Quercetin inhibits the tumorigenesis of colorectal cancer cells through downregulation of hsa_circ_0006990

doi:10.21203/rs.3.rs-1019635/v1

Download PDF

Primary research

Quercetin inhibits the tumorigenesis of colorectal cancer cells through downregulation of hsa_circ_0006990

https://doi.org/10.21203/rs.3.rs-1019635/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

This study was intended to investigate the function of Quercetin in chemoresistant colorectal cancer (CRC) cells. In addition, this research aimed to explore the mechanism by which Quercetin regulates the malignant behavior of CRC cells.

Methods

To induce THP-1 cells into M2 tumor-associated macrophages (M2-TAMs), THP-1 cells were stimulated by PMA and IL-4. MDC staining was used to investigate the autophagy in M2-TAMs. Meanwhile, cell proliferation was tested by colony formation assay. In addition, wound healing and transwell assay were performed to detect the cell migration and invasion, respectively. Dual luciferase assay was used to investigate the correlation between hsa_circ_0006990 and miR-132-3p/miR-532-3p. Furthermore, mRNA and protein levels were detected by RT-qPCR and western blot, respectively.

Results

Quercetin suppressed autophagy of M2-TAMs. In addition, M2-TAMs significantly inhibited the apoptosis and promoted the proliferation of CRC cells, while this phenomenon was reversed by Quercetin. Meanwhile, the expression of hsa_circ_0006990 in CRC cells was decreased by M2-TAMs, while Quercetin reversed this phenomenon. Furthermore, overexpression of hsa_circ_0006990 significantly reversed the anti-tumor effect of Quercetin on CRC.

Conclusion

Quercetin inhibited the tumorigenesis of colorectal cancer cells through downregulation of hsa_circ_0006990. Thus, our study might shed new lights on exploring the new strategies against CRC.

Cancer Biology

Oncology

tumor-associated macrophages

quercetin

hsa_circ_0006990-miR-132-3p/miR-342-3p network

autophagy

Colorectal cancer (CRC) ranks third among the most commonly diagnosed malignant tumors in females [1]. Genetic mutations, inflammation-pertinent malfunctions and bad habits could contribute to the progression of CRC [2, 3]. Despite great efforts have been made for the treatment of CRC, the outcomes remain not ideal [4]. Therefore, it is urgent to explore new strategies against CRC.

Quercetin is a natural bioflavonoid compound extracted from hippophase rhamnoides, and it could significantly inhibit the progression of multiple cancers. For example, Mohammed HA et al found Quercetin could induce the apoptosis and inhibit the invasion of breast cancer cells [5]; Soofiyani SR et al indicated that Quercetin could act as a novel agent for the treatment of lymphoma [6]. Meanwhile, Quercetin could inhibit the growth of CRC cells [7]. However, the detailed mechanism by which Quercetin regulates the progression of CRC remains largely unknown.

Tumor associated macrophages (TAMs) stands up 30%-50% of total cells in tumor tissues [8, 9], and it TAMs could promote the metastasis and lymphangiogenesis of malignant tumors through recruiting abundant monocytes into tumor matrix [10, 11]. For instance, TAMs-secreted epidermal growth factor (EGF), PDGF, TGF-β and basic fibroblast growth factor (bFGF) were expected to increase the growth and invasion of tumor cells [12]. More importantly, TAMs might exhibit the anti-tumor function by preventing macrophages from converting into type II TAMs (M2-TAMs) [13–15]. Meanwhile, it has been reported that inhibition of autophagy could inhibit the polarization of M2-TAMs [16], and autophagy of TAMs could affect the biological activity of CRC cells [17]. Thus, TAM autophagy might affect the progression of CRC by regulation of TAM polarization [18]. Nevertheless, the detailed correlation between Quercetin and TAM autophagy in the progression of CRC remains unclear.

Circular RNAs (circRNAs) are endogenous RNAs which have stable closed structure [19]. In addition, it has been reported that circRNAs could modulate the cellular process (cell proliferation, apoptosis, autophagy, et al) [20, 21]. Meanwhile, circRNAs could regulate the growth of CRC cells through sponging miRNAs [22, 23]. For example, hsa_circ_0026344 positively regulated the deterioration of CRC through sponging miR-21/miR-31 [24]. However, circRNAs that involved in Quercetin-mediated CRC progression need to be further explored.

Based on the above backgrounds, we sought to investigate the correlation among Quercetin, TAMs and circRNAs in the progression of CRC. We hope this study would supply a novel strategy for the treatment of CRC.

Cell culture

THP-1 cells were obtained from ATCC (USA). Cells were cultured in an incubator at 37 ℃ and 5% CO₂. THP-1 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 0.05 mmol/L β-mercaptoethanol. CRC cell lines (HCT116 and Lovo) were purchased from Shanghai Cell Bank of Chinese Academy of Sciences. They were seeded in RPMI-1640 medium supplemented with 10% FBS. CRC cells were cultured in the condition of 37°C and 5% CO₂.

