MicroRNA-15a-5p suppresses hypoxia-induced tumor growth and chemoresistance in bladder cancer by binding to eIF5A2

doi:10.21203/rs.3.rs-2870524/v1

Download PDF

Research Article

MicroRNA-15a-5p suppresses hypoxia-induced tumor growth and chemoresistance in bladder cancer by binding to eIF5A2

https://doi.org/10.21203/rs.3.rs-2870524/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

In various malignant tumors (including bladder cancer) poor prognosis is associated with hypoxia and therapeutic resistance. Evidence indicates that in bladder cancer, microRNAs (miRNAs) have vital functions in acquired drug resistance. However, the involvement of miRNAs in hypoxia-mediated bladder cancer doxorubicin (Dox) resistance is unknown. Herein, we showed that hypoxia and Dox treatment downregulated miR-15a-5p expression.

Methods and Results

Using UM-UC-3 and J82 bladder cancer cell lines, and in vivo mouse models of bladder cancer, we confirmed that miR-15a-5p arrests tumor cell growth and Dox resistance in vitro and in vivo. Furthermore, we determined the interaction between miR-15a-5p and eukaryotic translation initiation factor 5A-2 (eIF5A2) using dual luciferase reporters and quantitative real-time reverse transcription polymerase chain reaction assays. We also showed that an miR-15a-5p agomir repressed EIF5A2 expression in bladder cancer cells, thereby inhibiting the epithelial-mesenchymal transition (EMT) induced by Dox or hypoxia. Moreover, ectopic expression of miR-15a-5p abrogated eIF5A2-mediated Dox resistance in bladder cancer cells. Collectively, these data indicated that hypoxia promotes tumor growth and chemoresistance through the HIF-1α/miR-15a-5p/eIFTA2/EMT pathway.

Conclusion

This new finding not only has implications for improving our understanding of the Dox resistance process during bladder cancer progression, but also indicated that the miR-15a-5p agomir is a promising tool to prevent Dox resistance in patients with bladder cancer.

microRNA-15a-5p

bladder cancer

doxorubicin resistance

eIF5A2

EMT

Bladder cancer is a heterogeneous malignant tumor with numerous histological subtypes that can originate from anywhere in the urinary tract, with the majority of tumors starting from the epithelial cells in the bladder lining (urethral epithelium). According to the latest GLOBOCAN data, 3% of all cancers and 2.1% of cancer deaths are attributed to bladder cancer worldwide (1, 2). Moreover, patients with bladder cancer have a 70% 5-year survival rate, whereas in those with metastasis, the 5-year survival rate is only 5% (1, 2). Doxorubicin (Dox) is a commonly used chemotherapy for bladder cancer; however, the development of drug resistance markedly limits its long-term curative effects.(3) Hypoxia is characterized by low oxygen and has critical roles in the angiogenesis, metastasis, and drug resistance in most solid tumors, including bladder cancer (4–7). The bladder carbogen and nicotinamide (BCON) phase III clinical trial showed that hypoxia modification increased the survival of bladder cancer patients expressing high levels of hypoxia-related genes(8). Targeting hypoxia-mediated tumor metabolism is therefore a promising strategy to treat bladder cancer.

Hypoxia can regulate all the steps of metastasis, from the initial epithelial–mesenchymal transition (EMT) to the final organotropic colonization; therefore, it has been suggested that hypoxia acts as a master regulator in metastasis (9). Previous studies have demonstrated that a hypoxic environment could induce EMT during bladder cancer progression (10). In addition, EMT participates in the promotion of Dox resistance in bladder cancer cells.(11) Therefore, chronic treatment with Dox induces hypoxia, which leads to EMT, causing Dox resistance in bladder cancer. However, the molecular mechanisms that link hypoxia and EMT-induced Dox resistance in bladder cancer remain elusive.

