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Background Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored. Methods We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis. Results The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone. Conclusions The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.

Background Recent studies using single molecule, real-time (SMRT) sequencing revealed a substantial population of analyzable long cell-free DNA (cfDNA) in plasma. Potential clinical utilities of such long cfDNA in pregnancy and cancer have been demonstrated. However, the performance of different long-read sequencing platforms for the analysis of long cfDNA remains unknown. Methods Size biases of SMRT sequencing by Pacific Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies (ONT) were evaluated using artificial mixtures of sonicated human and mouse DNA of different sizes. cfDNA from plasma samples of pregnant women at different trimesters, hepatitis B carriers, and patients with hepatocellular carcinoma were sequenced with the 2 platforms. Results Both platforms showed biases to sequence longer (1500 bp vs 200 bp) DNA fragments, with PacBio showing a stronger bias (5-fold overrepresentation of long fragments vs 2-fold in ONT). Percentages of cfDNA fragments 500 bp were around 6-fold higher in PacBio compared with ONT. End motif profiles of cfDNA from PacBio and ONT were similar, yet exhibited platform-dependent patterns. Tissue-of-origin analysis based on single-molecule methylation patterns showed comparable performance on both platforms. Conclusions SMRT sequencing generated data with higher percentages of long cfDNA compared with nanopore sequencing. Yet, a higher number of long cfDNA fragments eligible for the tissue-of-origin analysis could be obtained from nanopore sequencing due to its much higher throughput. When analyzing the size and end motif of cfDNA, one should be aware of the analytical characteristics and possible biases of the sequencing platforms being used.

Background Jagged ends of plasma DNA are a recently recognized class of fragmentomic markers for cell-free DNA, reflecting the activity of nucleases. A number of recent studies have also highlighted the importance of jagged ends in the context of pregnancy and oncology. However, knowledge regarding the generation of jagged ends is incomplete. Methods Jaggedness of plasma DNA was analyzed based on Jag-seq, which utilized the differential methylation signals introduced by the DNA end-repair process. We investigated the jagged ends in plasma DNA using mouse models by deleting the deoxyribonuclease 1 (Dnase1), DNA fragmentation factor subunit beta (Dffb), or deoxyribonuclease 1 like 3 (Dnase1l3) gene. Results Aberrations in the profile of plasma DNA jagged ends correlated with the type of nuclease that had been genetically deleted, depending on nucleosomal structures. The deletion of Dnase1l3 led to a significant reduction of jaggedness for those plasma DNA molecules involving more than 1 nucleosome (e.g., size ranges 240-290 bp, 330-380 bp, and 420-470 bp). However, less significant effects of Dnase1 and Dffb deletions were observed regarding different sizes of DNA fragments. Interestingly, the aberration in plasma DNA jagged ends related to multinucleosomes was observed in human subjects with familial systemic lupus erythematosus with Dnase1l3 deficiency and human subjects with sporadic systemic lupus erythematosus. Conclusions Detailed understanding of the relationship between nuclease and plasma DNA jaggedness has opened up avenues for biomarker development.

Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5′ 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as “founder” end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.

The relationship between placental pathology and the maternal syndrome of preeclampsia is incompletely characterised. Mismatch between placental nutrient supply and fetal demands induces stress in the syncytiotrophoblast, the layer of placenta in direct contact with maternal blood. Such stress alters the content and increases the release of extracellular vesicles into the maternal circulation. 5’-tRNA fragments (5’-tRF) constitute the majority of short RNA in these vesicles. Here we characterise 5’-tRF profiles in syncytiotrophoblast vesicles in preeclampsia, reporting differential expression of >900 fragments. We detected preeclampsia-dysregulated 5’-tRF in peripheral plasma, where we identified a placentally-derived load. We demonstrated these 5’-tRF induce sterile inflammation in macrophages and monocytes. Our findings suggest syncytiotrophoblast-derived 5’-tRF may contribute to distant vascular inflammation, previously described in preeclampsia. This is the first demonstration of endocrine actions of vesicle-bound 5’-tRF, which may have relevance in other disciplines studying their functions, such as immunology and cancer biology.

