miR-5088-5p inhibitor reduces radiation-induced malignancy by decreasing Slug

doi:10.21203/rs.3.rs-1954909/v1

Download PDF

Research Article

miR-5088-5p inhibitor reduces radiation-induced malignancy by decreasing Slug

https://doi.org/10.21203/rs.3.rs-1954909/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Purpose

Radiotherapy is widely used for cancer treatment, but paradoxically, surviving cancer cells can become malignant, leading to tumor recurrence or metastasis. Therefore, in order to increase the effectiveness of radiotherapy, efforts to reduce radiation-induced malignancy are absolutely necessary. As a tool, microRNA simultaneously regulates the expression of multiple target mRNAs, so it has significant potential as an effective therapeutic agent. The main objective of this study is to elucidate the malignant mechanism of radiation-induced miR-5088-5p and to prove the efficacy of miR-5088-5p inhibitor for mitigating malignancy, thereby demonstrating its applicability as a therapeutic agent to increase the efficiency of radiotherapy.

Methods

To analyze the relationship between radiation and miR-5088-5p expression, miR-5088-5p levels were determined by qRT-PCR in the plasma of breast and lung cancer patients with or without radiotherapy. MSP and qMSP assays were used to confirm the methylation of radiation-induced miR-5088-5p. To determine whether miR-5088-5p inhibitor reduces radiation-induced malignancy by decreasing Slug, we used wound healing, invasion, sphere formation, Western blot, qRT-PCR assays, and in vivo mouse xenograft metastatic model.

Results

It was confirmed at both the cellular and animal model that miR-5088-5p, which showed higher expression in the plasma of breast cancer and lung cancer patients with radiotherapy, enhances tumor malignancy by enhancing its expression through hypomethylation of its promoter by radiation. On the other hand, it was shown that miR-5088-5p inhibitor reduced the mechanism of radiation-induced malignancy.

Conclusions

Collectively, miR-5088-5p inhibitors have shown potential as a combination therapy to enhance radiotherapy effectiveness by reducing radiation-induced malignancy.

miR-5088-5p inhibitor

radiation

malignancy

Slug

methylation

Radiotherapy is commonly used for treatment of about 50% of all cancer patients. In particular, radiotherapy is efficacious at the early stages of development of various tumor types, including skin, prostate, lung, cervical, lymphoma, and head and neck [1]. However, ionizing radiation (IR) is well established carcinogen [2] with occurrence of secondary malignant cancers documented in patients with primary carcinoma exposed to radiotherapy [3–5]. Increased risk of radiation-induced malignancy has additionally been reported in breast, prostate, gynecological tumor types, lymphoma, and pediatric malignancies [6–11]. To enhance the efficiency of radiotherapy, reduction of radiation-induced malignancy remains an urgent clinical requirement [12]. The current study explored the utility of microRNAs (miRNA) as therapeutic targets to inhibit the side-effects of radiation based on identification of radiation-associated target miRNAs and their underlying mechanisms.

MiRNA is a type of non-coding RNA composed of 18–24 nucleotides that regulates post-transcriptional expression of target genes. MiRNAs are involved in numerous biological pathways and cellular processes, including cell proliferation, apoptosis, development, and cell signaling [13]. In addition, IR-induced changes in miRNA expression have been reported in several gene expression profiling studies to date [14]. Several different miRNAs (including miR-17-3p [15], miR-22 [16], miR-29c [17], miR-34a [17], miR-145 [15], miR-193a-3p [18], miR-193b [17], and miR-365 [17]) are reported to be induced while the miR-15/16 (miR-15 a/b, miR-16, miR-195, miR-424, and miR-497) family [16, 17, 19] is inhibited by IR. In particular, radiation-upregulated miR-21 [20] and down-regulated miR-143 [21] are associated with radiation-induced carcinogenesis, suggesting critical roles in radiation-mediated malignancy. Depending on the tumor type, miRNAs can function as either onco-miRNAs or tumor suppressor genes (anti-onco-miRNAs) [22, 23]. In view of previous findings that deregulation or dysfunction of miRNAs contributes to cancer development, their efficacy as a cancer biomarker has been actively researched [13, 24–26]. For example, miR-224 [27], miR-130b [28], and miR-144-3p [29] are known to increase metastasis in hepatocellular carcinoma, non-small cell lung cancer, and renal cell carcinoma, respectively. In addition, miRNA-21 inhibitors are known to increase radio-sensitivity by inhibiting the PI3K/AKT pathway [30]. These molecules are released from cells and circulate in the blood, presenting an efficient tool for diagnosis of multiple diseases including cancer [13, 31].

Experiments by our group confirmed that miR-5088-5p, a miRNA identified in a previous study, promotes malignancy by inhibiting the expression of Deleted in Breast Cancer 2 (DBC2) in breast cancer [32]. The current study focused on the relationship between onco-miR-5088-5p and IR-induced malignancy. IR enhanced expression of miR-5088-5p through hypomethylation of its promoter, and eventually promoting tumor progression. Notably, IR-induced tumorigenicity was effectively suppressed by treatment with a miR-5088-5p inhibitor, supporting the potential of miR-5088-5p inhibitor as a therapeutic agent that can used in combination with radiotherapy to improve radiotherapy efficiency.

2.1 Cell Cultures

Highly metastatic MDA-MB-231 cells provided by S.J. Lee (FNCT Biotech, Korea) laboratory. MDA-MB-231 and H460 cells obtained from the Korea Cell Line Bank (KCLB). H460 and MDA-MB-231 cells were cultured in RPMI 1640 media (Mediatech, Inc., Manassas, VA) and DMEM (Mediatech, Inc., Manassas, VA) with 10% FBS (Tissue Culture Biologicals, CA, USA) and penicillin-streptomycin antibiotics (PAA Laboratories GmbH, Austria), respectively. All cells were cultured in a 5% CO₂ incubator at 37℃.