Recently, studies have reported that certain microRNAs (miRNAs) are directly regulated by hypoxia, participating in hypoxia-mediated survival signaling pathway (12, 13). MiRNAs (small, endogenous, and non-coding RNAs) are usually 20–22 nucleotides long. miRNAs regulate gene expression negatively at the post-transcriptional level by binding to their target mRNA via complementary sequences. miRNAs participate in many cancer physiological and pathological processes, such as radioresistance, apoptosis, infiltration, proliferation, differentiation, and chemoresistance(14, 15). In this study, we discovered that 20 miRNAs are downregulated by hypoxia in bladder cancer. Among them, miR-15a-5p was also reduced when bladder cancer cells were treated with Dox. Furthermore, we found that miR-15a-5p can improve hypoxia-induced Dox resistance and inhibit tumor growth in bladder cancer by inhibiting EMT. Therefore, we believe that in bladder cancer, miR-15a-5p has a vital function in the development of Dox sensitivity. Our findings providing new insights into therapeutic targets for enhancing Dox's clinical efficacy to treat patients with bladder cancer .

Cell culture

The ATCC (Rockville, MD, USA) provided the bladder cancer cell lines UM-UC-3 and J82. RMPI 1640 medium (Lonza, Switzerland) containing fetal bovine serum (10% (v/v)), penicillin (1%, Gibco, Grand Island, NY, USA), and streptomycin (1%; Gibco) was used to culture the cells under standard conditions. Dox was obtained from SigmaAldrich (Merck, Darmstadt, Germany) provided Dox, which was diluted in dimethyl sulfoxide.

Cell transfection

Cells (2 ×10⁵) were plated evenly on each well of a 6-well plate and allowed to attach to the bottom of the well. Then, we mixed the short interfering RNA (siRNA), miRNA mimics, or their inhibitors with Lipofectamine 2000, and then the mixture was added to cells grown in serum-free medium. Six hours later, the medium was replaced with normal medium for subsequent experiments. Ribobio (Guangzhou, China), Fulengen (Guangzhou, China), and Thermo Scientific (Waltham, MA, USA) provided the above reagents.

The miR-15a-5p mimics comprised: 5ʹ- UAGCAGCACAUAAUGGUUUGUG-3ʹ;

5ʹ- CAAACCAUUAUGUGCUGCUAUU-3ʹ;

The miR-15a-5p inhibitor comprised: 5ʹ- CACAAACCAUUAUGUGCUGCUA-3ʹ;

The negative control comprised: 5ʹ-CAGUACUUUUGUGUAGUACAA-3ʹ.

Cell Counting Kit-8 (CCK-8) assay

The CCK-8 assay for cell viability was carried out following the procedures detailed by Wang et al. (16).

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

The expression levels of mRNAs in bladder cancer cells was assessed using qRT-PCR according to the procedures detailed by Wang et al.^ref. The following primers were used:

EIF5A2 (encoding eukaryotic translation initiation factor 5A-2):

Forward: 5'-TATGCAGTGCTCGGCCTTG-3';

Reverse: 5'-TTGGAACATCCATGTTGTGAGTAGA-3';

CDH1 (encoding E-cadherin):

Forward: 5'-TACACTGCCCAGGAGCCAGA-3';

Reverse: 5'-TGGCACCAGTGTCCGGATTA-3';

VIM (encoding Vimentin):

Forward: 5'-TGAGTACCGGAGACAGGTGCAG-3';

Reverse: 5'-TAGCAGCTTCAACGGCAAAGTTC-3';

miR-15a-5p: 5'-GTGTTTGGTAATACACGACGAT − 3'.