Supplemental Tables S1-S5. Characteristics of the 45 normal tissues included in the PCDH10 expression analysis (S1); Characteristics of the 9 normal tissues included in the MALAT1 expression by ISH analysis (S2); Characteristics at primary treatment of 142 endometrial cancer patients from MoMaTEC* trial included in the MALAT1 investigation (S3); PCDH10 RNA expression from 142 prospectively collected endometrial cancer tumors, 19 metastatic lesions and 18 endometrial hyperplasia samples in relation to clinicopathologic data (S4); Oligos used for qRT-PCR, ChIP-PCR, Cloning and Bisulfate genomic sequencing (S5).

Background Human plasma contains RNA transcripts released by multiple cell types within the body. Single-cell transcriptomic analysis allows the cellular origin of circulating RNA molecules to be elucidated at high resolution and has been successfully utilized in the pregnancy context. We explored the application of a similar approach to develop plasma RNA markers for cancer detection. Methods Single-cell RNA sequencing was performed to decipher transcriptomic profiles of single cells from hepatocellular carcinoma (HCC) samples. Cell-type-specific transcripts were identified and used for deducing the cell-type-specific gene signature (CELSIG) scores of plasma RNA from patients with and without HCC. Results Six major cell clusters were identified, including hepatocyte-like, cholangiocyte-like, myofibroblast, endothelial, lymphoid, and myeloid cell clusters based on 4 HCC tumor tissues as well as their paired adjacent nontumoral tissues. The CELSIG score of hepatocyte-like cells was significantly increased in preoperative plasma RNA samples of patients with HCC (n = 14) compared with non-HCC participants (n = 49). The CELSIG score of hepatocyte-like cells declined in plasma RNA samples of patients with HCC within 3 days after tumor resection. Compared with the discriminating power between patients with and without HCC using the abundance of ALB transcript in plasma [area under curve (AUC) 0.72)], an improved performance (AUC: 0.84) was observed using the CELSIG score. The hepatocyte-specific transcript markers in plasma RNA were further validated by ddPCR assays. The CELSIG scores of hepatocyte-like cell and cholangiocyte trended with patients’ survival. Conclusions The combination of single-cell transcriptomic analysis and plasma RNA sequencing represents an approach for the development of new noninvasive cancer markers.

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5′ ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson’s absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.

Background Although the characterization of cell-free extrachromosomal circular DNA (eccDNA) has gained much research interest, the methylation status of these molecules is yet to be elucidated. We set out to compare the methylation densities of plasma eccDNA of maternal and fetal origins, and between small and large molecules. The clearance of fetal eccDNA from maternal circulation was also investigated. Methods We developed a sequencing protocol for eccDNA methylation analysis using tagmentation and enzymatic conversion approaches. A restriction enzyme-based approach was applied to verify the tagmentation results. The efficiency of cell-free fetal eccDNA clearance was investigated by fetal eccDNA fraction evaluations at various postpartum time points. Results The methylation densities of fetal eccDNA (median: 56.3%; range: 40.5–67.6%) were lower than the maternal eccDNA (median: 66.7%; range: 56.5–75.7%) (P = 0.02, paired t-test). In addition, eccDNA molecules from the smaller peak cluster (180–230 bp) were of lower methylation levels than those from the larger peak cluster (300–450 bp). Both of these findings were confirmed using the restriction enzyme approach. We also observed comparable methylation densities between linear and eccDNA of both maternal and fetal origins. The average half-lives of fetal linear and eccDNA in the maternal blood were 30.2 and 29.7 min, respectively. Conclusions We found that fetal eccDNA in plasma was relatively hypomethylated compared to the maternal eccDNA. The methylation densities of eccDNA were positively correlated with their sizes. In addition, fetal eccDNA was found to be rapidly cleared from the maternal blood after delivery, similar to fetal linear DNA.

BackgroundDouble-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends.MethodsJagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing.ResultsThe jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83.ConclusionsJagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.

