Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression

Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression | 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 Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression Avishek Kar, Kanchan Kumari, Sandip K. Mishra, Umakanta Subudhi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1464868/v2 This work is licensed under a CC BY 4.0 License Status: Under Review Version 2 posted 12 You are reading this latest preprint version Show more versions Abstract FOXO1 transcription factor is not only limit the cell cycle progression but also promote cell death as a tumor suppressor protein. Though the expression of FOXO1 is largely examined in breast cancer, the regulation of FOXO1 by miRNA is yet to be explored. In the current study, self-assembled branched DNA (bDNA) nanostructures containing oncogenic miRNAs were designed and transfected to MCF7 cell lines to decipher the FOXO1 expression. bDNA containing oncogenic miRNA 27a, 96 and 182 synergistically downregulate the expression of FOXO1 in MCF7 cells. The down-regulation is evident both in mRNA and protein level suggesting bDNA having miRNA sequences can selectively bind to mRNA and inhibit translation. Secondly, the downstream gene expression of P21 and P27 are also significantly downregulated in presence of miR-bDNA nanostructures. The cell proliferation activity was progressively increased in presence of miR-bDNA nanostructure which confirms the reduced tumor suppression activity of FOXO1 and the downstream gene expression. This finding can be explored to design novel bDNA structures which can downregulate the tumor suppressor protein in normal cells and induce cell proliferation activity to identify early phase markers of cancer. DNA nanostructure FOXO1 miRNAs Breast cancer MCF7 cell P21 P27 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The FOXO transcription factors play a major role in regulating the gene expression of cell cycle progression, apoptosis, cell differentiation, stress response, DNA damage control, and vital cellular functions [ 1 – 3 ]. Further, FOXO1 is identified as a key tumor suppressor protein in breast cancer that regulates cell proliferation, invasion, metastasis and survival [ 4 ]. Activation of FOXO members promotes cell cycle arrests at G1/S stage by upregulating cell cycle inhibitors p27 and p21 [ 5 , 6 ]. Thus, the downregulation of FOXO1 expression leads to dysregulation of cell cycle regulators which induce cell proliferation and play important role in the formation of cancer. Several study provides information regarding downregulated FOXO1 protein level in cancers, including glioblastoma [ 6 ], endometrial [ 7 ], ovarian carcinoma [ 8 ], prostate [ 9 ], and lung carcinoma [ 10 ]. Nevertheless, restoration of FOXO1 tumor suppressor protein in endometrial carcinoma leads to decrease in cell proliferation [ 7 ]. Further, Guttilla and White [ 11 ] demonstrated that the overexpression of FOXO1 strongly inhibits cell proliferation and induced apoptosis in breast cancer MCF7 cell lines. Thus, the activation of FOXO1 is regarded as a therapeutic strategy for cancer. Recently, microRNAs have been reported to modulate post-transcriptional regulation of mRNA resulting in the suppression of target gene expression [ 12 , 13 ]. OncomiRNAs like miR27a, miR96, and miR182 are upregulated in breast cancer and they collectively downregulate the expression of FOXO1 [ 12 ]. Thus, for the upregulation of tumor suppressor protein FOXO1, the oncomiRs need neutralization by antimiRNAs. Guttilla and White [ 11 ], demonstrated the downregulation of oncomiRs by administration of antimiRNAs and reported the overexpression of FOXO1 in MCF7 cell lines. Recently, self-assembled branched DNA (bDNA) nanostructures have been evolved as economic and efficient strategy for miRNA-based cancer therapy [ 14 – 20 ]. Recently, our group has also reported the synergistic downregulation of oncomiRs and upregulation of FOXO1 by antimiR-bDNA nanostructures which selectively binds to the oncomiRs 27a, 96 and 182 in MCF-7 cells [ 21 ]. In last two decades, substantial effort has been made for the delivery of antimiRs for upregulating tumor suppressor proteins or miRNA mimics for downregulating oncogenic proteins. Despite of restoring miRNAs and suppressing cancer cell proliferation using miRNA-based therapeutics very less attention has been made to initiate and activate cell proliferation in the presence of oncogenic miRNAs. Expression of oncogenic miRNA in normal cell is relatively low as compared to cancer cells. Once the oncogenic miRNAs and its target pathways are switched on in the normal cells they are transformed into cancer cells. Therefore, the transition from normal cell to cancer cell will be very useful in identifying early markers for cancer diagnostics. Keeping this as background, we hypothesize that if a group of oncogenic miRNAs can be delivered they will collectively downregulate tumor suppressor protein and activate cell proliferation. To validate the idea we prefer to target transcription factor FOXO1, which is a major tumor suppressor protein and found to be downregulated in breast cancer by oncogenic miRNAs 27a, 96 and 182 [ 11 , 21 ]. Though, downregulation of FOXO1 is a pivotal step for forming tumorigenesis, the mechanism of initiation of tumorigenesis by down regulated FOXO1 through exogenous miRNA is yet to be understood. Keeping this as background, the present study demonstrates the role of exogenous oncomiRs on the expression of FOXO1 in breast cancer cell line and its effect on cancer cell proliferation. In the current study, self-assembled bDNA nanostructure has been explored to carry miR27a, miR96, miR182 in the overhangs to downregulate the expression of FOXO1 in breast cancer cells. Thus, for the first time our data reports bDNA nanostructure can carry oncogenic miRNA sequences and selectively recognize the target mRNA resulting into down regulation of tumor suppressor protein and downstream cell cycle inhibitors p27 and p21in MCF7 cell lines. 2. Materials And Methods 2.1. Designing and self-assembly of bDNA nanostructures carrying microRNAs The designing of bDNA structure was performed as described previously [ 22 , 23 ] and purchased from Integrated DNA Technology (IDT), USA without any modification or purification (Table S1). In brief, the bDNA monomeric structure composed of four oligonucleotides namely, strands A, B, C and D. The external region of strands B and C are complementary to each other, whereas the internal regions are complementary to strands A and D respectively (Fig. 1 ). Nevertheless, the overhangs of strands A and D are not complementary to each other or to other oligonucleotides, thus they are replaced with either scramble sequences or miRNA sequences (Fig. 1 ). Since microRNA 27a, 96, and 182 binds to the 3’ UTR of FOXO1 (Fig. S1), it prompted us to design the self-assembled miR-bDNA structures that can also bind to the 3’ UTR of FOXO1. Thus, miR-bDNA nanostructures such as bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182 are generated containing respective miRNA sequences (Table S2, Fig. 1 ). Similarly, bDNAmiR-Mix contains all three miRNAs in the four overhangs whereas bDNA-Scramble is devoid of miRNA sequences (Fig. 1 ). 1 µM of each oligonucleotides were taken from a stock of 100 µM into 25 µl reaction mixture to prepare bDNA structures as mentioned earlier [ 21 , 22 ]. 2.2. Characterization of bDNA nanostructures The integrity of the self-assembled bDNA nanostructures was checked using 10% native polyacrylamide gel (nPAGE) as described previously [ 24 ]. The samples were electrophoresed for 2 h at 4°C in a vertical electrophoresis unit (SE260, Hofer, USA) at a constant voltage of 150 V by taking 1xTAE as running buffer. Then, the gels were stained with ethidium bromide solution (0.5 µg/ml) for 30 min and image was taken using FluroChem E system (Cell Biosciences). The conformation of bDNA-miRs was examined using Circular Dichroism (CD) spectrophotometer (Chirascan, Applied Photophysics) as mentioned earlier [ 25 ]. The scan rate of spectra was recorded at 60 nm/sec with bandwidth of 1 nm and a time per point of 0.5 sec. All measurements were done at 25°C between 320 to 200 nm by using a quartz cuvette of 1 mm path length. Three spectra per sample were averaged for getting the final spectrum and final spectra were corrected against buffer as background. 2.3. Serum stability and gel retardation assay To study the stability of different bDNA-miR structures, 1 µM of bDNA was taken in 10% Fetal bovine serum (FBS) . After mixing, the samples were incubated at 37°C for 0, 2, 4, 8, 12, 24 and 48 h. Similarly, free miRNA oligos were also examined for serum stability assay in presence of 10% FBS. Then the incubated products were analyzed by 1.5% agarose gel at 100 V for 30 min. Intensity of the DNA in gel images was measured through ImageJ software. The miRNA binding to 3’ UTR of FOXO1 was studied for binding scores using online miRNA target prediction tool TargetScan, Pictar, miRANDA (Fig. S2). Further, the integrity of microRNA binding to 3’UTR of FOXO1 region were evaluated using bDNA-miR and antimiR binding in gel retardation assay. AntimiR sequences were used to hybridize with corresponding bDNA-miRs in vitro . Different bDNA structures were incubated with respective antimiR sequence at 37°C for 2 h and then samples were run in 1.5% agarose gel at 100 V for 60 min. 2.4. Cell Culture and transfection with bDNA nanostructures The human breast cancer cell line MCF7 were obtained from the National Repository of Animal Cell Culture (NCCS Pune, Maharashtra, India). Cells were routinely maintained in Dulbecco’s Modified Eagles Medium (DMEM) (PAN Biotech, Germany) containing 10% (v/v) heat inactivated Fetal Bovine Serum (PAN Biotech, Germany) and 1% penicillin/streptomycin (PAN Biotech, Germany) in a humidified incubator with 95% humidity, 5% CO 2 at 37°C (SANYO). Cells were seeded in 6-well tissue culture plates (corning) with density 3×10 5 cells/well. When cells grown up to 50–80% confluence, the cells were transfected with 50 µl of self-assembled bDNA structures in 6-well plates with final volume of 2 ml Lipofectamine 3000 according to the manufacturer’s instruction (Invitrogen, USA). The cells were divided into five groups such as bDNA-Scr, bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182 and bDNAmiR-Mix for transfection study and incubated for 72 h. Similarly, groups of MCF7 cell lines were also transfected with oncogenic miRs (miR-27a, miR-96, miR-182 and miR-Mix) as control to observe the declined expression of FOXO1. 