Circular RNAs (circRNAs), a type of non-coding RNA, are generated by non-canonical back-splicing events and are highly expressed in the central nervous system (CNS). Numerous studies have shown that they are involved in many pathological and physiological processes. In April 2024, the research team led by Professor Yao Honghong from Southeast University published an article titled "Engagement of N6-methyladenisine methylation of Gng4 mRNA in astrocyte dysfunction regulated by CircHECW2" in the journal Acta Pharmaceutica Sinica B (IF = 14.5). In this article, the authors found that the level of circular RNA HECW2 (circHECW2) was significantly increased in the plasma of patients with major depressive disorder (MDD) and in the mouse model of chronic unpredictable stress (CUS). Notably, the downregulation of circHECW2 could alleviate astrocyte dysfunction and CUS-induced depression-like behaviors. In addition, it was further demonstrated that the downregulation of circHECW2 increased the expression of the methyltransferase WTAP, leading to an increase in the expression of Gng4 through m6A modification, which provided functional insights into the correlation between circHECW2 and m6A methylation. This indicates that circHECW2 may represent a potential target for the treatment of MDD. circHECW2 is upregulated in CUS mice and MDD patients It is noteworthy that LPS- and CUS-induced models of depression are well-documented in studies relevant to depression. The authord's previous study first demonstrated that circHECW2 levels were increased in the hippocampus of LPS treated mice. Thus, to investigate the potential involvement of circHECW2 in depression (Fig. 1C and D), the author isolated the hippocampus and collected plasma from CUS mice. Next, the author examined the levels of circHECW2 in the plasma of MDD patients and healthy control individuals (HCs), found that circHECW2 levels were markedly increased in MDD patients (Fig. 1E). Notably, our analysis revealed a positive correlation between circHECW2 levels and the scores of the Hamilton Rating Scale for depression 24 items (HAMD-24) (Fig. 1F), the scores of the Montgomery-Asberg Depression Rating Scale (MADRS) (Fig. 1G) and the scores of the Hamilton Anxiety Scale (HAMA) (Fig. 1H). Additionally, through linear regression analysis, the author uncovered that MDD patients with elevated circHECW2 expression levels and high scores on the childhood trauma questionnaire (CTQ) displayed more severe depression symptoms (Fig. 1J). To assess the predictive capacity of circHECW2 levels for MDD outcomes, the author examined the changes in circHECW2 level two weeks after treatment in MDD patient plasma and found that the level of circHECW2 was decreased 2 weeks after treatment in MDD patient plasma (Fig. 1K). The downregulation of circHECW2 ameliorates the behaviors induced by CUS After microinjection for 2 weeks, the author examined the efficacy of the lentiviral transduction and found that the expression of circHECW2 was decreased in shRNA-circHECW2-injected mice (Fig. 2C). Behavioural tests including sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST) were employed to evaluate the effect of circHECW2. The sucrose preference of the CUS-treated mice was decreased, indicative of anhedonia. Encouragingly, this deficit was significantly alleviated by the downregulation of circHECW2 expression (Fig. 2D). In both the FST and TST, the time of immobility was notably prolonged in the CUS mice, and these effects were markedly ameliorated in shRNA-circHECW2-injected mice (Fig. 2E and F). Role of circHECW2 on astrocyte dysfunction in CUS mice hippocampus Subsequently, the author investigated the cellular mechanism through which circHECW2 affects functional recovery after CUS. To further assess the cell types in which circHECW2 expression is upregulated, the author detected the expression of circHECW2 in astrocytes, microglia, neurons, and endothelial cells from the CUS mice brain (Fig. 3A). The results revealed a significant upregulation of astrocyte-derived circHECW2 in CUS compared to microglia-, neuronal-, or endothelial cell-derived circHECW2 (Fig. 3B). Additionally, the fluorescence in situ hybridization staining indicated that circHECW2 was abundant in astrocytes (Fig. 3C). Furthermore, shRNA-circHECW2 treatment significantly mitigated the decrease in GFAP expression