Dual luciferase reporter gene assay

J82 cells were co-transfected with negative control mimics or miR-15a-5p mimics and wildtype (WT) (pGL3-WT-EIF5A2-3' untranslated region [UTR]) or mutant (mut) (pGL3-mut-EIF5A2-3' UTR) reporter plasmids (GenePharma, Shanghai, China). The cells were incubated with the transfection mixtures for 48 h, collected by centrifugation, and lysed. The luciferase assays were then carried out following the instructions of the luciferase assay kit (Biovision, Milpitas, CA, USA) and the luciferase reporter assay system (Promega, Madison, WI, USA). The ratio of firefly luciferase activity to renilla luciferase activity determined the relative luciferase activity. The WT-EIF5A2-3' UTR and mut-EIF5A2-3' UTR were 5′-AAAATTATTAATCCGTGCTGCTT-3′ and 5′AAAATTATTAATCCGACGACGAT-3′, respectively.

Western blotting

Western blotting was carried out following the procedures detailed by Wang et al.^ref. Primary antibodies against the following proteins were used: eIF5A2 (1:1000; Abcam, Cambridge, MA, USA), HIF-1α (1:1000; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000; Abcam). Goat anti-rabbit IgG comprised the secondary antibody (1:2000, Abcam).

Tumor xenograft study

The Experimental Animal Center of Sun Yat-sen University (Guangzhou, China) provided BALB/c nude mice (male, 3–4 weeks old). Cells were collected by centrifugation and suspended in serum-free medium at 1 × 10⁷ cells/0.2 ml. UM-UC-3 cells were inoculated subcutaneously into the right flank of each mouse. Every 2 days, the tumor size was determined and mice were humanely killed after 4 weeks. For the in vivo chemosensitivity assays, Dox or saline were delivered to the mice via intraperitoneal injection (2mg/kg body weight Dox [once every 2 days]). MiR-15a-5p Angomir treatment was achieved via intratumor injection (30 µl per mouse [once every 3 days]). All animal experiments were approved by the Ethics Committee of Tongde Hospital of Zhejiang province in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Publications, No. 8023, revised 1978). We confirmed that all methods were performed in accordance with ARRIVE guidelines.

TUNEL and immunohistochemistry staining

Formalin-fixed, paraffin-embedded tumor specimens were sliced into 4 µm thick sections and mounted on glass slides. Terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL) (Roche, Basel, Switzerland) and Ki-67 (Santa Cruz, Dallas, TX, USA) staining were performed on three independent tumors from each group. The ratio of TUNEL- or Ki-67-positive nuclei among total nuclei was used to express the results. Image J (for windows, NIH, Bethesda, MD, USA) was used to carry out the quantitative analysis of the positive cells.

Statistical analyses

SPSS v18.0 (IBM, Armonk, NY, USA) was used to carry out all the statistical analyses. The mean ± the standard error of the mean (SEM) was used to present all the data. Differences between two groups were analyzed using two-tailed Student's t-tests. Oneway analysis of variance (ANOVA) was used to determine the statistically significant differences among multiple samples. Statistical significance was indicated by a P-value < 0.05.

MiR-15a-5p is transcriptionally inhibited by HIF-1α under hypoxia

Hypoxia, a salient feature of advanced stage bladder cancer, correlates with poor prognosis(17). To identify the miRNA signature regulated by hypoxia, we evaluated the expression of 20 different drug resistance-related miRNAs in J82 bladder cancer cells treated with hypoxia. The majority of the selected miRNAs were downregulated by hypoxia treatment (left panel of Fig. 1A). We then measured the expression of these 20 miRNAs in UM-UC-3 bladder cancer cells treated with Dox and found that miR-15a-5p expression was significantly reduced under both Dox and hypoxic conditions (Fig. 1A). Next, we explored whether transcription of miR-15a-5p is regulated by hypoxiainducible factor (HIF). The HIF-1α levels were greatly increased in UM-UC-3 and J82 cells under hypoxic conditions (Fig. 1B). Reducing HIF-1α levels using HIF-1αspecific siRNAs partially recovered the levels of miR-15a-5p after hypoxia (Fig. 1C). These results suggest that hypoxia, which is involved in the progression and chemoresistance of bladder cancer, activates HIF-1α, leading to miR-15a-5p downregulation.

