. These RFC complexes play crucial roles in DNA replication, repair pathways, activities post DNA damage, and ATP-dependent processes during DNA synthesis. However, the impact of RFC complexes proteins on CRC prognosis remains unclear. To explore this, we employed a computational analysis approach, utilizing platforms such as the DepMap portal, GEPIA, DAVID Bioinformatics for KEGG pathway analysis, Human Protein Atlas (HPA), STRING, and TIMER. Our results indicate that the mRNA levels of RFC1 and RFC5 were the least expressed among CRC cell lines compared to other RFC complex subunits. Notably, low RFC1 and RFC5 expression was correlated with poor prognosis in terms of CRC patients' overall survival (OS). Immunohistochemical results from the Human Protein Atlas demonstrated medium staining for RFC1, RFC2, and RFC5 in CRC tissues. Furthermore, the low expression of RFC1 and RFC5 showed a significant correlation with high expression levels of miR-26a-5p and miR-636, impacting cell proliferation through mismatch repair, DNA replication, and the nucleotide excision repair pathway. Although the precise functions of RFC1 in cancer are still unknown, our findings suggest that the small-molecule single target, CHEMBL430483, and multiple target molecules could be potential treatments for CRC. In conclusion, the elevated expression of miR-26a-5p and miR-636 targeting RFC1 and RFC5 expression holds promise as a potential biomarker for early-stage CRC detection. These insights provide novel directions and strategies for CRC therapies.

Differences between bacteria and eukaryotes in clamp loader mechanism, a conserved process underlying DNA replication.

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the Escherichia coli clamp loader at high resolution using cryo-electron microscopy. We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

Structure of the PCNA unloader Elg1-RFC.

During DNA replication, the proliferating cell nuclear antigen (PCNA) clamps are loaded onto primed sites for each Okazaki fragment synthesis by the AAA+ heteropentamer replication factor C (RFC). PCNA encircling duplex DNA is quite stable and is removed from DNA by the dedicated clamp unloader Elg1-RFC. Here, we show the cryo-EM structure of Elg1-RFC in various states with PCNA. The structures reveal essential features of Elg1-RFC that explain how it is dedicated to PCNA unloading. Specifically, Elg1 contains two external loops that block opening of the Elg1-RFC complex for DNA binding, and an "Elg1 plug" domain that fills the central DNA binding chamber, thereby reinforcing the exclusive PCNA unloading activity of Elg1-RFC. Elg1-RFC was capable of unloading PCNA using non-hydrolyzable AMP-PNP. Both RFC and Elg1-RFC could remove PCNA from covalently closed circular DNA, indicating that PCNA unloading occurs by a mechanism that is distinct from PCNA loading. Implications for the PCNA unloading mechanism are discussed.

Structural polymorphism of the nucleic acids in pentanucleotide repeats associated with the neurological disorder CANVAS.

Short tandem repeats are inherently unstable during DNA replication depending on repeat length, and the expansion of the repeat length in the human genome is responsible for repeat expansion disorders. Pentanucleotide AAGGG and ACAGG repeat expansions in intron 2 of the gene encoding replication factor C subunit 1 (RFC1) cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and other phenotypes of late-onset cerebellar ataxia. Herein, we reveal the structural polymorphism of the RFC1 repeats associated with CANVAS in vitro. Single-stranded AAGGG repeat DNA formed a hybrid-type G-quadruplex, whereas its RNA formed a parallel-type G-quadruplex with three layers. The RNA of the ACAGG repeat formed hairpin structure comprising C-G and G-C base pairs with A:A and GA:AG mismatched repeats. Furthermore, both pathogenic repeat RNAs formed more rigid structures than those of the nonpathogenic repeat RNAs. These findings provide novel insights into the structural polymorphism of the RFC1 repeats, which may be closely related to the disease mechanism of CANVAS.

Abnormal activation of RFC3, A YAP1/TEAD downstream target, promotes gastric cancer progression.

BACKGROUND: Gastric cancer (GC) is a malignant tumor with a high mortality rate, and thus, it is necessary to explore molecular mechanisms underlying its progression. While replication factor C subunit 3 (RFC3) has been demonstrated to function as an oncogene in many cancers, its role in GC remains unclear. METHODS: Tumor tissues were collected from clinical GC patients, and the expression of RFC3 was analyzed. NCI-N87 and HGC-27 cells were infected with lentivirus sh-RFC3 to knock down RFC3 expression. RFC3 expression levels were determined, in addition to cell biological behaviors both in vitro and in vivo. The relationship between RFC3 and the YAP1/TEAD signaling pathway was detected by dual luciferase reporter assay. RESULTS: RFC3 was upregulated in GC tumor tissues. RFC3 knockdown inhibited cell proliferation, promoted cell apoptosis of GC cells, and suppressed cell migration and invasion. Moreover, depleted RFC3 suppressed tumor growth and metastasis in vivo. Mechanistically, the YAP1/TEAD axis activated RFC3 expression transcriptionally by binding to the RFC3 promoter. CONCLUSIONS: RFC3 was transcriptional activated by the YAP1/TEAD signaling pathway, thus promoting GC progression. RFC3 may be a promising therapeutic target for GC.

