CCL2 supports colorectal cancer proliferation, invasion and metastasis through activating lncRNA GSTM3TV2/ATF4 signaling

CCL2 supports colorectal cancer proliferation, invasion and metastasis through activating lncRNA GSTM3TV2/ATF4 signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CCL2 supports colorectal cancer proliferation, invasion and metastasis through activating lncRNA GSTM3TV2/ATF4 signaling Yujun Rao, Xiaoying Wang, Hongyu Zhou, Ziqi Li, Shufang Ye, Zizhen Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7932268/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: Despite extensive research and advances in biological knowledge, therapeutic strategies, and prognostic outcomes, colorectal cancer (CRC) remains a common malignancy around the world. Elucidating molecular determinants promoting CRC progression will help develop novel, available, and effective treatment modalities. In this study, we proposed a novel C-C motif chemokine 2 (CCL2)/long noncoding RNA lncRNA glutathione S-transferase mu 3, transcript variant 2 ( GSTM3TV2 )/activating transcription factor 4 (ATF4) mechanism responsible for promoting CRC progression. Methods: CRC cells were exposed to 100 ng/ml recombinant CCL2, and/or transfected with GSTM3TV2 or ATF4 siRNA or pcDNA3.1 vector with the full-length of GSTM3TV2 or ATF4 sequence. Cell proliferation, invasion and metastasis were investigated through CCK-8, wound healing, Transwell, and subcutaneous mouse xenograft models. GSTM3TV2 and ATF4 expression levels were measured via qRT-PCR and immunofluorescence. Results: CCL2 treatment upregulated lncRNA GSTM3TV2 expression and conferred proliferative, invasive and metastatic potential of CRC cells, but such effects were effectively reversed by endogenous silencing of GSTM3TV2 . ATF4 was determined to be a downstream factor of GSTM3TV2 , whose expression levels were positively modulated by GSTM3TV2 . Endogenous silencing of ATF4 reversed the impact of overexpressing GSTM3TV2 or CCL2 treatment on heightening CRC cells proliferation, invasion and metastasis, thus preventing CRC progression. Conclusion: Altogether, our findings suggest that the CCL2/lncRNA GSTM3TV2 /ATF4 signaling could be an important mechanism underlying CRC progression, offering potential theoretical basis for the clinical therapy of CRC. colorectal cancer CCL2 GSTM3TV2 ATF4 tumor progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Colorectal cancer (CRC) remains the dominant cause of cancer mortality globally [1]. Although incidence has declined in patients with average age at onset in high-income economies, CRC remains the most common cancer globally, and its incidence is increasing in emerging economies [2]. As estimated, CRC burden will increase by 60% by 2030 [3]. In addition, early-onset CRC (age ≤50 years) is a growing concern worldwide. As CRC progresses, it becomes increasingly aggressive and metastatic [4]. Development of novel, available, and effective medicines and establishment of a personalized and tailored continuum of care for each patient will contribute to improvement in patient outcomes [5]. Thus, it is of importance to clarify molecular and cellular determinants promoting CRC progression. C-C motif chemokine 2 (CCL2) is critical for CRC progression because its expression is upregulated in CRC patients and CCL2 deficiency can prevent CRC progression [6]. In the tumor microenvironment, cancer-associated fibroblasts and macrophages are two key cell populations that are responsible for CCL2 secretion [7]. Circulating CCL2 is connected to tumor presence, with the potential as a diagnostic biomarker of CRC [8]. Long noncoding RNAs (LncRNAs) are a type of largely functional transcripts with a length of >200 nucleotides [9-11]. A grow body of evidence suggests that lncRNAs participate in the pathogenesis of cancer through governing transcription mechanism, gene-specific transcription, translation, and epigenetic modification, and more [12-14]. Especially, several lncRNAs function as key determinants of cancer progression, including CRC [15-17]. Hence, an in-depth understanding of the biology of lncRNAs may uncover novel treatment targets for CRC. Recently, lncRNA glutathione S-transferase mu 3, transcript variant 2 ( GSTM3TV2 ) has been found to contribute to proliferation and invasion of hepatocellular carcinoma [18], and gemcitabine resistance of pancreatic cancer [19]. Nonetheless, function and regulatory mechanism of GSTM3TV2 in CRC remain indistinct. Activating transcription factor 4 (ATF4) belongs to the ATF/cAMP response element-binding family and acts as a stress-responsive transcription factor [20-22], which is responsible for orchestrating cellular responses through regulation of many target genes, such as endoplasmic reticulum stress, starvation, and cysteine stress [23]. It has been demonstrated to mediate CRC progression through modulating distinct mechanisms such as glutamine metabolism and glycolysis [24], cysteine metabolism [25], amino acid metabolism [26], and immunogenic cell death [27]. Herein, on the basis of in vitro and in vivo experiments, our findings uncovered that CCL2 may be responsible for CRC cell proliferation, invasion and metastasis primarily through activation of lncRNA GSTM3TV2 /ATF4 signaling. Materials and Methods Ethical Approval and accordance Ethical approval for this study was obtained from Lishui University. All experiments involving animals were conducted according to the ethical policies and procedures approved by the Institutional Animal Care and Committee of Lishui University. All animal experiments were strictly implemented in compliance with the ARRIVE guidelines. Cell culture and treatment HCT116 cells (CTCC-085, CTCC, Zhejiang, China) were cultivated in McCoy’s 5A medium (PM150710, Pricella, Wuhan, China) containing 10% fetal bovine serum (S711-001, Lonsera, Shanghai, China), 100 units of penicillin-streptomycin (BL505A, Biosharp, USA) in a 5% CO 2 incubator at 37°C. HCT116 cells were treated with 100 ng/ml recombinant CCL2 (JN1524, BIO-LAB, Beijing, China) for 24 h. Transfection 5×10 5 cells/well were seeded into a 6-well plate. On the next day, each well was replaced with 2 ml fresh medium. 125 μl of McCoy’s 5A medium, 100 pmol of small interfering RNA (siRNA) of GSTM3TV2 or ATF4 /2.5μg of DNA, and 4 μl Lipo8000™ transfection reagent (C0533, Beyotime, Shanghai, China) were mixed. After preparation, the mixture was stored at room temperature for 6 h. Each well was added with 125 μl mixture. The full-length of GSTM3TV2 or ATF4 sequence was amplified by PCR and cloned into pcDNA3.1 vector (V012531, NovoPro, Shanghai, China). Through Lipo8000™ transfection reagent, the cells were transfected with pcDNA3.1 vector. After 48-h culture, transfection effect was evaluated. Quantitative reverse transcriptase PCR (qRT-PCR) Total RNA was extracted with Trizol reagent (15596018, Invitrogen, USA), with subsequent reverse transcription. PCR amplification was conducted, with the reaction system volume of 0.2 ml. The programs of PCR amplification included: pre-denaturation (95°C, 5 min), denaturation (95°C, 20 sec), annealing elongation (55 ℃, 60 sec; 72℃, 20 sec; 95℃, 15 sec; 40 cycles), and melting curve acquisition (60℃, 60 sec; 95℃, 30 sec; 60℃, 15 sec). This experiment was analyzed by ABI 7500 Real-time detector (ABI, USA) and ABI Prism 7500 SDS software. Relative target gene expression was calculated with 2 –ΔΔCt method with GAPDH as a reference control. The primer sequences included: GSTM3TV2 (human), 5’-CTCGGGTACTGGGATATTCGT-3’ (forward), 5’-AGGAGGTAGGGCAGATTAGGA-3’ (reverse); ATF4 (human), 5’-AGGTGTTCTCTGTGGGTC-3’ (forward), 5’-TAGTGGCTGCTGTCTTGT-3’ (reverse); Gapdh (human), 5’-GGAGCGAGATCCCTCCAAAAT-3’ (forward), 5’-GGCTGTTGTCATACTTCTCATGG-3’ (reverse). Cell Counting Kit-8 (CCK-8) 8×10 3 cells/well was seeded into a 96-well plate and cultured for 48 h at 37°C. 10 μl CCK-8 reagent (M007, CTCC) was added to each well and the cells were incubated for additional 2 h. Afterwards, absorbance at 450 nm was measured with a microplate reader (MK3, ThermoFisher, USA). Wound healing assay Two horizontal lines were marked evenly on the back of the 3.5cm dish utilizing a marker pen. 5×10 5 cells were inoculated in 3.5 cm dishes and cultivated overnight at an atmosphere of 37℃ and 5% CO 2 . Until the cell density was up to 90%, a 200 μl gun tip was adopted for making vertical horizontal scratches. The scratched cells were cleaned by PBS. The cells continued to be cultivated for 48 h. Cells were photographed using an IX71 microscope (Olympus, Japan) at 0 h and 48 h. Transwell A 24-well plate was pre-cooled with 800 μl 10% FBS-containing medium at 4℃ and placed in the Transwell (353097, FALCON, USA) chamber. 