Induction and collection of M2-TAMs

THP-1 cells were seeded in 6-well plates at the density of 1×10⁶/ml. After treated with 100 ng/mL PMA for 6 h, THP-1 cells were added with 20 ng/mL IL-4 for another 18 h. After that, THP-1 cells were cultured with 2 ml fresh serum-free medium for 24 h. Then, the supernatant was collected to be frozen at -80 ℃ for later use. The expressions of CD68, CD163 and CD206 were identified by flow cytometry.

Treatment of M2-TAMs with quercetin

Quercetin (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO), and then stored at -20 ℃ for later use. Subsequently, M2-TAMs were treated with Quercetin.

Co-culture of M2-TAMs and CRC cell lines

CRC cells at the logarithmic growth phase were seeded into 6-well culture plates at the density of 1×10⁵/ml, and M2-TAMs were seeded into the upper transwell chamber (semipermeable membrane pore size: 0.4 µm, model: 3450, Costar) at the density of 2×10⁵/ml. Then, CRC cells were treated with the chamber. After 48 h of incubation, total RNAs and proteins were extracted from CRC cells.

Monodansylcardeverine (MDC) staining

MDC staining was performed to determine autophagic vesicle (AV) of M2-TAMs according to the previous reference [25].

Cell transfection

CRC cells were seeded into 6-well plates. When the confluence reached 40%, cells were transfected by si-has_circ_0006990/NC, miR-132-3p inhibitor/mimic, miR-342-3p inhibitor/mimic or miR-NC for 48 h using Lipofectamine 2000.

Colony formation assay

CRC cells were seeded into 6-well plates for 12 days. After that, cells were fixed with 4% paraformaldehyde for 15 min and stained with Giemsa solution for 20 min after rinsed with PBS. Finally, the data was calculated.

TUNEL assay

Cells were washed and permeabilized. Then, TUNEL reaction mixtures (50 µl) were used to incubate the cells for 60 min with no light. After that, peroxidase (POD, 50 µl) was used to incubate the slides for 30 min at 37°C. Then, cells were rinsed with PBS, and then diaminobenzidine (DAB, 50 µl) substrate solution was used to incubate the cells for 10 min. Finally, the expression of apoptotic cells was observed under an optical microscope.

Wound healing assay

CRC cell lines (1.0×10⁵/ml) were seeded overnight. Then, cells were underlined perpendicular to the cell culture plate with a small pipette head. After washing with PBS 3 times, serum-free medium was used for further culture, and the scratch widths at 0 and 24 h were recorded under an optical microscope.

Transwell invasion assay

Matrigel (100 µl) was used to pre-treat the upper chamber. CRC cells (1.0×10⁶ cells per chamber) were seeded into the upper chamber in medium (1% FBS). In addition, the lower chamber was supplemented with RPMI1640 (10% FBS). Subsequently, the chamber was rinsed and fixed at 4°C. Then, crystal violet (0.1%) was used to stain the chamber for 20 minutes. The data was observed under a microscope after the chamber was washed.

Western blot

RIPA was applied to extract protein from cell lines. BCA kit was applied to quantify the total protein. SDS-PAGE (10%) was applied to separate the proteins (40 µg per lane), and then proteins were transferred onto PVDF membranes. Subsequently, the membranes were incubated overnight at 4˚C with primary antibodies targeted against: Bcl-2 (1:1,000), Bax (1:1,000), MUC13 (1:1,000), E2F1 (1:1,000) and GAPDH (1:1,000) after blocked with skimmed milk (5%) for 1 h. Following primary incubation, HRP-conjugated secondary antibodies (1:5,000) were used to incubate the membranes for 1 h. ECL kit was used to visualize the protein bands. GAPDH was regarded as internal control. The densitometry analysis was performed by using IPP 6.0 (Image-Pro Plus 6.0).

Real time polymerase chain reaction (RT-qPCR)

TRIzol® reagent was applied to extract total RNA. PrimeScript RT reagent kit was used in reverse transcription. Then, SYBR premix Ex Taq II kit (Takara) was used in RT-qPCR, and Real-Time qPCRs were used three times: 2 minutes at 94°C, followed by 35 cycles (94°C for 30 s and 55°C for 45 s). Primers of circRNAs and miRNAs were listed in Table 2 and Table 3. The data were quantified using 2^−ΔΔCt method. GAPDH was regarded as internal control.

