MicroRNA-532-3p modulates colorectal cancer cell proliferation and invasion via suppression of FOXM1

MicroRNA-532-3p modulates colorectal cancer cell proliferation and invasion via suppression of FOXM1 | 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 Article MicroRNA-532-3p modulates colorectal cancer cell proliferation and invasion via suppression of FOXM1 Ketakee Mahajan, Ramesh Pothuraju, Ani Das, Asha Nair This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4217992/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 Forkhead-box M1 (FOXM1) is a proliferation-associated transcription factor, overexpressed in almost all the cancers. Naturally, the mechanisms of FOXM1 regulation have been under investigation. Previously, we showed that FOXM1 binds to promoters of certain microRNAs. Database mining led to several microRNAs that might interact with FOXM1 3’UTR. The interactions between shortlisted microRNAs and FOXM1 3’UTR was quantitated by dual luciferase reporter assay. MicroRNA-532-3p interacted with 3’UTR of FOXM1 mRNA transcript most efficiently. MicroRNA-532-3p was ectopically overexpressed in colorectal cancer cell lines, leading to reduced transcript and protein levels of FOXM1 and cyclin B1, a direct transcriptional target of FOXM1. Flow cytometry analysis revealed an unaltered cell cycle. A clonogenic assay was conducted that revealed a decline in the ability of cells to form colonies. Additionally, wound healing assay and matrigel invasion assays were carried out. Significant reduction in migratory and invading cell numbers were observed, which were reinforced at molecular levels by the altered transcript and protein levels of the conventional EMT markers E-cadherin and vimentin. Overall, this study identifies the interaction between microRNA-532-3p and FOXM1 3’UTR, leading to reduced protein levels. Ectopic expression of miR-532-3p leads to suppression in cellular proliferation, migration and invasion in CRC cells. Biological sciences/Cancer Biological sciences/Cancer/Gastrointestinal cancer Biological sciences/Cancer/Tumour biomarkers MicroRNA Colorectal Cancer FOXM1 Metastasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Colorectal cancer (CRC) stands as the third most prevalent and second most lethal cancer worldwide. Its occurrence ranks second and third among women and men, respectively 1 . The incidence of CRC has seen a consistent increase over recent decades 2 . The causes of CRC can be either non-hereditary or sporadic, involving the downregulation of tumor suppressor genes and genes responsible for regulating cell cycle processes, DNA damage repair, and apoptosis 3 . Traditionally, CRC has been documented predominantly in the elderly population, affecting individuals aged 50 years and above 4 . The likelihood of developing CRC is notably higher among those with inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn’s disease 5 . Forkhead box M1 (FOXM1) stands as a pivotal member within the forkhead box proteins family, a group of transcription factors 6 . Functioning as a master regulator of the cell cycle, FOXM1 plays a crucial role in orchestrating the G2/M transition. It exerts direct control over the expression of key elements such as Aurora B kinase, Cyclin B, and Cdc25b phosphatase 7 . Notably, FOXM1 has demonstrated overexpression across a wide spectrum of cancers 8 , 9 . Elevated levels of FOXM1 expression have been associated with an unfavorable prognosis in breast cancer, esophageal adenocarcinoma 10 , and CRC 11 , 12 . Beyond its positive regulatory role in the cell cycle, FOXM1 engages in various cancer-related functions such as cellular proliferation 13 , migration, invasion 14 , and resistance to drugs 15 . The multifaceted nature of FOXM1 positions it as a potential therapeutic target, sparking interest in suppressing its expression for over a decade 16 . Within the eukaryotic genome, specific regions encode various types of non-coding RNAs, primarily categorized into short and long noncoding RNAs 17 . Noteworthy examples of short noncoding RNAs include microRNAs, piRNAs, and snoRNAs 18 , 19 . MicroRNAs represent a group of evolutionarily conserved, naturally occurring small nucleotides measuring approximately 19-24 bases in length. Their primary role involves post-transcriptional regulation of gene expression, accomplished through binding to mRNA transcripts. This interaction disrupts the translational complex, thereby impeding protein synthesis. Beyond translational inhibition, microRNAs are recognized for their ability to destabilize and degrade messenger RNA molecules 19 , 20 . MicroRNA-532-3p is an upcoming tumor suppressor in various cancers. In CRC, it has been reported to inhibit Wnt/β-catenin signaling, effectively impeding the disease's progression. The present study corroborates these findings by revealing its ability to enhance cleavage in PARP and Pro-caspase 7. Notably, ectopic overexpression of miR-532-3p in CRC cells resulted in a significant reduction in proliferative potential, migration, and invasion, corroborated with the previous study 21 . Similarly, its role in suppressing proliferation and facilitating apoptosis has been investigated in lymphoma, where it targets β-catenin 22 . In prostate cancer, miR-532-3p was found to impede bone metastasis by disabling NF-κB signaling 23 . In the present study, we have discussed the role of miR-532-3p in suppressing cellular proliferation, migration and invasion of CRC. MicroRNA-532-3p has been reported to act as a tumor suppressor in a renal cell carcinoma 24 and colorectal cancer 25 . Both the strands of microRNA-532; viz. miR-532-5p and miR-532-3p are known to positively and negatively impact the occurrence and prognosis of several types of cancers such as breast cancer, hepatocellular carcinoma, ovarian cancer 26 , 27 , 28 , 29 , 30 , 31 . Results MicroRNA-532-3p interacts with 3’UTR of FOXM1 transcript The database mining revealed several microRNAs potentially capable of regulating the expression by binding through the 3’UTR of FOXM1 transcript ( Fig. 1a ). Databases such as TargetScan and miRecords gave more than 450 hits each with possible complementary seed and target sequences in microRNAs and the 3’UTR, respectively ( Tables S1-S4 ). Network databases such as miRtargetLink also predicted several interactions, along with reporting a few validated ones. The microRNAs were analysed for the most frequently occurring hits by Venn analysis ( Fig. 1b ). The selected microRNAs, cloned in pRIP vector were co-transfected with Psicheck2 vector containing 3’UTR of FOXM1 insert, with a firefly luciferase gene in the downstream region ( Fig. S1-S3 ). The dual luciferase assay confirmed the interactions of miR-149-5p and -3p, miR-370-3p, miR-532-3p, miR-590-5p, miR-671-5p and miR-876-5p with FOXM1 3’UTR, indicated by the reduced luciferase activity. MicroRNA-532-3p showed nearly 50% of reduction in luciferase activity, suggesting a strong interaction ( Fig 1c ). Hence, miR-532-3p was selected for further investigation. A representation of a possible interaction between miR-532-3p and FOXM1 3’UTR was obtained from TargetScan database ( Fig. 1d ). miR-532-3p is down-regulated in Colorectal cancer Investigating The Cancer Genome Atlas (TCGA) datasets revealed that the expression of microRNA-532-3p was significantly lower in the malignant tissues compared to the normal cells and tissues ( Fig. 2a ). Also, the expression levels of miR-523-3p were significantly lower in colorectal tumors compared to the normal counterparts ( Fig. 2b ). Several CRC cell lines were screened for the expression of microRNA-532-3p, revealing that HCT116 showed the highest expression level ( Fig.2c) . However, Kaplan-Meier analysis showed no significant decrease in overall survival in colon and rectal patients with high miR-532-3p ( Fig. 2d ). miR-532-3p overexpression diminishes proliferation, migration and invasion of CRC cells To understand the functional significance of miR-532-3p, ectopic overexpression of miR-532-3p in various CRC cell lines was performed ( Fig. 3a ). An evaluation of potential cell death resulting from miR-532-3p overexpression was performed through a colorimetric analysis using the MTT assay ( Fig. 3b ). Intriguingly, no notable disparity in cell viability was observed upon miR-532-3p overexpression. However, a reduction in the expression of the cell proliferation marker PCNA was detected in CRC cell lines overexpressing