2.5. RNA isolation, cDNA and reverse transcriptase PCR Total RNA was isolated from MCF7 cell lines using miRNA Extraction Kit (217004, QIAGEN, USA) and reversely transcribed using cDNA synthesis kit (K1622, Thermo Scientific) according to manufacturer’s protocol and subsequently stored at -80°C. The reverse transcriptase PCR (BioRad, USA) was performed using gene specific primer (Table S3) and 18S-rRNA was used as internal control to normalize the gene expression. The relative expression change in RT images was quantified using ImageJ software and transcripts profile has been compared. 2.6. Western blotting analysis Whole cell lysate was prepared after 72 h of transfection in 6 well culture plates using RIPA buffer containing protease inhibitor. Extracted proteins from transfected cells were electrophoresed on 10% SDS-PAGE and were subsequently transferred onto PVDF membrane in a constant voltage. The immunoblot was blocked in 5% skimmed milk powder for 1.5 h and incubated overnight with primary antibody FOXO1, p21, p27 and GAPDH under shaking condition at 4°C. All antibodies were procured from Cell Signaling Technology, (Danver, MA, US). Next day the blot was washed five times with Tris-buffered saline containing Tween-20 (TBST) and the membrane was then incubated with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. After thorough ringing the membrane was developed using luminol on X-rays in the dark room. 2.7. Cell viability assay MTT (3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) assay was performed to study the cell viability in presence of bDNA structures. 3 x10 3 numbers of cells were seeded on 96-well culture plates. The cells were transfected with five different bDNA structures after 24 h of seeding. After stipulated time point, 100 µl of MTT (5 mg/ml, Sigma, USA) was added to each well and incubated for 3 h at 37ºC. The growth medium containing MTT was removed and the formazan crystals were dissolved in 150 µl of dimethyl sulphoxide (DMSO, Sigma, USA). The plates were read at 570 nm (Microplate Reader, BioRad, USA) for absorbance and the viable cells were calculated. 2.8. Statistical analysis Each experiment was tested for three biological repeats. One way ANOVA was performed to test the statistical significance followed by Duncan’s multiple range test. For all the analyses, statistically significant results were considered with a P -value P < 0.05. 3. Results 3.1. Characterization of self-assembled bDNA nanostructures The intensity of individual oligonucleotide showed a clear band whereas the complementary oligonucleotides (AB, BC, CD) were forming di-oligo complexes exhibiting decreased electrophoretic mobility (Fig. S2a,b,c). The di-oligos, tri-oligos and bDNA-scramble have different electrophoretic mobility suggesting migration of nucleic acids in PAGE is dependent on size, shape, and conformation (Fig. 2 a, Fig. S2d). Similarly, the bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182, and bDNAmiR-Mix were characterized with a clear intense band suggesting the formation of stable unimolecular bDNA structures (Fig. 2 b). Nevertheless, all the self-assembled bDNA structures showed B-form of DNA with characteristic positive peaks at ~ 280 nm and ~ 220 nm and a negative peak at ~ 250 nm (Fig. S3). 3.2. Stability of miR-bDNA and selective binding to mRNA Nuclease stability of bDNA nanostructures was examined in presence of FBS which comprises ~ 256 U/L of DNase 1. After incubation at 37°C the relative intensity of miR-bDNA was found to be stable for 12 h then slowly decreases upto 48 h (Fig. S4). On the contrary, naked miRNAs are stable in presence of nuclease. The gel retardation assay reveals a significant retardation of DNA suggesting the sequence specific binding between miR-bDNA and FOXO1 mRNA (Fig. S5). 3.3. Expression of FOXO1, p21, and p27 in response to miRNA A significant decreased expression of FOXO1 was observed in miR-182 transfected cells whereas no change in expression was noticed with miR-27a and 96 (Fig. 3 a,b). Interestingly the downregulation of FOXO1 was further decreased miR-Mix (Fig. 3 a,b). Further, the expression of well-known CDK inhibitors like p21 and p27 was examined in response to miR-bDNA structures. A significant (p < 0.05) reduction in expression of p21 was evident in cells transfected with miR-182 (Fig. 3 c). The expression was further reduced when cells were transfected with miR-Mix (Fig. 3 c). On the other hand, the expression of p27 was significantly (p < 0.05) repressed in presence of miR-96 and miR-182 and disappeared with miR-Mix (Fig. 3 d). 3.4. miRNA-bDNA modulates the expression profile of FOXO1, p21, and p27 On the contrary, no significant change in expression was found in cells transfected with bDNAmiR-27a, bDNAmiR-96 or bDNAmiR-182 (Fig. 4 ). However, a significant (p < 0.05) decrease in FOXO1 expression was evident when transfected with bDNAmiR-Mix. Nearly 40% downregulation of FOXO1 was observed as compared to bDNA-Scr (Fig. 4 a, b). Similarly, no significant change was observed in the expression of p21 and p27 in the presence of miR-bDNA nanostructures (Fig. 4 c,d). Nevertheless, a significant (p < 0.05) decrease in expression of p21 and p27 was noticed while transfected with bDNAmiR-Mix (Fig. 4 a,d). 3.5. Downregulation of FOXO1 and downstream gene expression The target site of miR-27a, miR-96, miR-182 on the 3’ UTR of FOXO1 has been confirmed using TargetScan, Pictar, and miRanda (Fig. S1). Thus, to examine the influence of bDNA-miRs on FOXO1, the endogenous expression of FOXO1 protein was evaluated using western blot analysis (Fig. S6). In comparison to bDNA-Scr, significant decrease in FOXO1 protein level was noticed when transfected with bDNAmiR-27a, and bDNA-Mix. However, an unaltered level of FOXO1 was observed in bDNAmiR-96 and an enhanced level was evident in cells transfected with bDNAmiR-182. FOXO1 is known to regulate the target gene expression of p21 and p27. Interestingly, the protein level of p21 and p27 was observed to be increased in all the bDNA-miRs transfected samples as compared to bDNA-Scr (Fig. S6). 3.6. Effect on cell proliferation by miR-bDNA nanostructures Cell proliferation was monitored in different time interval from 24 h, 48 h and 72 h. A significant (p \(<0.05\) ) increase in cell proliferation was found with bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182 and bDNAmiR-Mix as compared to bDNA-Scr after 24 h of incubation. Similarly, higher cell proliferation was noticed after 48 and 72 h of incubation with all bDNA nanostructures. Importantly, highest cell proliferation was noticed in MCF7 cells transfected with bDNAmiR-Mix structures (Fig. 5 ). 4. Discussion Breast cancer is the second leading cause of cancer death among women after lung cancer in the United States. It is estimated that 2,68,600 cases diagnosed with invasive breast cancer in the United States and annually 41,760 patients die in breast cancer [ 26 ]. Thus, the molecular mechanism of breast cancer development and progression deserve detailed investigations. FOXO1 is a master regulator that regulates cell cycle proliferation, invasion, metastasis and survival in several cancers including breast cancer [ 3 ]. Now it is well evident that miRNAs play a vital role in proliferation and metastasis of cancer in general and post-transcriptional regulation of tumor suppressor mRNAs in particular [ 12 ]. Currently, miR27a, miR96, and miR182 are known oncomiRs in breast cancer which coordinately reduce the expression of FOXO1 [ 11 ]. Nevertheless, in variety of other cancer miR27a, miR96, and miR182 act as oncomiRs to suppress FOXO1, including renal cell cancer [ 27 ], bladder cancer [ 28 ], Prostate cancer [ 29 ], colorectal cancer [ 30 ], thyroid carcinoma [ 31 ], ovarian cancer [ 32 ], liver cancer [ 33 ], gastric cancer [ 34 ], adenocarcinoma [ 35 ], and cervical cancer [ 36 ]. Thus, understanding the regulation of FOXO1 expression is vital in managing and monitoring the cancer in general and breast cancer in particular. Moreover, these findings strongly support that miR-27a, miR-96 and miR-182 act as oncogenic miRNAs for downregulating FOXO1 and cell cycle proliferation. Nevertheless, application of antimiRNAs 27a, 96, and 182 down regulate the expression of oncogenic miRNAs in MCF7 and synergistically upregulate the expression of FOXO1 [ 11 , 21 ]. However, the cellular response and FOXO1 expression was yet to be investigated when cells are subjected to the simultaneous transfection of multiple oncogenic miRNAs. Secondly, these model oncogenic miRNAs to be transfected to normal cell lines to initiate cell proliferation and find early diagnostic marker for cancer. Since self-assembled DNA nanostructures are biocompatible drug carrier, bDNAs were designed to carry multiple oncogenic miR-27a, miR-96, miR-182 and transected to MCF7 cell lines for understanding