Avenanthramide A Induces Cellular Senescence via miR-129-3p

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Bioactive Constituents, Metabolites, and Functions

Avenanthramide A induces cellular senescence via miR-129-3p/ Pirh2/p53 signaling pathway to suppress colon cancer growth Rong Fu, Peng Yang, Sajid Amin, and Zhuoyu li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00833 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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Journal of Agricultural and Food Chemistry

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Avenanthramide

A

induces

cellular

senescence

via

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miR-129-3p/Pirh2/p53 signaling pathway to suppress colon cancer

3

growth

4

Rong Fu †, #, ‡, Peng Yang †, #, ‡, Amin Sajid †, Zhuoyu Li †, §, *

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† Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular

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Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006,

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China

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# Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, China

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§ College of Life Science, Shanxi University, Taiyuan 030006, China

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‡ R.F. and P.Y. contributed equally to this work.

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*

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Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular

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Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006,

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PR China.

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Fax: +86 351 7018268.

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E-mail addresses: [email protected] (Zhuoyu. Li)

Corresponding Author: Prof. Zhuoyu Li

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ACS Paragon Plus Environment


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ABSTRACT

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Cellular senescence is the state of irreversible cell cycle arrest that provides a blockade

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during oncogenic transformation and tumor development. Avenanthramide A (AVN A) is an

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active ingredient exclusively extracted from oats, which possess antioxidant, anti-inflammatory

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and anticancer activities. However, the underlying mechanism(s) of AVN A in the prevention of

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cancer progression remains unclear. In the current study, we revealed that AVN A notably

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attenuated tumor formation in an azoxymethane/dextran sulfate sodium (AOM/DSS) mouse

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model. AVN A treatment triggered cellular senescence in human colon cancer cells, evidenced by

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enlarging cellular size, upregulating β-galactosidase activity, γ-H2AX positive staining and G1

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phase arrest. Moreover, AVN A treatment significantly increased the expression of miR-129-3p,

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which markedly repressed the E3 ubiquitin ligase Pirh2 and other two targets IGF2BP3 and

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CDK6. The Pirh2 silencing by miR-129-3p lead to a significant increase in protein levels of p53

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and its downstream target p21, which subsequently induced cell senescence. Taken together, our

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data indicates that miR-129-3p/Pirh2/p53 is a critical signaling pathway in AVN A-induced

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cellular senescence and it could be a potential chemopreventive strategy for cancer treatment.

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KEYWORDS: Avenanthramide A, miR-129-3p, Pirh2, cellular senescence, colorectal cancer


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INTRODUCTION

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Colorectal cancer (CRC) is the third most common cancer and the second largest cause of

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cancer-related deaths worldwide 1. At present, surgical resection and chemotherapy are the

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primary options for CRC treatment, which may increase the 5 years survival rate of patients.

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However, the outcomes of currently using chemotherapeutic drugs are not ideal due to obtrusive

41

drug resistance and toxic side effects 2. Natural products with their large structural diversity and

42

unique biological functionality without any apparent side effect remain a primary choice of drug

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development for cancer and several other diseases. Therefore, phytochemicals exhibiting

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anticancer properties with low toxicity and minimal side effects could be keen alternatives for

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cancer treatment.

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Oat (Avena sativa L.) is a whole grain cereal of the grass family Poaceae, which is

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recognized as a healthy food with high content of dietary fibers, phytochemicals and nutritional

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values 3. Oats contains a series of bioactive compound, including steroidal saponins, β-glucan,

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avenanthramides (AVNs) and flavonoids, contributes to health benefits 4. AVNs, a group of

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substituted N-cinnamoylanthranilic acids, are unique polyphenolic alkaloid exclusively extracted

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from oats 5. To date, more than 30 different forms of AVNs have been identified from oats, among

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which AVN A, AVN B and AVN C are the most abundant. Primarily, AVNs have been found to

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possess an array of bioactivities including antioxidation, anti-inflammation, anti-itching and

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immunomodulatory effects 6. Recent studies have revealed that AVNs may exert certain antitumor

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activities on numerous different types of cancer

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cell growth by reducing the expression of cyclin and activating the caspases 2, 8, 3 8-10. However,

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the possible mechanism(s) underlying AVN A-induced anticarcinogenic activities are still not

7-8.

It was reported that AVNs suppressed cancer



58

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clear.