miR-532-3p ( Fig. 3c ). Furthermore, ectopic overexpression of miR-532-3p in HT29 cells led to about 40% reduction in the number of colonies formed ( Fig. 3d) . A wound healing experiment conducted in SW480 cells with miR-532-3p overexpression revealed a slower rate of wound closure in the cells overexpressing miR-532-3p compared to the control at various time points ( Fig. 3e ). Additionally, a significant reduction was noted in the invasion of SW480 cells through matrigel in those ectopically overexpressing miR-532-3p ( Fig. 3f ). These observations were further supported by quantitative analysis of molecular markers at both transcript and protein levels. The said analyses revealed upregulated levels of the epithelial marker E-cadherin and reduced expression of vimentin in cells overexpressing miR-532-3p ( Fig. 3g and h ). These findings suggest that miR-532-3p influences cellular proliferation, migration, and invasion properties in CRC in vitro . miR-532-3p overexpression led to higher apoptosis, possibly via suppression of FOXM1 Primary findings from TCGA datasets revealed that the expression levels of FOXM1 are significantly altered between tumors and their disease-free counterparts in both colon and rectal tissues (Fig. 4a) . Furthermore, in terms of survival, TCGA revealed no significant difference between the survival rates of patients exhibiting high levels of FOXM1 against those with low levels (Dataset GSE12945) ( Fig. 4b ). Several CRC cell lines were screened for the expression of FOXM1 at transcript and protein levels, thereby showing the high transcript levels of FOXM1 in DLD1 and SW480 cells and high proteinlevel expression in HT29 cells ( Fig. 4c ). Ectopic overexpression of microRNA-532-3p resulted in reduced transcript and protein levels of FOXM1. This confirmed the ability of miR-532-3p to regulate FOXM1 at post-transcriptional level. Furthermore, the levels of cyclin B1 were analysed, which is a direct transcriptional target of FOXM1. The real-time PCR and western immunoblotting revealed that overexpressing miR-532-3p resulted in reduced transcript and protein levels of cyclin B1 in CRC cells ( Fig. 4d ). To determine whether overexpression of miR-532-3p may play role in cell cycle progression, we analyzed the DNA content by flow cytometry analysis of CRC cells overexpressing showed very little increase in the percentage of cells in G 2 /M phase ( Fig. 4e ). However, western immunoblotting analysis showed increased levels of several apoptotic markers such as activated form of caspase 7 and cleaved PARP levels. Additionally, an anti-apoptotic BCL2 marker was found to be downregulated ( Fig. 4f ). In essence, microRNA-532-3p interacts with FOXM1 3’UTR, leading to decreased protein levels, thereby suppressing proliferation, migration and invasion in colorectal cancer cells ( Fig. 4g ). Discussion MicroRNAs, recognized as one of the smallest classes of biomolecules, exert dynamic regulatory influence on developmental conditions and diseases through their distinctive mechanisms 32 , 33 , 34 . Their impact spans various facets of mammalian development, including the neural system, placental site, and hematopoiesis, operating at the minutest scale 35 , 36 , 37 , 38 . In the context of cancer, microRNAs play a pivotal role in governing processes ranging from the disease's onset and proliferation to the regulation of metastasis, as well as influencing sensitivity or resistance to chemotherapeutic drugs 39 , 40 , 41 , 42 , 43 . Their involvement extends to shaping the tumor microenvironment as well 44 . MicroRNAs execute these multifaceted functions by targeting components of several signaling pathways crucial to both development and cancer 45 , 46 , 47 . Recent years, MicroRNA-532-3p has emerged as a tumor suppressor in various cancers. In CRC, it has been documented to disrupt Wnt/β-catenin signaling, effectively impeding the disease's progression. The present study bolsters these findings and reveals the ability of miR-532-3p to upregulate p53 expression and enhance cleavage in PARP. Notably, ectopic overexpression of miR-532-3p in CRC cells resulted in a significant reduction in proliferative potential, migration, and invasion, corroborated with the previous study 25 . Similarly, its role in restraining proliferation and facilitating apoptosis has been investigated in lymphoma, where it targets β-catenin 22 . In prostate cancer, miR-532-3p hinders bone metastasis by inactivating NF-κB signaling 23 . Conversely, in renal cell carcinoma, clinical specimens showed a correlation between low expression levels of both mature strands of pre-miR-532, miR-532-5p, and miR-532-3p, and the presence of the disease 24 . Despite its well-established tumor-suppressive role in most cancers, microRNA-532-3p, along with its other mature form, microRNA-532-5p, has been recognized for exhibiting oncogenic roles in hepatocellular carcinoma and breast cancer 27 , 31 , 30 . FOXM1, the new target of miR-532-3p that we have discovered in this study is a known master regulator of cell cycle 7 . This has made FOXM1 a crucial factor in the occurrence and progression of cancer as a group of diseases that arises from dysregulation in cell cycle 48 , 49 . As mentioned before, FOXM1 has been of interest in reference to anticancer therapeutics as a key target 16 , 50 . Among the chemical inhibitors of FOXM1, thiazole antibiotics such as thiostrepton and siomycin A have been reported to induce apoptosis in human cancer cells by repressing the transcriptional activity of FOXM1 51 , 52 . Besides the cell cycle aspect, the higher expression levels of FOXM1 have been known to correlate with tumor invasion, leading to poor prognosis in colorectal cancer 11 . This ability of FOXM1 to aid and promote tumorigenesis and metastasis has been studied in ovarian carcinoma, breast cancer, non-small cell lung cancer as well 53 , 54 , 55 . These reports prove the importance studying FOXM1 and its regulation in the occurrence and progression of the disease. In the present study, we have demonstrated the ability of microRNA-532-3p to suppress the proliferation, migration, and invasion of CRC cells. However, we did not observe a direct effect of miR-532-3p on cell death and progression of cell cycle. Furthermore, we have established FOXM1 as a novel target of miR-532-3p, adding another important link in the events leading to malignancy. This finding suggests that miR-532-3p could be a significant predictive marker for metastasis and prognosis in CRC. Further studies in other types of cancers where miR-532-3p has been reported as a tumor suppressor molecule will help gain a better insight of the picture. This study validates the interaction between microRNA-532-3p and 3’UTR of FOXM1 transcript, which resulted in decreased protein levels of FOXM1. This consequently suppressed the proliferation, migration and invasion in colorectal cancer cells. The link between miR-532-3p and FOXM1 could offer an insight on the direct tumor suppressive function exhibited by miR-532-3p in colorectal cancer, among other types of the disease. Materials And Methods Cell lines and culture The human CRC cell lines (Caco2, Colo320, DLD1, HCT116, HT29, SW480, and SW620) and human embryonic kidney cells (HEK293T) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM), enriched with 10% fetal bovine serum (Gibco, Invitrogen, USA) and supplemented with antibiotics (100 X antibacterial-antimycotic solution, GibCo, USA). Cultures were maintained at 37°C in a 5% CO 2 atmosphere within a humidified chamber. In silico mining of databases and selection of microRNAs In the quest to identify microRNAs that may regulate the expression of FOXM1 by interacting with its 3’UTR transcript, multiple databases were utilized. These databases fell into four distinct categories: predictive interaction (TargetScan, miRecords, mirMap), network (Reactome and Biogrid), expression profile (TissueAtlas and dbDEMC 2.0), and literature (miRCancer). The results obtained from these searches were then subjected to Venn analysis to pinpoint the microRNAs that appeared most frequently. Subsequently, seven microRNAs were chosen from the compiled lists. Cloning The wild-type 3’UTR sequence and the sequences corresponding to the genes encoding the identified microRNAs were retrieved from the UCSC genome browser. Primers were designed, incorporating 100-base pair flanking regions for the specified genomic segments. Subsequently, amplicons