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures.

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts.

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.

RFC3 serves as a novel prognostic biomarker and target for head and neck squamous cell carcinoma.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer that originates from the squamous cells. The role of the replication factor C subunit 3 (RFC3) in HNSCC progression remains elusive. The aim of this study was to uncover RFC3 significance in HNSCC. METHODS: The Cancer Genome Atlas (TCGA-HNSCC) dataset was initially used to assess RFC3 expression and its association with HNSCC clinical features. Subsequently, quantitative reverse transcription PCR (RT-qPCR) confirmed RFC3 mRNA expression in oral squamous cell carcinoma (OSCC), a primary HNSCC type. Survival rates were evaluated using the Kaplan-Meier plot, while the Tumor Immune Estimation Resource (TIMER) database probed RFC3-immune cell interaction. Additionally, in silico tools were used to examine the RFC3 protein network and engagement in HNSCC pathways. RESULTS: RFC3 expression is significantly upregulated in HNSCC, including OSCC. Upregulated RFC3 expression was significantly correlated with the clinicopathological features of HNSCC, including tumor stage, grade, metastasis, and patient survival. RFC3 is also associated with immune cell infiltration. Functional analysis has highlighted its involvement in DNA replication, mismatch repair, and cell cycle pathways. Interestingly, RFC3 high expression is linked to well-known oncogenic signaling pathways, such as MYC/MYCN, HIPPO, and mTOR. CONCLUSIONS: In conclusion, RFC3 can be considered a novel prognostic biomarker for HNSCC, and further studies on its functional mechanisms may help to use RFC3 as a therapeutic target for HNSCC. CLINICAL RELEVANCE: The clinical relevance of this study lies in identifying RFC3 as a novel biomarker and prognostic indicator for HNSCC, offering insights that could impact future clinical approaches.

RFC2 promotes aerobic glycolysis and progression of colorectal cancer.

BACKGROUND: Replication factor C subunit 2 (RFC2) participates in the growth and metastasis of various malignancies. Our study investigated the roles of RFC2 in colorectal cancer (CRC). RESULTS: RFC2 expression was upregulated in CRC tissues and cells. High RFC2 expression was associated with poor prognosis. Knockdown RFC2 inhibited proliferation, induced apoptosis, and suppressed migration and invasion of CRC cells. CREB5 was a transcription factor of RFC2, and CREB5 knockdown suppressed RFC2 expression. Furthermore, RFC2 promoted aerobic glycolysis and MET/PI3K/AKT/mTOR pathway. CONCLUSION: RFC2 promoted the progression of CRC cells via activating aerobic glycolysis and the MET/PI3K/AKT/mTOR pathway.

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology.

Rad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' end, thus presumably leaving 9-1-1 on duplex 3' ss/double-stranded DNA (dsDNA) after Rad24-RFC ejects from DNA. We captured five Rad24-RFC-9-1-1 loading intermediates using a 10-nt gap DNA. We also determined the structure of Rad24-RFC-9-1-1 using a 5-nt gap DNA. The structures reveal that Rad24-RFC is unable to melt DNA ends and that a Rad24 loop limits the dsDNA length in the chamber. These observations explain Rad24-RFC's preference for a preexisting gap of over 5-nt ssDNA and suggest a direct role of the 9-1-1 in gap repair with various TLS (trans-lesion synthesis) polymerases in addition to signaling the ATR kinase.

RFC5, regulated by circ_0038985/miR-3614-5p, functions as an oncogene in the progression of colorectal cancer.