Background Analysis of circulating tumor DNA has become increasingly important as a tool for cancer care. However, the focus of previous studies has been on short fragments of DNA. Also, bisulfite sequencing, a conventional approach for methylation analysis, causes DNA degradation, which is not ideal for the assessment of long DNA properties and methylation patterns. This study attempted to overcome such obstacles by single-molecule sequencing. Methods Single-molecule real-time (SMRT) sequencing was used to sequence plasma DNA. We performed fragment size and direct methylation analysis for each molecule. A methylation score concerning single-molecule methylation patterns was used for cancer detection. Results A substantial proportion of plasma DNA was longer than 1 kb with a median of 16% in hepatocellular carcinoma (HCC) patients, hepatitis B virus carriers, and healthy individuals. The longest plasma DNA molecule in the HCC patients was 39.8 kb. Tumoral cell-free DNA (cfDNA) was generally shorter than nontumoral cfDNA. The longest tumoral cfDNA was 13.6 kb. Tumoral cfDNA had lower methylation levels compared with nontumoral cfDNA (median: 59.3% vs 76.9%). We developed and analyzed a metric reflecting single-molecule methylation patterns associated with cancer, named the HCC methylation score. HCC patients displayed significantly higher HCC methylation scores than those without HCC. Interestingly, compared to using short cfDNA (area under the receiver operating characteristic [ROC] curve, AUC: 0.75), the use of long cfDNA molecules greatly enhanced the discriminatory power (AUC: 0.91). Conclusions A previously unidentified long cfDNA population was revealed in cancer patients. The presence and direct methylation analysis of these molecules open new possibilities for cancer liquid biopsy.

Plasma DNA fragmentomics is an emerging area of research covering plasma DNA sizes, end points, and nucleosome footprints. In the present study, we found a significant increase in the diversity of plasma DNA end motifs in patients with hepatocellular carcinoma (HCC). Compared with patients without HCC, patients with HCC showed a preferential pattern of 4-mer end motifs. In particular, the abundance of plasma DNA motif CCCA was much lower in patients with HCC than in subjects without HCC. The aberrant end motifs were also observed in patients with other cancer types, including colorectal cancer, lung cancer, nasopharyngeal carcinoma, and head and neck squamous cell carcinoma. We further observed that the profile of plasma DNA end motifs originating from the same organ, such as the liver, placenta, and hematopoietic cells, generally clustered together. The profile of end motifs may therefore serve as a class of biomarkers for liquid biopsy in oncology, noninvasive prenatal testing, and transplantation monitoring. Significance: Plasma DNA molecules originating from the liver, HCC and other cancers, placenta, and hematopoietic cells each harbor a set of characteristic plasma DNA end motifs. Such markers carry tissue-of-origin information and represent a new class of biomarkers in the nascent field of fragmentomics. This article is highlighted in the In This Issue feature, p. 627

Background Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. Plasma EBV DNA is a validated screening tool for NPC. In screening, there are some individuals who do not have NPC but carry EBV DNA in plasma. Currently it is not known from screening if there may be any genotypic differences in EBV isolates from NPC and non-NPC subjects. Also, low concentrations of EBV DNA in plasma could pose challenge to such EBV genotypic analysis through plasma DNA sequencing. Methods In a training dataset comprised of plasma DNA sequencing data of NPC and non-NPC subjects, we studied the difference in the EBV single nucleotide variant (SNV) profiles between the two groups. The most differentiating SNVs across the EBV genome were identified. We proposed an NPC risk score to be derived from the genotypic patterns over these SNV sites. We subsequently analyzed the NPC risk scores in a testing set. Results A total of 661 significant SNVs across the EBV genome were identified from the training set. In the testing set, NPC plasma samples were shown to have high NPC risk scores, which suggested the presence of NPC-associated EBV SNV profiles. Among the non-NPC samples, there was a wide range of NPC risk scores. These results support the presence of diverse SNV profiles of EBV isolates from non-NPC subjects. Conclusion EBV genotypic analysis is feasible through plasma DNA sequencing. The NPC risk score may be used to inform the cancer risk based on the EBV genome-wide SNV profile.

Significance We observed the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. We found that the plasma eccDNA molecules were longer than their linear counterparts. Among such eccDNA molecules, those of fetal origin were shorter than those of maternal origin. Characteristic dual-repeat patterns of eccDNA junctions might shed light on their possible generation mechanisms and provide them with distinctive signatures over linear cell-free DNA. Furthermore, the closed circular structure of eccDNA might allow resistance to exonucleases and thus higher stability of these molecules over their linear counterparts. These features of eccDNA provide opportunities for research and biomarker development. This work represents an example in the nascent field of plasma DNA topologics., We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5′-untranslated regions (5′-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.

Significance We revealed a large population of long cell-free DNA molecules (up to 23,635 bp in length) in maternal plasma and developed an approach which leveraged the abundance of CpG sites on long molecules to deduce the tissue of origin of individual plasma DNA molecules based on single-molecule methylation analysis. We illustrated how such an approach may be utilized to achieve noninvasive prenatal testing of monogenic diseases. We also revealed a reduction in amounts of such long cell-free DNA molecules and a different end motif profile in maternal plasma DNA from pregnancies with preeclampsia. Hence, long cell-free DNA molecules represent a valuable resource of biomarker development for pregnancy-associated disorders., In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g.