the cell proliferation and FOXO1 expression. As expected, a significant decrease in FOXO1 expression was noticed in MCF7 while transfected with bDNA nanostructures. The molecular interaction between bDNA nanostructure and FOXO1 mRNA also caused translational inhibition of FOXO1. Further, FOXO1 expression was significantly decreased by miR-182 and miR-Mix transfection. Nevertheless, miR-Mix or bDNAmiR-Mix works better than individual miRNAs which supports earlier data on the multiple miRNA regulation to a particular gene expression [ 21 ]. A significant repression of p21 was also observed when cells transfected with miR-182 and miR-Mix. Similarly, p27 significantly suppressed by miR-96, miR-182 and miR-Mix. The results suggest that expression of p21 and p27 is largely dependent on miRNAs-mediated downregulation of FOXO1. The endogenous FOXO1 protein decreases when cells transfected with bDNA-miR27a and bDNA-Mix. Thus, the downregulated translated product of FOXO1 by bDNAmiR nanostructures support the hypothesis that FOXO1 protein expression is downregulated at the post-transcriptional level by multiple miRNAs. The expression of p21 and p27 protein was observed to be increased in all the bDNA-miRs transfected samples. Possibly, the expression of p21 and p27 was regulated by other miRNAs. However, miR-27a and miR-96 mediate cell proliferation by regulating cyclin D1, p21 and p27 [ 37 , 38 ]. In addition, miR-27a, miR-96 and miR-182 could promote migration and invasion by the regulation of PTPN9 in breast cancer [ 39 ], and PDCD4 in hepatocellular cancer [ 40 ]. Since overexpression of FOXO1 is associated with decreased proliferation and colony size in MCF7 cells, we expect increased cellular proliferation in MCF7 cells transfected with the miR-bDNA nanostructures. Therefore, cellular proliferation was analyzed in MCF7 cells transfected with bDNA-miRs at different incubation periods. A significant cell proliferation was noticed even after 24h of incubation. However, higher cell proliferation was noticed with bDNA-Mix nanostructures which suggest the synergistic effect of oncogenic miRNAs on cell proliferation. Secondly, these findings clearly suggest that miR-27a, miR-96 and mR-182 promote cell proliferation by targeting not only FOXO1 but also other tumor suppressor genes. 5. Conclusions In summary, the current communication for the first time reports the downregulation of FOXO1 by bDNA nanostructures carrying multiple miRNAs. The downregulation of FOXO1 and its downstream gene expression in breast cancer cell line is due to oncomiRs miR27a, miR96 and miR182. The designed bDNA acts as a single unit carrying three miRNAs at a time to synergistically regulate gene expression. Nevertheless, the enhanced cell proliferation by oncomiR-bDNA nanostructures is not only due to downregulation of FOXO1 but also associated tumor suppressor proteins. This proof of principle can be extended to multiple oncogenic miRNAs which can be transfected to the cell lines and coordinately downregulate tumor suppressor proteins and regulates cell proliferation, invasion, metastasis. Thus, the model oncogenic miRNAs like miR-27a, miR-96 and miR-182 can be transfected using bDNA nanostructures to various cell lines for switching cell proliferation and identifying early diagnostic markers for cancer. Declarations Ethics approval and consent to participate: Not applicable Consent for publication: C onsent for publication Not Applicable Availability of data and materials: The datasets generated and/or analysed during the current study does not contain any data that needs to be submitted in any database, thus given in materials and methods section of the manuscript. Competing financial interests: The authors declare no competing financial interests. Funding: Council of Scientific & Industrial Research (CSIR), Government of India, New Delhi and Department of Biotechnology (DBT), Government of India, New Delhi Author contributions: U.S initiated the project and designed the experiment. A.K, and K.K performed the experiments and acquired the data. All the authors drafted and critically reviewed the manuscript. Finally all the authors read and approved the manuscript submission. Acknowledgements: Authors are thankful to the Council of Scientific & Industrial Research (CSIR) and Department of Biotechnology (DBT), Government of India, New Delhi for supporting the work. Authors' information (optional): Not applicable References Huang, H., Tindall, D. J. (2007) Dynamic FoxO transcription factors. J. Cell Sci. 120:2479–2487. Fu, Z., Tindall, D. J. (2008) FOXOs, cancer and regulation of apoptosis. Oncogene. 27:2312–2319. Coomans de Brachène, A., Demoulin, J. B. (2016) FOXO transcription factors in cancer development and therapy. Cell. Mol. Life Sci. 73:1159–1172. Accili, D., Arden, K. C. (2004) FOXOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117:421–426. Dijkers, P. F., Medema, R. H., Pals, C., Banerji, L., Thomas, N. S., Lam, E. W., Burgering, B. M., Raaijmakers, J. A., Lammers, J. W., Koenderman, L., Coffer, P. J. (2000) Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol. Cell. Biol. 20:9138–9148. Seoane, J., Le, H. V., Shen, L., Anderson, S. A., Massagué, J. (2004) Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117:211–223. Goto, T., Takano, M., Albergaria, A., Briese, J., Pomeranz, K. M., Cloke, B., Fusi, L., Feroze-Zaidi, F., Maywald, N., Sajin, M., Dina, R. E., Ishihara, O., Takeda, S., Lam, E. W., Bamberger, A. M., Ghaem-Maghami, S., Brosens, J. J. (2008a) Mechanism and functional consequences of loss of FOXO1 expression in endometrioid endometrial cancer cells. Oncogene. 27:9–19. Goto, T., Takano, M., Hirata, J., Tsuda, H. (2008b) The involvement of FOXO1 in cytotoxic stress and drug-resistance induced by paclitaxel in ovarian cancers. Br. J. Cancer 98:1068–1075. Dong, X. Y., Chen, C., Sun, X., Guo, P., Vessella, R. L., Wang, R. X., Chung, L. W., Zhou, W., Dong, J. T. (2006) FOXO1A is a candidate for the 13q14 tumor suppressor gene inhibiting androgen receptor signaling in prostate cancer. Cancer Res. 66:6998–7006. Gao, Z., Liu, R., Ye, N., Liu, C., Li, X., Guo, X., Zhang, Z., Li, X., Yao, Y., Jiang, X. (2018) FOXO1 Inhibits Tumor Cell Migration via Regulating Cell Surface Morphology in Non-Small Cell Lung Cancer Cells. Cell. Physiol. Biochem. 48:138–148. Guttilla, I. K., White, B. A. (2009) Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells. J. Biol. Chem. 284:b23204–23216. Bartel D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116:281–297. O'Brien, J., Hayder, H., Zayed, Y., Peng, C. (2018) Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front. Endocrinol. 9:402. Wang, D., Fu, Y., Yan, J., Zhao, B., Dai, B., Chao, J., Liu, H., He, D., Zhang, Y., Fan, C., Song, S. (2014) Molecular logic gates on DNA origami nanostructures for microRNA diagnostics. Anal. Chem. 86:1932–1936. Zhou, W., Li, D., Xiong, C., Yuan, R., Xiang, Y. (2016) Multicolor-Encoded Reconfigurable DNA Nanostructures Enable Multiplexed Sensing of Intracellular MicroRNAs in Living Cells. ACS Appl. Mater. 8:13303–13308. Liu, Q., Wang, D., Yuan, M., He, B. F., Li, J., Mao, C., Wang, G. S., Qian, H. (2018) Capturing intracellular oncogenic microRNAs with self-assembled DNA nanostructures for microRNA-based cancer therapy. Chem. Sci. 9:7562–7568. Chandrasekaran, A. R., Punnoose, J. A., Zhou, L., Dey, P., Dey, B. K., Halvorsen, K. (2019) DNA nanotechnology approaches for microRNA detection and diagnosis. Nucleic Acids Res. 47:10489–10505. Jahanban-Esfahlan, R., Seidi, K., Jahanban-Esfahlan, A., Jaymand, M., Alizadeh, E., Majdi, H., Najjar, R., Javaheri, T., Zare, P. (2019) Static DNA nanostructures for cancer theranostics: Recent progress in design and applications. Nanotechnol. Sci. Appl. 12:25. Baral, B., Dutta, J., Subudhi, U. (2021) Biophysical interaction between self-assembled branched DNA nanostructures with bovine serum albumin and bovine liver catalase. Int. J. Biol. Macromol. 177:119–128. Kumari, K., Kar, A., Nayak, A. K., Mishra, S. K., Subudhi, U. (2021) MiRNA-mediated alteration of sulfatase modifying factor 1 expression using self-assembled branched DNA nanostructures. RSC Adv. 11:10670–10680. Nahar, S., Nayak, A. K., Ghosh, A., Subudhi, U., Maiti, S. (2017) Enhanced and synergistic downregulation of oncogenic miRNAs by self-assembled branched DNA. Nanoscale 10:195–202. Nayak, A. K., Subudhi, U. (2014) Directed self-assembly of genomic sequences into monomeric and polymeric branched DNA structures. RSC Adv. 4:54506–54511. Nayak, A. K., Rath, S. K., Subudhi, U. (2019) Preparation of stable branched DNA nanostructures: Process of cooperative self-assembly. J. Phys. Chem. B, 123:3591–3597. Bhanjadeo, M. M., Nayak, A. K., Subudhi, U. (2017) Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice. Biochem. Biophys. Res. 485:492–498. Bhanjadeo, M. M., Baral, B., Subudhi, U. (2019) Sequence-specific B-to-Z transition in self-assembled DNA: A biophysical and thermodynamic study. Int. J. Biol. Macromol. 137:337–345. DeSantis, C. E., Ma, J., Gaudet, M. M., Newman, L. A., Miller, K. D., Goding Sauer, A., Siegel, R. L. (2019) Breast cancer statistics, 2019. CA Cancer J. Clin. 69:438–451. Zhou, L., Yin, B., Liu, Y., Hong, Y., Zhang, C., Fan, J. (2012) Mechanism and function of decreased FOXO1 in renal cell carcinoma. J. Surg. Oncol. 105:841–847. Guo, Y., Liu, H., Zhang, H., Shang, C., Song, Y. (2012) MiR-96 regulates FOXO1-mediated cell apoptosis in bladder cancer. Oncol. Lett. 4:561–565. Wallis, C. J., Gordanpour, A., Bendavid, J. S., Sugar, L., Nam, R. K., Seth, A. (2015) MiR-182 Is Associated with Growth, Migration and Invasion in Prostate Cancer via Suppression of FOXO1. J. Cancer. 