MiR-15a-5p suppress tumor growth in bladder cancer

Previous studies have suggested that hypoxia plays major roles in bladder cancer chemoresistance, angiogenesis, and progression(18, 19). We first investigated whether miR-15a-5p is involved in tumor growth in bladder cancer. The expression of miR-15a-5p was evaluated using the online prediction software StarBase (version 3.0), which indicated high expression of miR-15a-5p in bladder cancer (Fig. 2A). A bladder cancer model was then established by inoculating 3 × 10⁶ cells (UM-UC-3) subcutaneously into athymic nude mice. The results showed that compared with those in the saline-treated control group, intratumoral administration of the miR-15a-5p angomir decreased the tumor sizes (Fig. 2B,C). Treatment with miR-15a-5p significantly slowed down tumor growth (Fig. 2D). We then examined cell proliferation using Ki-67 staining and apoptosis using TUNEL staining in the extracted tumor tissues. miR-15a-5p agomir treatment inhibited tumor cell proliferation and increased apoptosis (Fig. 2E-G). These results indicate that high miR-15a-5p expression arrests bladder cancer growth.

MiR-15a-5p enhances doxorubicin sensitivity of bladder cancer cells

Next, we examined the involvement of miR-15a-5p in hypoxia-mediated doxorubicin (Dox) resistance in UM-UC-3 and J82 cells. miR-15a-5p expression was highest in UM-UC-3 cells and lowest in JB2 cells (Fig. 3B). CCK-8 assays determined the viability of bladder cancer cells treated with different concentration of Dox for 48 hours. This result indicated that increased Dox concentrations reduced the viability of both cell lines in a dose-dependent manner, with different sensitivity to Dox in different bladder cancer cells (Fig. 3A).

To test whether miR-15a-5p suppresses Dox resistance, the effect on Dox resistance of miR-15a-5p expression was studied in the two bladder cancer cells: miR-15a-5p agomir transfection significantly enhanced miR-15a-5p levels (Fig. 3C), while transfection with the miR-15a-5p antagomir reduced miR-15a-5p levels in both cell lines (Fig. 3D). miR-15a-5p overexpression enhanced bladder cancer cell sensitivity to Dox (Fig. 3E). By contrast, miR-15a-5p downregulation using the miR-15a-5p antagomir enhanced Dox resistance in UM-UC-3 and J82 cells (Fig. 3F).

Next, the involvement of miR-15A-5p in hypoxia-mediated Dox resistance was investigated. In the two cell lines (J82 and UM-UC-3), hypoxic conditions (1% O₂) caused higher resistance to Dox compared with that under normal oxygen conditions (20% O₂) (Fig. 3G). miR-15a-5p upregulation using the miR-15a-5p agomir under hypoxia decreased Dox resistance to a similar level in both cell lines (Fig. 3H). These results suggested that hypoxia-induced Dox resistance is mediated through downregulation of miR-15a-5p and the miR-15a-5p agomir could raise the sensitivity of bladder cancer cells to Dox.

miR-15a-5p reverses the hypoxia-induced epithelial-mesenchymal transition

EMT is recognized as a critical regulator of chemoresistance in cancer; therefore, we explored whether in bladder cancer, miR-15a-5p suppresses Dox resistance by modulating the EMT process. Transfection of UM-UC-3 and J82cells with the miR-15a-5p agomir significantly elevated miR-15a-5p levels (Fig. 4A). Overexpression of miR-15a-5p in both cell lines decreased in the level of Vimentin (a mesenchymal markers) but increased the level of E-cadherin (an epithelial marker) at both the protein and mRNA levels (Fig. 4B-C), suggesting that miR-15a-5p can suppress EMT. Both Dox treatment and hypoxia facilitate EMT, as indicated by increased expression of Vimentin and decreased expression of E-cadherin (Fig. 4D). miR-15a-5p upregulation reversed the hypoxia and Dox-mediated changes in Vimentin and E-cadherin expression (Fig. 4D). Collectively, these results indicated that miR-15a-5p inhibits EMT of bladder cancer cells and reverses the increase in EMT caused by hypoxia and Dox treatment.