2.2 RNA Extraction and qRT-PCR

Plasma was isolated from normal and breast cancer patients and metastasis mouse model by 13,000 rpm, 15 min centrifugation. Total RNA was extracted with TRIzol reagent (Molecular Research Center Inc., OH, USA) from plasma obtained from patients and mice. The miRNA was synthesized using the Mir-X miRNA First-Stand cDNA Synthesis Kit (Takara, Japan) and the mRNA using the Tetro cDNA Synthesis Kit (BIOLINE, London, UK). Real time PCR was quantified with U6 (Takara, Japan) and GAPDH. All data were analyzed by the 2^−△△Cr method. The sequences of primers were as follows : primary-miR-5088-5p: (forward) 5’-CCTCTGCATGTTTGCTGCCA − 3’ and (reverse) 5’-TGAGGGCCCAGGAAGAAGGGA-3’, precursor-miR-5088-5p: (forward) 5’-CAGGGCT CAGGGATTGGATGGAGG-3’ and (reverse) 5’-TGAGGGCCCAGGAAGAAGGGA-3’, mature-miR-5088-5p: 5’-CAGGGCTCAGGGATTGGATGGAGG-3’, MMP-2: (forward) 5′-CATCAAGGGCATTCAGGAGC-3′ and (reverse) 5′-A GAACACAGCCTTCTCCTCC-3′, MMP-9: (forward) 5′-AGGACGACGTGAATGGCAT G-3′ and (reverse) 5′-ATCGTCCACCGGACTCAAAG-3′, Oct4: (forward) 5’-GAGCAAAACCCGGAGGAGT-3’ (reverse) 5’-TTCTCTTTCGGGCCTGCAC-3’, Nanog: (forward) 5’-GCTTGCCTTGCTTTGAAGCA-3’ (reverse) 5’-GTTCTTGCATCTGCTGGAGG-3’, Sox2: (forward) 5’-ATGCACCGCTACGACGTGA-3’ and (reverse) 5’-CTTTTGCACCCCTCCCATT-3’, CD133: (forward) 5’-GCCACCGCTCTAGATACTGC-3’ and (reverse) 5’-GCTTTTCCTATGCCAAACCA-3’, CD44: (forward) 5’-AAGGTGGAGCAAACACAACC-3’ and (reverse) 5’-ACTGCAATGCAAACTGCAAG-3’, DBC2: (forward) 5’-CAGAGCAGTAGACAGTGACC-3’ and (reverse) 5’-TGTAGTTGGTGCAGATGTGG-3’ and GAPDH: (forward) 5′-CATCTCTGCCCCCTCTGCT GA-3′ and (reverse) 5′-GGATGACCTTGCCCACAGCCT-3′.

2.3 Western blot analysis

Cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche, Indianapolis, USA). The extracted protein concentration was measured using Bradford assay (Bio-Rad Laboratories Inc, CA, USA). Proteins were separated using SDS-PAGE and then transferred to PVDF (Millipore Corporation, MA, USA). The membrane was blocked with 5% BSA (bovine serum albumin) in Tris-buffered saline-Tween 20 (TBST) (10 mM TrisHCl, pH 8.0, 150 mM NaCl and 0.05% Tween 20) and the primary antibody was treated. The secondary antibody was treated by dilution in 5% skim milk. After washing with TBST, it was detected with ECL solution (Thermo Scientific, Pierce, USA) using AmershamTM Imager 600 system equipment (GE Healthcare Bio-Sciences, PA, USA).

Antibodies against Slug and ß-actin were obtained from Santa Cruz Biotechnology (CA, USA). Zeb1 were purchased from Sigma (Sigma-Aldrich Company, MO, USA). Twist and N-cadherin were purchased from Abcam Inc. (Cambridge, MA, USA). Snail, Vimentin, DNMT1, DNMT3a, and DNMT3b were purchased from Cell Signaling technology (Beverly, MA, USA).

2.4 Irradiation

Ionizing radiation (IR) was performed H460 and MDA-MB-231 cells in 60 mm dishes as 5Gy and cells were harvested after 48 hours. We irradiated with a 3.81Gy/min dose rate using a 137Cs γ-rays source (Atomic Energy of Canada, Ltd, Canada). The mouse model is irradiated at 2Gy/min dose rate using X-RAD 320 irradiator (PXi, North Branford, CT) equipment.

2.5 Plasmid DNA, RNA oligoribonucleotides, and transfection

To make Slug-overexpressing vector, the Slug gene was inserted to the pCMV-taq2 vector. Each sequence of primers for plasmid constructs was as follows: Slug (forward) 5’-CGCGGATCCATGCCGCGCTCCTTCCTG-3’ and (reverse) 5’-CGGAATTCTCAGTGTGCTACACAGCAGCCA-3’. The inhibitor of miR-5088-5p was a 2’-O-methyl-modified oligoribonucleotide single strand with the sequence as 5’-CCUCCAUCCAAUCCCUGAGC CCU-3’ and was synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA). All siRNAs (Slug, Zeb1, and Snail) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). siRNAs (20 mM), miRNAs (10 mM), and plasmids (4 ug) were introduced into cells using G-fectin (Genolution, Seoul, Korea) or Lipofectamine 2000 reagent (Thermo Fisher Scientific, Invitrogen, USA) according to the manufacturer’s instructions, respectively.

2.6 Wound healing assay

Cells were seeded on 6-well culture plates and a plastic tip was scraped to mimic wound damage. Cells were washed with PBS to remove cell debris and moved for 16–24 hours. The recovery of wound damage was quantified by counting cells after observation with a light microscope (CKX53, OLYMPUS, Tokyo, Japan) [33].

2.7 Transwell invasion assay

After coating matrigel (BD Biosciences, CA, USA) in a transwell chamber (8µm pore) (Corning, Corning, NY), transfected cells were placed in the upper transwell chamber with opti-MEM (Gibco, USA) medium. After culture media was added to the lower chamber and cultured for 16 hours, the invaded cells in the lower transwell chamber were fixed and stained with Hemacolor solution (Merck, MA, USA). Stained cells were counted under a light microscope (CKX53, OLYMPUS, Tokyo, Japan).

2.8 Tube formation assay

The supernatant of MDA-MB-231 cells transfected with negative control (NC) or miR-5088-5p inhibitor using G-fectin (Genolution, Seoul, Korea) was harvested after 72 hours. After coating a 96-well plate with matrigel, 1x10⁴ cells/well of HUVECs and supernatant obtained from MDA-MB-231 cells were simultaneously added to the plate. After 16 hours, tube forming ability was observed under a microscope (Miotic AE31 series, Motic, Hong Kong).