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Cellular senescence refers to irreversible cell cycle arrest that occurs when cell is under

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potentially stress conditions such as telomere shortening, oncogene-induced senescence (OIS),

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metabolism dysfunction, oxidative stress, and genomic damages 11. Senescent cells display several

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morphological and biochemical hallmarks, including enlarged cellular size, enhanced

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senescence-associated β-galactosidase (SA-β-gal) activity, telomere shortening, induced

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expression of p53 and p16

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senescence responses are evolved at least in part to suppress their growth and proliferation, such

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as they remain highly sensitive to senescence induced by chemotherapy or radiotherapy

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addition, senescent cells also develop the senescence-associated secretory phenotype to secrete

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many secretory factors to modulate tumor microenvironment, which subsequently can inhibit the

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oncogenic transformation

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suppressive mechanism, and to identify natural compounds that induce cell senescence may

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represent a promising anticancer strategy.

14.

12.

Although cancer cells have unlimited replication potential, the

13.

In

Therefore, cellular senescence is considered as a potential tumor

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MicroRNAs (miRNAs) are a family of endogenous small noncoding RNAs, as silencers of

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various target genes through translational repression or mRNA degradation, has a critical role in

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regulating numerous biological processes including carcinogenesis 15. Growing evidence suggests

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that miRNAs could serve as unique proto-oncogene or tumor suppressor that regulates cellular

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senescence and its dysregulation leads to initiation and development of cancer

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has been reported that miR-129-3p to be downregulated in multiple cancer types where it

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functions as a tumor suppressor

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remains ambiguous.

17-18.

15-16.

Moreover, it

However, the role of miR-129-3p in cellular senescence


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80

Herein, we evaluated the suppressive effects of AVN A on an azoxymethane/dextran sodium

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sulfate (AOM/DSS)-induced colorectal carcinogenesis mouse model and demonstrated that AVN

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A significantly promoted the expression levels of p21 and p53 to induce CRC cellular senescence.

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Moreover, AVN A increased the expression level of miR-129-3p to degrade its direct target Pirh2

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(a negative regulator of p53) to trigger cellular senescence. In addition, IGF2BP3 and CDK6, the

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other two targets of miR-129-3p were all downregulated by AVN A treatment. The results

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revealed a novel role of miR-129-3p/Pirh2/p53 axis in regulation of cellular senescence and that

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AVN A could be a potential candidate for CRC trials.

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MATERIALS AND METHODS

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Chemicals and antibodies

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AVN A (purity > 99%) was obtained from Topharman (Shanghai, China). Cycloheximide

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(CHX), Azoxymethane (AOM) and Actinomycin D were purchased from Sigma (St. Louis, MO,

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USA). Dextran sulfate sodium (DSS) was purchased from MP Biomedicals Inc. (Irvine, CA,

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USA). Cell Cycle Detection Kit was obtained from Keygen Biotech (Nanjing, China). p21,

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γ-H2AX and GAPDH antibodies were obtained from Abcam (Cambridge, MA, USA). The

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antibodies against p16, p27, p53, MDM2 and Pirh2 were purchased from Proteintech (Wuhan,

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China). Ki67 and COP1 antibodies were obtained from Bioss (Beijing, China).

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Cell culture

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HEK293T, human colon carcinoma cell lines HCT-8, HCT-116 and normal colon epithelial

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cell line FHC were obtained from the ATCC (Manassas, VA, USA). HEK293T were cultured in

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DMEM in supplemented with 10% fetal bovine serum (FBS). HCT-8 and HCT-116 cells were

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cultured in RPMI-1640 and FHC cells were cultured in DMEM/F12 medium supplemented with



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10% FBS and 1% penicillin at 37 °C in a 5% CO2 incubator.

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Animal experiments

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Mouse colitis-associated cancer was carried out as previously described

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19.

Male mice

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C57BL/6J of age 6-weeks were purchased from Beijing Vital River Laboratories Co. (Beijing,

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China). All the animal experiments are conducted according to the Guidelines of the Committee

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on the Ethics of Animal Experiments of Shanxi University. After adaptation to new environment

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for one week, mice were randomly assigned to three groups: the control group, the AOM/DSS

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group and the AVN A-AOM/DSS group (n = 8 mice each group). For AVN A-AOM/DSS group,

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mice were orally given 30 mg/kg of AVN A dissolved in dimethylsulfoxide (DMSO) and diluted

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in distilled water for one week. Then, animals in the model and AVN A-AOM/DSS group

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received a single intraperitoneal injection of AOM (10 mg/kg). Next, mice were undergone four

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cycles of treatment. In each cycle, mice received 1.25% DSS in drinking water for 7 consecutive

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days followed by 14 days of regular drinking water and 30 mg/kg of AVN A daily for AOM/DSS

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group and AVN A-AOM/DSS group respectively. Body weight was measured twice a week.

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Finally, mice were sacrificed and tissues including colon, heart, liver, spleen and kidney were

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isolated for further analysis.