6:1295–1305. Gao, F., Wang, W. (2015) MicroRNA-96 promotes the proliferation of colorectal cancer cells and targets tumor protein p53 inducible nuclear protein 1, forkhead box protein O1 (FOXO1) and FOXO3a. Mol. Med. Rep. 11:1200–1206. Song, H. M., Luo, Y., Li, D. F., Wei, C. K., Hua, K. Y., Song, J. L., Xu, H., Maskey, N., Fang, L. (2015) MicroRNA-96 plays an oncogenic role by targeting FOXO1 and regulating AKT/FOXO1/Bim pathway in papillary thyroid carcinoma cells. Int. J. Clin. Exp. Pathol. 8:9889–9900. Zhang, L. Y., Chen, Y., Jia, J., Zhu, X., He, Y., Wu, L. M. (2019) MiR-27a promotes EMT in ovarian cancer through active Wnt/β-catenin signalling by targeting FOXO1. Cancer Biomarkers 24:31–42. Zheng, X., Zhang, F., Zhao, Y., Zhang, J., Dawulieti, J., Pan, Y., Cui, L., Sun, M., Shao, D., Li, M., He, K., Zhang, M., Li, J., Chen, L. (2018) Self-assembled dual fluorescence nanoparticles for CD44-targeted delivery of anti-miR-27a in liver cancer theranostics. Theranostics 8:3808–3823. Lang, C., Xu, M., Zhao, Z., Chen, J., Zhang, L. (2018) MicroRNA-96 expression induced by low-dose cisplatin or doxorubicin regulates chemosensitivity, cell death and proliferation in gastric cancer SGC7901 cells by targeting FOXO1. Oncol. Lett. 16:4020–4026. Ling, J., Dong, X., Wang, L., Xue, Y., Jia, X., Song, W., Li, Q. (2019) MiR-27a-regulated FOXO1 promotes pancreatic ductal adenocarcinoma cell progression by enhancing Wnt/β-catenin signaling activity. Am. J. Transl. Res. 11:3069. Yang, L., Liu, L., Zhang, X., Zhu, Y., Li, L., Wang, B., Liu, Y., Ren, C. (2020) Mir-96 enhances the proliferation of cervical cancer cells by targeting foxo1. Pathol. Res. Pract. 216:152854. Li, X., Xu, M., Ding, L., and Tang, J. (2019) MiR-27a: a novel biomarker and potential therapeutic target in tumors. J. Cancer. 10:2836. Lin, H., Dai, T., Xiong, H., Zhao, X., Chen, X., Yu, C., Li, J., Wang, X., Song, L. (2010) Unregulated miR-96 induces cell proliferation in human breast cancer by downregulating transcriptional factor FOXO3a. PloS One 5:e15797. Hong, Y., Liang, H., Wang, Y., Zhang, W., Zhou, Y., Chen, S. A., Chen, X. (2016) miR-96 promotes cell proliferation, migration and invasion by targeting PTPN9 in breast cancer. Sci. Rep. 6:1–16. Hu, J., Wang, Z., Wang, J., Jian, Y., Dai, J., Wang, X., Xiong, W. (2020) MicroRNA-182 Promotes Cell Migration by Targeting Programmed Cell Death 4 in Hepatocellular Carcinoma Cells. Onco Targets Ther. 13:9159. Additional Declarations No competing interests reported. Supplementary Files SupplemenatryInformationR1.docx Cite Share Download PDF Status: Under Review Version 2 posted Editorial decision: Major revision 03 Oct, 2022 Reviews received at journal 24 Sep, 2022 Reviewers agreed at journal 16 Sep, 2022 Reviews received at journal 01 Sep, 2022 Reviewers agreed at journal 23 Aug, 2022 Reviewers agreed at journal 21 Aug, 2022 Reviewers agreed at journal 14 Aug, 2022 Reviewers invited by journal 11 Aug, 2022 Editor assigned by journal 11 Aug, 2022 Editor invited by journal 03 Aug, 2022 Submission checks completed at journal 03 Aug, 2022 First submitted to journal 23 Jul, 2022 You are reading this latest preprint version Show more versions 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. 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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-1464868","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2022-03-21 14:47:37","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}}],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":130496802,"identity":"839cf7fd-f4e8-4535-8527-4c2036b0cea9","order_by":0,"name":"Avishek Kar","email":"","orcid":"","institution":"CSIR-Institute of Minerals \u0026 Materials Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Avishek","middleName":"","lastName":"Kar","suffix":""},{"id":130496803,"identity":"fec1912f-78bd-4fb0-9d3a-7b14c6622f7b","order_by":1,"name":"Kanchan Kumari","email":"","orcid":"","institution":"CSIR-Institute of Minerals \u0026 Materials Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kanchan","middleName":"","lastName":"Kumari","suffix":""},{"id":130496804,"identity":"9bf49344-4614-4f4c-a97e-9a25916d39b6","order_by":2,"name":"Sandip K. Mishra","email":"","orcid":"","institution":"Institute of Life Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sandip","middleName":"K.","lastName":"Mishra","suffix":""},{"id":130496805,"identity":"3f92370e-49d9-410d-ade6-03b95fba4ee3","order_by":3,"name":"Umakanta Subudhi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYHACNiA6wMPPDBdgbCBOi2QzkHmAFC0MBgfgWggAg/NnzB7zlN2RMT7Onbr5A8M9OfkG5rYHeLXcyDE35jn3jMfsMO+2GwcYio0NDjC2G+DXwmMmzdt2GKYlIXEDA2ObBCGHgbUYN0O01M9vIKTlQA5EiwEzREsCwwECWiRvpJUbzjl3mEcC5LAzBgmGGw4T0MJ3/vC2B2/KDtvz95/ddqOiIkFevr39GV4t6O4EYmaCqkbBKBgFo2AUEAIA5WJKGNhoQ+MAAAAASUVORK5CYII=","orcid":"","institution":"CSIR-Institute of Minerals \u0026 Materials Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Umakanta","middleName":"","lastName":"Subudhi","suffix":""}],"badges":[],"createdAt":"2022-03-18 08:59:13","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1464868/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1464868/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25504458,"identity":"e5a5f9b4-2858-4993-8f30-570468722fd6","added_by":"auto","created_at":"2022-08-22 16:54:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of self-assembled branched DNA (bDNA) nanostructures\u003c/strong\u003e. bDNA nanostructures containing scramble sequence in the overhangs of bDNA-Scramble (a) miR-27a sequence in bDNAmiR-27a (b) miR-96 sequence in bDNAmiR-96 (c) miR-182 sequence in bDNAmiR-182 (d) and miR 27a, miR 96 and miR 182 in bDNA-mix nanostructures (e).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1464868/v2/cc1f5b0898b13a393097487e.png"},{"id":25504459,"identity":"c97065e6-9895-4c42-871d-44a596734ba9","added_by":"auto","created_at":"2022-08-22 16:54:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":367888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of self-assembled bDNA nanostructures.\u003c/strong\u003e Gel image display the binding of oligonucleotides to form dioligo and trioligo complexes and bDNAmiR-Mix structures containing the sequence of miR-27a, miR-96, miR-182 in the four overhangs. Sample composition in each lane is mentioned in top of the lane. bDNAmiR-Mix shows decrease electrophoretic mobility with respect to di and tri-oligo complexes formation in 10% nPAGE (a). Migration of bDNA structures (bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182, bDNAmiR-Mix) having miR sequences in overhangs. bDNA structures (lane 6 to 10) shows less electrophoretic mobility with respect to mono, di and tri-oligo complexes. The single bands in each lane indicates sequence-specific base pairing among oligonucleotides (b).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1464868/v2/5203752bc968ccc7306660d6.png"},{"id":25504075,"identity":"6a80c302-0a13-4b62-bb9d-00e773fe04b9","added_by":"auto","created_at":"2022-08-22 16:49:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression in response to transfection of miRNAs. \u003c/strong\u003eRT-PCR products resolved in agarose gel showing the expression of 18S, FOXO1, p21, and p27 after transfected with miRs (a). Relative expression of FOXO1 (b) p21 (c) and p27 (d) on breast cancer cell lines transfected with miRs was quantified using ImageJ software against 18S as quantitative control. One way ANOVA was used for statistical significance at \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1464868/v2/1d5ac4b4f9f0791568931fe6.png"},{"id":25504460,"identity":"9c39b3e2-fc35-438a-941d-0dd15a520ce3","added_by":"auto","created_at":"2022-08-22 16:54:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscript profile after transfection with bDNAmiR nanostructures. \u003c/strong\u003eAgarose gel shows the transcripts level of FOXO1, p21, p27 and 18S in response to the transfection with bDNA-miRs (a). Relative expression change of FOXO1 (b) p21 (c) and p27 (d) on breast cancer cell line quantified using ImageJ software with 18S as quantitative control. One way ANOVA was used for statistical significance at \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1464868/v2/e9274eb8b351b571f0ca60b8.png"},{"id":25504077,"identity":"0221e243-c365-49a4-9d99-e505df0c77cf","added_by":"auto","created_at":"2022-08-22 16:49:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced cell proliferation of MCF7 breast cancer cell lines in presence of bDNAmiR-Mix\u003c/strong\u003e. MTT assay revealed the effect of bDNAmiRs on breast cancer cell growth after 24h, 48h and 72h in MCF cell lines. \u0026nbsp;One way ANOVA is used for statistical significance at P\u0026lt;0.05.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1464868/v2/2eee87768dc2fd0a503bec08.png"},{"id":25504461,"identity":"4d3d5cb4-4bf8-4de0-b79e-4495326a3f4d","added_by":"auto","created_at":"2022-08-22 16:54:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":830023,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1464868/v2/a01a4a2c-eeeb-4f4c-b65c-5bfb9fda880d.pdf"},{"id":25504080,"identity":"33bba099-c6c0-457c-8853-7030b0ba04f6","added_by":"auto","created_at":"2022-08-22 16:49:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7547741,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemenatryInformationR1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1464868/v2/e35e66e05b705b19ea524788.