were generated using Platinum high-fidelity DNA polymerase (Thermo Invitrogen, USA). The obtained amplicons underwent restriction digestion; XhoI and NotI for the PsiCheck2 vector and FOXM1 3’UTR, and BamHI and HindIII for the modified pRIP vector and microRNA amplicons. Ligation was performed at 4°C utilizing the T4 ligase kit (Thermo Invitrogen, USA). The ligated products underwent validation for inserts through PCR, restriction digestion, and Sanger sequencing. Dual luciferase reporter assay Dual luciferase reporter assay was performed as per instructed in the manufacturer’s protocol (Promega, Wisconsin, USA). HEK293T cells were transiently co-transfected with PsiCheck2 vector containing 3’UTR of FOXM1 and a modified pRIP vector containing the gene coding for one of the selected microRNAs. After 72 hours of transfection, the cells were lysed using Passive Lysis Buffer (1X). These lysates were used for dual luciferase reporter assay. Promega Glomax luminometer was used to record the luminescence. An empty PsiCheck2 vector was used as the internal control. Cell proliferation and colony formation assays CRC cells (5×10 4 cells/well) were plated in a 96-well cell culture plate and cultured at 37ºC with 5% CO2 in a humidified incubator (Thermo Scientific, USA) for 72 hours. Cell survival was assessed using the MTT assay. MTT reagent was applied to each well and incubated in darkness at 37ºC for 4 hours. Formazan crystals were then dissolved in isopropyl alcohol, and absorbance levels were measured. For the colony formation assay, cells (1×10 5 ) were initially plated in 35-mm culture dishes and cultured in standard DMEM. Upon reaching approximately 40-50% confluency, cells underwent transfection with miR-532-3p and the control plasmid, employing Lipofectamine 3000 as per the manufacturer’s instructions. Transfection was halted after 24 hours, followed by the addition of fresh media. Subsequently, cells were trypsinized and seeded at a density of 1000 cells per well in 6-well culture plates. The cells were then cultivated for a period of 14 days, with media renewal every 72 hours. After the incubation period, cells were rinsed with PBS and fixed in 4% paraformaldehyde (PFA) for 5 minutes. Following PFA removal, the fixed cells underwent three washes with PBS. A 0.5% crystal violet solution was applied to the cells and allowed to incubate for 30 minutes at room temperature. The crystal violet solution was then aspirated, and the plate was washed under tap water before being air-dried. Subsequently, colonies formed were enumerated, with those containing 50 or more cells considered as positive. Wound healing and cell invasion assays Cells (5×10 4 per well) were initially plated in a 12-well culture plate. Upon reaching 50% confluency, cells underwent transfection with miRNA-532-3p and the control plasmid as mentioned previously. Transfection was terminated after 24 hours, and fresh regular media were introduced to the cells. When the cells reached approximately 90% confluency, a wound was generated by gently scratching with a sterile micropipette tip. Subsequently, the cells were examined under an inverted microscope every 24 hours post-washing with PBS. For the matrigel invasion assay, CRC cells (1.5×10 5 ) were seeded in 60-mm dishes and transfected with the control plasmid and miR-532-3p mimic. The cells were then incubated at 37°C in OptiMEM. After 24 hours, the transfected cells were trypsinized and diluted to a suspension of 5×10 4 cells/mL. A 24-well plate with inserts containing matrix (BD Biosciences, USA) was pre-wet with regular medium for two hours at 37°C. Using sterile forceps, the inserts were carefully transferred onto these wells. The cell suspension was then added onto these inserts and incubated for 24 hours. The invaded cells on the lower side of the membrane were stained with 0.5% crystal violet stain, and counted in various fields. Immunoblotting analysis The cells were harvested and rinsed with PBS, followed by lysis in RIPA buffer at 4°C with agitation at 1400 RPM on a thermomixer for one hour and forty-five minutes. The resulting solution underwent centrifugation at 14000 RPM for 15 minutes at 4°C, and the supernatant, constituting whole cell protein, was collected. Subsequently, these whole cell proteins were separated on a 10-12% polyacrylamide gel and transferred electrophoretically onto a polyvinylidene difluoride membrane at 100 V for two hours. After transfer, the membrane was blocked with a 5% w/v skimmed milk solution in tris-buffered saline with 0.5% tween-20 (TBST) for one hour at room temperature. The membrane was then incubated with various primary antibodies. Following this, the membranes were washed in TBST and subsequently probed with the appropriate secondary antibodies for 1 hour at room temperature. Visualization was achieved using a chemiluminescence reagent (GE Healthcare Bio-Sciences, PA, USA) after washing the membrane with TBST. The following antibodies from various vendors were used for immunoblotting studies: anti-FOXM1 (Santa Cruz Biotechnology Inc. #sc-502, MA, USA), anti-PCNA (Cat. #sc-7907, MA, USA) from Santa Cruz, USA, E-Cadherin (Cat. #3195, CST, USA), vimentin (Cat. #5741S, CST, USA), cleaved caspase 7 (Cat. #9492S, CST, USA), cyclin B1 (Cat. #4138S, CST, USA) GAPDH (Cat. #5174T, CST, USA), cleaved PARP (Cat. #9542S, CST, USA) from Cell signaling Technology Inc. (MA, USA) and β-actin (Cat. #A5316, Sigma-Aldrich, USA). The secondary antibodies used were anti-mouse-HRP (Cat #7076P2, CST, USA) and anti-rabbit HRP (Cat #ab6721, Abcam, USA). RNA isolation and quantitative real time PCR Total RNA was extracted from CRC cell lines by using TRIzol (Invitrogen, USA). The Primescript RT reagent kit (Takara Biosciences, USA) was employed for the synthesis of single-strand cDNA. Real-time PCR detection was performed using SyBR TBgreen (Takara, USA). A specifically designed primer facilitated the synthesis of the stem-loop strand for miR-532-3p, which was subsequently detected using a specific forward primer and a universal reverse primer. RNU6B served as the internal control in this analysis. For the detection of transcripts from protein-coding genes, L19 was employed as the internal control. The quantification of expression fold change was determined using the 2 -ΔΔCt method. Cell cycle and apoptosis assays For cell cycle analysis, after overexpressing with miR-532-3p mimic and vector controls for 48 hours, cells were washed with PBS, and fixed with 70% ethanol. Ethanol was added drop by drop while gently vortexing to ensure a uniform cell suspension, followed by incubation at 4°C for one hour. Subsequently, the cells were washed and stained with propidium iodide solution, incubating for 10 minutes in the dark. The resulting suspension was passed through a sterile cell strainer and collected in a FACS tube. This suspension was then analyzed using the FACS ARIA machine. In case of apoptosis, the cells were harvested and washed with PBS. Then they were resuspended in binding buffer, to which of Annexin was added. This suspension was incubated in dark at room temperature for 15 minutes. Then 2 μL of propidium iodide solution was added to the suspension and it was incubated in the dark for 5 minutes. 400 μL of binding buffer was then added to this suspension, which was passed through a sterile cell strainer. This suspension was collected in a FACS tube and analyzed on FACS ARIA machine. Statistical analysis All experiments (apart from the matrigel invasion) were repeated three times independently. All data were expressed as mean ± SEM and analyzed with GraphPad Prism 8.0.2 software (GraphPad, Inc., La Jolla, CA, USA). Differences between two groups or more than two groups were analyzed by Student’s t-test or analysis of variance. p < 0.05 was considered to indicate statistically significant results. Declarations ACKNOWLEDGMENTS The authors thank Dr Ani Das and her team for providing vectors and guidance on designing cloning primers used in cloning. The authors thank Indu, Arya and Tanima for the flow cytometry facility. The authors thank Dr. Jackson James for the instrument used for luciferase activity. This work was supported by the annual extramural fund provided by Department of Biotechnology, Government of India. Ketakee Mahajan was supported by a research fellowship provided by Department of Biotechnology, Government of India. CONFLICTS OF INTEREST The authors declare no competing interest. AUTHORS' CONTRIBUTION KM, AN and AD conceived, designed and interpreted the study. KM and AN drafted the manuscript. KM conducted the experiments. 