miR193a-5p Mediated ZNF746 and c-Myc Signaling Axis Is Critically Involved in Morusin Induced Apoptosis in Colorectal Cancer Cells

Novel target therapy is on the spotlight for effective cancer therapy. Hence, in the present study, the underlying apoptotic mechanism of Morusin was explored in association with miR193a-5p mediated ZNF746/c-Myc signaling axis in colorectal cancer cells (CRCs). Herein, Morusin reduced the viability and the number of colonies in HCT116 and SW480 CRCs. Additionally, Morusin increased sub-G1 population, cleavages of poly (ADP-ribose) polymerase (PARP) and caspase-3 and inhibited the expression of zinc finger protein 746 (ZNF746) and c-Myc in HCT116 and SW480 cells. Conversely, overexpression of ZNF746 suppressed the ability of Morusin to abrogate the expression of c-Myc in HCT116 cells, as ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization in HCT116 cells by immunofluorescence and immunoprecipitation. Notably, Morusin upregulated miR193a-5p as a tumor suppressor, while miR193a-5p inhibitor masked the ability of Morusin to reduce the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells. To our knowledge, these findings provide the novel insight on miR193a-5p mediated inhibition of ZNF746/c-Myc signaling in Morusin induced apoptosis in CRCs.


Introduction
Colorectal cancer (CRC) is reported as the fourth most prevalent cancer in women and the third most common cancer in men worldwide [1,2]. Despite modern medicine, approximately 50% of CRC patients have experienced tumor recurrence, and their overall mortality rate is estimated up to approximately 40% [3]. Though chemotherapy, mainly with 5-FU, oxaliplatin (FOLFOX), Irinotecan and Cetuximab, radiotherapy and surgery. have been utilized for CRC treatment for years, recently molecular target therapy is of interest for EGFR, RAS, and VEGF [4,5].
It is well documented that c-Myc is a biomarker for poor prognosis of CRC patients, as it is a transcription factor that is critically associated with cell growth, cell adhesion, proliferation, and apoptosis [6]. Hence, to target c-Myc has been considered a good strategy for prevention or treatment of CRCs [7]. Additionally, ZNF746, with a C2HC/C2H2 type zinc finger protein at its C terminus [8,9] and a Kruppel-associated box at its N terminus [10,11], is known to act as a suppressor of PPAR gamma [12], regulate Parkin as a ubiquitin E3 ligase [13], and enhance the stability of c-Myc [1]. Additionally, accumulating evidence reveals that many miRNAs are critically involved in cancer biology as oncogenes or tumor suppressors [14][15][16].
Additionally, some natural compounds such as quercetin [17], ursolic acid [18], and curcumin [19] are being considered for combination therapy with classical anticancer agents. In the same line, Morusin, one of the prenylated flavonoids [20][21][22] derived from the root bark of Morus alba, is known to have anti-microbial [23], anti-inflammatory [24], and antitumor [3,25,26] effects. Nonetheless, its underlying molecular mechanism remains unclear in CRCs to date. Thus, in the present study, the molecular mechanism of Morusin was explored in CRCs, targeting c-Myc and ZNF746 signaling mediated by miRNA 193a-5p as a tumor suppressor.

Cell Viability Assay
Based on Jung et al.'s paper [1], cell viability assay was conducted in HCT116 and SW480 CRCs by using MTT assay. Briefly, HCT116 and SW480 cells (1 × 10 4 cells/well) were exposed to Morusin (0, 2.5, 5, and 10 µM) for 24 h and incubated with MTT (1 mg/mL) (Sigma-Aldrich, Saint Louis, MO, USA) for 2 h. The viability was calculated with optical density (OD) values as a percentage of viable cells in Morusin treated group versus untreated control. All assays were conducted in independent triplicates.

Colony Formation Assay
HCT116 and SW480 cells (3 × 10 3 /well) exposed to Morusin (0, 2.5, and 5 µM) for 24 h were distributed onto 6-well plates for a week. The cells were washed PBS, fixed, and stained with Diff quick solution (Sysmex, Kobe, Japan). Then, the colonies were counted under inverted microscope.