2.9 Sphere formation assay

After transfection of MDA-MB-231 cells (1 × 10⁵/dish) with negative control (NC) or miR-5088-5p inhibitor, cells seeded in Dulbecco's Modified Eagle Medium-F12 (Gibco, USA) containing B27(1:50) (Gibco, USA) Cellgro, Manassas, VA) and grown for 7–10 days. Spheres (> 20 µm in diameter) were counted in an inverted microscope (Miotic AE31 series, Motic, Hong Kong).

2.10 In vivo promoter methylation analysis

H460 cells were 1x10⁷ cells were injected into subcutaneously injected of a six-week-old mouse. After 14 days, 2.5Gy of radiation was fractionated to the mouse's chest every day for 3 days. After 56 days, the mouse was sacrificed, and tumors generated from the subcutaneous tissue were isolated and ground to extract genomic DNA and mRNA. Methylation of miR-5088-5p promoter was confirmed using genomic DNA by phenol-chloroform extraction method, and the level of miR-5088-5p was confirmed using mRNA by TRIzol method. This experiment was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Institute of Radiological & Medical Science.

2.11 Pyrosequencing analysis

To confirm the methylation of miR-5088-5p at 10 sites on the CpG islands of the miR-5088-5p promoter, pyrosequencing analysis was performed. PCR was performed with taq polymerase (TaKaRa, Kyoto, Japan) using the primers of miR-5088-5p (methy-miR-5088-5p: 5’-ATTTTTGTTTTAGGAGATGTTGAAG-3’ (forward) and 5’-CCTAACCACCCTAAAAACTCAC-3’ (reverse)) [32]. Pyrosequencing was conducted on a PyroMark ID system (Qiagen, Hilden, Germany) using streptavidin Sepharose HP beads (Amersham Biosciences, Piscataway, NJ) and a Pyro Gold Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

2.12 Methylation specific PCR (MSP) and quantitative methylation-specific PCR (qMSP) analysis

After genomic DNA was extracted according to a standard phenol-chloroform extraction method, bisulfite modification of genomic DNA was performed using an EZ DNA methylation kit (Zymo Research, USA). The methylation analysis of the miR-5088-5p promoters was performed using MSP primer pairs covering the putative transcriptional start site in the 5′ CpG islands with 1 µℓ of bisulfite-treated DNA as template and JumpStart Red Taq DNA Polymerase (Sigma-Aldrich Company, MO, USA) for amplification as previously described [34]. DKO (DNMT1(-/-), DNMT3B(-/-) double knockout in HCT116 cells) as unmethylation control, IVD (in vitro methylated DNA) as methylation control, and ddH2O as PCR negative control were used. Primers of the miR-5088-5p promoter region across the upstream from − 225 to 34 from mature form sites (Fig. E2A). The unmethylation forward primer of the miR-5088-5p; 5’-GTTTTAGGAGATGTTGAAGGATGA-3’ and unmethylation reverse primer; 5’-CATACAAAAAATACCAATACCCAAA-3’. The methylation forward primer of the miR-5088-5p; 5’-TTTGTTTTAGGAGATGTTGAAGGAC-3’ and the methylation reverse primer; 5’-ATACAAAAAATACCGATACCCGAA-3’. MSP amplification was performed on bisulfite treated samples and normalized using the Alu element. Real-time PCR was performed by a CFX96TM real-time system (Bio-Rad, Hercules, CA, USA). Alu primer sequence information is previously described [35].

2.13 In vivo metastasis assay

Human lung cancer cells, H460 cells were transfected with negative control (NC) and anti-miR-5088-5p. 2x10⁶ cells were injected into tail vein of a six-week-old mouse. After 14 days, 2.5Gy of radiation was fractionated to the mouse's chest every day for 3 days. After 56 days, the mouse was sacrificed, and blood and lungs were collected. The lungs were fixed with 4% paraformaldehyde, metastatic nodules were counted, and a block was made to perform hematoxylin and eosin (H&E) staining (shandon™, Thermo Scientific, Pierce, USA). This experiment was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Institute of Radiological & Medical Science.

2.14 Colony formation assay

Cells (5x10²) were seeded in 60mm dishes cultured for 3 weeks. When visible colonies appeared, the cells were washed twice with PBS, fixed in methanol for 15 minutes, stained with crystal violet for 20 minutes, washed with water, and air dried. The number of dishes repeated 3 times was counted. The number of visible colonies is the rate of colony formation = (number of colonies / number of seeding cells) × 100%.

2.15 MTT assay

H460 and MDA-MB-231 cells (5x10³ cells per well) were seeding on 96-well plates in triplicate wells. The next day, the cells were treated with 50µM of cisplatin (Sigma-Aldrich Company, MO, USA) or 5Gy radiation. MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich Company, MO, USA) was added at 24h, 48h, and 72h, and proliferation was measured at 450nm after 4h.

2.16 Clinical specimens

Biospecimens and data used in this study were provided by the Korea Institute of Radiological and Medical Sciences (KIRAMS) Radiation Biobank (KRB) of Korea Cancer Center Hospital in Republic of Korea. Breast cancer patient plasma was provided in samples from normal (n = 26) and breast cancer patients with (n = 15) and without (n = 21) radiotherapy, respectively (KRB-2016-I005). Lung cancer patient plasma was provided in samples from cancer patients with (n = 26) and without (n = 26) radiotherapy (KRB-2021-I002). All samples used in this experiment have Institutional Review Board (IRB) approval (K-1907-002-002, KIRAMS 2021-04-002-001) in KIRAMS.

2.17 Statistical Analysis

All experiments were performed in triplicate. Data are presented as mean standard deviations and, where appropriate, Student's t tests were analyzed using GraphPad Prism software. P values of P < 0.05 were considered by Student t test to be statistically significant.