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Immunohistochemistry

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For immunohistochemistry (IHC) analysis, paraffin sections (5 μm thick) were incubated

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with specific antibodies against γ-H2AX and Ki67 overnight at 4 °C. A universal labeled

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streptavidin-biotin kit (Maixin Biotechnology, Fuzhou, China) was used as a standard protocol for

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staining. Cells were visualized using a microscope (Zeiss, Oberkochen, German). The organ

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tissues were with hematoxylin (Shanghai Ruji Biotechnology Development Co., Ltd., Shanghai,


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China) and eosin (Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjin, China).

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Cell cycle analysis

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The Cell Cycle Detection Kit (KeyGEN BioTECH, Nanjing, China) was used for cell cycle

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analysis according to the manufacturer’s instructions. Cells were seeded in six-well plates and

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incubated with AVN A. After harvest and fixed in 70% ethanol, cells were resuspended in

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ice-cold PBS supplemented with RNase A (25 ng/mL), and stained with 0.5 mg/mL propidium

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iodide (PI) for 30 min at 37 °C. The PI fluorescence was analyzed by flow cytometry (BD

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Biosciences, Oxford, UK).

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SA-β-galactosidase staining

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SA-β-galactosidase detection was determined following the manufacturer’s instructions

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(Beyotime Biotech, Beijing, China). Briefly, the indicated cells were seeded in 6-well plates and

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treated with AVN A for 3, 5 or 7 days. After fixed in 4% paraformaldehyde, cells were then

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incubated in a freshly prepared β-gal Staining Solution overnight. SA-β-gal positive stained cells

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were photographed and quantified based on 3 independent images.

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Western blot analysis

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Protein concentrations of whole cell lysates were detected using the BCA Protein Assay Kit

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(Beyotime Biotech, Beijing, China). Equal amounts of protein were subjected to SDS–PAGE,

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transferred to PVDF membrane and incubated with the indicated antibodies. The enhanced

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chemiluminescence signal was determined by radiographic film.

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Cycloheximide and Actinomycin D chase assays

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HCT-8 cells were treated with AVN A in the presence of cycloheximide. Cells were lysed at

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various time points to measure changes in post-translational turnover by western blot analysis



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using p21 and p53 antibodies. Actinomycin D was used for Actinomycin D chase assays as

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previously described 20.

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Quantitative reverse transcription PCR

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Total RNA was extracted from cells with TransZol Up (TransGen Biotech, Beijing, China)

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for miRNA or mRNA analysis. cDNAs of miRNA were polyadenylated using All-in-One miRNA

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qRT‐PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) following the manufacturer’s

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product manual. Next, qPCR was performed using TransStart Top Green qPCR SuperMix

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(TransGen Biotech, Beijing, China). U6 and GAPDH were used as internal control for miRNA

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and mRNA detection, respectively. The primers used in the study were as follows:

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p21:

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p53: 5’- CTTTGAGGTGCGTGTTTGTGCC-3’, 5’-GGTTTCTTCTTTGGCTGGGGA-3’;

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GAPDH: 5’- AAGGTCGGAGTCAACGGATTT-3’, 5’- CCTGGAAGATGGTGATGGGATT-3’;

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miR-129-3p: 5’- ACACTCCAGCTGGGAAGCCCTTACCCCAAA -3’;

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U6: 5’-CGCTTCGGCAGCACATATAC-3’, 5’-AAAATATGGAACGCTTCACGA-3’.

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siRNA transfection

5’- CGTCAAATCCTCCCCTTCCTG-3’, 5’-CCTGCCTCCTCCCAACTCATC-3’;

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Two sets of siRNAs against Pirh2 (5’-CAUGCCCAACAGACUUGUGdTdT-3’ and 5’-GGA

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AGUGCAGUGCAUAAACdTdT-3’) and scrambled siRNA were purchased from GenePharma

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(Shanghai, China). Mature miR-129-3p mimics and its negative control (NC) were designed and

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synthesized by GenePharma (Shanghai, China). HCT-116 and HCT-8 cells were seeded in 6-well

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plate and then transfected until 70 % confluence using Lipofectamine 3000 reagents (Invitrogen,

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Carlsbad, CA, USA) following the manufacturer’s protocol. Further assays were performed after

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48 h of transfection.


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Luciferase reporter assay

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The miR-129-3p response element in the Pirh2 3’-UTR (wildtype) was amplified and cloned

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into the pGL4.23 vector. Site-directed mutagenesis (mutant) of the miR-129-3p binding site was

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carried out using the Easy Mutagenesis System produced by Transgen Biotech (Beijing, China).

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The primers for plasmid construction were as follows:

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Pirh2 3’UTR-WT: 5’-CCGCTCGAGTCGTGTTATCATGTGTCGTCA -3’,

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5’-GGAAGATCTCCACTCATCCATCCCCTATTT -3’;

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Pirh2-3’UTR-Mutant: 5’-TCTTGACTTATTATGCGGTGTGTTATATTA -3’,

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5’-CCGCATAATAAGTCAAGA CTACTACTGAAA -3’.