Cell Cycle Analysis
HCT116 and SW480 cells (2 × 10 5 cells/mL) exposed to Morusin (0, 2.5, or 5 µM) for 24 h were incubated with RNase A (10 mg/mL) for 1 h at 37 • C and stained with propidium iodide (50 µg/mL) in dark. The stained cells were analyzed for the DNA content by FACS Calibur with CellQuest Software.

TUNEL Assay
Based on Park et al.'s paper [6], to detect cell death, HCT116 or SW480 cells were treated with Morusin (5 µM) for 24 h, and incubated with TUNEL assay mixture for 60 min by using the DeadEnd™ Fluorometric TUNEL system kit (Promega, Madison, WI, USA).
Then, TUNEL-stained cells were visualized by a Delta Vision imaging system (Applied Precision, Issaquah, WA, USA).

Co-Immunoprecipitation
HCT116 cells exposed to Morusin (5 µM) for 24 h were lyzed in lysis buffer and immunoprecipitated with ZNF746 antibody or normal immunoglobulin G antibody, then protein A/G sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The precipitated proteins were subjected to immunoblotting with the antibodies of ZNF746 and c-Myc.

Statistical Analysis
All data represent means ± standard deviation (SD). For statistical analysis Student's t-test was used for comparison of two groups by using GraphPad Prism software (Version 5.0, CA, USA). The statistical significance was determined at p value of < 0.05 between control and Morusin treated groups.

Cytotoxic Effect of Morusin in Colorectal Cancer Cells
To explore the cytotoxic effect of Morusin ( Figure 1A), a cell viability assay was carried out in HCT116 and SW480 CRCs by MTT assay. The cells were exposed to Morusin (0, 2.5, 5, and 10 µM) for 24 h. Here, Morusin inhibited the viability in HCT116 and SW480 cells ( Figure 1B), while HCT116 cells were more susceptible to Morusin compared to SW480 cells. Likewise, Morusin reduced the number of colonies in HCT116 and SW480 cells by the colony formation assay ( Figure 1C).

Morusin Induced Apoptosis in HCT116 and SW480 Cells
To confirm the apoptotic effect of Morusin, cell cycle assay and Western blotting were conducted in Morusin treated HCT116 and SW480 cells. Herein, Morusin increased sub-G1 population in HCT116 and SW480 cells compared to untreated control (Figure 2A

Morusin Induced Apoptosis in HCT116 and SW480 Cells
To confirm the apoptotic effect of Morusin, cell cycle assay and Western blotting were conducted in Morusin treated HCT116 and SW480 cells. Herein, Morusin increased sub-G1 population in HCT116 and SW480 cells compared to untreated control (Figure 2A,B). (B) HCT116 and SW480 cells were exposed to various concentrations of Morusin (0, 2.5, 5, and 10 µM) for 24 h and cell viability was evaluated by MTT assay. Data stand for means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 versus untreated control. (C) Photos for colony formation of Morusin (0, 2.5, and 5 µM) treated HCT116 and SW480 cells. The colonies were visualized by staining with Diff-Quick solution (Sysmex, Japan). Data represent means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated control.
Consistently, Morusin enhanced the cleavage of PARP, and reduced the expression of pro-PARP and pro-caspase 3 in HCT116 and SW480 cells ( Figure 2C,D). Bar graphs showed quantification of cell cycle population (%) of three independent experiments. * p < 0.05, *** p < 0.001 vs. untreated control. (C,D) HCT116 and SW480 cells were exposed to Morusin for 24 h and subjected to Western blotting for PARP and caspase-3. Graphs stand for relative level of protein/β-Actin as means ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 versus untreated control. Bar graphs showed quantification of cell cycle population (%) of three independent experiments. * p < 0.05, *** p < 0.001 vs. untreated control. (C,D) HCT116 and SW480 cells were exposed to Morusin for 24 h and subjected to Western blotting for PARP and caspase-3. Graphs stand for relative level of protein/β-Actin as means ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 versus untreated control.

Morusin Attenuated the Expression of ZNF746 and c-Myc in HCT116 and SW480 Cells
To determine the role of c-Myc and ZNF746 in Morusin induced apoptosis, Western blotting was performed in HCT116 and SW480 cells. As shown in Figure 3A,B, Morusin significantly attenuated the protein expression of c-Myc and ZNF746 in HCT116 and SW480 cells.