3.1 IR upregulates biogenesis of miR-5088-5p through inhibiting methylation

Radiotherapy is one of the most common treatment options for cancer. A previous study by our group showed that miR-5088-5p promotes tumorigenic properties in breast cancer [32]. Here, we further examined the expression patterns and mechanisms of action of miR-5088-5p in tumor malignancy during radiotherapy. First, we compared the expression patterns of miR-5088-5p in plasma from breast and lung cancer patients with and without radiotherapy. As a result, miR-5088-5p was upregulated in plasma of breast or lung cancer patients treated with radiation compared to the non-radiated groups (Fig. 1A, and Supplementary Fig. S1). In addition, expression of miR-5088-5p in the tumor tissues of irradiated mice after injection of H460 or MDA-MB-231 cells was higher than that of the control group (Fig. 1B). In our previous study, our group identified the mechanism of DBC2 (Deleted in Breast Cancer 2) as a target of miR-5088-5p [32]. Expression of DBC2 was dramatically decreased in the tumor tissue exposed to radiation in the xenograft mouse model (Supplementary Fig. S2). Our results suggest that miR-5088-5p is upregulated while its target DBC2 is downregulated under conditions of irradiation.

At the cellular level, the effect of radiation on biogenesis of miR-5088-5p was confirmed in lung and breast cancer cells, H460 and MDA-MB-231 cells, respectively. Ionizing radiation (IR, 5Gy) enhanced biogenesis of miR-5088-5p by increasing the primary and precursor as well as mature forms of miR-5088-5p (Fig. 1C). Radiation is known to induce various epigenetic mutations in cancer [36]. Accordingly, epigenetic alterations of the miR-5088-5p promoter were further examined using MSP (methylation-specific PCR) and qMSP (quantitative methylation-specific PCR) to identify the underlying cause of upregulation. Reduction in methylation of the miR-5088-5p promoter via radiation was detected at the cellular level (Fig. 1D). The methylation of the miR-5088-5p promoter, including location of CpG islands, was additionally confirmed using pyrosequencing (Supplementary Fig. S3A). As a result, it was confirmed that IR reduced the methylation of 10 CpG islands in the miR-5088-5p promoter (Supplementary Fig. S3B). On the other hand, miR-5088-5p promoter methylation was decreased (Supplementary Fig. S4A) and expression of miR-5088-5p was increased (Supplementary Fig. S4B) in metastatic MDA-MB-231 relative to in parental MDA-MB-231 cells. Metastatic MDA-MB-231 (MDA-MB-231 LM2) cells constitute MDA-MB-231 cells that have metastasized to the lungs and are organ-specific metastatic clones with strong migration and invasive properties [37]. These results support our hypothesis that miR-5088-5p expression is upregulated via suppression of its promoter methylation, which, in turn, promotes tumor malignancy. For confirmation of our findings in an animal model, methylation of the miR-5088-5p promoter was examined in tumor tissues of mice with and without radiation treatment after subcutaneous injection of H460 cells. Decreased miR-5088-5p promoter methylation was confirmed in tumor tissues of radiation-treated relative to the non-irradiated mice (Fig. 1E).

In addition, IR induced a decrease in DNA methyl transferases (DNMT), such as DNMT1, DNMT3a, and DNMT3b, involved in methylation of H460 and MDA-MB-231 cells (Fig. 1F). Taken together, the data clearly indicate that IR decreased methylation of CpG islands in the miR-5088-5p promoter region via reducing DNMT, eventually inducing an increase in miR-5088-5p biosynthesis.

3.2 The miR-5088-5p inhibitor suppress IR-induced tumorigenic properties

We further examined whether miR-5088-5p is involved in the mechanism of IR-induced malignant transformation with the aid of an miR-5088-5p inhibitor. The malignant phenotype was confirmed after IR (5Gy) exposure of H460 and MDA-MB-231 cells transfected with miR-5088-5p inhibitor (anti-miR-5088-5p) or negative control. The miR-5088-5p inhibitor reduced IR-induced mesenchymal markers, such as Slug, Zeb1, Snail, Twist, Vimentin, and N-cadherin (Fig. 2A), mobility (Fig. 2B), and invasiveness by suppressing matrix metalloproteinase (MMP)-2/9 levels (Fig. 2C and 2D). Additionally, the miR-5088-5p inhibitor induced a decrease in IR-induced tube formation capacity (Fig. 3A) by inhibiting angiogenesis-related factors such as vascular endothelial growth factor (VEGF) and angiopoietin (Ang) 2 (Fig. 3B and 3C), and sphere formation ability (Fig. 3D) by reducing cancer stem-like cell (CSC) marker proteins, such as Oct4, Nanog, Sox2, CD133, and CD44 (Fig. 3E). In addition, non-metastatic MCF-7 cells obtained the same results as H460 and MDA-MB-231 cells, reconfirming the mechanisms of radiation and miR-5088-5p inhibitors (Supplementary Fig. S5). The collective results clearly demonstrate that IR-induced miR-5088-5p enhances epithelial-mesenchymal transition (EMT), migration, invasion, angiogenesis, and stemness maintenance.

3.3 The miR-5088-5p inhibitor reduces IR-induced pulmonary metastasis

To validate the relevance of miR-5088-5p in the mechanism of IR-induced metastasis, a mouse xenograft model using H460 cells transfected with miR-5088-5p inhibitor (anti-miR-5088-5p) was employed. Six-week-old BALB/C nude mice were injected with H460 cells transfected with miR-5088-5p inhibitor into the tail vein. After 14 days, mice were subjected to IR (2.5 Gy/day) in the chest for 3 days (Fig. 4A). The radiation dose to induce malignancy was selected by referring to several previous papers [38–41]. Histogram and H&E staining results showed that the lung metastasis pattern of the group of mice treated with radiation after injection of cells transfected cells with miR-5088-5p inhibitor was further reduced relative to the group exposed to radiation alone (Fig. 4B). The number of nodules in lung was counted and graphs generated (Fig. 4C). Based on the collective findings, we concluded that the miR-5088-5p inhibitor could effectively suppress IR-induced lung metastasis.

MiRNAs are important circulating factor in blood and extensive studies have focused on their potential as biomarkers for tumor diagnosis and treatment [42–44]. In our experiments, expression of miR-5088-5p was examined in plasma of mice, with a view to determining its potential as a diagnostic marker. Comparison of the levels of miR-5088-5p in each group revealed the most significant increase in IR-treated mice and decrease in mice co-treated with IR and miR-5088-5p inhibitor (Fig. 4D), supporting its clinical applicability as a biomarker for cancer metastasis. Thus, inhibition of miR-5088-5p presents an effective strategy to suppress metastasis due to radiation resistance and may be concomitantly utilized to improve the efficiency of radiotherapy.