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The wildtype or mutant 3’UTR vector was cotransfected with miR-129-3p mimics or control

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into HEK293T cells. Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was

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performed as the manufacturer’s instructions. The data was normalized to Renilla luciferase

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activity for each well.

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Statistical analysis

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Statistical analysis was performed using GraphPad Prism Software (San Diego, CA, USA).

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Data are displayed as the mean ± SD or mean ± SEM. p-values < 0.05 were considered to be

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statistically significant.

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Results

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AVN A suppresses tumor growth in AOM/DSS-induced colon carcinogenesis.

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As a well-recognized model for studying CRC progression, AOM/DSS-induced

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colitis-associated carcinogenesis can be effectively used in the evaluation of chemopreventive and

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chemotherapeutic activities of phytochemicals

21.

To determine whether AVN A attenuate the



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formation of CRC, AOM/DSS model and AVN A treatment with daily intake about 30mg/kg by

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drinking water were established as shown in Fig. 1A. The mice in AOM/DSS group showed a

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high tumor burden in colon tissues, while AVN A treatment obviously suppressed AOM/DSS

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induced tumors (Fig. 1B). As shown in Fig. 1C, AOM/DSS group exhibited significant weight

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loss when compared to the control group. In contrast, the body weight of AVN A fed mice was

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similar to the healthy group and did not cause any observable toxicity. Although colon length

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remains the same (Fig. 1D), the tumor incidence was decreased (Fig. 1E) and tumor diameter (Fig.

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1F) of macroscopic polyps were significantly lesser in AVN A-fed mice than that of AOM/DSS

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group. Together, these results suggested that oral administration of AVN A impeded

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AOM/DSS-induced carcinogenesis.

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AVN A causes CRC cells senescence.

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To check the direct effects of AVN A on tumor growth, the expression of Ki67 and γH2AX

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were detected by immunohistochemistry. As shown in Fig. 2A, AVNA treatment notably

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decreased the expression of Ki67, and increased the level of γ-H2AX, which indicated that tumor

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growth was suppressed and caused DNA damage. Cell cycle analysis revealed an obvious G1

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phase arrest in CRC cells after treated by AVN A for 3 days (Fig. 2B). Cellular senescence is

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considered to be a potent tumor suppressor mechanism with properties of irreversible arrest of cell

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proliferation and usually accompanied by upregulation of γ-H2AX, G1 arrest and β-galactosidase

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activity 22. We suspected that AVN A caused tumor cells senescence. To this end, we checked the

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senescence-associated β-galactosidase acidic activity. As shown in Fig. 2C and 2D, the proportion

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of cells with senescent cell morphology and SA-β-gal staining were increased in dose and

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time-dependent manner after exposed to indicated concentration of AVN A for 3, 5 or 7 days.


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Hence, these data indicated that AVN A significantly triggered cellular senescence in CRC cells.

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AVN A induces p21 expression by promoting its transcription

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Growing number of studies showed that cyclin dependent kinase inhibitors (CDKI) such as

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p16, p21 and p27 could be key effectors of cellular senescence

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influence of AVN A on CDKI to promote cellular senescence. As shown in Fig. 3A and 3B, the

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protein level of p21 increased in a dose-dependent manner when treated with AVN A for 3 days.

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On contrary, expression levels of p16 and p27 were unaffected by AVN A treatment. Next, we

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performed Actinomycin D chase assays to explore whether AVN A modulate the transcriptional

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or post-transcriptional regulation of p21. The mRNA decay rate of p21 in cells treated with AVN

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A was consistent with that of control cells by Actinomycin D treatment at any intervals (Fig. 3C).

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Furthermore, protein synthesis inhibitor cycloheximide (CHX) was applied to compare protein

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stability with AVN A treatment. As shown in Fig. 3D and 3E, the half-life of p21 was not changed

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in cells treated with AVN A compared to control cells, indicated that AVN A did not affect the

225

stability of p21 protein. Thus, these data suggested that AVN A seemed to promote p21

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expression by augmenting its transcription levels.

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The E3 ubiquitin ligase of p53, Pirh2 is critical for AVN A-induced p53 expression.

11, 23.

Herein, we examined the

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Previous report has shown that tumor suppressor p53 transcriptionally regulates the

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expression of p21 and that the activated p53/p21 axis plays a crucial role in the senescent

230

phenotype 24. Therefore, we questioned whether AVN A affects the expression of p53. As shown

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in Fig. 4A and 4B, p53 protein levels were strikingly upregulated after AVN A treatment. We

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further explored the underlying mechanism of AVN A-mediated increased in p53 protein level.