Table 2

**Primers of colorectal cancer-relevant circRNAs.**
CircRNAs	Primers
CircRNAs	Sense	Anti-sense
hsa_circ_0096088	5'-TGGAGGAGCCCAGGTATAAA-3'	5'-TCCATGTCGGGATCTTCTTC-3'
hsa_circ_0006990	5'-TGAAATGCCCAATGAAAATG3'	5'-GGCGAGGTGCTGTAGTCTTC-3'
hsa_circ_0004380	5'-GAATGCGGGAGTGATTGAGT-3'	5'-TCTTAGCACGTCCGATCTCA-3'
hsa_circ_0001946	5'-CATGTCTTCCAACGTCTCCA-3'	5'-CTGGAAGACCCGGAGTTGT-3'

Table 3

**Primers of hsa_circ_0006990-sponged miRNAs.**
MiRNAs	Primers
MiRNAs	Sense	Anti-sense
hsa-miR-3611	5'-GCGGCGGTTGTGAAGAAAGAAA-3'	5'-ATCCAGTGCAGGGTCCGAGG-3'
hsa-miR-377-3p	5'-GCGGCGGATCACACACAAAGGC-3'	5'-ATCCAGTGCAGGGTCCGAGG-3'
hsa-miR-342-3p	5'-GCGGCGGTCTCACACAGAAATC-3'	5'-ATCCAGTGCAGGGTCCGAGG-3'
hsa-miR-132-3p	5'-GCGGCGGTAACAGTCTACAGCC-3'	5'-ATCCAGTGCAGGGTCCGAGG-3'

Dual luciferase reporter gene assay

Hsa_circ_0006990 containing the binding sites of miR-132-3p/miR-342-3p was cloned into the pmirGLO vectors for establishment of hsa_circ_0006990 (WT/MT). Hsa_circ_0006990 (WT/MT) was transfected into CRC cells with miR-132-3p/miR-342-3p/NC mimics using Lipofectamine 2000. Dual-Glo Luciferase Assay System was used to analyze the result.

3’-UTR of E2F1/MUC13 containing the putative binding sites of miR-342-3p/miR-132-3p were obtained from Beyotime (Shanghai, China), then were cloned into the pmirGLO vectors to construct wild type/mutant type vectors E2F1/MUC13 (WT/MT). E2F1/MUC13 (WT/MT) was transfected into cells using Lipofectamine 2000 (Thermo Fisher Scientific). Dual-Glo Luciferase Assay System was used to analyze the result.

Statistical analyses

Three independent experiments were performed in each group. In addition, the mean ± standard deviation (SD) was used to express all data. The comparisons between two groups were analyzed using Student’s t-test, and the differences between multiple groups (more than 2 groups) were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test (Graphpad Prism7). P<0.05 indicates a significant change.

Quercetin suppressed the polarization of M2-TAMs via inhibiting the autoiphagy

In order to induce THP-1 cells into M2-TAMs, THP-1 cells were treated with PMA and IL-4. As revealed in Figure 1A, cells exhibited more pseudopods and round-shaped after 48 h of treatment. (Figure 1A). This phenomenon was in consistent with the feature of M2-TAMs [26]. Furthermore, the percentage of CD68, CD163 and CD206 in THP-1 cells was significantly increased by PMA and IL-4 (Figure 1B). These data indicated that THP-1 cells were successfully induced into M2-TAMs.

Additionally, the data of MDC staining revealed that autophagy of M2-TAMs was promoted by rapamycin, while it was obviously inhibited by bafilomycina1 and quercetin (Figure 1C). Meanwhile, bafilomycin A1 and Quercetin notably decreased the percentage of CD163 and CD206 (Figure 1D). Taken together, Quercetin suppressed autophagy of M2-TAMs and induced the differentiation into M1-TAMs.

Quercetin significantly reverses M2-TAMs-induced proliferation of CRC cells via inhibiting the autophagy

CRC cells were co-cultured with M2-TAMs. Then, colony formation assay was performed. The data revealed that proliferation of CRC cells was significantly promoted by M2-TAMs, which was further increased by rapamycin (Figure 2A). In contrast, M2-TAMs-induced CRC cell proliferation was significantly reversed in the presence of Bafilomycin A1 or Quercetin (Figure 2A). Moreover, Quercetin or Bafilomycin A1 greatly induced the apoptosis of M2-TAMs-treated CRC cells (Figure 2B). Meanwhile, M2-TAMs significantly inhibited the level of Bax and upregulated the expression of Bcl-2 in CRC cells, while this phenomenon was greatly restored by Bafilomycin A1 or Quercetin (Figure 2C and 2D). In summary, Quercetin significantly reversed M2-TAMs-induced proliferation of CRC cells via inhibiting the autophagy.

Quercetin significantly restores M2-TAMs-induced migration and invasion of CRC cells via inhibiting the autophagy

To investigate the cell migration, wound healing assay was used. As shown in Figure 3A, M2-TAMs significantly promoted the migration of CRC cells, while the effect of M2-TAMs was obviously reversed by Bafilomycin A1 or Quercetin. Consistently, M2-TAMs-indcued CRC cell invasion was notably inhibited by Bafilomycin A1 or Quercetin (Figure 3B). To sum up, Quercetin significantly restored M2-TAMs-induced migration and invasion of CRC cells via inhibiting the autophagy.