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Worrisome trends and oncogenic features. Dig. Liver Dis. (2018) doi:10.1016/j.dld.2018.02.009. Tian, L., Zhao, Z., Xie, L. & Zhu, J. P. MiR-361-5p suppresses chemoresistance of gastric cancer cells by targeting FOXM1 via the PI3K/Akt/mTOR pathway. Oncotarget 9 , 4886–4896 (2018). Signs, S. A. et al. Stromal miR-20a controls paracrine CXCL8 secretion in colitis and colon cancer. Oncotarget 9 , 13048–13059 (2018). Zhang, N. A. N., Lu, C. & Chen, L. I. N. miR-217 regulates tumor growth and apoptosis by targeting the MAPK signaling pathway in colorectal cancer. 4589–4597 (2016) doi:10.3892/ol.2016.5249. Yan, S. et al. MicroRNA-6869-5p acts as a tumor suppressor via targeting TLR4/NF-κB signaling pathway in colorectal cancer. Journal of Cellular Physiology vol. 233 6660–6668 at https://doi.org/10.1002/jcp.26316 (2018). Feng, J., Wang, X., Zhu, W., Chen, S. & Feng, C. MicroRNA-630 suppresses epithelial-to-mesenchymal transition by regulating FoxM1 in gastric cancer cells. Biochem. 82 , 707–714 (2017). Whitfield, M. L., George, L. K., Grant, G. D. & Perou, C. M. Common markers of proliferation. Nat. Rev. Cancer 6 , 99–106 (2006). Koo, C. Y., Muir, K. W. & Lam, E. W. F. FOXM1: From cancer initiation to progression and treatment. Biochim. Biophys. Acta - Gene Regul. Mech. 1819 , 28–37 (2012). Borhani, S. & Gartel, A. L. Expert Opinion on Therapeutic Targets FOXM1 : a potential therapeutic target in human solid cancers FOXM1 : a potential therapeutic target in human solid cancers. Expert Opin. Ther. Targets 24 , 205–217 (2020). Bhat, U. G., Halasi, M. & Gartel, A. L. Thiazole Antibiotics Target FoxM1 and Induce Apoptosis in Human Cancer Cells. 4 , 1–7 (2009). Gartel, A. L. Thiazole antibiotics siomycin A and thiostrepton inhibit the transcriptional activity of FOXM1. 3 , 3389 (2013). Wang, L. et al. MiR-216b suppresses cell proliferation , migration , invasion , and epithelial – mesenchymal transition by regulating FOXM1 expression in human non-small cell lung cancer. (2022) doi:10.2147/OTT.S202523. Ngan, H. Y. S. Aberrant Activation of ERK / FOXM1 Signaling Cascade Triggers the Cell Migration / Invasion in Ovarian Cancer Cells. 6 , (2011). Hamurcu, Z., Ashour, A., Kahraman, N. & Ozpolat, B. FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation , invasion and tumorgenesis of human triple negative breast cancer cells. 7 , (2015). Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.pdf 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. 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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-4217992","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":294829968,"identity":"82ac2bb4-fd68-47c5-a3cf-00138efb193f","order_by":0,"name":"Ketakee Mahajan","email":"","orcid":"","institution":"Rajiv Gandhi Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Ketakee","middleName":"","lastName":"Mahajan","suffix":""},{"id":294829970,"identity":"39581475-dcc2-4207-a9b3-58fa293e4373","order_by":1,"name":"Ramesh Pothuraju","email":"","orcid":"","institution":"Rajiv Gandhi Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Ramesh","middleName":"","lastName":"Pothuraju","suffix":""},{"id":294829971,"identity":"f786f70e-b0de-4b18-9d0f-7b9c3923d5c9","order_by":2,"name":"Ani Das","email":"","orcid":"","institution":"Rajiv Gandhi Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Ani","middleName":"","lastName":"Das","suffix":""},{"id":294829972,"identity":"4bda9756-4d8d-42f0-93cd-158cbaea0f76","order_by":3,"name":"Asha Nair","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYHACNgaGCiAlAeHxEKnlDMlaGNsQWggD+fbjzx78nHfPrn92A9uHD38YZMwJaTE4k2Nu2LutOHnGnQPMM2e2MfBYNhDSwpDDJsG7LSHZQCKBmZm3gYHH4AAhh/U/fyb5dw5Uy58/RGhhuJFgJs3bkGAH1gIMCsJaDG68MZOWOZaQIHHnYDNjb5sEMQ5Lfyb5pibBnn9282GGH39s7Ak7DAoSGxgYGxhIiB0GBnvilY6CUTAKRsGIAwBynzp3rd98cgAAAABJRU5ErkJggg==","orcid":"","institution":"Rajiv Gandhi Centre for Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Asha","middleName":"","lastName":"Nair","suffix":""}],"badges":[],"createdAt":"2024-04-04 12:50:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4217992/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4217992/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55433641,"identity":"8a47563c-30b6-4e24-a8a0-4c4e095154ea","added_by":"auto","created_at":"2024-04-27 14:19:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":402147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelection of candidate microRNA that might bind to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFOXM1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e 3’UTR. a)\u003c/strong\u003e Mining of prediction, pathway and expression databases to find microRNAs that might bind to \u003cem\u003eFOXM1\u003c/em\u003e 3’UTR. \u003cstrong\u003eb) \u003c/strong\u003eAnalysis of hits obtained from several prediction algorithms. \u003cstrong\u003ec) \u003c/strong\u003eDual luciferase reporter assay to evaluate the predicted interaction between \u003cem\u003eFOXM1\u003c/em\u003e 3’UTR and selected microRNAs. \u003cstrong\u003ed)\u003c/strong\u003e A prediction of binding between microRNA-532-3p and \u003cem\u003eFOXM1\u003c/em\u003e 3’UTR. (*: p=0.05, **: p\u0026lt;0.01, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4217992/v1/ea31b10b837a9cb99baf34dc.png"},{"id":55433643,"identity":"80c0df5a-6e21-4368-b748-68d70da77d2f","added_by":"auto","created_at":"2024-04-27 14:19:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":939720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroRNA-532-3p in Cancers and in CRC. a) \u003c/strong\u003eExpression of miR-532-3p in Human tissues: cancer vs. normal (TCGA database). \u003cstrong\u003eb) \u003c/strong\u003eExpression of miR-532-3p in tumors against normal tissues of colon and rectum (TCGA). \u003cstrong\u003ec) \u003c/strong\u003eExpression of miR-532-3p in CRC cells. \u003cstrong\u003ed)\u003c/strong\u003e Survival analyses of colon adenocarcinoma and rectal adenocarcinoma cases against expression levels of miR-532-3p (TCGA).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4217992/v1/0e03e27772f4f98d3726c523.png"},{"id":55433642,"identity":"212ce092-b8d8-40a0-9ea9-8f749f49d1e9","added_by":"auto","created_at":"2024-04-27 14:19:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4990728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of miR-532-3p in CRC cells.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e Ectopic overexpression of microRNA-532-3p in CRC cells. (p=0.0012) \u003cstrong\u003eb) \u003c/strong\u003eEffect of ectopic overexpression of miR-532-3p on cell viability in HCT116, HT29 and SW480 cells. Effect of ectopic overexpression of miR-532-3p on cellular proliferation by \u003cstrong\u003ec)\u003c/strong\u003eexpression of PCNA, a cell proliferation marker (p=0.0004) in CRC cells and \u003cstrong\u003ed)\u003c/strong\u003ecolony formation assay (p=0.0068) in HT29 cells. Effect of ectopic overexpression of miR-532-3p on \u003cstrong\u003ee)\u003c/strong\u003e cellular migration (p=0.011) and \u003cstrong\u003ef)\u003c/strong\u003ematrigel invasion (p\u0026lt;0.0001) in SW480 cells. Levels of EMT biomarkers E-cadherin and vimentin at \u003cstrong\u003eg)\u003c/strong\u003e transcript (p=0.0315, p\u0026lt;0/0001 respectively) and\u003cstrong\u003e h)\u003c/strong\u003e protein (p=0.0589, p=0.0038 respectively) level on ectopic overexpression of miR-532-3p. (*: p=0.05, **: p\u0026lt;0.01, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4217992/v1/6edc93d91a8fea753ca7c817.png"},{"id":55433644,"identity":"192d3d60-aab9-4de2-8184-0217e760dc64","added_by":"auto","created_at":"2024-04-27 14:19:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3112794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroRNA-532-3p regulates the expression of FOXM1.