observed in CUS mice (Fig. 3D and E). Then, the author detected the function of shRNA-circHECW2 on the astrocyte’s morphology using GFAP and 3D reconstruction (Fig. 3F). Sholl analysis indicated that astrocyte dysfunction was induced by CUS, as evidenced by a reduction in branch numbers, length, and volume of astrocytes. Importantly, these deficits were markedly improved by shRNA-circHECW2 treatment (Fig. 3GeI). Taken together, these results suggest that the abnormal upregulation of circHECW2 in astrocytes may represent a critical molecular event in the progression of depression. circHECW2 inhibits m6A methylation by downregulating WTAP Given the potential role of m6A methylation in MDD and the mutual regulation between circRNAs and m6A methylation, we embarked on an investigation to determine whether circRNA’s regulatory role in the pathological processes of depression, particularly in astrocyte-mediated mechanisms, involves m6A modifications. CUS led to a decrease in m6A levels in the hippocampus, an effect that was significantly mitigated by shRNAcircHECW2, indicating a regulation of circHECW2 on m6A methylation (Fig. 4A). We next measured the expression of m6Amodifying enzymes above in CUS mouse models both in mRNA and protein levels, found that only the protein level of WTAP was reduced in the hippocampus of CUS mice (Fig. 4B). Subsequently, we used mouse primary astrocytes transduced with shRNA-circHECW2 lentivirus or circHECW2-overexpressed plasmid for further investigations(Fig. 4C and D). The expression of WTAP was significantly increased in shRNA-circHECW2-treated cells (Fig. 4E), whereas the Western blot analysis indicated that circHECW2 did not alter the levels of METTL3, METTL14, FTO, and ALKBH5, suggesting a specific association between circHECW2 and WTAP. To confirm these findings, primary astrocytes were transduced with the circHECW2 circHECW2-overexpressed plasmid and WTAP was significantly decreased in astrocytes (Fig. 4F). Next, pulldown assay was used to explore the interaction between circHECW2 and WTAP, and circHECW2 showed a stronger affinity to WTAP (Fig. 4G). Furthermore, in vivo experiments demonstrated that shRNA-circHECW2 significantly improved the decrease in WTAP expression induced by the CUS model (Fig. 4H). To explore why WTAP decreased after CUS, we employed immunoprecipitation to detect ubiquitination. The lysine 48-linked ubiquitination (Ub-K48) level of WTAP was significantly decreased by circHECW2 knockdown in the CUS model (Fig. 4I). Next, we investigated the effect of circHECW2 and WTAP on the survival of astrocytes. Corticosterone was used to mimic the depression in vitro. Astrocytes transduced with shRNAcircHECW2 showed an amelioration of the decreased viability induced by corticosterone (Fig. 4J). In contrast, knockdown the expression of WTAP significantly aggravated the decreased viability of astrocytes treated with corticosterone (Fig. 4K). Furthermore, WTAP siRNA decreased viability of astrocytes was ameliorated by shRNA-circHECW2, further indicating a close relationship between circHECW2 and WTAP (Fig. 4L). Additionally, we constructed a brain astrocyte-specific AAVGFAP-WATP knockdown (KD) virus. Three weeks after the microinjection of AAV-GFAP-WATP KD and shRNAcircHECW2 lentivirus in the hippocampus, the mice were subjected to CUS or control. Behavioral experiments including SPT, FST, and TST were examined after 4 weeks of CUS exposure. WTAP regulates m6A modification of Gng4 mRNA in depression To seek the potential downstream molecule that participated in the CUS mouse, the author posted a transcriptome-wide detection of m6A modification in the hippocampus of the CUS mouse in previous study. The gene ontology biological processes (GO-BP) analysis showed that the downregulated genes were enriched in gene terms associated with the cellular process. The region of mRNA with altered m6A modification (downregulated genes) (Fig. 5B). Next, RNA-seq analysis was performed in the hippocampus of the CUS mouse model. We identified 288 genes that were differentially expressed in RNA-seq analysis (Cuffdiff adjusted P-value<0.05) (Fig. 5C). Of particular interest, there were 19 overlapping transcripts between the two comparisons (Fig. 5D), suggesting that these genes may be the target genes involved in astrocyte dysfunction after CUS. Notably, these 19 overlapping transcripts