100 μl of 1 mg/ml Matrigel (356234, Corning, USA) was added vertically into the center of the upper chamber, which was then dried at 37℃ for 4 h. 200 μl cell suspension (3×10 5 /ml) was inserted into the upper chamber of the Transwell and cultivated for 48 h at 37℃ in a 5% CO 2 incubator. The Transwell chamber was cleaned with PBS, with subsequent cell fixation utilizing 70% icy ethanol solution for 1 h. 0.5% crystal violet reagent was used for staining. After 20 min at room temperature, the cells were washed by PBS, and uninvaded cells were removed with a clean cotton ball. Invaded cells were photographed under an IX71 microscope (Olympus, Japan). Subcutaneous mouse xenograft models The animal experimental protocols were conducted strictly following the ethical approval of the Institutional Animal Care and Committee of Lishui University. BALB/c nude mice (4~6 weeks old) were purchased from CAVENS (Changzhou, China) and observed in a SPF environment for 3 days. The skin at the injection site of nude mice was disinfected with 75% alcohol, and cell suspension with 1×10 7 cells was injected into the underarm of nude mice. The piercing point was ~1 cm away from the injection point, forming a raised skin mound to prevent liquid leakage, followed by skin disinfection. The tumor size was measured every 3 days. The maximal tumor size permitted by the Experimental Animal Ethics Committee is 1 cm in diameter. The longest (a) and shortest (b) diameters of tumor were measured with a vernier caliper, followed by calculation of the tumor volume (mm 3 ) = ab 2 /2. The tumor growth curve was plotted. None of our animals had tumors larger than 1 cm in diameter. The nude mice were euthanized 28 days later, and the subcutaneous tumor was dissected and photographed. Immunofluorescence (IF) Tissues were fixed in 4% paraformaldehyde for 24 h, embedded in wax molds, and cut into 5-μm-thick sections. The paraffin sections were deparaffinized, and baked at 60°C for 3 h. After antigen retrieval (P0086, Beyotime), the sections were blocked with 5% BSA (4240GR500, BioFROXX, Germany) at 37℃ for 1 h. Antibody of ATF4 (1:200, 10835-1-AP, Proteintech) was added to the sections, with subsequent 2-h incubation at 37℃. After cleaning with PBS, the sections were incubated with fluorescein (FITC)-conjugated goat anti-mouse IgG(H+L) (1:100, SA00003-1, Proteintech) for additional 1 h. Afterwards, they were cleaned with PBS. Hoechst (C1022, Beyotime) was added to the sections, with subsequent 15-min incubation at room temperature. Immunofluorescent images were acquired by a fluorescence microscope (Olympus, Japan). Statistical analysis All data are expressed as the mean ± standard deviation (SD) from three independently repeated experiments. All the analyses were conducted by use of GraphPad Prism software v9.0.0. Statistical difference was evaluated by one- or two-way analysis of variance (ANOVA) with Tukey’s post hoc test. P<0.05 was considered statistically significant. Results CCL2 upregulates lncRNA GSTM3TV2 expression to support CRC cell proliferation, invasion and metastasis Treatment of recombinant CCL2 (100 ng/ml) notably elevated the transcript level of GSTM3TV2 in HCT116 cells, which was similar to the effect of transfection with pcDNA3.1 vector with full-length of GSTM3TV2 ( OE-GSTM3TV2 ) ( Figure 1A ). This suggested that CCL2 was effective in activating the transcription of GSTM3TV2 in CRC cells. Nevertheless, transfection with GSTM3TV2 siRNA ( si-GSTM3TV2 ) prominently attenuated the CCL2-induced elevation in the transcript level of GSTM3TV2 ( Figure 1A ). Next, we investigated whether CCL2 affected CRC progression through transcriptionally activating GSTM3TV2 . As shown in the CCK-8 results, both CCL2 treatment and overexpressing GSTM3TV2 led to a remarkable enhancement in HCT116 cell proliferation ( Figure 1B, C ). Endogenous silencing of GSTM3TV2 reversed the impact of CCL2 treatment on heightening HCT116 cell proliferation. Through Transwell (with Matrigel), cellular invasion was tested. Upon CCL2 treatment, the invasive ability of HCT116 cells showed notable improvement, with the similar effect to overexpressing GSTM3TV2 ( Figure 1D, E ). The improvement in the invasive ability was prominently weakened in the context of endogenous GSTM3TV2 silencing. Wound healing assay was also conducted. Consequently, cellular mobility was strengthened by both CCL2 treatment and overexpressing GSTM3TV2 ( Figure 1F, G ). Endogenous silencing of GSTM3TV2 effectively weakened the impact of CCL2 treatment on cellular mobility. Hence, the above data uncovered that CCL2 may upregulate GSTM3TV2 to expression support CRC cell proliferation, invasion and metastasis. CCL2 accelerates CRC growth through elevating lncRNA GSTM3TV2 expression Through injection of 1×10 7 HCT116 cells (transfected with si-GSTM3TV2 or pcDNA3.1 vector with OE-GSTM3TV2 , with or without subsequent treatment of 100 ng/ml recombinant CCL2) into the underarm of nude mice, subcutaneous mouse xenograft models were developed. 28 days later, we dissected and photographed subcutaneous tumors. Figure 2A, B show the tumor-bearing nude mice and subcutaneous tumors. Clearly, both CCL2 treatment and overexpressing GSTM3TV2 led to accelerated tumor growth. Suppressing GSTM3TV2 weakened the impact of CCL2 treatment on accelerating tumor growth. In addition, tumor volume was measured regularly. Consequently, both CCL2 treatment and overexpressing GSTM3TV2 notably increased tumor volume, while GSTM3TV2 suppression attenuated the impact of CCL2 treatment on increasing tumor volume ( Figure 2C ). Collectively, CCL2 may accelerate CRC growth via elevating lncRNA GSTM3TV2 expression. GSTM3TV2 supports CRC cell proliferation, invasion and metastasis through transcriptionally activating ATF4 Overexpressing GSTM3TV2 through transfection of pcDNA3.1 vector with OE-GSTM3TV2 led to the remarkable elevation in ATF4 transcript level in HCT116 cells ( Figure 3A, B ). Additionally, endogenous silencing of GSTM3TV2 through transfecting si-GSTM3TV2 prominently reduced transcript level of ATF4 in HCT116 cells. This suggested that GSTM3TV2 may transcriptionally activate ATF4 in CRC cells. On the basis of the CCK-8 results, HCT116 cell proliferation was heightened by overexpressing GSTM3TV2 , which was impaired by endogenous silencing of GSTM3TV2 ( Figure 3C, D ). As shown in the Transwell (with Matrigel) results, overexpressing GSTM3TV2 led to the enhancement in HCT116 cell invasion, with endogenous silencing of GSTM3TV2 impairing HCT116 cell invasion ( Figure 3E, F ). In addition, wound healing assay was carried out. HCT116 cell mobility was heightened by overexpressing GSTM3TV2 and was suppressed by endogenous silencing of GSTM3TV2 ( Figure 3G, H ) . These data indicated that GSTM3TV2 may support CRC cell proliferation, invasion and metastasis via transcriptionally activating ATF4 . GSTM3TV2 supports CRC growth through elevating ATF4 expression 1×10 7 HCT116 cells with si-GSTM3TV2 or pcDNA3.1 vector with OE-GSTM3TV2 transfection was infected into the underarm of nude mice to develop subcutaneous mouse xenograft models. 