Cell-free extrachromosomal circular DNA (eccDNA) as a distinct topological form from linear DNA has recently gained increasing research interest, with possible clinical applications as a class of biomarkers. In this study, we aimed to explore the relationship between nucleases and eccDNA characteristics in plasma. By using knockout mouse models with deficiencies in deoxyribonuclease 1 (DNASE1) or deoxyribonuclease 1 like 3 (DNASE1L3), we found that cell-free eccDNA in Dnase1l3-/- mice exhibited larger size distributions than that in wild-type mice. Such size alterations were not found in tissue eccDNA of either Dnase1-/- or Dnase1l3-/- mice, suggesting that DNASE1L3 could digest eccDNA extracellularly but did not seem to affect intracellular eccDNA. Using a mouse pregnancy model, we observed that in Dnase1l3-/- mice pregnant with Dnase1l3+/- fetuses, the eccDNA in the maternal plasma was shorter compared with that of Dnase1l3-/- mice carrying Dnase1l3-/- fetuses, highlighting the systemic effects of circulating fetal DNASE1L3 degrading the maternal eccDNA extracellularly. Furthermore, plasma eccDNA in patients with DNASE1L3 mutations also exhibited longer size distributions than that in healthy controls. Taken together, this study provided a hitherto missing link between nuclease activity and the biological manifestations of eccDNA in plasma, paving the way for future biomarker development of this special form of DNA molecules.

Single-stranded ends of double-stranded DNA (jagged ends) are more abundant in urinary DNA than in plasma DNA. However, the lengths of jagged ends in urinary DNA remained undetermined, as a previous method used for urinary DNA jagged end sequencing analysis (Jag-seq) relied on unmethylation at CpG sites, limiting the resolution. Here, we performed high-resolution Jag-seq analysis using methylation at non-CpG cytosine sites, allowing determination of exact length of jagged ends. The urinary DNA bore longer jagged ends (~26-nt) than plasma DNA (~17-nt). The jagged end length distribution displayed 10-nt periodicities in urinary DNA, which were much less observable in plasma DNA. Amplitude of the 10-nt periodicities increased in patients with renal cell carcinoma. Heparin treatment of urine diminished the 10-nt periodicities. The urinary DNA jagged ends often extended into nucleosomal cores, suggesting potential interactions with histones. This study has thus advanced our knowledge of jagged ends in urine DNA.

Circular RNA (circRNA) is an important factor for regulating the progression of many cardiovascular diseases, including acute myocardial infarction (AMI). However, the role of circ_0124644 in AMI progression remains unclear. Hypoxia was used to induce cardiomyocytes injury. The expression of circ_0124644, microRNA (miR)-590-3p, and SRY-box transcription factor 4 (SOX4) mRNA was measured by qRT-PCR. Cell counting kit 8 (CCK8) assay and flow cytometry were utilized to detect cell viability, cell cycle progression, and apoptosis. The protein levels of apoptosis markers and SOX4 were determined by western blot (WB) analysis, and the levels of oxidative stress markers were assessed using commercial Assay Kits. Dual-luciferase reporter assay, RIP assay, and RNA pull-down assay were employed to confirm the interaction between miR-590-3p and circ_0124644 or SOX4. Circ_0124644 was upregulated in AMI patients and hypoxia-induced cardiomyocytes. Hypoxia could inhibit cardiomyocytes viability, cell cycle process, and promote apoptosis and oxidative stress, while silencing circ_0124644 could alleviate hypoxia-induced cardiomyocytes injury. In terms of mechanism, circ_0124644 could target miR-590-3p. MiR-590-3p overexpression could relieve hypoxia-induced cardiomyocytes injury. Also, the suppressive effect of circ_0124644 knockdown on hypoxia-induced cardiomyocytes injury could be reversed by miR-590-3p inhibitor. Moreover, SOX4 was found to be a target of miR-590-3p, and its overexpression also could reverse the regulation of miR-590-3p on hypoxia-induced cardiomyocytes injury. Circ_0124644 silencing could alleviate hypoxia-induced cardiomyocytes injury by regulating the miR-590-3p/SOX4 axis, suggesting that it might be a target for alleviating AMI.