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e Relative expression levels of FOXM1 in colon and rectal adenocarcinoma; tumor vs. normal tissues. \u003cstrong\u003eb)\u003c/strong\u003e Overall survival of CRC patients with respect to FOXM1 expression levels (Dataset GSE12945, Prognoscan). Constitutive expression of FOXM1 at \u003cstrong\u003ec)\u003c/strong\u003e protein and transcript level in CRC cells. \u003cstrong\u003ed)\u003c/strong\u003e Effect of ectopic overexpression of miR-532-3p on FOXM1 expression at transcript (p=0.0185) and protein (p=0.0001) levels and on cyclin B1 protein level (p=0.002) in CRC cells. \u003cstrong\u003ee)\u003c/strong\u003eEffect of ectopic overexpression of miR-532-3p on cell cycle progression in CRC cells. \u003cstrong\u003ef\u003c/strong\u003e) Effect of ectopic overexpression of miR-532-3p on expression of apoptotic biomarkers cleaved caspase-7 (p=0.0876), cleaved PARP (p=0.0488) and anti-apoptotic marker BCL2 (p\u0026lt;0.0001) in CRC cells. \u003cstrong\u003eg)\u003c/strong\u003e MicroRNA-532-3p diminishes the expression of FOXM1 post-transcriptionally, resulting in suppressed proliferation, migration and invasion in CRC cells. (*: p=0.05, **: p\u0026lt;0.01, ***: p\u0026lt;0.001, ****: p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4217992/v1/4af433bae6276159907e2c73.png"},{"id":59286805,"identity":"a257027a-b300-4d02-b162-3f64d130f6e4","added_by":"auto","created_at":"2024-06-28 16:48:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12915495,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4217992/v1/8702067a-3f20-4ebd-93f6-32795c331bcf.pdf"},{"id":55433646,"identity":"e86ca74e-fe46-4526-a4f6-06218e4ff4f0","added_by":"auto","created_at":"2024-04-27 14:19:16","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1624399,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4217992/v1/818c9f17baea5348bf32d029.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MicroRNA-532-3p modulates colorectal cancer cell proliferation and invasion via suppression of FOXM1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) stands as the third most prevalent and second most lethal cancer worldwide. Its occurrence ranks second and third among women and men, respectively\u003csup\u003e1\u003c/sup\u003e. The incidence of CRC has seen a consistent increase over recent decades\u003csup\u003e2\u003c/sup\u003e. \u0026nbsp;The causes of CRC can be either non-hereditary or sporadic, involving the downregulation of tumor suppressor genes and genes responsible for regulating cell cycle processes, DNA damage repair, and apoptosis\u003csup\u003e3\u003c/sup\u003e. Traditionally, CRC has been documented predominantly in the elderly population, affecting individuals aged 50 years and above\u003csup\u003e4\u003c/sup\u003e. The likelihood of developing CRC is notably higher among those with inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn\u0026rsquo;s disease\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eForkhead box M1 (FOXM1) stands as a pivotal member within the forkhead box proteins family, a group of transcription factors\u003csup\u003e6\u003c/sup\u003e. Functioning as a master regulator of the cell cycle, FOXM1 plays a crucial role in orchestrating the G2/M transition. It exerts direct control over the expression of key elements such as Aurora B kinase, Cyclin B, and Cdc25b phosphatase\u003csup\u003e7\u003c/sup\u003e. Notably, FOXM1 has demonstrated overexpression across a wide spectrum of cancers\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e9\u003c/sup\u003e. Elevated levels of FOXM1 expression have been associated with an unfavorable prognosis in breast cancer, esophageal adenocarcinoma\u003csup\u003e10\u003c/sup\u003e, and CRC\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e12\u003c/sup\u003e. Beyond its positive regulatory role in the cell cycle, FOXM1 engages in various cancer-related functions such as cellular proliferation\u003csup\u003e13\u003c/sup\u003e, migration, invasion\u003csup\u003e14\u003c/sup\u003e, and resistance to drugs\u003csup\u003e15\u003c/sup\u003e. The multifaceted nature of FOXM1 positions it as a potential therapeutic target, sparking interest in suppressing its expression for over a decade\u003csup\u003e16\u003c/sup\u003e. Within the eukaryotic genome, specific regions encode various types of non-coding RNAs, primarily categorized into short and long noncoding RNAs\u003csup\u003e17\u003c/sup\u003e. Noteworthy examples of short noncoding RNAs include microRNAs, piRNAs, and snoRNAs\u003csup\u003e18\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMicroRNAs represent a group of evolutionarily conserved, naturally occurring small nucleotides measuring approximately 19-24 bases in length. Their primary role involves post-transcriptional regulation of gene expression, accomplished through binding to mRNA transcripts. This interaction disrupts the translational complex, thereby impeding protein synthesis. Beyond translational inhibition, microRNAs are recognized for their ability to destabilize and degrade messenger RNA molecules\u003csup\u003e19\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicroRNA-532-3p is an upcoming tumor suppressor in various cancers. In CRC, it has been reported to inhibit Wnt/\u0026beta;-catenin signaling, effectively impeding the disease\u0026apos;s progression. The present study corroborates these findings by revealing its ability to enhance cleavage in PARP and Pro-caspase 7. Notably, ectopic overexpression of miR-532-3p in CRC cells resulted in a significant reduction in proliferative potential, migration, and invasion, corroborated with the previous study\u003csup\u003e21\u003c/sup\u003e. Similarly, its role in suppressing proliferation and facilitating apoptosis has been investigated in lymphoma, where it targets \u0026beta;-catenin\u003csup\u003e22\u003c/sup\u003e. In prostate cancer, miR-532-3p was found to impede bone metastasis by disabling NF-\u0026kappa;B signaling\u003csup\u003e23\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, we have discussed the role of miR-532-3p in suppressing cellular proliferation, migration and invasion of CRC. MicroRNA-532-3p has been reported to act as a tumor suppressor in a renal cell carcinoma\u003csup\u003e24\u003c/sup\u003e and colorectal cancer\u003csup\u003e25\u003c/sup\u003e. Both the strands of microRNA-532; viz. miR-532-5p and miR-532-3p are known to positively and negatively impact the occurrence and prognosis of several types of cancers such as breast cancer, hepatocellular carcinoma, ovarian cancer\u003csup\u003e26\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e27\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e28\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e29\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e30\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e31\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMicroRNA-532-3p interacts with 3\u0026rsquo;UTR of \u003cem\u003eFOXM1\u003c/em\u003e transcript\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe database mining revealed several microRNAs potentially capable of regulating the expression by binding through the 3\u0026rsquo;UTR of \u003cem\u003eFOXM1\u003c/em\u003e transcript (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). Databases such as TargetScan and miRecords gave more than 450 hits each with possible complementary seed and target sequences in microRNAs and the 3\u0026rsquo;UTR, respectively (\u003cstrong\u003eTables S1-S4\u003c/strong\u003e). Network databases such as miRtargetLink also predicted several interactions, along with reporting a few validated ones. The microRNAs were analysed for the most frequently occurring hits by Venn analysis (\u003cstrong\u003eFig. 1b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe selected microRNAs, cloned in pRIP vector were co-transfected with Psicheck2 vector containing 3\u0026rsquo;UTR of FOXM1 insert, with a firefly luciferase gene in the downstream region (\u003cstrong\u003eFig. S1-S3\u003c/strong\u003e). The dual luciferase assay confirmed the interactions of miR-149-5p and -3p, miR-370-3p, miR-532-3p, miR-590-5p, miR-671-5p and miR-876-5p with \u003cem\u003eFOXM1\u003c/em\u003e 3\u0026rsquo;UTR, indicated by the reduced luciferase activity. MicroRNA-532-3p showed nearly 50% of reduction in luciferase activity, suggesting a strong interaction (\u003cstrong\u003eFig 1c\u003c/strong\u003e). Hence, miR-532-3p was selected for further investigation. A representation of a possible interaction between miR-532-3p and \u003cem\u003eFOXM1\u003c/em\u003e 3\u0026rsquo;UTR was obtained from TargetScan database (\u003cstrong\u003eFig. 1d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-532-3p is down-regulated in Colorectal cancer\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigating The Cancer Genome Atlas (TCGA) datasets revealed that the