The isoflavone fraction from soybean presents mRNA maturation inhibition activity

Abstract

Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.

Soyaflavone HG, luciferase, RNA-FISH

The mRNA maturation process (5′ capping, splicing, and 3′ end-processing) in the nucleus is a critical step for gene expression, cell proliferation, and survival.^1−6,) Of these steps, splicing events have received much attention recently as they were found to be central to the pathology of numerous diseases especially cancer, therefore making mRNA splicing inhibition an important therapeutic strategy.^4−9,) Previously, several compounds with mRNA inhibitory activity were reported to be derived from the culture of micro-organisms.^10−12,) They interfered with the mRNA splicing through directly inhibiting the recruitment of key component(s) required for mRNA splicing or indirectly by modulating the phosphorylation status of essential SR proteins by inhibiting the kinase activity.^13−17,) However, a limited number of inhibitors are available up to date. In addition, the active compounds identified so far are not of dietary origins except for resveratrol,^18,) although a lot of active compounds like PPARγ agonists of plant and dietary origins are discovered.¹⁹⁻²¹⁾ Inhibiting the mRNA maturation process by taking in the active compound of dietary origins may hamper the tumor growth. It becomes a support for the better quality of life.

To investigate the various aspects of mRNA processing, we have established a monitoring system which is suitable for the screening of a wide range of chemical compounds that regulate mRNA processing.^22,) The first assay is designed to assess whether the expression of intron-containing Renilla luciferase reporter is affected. The second assay measures the subcellular localization of mRNA (i.e. retention of pre-mRNA).^22,23) Using this mRNA quality control system, compounds in the dietary sample can be examined for their potential inhibitory activity on mRNA maturation by RNA-fluorescence in situ hybridization (FISH). Here, we examined the inhibitory activity on mRNA maturation of dietary origins. These include wheat, cumin, turmeric, carrot, soybean, and various kinds of miso samples. We found that the isoflavone fraction (i.e. Soya-flavone HG, its content is shown in the reference²⁴⁾) extracted from soybean possessed mRNA processing inhibitory activity.

For the measurement of Renilla luciferase activity, 2 × 10⁴ HeLa cells expressing Renilla luciferase (RLM1 cells) cells were inoculated in a 24-well plate.^22,25) The isoflavone fraction (0–0.5% weight/vol) dissolved in PBS was added in the culture media for 24 h. The cells were lysed by Passive Lysis Buffer (Promega, Madison, WI) for 30 min. The clear lysate was obtained after spinning. The Renilla luciferase activity was normalized with total protein content (Nacalai, Kyoto, Japan). From the Renilla luciferase screening, the addition of isoflavone fraction from soybean decreased the Renilla luciferase activity in a dose-dependent manner (Fig. 1).

Fig. 1.

Isoflavone fraction from soybean decreased.

Notes: Renilla luciferase activity. Renilla luciferase activity was measured and normalized by total protein using RLM1 cells stably expressing Renilla luciferase. Luciferase activity was shown as relative activity to control sample. Gex1A (10 ng/mL) was used as positive control. Each value is the mean ± SD of triplicate experiments. Statistical analysis was performed using One-way ANOVA followed by Dunnett’s test. p-values, *p < 0. 05, **p < 0. 01, ***p < 0. 001.

We next observed the mRNA localization by RNA-FISH. U2OS cells (5 × 10⁴ cells/mL) on coverslips in 12-well plate were treated with test samples for 24 h, fixed in 10% formaldehyde in PBS for 20 min, and permeabilized in 0.1% Triton X-100 in PBS for 10 min. Cells were washed once with 2× standard sodium citrate (SSC) for 5 min to exchange the buffer content, prehybridized with ULTRAhyb-Oligo Hybridization Buffer (Ambion, Austin, TX) for 1 h at 42 °C in a humidified chamber, and then incubated overnight with 20 pmol Cy3-labeled oligo-dT₄₅ probe diluted with hybridization buffer. Cells were washed for 20 min at 42 °C with 2× SSC, 0.5× SSC, and 0.1× SSC, respectively. The nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI).^22,26) In control cells, mRNA signal was mainly detected in the cytoplasm (Fig. 2(A)). By contrast, the addition of isoflavone fraction to the cell resulted in the accumulation of mRNA in the nucleus (Fig. 2(A)). The increase in mRNA localization in the nucleus seems to be less than that of Gex1A, an inhibitor of mRNA splicing. To quantify mRNA localization, the ratio of mRNA signal in the nucleus to that in the cytoplasm was calculated using ImageJ software (https://imagej.nih.gov/ij/) according to the instructions. In control cells, the population of mRNA in the nucleus was 31% of total mRNA (Fig. 2(B)). When the isoflavone fraction was added in the cell culture, the subcellular distribution of mRNA in the nucleus has gradually but steadily increased. The addition of the isoflavone fraction with a final concentration of 0.5% resulted in a nucleus/cytoplasm mRNA signal of 42%. These results indicate that the isoflavone fraction contains the compound(s) that inhibit the mRNA processing.