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe FOXO transcription factors play a major role in regulating the gene expression of cell cycle progression, apoptosis, cell differentiation, stress response, DNA damage control, and vital cellular functions [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Further, FOXO1 is identified as a key tumor suppressor protein in breast cancer that regulates cell proliferation, invasion, metastasis and survival [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Activation of FOXO members promotes cell cycle arrests at G1/S stage by upregulating cell cycle inhibitors p27 and p21 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, the downregulation of FOXO1 expression leads to dysregulation of cell cycle regulators which induce cell proliferation and play important role in the formation of cancer. Several study provides information regarding downregulated FOXO1 protein level in cancers, including glioblastoma [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], endometrial [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], ovarian carcinoma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], prostate [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and lung carcinoma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nevertheless, restoration of FOXO1 tumor suppressor protein in endometrial carcinoma leads to decrease in cell proliferation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Further, Guttilla and White [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] demonstrated that the overexpression of FOXO1 strongly inhibits cell proliferation and induced apoptosis in breast cancer MCF7 cell lines. Thus, the activation of FOXO1 is regarded as a therapeutic strategy for cancer.\u003c/p\u003e \u003cp\u003eRecently, microRNAs have been reported to modulate post-transcriptional regulation of mRNA resulting in the suppression of target gene expression [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. OncomiRNAs like miR27a, miR96, and miR182 are upregulated in breast cancer and they collectively downregulate the expression of FOXO1 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, for the upregulation of tumor suppressor protein FOXO1, the oncomiRs need neutralization by antimiRNAs. Guttilla and White [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], demonstrated the downregulation of oncomiRs by administration of antimiRNAs and reported the overexpression of FOXO1 in MCF7 cell lines. Recently, self-assembled branched DNA (bDNA) nanostructures have been evolved as economic and efficient strategy for miRNA-based cancer therapy [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recently, our group has also reported the synergistic downregulation of oncomiRs and upregulation of FOXO1 by antimiR-bDNA nanostructures which selectively binds to the oncomiRs 27a, 96 and 182 in MCF-7 cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn last two decades, substantial effort has been made for the delivery of antimiRs for upregulating tumor suppressor proteins or miRNA mimics for downregulating oncogenic proteins. Despite of restoring miRNAs and suppressing cancer cell proliferation using miRNA-based therapeutics very less attention has been made to initiate and activate cell proliferation in the presence of oncogenic miRNAs. Expression of oncogenic miRNA in normal cell is relatively low as compared to cancer cells. Once the oncogenic miRNAs and its target pathways are switched on in the normal cells they are transformed into cancer cells. Therefore, the transition from normal cell to cancer cell will be very useful in identifying early markers for cancer diagnostics. Keeping this as background, we hypothesize that if a group of oncogenic miRNAs can be delivered they will collectively downregulate tumor suppressor protein and activate cell proliferation. To validate the idea we prefer to target transcription factor FOXO1, which is a major tumor suppressor protein and found to be downregulated in breast cancer by oncogenic miRNAs 27a, 96 and 182 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Though, downregulation of FOXO1 is a pivotal step for forming tumorigenesis, the mechanism of initiation of tumorigenesis by down regulated FOXO1 through exogenous miRNA is yet to be understood. Keeping this as background, the present study demonstrates the role of exogenous oncomiRs on the expression of FOXO1 in breast cancer cell line and its effect on cancer cell proliferation. In the current study, self-assembled bDNA nanostructure has been explored to carry miR27a, miR96, miR182 in the overhangs to downregulate the expression of FOXO1 in breast cancer cells. Thus, for the first time our data reports bDNA nanostructure can carry oncogenic miRNA sequences and selectively recognize the target mRNA resulting into down regulation of tumor suppressor protein and downstream cell cycle inhibitors p27 and p21in MCF7 cell lines.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Designing and self-assembly of bDNA nanostructures carrying microRNAs\u003c/h2\u003e \u003cp\u003eThe designing of bDNA structure was performed as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and purchased from Integrated DNA Technology (IDT), USA without any modification or purification (Table S1). In brief, the bDNA monomeric structure composed of four oligonucleotides namely, strands A, B, C and D. The external region of strands B and C are complementary to each other, whereas the internal regions are complementary to strands A and D respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nevertheless, the overhangs of strands A and D are not complementary to each other or to other oligonucleotides, thus they are replaced with either scramble sequences or miRNA sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Since microRNA 27a, 96, and 182 binds to the 3\u0026rsquo; UTR of FOXO1 (Fig. S1), it prompted us to design the self-assembled miR-bDNA structures that can also bind to the 3\u0026rsquo; UTR of FOXO1. Thus, miR-bDNA nanostructures such as bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182 are generated containing respective miRNA sequences (Table S2, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similarly, bDNAmiR-Mix contains all three miRNAs in the four overhangs whereas bDNA-Scramble is devoid of miRNA sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 1 \u0026micro;M of each oligonucleotides were taken from a stock of 100 \u0026micro;M into 25 \u0026micro;l reaction mixture to prepare bDNA structures as mentioned earlier [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Characterization of bDNA nanostructures\u003c/h2\u003e \u003cp\u003eThe integrity of the self-assembled bDNA nanostructures was checked using 10% native polyacrylamide gel (nPAGE) as described previously [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The samples were electrophoresed for 2 h at 4\u0026deg;C in a vertical electrophoresis unit (SE260, Hofer, USA) at a constant voltage of 150 V by taking 1xTAE as running buffer. Then, the gels were stained with ethidium bromide solution (0.5 \u0026micro;g/ml) for 30 min and image was taken using FluroChem E system (Cell Biosciences).\u003c/p\u003e \u003cp\u003eThe conformation of bDNA-miRs was examined using Circular Dichroism (CD) spectrophotometer (Chirascan, Applied Photophysics) as mentioned earlier [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The scan rate of spectra was recorded at 60 nm/sec with bandwidth of 1 nm and a time per point of 0.5 sec. All measurements were done at 25\u0026deg;C between 320 to 200 nm by using a quartz cuvette of 1 mm path length. Three spectra per sample were averaged for getting the final spectrum and final spectra were corrected against buffer as background.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Serum stability and gel retardation assay\u003c/h2\u003e \u003cp\u003eTo study the stability of different bDNA-miR structures, 1 \u0026micro;M of bDNA was taken in 10% \u003cem\u003eFetal bovine serum (FBS)\u003c/em\u003e. After mixing, the samples were incubated at 37\u0026deg;C for 0, 2, 4, 8, 12, 24 and 48 h. Similarly, free miRNA oligos were also examined for serum stability assay in presence of 10% FBS. Then the incubated products were analyzed by 1.5% agarose gel at 100 V for 30 min. Intensity of the DNA in gel images was measured through ImageJ software.\u003c/p\u003e \u003cp\u003eThe miRNA binding to 3\u0026rsquo; UTR of FOXO1 was studied for binding scores using online miRNA target prediction tool TargetScan, Pictar, miRANDA (Fig. S2). Further, the integrity of microRNA binding to 3\u0026rsquo;UTR of FOXO1 region were evaluated using bDNA-miR and antimiR binding in gel retardation assay. AntimiR sequences were used to hybridize with corresponding bDNA-miRs \u003cem\u003ein vitro\u003c/em\u003e. Different bDNA structures were incubated with respective antimiR sequence at 37\u0026deg;C for 2 h and then samples were run in 1.5% agarose gel at 100 V for 60 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell Culture and transfection with bDNA nanostructures\u003c/h2\u003e \u003cp\u003eThe human breast cancer cell line MCF7 were obtained from the National Repository of Animal Cell Culture (NCCS Pune, Maharashtra, India). Cells were routinely maintained in Dulbecco\u0026rsquo;s Modified Eagles Medium (DMEM) (PAN Biotech, Germany) containing 10% (v/v) heat inactivated Fetal Bovine Serum (PAN Biotech, Germany) and 1% penicillin/streptomycin (PAN Biotech, Germany) in a humidified incubator with 95% humidity, 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C (SANYO). Cells were seeded in 6-well tissue culture plates (corning) with density 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well. When cells grown up to 50\u0026ndash;80% confluence, the cells were transfected with 50 \u0026micro;l of self-assembled bDNA structures in 6-well plates with final volume of 2 ml Lipofectamine 3000 according to the manufacturer\u0026rsquo;s instruction (Invitrogen, USA). The cells were divided into five groups such as