expression of microRNA-532-3p was significantly lower in the malignant tissues compared to the normal cells and tissues (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). Also, the expression levels of miR-523-3p were significantly lower in colorectal tumors compared to the normal counterparts (\u003cstrong\u003eFig. 2b\u003c/strong\u003e). Several CRC cell lines were screened for the expression of microRNA-532-3p, revealing that HCT116 showed the highest expression level (\u003cstrong\u003eFig.2c)\u003c/strong\u003e. \u0026nbsp;However, Kaplan-Meier analysis showed no significant decrease in overall survival in colon and rectal patients with high miR-532-3p (\u003cstrong\u003eFig. 2d\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-532-3p overexpression diminishes proliferation, migration and invasion of CRC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the functional significance of miR-532-3p, ectopic overexpression of miR-532-3p in various CRC cell lines was performed (\u003cstrong\u003eFig. 3a\u003c/strong\u003e). An evaluation of potential cell death resulting from miR-532-3p overexpression was performed through a colorimetric analysis using the MTT assay (\u003cstrong\u003eFig. 3b\u003c/strong\u003e). Intriguingly, no notable disparity in cell viability was observed upon miR-532-3p overexpression. However, a reduction in the expression of the cell proliferation marker PCNA was detected in CRC cell lines overexpressing miR-532-3p (\u003cstrong\u003eFig. 3c\u003c/strong\u003e). Furthermore, ectopic overexpression of miR-532-3p in HT29 cells led to about 40% reduction in the number of colonies formed (\u003cstrong\u003eFig. 3d)\u003c/strong\u003e. A wound healing experiment conducted in SW480 cells with miR-532-3p overexpression revealed a slower rate of wound closure in the cells overexpressing miR-532-3p compared to the control at various time points (\u003cstrong\u003eFig. 3e\u003c/strong\u003e). Additionally, a significant reduction was noted in the invasion of SW480 cells through matrigel in those ectopically overexpressing miR-532-3p (\u003cstrong\u003eFig. 3f\u003c/strong\u003e). These observations were further supported by quantitative analysis of molecular markers at both transcript and protein levels. The said analyses revealed upregulated levels of the epithelial marker E-cadherin and reduced expression of vimentin in cells overexpressing miR-532-3p (\u003cstrong\u003eFig. 3g and h\u003c/strong\u003e). These findings suggest that miR-532-3p influences cellular proliferation, migration, and invasion properties in CRC \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-532-3p overexpression led to higher apoptosis, possibly via suppression of FOXM1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary findings from TCGA datasets revealed that the expression levels of FOXM1 are significantly altered between tumors and their disease-free counterparts in both colon and rectal tissues \u003cstrong\u003e(Fig. 4a)\u003c/strong\u003e. Furthermore, in terms of survival, TCGA revealed no significant difference between the survival rates of patients exhibiting high levels of FOXM1 against those with low levels (Dataset GSE12945) (\u003cstrong\u003eFig. 4b\u003c/strong\u003e). Several CRC cell lines were screened for the expression of FOXM1 at transcript and protein levels, thereby showing the high transcript levels of FOXM1 in DLD1 and SW480 cells and high proteinlevel expression in HT29 cells (\u003cstrong\u003eFig. 4c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eEctopic overexpression of microRNA-532-3p resulted in reduced transcript and protein levels of FOXM1. This confirmed the ability of miR-532-3p to regulate FOXM1 at post-transcriptional level. Furthermore, the levels of cyclin B1 were analysed, which is a direct transcriptional target of FOXM1. The real-time PCR and western immunoblotting revealed that overexpressing miR-532-3p resulted in reduced transcript and protein levels of cyclin B1 in CRC cells (\u003cstrong\u003eFig. 4d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo determine whether overexpression of miR-532-3p may play role in cell cycle progression, we analyzed the DNA content by flow cytometry analysis of CRC cells overexpressing showed very little increase in the percentage of cells in G\u003csub\u003e2\u003c/sub\u003e/M phase (\u003cstrong\u003eFig. 4e\u003c/strong\u003e). However, western immunoblotting analysis showed increased levels of several apoptotic markers such as activated form of caspase 7 and cleaved PARP levels. Additionally, an anti-apoptotic BCL2 marker was found to be downregulated (\u003cstrong\u003eFig. 4f\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn essence, microRNA-532-3p interacts with \u003cem\u003eFOXM1\u003c/em\u003e 3\u0026rsquo;UTR, leading to decreased protein levels, thereby suppressing proliferation, migration and invasion in colorectal cancer cells (\u003cstrong\u003eFig. 4g\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicroRNAs, recognized as one of the smallest classes of biomolecules, exert dynamic regulatory influence on developmental conditions and diseases through their distinctive mechanisms\u003csup\u003e32\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e33\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e34\u003c/sup\u003e. Their impact spans various facets of mammalian development, including the neural system, placental site, and hematopoiesis, operating at the minutest scale\u003csup\u003e35\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e36\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e37\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e38\u003c/sup\u003e. In the context of cancer, microRNAs play a pivotal role in governing processes ranging from the disease\u0026apos;s onset and proliferation to the regulation of metastasis, as well as influencing sensitivity or resistance to chemotherapeutic drugs\u003csup\u003e39\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e40\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e41\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e42\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e43\u003c/sup\u003e. Their involvement extends to shaping the tumor microenvironment as well\u003csup\u003e44\u003c/sup\u003e. MicroRNAs execute these multifaceted functions by targeting components of several signaling pathways crucial to both development and cancer\u003csup\u003e45\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e46\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRecent years, MicroRNA-532-3p has emerged as a tumor suppressor in various cancers. In CRC, it has been documented to disrupt Wnt/\u0026beta;-catenin signaling, effectively impeding the disease\u0026apos;s progression. The present study bolsters these findings and reveals the ability of miR-532-3p to upregulate p53 expression and enhance cleavage in PARP. Notably, ectopic overexpression of miR-532-3p in CRC cells resulted in a significant reduction in proliferative potential, migration, and invasion, corroborated with the previous study\u003csup\u003e25\u003c/sup\u003e. Similarly, its role in restraining proliferation and facilitating apoptosis has been investigated in lymphoma, where it targets \u0026beta;-catenin\u003csup\u003e22\u003c/sup\u003e. In prostate cancer, miR-532-3p hinders bone metastasis by inactivating NF-\u0026kappa;B signaling\u003csup\u003e23\u003c/sup\u003e. Conversely, in renal cell carcinoma, clinical specimens showed a correlation between low expression levels of both mature strands of pre-miR-532, miR-532-5p, and miR-532-3p, and the presence of the disease\u003csup\u003e24\u003c/sup\u003e. \u0026nbsp;Despite its well-established tumor-suppressive role in most cancers, microRNA-532-3p, along with its other \u0026nbsp; mature form, microRNA-532-5p, has been recognized for exhibiting oncogenic roles in hepatocellular carcinoma and breast cancer\u003csup\u003e27\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e31\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e30\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFOXM1, the new target of miR-532-3p that we have discovered in this study is a known master regulator of cell cycle\u003csup\u003e7\u003c/sup\u003e. This has made FOXM1 a crucial factor in the occurrence and progression of cancer as a group of diseases that arises from dysregulation in cell cycle\u003csup\u003e48\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e49\u003c/sup\u003e. As mentioned before, FOXM1 has been of interest in reference to anticancer therapeutics as a key target\u003csup\u003e16\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e50\u003c/sup\u003e. Among the chemical inhibitors of FOXM1, thiazole antibiotics such as thiostrepton and siomycin A have been reported to induce apoptosis in human cancer cells by repressing the transcriptional activity of FOXM1\u003csup\u003e51\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e52\u003c/sup\u003e. Besides the cell cycle aspect, the higher expression levels of FOXM1 have been known to correlate with tumor invasion, leading to poor prognosis in colorectal cancer\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e. This ability of FOXM1 to aid and promote tumorigenesis and metastasis has been studied in ovarian carcinoma, breast cancer, non-small cell lung cancer as well\u003csup\u003e53\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e54\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e55\u003c/sup\u003e. These reports prove the importance studying FOXM1 and its regulation in the occurrence and progression of the disease.