EIF5A2 is the downstream target of the miR-15a-5p

Previously, we showed that eIF5A2 promotes EMT and increases Dox resistance in bladder cancer cells (20). Consistent with our previous findings, in bladder cancer cells, overexpression of EIF5A2 enhanced Dox resistance, while EIF5A2 knockdown inhibited Dox resistance (Supplementary Fig. 1A-B). In addition, in bladder cancer cells, overexpression of EIF5A2 promoted EMT, while EIF5A2 knockdown inhibited EMT (Supplementary Fig. 1C-D). We further demonstrated that hypoxia increased eIF5A2 expression by 2- to 4-fold in J82 cells (Supplementary Fig. 2A). EIF5A2 knockdown inhibited hypoxia-induced EMT and thus rescued hypoxia-mediated Dox resistance (Supplementary Fig. 2B-C).

We next attempted to determine whether eIF5A2 lies downstream of miR-15a-5p during EMT regulation and Dox resistance. Using TargetScan, we found that there is a consensus-binding site for miR-15a-5p in the EIF5A2 mRNA (Fig. 5A). In addition, the expression of eIF5A2 correlated negatively with that of miR-15a-5p (Fig. 5A). Furthermore, eIF5A2 protein and mRNA levels were downregulated significantly in miR-15a-5p agomir-transfected UM-UC-3 and J82 cells compared those in cells transfected with the negative control miRNA (Fig. 5B-C). In addition, eIF5A2 protein and mRNA levels were markedly elevated in bladder cancer cells transfected with the miR-15a-5p antagomir (Fig. 5B-C).

Next, the effect of miR-15a-5p on eIF5A2 expression was assessed using the psiCKeIF5A2 luciferase reporter system. miR-15a-5p agomir co-transfection with the psiCK-wt-eIF5A2 reporter significantly downregulated the luciferase activity; however, the miR-15a-5p agomir had no effect on luciferase activity when it was co-transfected with the reporter vector expressing the mutated EIF5A2 sequence that cannot be recognized by miR-15a-5p (Fig. 5D-E). Thus, miR-15a-5p targets EIF5A2 and reduces its expression.

We then studied whether overexpression of miR-15-5p with eIF5A2 would affect bladder cancer cell Dox resistance. Transfection of the miR-15a-5p agomir into bladder cancer cells ameliorated eIF5A2-induced Dox resistance (Fig. 5F). The miR-15a-5p agomir only slightly reduced eIF5A2-induced Dox resistance in UM-UC-3 and J82 cells (Fig. 5F), which might have been caused by the high endogenous expression of eIF5A2 in these two cell liens (Fig. 5B). Collectively, these findings showed that miR-15a-5p might inhibit the hypoxia-induced Dox resistance by specifically downregulating EIF5A2 expression.

In the advanced stage of bladder cancer, hypoxia stimulates HIF-1αmediated signaling pathways, permitting cancer cell survival under hypoxic conditions by promoting tumor growth, metastasis, drug resistance, and radio resistance(21, 22). Generally, intratumoral hypoxia-induced HIF-1α is considered an oncoprotein (23). Experimental and clinical evidence supports the view that hypoxia adaptation contributes to chemotherapy resistance development in bladder cancer, and overexpression of HIF-1α is associated with poor patient prognosis(18, 24). Our findings not only provide further evidence supporting the key roles of hypoxia in bladder cancer progression and chemoresistance, but also revealed a new molecular mechanism by which hypoxia activates the miR-15a-5p/eIF5A2/EMT signaling pathway to facilitate tumor growth and Dox resistance in bladder cancer.