bDNA-Scr, bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182 and bDNAmiR-Mix for transfection study and incubated for 72 h. Similarly, groups of MCF7 cell lines were also transfected with oncogenic miRs (miR-27a, miR-96, miR-182 and miR-Mix) as control to observe the declined expression of FOXO1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. RNA isolation, cDNA and reverse transcriptase PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from MCF7 cell lines using miRNA Extraction Kit (217004, QIAGEN, USA) and reversely transcribed using cDNA synthesis kit (K1622, Thermo Scientific) according to manufacturer\u0026rsquo;s protocol and subsequently stored at -80\u0026deg;C. The reverse transcriptase PCR (BioRad, USA) was performed using gene specific primer (Table S3) and 18S-rRNA was used as internal control to normalize the gene expression. The relative expression change in RT images was quantified using ImageJ software and transcripts profile has been compared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western blotting analysis\u003c/h2\u003e \u003cp\u003eWhole cell lysate was prepared after 72 h of transfection in 6 well culture plates using RIPA buffer containing protease inhibitor. Extracted proteins from transfected cells were electrophoresed on 10% SDS-PAGE and were subsequently transferred onto PVDF membrane in a constant voltage. The immunoblot was blocked in 5% skimmed milk powder for 1.5 h and incubated overnight with primary antibody FOXO1, p21, p27 and GAPDH under shaking condition at 4\u0026deg;C. All antibodies were procured from Cell Signaling Technology, (Danver, MA, US). Next day the blot was washed five times with Tris-buffered saline containing Tween-20 (TBST) and the membrane was then incubated with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. After thorough ringing the membrane was developed using luminol on X-rays in the dark room.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cell viability assay\u003c/h2\u003e \u003cp\u003eMTT (3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) assay was performed to study the cell viability in presence of bDNA structures. 3 x10\u003csup\u003e3\u003c/sup\u003e numbers of cells were seeded on 96-well culture plates. The cells were transfected with five different bDNA structures after 24 h of seeding. After stipulated time point, 100 \u0026micro;l of MTT (5 mg/ml, Sigma, USA) was added to each well and incubated for 3 h at 37\u0026ordm;C. The growth medium containing MTT was removed and the formazan crystals were dissolved in 150 \u0026micro;l of dimethyl sulphoxide (DMSO, Sigma, USA). The plates were read at 570 nm (Microplate Reader, BioRad, USA) for absorbance and the viable cells were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eEach experiment was tested for three biological repeats. One way ANOVA was performed to test the statistical significance followed by Duncan\u0026rsquo;s multiple range test. For all the analyses, statistically significant results were considered with a \u003cem\u003eP\u003c/em\u003e-value \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of self-assembled bDNA nanostructures\u003c/h2\u003e \u003cp\u003eThe intensity of individual oligonucleotide showed a clear band whereas the complementary oligonucleotides (AB, BC, CD) were forming di-oligo complexes exhibiting decreased electrophoretic mobility (Fig. S2a,b,c). The di-oligos, tri-oligos and bDNA-scramble have different electrophoretic mobility suggesting migration of nucleic acids in PAGE is dependent on size, shape, and conformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Fig. S2d). Similarly, the bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182, and bDNAmiR-Mix were characterized with a clear intense band suggesting the formation of stable unimolecular bDNA structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Nevertheless, all the self-assembled bDNA structures showed B-form of DNA with characteristic positive peaks at ~\u0026thinsp;280 nm and ~\u0026thinsp;220 nm and a negative peak at ~\u0026thinsp;250 nm (Fig. S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Stability of miR-bDNA and selective binding to mRNA\u003c/h2\u003e \u003cp\u003eNuclease stability of bDNA nanostructures was examined in presence of \u003cem\u003eFBS\u003c/em\u003e which comprises\u0026thinsp;~\u0026thinsp;256 U/L of DNase 1. After incubation at 37\u0026deg;C the relative intensity of miR-bDNA was found to be stable for 12 h then slowly decreases upto 48 h (Fig. S4). On the contrary, naked miRNAs are stable in presence of nuclease. The gel retardation assay reveals a significant retardation of DNA suggesting the sequence specific binding between miR-bDNA and FOXO1 mRNA (Fig. S5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Expression of FOXO1, p21, and p27 in response to miRNA\u003c/h2\u003e \u003cp\u003eA significant decreased expression of FOXO1 was observed in miR-182 transfected cells whereas no change in expression was noticed with miR-27a and 96 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). Interestingly the downregulation of FOXO1 was further decreased miR-Mix (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). Further, the expression of well-known CDK inhibitors like p21 and p27 was examined in response to miR-bDNA structures. A significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction in expression of p21 was evident in cells transfected with miR-182 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The expression was further reduced when cells were transfected with miR-Mix (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). On the other hand, the expression of p27 was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) repressed in presence of miR-96 and miR-182 and disappeared with miR-Mix (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. miRNA-bDNA modulates the expression profile of FOXO1, p21, and p27\u003c/h2\u003e \u003cp\u003eOn the contrary, no significant change in expression was found in cells transfected with bDNAmiR-27a, bDNAmiR-96 or bDNAmiR-182 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in FOXO1 expression was evident when transfected with bDNAmiR-Mix. Nearly 40% downregulation of FOXO1 was observed as compared to bDNA-Scr (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Similarly, no significant change was observed in the expression of p21 and p27 in the presence of miR-bDNA nanostructures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d). Nevertheless, a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in expression of p21 and p27 was noticed while transfected with bDNAmiR-Mix (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Downregulation of FOXO1 and downstream gene expression\u003c/h2\u003e \u003cp\u003eThe target site of miR-27a, miR-96, miR-182 on the 3\u0026rsquo; UTR of FOXO1 has been confirmed using TargetScan, Pictar, and miRanda (Fig. S1). Thus, to examine the influence of bDNA-miRs on FOXO1, the endogenous expression of FOXO1 protein was evaluated using western blot analysis (Fig. S6). In comparison to bDNA-Scr, significant decrease in FOXO1 protein level was noticed when transfected with bDNAmiR-27a, and bDNA-Mix. However, an unaltered level of FOXO1 was observed in bDNAmiR-96 and an enhanced level was evident in cells transfected with bDNAmiR-182. FOXO1 is known to regulate the target gene expression of p21 and p27. Interestingly, the protein level of p21 and p27 was observed to be increased in all the bDNA-miRs transfected samples as compared to bDNA-Scr (Fig. S6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effect on cell proliferation by miR-bDNA nanostructures\u003c/h2\u003e \u003cp\u003eCell proliferation was monitored in different time interval from 24 h, 48 h and 72 h. A significant (p\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\u0026lt;0.05\\)\u003c/span\u003e\u003c/span\u003e) increase in cell proliferation was found with bDNAmiR-27a, bDNAmiR-96, bDNAmiR-182 and bDNAmiR-Mix as compared to bDNA-Scr after 24 h of incubation. Similarly, higher cell proliferation was noticed after 48 and 72 h of incubation with all bDNA nanostructures. Importantly, highest cell proliferation was noticed in MCF7 cells transfected with bDNAmiR-Mix structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eBreast cancer is the second leading cause of cancer death among women after lung cancer in the United States. It is estimated that 2,68,600 cases diagnosed with invasive breast cancer in the United States and annually 41,760 patients die in breast cancer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, the molecular mechanism of breast cancer development and progression deserve detailed investigations. FOXO1 is a master regulator that regulates cell cycle proliferation, invasion, metastasis and survival in several cancers including breast cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Now it is well evident that miRNAs play a vital role in proliferation and metastasis of cancer in general and post-transcriptional regulation of tumor suppressor mRNAs in particular [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Currently, miR27a, miR96, and miR182 are known oncomiRs in breast cancer which coordinately reduce the expression of FOXO1 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Nevertheless, in variety of other cancer miR27a, miR96, and miR182 act as oncomiRs to suppress FOXO1, including renal cell cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], bladder cancer [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], Prostate cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], thyroid carcinoma [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], ovarian cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], liver cancer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], gastric cancer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], adenocarcinoma [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and cervical cancer [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Thus, understanding the regulation of FOXO1 expression is vital in managing and monitoring the cancer in general and breast cancer in particular. Moreover, these findings strongly support that miR-27a, miR-96 and miR-182 act as oncogenic miRNAs for downregulating FOXO1 and cell cycle proliferation. Nevertheless, application of antimiRNAs 27a, 96, and 182 down regulate the expression of oncogenic miRNAs in MCF7 and synergistically upregulate the expression of FOXO1 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the cellular response and FOXO1 expression was yet to be investigated when cells are subjected to the simultaneous transfection of multiple oncogenic miRNAs. Secondly, these model oncogenic miRNAs to be transfected to normal cell lines to initiate cell proliferation and find early diagnostic marker for cancer. Since self-assembled DNA nanostructures are biocompatible drug carrier, bDNAs were designed to carry multiple oncogenic miR-27a, miR-96, miR-182 and transected to MCF7 cell lines for understanding the cell proliferation and FOXO1 expression.