3.4 The miR-5088-5p inhibitor reduces resistance to IR and drug therapy

Since surgery, radiotherapy, and chemotherapeutic drugs constitute common cancer treatments, the effects of miR-5088-5p inhibitor on cancer cell sensitivity to radiation and anticancer drug cisplatin were examined via colony formation and methylthiazole tetrazolium (MTT) analyses, respectively. In H460 and MDA-MB-231 cells transfected with the miR-5088-5p inhibitor, colony forming ability (Supplementary Fig. S6A) and cancer cell proliferation (Supplementary Fig. S6B) were reduced compared to the corresponding control group. Additionally, the proliferation rate was reduced in cells co-treated with miR-5088-5p inhibitor and radiation/cisplatin relative to those exposed to radiation/anticancer drug alone (Supplementary Fig. S6B and S6C). Our findings clearly suggest that miR-5088-5p inhibitor effectively promotes cancer cell death by increasing sensitivity to radiation and chemotherapy drugs.

3.5 The miR-5088-5p inhibitor attenuates migratory ability, invasiveness, and stemness maintenance via suppression of Slug

In order to discover a major factor involved in the suppression mechanism of miR-5088-5p inhibitors against IR-induced malignancy, mRNA expression of malignancy-related factors was compared using siRNA of Slug, Snail, and Zeb1 (Fig. 2A), which are transcription factors for EMT markers increased by IR. As a result, siRNA against Slug were most effectively reduced the mRNA expression of Vimentin and N-cadherin as EMT markers, CD44 as a stemness marker, VEGF as an angiogenesis marker, and MMP9 as an invasion-related marker MMP-9 (Supplementary Fig. S7). Therefore, it was expected that Slug acts as a key factor in the inhibitory mechanism of IR-induced malignancy by miR-5088-5p inhibitor.

In addition, data from the available public dataset showed high expression of Slug in tumor tissues from lung and breast cancer patients relative to their normal tissue counterparts (Fig. 5A). To investigate the effect of Slug on IR-induced malignancy, we examined the expression of mesenchymal marker proteins, migration, invasion, and sphere formation assay using siRNA against Slug. As a result, IR-induced EMT markers such as Zeb1, Snail, Twist, Vimentin and N-cadherin were reduced by Slug siRNA (Fig. 5B). In addition, IR-induced increased migration, invasion, and sphere formation ability were all reduced by siSlug (Fig. 5C-E). These results showed that reduction of Slug decreased IR-induced malignancy such as migration, invasion, stemness, and expression of theirs related factors.

Next, we tested the involvement of Slug in the inhibitory mechanism of IR-induced malignancy by miR-5088-5p inhibitor using miR-5088-5p inhibitor and Slug overexpressing vector. As a result, the miR-5088-5p inhibitor effectively reduced Slug-induced mesenchymal trait (Fig. 6A), migration capacity (Fig. 6B), invasiveness (Fig. 6C), the expression of angiogenesis-related factors, VEGF and Ang2 (Fig. 6D), sphere formation ability (Fig. 6E), and mRNA levels of the cancer stem-like cell markers Oct4, Nanog, Sox2, CD133, and CD44 (Fig. 6F). Accordingly, we propose that the miR-5088-5p inhibitor decreases IR-induced tumorigenicity through suppression of the miR-5088-5p-Slug signaling pathway (Fig. 7).

Ionizing radiation (IR) is a commonly used effective therapeutic tool for cancer [45, 46]. However, cancer cells that survive after radiotherapy can develop malignant characteristics, including EMT and stemness [47–50], promoting tumor recurrence, and metastasis [47, 51, 52]. Therefore, considerable research efforts to reduce the side-effects triggered by radiation are underway to enhance treatment efficiency. Here, we focused on miRNAs as a tool to understand the mechanisms underlying radiation-induced malignancy and optimize the therapeutic effects of radiation.

MiRNA regulate the post-transcriptional expression of target mRNA molecules and are involved in various biological processes and diseases [42–44]. Since miRNAs are differentially expressed in various diseases and targets multiple mRNAs, their utility as novel therapeutic targets is a hot topic of interest [53]. At present, small-interfering RNA (siRNA) approved by the FDA in 2018 as a therapeutic small RNA molecules [54, 55] and miRNAs (including miR-34 (MRX34), miR-92 (MRG 110), miR-16 (MesomiR-1), miR-122 (Miravirsen), miR-29 (MRG-201), miR-21 (RG-012), and miR-155 (Cobomarsen-MRG-106)) are in clinical trials for various diseases [55, 56]. Studies are further underway to transform miRNA into stable forms for use as therapeutic agents for diseases [57, 58].0

Previously, we showed that miR-5088-5p is more highly expressed in breast cancer patients and increases malignancy and metastasis via downregulating target DBC2 (Deleted in Breast Cancer 2; RHOBTB2) [32]. Since overexpression of miR-5088-5p has a positive relationship with tumorigenicity and metastasis in breast cancer patients, the expression pattern of miR-5088-5p in plasma showed potential as a target for diagnosing tumor malignancy. In this current study, a correlation between radiation-induced malignancy and overexpression of miR-5088-5p as an onco-miRNA in lung and breast cancer was established. The expression levels of miR-5088-5p were increased in both animal models (Fig. 1B) and cells (Fig. 1C) as well as breast and lung cancer patients subjected to radiotherapy (Fig. 1A, S1) while levels of target DBC2 were relatively decreased (Supplementary Fig. S2), supporting our earlier findings.

Additionally, to analyze whether change in miR-5088-5p expression under conditions of radiation occur as a result of epigenetic modification, methylation of the miR-5088-5p promoter was assessed. IR induced hypomethylation of the miR-5088-5p promoter through suppression of DNMT1, DNMT3a, and DNMT3b in vivo and in vitro (Fig. 1D-F, S3-4), consistent with earlier reports that radiation induces DNA hypomethylation [32].

To elucidate the specific mechanisms underlying upregulation of miR-5088-5p upon radiation treatment in lung and breast cancer cells, a miR-5088-5p inhibitor was used. At the cellular level, miR-5088-5p inhibitor reduced IR-induced EMT, migration, invasion, angiogenesis, and stemness maintenance (Fig. 2–3), and increased sensitivity to radiation and cisplatin, thereby decreasing cell viability (Supplementary Fig. S6). Additionally, in an experimental animal model, IR-induced lung metastasis was reduced by miR-5088-5p inhibitor (Fig. 4), supporting the results at the cellular level (Fig. 2–3). Taken together, this study showed the potential as a new targeted therapeutic agent that can increase the efficiency of radiation therapy when the miR-5088-5p inhibitor is used in combination with radiation therapy.