Quercetin could regulate hsa_circ_0006990 in M2-TAMs-induced CRC cells

The differentially expressed circRNAs in CRC were presented in Table 1 and Supplementary Figure 1A. Among these differentially expressed circRNAs, hsa_circ_0006990 was reported to regulate the tumorigenesis of CRC [27]. Thus, hsa_circ_0006990 was selected in our research. Furthermore, downstream miRNAs of hsa_circ_0006990, as predicted by bioinformatics tool (https://circinteractome.nia.nih.gov/rna_binding_protein.html), were also measured in CRC cells (Supplementary Figure 1B). It was indicated that miR-132-3p and miR-342-3p were both downregulated in CRC cells co-cultured with M2-TAMs. In addition, quercetin and Bafilomycin A1 reversed the effect of M2-TAMs on miR-132-3p and miR-342-3p levels (Supplementary Figure 1B). Based on the above results, miR-132-3p and miR-342-3p were selected in our study.

Table 1

**Screening of oncogenic circRNAs in CRC**
CircRNAs	Gene symbol	Position*	Genomic length*	Spliced length*	Expression change in CRC	Reference
hsa_circ_0096088	MACROD1	chr11:63918710-63919865	1155	219	↑	[PMID: 32564659]
hsa_circ_0006990	VAPA	chr18:9931806-9937063	5257	338	↑	[PMID: 32564659]
hsa_circ_0004380	TRAPPC9	chr8:141407718-141415797	8079	248	↑	[PMID: 32564659]
hsa_circ_0001946	CDR1	chrX:139865339-139866824	1485	1485	↑	[PMID: 32508871]
*CircBase (http://circrna.org/).

The level of miR-132-3p/miR-342-3p in CRC cells was significantly upregulated by miR-132-3p/miR-342-3p mimics but inhibited by miR-132-3p/ miR-342-3p inhibitor (Supplementary Figure 2A and 2B). Meanwhile, the luciferase activity in WT-hsa_circ_0006990 was significantly reduced by miR-132-3p/miR-342-3p mimics (Supplementary Figure 2C and 2D). Furthermore, the expression of miR-132-3p or miR-342-3p in CRC cells was negatively regulated by hsa_circ_0006990 (Supplementary Figure 2E and 2F).

Quercetin inhibited the proliferation and invasion of CRC cells via mediation of hsa_circ_0006990

To investigate the effect of Quercetin on hsa_circ_0006990 expression, RT-qPCR was performed. As demonstrated in Figure 4A, Quercetin significantly inhibited the level of hsa_circ_0006990 in CRC cells. In addition, the inhibitory effect of Quercetin on CRC cell proliferation was significantly rescued by pcDNA3.1-hsa_circ_0006990 (Figure 4B). Consistently, overexpression of hsa_circ_0006990 significantly reversed Quercetin-induced cell apoptosis (Figure 4C). Furthermore, the migration and invasion of CRC cells was significantly inhibited by Quercetin, which was obviously restored by pcDNA3.1-hsa_circ_0006990 (Figure 5A and 5B). To sum up, Quercetin inhibited the proliferation and invasion of CRC cells via mediation of hsa_circ_0006990.

MiR-342-3p directly targets E2F1 in CRC cells

To explore the downstream mRNA of miR-342-3p, targetscan was used. As revealed in Figure 6A, miR-342-3p had binding sites with E2F1, and the luciferase activity in WT-E2F1 was notably decreased by miR-342-3p mimics (Figure 6B). Meanwhile, miR-342-3p could negatively regulate the level of E2F1 in CRC cells (Figure 6C). Furthermore, the effect of hsa_circ_0006990 silencing on E2F1 level was markedly reversed by miR-342-3p inhibitor (Figure 6D). Altogether, MiR-342-3p directly targets E2F1 in CRC cells.

MiR-132-3p directly targets MUC13 in CRC cells

To explore the downstream mRNA of miR-132-3p, targetscan was used. As revealed in Figure 7A, miR-132-3p had binding sites with MUC13, and the luciferase activity in WT-MUC13 was notably decreased by miR-132-3p mimics (Figure 7B). Meanwhile, miR-132-3p could negatively regulate the level of MUC13 in CRC cells (Figure 7C). Furthermore, the effect of hsa_circ_0006990 silencing on MUC13 level was markedly reversed by miR-132-3p inhibitor (Figure 7D). Altogether, MiR-132-3p directly targets MUC13 in CRC cells.