Morusin Attenuated the Expression of ZNF746 and c-Myc in HCT116 and SW480 Cells
To determine the role of c-Myc and ZNF746 in Morusin induced apoptosis, Western blotting was performed in HCT116 and SW480 cells. As shown in Figure 3A,B, Morusin significantly attenuated the protein expression of c-Myc and ZNF746 in HCT116 and SW480 cells.  SW480 cells (B) were exposed to Morusin (0, 2.5, and 5 µM) for 24 h and subjected to Western blotting for ZNF746, c-Myc, Bcl-2, and Bcl-xL. Bar graphs stand for means ± SD for relative ZNF746, c-Myc, Bcl-2, and Bcl-xL protein expression. Data represent means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus untreated control.

Morusin Reduced the Stability of ZNF746 and c-Myc in HCT116 and SW480 Cells in the Presence of Cycloheximide
To confirm whether Morusin regulates the stability of ZNF746 and c-Myc, a cycloheximide assay was carried out in HCT116 and SW480 cells. As shown in Figure 4A,B, Morusin reduced the half-life stability of ZNF746 and c-Myc from 15 min in HCT116 or SW480 cells exposed to DNA synthesis inhibitor cycloheximide.

Morusin Reduced the Stability of ZNF746 and c-Myc in HCT116 and SW480 Cells in the Presence of Cycloheximide
To confirm whether Morusin regulates the stability of ZNF746 and c-Myc, a cycloheximide assay was carried out in HCT116 and SW480 cells. As shown in Figure  4A,B, Morusin reduced the half-life stability of ZNF746 and c-Myc from 15 min in HCT116 or SW480 cells exposed to DNA synthesis inhibitor cycloheximide.

Ectopic Expression of ZNF746 Reduces Apoptotic Effect of Morusin in HCT116 Cells
To confirm the important role of ZNF746, Western blotting and TUNEL assays were carried out in HCT116 cells transfected with overexpression plasmid of ZNF746. As shown in Figure 5A

Ectopic Expression of ZNF746 Reduces Apoptotic Effect of Morusin in HCT116 Cells
To confirm the important role of ZNF746, Western blotting and TUNEL assays were carried out in HCT116 cells transfected with overexpression plasmid of ZNF746. As shown in Figure 5A,B, overexpression of ZNF746 reversed the ability of Morusin to attenuate the expression of c-Myc and ZNF746 and the number of TUNEL positive cells in HCT116 cells.

Morusin Disrupted the Binding of c-Myc and ZNF746 in HCT116 Cells
To confirm whether Morusin disrupts interaction between ZNF746 and c-Myc, immunoprecipitation was carried out in Morusin treated HCT116 cells. Here, Morusin suppressed the binding of c-Myc and ZNF746 in HCT116 cells ( Figure 6A). Consistently, immunofluorescence reveals that c-Myc (red) was completely merged to ZNF746 (green) in HCT116 cells ( Figure 6B).

Morusin Disrupted the Binding of c-Myc and ZNF746 in HCT116 Cells
To confirm whether Morusin disrupts interaction between ZNF746 and c-Myc, immunoprecipitation was carried out in Morusin treated HCT116 cells. Here, Morusin suppressed the binding of c-Myc and ZNF746 in HCT116 cells ( Figure 6A). Consistently, immunofluorescence reveals that c-Myc (red) was completely merged to ZNF746 (green) in HCT116 cells ( Figure 6B).

miR193a-5p Plays a Pivotal Role in Morusin-Induced Apoptosis in HCT116 Cells
To determine the role of miR193a-5p in Morusin-induced apoptosis, Western blotting was performed in HCT116 cells. Interestingly, TargetScan web server predicts that miR-193a-5p directly binds to the 3 -Untranslated region of c-Myc and ZNF746 by bioinformatics analysis ( Figure 7A). Additionally, Morusin increased the mRNA expression of miR193a-5p in HCT116 cells ( Figure 7B). Furthermore, miR193a-5p mimic reduced the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells ( Figure 7C). In contrast, miR193a-5p inhibitor masked the ability of Morusin to suppress the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells ( Figure 7D), implying the partial role of miR193a-5p in Morusin-induced apoptosis.