\u003c/p\u003e \u003cp\u003eAs expected, a significant decrease in FOXO1 expression was noticed in MCF7 while transfected with bDNA nanostructures. The molecular interaction between bDNA nanostructure and FOXO1 mRNA also caused translational inhibition of FOXO1. Further, FOXO1 expression was significantly decreased by miR-182 and miR-Mix transfection. Nevertheless, miR-Mix or bDNAmiR-Mix works better than individual miRNAs which supports earlier data on the multiple miRNA regulation to a particular gene expression [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. A significant repression of p21 was also observed when cells transfected with miR-182 and miR-Mix. Similarly, p27 significantly suppressed by miR-96, miR-182 and miR-Mix. The results suggest that expression of p21 and p27 is largely dependent on miRNAs-mediated downregulation of FOXO1. The endogenous FOXO1 protein decreases when cells transfected with bDNA-miR27a and bDNA-Mix. Thus, the downregulated translated product of FOXO1 by bDNAmiR nanostructures support the hypothesis that FOXO1 protein expression is downregulated at the post-transcriptional level by multiple miRNAs. The expression of p21 and p27 protein was observed to be increased in all the bDNA-miRs transfected samples. Possibly, the expression of p21 and p27 was regulated by other miRNAs. However, miR-27a and miR-96 mediate cell proliferation by regulating cyclin D1, p21 and p27 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, miR-27a, miR-96 and miR-182 could promote migration and invasion by the regulation of PTPN9 in breast cancer [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and PDCD4 in hepatocellular cancer [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Since overexpression of FOXO1 is associated with decreased proliferation and colony size in MCF7 cells, we expect increased cellular proliferation in MCF7 cells transfected with the miR-bDNA nanostructures. Therefore, cellular proliferation was analyzed in MCF7 cells transfected with bDNA-miRs at different incubation periods. A significant cell proliferation was noticed even after 24h of incubation. However, higher cell proliferation was noticed with bDNA-Mix nanostructures which suggest the synergistic effect of oncogenic miRNAs on cell proliferation. Secondly, these findings clearly suggest that miR-27a, miR-96 and mR-182 promote cell proliferation by targeting not only FOXO1 but also other tumor suppressor genes.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, the current communication for the first time reports the downregulation of FOXO1 by bDNA nanostructures carrying multiple miRNAs. The downregulation of FOXO1 and its downstream gene expression in breast cancer cell line is due to oncomiRs miR27a, miR96 and miR182. The designed bDNA acts as a single unit carrying three miRNAs at a time to synergistically regulate gene expression. Nevertheless, the enhanced cell proliferation by oncomiR-bDNA nanostructures is not only due to downregulation of FOXO1 but also associated tumor suppressor proteins. This proof of principle can be extended to multiple oncogenic miRNAs which can be transfected to the cell lines and coordinately downregulate tumor suppressor proteins and regulates cell proliferation, invasion, metastasis. Thus, the model oncogenic miRNAs like miR-27a, miR-96 and miR-182 can be transfected using bDNA nanostructures to various cell lines for switching cell proliferation and identifying early diagnostic markers for cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u003cstrong\u003e\u003cbr\u003e Consent for publication: C\u003cstrong\u003eonsent for publication Not Applicable\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Availability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets generated and/or analysed during the current study does not contain any data that needs to be submitted in any database, thus given in materials and methods section of the manuscript.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting financial interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eCouncil of Scientific \u0026amp; Industrial Research (CSIR), Government of India, New \u0026nbsp; \u0026nbsp;Delhi and Department of Biotechnology (DBT), Government of India, New Delhi\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;Author contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eU.S initiated the project and designed the experiment. A.K, and K.K performed the experiments and acquired the data. All the authors drafted and critically reviewed the manuscript. Finally all the authors read and approved the manuscript submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are thankful to the Council of Scientific \u0026amp; Industrial Research (CSIR) and Department of Biotechnology (DBT), Government of India, New Delhi for supporting the work.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information (optional):\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eHuang, H., Tindall, D. J. (2007) Dynamic FoxO transcription factors. J. Cell Sci. 120:2479\u0026ndash;2487.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFu, Z., Tindall, D. J. (2008) FOXOs, cancer and regulation of apoptosis. Oncogene. 27:2312\u0026ndash;2319.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCoomans de Brach\u0026egrave;ne, A., Demoulin, J. B. (2016) FOXO transcription factors in cancer development and therapy. Cell. Mol. Life Sci. 73:1159\u0026ndash;1172.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAccili, D., Arden, K. C. (2004) FOXOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117:421\u0026ndash;426.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDijkers, P. F., Medema, R. H., Pals, C., Banerji, L., Thomas, N. S., Lam, E. W., Burgering, B. M., Raaijmakers, J. A., Lammers, J. W., Koenderman, L., Coffer, P. J. (2000) Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol. Cell. Biol. 20:9138\u0026ndash;9148.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSeoane, J., Le, H. V., Shen, L., Anderson, S. A., Massagu\u0026eacute;, J. (2004) Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117:211\u0026ndash;223.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGoto, T., Takano, M., Albergaria, A., Briese, J., Pomeranz, K. M., Cloke, B., Fusi, L., Feroze-Zaidi, F., Maywald, N., Sajin, M., Dina, R. E., Ishihara, O., Takeda, S., Lam, E. W., Bamberger, A. M., Ghaem-Maghami, S., Brosens, J. J. (2008a) Mechanism and functional consequences of loss of FOXO1 expression in endometrioid endometrial cancer cells. Oncogene. 27:9\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGoto, T., Takano, M., Hirata, J., Tsuda, H. (2008b) The involvement of FOXO1 in cytotoxic stress and drug-resistance induced by paclitaxel in ovarian cancers. Br. J. Cancer 98:1068\u0026ndash;1075.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDong, X. Y., Chen, C., Sun, X., Guo, P., Vessella, R. L., Wang, R. X., Chung, L. W., Zhou, W., Dong, J. T. (2006) FOXO1A is a candidate for the 13q14 tumor suppressor gene inhibiting androgen receptor signaling in prostate cancer. Cancer Res. 66:6998\u0026ndash;7006.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGao, Z., Liu, R., Ye, N., Liu, C., Li, X., Guo, X., Zhang, Z., Li, X., Yao, Y., Jiang, X. (2018) FOXO1 Inhibits Tumor Cell Migration via Regulating Cell Surface Morphology in Non-Small Cell Lung Cancer Cells. Cell. Physiol. Biochem. 48:138\u0026ndash;148.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGuttilla, I. K., White, B. A. (2009) Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells. J. Biol. Chem. 284:b23204\u0026ndash;23216.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBartel D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116:281\u0026ndash;297.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eO\u0026apos;Brien, J., Hayder, H., Zayed, Y., Peng, C. (2018) Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front. Endocrinol. 9:402.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWang, D., Fu, Y., Yan, J., Zhao, B., Dai, B., Chao, J., Liu, H., He, D., Zhang, Y., Fan, C., Song, S. (2014) Molecular logic gates on DNA origami nanostructures for microRNA diagnostics. Anal. Chem. 86:1932\u0026ndash;1936.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhou, W., Li, D., Xiong, C., Yuan, R., Xiang, Y. (2016) Multicolor-Encoded Reconfigurable DNA Nanostructures Enable Multiplexed Sensing of Intracellular MicroRNAs in Living Cells. ACS Appl. Mater. 