IR, ionizing radiation; miRNA, microRNAs; DBC2, deleted in breast cancer 2; DNMT, DNA methyl transferases; MSP, methylation-specific PCR; VEGF, vascular endothelial growth factor; Ang, angiopoietin; CSC, cancer stem-like cell; EMT, epithelial-mesenchymal transition; H&E, hematoxylin and eosin; MTT, methylthiazole tetrazolium; siRNA, small-interfering RNA

Ethics approval and consent to participate All animal studies were reviewed and approved by the Animal Experimentation Committee (IACUC) of the Korea Institute of Radiological & Medical Sciences. All animals were monitored to minimize pain and euthanized after anesthesia to minimize pain.

Human Ethics The patient samples used in this study were provided by the Radiation Biobank (KRB) of Korea Cancer Center Hospital in Korea Institute of Radiological & Medical Sciences (KIRAMS). All samples used in this experiment were approved by the Institutional Review Board (IRB) of KIRAMS (K-1907-002-002, KIRAMS 2021-04-002-001).

Consent for publication Not applicable.

Availability of supporting data Data information generated or analyzed during this study, including resources, is included in the article.

Competing interests The authors declare no conflict of interest.

Funding This study was supported by grants of the National Research Foundation of Korea and the Korea Institute of Radiological & Medical Sciences (KIRAMS), funded by Ministry of Science and ICT (MSIT) in Republic of Korea [No. NRF-2021R1A2C2005966(50698-2022), NRF-2017M2A2A7A01018542(50035-2019), and 50531-2022].

Author contributions I.H.B. supervised the work; I.H.B. and H.J.S. designed the experiments and drafted the manuscript. I.H.B., H.J.S., and J.Y.C. designed and performed animal experiments. I.H.B., H.J.S., and J.M.Y. designed and performed MSP and qMSP. All authors discussed the results and commented on the manuscript.

Acknowledgements Not applicable.