Replication factor C 5 (RFC5) is involved in a variety of biological functions of cancer. However, the expression pattern of RFC5 and the underlying mechanisms in colorectal cancer (CRC) remain elusive. Here, we show that RFC5 is significantly upregulated in CRC tissues and cells. Patients with CRC and increased RFC5 levels have an unfavorable prognosis. RFC5 can promote the proliferation, migration, and invasion of CRC cells and inhibit the apoptosis of CRC cells. Additionally, upstream of RFC5, we constructed the competing endogenous RNA network and confirmed that RFC5 in this network was inhibited by miR-3614-5p by directly targeting its 3'-untranslated regions. We verified that circ_0038985, which is positively correlated with RFC5, directly targeted miR-3614-5p. Overexpression of circ_0038985 promoted CRC cell migration and invasion, and these effects were partially reversed by the reintroduction of miR-3614-5p. Moreover, we found that RFC5 may promote the vascular endothelial growth factor A (VEGFa)/vascular endothelial growth factor receptor 2 (VEGFR2)/extracellular signal-regulated protein kinase (ERK) pathway. The knockdown of RFC5 reduced CRC tumorigenesis in vivo. Collectively, these data demonstrate that the circ_0038985/miR-3614-5p/RFC5 axis plays a critical role in the progression of CRC, and RFC5 may promote CRC progression by affecting the VEGFa/VEGFR2/ERK pathway.

Yeast 9-1-1 complex acts as a sliding clamp for DNA synthesis by DNA polymerase ε.

Eukaryotic cells harbor two DNA-binding clamps, proliferating cell nuclear antigen (PCNA), and another clamp commonly referred to as 9-1-1 clamp. In contrast to the essential role of PCNA in DNA replication as a sliding clamp for DNA polymerase (Pol) δ, no such role in DNA synthesis has been identified for the human 9-1-1 clamp or the orthologous yeast 17-3-1 clamp. The only role identified for either the 9-1-1 or 17-3-1 clamp is in the recruitment of signal transduction kinases, which affect the activation of cell cycle checkpoints in response to DNA damage. However, unlike the loading of PCNA by the replication factor C (RFC) clamp loader onto 3'-recessed DNA junctions for processive DNA synthesis by Polδ, the 17-3-1 clamp or the 9-1-1 clamp is loaded by their respective clamp loader Rad24-RFC or RAD17-RFC onto the 5'-recessed DNA junction of replication protein A-coated DNA for the recruitment of signal transduction kinases. Here, we identify a novel role of 17-3-1 clamp as a sliding clamp for DNA synthesis by Polε. We provide evidence that similar to the loading of PCNA by RFC, the 17-3-1 clamp is loaded by the Rad24-RFC clamp loader at the 3'-recessed DNA junction in an ATP-dependent manner. However, unlike PCNA, the 17-3-1 clamp does not enhance the processivity of DNA synthesis by Polε; instead, it greatly increases the catalytic efficiency of Polε for correct nucleotide incorporation. Furthermore, we show that the same PCNA-interacting peptide domain in the polymerase 2 catalytic subunit mediates Polε interaction with the 17-3-1 clamp and with PCNA.

Knockdown of RFC4 inhibits the cell proliferation of nasopharyngeal carcinoma in vitro and in vivo.

Nasopharyngeal carcinoma (NPC) is a malignant tumor that mainly occurs in East and Southeast Asia. Although patients benefit from the main NPC treatments (e.g., radiotherapy and concurrent chemotherapy), persistent and recurrent diseases still occur in some NPC patients. Therefore, investigating the pathogenesis of NPC is of great clinical significance. In the present study, replication factor c subunit 4 (RFC4) is a key potential target involved in NPC progression via bioinformatics analysis. Furthermore, the expression and mechanism of RFC4 in NPC were investigated in vitro and in vivo. Our results revealed that RFC4 was more elevated in NPC tumor tissues than in normal tissues. RFC4 knockdown induced G2/M cell cycle arrest and inhibited NPC cell proliferation in vitro and in vivo. Interestingly, HOXA10 was confirmed as a downstream target of RFC4, and the overexpression of HOXA10 attenuated the silencing of RFC4-induced cell proliferation, colony formation inhibition, and cell cycle arrest. For the first time, this study reveals that RFC4 is required for NPC cell proliferation and may play a pivotal role in NPC tumorigenesis.

Knockdown of RFC4 inhibits the cell proliferation of nasopharyngeal carcinoma in vitro and in vivo / 医学前沿

Nasopharyngeal carcinoma (NPC) is a malignant tumor that mainly occurs in East and Southeast Asia. Although patients benefit from the main NPC treatments (e.g., radiotherapy and concurrent chemotherapy), persistent and recurrent diseases still occur in some NPC patients. Therefore, investigating the pathogenesis of NPC is of great clinical significance. In the present study, replication factor c subunit 4 (RFC4) is a key potential target involved in NPC progression via bioinformatics analysis. Furthermore, the expression and mechanism of RFC4 in NPC were investigated in vitro and in vivo. Our results revealed that RFC4 was more elevated in NPC tumor tissues than in normal tissues. RFC4 knockdown induced G2/M cell cycle arrest and inhibited NPC cell proliferation in vitro and in vivo. Interestingly, HOXA10 was confirmed as a downstream target of RFC4, and the overexpression of HOXA10 attenuated the silencing of RFC4-induced cell proliferation, colony formation inhibition, and cell cycle arrest. For the first time, this study reveals that RFC4 is required for NPC cell proliferation and may play a pivotal role in NPC tumorigenesis.