\u003c/p\u003e\n\u003cp\u003eIn the present study, we have demonstrated the ability of microRNA-532-3p to suppress the proliferation, migration, and invasion of CRC cells. However, we did not observe a direct effect of miR-532-3p on cell death and progression of cell cycle. Furthermore, we have established FOXM1 as a novel target of miR-532-3p, adding another important link in the events leading to malignancy. This finding suggests that miR-532-3p could be a significant predictive marker for metastasis and prognosis in CRC. Further studies in other types of cancers where miR-532-3p has been reported as a tumor suppressor molecule will help gain a better insight of the picture.\u003c/p\u003e\n\u003cp\u003eThis study validates the interaction between microRNA-532-3p and 3\u0026rsquo;UTR of \u003cem\u003eFOXM1\u003c/em\u003e transcript, which resulted in decreased protein levels of FOXM1. This consequently suppressed the proliferation, migration and invasion in colorectal cancer cells. The link between miR-532-3p and FOXM1 could offer an insight on the direct tumor suppressive function exhibited by miR-532-3p in colorectal cancer, among other types of the disease.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell lines and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human CRC cell lines (Caco2, Colo320, DLD1, HCT116, HT29, SW480, and SW620) and human embryonic kidney cells (HEK293T) were grown in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM), enriched with 10% fetal bovine serum (Gibco, Invitrogen, USA) and supplemented with antibiotics (100\u0026thinsp;X antibacterial-antimycotic solution, GibCo, USA). Cultures were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere within a humidified chamber.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn silico mining of databases and selection of microRNAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the quest to identify microRNAs that may regulate the expression of FOXM1 by interacting with its 3\u0026rsquo;UTR transcript, multiple databases were utilized. These databases fell into four distinct categories: predictive interaction (TargetScan, miRecords, mirMap), network (Reactome and Biogrid), expression profile (TissueAtlas and dbDEMC 2.0), and literature (miRCancer). The results obtained from these searches were then subjected to Venn analysis to pinpoint the microRNAs that appeared most frequently. Subsequently, seven microRNAs were chosen from the compiled lists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild-type 3\u0026rsquo;UTR sequence and the sequences corresponding to the genes encoding the identified microRNAs were retrieved from the UCSC genome browser. Primers were designed, incorporating 100-base pair flanking regions for the specified genomic segments. Subsequently, amplicons were generated using Platinum high-fidelity DNA polymerase (Thermo Invitrogen, USA). The obtained amplicons underwent restriction digestion; XhoI and NotI for the PsiCheck2 vector and FOXM1 3\u0026rsquo;UTR, and BamHI and HindIII for the modified pRIP vector and microRNA amplicons. Ligation was performed at 4\u0026deg;C utilizing the T4 ligase kit (Thermo Invitrogen, USA). The ligated products underwent validation for inserts through PCR, restriction digestion, and Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDual luciferase reporter assay was performed as per instructed in the manufacturer\u0026rsquo;s protocol (Promega, Wisconsin, USA). HEK293T cells were transiently co-transfected with PsiCheck2 vector containing 3\u0026rsquo;UTR of FOXM1 and a modified pRIP vector containing the gene coding for one of the selected microRNAs. After 72 hours of transfection, the cells were lysed using Passive Lysis Buffer (1X). These lysates were used for dual luciferase reporter assay. Promega Glomax luminometer was used to record the luminescence. An empty PsiCheck2 vector was used as the internal control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation and colony formation assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRC cells (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) were plated in a 96-well cell culture plate and cultured at 37\u0026ordm;C with 5% CO2 in a humidified incubator (Thermo Scientific, USA) for 72 hours. Cell survival was assessed using the MTT assay. MTT reagent was applied to each well and incubated in darkness at 37\u0026ordm;C for 4 hours. Formazan crystals were then dissolved in isopropyl alcohol, and absorbance levels were measured. For the colony formation assay, cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were initially plated in 35-mm culture dishes and cultured in standard DMEM. Upon reaching approximately 40-50% confluency, cells underwent transfection with miR-532-3p and the control plasmid, employing Lipofectamine 3000 as per the manufacturer\u0026rsquo;s instructions. Transfection was halted after 24 hours, followed by the addition of fresh media. Subsequently, cells were trypsinized and seeded at a density of 1000 cells per well in 6-well culture plates. The cells were then cultivated for a period of 14 days, with media renewal every 72 hours. After the incubation period, cells were rinsed with PBS and fixed in 4% paraformaldehyde (PFA) for 5 minutes. Following PFA removal, the fixed cells underwent three washes with PBS. A 0.5% crystal violet solution was applied to the cells and allowed to incubate for 30 minutes at room temperature. The crystal violet solution was then aspirated, and the plate was washed under tap water before being air-dried. Subsequently, colonies formed were enumerated, with those containing 50 or more cells considered as positive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing and cell invasion assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e per well) were initially plated in a 12-well culture plate. Upon reaching 50% confluency, cells underwent transfection with miRNA-532-3p and the control plasmid as mentioned previously. Transfection was terminated after 24 hours, and fresh regular media were introduced to the cells. When the cells reached approximately 90% confluency, a wound was generated by gently scratching with a sterile micropipette tip. Subsequently, the cells were examined under an inverted microscope every 24 hours post-washing with PBS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the matrigel invasion assay, CRC cells (1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were seeded in 60-mm dishes and transfected with the control plasmid and miR-532-3p mimic. The cells were then incubated at 37\u0026deg;C in OptiMEM. After 24 hours, the transfected cells were trypsinized and diluted to a suspension of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL. A 24-well plate with inserts containing matrix (BD Biosciences, USA) was pre-wet with regular medium for two hours at 37\u0026deg;C. Using sterile forceps, the inserts were carefully transferred onto these wells. The cell suspension was then added onto these inserts and incubated for 24 hours. The invaded cells on the lower side of the membrane were stained with 0.5% crystal violet stain, and counted in various fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were harvested and rinsed with PBS, followed