28 days later, subcutaneous tumors were dissected and photographed ( Figure 4A, B ). As a result, overexpressing GSTM3TV2 facilitated tumor growth, whereas endogenous silencing of GSTM3TV2 prevented tumor growth. Additionally, overexpressing GSTM3TV2 notably elevated tumor volume, with opposite findings when GSTM3TV2 was endogenously silenced ( Figure 4C ). Through IF, ATF4 expression levels were measured in mouse tumors. Consistent with the in vitro findings, overexpressing GSTM3TV2 led to the elevation in ATF4 expression in mouse tumors, while endogenous silencing of GSTM3TV2 led to the reduction in ATF4 expression ( Figure 4D, E ). Altogether, GSTM3TV2 may support CRC growth through elevating ATF4 expression. CCL2 supports CRC cell proliferation, invasion and metastasis through activating lncRNA GSTM3TV2 /ATF4 signaling Further analysis showed that endogenous silencing of ATF4 did not alter the upregulation in GSTM3TV2 expression induced by CCL2 treatment or transfection of pcDNA3.1 vector with OE-GSTM3TV2 in HCT116 cells ( Figure 5A ). Furthermore, overexpressing ATF4 did not alter the reduction in GSTM3TV2 expression caused by si-GSTM3TV2 transfection. It was also observed that neither CCL2 treatment nor overexpressing GSTM3TV2 reversed the alterations in ATF4 expression caused by si-ATF4 or pcDNA3.1 vector with OE-ATF4 ( Figure 5B ) . As depicted in the CCK-8 results, endogenous silencing of GSTM3TV2 did not reverse the impact of overexpressing ATF4 on strengthening HCT116 cell proliferation ( Figure 5C, D ) . Additionally, neither CCL2 treatment nor overexpressing GSTM3TV2 altered the suppression in cell proliferation caused by endogenous silencing of ATF4 . The Transwell (with Matrigel) results showed that endogenous silencing of GSTM3TV2 did not attenuate the impact of overexpressing ATF4 on strengthening HCT116 cell invasion ( Figure 5E, F ) . Neither CCL2 treatment nor overexpressing GSTM3TV2 reversed the invasion suppression caused by endogenous silencing of ATF4 . The results from the wound healing assay also demonstrated that endogenous silencing of GSTM3TV2 did not weaken the improvement in HCT116 cell mobility triggered by overexpressing ATF4 ( Figure 5G, H ). Neither CCL2 treatment nor overexpressing GSTM3TV2 reversed the cell mobility inhibition triggered by endogenous silencing of ATF4 . Altogether, our findings uncovered that CCL2 may support CRC cell proliferation, invasion and metastasis via activating lncRNA GSTM3TV2 /ATF4 signaling. Discussion Despite extensive research and improvement in biological knowledge, therapeutic strategies, and prognostic outcomes, CRC remains the most diagnosed and deadliest malignancy around the world [28]. Determining new, available and effective therapeutic strategies in this large and expanding patient population is a critical and largely unmet medical need [29, 30]. This study for the first time proposed the CCL2/GSTM3TV2/ATF4 mechanism facilitating CRC cell proliferation, invasion and metastasis. To comprehend how new therapeutic options target CRC, it is of importance to discern the intricate molecular mechanisms underlying CRC [31, 32]. Recognizing the factors that modulate proliferation, invasion and metastasis will offer the basis of which new treatment modalities can be assessed in clinical trials and eventually improve patient prognosis [33-35]. Herein, we observed that CCL2 effectively elevated lncRNA GSTM3TV2 expression in CRC cells. In addition, CCL2 treatment resulted in a remarkable enhancement in CRC cell proliferation, invasion and metastasis, but endogenous silencing of GSTM3TV2 could effectively weaken the impact of CCL2 treatment on the malignant behaviors of CRC cells, suggesting that CCL2 may elevate lncRNA GSTM3TV2 expression to support CRC progression. CCL2 is primarily secreted by cancer-associated fibroblasts and macrophages in the tumor microenvironment. Numerous studies have reported the involvement of CCL2 in CRC progression, and targeted suppression of CCL2 can hinder CRC progression and metastasis [6, 36, 37]. LncRNA GSTM3TV2 was determined as a regulatory target of CCL2, but detailed regulatory mechanism needs to be in-depth explored. Our study for the first time proposed the involvement of lncRNA GSTM3TV2 in heightening proliferation, invasion and metastasis of CRC cells. LncRNA GSTM3TV2 conferred malignant behaviors of CRC cells through elevating ATF4 expression. ATF4 acts as a stress-responsive transcription factor, which is activated and promotes cell adaptation for survival in response to cellular stress [38-40]. For instance, ATF4 prevents hepatocellular carcinoma occurrence through activation of SLC7A11 to inhibit stress-associated ferroptosis [21]. ATF4 links endoplasmic reticulum stress with reticulophagy in glioblastoma cells [41]. ATF4-mediated integrated stress response drives angiogenesis and cancer development through activation of perivascular cancer-associated fibroblasts [42]. Inducing endoplasmic reticulum stress through ATF4-SPHK1 signaling contributes to glioblastoma aggressiveness and resistance to chemotherapy [43]. ATF4 has been extensively studied in CRC pathogenesis, and targeted inhibition of ATF4 has been evidenced to prevent CRC progression [26, 44, 45]. In the present study, ATF4 was determined as a potential downstream target of GSTM3TV2 in CRC cells upon CCL2 treatment, with more studies being required for proving the direct interaction between GSTM3TV2 and ATF4. Although our study demonstrated that CCL2 accelerated CRC cell proliferation, invasion and metastasis via activating lncRNA GSTM3TV2 /ATF4 signaling, the regulatory mechanisms and binding structures of CCL2/lncRNA GSTM3TV2 and lncRNA GSTM3TV2 /ATF4 need to be in-depth explored. The clinical significance of the CCL2/lncRNA GSTM3TV2 /ATF4 mechanism in CRC patients’ survival outcomes needs to be investigated in large clinical cohorts. Conclusion Collectively, our study revealed that CCL2 may be responsible for accelerating CRC cell proliferation, invasion and metastasis through activation of lncRNA GSTM3TV2 /ATF4 signaling, hopefully offering potential therapeutic targets for the clinical treatment of CRC. Abbreviations CRC: colorectal cancer; CCL2: C-C motif chemokine 2; lncRNAs: long noncoding RNAs; GSTM3TV2: glutathione S-transferase mu 3, transcript variant 2; ATF4: activating transcription factor 4; siRNA: small interfering RNA; qRT-PCR: quantitative reverse transcriptase PCR; CCK-8: Cell Counting Kit-8; FITC: fluorescein; SD: standard deviation; ANOVA: analysis of variance. Declarations Author contributions YL and XF designed the study. YR, XW, and HZ completed the animal experiment and collated the data, conducted data analysis and produced the first draft of the male man uscript. ZL, SY and ZZ participated in the guidance of this project. All authors have read and approved the final submitted manuscript. Consent to Publish declaration Not applicable. Consent to Participate declaration Not applicable. Clinical trial number Not applicable. Conflict of interest We declare that we have no conflicts of interest. Ethics approval All experiments involving animals were conducted according to the ethical policies and procedures approved by the Institutional Animal Care and Use Committees of the Lishui University. All animal experiments were strictly implemented in compliance with the ARRIVE guidelines. The maximum subcutaneous tumor diameter allowed by the ethics committee of our hospital in nude mice was 1 cm, and this study met this criterion. Data availability The data generated and/or analyzed during the current study can be provided based on the request from the corresponding author. Funding This work was supported by the Science and Technology Plan of Medicine and Health of Zhejiang Province (2025KY1968), the grants from the National Natural Science Foundation of China (No. 82200617), the Public Welfare Technology Research Program of Lishui City (No. 2022GYX51), and Zhejiang Provincial Natural Science Foundation of China under Grant No.LY21H160001 . References Zhou J, Yang Q, Zhao S et al. Evolving landscape of colorectal cancer: Global and regional burden, risk factor dynamics, and future scenarios (the Global Burden of Disease 1990-2050). Ageing Res Rev 2025; 104:102666. Eng C, Yoshino T, Ruíz-García E et al. Colorectal cancer. Lancet 2024; 404:294-310. Arnold M, Sierra MS, Laversanne M et al. Global patterns and trends in colorectal cancer incidence and mortality. Gut 2017; 66:683-691. 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Zheng T, Zhang D, Fu Q et al. DNA methylation-driven gene FAM3D promotes colorectal cancer growth via the ATF4-SESN2-mTORC1 pathway. Aging (Albany NY) 2024; 16:12866-12892. Liu L, Liu T, Tao W et al. Flavonoids from Scutellaria barbata D. Don exert antitumor activity in colorectal cancer through inhibited autophagy and promoted apoptosis via ATF4/sestrin2 pathway. Phytomedicine 2022; 99:154007. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7932268","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550557308,"identity":"34173632-8384-4172-94f3-38dcc5db174b","order_by":0,"name":"Yujun Rao","email":"","orcid":"","institution":"Lishui Hospital of Wenzhou Medical University, The first affiliated hospital of Lishui University, Lishui People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yujun","middleName":"","lastName":"Rao","suffix":""},{"id":550557309,"identity":"e8e003b9-08d0-447f-95fb-4cf542a9454d","order_by":1,"name":"Xiaoying Wang","email":"","orcid":"","institution":"Lishui Hospital of Wenzhou Medical University, The first affiliated hospital of Lishui University, Lishui People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Wang","suffix":""},{"id":550557310,"identity":"1c04612d-1eee-4b48-be05-fc1f34e291f9","order_by":2,"name":"Hongyu Zhou","email":"","orcid":"","institution":"Lishui Hospital of Wenzhou Medical University, The first affiliated hospital of Lishui University, Lishui People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Zhou","suffix":""},{"id":550557311,"identity":"99fa4ad5-ec5c-4539-970b-51a88d80c056","order_by":3,"name":"Ziqi Li","email":"","orcid":"","institution":"Lishui Hospital of Wenzhou Medical University, The first affiliated hospital of Lishui University, Lishui People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ziqi","middleName":"","lastName":"Li","suffix":""},{"id":550557312,"identity":"e72220f2-e7f9-4deb-9682-cc138b07936b","order_by":4,"name":"Shufang Ye","email":"","orcid":"","institution":"Lishui Hospital of Wenzhou Medical University, The first affiliated hospital of Lishui University, Lishui People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shufang","middleName":"","lastName":"Ye","suffix":""},{"id":550557313,"identity":"e0f1916f-d106-4c3d-9761-fc41d6f503dc","order_by":5,"name":"Zizhen Zhang","email":"","orcid":"","institution":"Peking University Cancer Hospital \u0026 Institute","correspondingAuthor":false,"prefix":"","firstName":"Zizhen","middleName":"","lastName":"Zhang","suffix":""},{"id":550557314,"identity":"4c369a09-cf1a-4c77-9773-3fc564f2666f","order_by":6,"name":"Xin Fu","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Fu","suffix":""},{"id":550557315,"identity":"70b502c1-88a6-4562-a0a9-ad8dc6800712","order_by":7,"name":"Yangyang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACPmYgwdggwcAP4TMT1sIG1SIh2UC0FgawFgYJgwNEa2HnMWD4ucOizvhGdpoEQ4V1YgP72QMEHMZjwNh7RkLC7EbuNgmGM+mJDTx5CQS1MDO2AbXcBmphbDuc2CABtJcoLcazQVr+kaLFQBqkpYEoLWwFjL1tEpIz7r/dbJFwLN24jScHvxZ+/sMbGH621fHz95zdeONDjbVsP/sZ/FqAgP0HnJnAAI2pUTAKRsEoGAWUAQDWyjaIOAboSAAAAABJRU5ErkJggg==","orcid":"","institution":"Lishui Hospital of Wenzhou Medical University, The first affiliated hospital of Lishui University, Lishui People’s Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yangyang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-10-23 12:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7932268/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7932268/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96855010,"identity":"c2fd0b06-e539-4acd-82b9-7632d34b9b1f","added_by":"auto","created_at":"2025-11-26 18:55:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":585590,"visible":true,"origin":"","legend":"\u003cp\u003eCCL2 transcriptionally activates \u003cem\u003eGSTM3TV2 \u003c/em\u003eto support CRC cell proliferation, invasion and metastasis. (A) qRT-PCR for transcript level of \u003cem\u003eGSTM3TV2\u003c/em\u003e in HCT116 cells transfected with\u003cem\u003e GSTM3TV2\u003c/em\u003e siRNA (\u003cem\u003esi-GSTM3TV2\u003c/em\u003e) or pcDNA3.1 vector with full-length of \u003cem\u003eGSTM3TV2\u003c/em\u003e (\u003cem\u003eOE-GSTM3TV2\u003c/em\u003e), with or without subsequent treatment of 100 ng/ml recombinant CCL2. (B) Photographs of HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. Scale bar, 100 μm. (C) Cell viability of HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003eor pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2 through CCK-8. (D) Transwell (with Matrigel) photographs of HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. Scale bar, 50 μm. (E) Number of invasive HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. (F) 0-h and 48-h wound healing photographs of HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003eor pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. Scale bar, 100 μm. (G) Quantification of mobility rate of HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001, from one-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7932268/v1/d05edb982da158f4882e36d5.png"},{"id":96855008,"identity":"e32edce0-d65b-4b38-9e19-5b2f195037ed","added_by":"auto","created_at":"2025-11-26 18:55:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":787375,"visible":true,"origin":"","legend":"\u003cp\u003eCCL2 accelerates CRC growth through elevating \u003cem\u003eGSTM3TV2\u003c/em\u003e expression. (A, B) Photographs of tumor-bearing nude mice and tumors (n=3 per group). Cell suspension with 1×10\u003csup\u003e7\u003c/sup\u003e HCT116 cells (transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2) was injected into the underarm of nude mice. 28 days later, the subcutaneous tumor was dissected and photographed. (C) Measurement of tumor volume at the indicated time. ****, p\u0026lt;0.0001, from one- or two-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7932268/v1/8714494580deab5184649315.png"},{"id":96855009,"identity":"d73b974f-ed92-47b2-96fe-d61493626724","added_by":"auto","created_at":"2025-11-26 18:55:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":460076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGSTM3TV2\u003c/em\u003e supports CRC cell proliferation, invasion and metastasis through transcriptionally activating \u003cem\u003eATF4\u003c/em\u003e. (A) qRT-PCR for transcript level of \u003cem\u003eGSTM3TV2\u003c/em\u003e in HCT116 cells transfected with\u003cem\u003esi-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e. (B) qRT-PCR for transcript level of \u003cem\u003eATF4\u003c/em\u003e in HCT116 cells with transfection of\u003cem\u003esi-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e. (C) Photographs of HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e. Scale bar, 100 μm. (D) Cell viability of HCT116 cells transfected with\u003cem\u003esi-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e by use of CCK-8. (E) Transwell (with Matrigel) photographs of HCT116 cells with\u003cem\u003e si-GSTM3TV2\u003c/em\u003eor pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e transfection. Scale bar, 50 μm. (F) Number of invasive HCT116 cells with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e transfection. (G) 0-h and 48-h wound healing photographs of HCT116 cells with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003etransfection. Scale bar, 100 μm. (H) Quantification of mobility rate of HCT116 cells with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003etransfection. **, p\u0026lt;0.01; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001, from one-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7932268/v1/4c1c5f263daca6b0b20ac43f.png"},{"id":96917968,"identity":"0f4e8a1a-1389-457d-9c6c-c441c78fe822","added_by":"auto","created_at":"2025-11-27 14:10:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":818083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGSTM3TV2\u003c/em\u003e supports CRC growth through elevating ATF4 expression. (A, B) Photographs of tumor-bearing nude mice and tumors (n=3 per group). Cell suspension with 1×10\u003csup\u003e7\u003c/sup\u003e HCT116 cells (transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e) was injected into the underarm of nude mice. 28 days later, the subcutaneous tumor was dissected and photographed. (C) Tumor volume measurement at the indicated time. (D) IF photographs of ATF4 (red) in mouse tumors. The cell nucleus was stained with Hoechst (blue). Scale bar, 50 μm. (E) Quantification of average density of ATF4 in mouse tumors. *, p\u0026lt;0.05; **, p\u0026lt;0.01; ****, p\u0026lt;0.0001, from one- or two-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7932268/v1/b8303316862f38548133cab3.png"},{"id":96855011,"identity":"89232001-3a39-46af-bd30-d09fdb35b9e2","added_by":"auto","created_at":"2025-11-26 18:55:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":620551,"visible":true,"origin":"","legend":"\u003cp\u003eCCL2 supports CRC cell proliferation, invasion and metastasis through activating \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 signaling. (A) qRT-PCR for