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Notably, AVN A treatment did not affect thep53 mRNA expression (Fig. 4C). The results of CHX



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chase experiment showed that p53 was degraded much slower in cells treated with AVN A

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compared to that of control cells in the presence of CHX (Fig. 4D and 4E). These data suggested

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that AVN A mitigated the p53 protein degradation rather than transcriptional regulation to induce

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p53 expression. A large number of studies showed that diverse E3 ligases, including MDM2,

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COP1 and Pirh2 play important role in regulating p53 degradation 25. To this end, we tested that

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which one of these E3 ligases is involved in p53 degradation. The results showed that AVN A

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treatment significantly abrogated Pirh2 expression, whereas the expression levels of MDM2 and

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COP1 remain unchanged after AVN A treatment (Fig. 4F and 4G). Moreover, we knocked down

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Pirh2 expression in CRC cells to determine whether the elevation of p53 and p21 were driven by

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Pirh2. Results showed that depletion of Pirh2 remarkably induced expression of both p53 and p21

244

(Fig. 4H and 4I). In addition, Actinomycin D chase assays showed that Pirh2 mRNA was decayed

245

more quickly in cells treated with AVN A compared to the control cells (Fig. 4J). Taken together,

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these results suggested that Pirh2 downregulation by AVN A treatment is achieved by mRNA

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destabilization, and that Pirh2 may have a critical role in AVN A-induced p53/p21 axis.

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AVN A activates miR-129-3p/Pirh2/p53 signal pathway to drive cellular senescence.

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It has been reported that miRNA suppressed gene expression via translational repression,

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mRNA degradation and destabilization

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carried out to predict miRNAs possessing potential binding affinity to Pirh2, and it was found that

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miR-129-3p could conservatively target to Pirh2 which also strongly related to tumor progress and

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cell cycle (Fig. 5E). To assess the effect of AVN A on miR-129-3p, HCT-8 and HCT-116 cells

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were treated with different doses of AVN A, and then the level of miR-129-3p was analysed by

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qPCR. As shown in Fig. 5A, AVN A treatment significantly elevated miR-129-3p expression in a

26.

The online TargetScan (v7.0; targetscan.org) was


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dose-dependent manner. Overexpression of miR-129-3p dramatically suppressed mRNA level of

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Pirh2 in CRC cells, which was in agreement with our observations that AVN A treatment

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dramatically decreased the Pirh2 mRNA level (Fig. 5B). Moreover, western blot analysis also

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confirmed that the expression of Pirh2 was downregulated in miR-129-3p overexpressing cells

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(Fig. 5C and 5D). To further evaluate whether Pirh2 is a target gene of miR-129-3p, we

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constructed wildtype and mutant Pirh2 3’-UTR into dual-luciferase reporter vector pGL4.23. The

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results showed that miR-129-3p overexpression caused robust suppression in the relative

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luciferase activity of 3’-UTR binding site of the wildtype Pirh2, whereas luciferase activity was

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not affected in the mutant 3’-UTR (Fig. 5E). Subsequently, we examined the effect of miR-129-3p

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overexpression and Pirh2 knockdown on CRC cell senescence. As shown in Fig. 5F and 5G,

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miR-129-3p overexpression or Pirh2 depletion resulted in a significant increase of SA-β-gal

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activity and senescent cell morphology. It has been reported that miR-129-3p functions as a

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negative regulator of IGF2BP3 and CDK6 to induce cell cycle arrest of glioblastoma multiforme

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27.

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observed that the expression of both IGF2BP3 and CDK6 was markedly decreased in miR-129-3p

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overexpressing or AVN A treated cells (Fig. 5H and 5I). Taken together, these results

272

demonstrated that miR-129-3p is critical for AVN A induced CRC cellular senescence.

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Biosafety Evaluation of AVN A.

Consistent with the previous study that miR-129-3p directly targets IGF2BP3 and CDK6 27, we

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Natural compounds featured by low toxicity and minimal side effects may raise the

275

probability of drug discovery success 28. Since AVN A exerted remarkable antitumor properties in

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AOM/DSS model, the potential side effects of AVN A were evaluated. As shown in Fig. 6A, the

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microscopic examination of the organs including the heart, liver, spleen and kidney in mice which



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orally administrated with AVN A did not showed any detectable morphological changes.

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Additionally, we further evaluated whether human normal colonic epithelial FHC cells undergo

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senescence by AVN A treatment. The results showed that AVN A exhibited almost no effect on

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SA-β-gal activity and did not caused morphological changes in FHC cells (Fig. 6B and 6C).

282

Together, these results indicated that AVN A can be considered as a safe and potential

283

chemopreventive candidate for CRC trials.