Recently, miR-15a-5p was identified as a cancer-associated miRNA that participates in cancer progression. For example, miR-15a-5p overexpression was reported to be oncogenic in hepatocellular carcinoma and renal cell carcinoma (25, 26). According to the microRNA target gene database StarBase, in bladder cancer, miR-15a-5p functions as an oncogene (Fig. 2A). However, in this paper, we found that the roles of miR-15a-5p in bladder cancer are opposite to previously reported findings. Our results revealed that miR-15a-5p expression was downregulated by both hypoxia and Dox treatment (Fig. 1). In bladder cancer, miR-15a-5p knockdown conferred in vivo and in vitro chemotherapeutic resistance, while the miR-15a-5p agomir decreased hypoxia-induced tumor growth and enhanced Dox resistance. Thus, our results indicated that miR-15a-5p is a therapeutic target to enhancing doxorubicin cytotoxicity in patients with bladder cancer.

MiRNAs exert their functions through repressing gene expression posttranscriptionally by binding to the 3′ UTR of their target mRNAs(27). Herein, we determined that EIF5A2 is the target of miR-15a-5p: miR-15a-5p binding to the EIF5A2 3′ UTR decreased its expression. Previously, high expression of eIF5A2 was reported in bladder cancer, which is capable of elevating Dox resistance (20); therefore, we speculated that miR-15a-5p-mediated downregulation of EIF5A2 would decrease Dox resistance in bladder cancer.

EMT plays pivotal roles in the induction of Dox resistance under hypoxia in bladder cancer (28, 29). In the present study, we found that rescue of miR-15a-5p expression in hypoxia-treated bladder cancer cells disturbed the EMT phenotype induced by hypoxia, i.e., increased HIF-1α and Vimentin (a mesenchymal marker) levels and decreased Ecadherin levels (an epithelial marker). Additionally, knockdown of EIF5A2 prevented the EMT phenotype, while high expression of EIF5A2 induced EMT, in hypoxic bladder cancer cells. Furthermore, the miR-15a-5p agomir inhibited eIF5A2-mediated EMT. Notably, EIF5A2 overexpression in UM-UC-3 and J82 cells only slightly increase Dox resistance (Fig. 5F), because the control cells also have high endogenous eIF5A2 expression (Fig. 5B). Consequently, miR-15a-5p cannot effectively repress EIF5A2 expression and reduce Dox resistance in these cell lines(Fig. 5F). Our results suggest that inhibiting the downregulation of miR-15a-5p in hypoxic tumor regions could reduce HIF signaling and EMT in bladder cancer.

Our findings identified miR-15a-5p as a new molecular player involved in hypoxiainduced tumor growth and Dox resistance in bladder cancer. We revealed that hypoxia, which occurs during tumor progression and chemoresistance, activates HIF-1α expression, resulting in downregulation of miR-15a-5p. This causes a reversal of miR-15a-5p-mediated inhibition of EIF5A2 expression and EMT, thereby promoting tumor growth and decreasing Dox sensitivity. Therefore, specific upregulation of miR-15a-5p could represent therapeutic strategy to ameliorate hypoxia-induced Dox resistance in the treatment of bladder cancer.

Ethics State

All animal experiments were approved by the Ethics Committee of Tongde Hospital of Zhejiang province in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Publications, No. 8023, revised 1978).

Consent for publication

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Funding

This study was supported by the Zhejiang Provincial Nature Science Foundation of China (LSY19H160003 and LY21H160033), and the National Natural Science Foundation of China (81802085).

Author contribution

Dajin Chen and Wei Chen designed the study and collected data. Jinsong Yang, Haoyi Mengjing Cheng and Xue Jiang did the experiments. Ying Chen, Lingyan Zheng, Shufen Zhang and Senxiang Yan analyzed the data. Dajin Chen wrote the manuscript. WC and JW reviewed and edited he manuscript. All authors read and approved the final manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Acknowledgments