by lysis in RIPA buffer at 4\u0026deg;C with agitation at 1400 RPM on a thermomixer for one hour and forty-five minutes. The resulting solution underwent centrifugation at 14000 RPM for 15 minutes at 4\u0026deg;C, and the supernatant, constituting whole cell protein, was collected. Subsequently, these whole cell proteins were separated on a 10-12% polyacrylamide gel and transferred electrophoretically onto a polyvinylidene difluoride membrane at 100 V for two hours. After transfer, the membrane was blocked with a 5% w/v skimmed milk solution in tris-buffered saline with 0.5% tween-20 (TBST) for one hour at room temperature. The membrane was then incubated with various primary antibodies. Following this, the membranes were washed in TBST and subsequently probed with the appropriate secondary antibodies for 1 hour at room temperature. Visualization was achieved using a chemiluminescence reagent (GE Healthcare Bio-Sciences, PA, USA) after washing the membrane with TBST. The following antibodies from various vendors were used for immunoblotting studies: anti-FOXM1 (Santa Cruz Biotechnology Inc. #sc-502, MA, USA), anti-PCNA (Cat. #sc-7907, MA, USA) from Santa Cruz, USA, E-Cadherin (Cat. #3195, CST, USA), vimentin (Cat. #5741S, CST, USA), cleaved caspase 7 (Cat. #9492S, CST, USA), cyclin B1 (Cat. #4138S, CST, USA) GAPDH (Cat. #5174T, CST, USA), cleaved PARP (Cat. #9542S, CST, USA) from Cell signaling Technology Inc. (MA, USA) and \u0026beta;-actin (Cat. #A5316, Sigma-Aldrich, USA). The secondary antibodies used were anti-mouse-HRP (Cat #7076P2, CST, USA) and anti-rabbit HRP (Cat #ab6721, Abcam, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and quantitative real time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from CRC cell lines by using TRIzol (Invitrogen, USA). The Primescript RT reagent kit (Takara Biosciences, USA) was employed for the synthesis of single-strand cDNA. Real-time PCR detection was performed using SyBR TBgreen (Takara, USA). A specifically designed primer facilitated the synthesis of the stem-loop strand for miR-532-3p, which was subsequently detected using a specific forward primer and a universal reverse primer. RNU6B served as the internal control in this analysis. For the detection of transcripts from protein-coding genes, L19 was employed as the internal control. The quantification of expression fold change was determined using the 2\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u0026nbsp;\u003c/sup\u003emethod.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle and apoptosis assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cell cycle analysis, after overexpressing with miR-532-3p mimic and vector controls for 48 hours, cells were washed with PBS, and fixed with 70% ethanol. Ethanol was added drop by drop while gently vortexing to ensure a uniform cell suspension, followed by incubation at 4\u0026deg;C for one hour. Subsequently, the cells were washed and stained with propidium iodide solution, incubating for 10 minutes in the dark. The resulting suspension was passed through a sterile cell strainer and collected in a FACS tube. This suspension was then analyzed using the FACS ARIA machine. In case of apoptosis, the cells were harvested and washed with PBS. Then they were resuspended in binding buffer, to which of Annexin was added. This suspension was incubated in dark at room temperature for 15 minutes. Then 2 \u0026mu;L of propidium iodide solution was added to the suspension and it was incubated in the dark for 5 minutes. 400 \u0026mu;L of binding buffer was then added to this suspension, which was passed through a sterile cell strainer. This suspension was collected in a FACS tube and analyzed on FACS ARIA machine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments (apart from the matrigel invasion) were repeated three times independently. All data were expressed as mean \u0026plusmn; SEM and analyzed with GraphPad Prism 8.0.2 software (GraphPad, Inc., La Jolla, CA, USA). Differences between two groups or more than two groups were analyzed by Student\u0026rsquo;s t-test or analysis of variance. p \u0026lt; 0.05 was considered to indicate statistically significant results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr Ani Das and her team for providing vectors and guidance on designing cloning primers used in cloning. The authors thank Indu, Arya and Tanima for the flow cytometry facility. The authors thank Dr. Jackson James for the instrument used for luciferase activity. This work was supported by the annual extramural fund provided by Department of Biotechnology, Government of India. Ketakee Mahajan was supported by a research fellowship provided by Department of Biotechnology, Government of India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026apos; CONTRIBUTION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKM, AN and AD conceived, designed and interpreted the study. KM and AN drafted the manuscript. KM conducted the experiments. RP reviewed the manuscript and made significant revisions on the draft. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILIBILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung, H. \u003cem\u003eet al.\u003c/em\u003e Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA. Cancer J. Clin.\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 209\u0026ndash;249 (2021).\u003c/li\u003e\n\u003cli\u003eCenter, M. M., Jemal, A., Smith, R. A. \u0026amp; Ward, E. Worldwide variations in colorectal cancer. \u003cem\u003eDis. Colon Rectum\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1099 (2010).\u003c/li\u003e\n\u003cli\u003eGranados-Romero, J. 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FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation , invasion and tumorgenesis of human triple negative breast cancer cells. \u003cstrong\u003e7\u003c/strong\u003e, (2015).\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":"MicroRNA, Colorectal Cancer, FOXM1, Metastasis","lastPublishedDoi":"10.21203/rs.3.rs-4217992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4217992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Forkhead-box M1 (FOXM1) is a proliferation-associated transcription factor, overexpressed in almost all the cancers. Naturally, the mechanisms of FOXM1 regulation have been under investigation. Previously, we showed that FOXM1 binds to promoters of certain microRNAs. Database mining led to several microRNAs that might interact with FOXM1 3’UTR. The interactions between shortlisted microRNAs and FOXM1 3’UTR was quantitated by dual luciferase reporter assay. MicroRNA-532-3p interacted with 3’UTR of FOXM1 mRNA transcript most efficiently. MicroRNA-532-3p was ectopically overexpressed in colorectal cancer cell lines, leading to reduced transcript and protein levels of FOXM1 and cyclin B1, a direct transcriptional target of FOXM1. Flow cytometry analysis revealed an unaltered cell cycle. A clonogenic assay was conducted that revealed a decline in the ability of cells to form colonies. Additionally, wound healing assay and matrigel invasion assays were carried out. Significant reduction in migratory and invading cell numbers were observed, which were reinforced at molecular levels by the altered transcript and protein levels of the conventional EMT markers E-cadherin and vimentin. Overall, this study identifies the interaction between microRNA-532-3p and FOXM1 3’UTR, leading to reduced protein levels. Ectopic expression of miR-532-3p leads to suppression in cellular proliferation, migration and invasion in CRC cells.","manuscriptTitle":"MicroRNA-532-3p modulates colorectal cancer cell proliferation and invasion via suppression of FOXM1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-27 14:19:11","doi":"10.21203/rs.3.rs-4217992/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":"34df8581-5446-4ceb-b993-11c0af68d272","owner":[],"postedDate":"April 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31082231,"name":"Biological sciences/Cancer"},{"id":31082232,"name":"Biological sciences/Cancer/Gastrointestinal cancer"},{"id":31082233,"name":"Biological sciences/Cancer/Tumour biomarkers"}],"tags":[],"updatedAt":"2024-06-28T16:40:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-27 14:19:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4217992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4217992","identity":"rs-4217992","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}