R. Baskar, K.A. Lee, R. Yeo, K.W. Yeoh, Cancer and radiation therapy: current advances and future directions. Int. J. Med. Sci. 9, 193–199 (2012)
J.B. Little, Radiation Carcinog. Carcinog. 21, 397–404 (2000)
H. Inano, M. Onoda, Role of nitric oxide in radiation-induced initiation of mammary tumorigenesis in rats. Nitric Oxide 8, 144–148 (2003)
C. Boltze, G. Brabant, H. Dralle, R. Gerlach, A. Roessner, C. Hoang-Vu, Radiation-induced thyroid carcinogenesis as a function of time and dietary iodine supply:. an in vivo model of tumorigenesis in the rat Endocrinology 143, 2584–2592 (2002)
S. Braunstein, J.L. Nakamura, Radiotherapy-induced malignancies: review of clinical features, pathobiology, and evolving approaches for mitigating risk. Front. Oncol. 3, 73 (2013)
C.B. Dracham, A. Shankar, R. Madan, Radiation induced secondary malignancies: a review article. Radiat. Oncol. J. 36, 85–94 (2018)
T. Grantzau, L. Mellemkjaer, J. Overgaard, Second primary cancers after adjuvant radiotherapy in early breast cancer patients: a national population based study under the Danish Breast Cancer Cooperative Group (DBCG). Radiother Oncol. 106, 42–49 (2013)
D.J. Brenner, R.E. Curtis, E.J. Hall, E. Ron, Second malignancies in prostate carcinoma patients after radiotherapy compared with surgery Cancer 88, 398–406 (2000)
A.K. Chaturvedi, E.A. Engels, E.S. Gilbert, B.E. Chen, H. Storm, C.F. Lynch, P. Hall, F. Langmark, E. Pukkala, M. Kaijser, Second cancers among 104760 survivors of cervical cancer: evaluation of long-term risk. J. Natl Cancer Inst. 99, 1634–1643 (2007)
M.M. O'Brien, S.S. Donaldson, R.R. Balise, A.S. Whittemore, M.P. Link, Second malignant neoplasms in survivors of pediatric Hodgkin's lymphoma treated with low-dose radiation and chemotherapy. J. Clin. Oncol. 28, 1232–1239 (2010)
R.C. Reulen, C. Frobisher, D.L. Winter, J. Kelly, E.R. Lancashire, C.A. Stiller, K. Pritchard-Jones, H.C. Jenkinson, M.M. Hawkins and G. British Childhood Cancer Survivor Study Steering, Long-term risks of subsequent primary neoplasms among survivors of childhood cancer JAMA 305, 2311–2319 (2011)
C. Oliai, L.X. Yang, Radioprotectants to reduce the risk of radiation-induced carcinogenesis. Int. J. Radiat. Biol. 90, 203–213 (2014)
H. Zhu, G.C. Fan, Extracellular/circulating microRNAs and their potential role in cardiovascular disease. Am. J. Cardiovasc. Dis. 1, 138–149 (2011)
J.R. Czochor, P.M. Glazer, microRNAs in cancer cell response to ionizing radiation Antioxid Redox Signal 21, 293–312 (2014)
M.A. Chaudhry, R.A. Omaruddin, Differential regulation of microRNA expression in irradiated and bystander cells. Mol. Biol. (Mosk) 46, 634–643 (2012)
M.A. Chaudhry, R.A. Omaruddin, C.D. Brumbaugh, M.A. Tariq, N. Pourmand, Identification of radiation-induced microRNA transcriptome by next-generation massively parallel sequencing. J. Radiat. Res. 54, 808–822 (2013)
H.J. Cha, S. Shin, H. Yoo, E.M. Lee, S. Bae, K.H. Yang, S.J. Lee, I.C. Park, Y.W. Jin, S. An, Identification of ionizing radiation-responsive microRNAs in the IM9 human B lymphoblastic cell line. Int. J. Oncol. 34, 1661–1668 (2009)
J.E. Kwon, B.Y. Kim, S.Y. Kwak, I.H. Bae, Y.H. Han, Ionizing radiation-inducible microRNA miR-193a-3p induces apoptosis by directly targeting. Mcl-1 Apoptosis 18, 896–909 (2013)
S. Shin, H.J. Cha, E.M. Lee, S.J. Lee, S.K. Seo, H.O. Jin, I.C. Park, Y.W. Jin, S. An, Alteration of miRNA profiles by ionizing radiation in A549 human non-small cell lung cancer cells. Int. J. Oncol. 35, 81–86 (2009)
C. Liu, B. Li, Y. Cheng, J. Lin, J. Hao, S. Zhang, R.E. Mitchel, D. Sun, J. Ni, L. Zhao, F. Gao, J. Cai, MiR-21 plays an important role in radiation induced carcinogenesis in BALB/c mice by directly targeting the tumor suppressor gene Big-h3. Int. J. Biol. Sci. 7, 347–363 (2011)
H. Zhao, Y. Cheng, S. Dong, J. Du, F. Gao, D. Sun, J. Cui, J. Ni, J. Cai, Down regulation of miR-143 promotes radiation - Induced thymic lymphoma by targeting B7H1. Toxicol. Lett. 280, 116–124 (2017)
J. Manikandan, J.J. Aarthi, S.D. Kumar, P.N. Pushparaj, Oncomirs: the potential role of non-coding microRNAs. Underst. cancer Bioinformation 2, 330–334 (2008)
S.M. Hammond, MicroRNAs as tumor suppressors Nature genetics 39, 582–583 (2007)
T. Aggarwal, R. Wadhwa, R. Gupta, T. Collet, D.K. Chellapan, G. Gupta, H. Perumalsamy, M. Mehta, S. Satija, P.M. Hansbro, K. Dua, P.K. Maurya, MicroRNAs as Biomarker for Breast Cancer Endocr Metab Immune Disord Drug Targets, (2020)
H. Zhang, B. Lu, microRNAs as biomarkers of ovarian cancer Expert Rev Anticancer Ther, 1–13 (2020)
O. Rapado-Gonzalez, A. Alvarez-Castro, R. Lopez-Lopez, J. Iglesias-Canle, M.M. Suarez-Cunqueiro, L. Muinelo-Romay, Circulating microRNAs as Promising Biomarkers in Colorectal Cancer Cancers (Basel) 11, (2019)
D. Ma, X. Tao, F. Gao, C. Fan, D. Wu, miR-224 functions as an onco-miRNA in hepatocellular carcinoma cells by activating. AKT Signal. Oncol. Lett. 4, 483–488 (2012)
T. Hirono, K. Jingushi, T. Nagata, M. Sato, K. Minami, M. Aoki, A.H. Takeda, T. Umehara, H. Egawa, Y. Nakatsuji, K. Kitae, Y. Ueda, H. Hase, M. Yamamoto, Y. Shinsato, K. Kawahara, T. Furukawa, K. Tsujikawa, MicroRNA-130b functions as an oncomiRNA in non-small cell lung cancer by targeting tissue inhibitor of metalloproteinase-2. Sci. Rep. 9, 6956 (2019)
W. Xiao, N. Lou, H. Ruan, L. Bao, Z. Xiong, C. Yuan, J. Tong, G. Xu, Y. Zhou, Y. Qu, W. Hu, Y. Gao, Z. Ru, L. Liu, H. Xiao, K. Chen, H. Yang, X. Zhang, Mir-144-3p Promotes Cell Proliferation, Metastasis, Sunitinib Resistance in Clear Cell Renal Cell Carcinoma by Downregulating ARID1A Cell. Physiol. Biochem. 43, 2420–2433 (2017)
H.-S. Gwak, T.H. Kim, G.H. Jo, Y.-J. Kim, H.-J. Kwak, J.H. Kim, J. Yin, H. Yoo, S.H. Lee, J.B. Park, Silencing of microRNA-21 confers radio-sensitivity through inhibition of the PI3K/AKT pathway and enhancing autophagy in malignant glioma cell lines, (2012)
A.L. Teixeira, F. Dias, M. Gomes, M. Fernandes, R. Medeiros, Circulating biomarkers in renal cell carcinoma: the link between microRNAs and extracellular vesicles, where are we now? J. Kidney Cancer VHL 1, 84–98 (2014)
H.J. Seok, Y.E. Choi, J.Y. Choi, J.M. Yi, E.J. Kim, M.Y. Choi, S.-J. Lee, I.H. Bae, Novel miR-5088-5p promotes malignancy of breast cancer by inhibiting DBC2 Molecular (Therapy-Nucleic Acids, 2021)
E.S. Kim, Y.E. Choi, S.J. Hwang, Y.H. Han, M.J. Park, I.H. Bae, IL-4, a direct target of miR-340/429, is involved in radiation-induced aggressive tumor behavior. Hum. carcinoma cells Oncotarget 7, 86836–86856 (2016)
J.G. Herman, J.R. Graff, S. Myohanen, B.D. Nelkin, S.B. Baylin, Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. U S A 93, 9821–9826 (1996)
J.M. Yi, E.J. Kang, H.M. Kwon, J.H. Bae, K. Kang, N. Ahuja, K. Yang, Epigenetically altered miR-1247 functions as a tumor suppressor. Pancreat. cancer Oncotarget 8, 26600–26612 (2017)
D.A. Antwih, K.M. Gabbara, W.D. Lancaster, D.M. Ruden, S.P. Zielske, Radiation-induced epigenetic DNA methylation modification of radiation-response pathways Epigenetics 8, 839–848 (2013)
B. Firatligil-Yildirir, G. Bati-Ayaz, I. Tahmaz, M. Bilgen, D. Pesen-Okvur, and O. Yalcin-Ozuysal, Homing Choices of Breast Cancer Cells Revealed by Tissue Specific Invasion (and Extravasation Lab-on-a-chip Platforms bioRxiv, 2020)
S. Filipów, Ł Łaczmański, Blood circulating miRNAs as cancer biomarkers for diagnosis and surgical treatment response Frontiers in genetics 10, (2019)
K. Alhallak, S.V. Jenkins, D.E. Lee, N.P. Greene, K.P. Quinn, R.J. Griffin, R.P.M. Dings, N. Rajaram, Optical imaging of radiation-induced metabolic changes in radiation-sensitive and resistant cancer cells. J. Biomed. Opt. 22, 60502 (2017)
K.C. Yoo, J.H. Kang, M.Y. Choi, Y. Suh, Y. Zhao, M.J. Kim, J.H. Chang, J.K. Shim, S.J. Yoon, S.G. Kang, Soluble ICAM-1 a Pivotal Communicator between Tumors and Macrophages, 2102768 (Promotes Mesenchymal Shift of Glioblastoma Advanced Science, 2021)
L. Rivina, R. Schiestl, Mouse models of radiation-induced cancers Advances in genetics 84, 83–122 (2013)
L. Jiang, J. Zhu, Review of MiRNA-Disease Association (prediction Curr Protein Pept Sci, 2020)
T. Powrozek, T. Malecka-Massalska, MiRNA and lung cancer radiosensitivity: a mini-review. Eur. Rev. Med. Pharmacol. Sci. 23, 8422–8428 (2019)
S. Swarbrick, N. Wragg, S. Ghosh, A. Stolzing, Systematic Review of miRNA as Biomarkers in Alzheimer's. Disease Mol. Neurobiol. 56, 6156–6167 (2019)
H. Bartelink, J.C. Horiot, P. Poortmans, H. Struikmans, W. Van den Bogaert, I. Barillot, A. Fourquet, J. Borger, J. Jager, W. Hoogenraad, L. Collette, M. Pierart, R. European Organization for, R. Treatment of Cancer and G. Breast Cancer, Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation. N Engl. J. Med. 345, 1378–1387 (2001)
J. Bernier, E.J. Hall, A. Giaccia, Radiation oncology: a century of achievements. Nat. Rev. Cancer 4, 737–747 (2004)
C. Moncharmont, A. Levy, J.B. Guy, A.T. Falk, M. Guilbert, J.C. Trone, G. Alphonse, M. Gilormini, D. Ardail, R.A. Toillon, C. Rodriguez-Lafrasse, N. Magne, Radiation-enhanced cell migration/invasion process: a review. Crit. Rev. Oncol. Hematol. 92, 133–142 (2014)
I. Madani, W. De Neve, M. Mareel, Does ionizing radiation stimulate cancer invasion and metastasis? Bull Cancer 95, 292–300 (2008)
C.M. Park, M.J. Park, H.J. Kwak, H.C. Lee, M.S. Kim, S.H. Lee, I.C. Park, C.H. Rhee, S.I. Hong, Ionizing radiation enhances matrix metalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidylinositol 3-kinase/Akt signaling pathways. Cancer Res. 66, 8511–8519 (2006)
Y.C. Zhou, J.Y. Liu, J. Li, J. Zhang, Y.Q. Xu, H.W. Zhang, L.B. Qiu, G.R. Ding, X.M. Su, S. Mei, G.Z. Guo, Ionizing radiation promotes migration and invasion of cancer cells through transforming growth factor-beta-mediated epithelial-mesenchymal transition. Int. J. Radiat. Oncol. Biol. Phys. 81, 1530–1537 (2011)
C.F. von Essen, Radiation enhancement of metastasis: a review. Clin. Exp. Metastasis 9, 77–104 (1991)
C. Wild-Bode, M. Weller, A. Rimner, J. Dichgans, W. Wick, Sublethal irradiation promotes migration and invasiveness of glioma cells: implications for radiotherapy of human glioblastoma. Cancer Res. 61, 2744–2750 (2001)
A.F. Christopher, R.P. Kaur, G. Kaur, A. Kaur, V. Gupta, and P. Bansal, MicroRNA therapeutics: Discovering novel targets and developing specific therapy. Perspect. Clin. Res. 7, 68–74 (2016)
A.V. Kristen, S. Ajroud-Driss, I. Conceicao, P. Gorevic, T. Kyriakides, L. Obici, Patisiran, an RNAi therapeutic for the treatment of hereditary transthyretin-mediated amyloidosis. Neurodegener Dis. Manag 9, 5–23 (2019)
J. Hanna, G.S. Hossain, J. Kocerha, The Potential for microRNA. Ther. Clin. Res. Front Genet 10, 478 (2019)
C. Chakraborty, A.R. Sharma, G. Sharma, C.G.P. Doss, S.S. Lee, Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine Mol. Ther. Nucleic Acids 8, 132–143 (2017)
S.H. Hsu, B. Yu, X. Wang, Y. Lu, C.R. Schmidt, R.J. Lee, L.J. Lee, S.T. Jacob, K. Ghoshal, Cationic lipid nanoparticles for therapeutic delivery of siRNA and miRNA to murine liver tumor Nanomedicine 9, 1169–1180 (2013)
Y. Fu, J. Chen, Z. Huang, Recent progress in microRNA-based delivery systems for the treatment of human disease ExRNA 1, 1–14 (2019)