transcript level of \u003cem\u003eGSTM3TV2\u003c/em\u003e in HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003eor \u003cem\u003eOE-ATF4\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. (B) qRT-PCR for transcript level of \u003cem\u003eATF4\u003c/em\u003e in HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e or \u003cem\u003eOE-ATF4\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. (C) Photographs of HCT116 cells transfected with\u003cem\u003esi-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e or \u003cem\u003eOE-ATF4\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2. Scale bar, 100 μm. (D) Cell viability of HCT116 cells transfected with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e or \u003cem\u003eOE-ATF4\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2 by use of CCK-8. (E) Transwell (with Matrigel) photographs of HCT116 cells with\u003cem\u003esi-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e or \u003cem\u003eOE-ATF4 \u003c/em\u003etransfection, with or without subsequent treatment of 100 ng/ml recombinant CCL2. Scale bar, 50 μm. (F) Number of invasive HCT116 cells with \u003cem\u003esi-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e or \u003cem\u003eOE-ATF4\u003c/em\u003etransfection, with or without subsequent treatment of 100 ng/ml recombinant CCL2. (G) 0-h and 48-h wound healing photographs of HCT116 cells with\u003cem\u003esi-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e or \u003cem\u003eOE-ATF4\u003c/em\u003etransfection, with or without subsequent treatment of 100 ng/ml recombinant CCL2. Scale bar, 100 μm. (H) Quantification of mobility rate of HCT116 cells with\u003cem\u003e si-GSTM3TV2\u003c/em\u003e, \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003eor \u003cem\u003eOE-ATF4\u003c/em\u003e transfection, with or without subsequent treatment of 100 ng/ml recombinant CCL2. ns, p\u0026gt;0.05; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001, from one-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7932268/v1/54fe5a574f4ea2c41675c61a.png"},{"id":100366914,"identity":"086b1717-02b4-4131-b0f4-dcbb62ce4dee","added_by":"auto","created_at":"2026-01-16 07:56:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4289915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7932268/v1/bde535a5-e80d-4df7-8956-8e2d957711c5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CCL2 supports colorectal cancer proliferation, invasion and metastasis through activating lncRNA GSTM3TV2/ATF4 signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) remains the dominant cause of cancer mortality\u0026nbsp;globally [1]. Although incidence has declined in patients with average age at onset in high-income economies, CRC remains the most common cancer globally, and its incidence is increasing in emerging economies [2]. As estimated, CRC burden will increase by 60% by 2030\u0026nbsp;[3]. In addition, early-onset CRC (age \u0026le;50 years) is a growing concern worldwide. As CRC progresses, it becomes increasingly aggressive and metastatic\u0026nbsp;[4]. Development of novel, available, and effective medicines and establishment of a personalized and tailored continuum of care for each patient will contribute to improvement in patient outcomes\u0026nbsp;[5]. Thus, it is of importance to clarify molecular and cellular determinants promoting CRC progression.\u003c/p\u003e\n\u003cp\u003eC-C motif chemokine 2 (CCL2) is critical for CRC progression because its expression is upregulated in CRC patients and CCL2 deficiency can prevent CRC progression [6]. In the tumor microenvironment, cancer-associated fibroblasts and macrophages are two key cell populations that are responsible for CCL2 secretion [7]. Circulating CCL2 is connected to tumor presence, with the potential as a diagnostic biomarker of CRC [8]. Long noncoding RNAs (LncRNAs) are a type of largely functional transcripts with a length of \u0026gt;200 nucleotides [9-11]. A grow body of evidence suggests that lncRNAs participate in\u0026nbsp;the pathogenesis of cancer through governing transcription mechanism, gene-specific transcription, translation, and epigenetic modification, and more [12-14]. Especially, several lncRNAs function as key determinants of cancer progression, including CRC [15-17]. Hence, an in-depth understanding of the biology of lncRNAs may uncover novel treatment targets for CRC. Recently, lncRNA glutathione S-transferase mu 3, transcript variant 2 (\u003cem\u003eGSTM3TV2\u003c/em\u003e) has been found to contribute to proliferation and invasion of hepatocellular carcinoma [18], and gemcitabine resistance of pancreatic cancer [19]. Nonetheless, function and regulatory mechanism of\u003cem\u003e\u0026nbsp;GSTM3TV2\u003c/em\u003e in CRC remain indistinct. Activating transcription factor 4 (ATF4) belongs to the ATF/cAMP response element-binding family and acts as a stress-responsive transcription factor [20-22], which is responsible for orchestrating cellular responses through regulation of many target genes, such as endoplasmic reticulum stress, starvation, and cysteine stress [23]. It has been demonstrated to mediate CRC progression through modulating distinct mechanisms such as glutamine metabolism and glycolysis [24], cysteine metabolism [25],\u0026nbsp;amino acid metabolism\u0026nbsp;[26], and immunogenic cell death\u0026nbsp;[27]. Herein, on the basis of \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e experiments, our findings uncovered that CCL2 may be responsible for CRC cell proliferation, invasion and metastasis primarily through activation of lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 signaling.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and accordance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from Lishui University. All experiments involving animals were conducted according to the ethical policies and procedures approved by the Institutional Animal Care and Committee of Lishui University. All animal experiments were strictly implemented in compliance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCT116 cells (CTCC-085, CTCC, Zhejiang, China) were cultivated in McCoy\u0026rsquo;s 5A medium (PM150710, Pricella, Wuhan, China) containing 10% fetal bovine serum (S711-001, Lonsera, Shanghai, China), 100 units of penicillin-streptomycin (BL505A, Biosharp, USA) in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. HCT116 cells were treated with 100 ng/ml recombinant CCL2 (JN1524, BIO-LAB, Beijing, China) for 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well were seeded into a 6-well plate. On the next day, each well was replaced with 2 ml fresh medium. 125 \u0026mu;l of McCoy\u0026rsquo;s 5A medium, 100 pmol of small interfering RNA (siRNA) of \u003cem\u003eGSTM3TV2\u003c/em\u003e or\u003cem\u003e\u0026nbsp;ATF4\u003c/em\u003e/2.5\u0026mu;g of DNA, and 4 \u0026mu;l Lipo8000\u0026trade; transfection reagent (C0533, Beyotime, Shanghai, China) were mixed. After preparation, the mixture was stored at room temperature for 6 h. Each well was added with 125 \u0026mu;l mixture. The full-length of\u003cem\u003e\u0026nbsp;GSTM3TV2\u003c/em\u003e or \u003cem\u003eATF4\u0026nbsp;\u003c/em\u003esequence was amplified by PCR and cloned into pcDNA3.1 vector (V012531, NovoPro, Shanghai, China). Through Lipo8000\u0026trade; transfection reagent, the cells were transfected with pcDNA3.1 vector. After 48-h culture, transfection effect was evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative reverse transcriptase PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted with Trizol reagent (15596018, Invitrogen, USA), with subsequent reverse transcription. PCR amplification was conducted, with the reaction system volume of 0.2 ml. The programs of PCR amplification included: pre-denaturation (95\u0026deg;C, 5\u0026thinsp;min), denaturation (95\u0026deg;C, 20 sec),\u0026nbsp;annealing elongation (55 ℃, 60 sec; 72℃, 20 sec; 95℃, 15 sec; 40 cycles), and melting curve acquisition (60℃, 60 sec; 95℃, 30 sec; 60℃, 15 sec).