Quercetin has been reported to inhibit the progression of malignant tumors (including breast cancer, ovarian cancer and gastric cancer) [28–30]. Consistently, we found Quercetin could inhibit the progression of CRC. Moreover, this research revealed Quercetin could inhibit the M2 polarization of macrophages, and it could inhibit the autophagy. Thus, this study firstly explored the function of Quercetin in M2-TAMs and autophagy during the progression of CRC, suggesting that Quercetin could act as an inhibitor in M2-TAMs.

It has been confirmed that M2-TAMs could play a vital role in tumor microenvironment [31, 32]. For instance, cytokines generated by M2-TAMs (including TNF-α and TGF-ß) could induce the angiogenesis in breast cancer [33]; the activation of M2-TAMs could lead to the poor prognosis among patients with tumors (lymphoma, breast cancer, et al) [34, 35]. Consistently, our data found that Quercetin could inhibit the tumorigenesis of CRC via inhibiting the polarization of M2 macrophages. On the other hand, it has been reported that autophagy activation could lead to the polarization of macrophages [36, 37]. Our research indicated that autophagy could induce the polarization of M2 macrophages, and Quercetin could inhibit the autophagy in M2 macrophages. Thus, it could be suggested that Quercetin could inhibit the polarization of M2 macrophages via inhibiting the autophagy. Notably, the mechanism by which Quercetin regulates the autophagy remains unclear, and it is needed to be further explored in future.

This study found that hsa_circ_0006990 was upregulated in CRC cells co-cultured with M2-TAMs. It has been indicated that hsa_circ_0006990 might acts as a promoter in CRC [27]. Thus, our data was in consistent to this previous study. In addition, our study firstly explored the relation between Quercetin and hsa_circ_0006990 in CRC, suggesting that Quercetin could inhibit the tumorigenesis of CRC via downregulation of hsa_circ_0006990. Meanwhile, the detailed correlation among hsa_circ_0006990, M2-TAMs and autophagy remains further investigating.

MiR-342-3p and miR-132-3p were found to be sponged by hsa_circ_0006990 in this study. Thus, our research firstly explored the relation between hsa_circ_0006990 and miR-342-3p/miR-132-3p in CRC. In addition, miR⁃342⁃3p/E2F1 and miR-132/MUC13 were also found to be involved in Quercetin-mediated CRC progression. It has been reported that miR⁃342⁃3p could inhibit the progression of lung cancer [38], prostate cancer [39], osteosarcoma [40], glioma [41] and CRC [42, 43]. In addition, downregulation E2F1 was reported to enhance the sensitivity of CRC cells to oxaliplatin treatment [44]. Based on these backgrounds, it could be concluded that Quercetin significantly inhibited the tumorigenesis of CRC via mediation of hsa_circ_0006990/miR-342-3p/E2F1 axis. On the other hand, miR-132-3p was reported to act as an inhibitor in cancer progression. In detail, miR-132-3p could inhibit the progression of lung cancer [45], glioma [46], pituitary tumor [47], CRC [48] and bladder cancer [49]. More importantly, high expressed miR-132-3p could sensitize CRC cells to adriamycin treatment [50]. Besides, MUC13 was identified to be the downstream of miR-132, and it was confirmed to be upregulated in CRC tissues [51]. Thereby, our data confirmed that Quercetin notably suppressed the progression of CRC via mediation of hsa_circ_0006990/miR-132-3p/MUC13 axis

In conclusion, Quercetin could inhibit the tumorigenesis of CRC via inhibiting the polarization of M2 macrophages and downregulating hsa_circ_0006990. Thus, our study might shed new lights on exploring the new methods against CRC.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

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

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Health System Innovation Project of Shanghai Putuo Science and Technology Commission (No. ptkwws202002), Traditional Chinese Medicine Clinical Key Specialty Construction Project of Shanghai Putuo District (No. ptzyzk2101), Traditional Chinese Medicine Research Project of Shanghai Municipal Commission of Health and Family Planning (No. 2018JP004), Traditional Chinese Medicine Research Project of Shanghai Municipal Health Commission (No. 2020JP004), Research Project of Shanghai Putuo District Central Hospital (No. 2020364A) and National Natural Science Foundation of Shanghai (No. 19ZR1410500).

Authors' contributions

BC, HX and LW made major contributions to the conception, design and manuscript drafting of this study. TW, SW, HY, GW, MX and XH were responsible for data acquisition, data analysis, data interpretation and manuscript revision. WX, RZ and WD made substantial contributions to conception and design of the study and revised the manuscript. All authors agreed to be accountable for all aspects of the work. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

1. Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, Stein KD, Alteri R, Jemal A: Cancer treatment and survivorship statistics, 2016. CA: a cancer journal for clinicians 2016, 66(4):271-289.

2. Vasen HF, Tomlinson I, Castells A: Clinical management of hereditary colorectal cancer syndromes. Nature reviews Gastroenterology & hepatology 2015, 12(2):88-97.

3. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F: Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer 2015, 136(5):E359-386.