were verified at the mRNA level. In the 4 upregulated transcripts, no gene was validated. Additionally, GNG4 was significantly decreased in the plasma of MDD patients. Additionally, there was a negative correlation between GNG4 and the scores of the HAMD-Cognitive impairment. Based on these findings, GNG4 emerged as a central focus in our study of MDD. Furthermore, we analyzed the pathway involving GNG4 and its association with decreased m6A modification by Kyoto Encyclopedia of Genes and Genomes (KEGG) (Fig. 5E). Next, further results indicated that m6A modification was decreased in the peak region (chr13: 13825437e13825707) in the 30-UTR of Gng4 mRNA (Fig. 5F and G). The author employed luciferase assays to compare the wild-type (WT) and mutant m6A sites with and without WTAP siRNA treatment. These assays demonstrated that the mutation prevented methylation and increased the stability of Gng4 mRNA (Fig. 5H). As expected, overexpression of Gng4 markedly ameliorated the declined viability of astrocytes treated with corticosterone (Fig. 5I). CircHECW2 regulates m6A methylation of Gng4 mRNA via WTAP Furthermore, the author examined the expression and function of GNG4 in the CUS mouse model. The mRNA and protein levels of Gng4 were significantly decreased in the hippocampus of CUS mice, which was consistent with the decreased m6A modification of Gng4 (Fig. 6A and B). In addition, to understand the relationship between Gng4 levels and the WTAP, WTAP siRNA was transfected in mouse primary astrocytes. The expression of GNG4 and Gng4 mRNA are all decreased (Fig. 6C). Moreover, the upregulation of circHECW2 led to reduced mRNA and protein levels of Gng4 (Fig. 6D). Conversely, the downregulation of circHECW2 in astrocytes increased the mRNA and protein levels of Gng4 (Fig. 6E). Co-transfection of WTAP siRNA and shRNAcircHECW2 indicated that WTAP siRNA decreased the GNG4 level, which was markedly ameliorated by shRNA-circHECW2 (Fig. 6F). To validate these in vitro findings, Western blot analysis was performed to assess the levels of GNG4 in shRNA-circHECW2-treated CUS mice. The results showed that shRNA-circHECW2 treatment significantly mitigated the decrease in GNG4 expression observed in CUS mice (Fig. 6G and H). We also constructed a brain astrocyte-specific AAV-GFAP-Gng4 KD virus. Three weeks after the microinjection of AAV-GFAP-Gng4 KD and shRNA-circHECW2 lentivirus into the hippocampus, the mice were exposed to CUS or kept in a control condition. The results of SPT, FSF, and TST revealed that astrocytic Gng4 is involved in the regulation of circHECW2 in depression (Fig. 6IeK). Hippocampus functional connectivity to the prefrontal cortex was positively correlated with GNG4 in MDD patients Finally, the seed-to-voxel analysis (hippocampal functional connectivity) was performed and showed hippocampus functional connectivity (FC) with prefrontal cortex (PFC) in MDD patients. As shown in Fig. 7A-C, the FC between the hippocampus and the prefrontal cortex was significantly reduced in MDD patients when compared to HCs. We next identified a noteworthy decrease in the resting-state functional connectivity (rsFC) between the hippocampus and the dorsolateral prefrontal cortex (Fig. 7D), whereas other brain regions, such as the visual cortex and inferior temporal lobe, exhibited no significant difference. Moreover, we found a positive correlation between GNG4 levels and rsFC, suggesting that GNG4 may play a role in the cognitive brain function of individuals with MDD (Fig. 7E). Summary Our study demonstrated that upregulation of circHECW2 led to the decrease in Gng4 mRNA via WTAP-mediated m6A modification, and caused subsequent astrocyte dysfunction. Specifically, circHECW2 promoted the ubiquitin-mediated degradation of WTAP in the CUS mouse model, and the effect of GNG4 on maintaining normal astrocyte functions is abolished when WTAP is downregulated in astrocytes. Therefore, the circHECW2/WTAP/GNG4 axis regulates astrocyte dysfunction by decreasing GNG4 stability via WTAP-mediated m6A modification. In conclusion, our findings indicate that circHECW2 holds promise as a therapeutic target for the treatment of depression. In addition, our study sheds light on the functional link between circHECW2 and m6A methylation, offering novel insights for the development of preventive strategies and effective treatments for MDD.