\u0026nbsp;This experiment was analyzed by ABI 7500\u0026nbsp;Real-time detector (ABI, USA) and\u0026nbsp;ABI Prism 7500 SDS software. Relative target gene expression was calculated with 2\u003csup\u003e\u0026ndash;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method with GAPDH as a reference control. The primer sequences included: \u003cem\u003eGSTM3TV2\u003c/em\u003e (human), 5\u0026rsquo;-CTCGGGTACTGGGATATTCGT-3\u0026rsquo; (forward), 5\u0026rsquo;-AGGAGGTAGGGCAGATTAGGA-3\u0026rsquo; (reverse); \u003cem\u003eATF4\u003c/em\u003e (human), 5\u0026rsquo;-AGGTGTTCTCTGTGGGTC-3\u0026rsquo; (forward), 5\u0026rsquo;-TAGTGGCTGCTGTCTTGT-3\u0026rsquo; (reverse);\u003cem\u003e\u0026nbsp;Gapdh\u003c/em\u003e (human), 5\u0026rsquo;-GGAGCGAGATCCCTCCAAAAT-3\u0026rsquo; (forward), 5\u0026rsquo;-GGCTGTTGTCATACTTCTCATGG-3\u0026rsquo; (reverse).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Counting Kit-8 (CCK-8)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e8\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well was seeded into a 96-well plate and cultured for 48 h at 37\u0026deg;C. 10 \u0026mu;l CCK-8 reagent (M007, CTCC) was added to each well and the cells were incubated for additional 2 h. Afterwards, absorbance at 450 nm was measured with a microplate reader (MK3, ThermoFisher, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo horizontal lines were marked evenly on the back of the 3.5cm dish utilizing a marker pen. 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were inoculated in 3.5 cm dishes and cultivated overnight at an atmosphere of 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. Until the cell density was up to 90%, a 200 \u0026mu;l gun tip was adopted for making vertical horizontal scratches. The scratched cells were cleaned by PBS. The cells continued to be cultivated for 48 h. Cells were photographed using an IX71 microscope (Olympus, Japan) at 0 h and 48 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 24-well plate was pre-cooled with 800 \u0026mu;l 10% FBS-containing medium at 4℃ and placed in the Transwell (353097, FALCON, USA) chamber. 100 \u0026mu;l of 1 mg/ml Matrigel (356234, Corning, USA) was added vertically into the center of the upper chamber, which was then dried at 37℃ for 4 h. 200 \u0026mu;l cell suspension (3\u0026times;10\u003csup\u003e5\u003c/sup\u003e/ml) was inserted into the upper chamber of the Transwell and cultivated for 48 h at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The Transwell chamber was cleaned with PBS, with subsequent cell fixation utilizing 70% icy ethanol solution for 1 h. 0.5% crystal violet reagent was used for staining. After 20 min at room temperature, the cells were washed by PBS, and uninvaded cells were removed with a clean cotton ball. Invaded cells were photographed under an IX71 microscope (Olympus, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcutaneous mouse xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experimental protocols were conducted strictly following the ethical approval of the Institutional Animal Care and Committee of Lishui University. BALB/c nude mice (4~6 weeks old) were purchased from CAVENS (Changzhou, China) and observed in a SPF environment for 3 days. The skin at the injection site of nude mice was disinfected with 75% alcohol, and cell suspension with 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells was injected into the underarm of nude mice. The piercing point was ~1 cm away from the injection point, forming a raised skin mound to prevent liquid leakage, followed by skin disinfection. The tumor size was measured every 3 days. The maximal tumor size permitted by the Experimental Animal Ethics Committee is 1 cm in diameter. The longest (a) and shortest (b) diameters of tumor were measured with a vernier caliper, followed by calculation of the tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) = ab\u003csup\u003e2\u003c/sup\u003e/2. The tumor growth curve was plotted. None of our animals had tumors larger than 1\u0026nbsp;cm in diameter. The nude mice were euthanized 28 days later, and the subcutaneous tumor was dissected and photographed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence (IF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were fixed in 4% paraformaldehyde for 24\u0026thinsp;h, embedded in wax molds, and cut into 5-\u0026mu;m-thick sections. The paraffin sections were deparaffinized, and baked at 60\u0026deg;C for 3\u0026thinsp;h. After antigen retrieval (P0086, Beyotime), the sections were blocked with 5% BSA (4240GR500, BioFROXX, Germany) at 37℃ for 1 h. Antibody of ATF4 (1:200, 10835-1-AP, Proteintech) was added to the sections, with subsequent 2-h incubation at 37℃. After cleaning with PBS, the sections were incubated with fluorescein (FITC)-conjugated goat anti-mouse IgG(H+L) (1:100, SA00003-1, Proteintech) for additional 1 h. Afterwards, they were cleaned with PBS. Hoechst (C1022, Beyotime) was added to the sections, with subsequent 15-min incubation at room temperature.\u0026nbsp;Immunofluorescent images were acquired by a fluorescence\u0026nbsp;microscope (Olympus, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independently repeated experiments. All the analyses were conducted by use of GraphPad Prism software v9.0.0. Statistical difference was evaluated by one- or two-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post hoc test. P\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCCL2 upregulates lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e expression to support CRC cell proliferation, invasion and metastasis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment of recombinant CCL2 (100 ng/ml) notably elevated the transcript level of \u003cem\u003eGSTM3TV2\u003c/em\u003e in HCT116 cells, which was similar to the effect of transfection with pcDNA3.1 vector with full-length of \u003cem\u003eGSTM3TV2\u003c/em\u003e (\u003cem\u003eOE-GSTM3TV2\u003c/em\u003e) (\u003cstrong\u003eFigure 1A\u003c/strong\u003e). This suggested that CCL2 was effective in activating the transcription of \u003cem\u003eGSTM3TV2\u003c/em\u003e in CRC cells. Nevertheless, transfection with \u003cem\u003eGSTM3TV2\u003c/em\u003e siRNA (\u003cem\u003esi-GSTM3TV2\u003c/em\u003e) prominently attenuated the CCL2-induced elevation in the transcript level of \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFigure 1A\u003c/strong\u003e). Next, we investigated whether CCL2 affected CRC progression through transcriptionally activating \u003cem\u003eGSTM3TV2\u003c/em\u003e. As shown in the CCK-8 results, both CCL2 treatment and overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e led to a remarkable enhancement in HCT116 cell proliferation\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFigure 1B, C\u003c/strong\u003e). Endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e reversed the impact of CCL2 treatment on heightening HCT116 cell proliferation. Through Transwell (with Matrigel), cellular invasion was tested. Upon CCL2 treatment, the invasive ability of HCT116 cells showed notable improvement, with the similar effect to overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e (\u003cstrong\u003eFigure 1D, E\u003c/strong\u003e). The improvement in the invasive ability was prominently weakened in the context of endogenous \u003cem\u003eGSTM3TV2\u003c/em\u003e silencing. Wound healing assay was also conducted. Consequently, cellular mobility was strengthened by both CCL2 treatment and overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e (\u003cstrong\u003eFigure 1F, G\u003c/strong\u003e). Endogenous silencing of \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003eeffectively weakened the impact of CCL2 treatment on cellular mobility. Hence, the above data uncovered that CCL2 may upregulate \u003cem\u003eGSTM3TV2\u003c/em\u003e to expression support CRC cell proliferation, invasion and metastasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCL2 accelerates CRC growth through elevating lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough injection of 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e HCT116 cells (transfected with\u003cem\u003e\u0026nbsp;si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e, with or without subsequent treatment of 100 ng/ml recombinant CCL2) into the underarm of nude mice, subcutaneous mouse xenograft models were developed. 28 days later, we dissected and photographed subcutaneous tumors.