8:13303\u0026ndash;13308.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLiu, Q., Wang, D., Yuan, M., He, B. F., Li, J., Mao, C., Wang, G. S., Qian, H. (2018) Capturing intracellular oncogenic microRNAs with self-assembled DNA nanostructures for microRNA-based cancer therapy. Chem. Sci. 9:7562\u0026ndash;7568.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChandrasekaran, A. R., Punnoose, J. A., Zhou, L., Dey, P., Dey, B. K., Halvorsen, K. (2019) DNA nanotechnology approaches for microRNA detection and diagnosis. Nucleic Acids Res. 47:10489\u0026ndash;10505.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJahanban-Esfahlan, R., Seidi, K., Jahanban-Esfahlan, A., Jaymand, M., Alizadeh, E., Majdi, H., Najjar, R., Javaheri, T., Zare, P. (2019) Static DNA nanostructures for cancer theranostics: Recent progress in design and applications. Nanotechnol. Sci. Appl. 12:25.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBaral, B., Dutta, J., Subudhi, U. (2021) Biophysical interaction between self-assembled branched DNA nanostructures with bovine serum albumin and bovine liver catalase. Int. J. Biol. Macromol. 177:119\u0026ndash;128.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKumari, K., Kar, A., Nayak, A. K., Mishra, S. K., Subudhi, U. (2021) MiRNA-mediated alteration of sulfatase modifying factor 1 expression using self-assembled branched DNA nanostructures. RSC Adv. 11:10670\u0026ndash;10680.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNahar, S., Nayak, A. K., Ghosh, A., Subudhi, U., Maiti, S. (2017) Enhanced and synergistic downregulation of oncogenic miRNAs by self-assembled branched DNA. Nanoscale 10:195\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNayak, A. K., Subudhi, U. (2014) Directed self-assembly of genomic sequences into monomeric and polymeric branched DNA structures. RSC Adv. 4:54506\u0026ndash;54511.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNayak, A. K., Rath, S. K., Subudhi, U. (2019) Preparation of stable branched DNA nanostructures: Process of cooperative self-assembly. J. Phys. Chem. B, 123:3591\u0026ndash;3597.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBhanjadeo, M. M., Nayak, A. K., Subudhi, U. (2017) Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice. Biochem. Biophys. Res. 485:492\u0026ndash;498.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBhanjadeo, M. M., Baral, B., Subudhi, U. (2019) Sequence-specific B-to-Z transition in self-assembled DNA: A biophysical and thermodynamic study. Int. J. Biol. Macromol. 137:337\u0026ndash;345.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDeSantis, C. E., Ma, J., Gaudet, M. M., Newman, L. A., Miller, K. D., Goding Sauer, A., Siegel, R. L. (2019) Breast cancer statistics, 2019. CA Cancer J. Clin. 69:438\u0026ndash;451.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhou, L., Yin, B., Liu, Y., Hong, Y., Zhang, C., Fan, J. (2012) Mechanism and function of decreased FOXO1 in renal cell carcinoma. J. Surg. Oncol. 105:841\u0026ndash;847.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGuo, Y., Liu, H., Zhang, H., Shang, C., Song, Y. (2012) MiR-96 regulates FOXO1-mediated cell apoptosis in bladder cancer. Oncol. Lett. 4:561\u0026ndash;565.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWallis, C. J., Gordanpour, A., Bendavid, J. S., Sugar, L., Nam, R. K., Seth, A. (2015) MiR-182 Is Associated with Growth, Migration and Invasion in Prostate Cancer via Suppression of FOXO1. J. Cancer. 6:1295\u0026ndash;1305.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGao, F., Wang, W. (2015) MicroRNA-96 promotes the proliferation of colorectal cancer cells and targets tumor protein p53 inducible nuclear protein 1, forkhead box protein O1 (FOXO1) and FOXO3a. Mol. Med. Rep. 11:1200\u0026ndash;1206.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSong, H. M., Luo, Y., Li, D. F., Wei, C. K., Hua, K. Y., Song, J. L., Xu, H., Maskey, N., Fang, L. (2015) MicroRNA-96 plays an oncogenic role by targeting FOXO1 and regulating AKT/FOXO1/Bim pathway in papillary thyroid carcinoma cells. Int. J. Clin. Exp. Pathol. 8:9889\u0026ndash;9900.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhang, L. Y., Chen, Y., Jia, J., Zhu, X., He, Y., Wu, L. M. (2019) MiR-27a promotes EMT in ovarian cancer through active Wnt/\u0026beta;-catenin signalling by targeting FOXO1. Cancer Biomarkers 24:31\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZheng, X., Zhang, F., Zhao, Y., Zhang, J., Dawulieti, J., Pan, Y., Cui, L., Sun, M., Shao, D., Li, M., He, K., Zhang, M., Li, J., Chen, L. (2018) Self-assembled dual fluorescence nanoparticles for CD44-targeted delivery of anti-miR-27a in liver cancer theranostics. Theranostics 8:3808\u0026ndash;3823.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLang, C., Xu, M., Zhao, Z., Chen, J., Zhang, L. (2018) MicroRNA-96 expression induced by low-dose cisplatin or doxorubicin regulates chemosensitivity, cell death and proliferation in gastric cancer SGC7901 cells by targeting FOXO1. Oncol. Lett. 16:4020\u0026ndash;4026.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLing, J., Dong, X., Wang, L., Xue, Y., Jia, X., Song, W., Li, Q. (2019) MiR-27a-regulated FOXO1 promotes pancreatic ductal adenocarcinoma cell progression by enhancing Wnt/\u0026beta;-catenin signaling activity. Am. J. Transl. Res. 11:3069.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYang, L., Liu, L., Zhang, X., Zhu, Y., Li, L., Wang, B., Liu, Y., Ren, C. (2020) Mir-96 enhances the proliferation of cervical cancer cells by targeting foxo1. Pathol. Res. Pract. 216:152854.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi, X., Xu, M., Ding, L., and Tang, J. (2019) MiR-27a: a novel biomarker and potential therapeutic target in tumors. J. Cancer. 10:2836.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLin, H., Dai, T., Xiong, H., Zhao, X., Chen, X., Yu, C., Li, J., Wang, X., Song, L. (2010) Unregulated miR-96 induces cell proliferation in human breast cancer by downregulating transcriptional factor FOXO3a. PloS One 5:e15797.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHong, Y., Liang, H., Wang, Y., Zhang, W., Zhou, Y., Chen, S. A., Chen, X. (2016) miR-96 promotes cell proliferation, migration and invasion by targeting PTPN9 in breast cancer. Sci. Rep. 6:1\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHu, J., Wang, Z., Wang, J., Jian, Y., Dai, J., Wang, X., Xiong, W. (2020) MicroRNA-182 Promotes Cell Migration by Targeting Programmed Cell Death 4 in Hepatocellular Carcinoma Cells. Onco Targets Ther. 13:9159.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DNA nanostructure, FOXO1, miRNAs, Breast cancer, MCF7 cell, P21, P27","lastPublishedDoi":"10.21203/rs.3.rs-1464868/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1464868/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFOXO1 transcription factor is not only limit the cell cycle progression but also promote cell death as a tumor suppressor protein. Though the expression of FOXO1 is largely examined in breast cancer, the regulation of FOXO1 by miRNA is yet to be explored. In the current study, self-assembled branched DNA (bDNA) nanostructures containing oncogenic miRNAs were designed and transfected to MCF7 cell lines to decipher the FOXO1 expression. bDNA containing oncogenic miRNA 27a, 96 and 182 synergistically downregulate the expression of FOXO1 in MCF7 cells. The down-regulation is evident both in mRNA and protein level suggesting bDNA having miRNA sequences can selectively bind to mRNA and inhibit translation. Secondly, the downstream gene expression of P21 and P27 are also significantly downregulated in presence of miR-bDNA nanostructures. The cell proliferation activity was progressively increased in presence of miR-bDNA nanostructure which confirms the reduced tumor suppression activity of FOXO1 and the downstream gene expression. This finding can be explored to design novel bDNA structures which can downregulate the tumor suppressor protein in normal cells and induce cell proliferation activity to identify early phase markers of cancer.\u003c/p\u003e","manuscriptTitle":"Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-08-22 16:49:37","doi":"10.21203/rs.3.rs-1464868/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-10-04T03:13:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-09-24T21:07:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32985a5d-2c5e-4f9b-a42a-1c0da41ee8cf","date":"2022-09-16T18:42:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-09-01T07:41:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3adec751-6532-42ba-a6f9-3eb94278cf8a","date":"2022-08-23T22:51:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6fd33a53-f011-4019-aa37-f8627508dec1","date":"2022-08-22T00:00:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91b6cc45-ccd9-4220-a7aa-117d05079814","date":"2022-08-15T00:37:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-08-11T14:20:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-08-11T13:59:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-08-03T05:29:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-08-03T05:16:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2022-07-23T14:52:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d40260d-a098-4a4f-94ab-376820e7dc55","owner":[],"postedDate":"August 22nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-12-07T10:44:27+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-22 16:49:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1464868","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1464868","identity":"rs-1464868","version":["v2"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}