Sequential gene expression analysis of cervical malignant transformation identifies RFC4 as a novel diagnostic and prognostic biomarker.

BACKGROUND: Cervical squamous cell carcinoma (SCC) is known to arise through increasingly higher-grade squamous intraepithelial lesions (SILs) or cervical intraepithelial neoplasias (CINs). This study aimed to describe sequential molecular changes and identify biomarkers in cervical malignant transformation. METHODS: Multidimensional data from five publicly available microarray and TCGA-CESC datasets were analyzed. Immunohistochemistry was carried out on 354 cervical tissues (42 normal, 62 CIN1, 26 CIN2, 47 CIN3, and 177 SCC) to determine the potential diagnostic and prognostic value of identified biomarkers. RESULTS: We demonstrated that normal epithelium and SILs presented higher molecular homogeneity than SCC. Genes in the region (e.g., 3q, 12q13) with copy number alteration or HPV integration were more likely to lose or gain expression. The IL-17 signaling pathway was enriched throughout disease progression with downregulation of IL17C and decreased Th17 cells at late stage. Furthermore, we identified AURKA, TOP2A, RFC4, and CEP55 as potential causative genes gradually upregulated during the normal-SILs-SCC transition. For detecting high-grade SIL (HSIL), TOP2A and RFC4 showed balanced sensitivity (both 88.2%) and specificity (87.1 and 90.1%), with high AUC (0.88 and 0.89). They had equivalent diagnostic performance alone to the combination of p16INK4a and Ki-67. Meanwhile, increased expression of RFC4 significantly and independently predicted favorable outcomes in multi-institutional cohorts of SCC patients. CONCLUSIONS: Our comprehensive study of gene expression profiling has identified dysregulated genes and biological processes during cervical carcinogenesis. RFC4 is proposed as a novel surrogate biomarker for determining HSIL and HSIL+, and an independent prognostic biomarker for SCC.

Unexpected new insights into DNA clamp loaders: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage.

Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3' primed DNA by the replication factor C (RFC) hetero-pentameric clamp loader. Eukaryotes also have three alternative RFC-like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24-RFC (Rad17-RFC in human) that loads a 9-1-1 heterotrimer clamp onto a recessed 5' end of DNA. Recent structural studies of Rad24-RFC have discovered an unexpected 5' DNA binding site on the outside of the clamp loader and reveal how a 5' end can be utilized for loading the 9-1-1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5' DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism.

Multistep loading of a DNA sliding clamp onto DNA by replication factor C.

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

CircCOL1A2 Sponges MiR-1286 to Promote Cell Invasion and Migration of Gastric Cancer by Elevating Expression of USP10 to Downregulate RFC2 Ubiquitination Level.

Gastric cancers (GC) are generally malignant tumors, occurring with high incidence and threatening public health around the world. Circular RNAs (circRNAs) play crucial roles in modulating various cancers, including GC. However, the functions of circRNAs and their regulatory mechanism in colorectal cancer (CRC) remain largely unknown. This study focuses on both the role of circCOL1A2 in CRC progression as well as its downstream molecular mechanism. Quantitative polymerase chain reaction (qPCR) and western blot were adopted for gene expression analysis. Functional experiments were performed to study the biological functions. Fluorescence in situ hybridization (FISH) and subcellular fraction assays were employed to detect the subcellular distribution. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), RNA pull-down, and immunofluorescence (IF) and immunoprecipitation (IP) assays were used to explore the underlying mechanisms. Our results found circCOL1A2 to be not only upregulated in GC cells, but that it also propels the migration and invasion of GC cells. CircCOL1A2 functions as a competing endogenous RNA (ceRNA) by sequestering microRNA-1286 (miR-1286) to modulate ubiquitin-specific peptidase 10 (USP10), which in turn spurs the migration and invasion of GC cells by regulating RFC2. In sum, CircCOL1A2 sponges miR-1286 to promote cell invasion and migration of GC by elevating the expression of USP10 to downregulate the level of RFC2 ubiquitination. Our study offers a potential novel target for the early diagnosis and treatment of GC.