No competing interests reported.

SupplementalFigure202207143.pptx

Download PDF

Version 1

posted

You are reading this latest preprint version

miR-5088-5p inhibitor reduces radiation-induced malignancy by decreasing Slug

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusions

Figures

1 Introduction

2 Materials And Methods

2.1 Cell Cultures

2.2 RNA Extraction and qRT-PCR

2.3 Western blot analysis

2.4 Irradiation

2.5 Plasmid DNA, RNA oligoribonucleotides, and transfection

2.6 Wound healing assay

2.7 Transwell invasion assay

2.8 Tube formation assay

2.9 Sphere formation assay

2.10 In vivo promoter methylation analysis

2.11 Pyrosequencing analysis

2.12 Methylation specific PCR (MSP) and quantitative methylation-specific PCR (qMSP) analysis

2.13 In vivo metastasis assay

2.14 Colony formation assay

2.15 MTT assay

2.16 Clinical specimens

2.17 Statistical Analysis

3 Results

3.1 IR upregulates biogenesis of miR-5088-5p through inhibiting methylation

3.2 The miR-5088-5p inhibitor suppress IR-induced tumorigenic properties

3.3 The miR-5088-5p inhibitor reduces IR-induced pulmonary metastasis

3.4 The miR-5088-5p inhibitor reduces resistance to IR and drug therapy

3.5 The miR-5088-5p inhibitor attenuates migratory ability, invasiveness, and stemness maintenance via suppression of Slug

4 Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1