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Discussion

285

Numerous epidemiological studies have been shown that the long-term intake of whole grain

286

is beneficial for the health function of digestive tract and associated with lower risk of CRC

287

Oats are whole grain cereals that contain a variety of bioactive ingredients and are particularly rich

288

in AVNs. AVNs, possess antioxidant, anti-inflammatory and anticancer properties

289

AVN A is one of the most abundant and active constituents of AVNs, the anticarcinogenic

290

activities of AVN A and the underlying molecular mechanism(s) remains elusive. Here, we

291

investigated the chemopreventive effects of dietary AVN A in an AOM/DSS induced

292

colitis-associated carcinogenesis model. Our data showed that AVN A noticeably suppressed

293

tumor incidence and growth in AOM/DSS mice by inducing CRC cellular senescence. We

294

provided the evidence that AVN A induced miR-129-3p directly targeted Pirh2 to activate

295

p53/p21 axis, which in turn triggers cell senescence. In addition, miR-129-3p also negatively

296

regulates both IGF2BP3 and MAPK1 to induce senescence. To the best of our knowledge, this

297

work represents the first to demonstrate induction of senescence by increasing miR-129-3p levels

298

as a result of AVN A treatment in CRC cells.

299

30.

29.

Although

Cell senescence has been considered as an effective tumor-suppressor mechanism, which


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300

results in the permanent cell cycle arrest

301

senescence remain unclear, many studies have demonstrated that miRNAs function as potential

302

regulators of cellular senescence 15-16. The expression of miR-129-3p is downregulated in various

303

tumors, including endometrial, hepatic, gastrointestinal and colon cancer. Evidence from the

304

previous literature suggests that miR-129-3p serve as a tumor suppressor and closely related to the

305

activation of apoptotic cell death, autophagy, cell cycle arrest and sensitize CRC cells to

306

chemotherapy

307

demonstrating that miR-129-3p directly targets IGF2BP3 and MAPK1 to arrest G1/S transition 27.

308

However, the relationship between miR-129-3p and cell senescence was not clear till to date. In

309

the present study, AVN A treatment significantly promoted miR-129-3p expression and

310

miR-129-3p overexpression was observed to be exclusively sufficient to trigger cellular

311

senescence in CRC cells (Fig. 5A 5F and 5G). Moreover, we deduced that the miR-129-3p

312

induction could be employed as a biomarker to detect senescence in CRC cells. Several available

313

reports have demonstrated that the promoter of miR-129-3p was hypermethylated in CRC cells

314

and clinical samples as compared to normal colon epithetical cells and tissue samples 32. Bandres

315

E et al. also identified miR-129-3p located around/on a CpG island to be down-regulated in

316

patients with colorectal cancer, and methyltransferase inhibitor was shown to significantly restore

317

the expression of miR-129-3p

318

region could lead to the downregulation of miR-129-3p. One outstanding question is that how

319

AVN A facilitate the expression of miR-129-3p? A plausible explanation for this question should

320

be that AVN A may modulate the key enzymes involved in miR-129-3p methylation to induce its

321

expression, which obviously requires further investigations.

31-32.

12.

Although the factors that provoke tumor cell

Notably, our findings are in the agreement with the previous data

33.

These studies suggested that hypermethylation in the promoter



Page 16 of 33

322

Senescent cells are unique in gene signatures, chromatin structure, altered metabolism and

323

senescence associated secretory phenotype (SASP), which further render them vulnerable to some

324

sort of drugs that have limited efficacy in their proliferating counterparts. To date, several efforts

325

have been made to develop safe and efficient strategies to target senescent cells. Wang et al.

326

recently proposed a one-two punch approach in which a first drug selectively induce senescence in

327

cancer cells and senescent cancer cells were subsequently eliminated by pro-apoptotic agent

328

Previous study demonstrated that the combination of natural compound emodin with

329

5-fluorouracil significantly sensitized breast cancer chemotherapy via inducing tumor senescence

330

34.

331

pathways 35. Based on these findings, it is proposed that the combination of AVN A with agents

332

which selectively target vulnerable Achilles’ heels of senescent cancer cells could be employed

333

for cancer therapy.

14.

In addition, senescent cancer cells were sensitive to inhibition of autophagy or pro-survival

334

At present, several first-line clinical drugs for colon cancer, including 5-fluorouracil and

335

oxaliplatin are commercially available 2. However, their side effects are severe due to their poor

336

specificity and toxicity, which obviously restrict the therapeutic efficacy of CRC. Since natural

337

products are considered as invaluable sources for drug discovery, therefore to exploit natural

338

compounds with low toxicity and anticancer activities has attracted considerable attention.