\u003cstrong\u003e\u0026nbsp;Figure 2A, B\u003c/strong\u003e show the tumor-bearing nude mice and subcutaneous tumors. Clearly, both CCL2 treatment and overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e led to accelerated tumor growth. Suppressing \u003cem\u003eGSTM3TV2\u003c/em\u003e weakened the impact of CCL2 treatment on accelerating tumor growth. In addition, tumor volume was measured regularly. Consequently, both CCL2 treatment and overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e notably increased tumor volume, while \u003cem\u003eGSTM3TV2\u003c/em\u003e suppression attenuated the impact of CCL2 treatment on increasing tumor volume (\u003cstrong\u003eFigure 2C\u003c/strong\u003e). Collectively, CCL2 may accelerate CRC growth via elevating lncRNA \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003eexpression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGSTM3TV2\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003esupports CRC cell proliferation, invasion and metastasis through transcriptionally activating \u003cem\u003eATF4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverexpressing \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003ethrough transfection of pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e led to the remarkable elevation in \u003cem\u003eATF4\u003c/em\u003e transcript level in HCT116 cells (\u003cstrong\u003eFigure 3A, B\u003c/strong\u003e). Additionally, endogenous silencing of \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003ethrough transfecting \u003cem\u003esi-GSTM3TV2\u003c/em\u003e prominently reduced transcript level of \u003cem\u003eATF4\u003c/em\u003e in HCT116 cells. This suggested that\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eGSTM3TV2\u003c/em\u003e may transcriptionally activate \u003cem\u003eATF4\u003c/em\u003e in CRC cells. On the basis of the CCK-8 results, HCT116 cell proliferation was heightened by overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e, which was impaired by endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e (\u003cstrong\u003eFigure 3C, D\u003c/strong\u003e). As shown in the Transwell (with Matrigel) results, overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e led to the enhancement in HCT116 cell invasion, with endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e impairing HCT116 cell invasion (\u003cstrong\u003eFigure 3E, F\u003c/strong\u003e). In addition, wound healing assay was carried out. HCT116 cell mobility was heightened by overexpressing \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003eand was suppressed by endogenous silencing of \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFigure 3G, H\u003c/strong\u003e)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThese data indicated that \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003emay support CRC cell proliferation, invasion and metastasis via transcriptionally activating \u003cem\u003eATF4\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGSTM3TV2\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003esupports CRC growth through elevating ATF4 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1\u0026times;10\u003csup\u003e7\u003c/sup\u003e HCT116 cells with\u003cem\u003e\u0026nbsp;si-GSTM3TV2\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e transfection was infected into the underarm of nude mice to develop subcutaneous mouse xenograft models. 28 days later, subcutaneous tumors were dissected and photographed (\u003cstrong\u003eFigure 4A, B\u003c/strong\u003e). As a result, overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e facilitated tumor growth, whereas endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e prevented tumor growth. Additionally, overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e notably elevated tumor volume, with opposite findings when \u003cem\u003eGSTM3TV2\u003c/em\u003e was endogenously silenced (\u003cstrong\u003eFigure 4C\u003c/strong\u003e). Through IF, ATF4 expression levels were measured in mouse tumors. Consistent with the \u003cem\u003ein vitro\u003c/em\u003e findings, overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e led to the elevation in ATF4 expression in mouse tumors, while endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e led to the reduction in ATF4 expression (\u003cstrong\u003eFigure 4D, E\u003c/strong\u003e). Altogether, \u003cem\u003eGSTM3TV2\u003c/em\u003e may support CRC growth through elevating ATF4 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCL2 supports CRC cell proliferation, invasion and metastasis through activating lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther analysis showed that endogenous silencing of \u003cem\u003eATF4\u003c/em\u003e did not alter the upregulation in \u003cem\u003eGSTM3TV2\u003c/em\u003e expression induced by CCL2 treatment or transfection of pcDNA3.1 vector with \u003cem\u003eOE-GSTM3TV2\u003c/em\u003e in HCT116 cells (\u003cstrong\u003eFigure 5A\u003c/strong\u003e). Furthermore, overexpressing \u003cem\u003eATF4\u003c/em\u003e did not alter the reduction in \u003cem\u003eGSTM3TV2\u003c/em\u003e expression caused by \u003cem\u003esi-GSTM3TV2\u003c/em\u003e transfection. It was also observed that neither CCL2 treatment nor overexpressing \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003ereversed the alterations in \u003cem\u003eATF4\u003c/em\u003e expression caused by \u003cem\u003esi-ATF4\u003c/em\u003e or pcDNA3.1 vector with \u003cem\u003eOE-ATF4\u003c/em\u003e (\u003cstrong\u003eFigure 5B\u003c/strong\u003e)\u003cem\u003e.\u003c/em\u003e As depicted in the CCK-8 results, endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e did not reverse the impact of overexpressing \u003cem\u003eATF4\u003c/em\u003e on strengthening HCT116 cell proliferation (\u003cstrong\u003eFigure 5C, D\u003c/strong\u003e)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eAdditionally, neither CCL2 treatment nor overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e altered the suppression in cell proliferation caused by endogenous silencing of \u003cem\u003eATF4\u003c/em\u003e. The Transwell (with Matrigel) results showed that endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e did not attenuate the impact of overexpressing \u003cem\u003eATF4\u003c/em\u003e on strengthening HCT116 cell invasion (\u003cstrong\u003eFigure 5E, F\u003c/strong\u003e)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eNeither CCL2 treatment nor overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e reversed the invasion suppression caused by endogenous silencing of \u003cem\u003eATF4\u003c/em\u003e. The results from the wound healing assay also demonstrated that endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e did not weaken the improvement in HCT116 cell mobility triggered by overexpressing \u003cem\u003eATF4\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFigure 5G, H\u003c/strong\u003e). Neither CCL2 treatment nor overexpressing \u003cem\u003eGSTM3TV2\u003c/em\u003e reversed the cell mobility inhibition triggered by endogenous silencing of \u003cem\u003eATF4\u003c/em\u003e. Altogether, our findings uncovered that CCL2 may support CRC cell proliferation, invasion and metastasis via activating lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 signaling.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite extensive research and improvement in biological knowledge, therapeutic strategies, and prognostic outcomes, CRC remains the most diagnosed and deadliest malignancy around the world [28]. Determining new, available and effective therapeutic strategies in this large and expanding patient population is a critical and largely unmet medical need [29, 30]. This study for the first time proposed the CCL2/GSTM3TV2/ATF4 mechanism facilitating CRC cell proliferation, invasion and metastasis.