Extracellular vesicles (EVs) are various membrane-structured vesicular structures (40-100 nm) released by cells. Due to their low immunogenicity, biodegradability, low toxicity, and the ability to cross the blood-brain barrier, EVs have become prospective drug carriers in the fields of immunotherapy, regenerative medicine, etc., and have emerged as a vehicle for novel drug delivery systems. Circular RNA DYM (circDYM) is derived from exons 4, 5, and 6 of the DYM gene. It can act as a "sponge" for miRNA 9 to adsorb and inhibit miRNA, thus playing a regulatory role. In 2018, the research team led by Yao Honghong from the Department of Pharmacology, School of Medicine, Southeast University discovered that the overexpression of circDYM in the hippocampal region could inhibit the activity of miRNA 9, ultimately reducing microglial cell activation and alleviating depression-like behaviors. On January 13, 2022, based on the above research achievements, the research team led by Yao Honghong constructed extracellular vesicles that target the central nervous system and encapsulate circDYM (RVG-circDYM-EVs), which effectively alleviated the depression-like behavioral disorders caused by chronic stress. They successfully developed circular RNA into a nucleic acid drug. In 2011, a new method for targeted delivery of siRNA using exosomes was developed. The surface membrane protein lamp2b of exosomes was modified and fused with the neuron-specific rabies virus glycoprotein (RVG) to form exosomes with the RVG-lamp2b fusion protein. Through the method of electroporation, exogenous short-chain RNA interference was made to enter the exosomes. The RVG on the surface of the exosomes binds to the acetylcholine receptor to specifically release the short-chain RNA interference into neurons, significantly down-regulating the expression of BACE1, a protein related to Alzheimerd's disease. Through a similar method, the researchers constructed the RVG - circDYM - EVs system. Using exosomes as carriers, they achieved the over - expression of circDYM with nicotinic acetylcholine receptor targeting (Figure 1). Two hours after the tail - vein injection of 200 μg of RVG - circDYM - EVs, this exosome system began to enrich in the liver, kidneys, heart, spleen, brain and other parts of the mice (Figure 2). Among them, the liver had the highest enrichment and expression. Over time, the content of exosomes gradually decreased. Furthermore, RVG - circDYM - EVs can be specifically expressed on microglia, neurons and astrocytes in the hippocampal region. This indicates that the exosomes with nicotinic acetylcholine receptor - targeted over - expression of circDYM can penetrate the blood - brain barrier and enter the mouse brain, and are evenly distributed. In vitro cell experiments revealed that RVG - circDYM - EVs could reduce the activation of microglia induced by lipopolysaccharide: the expressions of iNOS, IL - 6, IL - 1β and MCP - 1 were decreased. Subsequently, after tail - vein injection of 100, 200, 300 and 400 μg of RVG - circDYM - EVs to mice with depression - like behavior caused by chronic stress, the 200, 300 and 400 μg dose groups could significantly improve the depression - like behavior of the mice (Figure 3). To further explore the molecular mechanism by which RVG - circDYM - EVs regulate the activity of microglia, the researchers found through single - cell sequencing that the transcription factor TATA - box binding protein - associated factor 1 (TAF1) protein could regulate more than 10 differentially expressed genes and interact with circDYM, co - localizing in the cytoplasm of microglia. In vitro experiments showed that over - expression of TAF1 promoted the expressions of Trpm6 and Cyp39a1, and over - expression of circDYM could inhibit this promoting effect. Lipopolysaccharide could promote the binding of TAF1 to the promoters of Trpm6 and Cyp39a1, and knockdown of either Trpm6 or Cyp39a1 could block the increase in iNOS levels caused by lipopolysaccharide. These molecular experiments indicated that circDYM could directly regulate the activation of microglia through TAF1. Chronic stress could cause a decrease in the expression of tight - junction proteins in the hippocampal region, an increase in the permeability of the blood - brain barrier, and ultimately lead to the infiltration of peripheral immune cells such as CD4 - positive T cells, CD8 - positive T cells and B220 - positive B cells into the brain parenchyma. Treatment with RVG - circDYM - EVs could partially repair the blood - brain barrier and reduce the infiltration of peripheral immune cells. It is reported that the RVG - circDYM - EVs developed in this paper was applied for a patent on February 13, 2020. The application name is: Exosomes with nicotinic acetylcholine receptor - targeted over - expression of circDYM and their preparation methods and applications.