4. Ulaganathan V, Kandiah M, Mohd Shariff Z: A case-control study of the association between metabolic syndrome and colorectal cancer: a comparison of International Diabetes Federation, National Cholesterol Education Program Adults Treatment Panel III, and World Health Organization definitions. Journal of gastrointestinal oncology 2018, 9(4):650-663.

5. Mohammed HA, Sulaiman GM, Anwar SS, Tawfeeq AT, Khan RA, Mohammed SAA, Al-Omar MS, Alsharidah M, Rugaie OA, Al-Amiery AA: Quercetin against MCF7 and CAL51 breast cancer cell lines: apoptosis, gene expression and cytotoxicity of nano-quercetin. Nanomedicine (Lond) 2021, 16(22):1937-1961.

6. Soofiyani SR, Hosseini K, Forouhandeh H, Ghasemnejad T, Tarhriz V, Asgharian P, Reiner Z, Sharifi-Rad J, Cho WC: Quercetin as a Novel Therapeutic Approach for Lymphoma. Oxid Med Cell Longev 2021, 2021:3157867.

7. Qi X, Xu H, Zhang P, Chen G, Chen Z, Fang C, Lin L: Investigating the Mechanism of Scutellariae barbata Herba in the Treatment of Colorectal Cancer by Network Pharmacology and Molecular Docking. Evid Based Complement Alternat Med 2021, 2021:3905367.

8. Pollard JW: Tumour-educated macrophages promote tumour progression and metastasis. Nature reviews Cancer 2004, 4(1):71-78.

9. Coussens LM, Werb Z: Inflammation and cancer. Nature 2002, 420(6917):860-867.

10. Salmaninejad A, Valilou SF, Soltani A, Ahmadi S, Abarghan YJ, Rosengren RJ, Sahebkar A: Tumor-associated macrophages: role in cancer development and therapeutic implications. Cell Oncol (Dordr) 2019, 42(5):591-608.

11. Yue ZQ, Liu YP, Ruan JS, Zhou L, Lu Y: Tumor-associated macrophages: a novel potential target for cancer treatment. Chinese medical journal 2012, 125(18):3305-3311.

12. Rigo A, Gottardi M, Zamo A, Mauri P, Bonifacio M, Krampera M, Damiani E, Pizzolo G, Vinante F: Macrophages may promote cancer growth via a GM-CSF/HB-EGF paracrine loop that is enhanced by CXCL12. Molecular cancer 2010, 9:273.

13. Porta C, Riboldi E, Ippolito A, Sica A: Molecular and epigenetic basis of macrophage polarized activation. Seminars in immunology 2015, 27(4):237-248.

14. Haabeth OA, Lorvik KB, Hammarstrom C, Donaldson IM, Haraldsen G, Bogen B, Corthay A: Inflammation driven by tumour-specific Th1 cells protects against B-cell cancer. Nature communications 2011, 2:240.

15. Corthay A, Skovseth DK, Lundin KU, Rosjo E, Omholt H, Hofgaard PO, Haraldsen G, Bogen B: Primary antitumor immune response mediated by CD4+ T cells. Immunity 2005, 22(3):371-383.

16. Chen TA, Wang JL, Hung SW, Chu CL, Cheng YC, Liang SM: Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production. PloS one 2011, 6(8):e23317.

17. Shao LN, Zhu BS, Xing CG, Yang XD, Young W, Cao JP: Effects of autophagy regulation of tumor-associated macrophages on radiosensitivity of colorectal cancer cells. Molecular medicine reports 2016, 13(3):2661-2670.

18. Chen P, Cescon M, Bonaldo P: Autophagy-mediated regulation of macrophages and its applications for cancer. Autophagy 2014, 10(2):192-200.

19. Cocquerelle C, Mascrez B, Hetuin D, Bailleul B: Mis-splicing yields circular RNA molecules. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 1993, 7(1):155-160.

20. Xia S, Feng J, Lei L, Hu J, Xia L, Wang J, Xiang Y, Liu L, Zhong S, Han L et al: Comprehensive characterization of tissue-specific circular RNAs in the human and mouse genomes. Briefings in bioinformatics 2017, 18(6):984-992.

21. Jeck WR, Sorrentino JA, Wang K, Slevin MK, Burd CE, Liu J, Marzluff WF, Sharpless NE: Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 2013, 19(2):141-157.

22. Pervouchine DD: Circular exonic RNAs: When RNA structure meets topology. Biochimica et biophysica acta Gene regulatory mechanisms 2019, 1862(11-12):194384.

23. Guo JU, Agarwal V, Guo H, Bartel DP: Expanded identification and characterization of mammalian circular RNAs. Genome biology 2014, 15(7):409.

24. Yuan Y, Liu W, Zhang Y, Sun S: CircRNA circ_0026344 as a prognostic biomarker suppresses colorectal cancer progression via microRNA-21 and microRNA-31. Biochemical and biophysical research communications 2018, 503(2):870-875.