\u003c/p\u003e\n\u003cp\u003eTo comprehend how new therapeutic options target CRC, it is of importance to discern the intricate molecular mechanisms underlying CRC [31, 32]. Recognizing the factors that modulate proliferation, invasion and metastasis will offer the basis of which new treatment modalities can be assessed in clinical trials and eventually improve patient prognosis [33-35]. Herein, we observed that CCL2 effectively elevated lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e expression in CRC cells. In addition, CCL2 treatment resulted in a remarkable enhancement in CRC cell proliferation, invasion and metastasis, but endogenous silencing of \u003cem\u003eGSTM3TV2\u0026nbsp;\u003c/em\u003ecould effectively weaken the impact of CCL2 treatment on the malignant behaviors of CRC cells, suggesting that CCL2 may elevate lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e expression to support CRC progression. CCL2 is primarily secreted by cancer-associated fibroblasts and macrophages in the tumor microenvironment. Numerous studies have reported the involvement of CCL2 in CRC progression, and targeted suppression of CCL2 can hinder CRC progression and metastasis [6, 36, 37]. LncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e was determined as a regulatory target of CCL2, but detailed regulatory mechanism needs to be in-depth explored. Our study for the first time proposed the involvement of lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e in heightening proliferation, invasion and metastasis of CRC cells.\u003c/p\u003e\n\u003cp\u003eLncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e conferred malignant behaviors of CRC cells through elevating ATF4 expression. ATF4 acts as a stress-responsive transcription factor, which is activated and promotes cell adaptation for survival in response to cellular stress [38-40]. For instance,\u0026nbsp;ATF4 prevents hepatocellular carcinoma occurrence through activation of SLC7A11 to inhibit stress-associated ferroptosis\u0026nbsp;[21]. ATF4 links endoplasmic reticulum stress with reticulophagy in glioblastoma cells\u0026nbsp;[41]. ATF4-mediated integrated stress response drives angiogenesis and cancer development through activation of perivascular cancer-associated fibroblasts\u0026nbsp;[42]. Inducing endoplasmic reticulum stress through ATF4-SPHK1 signaling contributes to glioblastoma aggressiveness and resistance to chemotherapy\u0026nbsp;[43]. ATF4 has been extensively studied in CRC pathogenesis, and targeted inhibition of ATF4 has been evidenced to prevent CRC progression\u0026nbsp;[26, 44, 45]. In the present study, ATF4 was determined as a potential downstream target of GSTM3TV2 in CRC cells upon CCL2 treatment, with more studies being required for proving the direct interaction between \u003cem\u003eGSTM3TV2\u003c/em\u003e and ATF4.\u003c/p\u003e\n\u003cp\u003eAlthough our study demonstrated that CCL2 accelerated CRC cell proliferation, invasion and metastasis via activating lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 signaling, the regulatory mechanisms and binding structures of CCL2/lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e and lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 need to be in-depth explored. The clinical significance of the CCL2/lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 mechanism in CRC patients\u0026rsquo; survival outcomes needs to be investigated in large clinical cohorts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCollectively, our study revealed that CCL2 may be responsible for accelerating CRC cell proliferation, invasion and metastasis through activation of lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 signaling, hopefully offering potential therapeutic targets for the clinical treatment of CRC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRC: colorectal cancer; CCL2: C-C motif chemokine 2; lncRNAs: long noncoding RNAs; GSTM3TV2: glutathione S-transferase mu 3, transcript variant 2; ATF4: activating transcription factor 4; siRNA: small interfering RNA; qRT-PCR: quantitative reverse transcriptase PCR; CCK-8: Cell Counting Kit-8; FITC: fluorescein; SD: standard deviation; ANOVA: analysis of variance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYL and XF designed the study. YR, XW, and HZ completed the animal experiment and collated the data, conducted data analysis and produced the first draft of the male man uscript. ZL, SY and ZZ participated in the guidance of this project. All authors have read and approved the final submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that we have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments involving animals were conducted according to the ethical policies and procedures approved by the Institutional Animal Care and Use Committees of the Lishui University. All animal experiments were strictly implemented in compliance with the ARRIVE guidelines. The maximum subcutaneous tumor diameter allowed by the ethics committee of our hospital in nude mice was 1 cm, and this study met this criterion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated and/or analyzed during the current study can be provided based on the request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Plan of Medicine and Health of Zhejiang Province (2025KY1968), the grants from the National Natural Science Foundation of China (No. 82200617), the Public Welfare Technology Research Program of Lishui City (No. 2022GYX51), and Zhejiang Provincial Natural Science Foundation of China under Grant No.LY21H160001 .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhou J, Yang Q, Zhao S\u003cem\u003e et al.\u003c/em\u003e Evolving landscape of colorectal cancer: Global and regional burden, risk factor dynamics, and future scenarios (the Global Burden of Disease 1990-2050). 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Phytomedicine 2022; 99:154007.\u003cstrong\u003e\u003cbr\u003e \u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"colorectal cancer, CCL2, GSTM3TV2, ATF4, tumor progression","lastPublishedDoi":"10.21203/rs.3.rs-7932268/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7932268/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e Despite extensive research and advances in biological knowledge, therapeutic strategies, and prognostic outcomes, colorectal cancer (CRC) remains a common malignancy around the world. Elucidating molecular determinants promoting CRC progression will help develop novel, available, and effective treatment modalities. In this study, we proposed a novel C-C motif chemokine 2 (CCL2)/long noncoding RNA lncRNA glutathione S-transferase mu 3, transcript variant 2 (\u003cem\u003eGSTM3TV2\u003c/em\u003e)/activating transcription factor 4 (ATF4) mechanism responsible for promoting CRC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eCRC cells were exposed to 100 ng/ml recombinant CCL2, and/or transfected with \u003cem\u003eGSTM3TV2\u003c/em\u003eor\u003cem\u003e ATF4\u003c/em\u003e siRNA or pcDNA3.1 vector with the full-length of\u003cem\u003e GSTM3TV2\u003c/em\u003eor \u003cem\u003eATF4 \u003c/em\u003esequence. Cell proliferation, invasion and metastasis were investigated through CCK-8, wound healing, Transwell, and subcutaneous mouse xenograft models. \u003cem\u003eGSTM3TV2\u003c/em\u003e and ATF4 expression levels were measured via qRT-PCR and immunofluorescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCCL2 treatment upregulated lncRNA\u003cem\u003e GSTM3TV2\u003c/em\u003e expression and conferred proliferative, invasive and metastatic potential of CRC cells, but such effects were effectively reversed by endogenous silencing of \u003cem\u003eGSTM3TV2\u003c/em\u003e. ATF4 was determined to be a downstream factor of \u003cem\u003eGSTM3TV2\u003c/em\u003e, whose expression levels were positively modulated by \u003cem\u003eGSTM3TV2\u003c/em\u003e. Endogenous silencing of\u003cem\u003e ATF4\u003c/em\u003ereversed the impact of overexpressing\u003cem\u003e GSTM3TV2 \u003c/em\u003eor CCL2 treatment on heightening CRC cells proliferation, invasion and metastasis, thus preventing CRC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Altogether, our findings suggest that the CCL2/lncRNA \u003cem\u003eGSTM3TV2\u003c/em\u003e/ATF4 signaling could be an important mechanism underlying CRC progression, offering potential theoretical basis for the clinical therapy of CRC.\u003c/p\u003e","manuscriptTitle":"CCL2 supports colorectal cancer proliferation, invasion and metastasis through activating lncRNA GSTM3TV2/ATF4 signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 18:55:26","doi":"10.21203/rs.3.rs-7932268/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b26bbdf-a891-402d-917b-bb8ad274dbf5","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-13T09:25:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 18:55:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7932268","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7932268","identity":"rs-7932268","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}