We sincerely acknowledge all members in the Wei Chen laboratory for their technical assistance and helpful advices.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. Bladder Cancer Incidence and Mortality: A Global Overview and Recent Trends. European urology. 2017;71(1):96-108.
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2018;68(6):394-424.
Barocas DA, Clark PE. Bladder cancer. Current opinion in oncology. 2008;20(3):307-14.
de Heer EC, Jalving M, Harris AL. HIFs, angiogenesis, and metabolism: elusive enemies in breast cancer. The Journal of clinical investigation. 2020;130(10):5074-87.
Saxena K, Jolly MK, Balamurugan K. Hypoxia, partial EMT and collective migration: Emerging culprits in metastasis. Translational oncology. 2020;13(11):100845.
Erin N, Grahovac J, Brozovic A, Efferth T. Tumor microenvironment and epithelial mesenchymal transition as targets to overcome tumor multidrug resistance. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy. 2020;53:100715.
Tickoo SK, Milowsky MI, Dhar N, Dudas ME, Gallagher DJ, Al-Ahmadie H, et al. Hypoxia-inducible factor and mammalian target of rapamycin pathway markers in urothelial carcinoma of the bladder: possible therapeutic implications. BJU Int. 2011;107(5):844-9.
Song YP, Mistry H, Irlam J, Valentine H, Yang L, Lane B, et al. Long-Term Outcomes of Radical Radiation Therapy with Hypoxia Modification with Biomarker Discovery for Stratification: 10-Year Update of the BCON (Bladder Carbogen Nicotinamide) Phase 3 Randomized Trial (ISRCTN45938399). Int J Radiat Oncol Biol Phys. 2021;110(5):1407-15.
Lu X, Kang Y. Hypoxia and hypoxia-inducible factors: master regulators of metastasis. Clin Cancer Res. 2010;16(24):5928-35.
Lv W-L, Liu Q, An J-H, Song X-Y. Scutellarin inhibits hypoxia-induced epithelial-mesenchymal transition in bladder cancer cells. Journal of cellular physiology. 2019;234(12):23169-75.
Zareinejad M, Faghih Z, Ariafar A, Safaei A, Ghaderi A. Establishment of a bladder cancer cell line expressing both mesenchymal and epithelial lineage-associated markers. Human cell. 2021;34(2):675-87.
Costales MG, Haga CL, Velagapudi SP, Childs-Disney JL, Phinney DG, Disney MD. Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit. J Am Chem Soc. 2017;139(9):3446-55.
Shen G, Li X, Jia YF, Piazza GA, Xi Y. Hypoxia-regulated microRNAs in human cancer. Acta Pharmacol Sin. 2013;34(3):336-41.
Lee YS, Dutta A. MicroRNAs in cancer. Annual review of pathology. 2009;4:199-227.
Gebert LFR, MacRae IJ. Regulation of microRNA function in animals. Nat Rev Mol Cell Biol. 2019;20(1):21-37.
Wang S, Cheng M, Zheng X, Zheng L, Liu H, Lu J, et al. Interactions Between lncRNA TUG1 and miR-9-5p Modulate the Resistance of Breast Cancer Cells to Doxorubicin by Regulating eIF5A2. Onco Targets Ther. 2020;13:13159-70.
Peixoto A, Fernandes E, Gaiteiro C, Lima L, Azevedo R, Soares J, et al. Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension. Oncotarget. 2016;7(39):63138-57.
Theodoropoulos VE, Lazaris A, Sofras F, Gerzelis I, Tsoukala V, Ghikonti I, et al. Hypoxia-inducible factor 1 alpha expression correlates with angiogenesis and unfavorable prognosis in bladder cancer. Eur Urol. 2004;46(2):200-8.
Theodoropoulos VE, Lazaris AC, Kastriotis I, Spiliadi C, Theodoropoulos GE, Tsoukala V, et al. Evaluation of hypoxia-inducible factor 1alpha overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma. BJU Int. 2005;95(3):425-31.
Yang J, Yu H, Shen M, Wei W, Xia L, Zhao P. N1-guanyl-1,7-diaminoheptane sensitizes bladder cancer cells to doxorubicin by preventing epithelial-mesenchymal transition through inhibition of eukaryotic translation initiation factor 5A2 activation. Cancer Sci. 2014;105(2):219-27.
Yong Y, Zhang C, Gu Z, Du J, Guo Z, Dong X, et al. Polyoxometalate-Based Radiosensitization Platform for Treating Hypoxic Tumors by Attenuating Radioresistance and Enhancing Radiation Response. ACS nano. 2017;11(7):7164-76.
Liu Z, Wang Y, Dou C, Xu M, Sun L, Wang L, et al. Hypoxia-induced up-regulation of VASP promotes invasiveness and metastasis of hepatocellular carcinoma. Theranostics. 2018;8(17):4649-63.
Pawlus MR, Hu CJ. Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response. Cellular signalling. 2013;25(9):1895-903.
Yang L, Roberts D, Takhar M, Erho N, Bibby BAS, Thiruthaneeswaran N, et al. Development and Validation of a 28-gene Hypoxia-related Prognostic Signature for Localized Prostate Cancer. EBioMedicine. 2018;31:182-9.
He Y, Huang H, Jin L, Zhang F, Zeng M, Wei L, et al. CircZNF609 enhances hepatocellular carcinoma cell proliferation, metastasis, and stemness by activating the Hedgehog pathway through the regulation of miR-15a-5p/15b-5p and GLI2 expressions. Cell Death Dis. 2020;11(5):358.
Jin L, Li Y, He T, Hu J, Liu J, Chen M, et al. miR15a5p acts as an oncogene in renal cell carcinoma. Mol Med Rep. 2017;15(3):1379-86.
Ivey KN, Srivastava D. microRNAs as Developmental Regulators. Cold Spring Harbor perspectives in biology. 2015;7(7):a008144.
Xue M, Chen W, Xiang A, Wang R, Chen H, Pan J, et al. Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1. Molecular cancer. 2017;16(1):143.
Wang L, Saci A, Szabo PM, Chasalow SD, Castillo-Martin M, Domingo-Domenech J, et al. EMT- and stroma-related gene expression and resistance to PD-1 blockade in urothelial cancer. Nature communications. 2018;9(1):3503.