25. Biederbick A, Kern HF, Elsasser HP: Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. European journal of cell biology 1995, 66(1):3-14.

26. Yunna C, Mengru H, Lei W, Weidong C: Macrophage M1/M2 polarization. Eur J Pharmacol 2020, 877:173090.

27. Li XN, Wang ZJ, Ye CX, Zhao BC, Huang XX, Yang L: Circular RNA circVAPA is up-regulated and exerts oncogenic properties by sponging miR-101 in colorectal cancer. Biomed Pharmacother 2019, 112:108611.

28. Nguyen LT, Lee YH, Sharma AR, Park JB, Jagga S, Sharma G, Lee SS, Nam JS: Quercetin induces apoptosis and cell cycle arrest in triple-negative breast cancer cells through modulation of Foxo3a activity. The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology 2017, 21(2):205-213.

29. Liu Y, Gong W, Yang ZY, Zhou XS, Gong C, Zhang TR, Wei X, Ma D, Ye F, Gao QL: Quercetin induces protective autophagy and apoptosis through ER stress via the p-STAT3/Bcl-2 axis in ovarian cancer. Apoptosis : an international journal on programmed cell death 2017, 22(4):544-557.

30. Zhou J, Li LU, Fang LI, Xie H, Yao W, Zhou X, Xiong Z, Wang LI, Li Z, Luo F: Quercetin reduces cyclin D1 activity and induces G1 phase arrest in HepG2 cells. Oncology letters 2016, 12(1):516-522.

31. Chen P, Bonaldo P: Role of macrophage polarization in tumor angiogenesis and vessel normalization: implications for new anticancer therapies. International review of cell and molecular biology 2013, 301:1-35.

32. Qian BZ, Pollard JW: Macrophage diversity enhances tumor progression and metastasis. Cell 2010, 141(1):39-51.

33. Bingle L, Lewis CE, Corke KP, Reed MW, Brown NJ: Macrophages promote angiogenesis in human breast tumour spheroids in vivo. British journal of cancer 2006, 94(1):101-107.

34. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL, Walker MG, Watson D, Park T et al: A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. The New England journal of medicine 2004, 351(27):2817-2826.

35. Dave SS, Wright G, Tan B, Rosenwald A, Gascoyne RD, Chan WC, Fisher RI, Braziel RM, Rimsza LM, Grogan TM et al: Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. The New England journal of medicine 2004, 351(21):2159-2169.

36. Yang Y, Wei S, Chu K, Li Q, Zhou Y, Ma Y, Xue L, Tian H, Tao S: Upregulation of autophagy in M2 macrophage by vitamin D alleviates crystalline silica-induced pulmonary inflammatory damage. Ecotoxicol Environ Saf 2021, 225:112730.

37. Yang S, Zhang X, Zhang H, Lin X, Chen X, Zhang Y, Lin X, Huang L, Zhuge Q: Dimethyl itaconate inhibits LPSinduced microglia inflammation and inflammasomemediated pyroptosis via inducing autophagy and regulating the Nrf2/HO1 signaling pathway. Mol Med Rep 2021, 24(3).

38. Xue X, Fei X, Hou W, Zhang Y, Liu L, Hu R: miR-342-3p suppresses cell proliferation and migration by targeting AGR2 in non-small cell lung cancer. Cancer letters 2018, 412:170-178.

39. Hu K, Mu X, Kolibaba H, Yin Q, Liu C, Liang X, Lu J: Metadherin is an apoptotic modulator in prostate cancer through miR-342-3p regulation. Saudi journal of biological sciences 2018, 25(5):975-981.

40. Zhang S, Liu L, Lv Z, Li Q, Gong W, Wu H: MicroRNA-342-3p Inhibits the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting Astrocyte-Elevated Gene-1 (AEG-1). Oncology research 2017, 25(9):1505-1515.

41. Wang Q, Li P, Li A, Jiang W, Wang H, Wang J, Xie K: Plasma specific miRNAs as predictive biomarkers for diagnosis and prognosis of glioma. Journal of experimental & clinical cancer research : CR 2012, 31:97.

42. Weng W, Okugawa Y, Toden S, Toiyama Y, Kusunoki M, Goel A: FOXM1 and FOXQ1 Are Promising Prognostic Biomarkers and Novel Targets of Tumor-Suppressive miR-342 in Human Colorectal Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2016, 22(19):4947-4957.

43. Tao K, Yang J, Guo Z, Hu Y, Sheng H, Gao H, Yu H: Prognostic value of miR-221-3p, miR-342-3p and miR-491-5p expression in colon cancer. American journal of translational research 2014, 6(4):391-401.