On April 21, 2025, the inaugural meeting and the first working meeting of the University Health Management Professional Committee of the 9th Council of the Shanghai Public Health Care Management Association were grandly held at Shanghai Jiao Tong University (Xuhui branch, https://en.sjtu.edu.cn/). Nanjing Healed Gene Biotechnology Co., Ltd. (hereinafter referred to as Healed Gene), as an enterprise focusing on precision treatment in the mental field, was invited to attend the meeting and discuss the seminar on university students' mental health with Shanghai University health management experts.   In the training lecture session of this conference, "University Students' Mental Health Construction Program" became the focus topic. The "CircRNA RT-PCR Detection Kit" program brought by Healed Gene was reported and discussed in detail in this session. At a time when the mental health issues of university students are increasingly valued, Healed Gene's "objective biomarker" test can help diagnose depression more objectively and reduce misdiagnosis and missed diagnosis; when the symptoms are not obvious, biomarkers can be used to detect problems earlier and intervene in time.   The Healed Gene "CircRNA RT-PCR Detection Kit" can be tested using non-invasive samples such as saliva, which greatly reduces the psychological burden and physical discomfort of the test subjects. In terms of accessibility, it is supplied through vending machines on campus and e-commerce platforms, and students can place orders on their own, which effectively protects privacy. This innovative solution was highly recognized by the experts present. Experts said that the Healed Gene "CircRNA RT-PCR Detection Kit" provides a new, efficient and feasible solution for the mental health screening of university students, and improves the objectivity and scientificity of depression risk identification. It is expected to be widely used in the mental health survey of university students in the future, helping universities to promptly identify potential high-risk groups for depression and safeguard the mental health of students.   Healed Gene has always adhered to the concept of technological innovation driving the progress of diagnosis and treatment, and is committed to developing more accurate, more convenient and more humane mental testing solutions. The recognition obtained at the founding conference of the Professional Committee of Health Management of Colleges and Universities is an affirmation of our past efforts and a driving force for us to move forward.   In the future, Healed Gene will continue to increase its investment in research and development, continuously optimize product performance, and work closely with university health management departments and related institutions to contribute to the mental health of students, so that the light of science and technology can illuminate every heart in need of care.

"The 7th circRNA Research and Industry Forum" was successfully held in Guangzhou on November 11-12, 2023! We aim to provide an exchange platform to share the latest circRNA research results, discuss cutting-edge technical methods, and promote the development and application of circRNA in the biomedical industry. Over the past few years, there has been remarkable progress in circRNA research, revealing its important role in gene expression and regulation. Today, with the advancement of technology and in-depth research, we have begun to use circRNA as a biomarker for disease diagnosis, as well as a new target for drug development. At the same time, by combining circRNA research with advanced technologies such as artificial intelligence, we are starting a new wave of biomedical innovation. In this context, we specially planned a roundtable discussion to bring together top experts and industry leaders across disciplines to discuss circRNAd's future development strategies, technology challenges and transformation opportunities. We are convinced that such dialogue and collision will not only promote the basic research of circRNA, but also accelerate its practical application in medicine, health, biotechnology and other fields. Letd's take a step into this roundtable discussion, listen to the insights and insights of experts, and explore the infinite possibilities of circRNA. The high-quality development of circRNA from basic research to industrial transformation The high-quality development of circRNA from basic research to industrial transformation On the 11th, Chief Professor, Southeast University Prof. Yao Honghong, Prof. Yang Baihua, Professor of Experimental Medicine and Pathology, University of Toronto, Canada; Prof. Li Xiangdong, Professor of College of Biological Sciences and Medicine, University of Science and Technology of China; Prof. Peng Yong, Professor of State Key Laboratory of Biological Therapy, West China Hospital, Sichuan University; The topic of "High-quality development of circRNA from basic research to industrial transformation" was discussed.   The meeting mainly discussed the problems of circular RNA in basic research, application and industrial transformation. In terms of basic research, the key issues to be solved include the specificity of protein expression and the dynamic regulation of RNA structure. In terms of applications, circular RNA can be used as a diagnostic marker and is expected to be a tool for minimally invasive or non-invasive diagnosis. In terms of industrial transformation, problems such as delivery and scale production need to be solved, and the emergence of circular RNA drugs may be predicted in the future. Opportunities and challenges of circular RNA therapy On the 12th, Mr. Zhang Maolei, CTO of Geisai Biology, Mr. Yang Yun, CTO of Ring Code Biology, Mr. Gao Lu, CEO of Yuanyin Biology, Mr. Dai Dongsheng, CEO of Youhuan Biology, and Mr. Xu Congcong, young Distinguished Professor of Suzhou University, had a lively and multi-dimensional discussion on the topic of "Opportunities and Challenges of circular RNA therapy".   The meeting mainly discussed key issues such as the industrialization prospect of circular RNA, industrial chain construction, and delivery, as well as specific practices such as how to find blockbuster drug pipelines and promote clinical progress. The teachers believe that the industrialization of circular RNA has broad development prospects, but it needs to solve the problems at the process level and the bottleneck of industrial chain construction. They suggested strengthening industry-university-research-medical cooperation, promoting innovative drug research and development, and solving circulation problems to promote the development and application of circular RNA therapy.