339

Mounting evidence suggested that dietary phytochemicals, such as apigenin, resveratrol and

340

curcumin exert anticancer properties by targeting senescence-associated cellular signalling to

341

induce cell senescence

342

cancer cellular senescence may represent an alternative strategy for cancer therapy. In this study,

343

we showed that AVN A exhibits potent anticarcinogenic activity featured by inducing cell

12-13, 36.

Thus, the discovery of natural compounds which could trigger


Page 17 of 33


344

senescence. Our study investigated the safety of AVNA in vitro and in vivo. Of note, the data

345

displayed no obvious toxicity as detected in AVNA group by pathological review of sections of

346

heart, liver, spleen and kidneys (Fig. 6A). AVN A treatment even improved organ lesions caused

347

by AOM/DSS administration and abrogated body weight loss induced by AOM/DSS (Fig. 1B, 1C

348

and 6A). It is important to note that AVN A selectively induced senescence in CRC cells but not

349

in non-cancerous counterpart FHC cells (Fig. 6B and 6C). These results indicated that dietary

350

AVN A exerted it chemopreventive effects against colorectal carcinogenesis and may be

351

developed as safe and adjuvant chemotherapeutic agent for CRC treatment.

352

In summary, our findings provide evidence that AVN A exhibits potential chemopreventive

353

effects against colorectal carcinogenesis. AVN A notably induces miR-129-3p expression which

354

directly degrades Pirh2 to activate p53/p21 axis, and ultimately leads to the cellular senescence in

355

CRC cells. Moreover, IGF2BP3 and MAPK1 are also involved in senescence induced by

356

miR-129-3p. These findings suggest that AVN A is a unique bioactive constituent of oats that

357

seems to be more promising auxiliary agent for the treatment of CRC.

358

ABBREVIATIONS USED

359

CRC, colorectal cancer; AVN A, Avenanthramide A; AVNs, Avenanthramides; OIS,

360

oncogene-induced

361

Cycloheximide; CDKI, cyclin dependent kinase inhibitors; AOM/DSS, azoxymethane/dextran

362

sulfate sodium; SASP, senescence associated secretory phenotype.

363

NOTES

364 365

senescence;

SA-β-gal,

senescence-associated

The authors declare no competing financial interest.

FUNDING SOURCES


β-galactosidase;

CHX,


366

This work was supported by the National Natural Science Foundation of China (No.

367

31770382, 31800657), Shanxi Province Science Foundation for Key Projects (No.

368

201801D111001), Shanxi Province Science Foundation for Youths (No. 201601D021107),

369

Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (No.

370

2016121) and “1331 Project” Key Innovation Team of Shanxi Province (Prof. Zhuoyu Li).

371

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35. Ovadya, Y.; Krizhanovsky, V., Strategies targeting cellular senescence. J. Clin. Invest. 2018,

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475


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Figure Legends

477

Figure 1. The preventive effects of AVN A on AOM/DSS-induced colitis associated

478

tumorigenesis.

479

(A) Animal study design. Molecular structure of AVN A (left panel). Experimental procedure

480

used for the control group, AOM/DSS group and AVN A-AOM/DSS group (right panel). (B)

481

Representative pictures of colon from control, AOM/DSS or AVN A groups. (C) Effects of AVN

482

A on body weight changes of control, AOM/DSS or AVN A groups. (D-F) The quantification of

483

colon length (D) tumor incidence (E) and tumor diameter (F) in AVN A fed and control mice are

484

shown at the experiment. Data are presented as mean ± SEM and were assessed using the

485

Mann-Whitney U-test, * p < 0.05; ** p < 0.01; *** p < 0.001; n = 8/group.

486

Figure 2. AVN A causes G1-phase cell cycle arrest and increases expression of

487

senescence-associated markers in CRC cells.

488

(A) Left panel: immunohistochemical staining with antibodies against Ki67 and γH2AX in tumor

489

sections from AOM/DSS group and AVN A group. Right panel: IHC scoring of Ki67 and γH2AX

490

were performed in both groups according to the staining intensity. (n = 3, mean ± SD). (B)

491

HCT116 and HCT-8 cells were treated with 7.5 μM AVN A for 3 days and cell-cycle distribution

492

was determined by flow cytometry analysis (cell count vs DNA content). (C) HCT-8 cells were

493

treated with indicated concentration of AVN A for 3, 5 or 7 days and then subjected to SA-β-gal

494

staining. (D) Bar diagram represents percentage of SA-β-gal positive cells from (C). Data shown

495

are means ± SEM (n = 3) * p < 0.05; ** p < 0.01; *** p < 0.001.

496

Figure 3. AVN A promotes transcription of p21.