44. Fang Z, Gong C, Yu S, Zhou W, Hassan W, Li H, Wang X, Hu Y, Gu K, Chen X et al: NFYB-induced high expression of E2F1 contributes to oxaliplatin resistance in colorectal cancer via the enhancement of CHK1 signaling. Cancer letters 2018, 415:58-72.

45. Guo H, Zhang X, Chen Q, Bao Y, Dong C, Wang X: miR-132 suppresses the migration and invasion of lung cancer cells by blocking USP9X-induced epithelial-mesenchymal transition. American journal of translational research 2018, 10(1):224-234.

46. Wang YZ, Han JJ, Fan SQ, Yang W, Zhang YB, Xu TJ, Xu GM: miR-132 weakens proliferation and invasion of glioma cells via the inhibition of Gli1. European review for medical and pharmacological sciences 2018, 22(7):1971-1978.

47. Renjie W, Haiqian L: MiR-132, miR-15a and miR-16 synergistically inhibit pituitary tumor cell proliferation, invasion and migration by targeting Sox5. Cancer letters 2015, 356(2 Pt B):568-578.

48. Zheng YB, Luo HP, Shi Q, Hao ZN, Ding Y, Wang QS, Li SB, Xiao GC, Tong SL: miR-132 inhibits colorectal cancer invasion and metastasis via directly targeting ZEB2. World journal of gastroenterology 2014, 20(21):6515-6522.

49. Wei XC, Lv ZH: MicroRNA-132 inhibits migration, invasion and epithelial-mesenchymal transition via TGFbeta1/Smad2 signaling pathway in human bladder cancer. OncoTargets and therapy 2019, 12:5937-5945.

50. Liu Y, Zhang M: miR-132 Regulates Adriamycin Resistance in Colorectal Cancer Cells Through Targeting Extracellular Signal-Regulated Kinase 1. Cancer biotherapy & radiopharmaceuticals 2019, 34(6):398-404.

51. Walsh MD, Young JP, Leggett BA, Williams SH, Jass JR, McGuckin MA: The MUC13 cell surface mucin is highly expressed by human colorectal carcinomas. Human pathology 2007, 38(6):883-892.

SupplementaryFigure1.jpg
Quercetin could regulate hsa_circ_0006990 in M2-TAMs-induced CRC cells. (A) CRC cells were co-cultured with M2-TAMs. The differentially expressed circRNAs were presented. (B) The differentially expressed miRNAs (downstream of has_circ_0006990) were presented.
SupplementaryFigure2.jpg
Quercetin could regulate hsa_circ_0006990 in M2-TAMs-induced CRC cells. (A) CRC cells were transfected with miR-132-3p mimics/inhibitor. The level of miR-132-3p in CRC cells was tested by RT-qPCR. *P<0.05 compared to miR-NC. (B) CRC cells were transfected with miR-342-3p mimics/inhibitor. The level of miR-342-3p in CRC cells was tested by RT-qPCR. *P<0.05 compared to miR-NC. (C) MiR-132-3p was predicted to be the downstream miRNA of hsa_circ_0006990. The relative luciferase activity of WT-hsa_circ_0006990 was measured by dual luciferase assay. *P<0.05. (D) MiR-132-3p was predicted to be the downstream miRNA of hsa_circ_0006990. The relative luciferase activity of WT-hsa_circ_0006990 was measured by dual luciferase assay. *P<0.05. (E, F) CRC cells were transfected with si-hsa_circ_0006990 or pcDNA3.1-hsa_circ_0006990. The level of miR-132-3p or miR-342-3p in CRC cells was tested by RT-qPCR. *P<0.05.

Download PDF

Version 1

posted

You are reading this latest preprint version

Quercetin inhibits the tumorigenesis of colorectal cancer cells through downregulation of hsa_circ_0006990

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Cell culture

Induction and collection of M2-TAMs

Treatment of M2-TAMs with quercetin

Co-culture of M2-TAMs and CRC cell lines

Monodansylcardeverine (MDC) staining

Cell transfection

Colony formation assay

TUNEL assay

Wound healing assay

Transwell invasion assay

Western blot

Real time polymerase chain reaction (RT-qPCR)

Dual luciferase reporter gene assay

Statistical analyses

Results

Quercetin suppressed the polarization of M2-TAMs via inhibiting the autoiphagy

Quercetin significantly reverses M2-TAMs-induced proliferation of CRC cells via inhibiting the autophagy

Quercetin significantly restores M2-TAMs-induced migration and invasion of CRC cells via inhibiting the autophagy

Quercetin could regulate hsa_circ_0006990 in M2-TAMs-induced CRC cells

Quercetin inhibited the proliferation and invasion of CRC cells via mediation of hsa_circ_0006990

MiR-342-3p directly targets E2F1 in CRC cells

MiR-132-3p directly targets MUC13 in CRC cells

Discussion

Declarations

References

Supplementary Files

Status:

Version 1