floatimage6.png
Supplementary Figure 1 A-B. CCK-8 assay the viability of Dox-treated cells transfected with an EIF5A2 siRNA or OV-eIF5A2. C-D. Vimentin, E-cadherin and eIF5A2 expression levels in cells transfected with eIF5A2 siRNA or OV-eIF5A2.
floatimage7.png
Supplementary Figure 2 A-B. Vimentin, E-cadherin, and eIF5A2 expression levels examined following transfection with or without an EIF5A2 siRNA under hypoxic condition. C. CCK-8 assay of cell viability following transfection with or without the EIF5A2 siRNA under hypoxic condition.

Download PDF

Editorial decision: Returned Without Review
05 Sep, 2023
Reviewers agreed at journal
25 Jul, 2023
Reviewers invited by journal
16 May, 2023
Editor assigned by journal
04 May, 2023
First submitted to journal
03 May, 2023

You are reading this latest preprint version

MicroRNA-15a-5p suppresses hypoxia-induced tumor growth and chemoresistance in bladder cancer by binding to eIF5A2

Status:

Version 1

Abstract

Background

Methods and Results

Conclusion

Figures

Introduction

Materials and Methods

Cell culture

Cell transfection

Cell Counting Kit-8 (CCK-8) assay

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Dual luciferase reporter gene assay

Western blotting

Tumor xenograft study

TUNEL and immunohistochemistry staining

Statistical analyses

Results

MiR-15a-5p is transcriptionally inhibited by HIF-1α under hypoxia

MiR-15a-5p suppress tumor growth in bladder cancer

MiR-15a-5p enhances doxorubicin sensitivity of bladder cancer cells

miR-15a-5p reverses the hypoxia-induced epithelial-mesenchymal transition

EIF5A2 is the downstream target of the miR-15a-5p

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1