497

(A) HCT116 and HCT-8 cells were treated with indicated concentrations of AVN A for 3 days,



498

and then the expression levels of p16, p27 and p21 were determined by western blot. (B)

499

Normalized intensity of p16, p27 and p21 protein versus GAPDH in HCT116 (upper panel) and

500

HCT-8 cells (lower panel). (n = 3, mean ± SD). (C) HCT-8 cells were treated with 15 μM AVN A,

501

then cultured with Actinomycin D (1 μg/ml) for 7 h and qPCR analysis was applied to determine

502

the stability of the p21 mRNAs (n = 3, mean ± SD). (D) HCT-8 cells were treated with 15μM

503

AVN A in the presence of 20 μM CHX for 0, 1, 2, 3 h respectively. The protein level of p21 was

504

detected by western blot. (E) The relative band intensity of p21 protein was normalized to

505

GAPDH. Data shown are means ± SD (n = 3) * p < 0.05; ** p < 0.01; *** p < 0.001.

506

Figure 4. Pirh2, an E3 ubiqutin ligase for p53, was suppressed by AVN A.

507

(A) HCT116 and HCT-8 cells were treated with 15 μM AVN A for 3 days. (B) The protein level

508

of p53 was detected by western blot and the intensity of p53 was normalized to GAPDH. (C) The

509

effect of AVN A treatment on the mRNA expression levels of p53 was analyzed by qPCR. (D)

510

HCT-8 cells treated with AVN A in the presence of 20 μM CHX for 0, 1, 3 h respectively. Then,

511

p53 protein levels were detected by western blot and the intensity of p53 was normalized to

512

GAPDH (E). (F) Western blot analysis was performed to determine the protein levels of MDM2,

513

Pirh2 and COP1 in HCT116 and HCT-8 cells after treated by AVN A for 3 days. (G) Normalized

514

intensity of MDM2, Pirh2 and COP1 protein versus GAPDH. (H) The expression of Pirh2, p21

515

and p53 were analyzed by western blot in HCT-8 cells transfected with control and Pirh2 siRNAs

516

for 48h and the intensity of Pirh2, p21 and p53 was normalized to GAPDH (I). (J) The mRNA

517

decay rate of Pirh2 mRNA was analyzed by qPCR in HCT-8 cells which treated with AVN A in

518

the presence of Actinomycin D (1 μg/ml) for 7 h. The data are shown as the mean ± SD of three

519

independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.


Page 24 of 33

Page 25 of 33


520

Figure 5. miR-129-3p is critical in AVN A induced cell senescence.

521

(A) The effect of AVN A treatment on the expression level of miR-129-3p was analyzed by qPCR

522

analysis. (n = 3, mean ± SD). (B) Expression of Pirh2 in HCT116 and HCT-8 cells treated with

523

AVN A or miR-129-3p mimics were determined by qPCR analysis. (n = 3, mean ± SD). (C) The

524

protein level of pirh2 treated by miR-129-3p mimics or control was detected by western blot. (D)

525

Normalized intensity of Pirh2 protein versus GAPDH. (E) Schematic diagram of the putative

526

binding sites of miR-129-3p in the wildtype (WT) Pirh2 3’untranslated regions (UTR). The

527

miR-129-3p seed matches in the Pirh2 3’UTR are mutated at the positions as indicated (upper

528

panel). Luciferase activity assays of wildtype (WT) and mutated Pirh2 3’UTR luciferase reporters

529

after co-transfection with miR-129-3p mimic or miRNA mimic control in HEK293T cells. The

530

Luciferase activity was detected at 48 h after transfection and normalized to the Renilla luciferase

531

activity (lower panel). (F) HCT-8 cells were treated with AVN A, miR-129-3p mimics or

532

Si-Pirh2-2 and then subjected to SA-β-gal staining. (G) Bar diagram represents percentage of

533

SA-β-gal positive cells from (F). Data shown are means ± SEM (n = 3) * p < 0.05; ** p < 0.01;

534

*** p < 0.001. (H) Expressions of IGF2BP3 and CDK6 in HCT-8 cells treated with AVN A or

535

miR-129-3p mimics were determined by western blot and the intensity of IGF2BP3 and CDK6

536

was normalized to GAPDH (I). Data shown are means ± SD (n = 3) * p < 0.05; ** p < 0.01; *** p

537

< 0.001.

538

Figure 6. Evaluation of the toxicity of ANV A.

539

(A) Representative H&E staining of various organs including hearts, livers, spleens and kidneys

540

harvested from the control group, AOM/DSS group and AVN A-AOM/DSS group. The scale bar

541

was 100 μm. (B) FHC cells were incubated with AVN A for 7 days and stained for SA-β-gal



542

activity. (C) Quantification of SA-β-gal activity shown in (B). Data shown are means ± SEM (n =

543

3) * p < 0.05; ** p < 0.01; *** p < 0.001.

544


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For Table of Contents Only

546



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Fig. 6


Avenanthramide A Induces Cellular Senescence via miR-129-3p

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