Fig. 2.

Isoflavone fraction inhibited mRNA maturation process.

Notes: (A) The bulk mRNA was visualized by Cy3 labeled olio dT₄₅ probe in U2OS cells. Cells were treated with indicated concentration of isoflavone for 24 h. Gex1A (30 ng/mL) was used as positive control. The nuclei were stained with DAPI. (B) Subcellular distribution of mRNA. The signal intensity of the whole cell and that in the nucleus was quantified using ImageJ. The cytoplasmic intensity was calculated by subtracting the nuclear intensity from the whole cellular intensity. Each value is the mean ± SD (n = 8). Statistical analysis was performed using One-way ANOVA followed by Dunnett’s test. p-values, **p < 0. 01, ***p < 0. 001. Scale bar, 10 μm.

To investigate whether the addition of the isoflavone fraction inhibits the cell metabolism, we performed the cell proliferation assay using MTT reagent. The cells were inoculated at 8 × 10³ cells/mL in a 96-well plate and cultured for 20 h. The test samples were added to each U2OS cell cultured wells and incubated for 20 h. Then, 5 μL of MTT reagent (5 mg/mL) (Sigma-Aldrich Japan, Tokyo, Japan) was added to the cells. And the cells were incubated for 4 h and solubilized with 10 mM NH₄Cl containing 10% SDS (pH 7.0). Cell proliferation was estimated by measuring the optical absorbance at 600 nm. When the dose of isoflavone fraction was 0–0.1%, the cell proliferation activity was stable, but it decreased with an increasing concentration of 0.1–0.5% (Fig. 3). The reduction in cell proliferation was positively correlated with the accumulation of mRNA in the nucleus, suggesting that the inhibition of mRNA maturation in the nucleus might affect the cell proliferation.

Fig. 3.

Isoflavone fraction inhibited cell proliferation.

Notes: The effect for the cell proliferation by isoflavone was estimated by MTT assay. Isoflavone fraction was added to U2OS cells for 24 h, and then, MTT regent was added to cell culture media for 4 h. The produced formazan was solubilized with the buffer containing 10 mM NH₄Cl and 10% SDS. Absorbance was measured at 600 nm. Relative intensity of control absorbance was calculated as 100%. Each value is the mean ± SD of triplicate experiments. Statistical analysis was performed using One-way ANOVA followed by Dunnett’s test. p-values, **p < 0. 01.

In the present study, we have screened the active compounds of dietary origin which inhibit mRNA processing and found that the isoflavone fraction showed anti-mRNA maturation activity. Compared with the treatment of mRNA splicing inhibitor, Gex1A, isoflavone fraction exhibited weak inhibitory effect. If the activity of active compound(s) were equally strong in a similar manner to that of Gex1A, they might not be suitable to be ingested as food. It is, therefore, reasonable that their activity is less than that of pharmacological agents. In addition, it has been shown that resveratrol can influence the alternative splicing of certain types of mRNA.¹⁸⁾ These findings implicate that a novel anti-mRNA maturation activity can be attained from a wide range of resources of dietary origins. Daily incorporation of such weak compounds of dietary origin may have a benefit for retarding the undesired cell growth.

As a conclusion, we demonstrated that our mRNA monitoring system is suitable to detect the mRNA maturation-inhibitory activity. Further research is required to identify active compound(s) in the isoflavone fraction and to determine the target process for mRNA maturation (capping, splicing, 3′ end-processing) and the target protein(s).

Author contributions

K.M., M.Y. carried out the experiments. M.K carried out the statistical analysis. K.M. and M.S. designed the study in cooperation with M.Y., K.T., N.M., N.H., and S.Y. M.I.F.A., T.M., and M.S. wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported in part by “grants-in-aid” from Central Miso Research Institute, The Skylark Food Science Institute, Fuji Foundation for Protein Research, Takano Life Science Research Foundation, Asahi Group Foundation, The Tojuro Iijima Foundation for Food Science and Technology, The Foundation for Dietary Scientific Research and JSBBA Innovative Research Program Award to S.M.; JSPS Kakenhi [grant number 15K11299 to Y.S.].

Footnotes

Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; PBS, phosphate buffered saline; SSC, standard sodium citrate.

Acknowledgment

We thank Fuji Foundation for Protein Research for providing Soyaflavone HG for the test compound and Kyowahakko for providing Gex1A as positive control reagent.