miR193a-5p Plays a Pivotal Role in Morusin-Induced Apoptosis in HCT116 Cells
To determine the role of miR193a-5p in Morusin-induced apoptosis, Western blotting was performed in HCT116 cells. Interestingly, TargetScan web server predicts that miR-193a-5p directly binds to the 3′-Untranslated region of c-Myc and ZNF746 by bioinformatics analysis ( Figure 7A). Additionally, Morusin increased the mRNA expression of miR193a-5p in HCT116 cells ( Figure 7B). Furthermore, miR193a-5p mimic reduced the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells ( Figure 7C). In contrast, miR193a-5p inhibitor masked the ability of Morusin to suppress the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells ( Figure 7D), implying the partial role of miR193a-5p in Morusin-induced apoptosis.

Discussion
Recently, several natural compounds, including phenolic compounds, phytost nitrogen compounds, carotenoids, iridoids, organosulfur compounds, essential oils dietary fibers [27], are gaining interest due to significant antitumor effects and low to in colorectal cancers (CRCs) with combination therapy potential [28]. Though pre evidence reveals that Morusin suppresses the growth of colorectal cancer stem-like [3] and induces apoptosis in HT-29 CRCs via inhibition of NF-kB [29], the under antitumor mechanism of Morusin still remains unclear to date. Thus, in the current w the antitumor mechanism of Morusin was investigated in association with C-My ZNF746 mediated by miR193a-5p in HCT116 and SW480 cells. Herein, Morusin red the viability and the number of colonies in HCT116 and SW480 cells, implyin cytotoxic and anti-proliferative effect of Morusin. It is well known that s accumulation represents apoptosis in the cells [30], and cleavages of PARP and cas indicate intrinsic or extrinsic apoptosis [31][32][33]. Consistently, Morusin increased su population, and cleaved PARP and caspase-3 in HCT116 and SW480 cells, indicatin apoptotic effect of Morusin in CRCs.
Emerging evidence indicates that c-Myc is a known nuclear transcription oncogene among the Myc family, comprising of n-Myc, c-Myc, and l-Myc, in se cancers [34,35]. Thus, c-Myc inhibitors are considered to control tumor initiation

Discussion
Recently, several natural compounds, including phenolic compounds, phytosterols, nitrogen compounds, carotenoids, iridoids, organosulfur compounds, essential oils, and dietary fibers [27], are gaining interest due to significant antitumor effects and low toxicity in colorectal cancers (CRCs) with combination therapy potential [28]. Though previous evidence reveals that Morusin suppresses the growth of colorectal cancer stem-like cells [3] and induces apoptosis in HT-29 CRCs via inhibition of NF-kB [29], the underlying antitumor mechanism of Morusin still remains unclear to date. Thus, in the current work, the antitumor mechanism of Morusin was investigated in association with C-Myc and ZNF746 mediated by miR193a-5p in HCT116 and SW480 cells. Herein, Morusin reduced the viability and the number of colonies in HCT116 and SW480 cells, implying the cytotoxic and anti-proliferative effect of Morusin. It is well known that subG1 accumulation represents apoptosis in the cells [30], and cleavages of PARP and caspases indicate intrinsic or extrinsic apoptosis [31][32][33]. Consistently, Morusin increased sub-G1 population, and cleaved PARP and caspase-3 in HCT116 and SW480 cells, indicating the apoptotic effect of Morusin in CRCs.
Emerging evidence indicates that c-Myc is a known nuclear transcription factor oncogene among the Myc family, comprising of n-Myc, c-Myc, and l-Myc, in several cancers [34,35]. Thus, c-Myc inhibitors are considered to control tumor initiation and progression [36,37]. Additionally, ZNF746 is known to promote cancer progression via c-Myc stability in CRC [1], bladder cancer [38], and lung cancer [12]. Herein, Morusin inhibited the expression of c-Myc and ZNF746, and the stability of c-Myc, and disrupted the direct binding through nuclear colocalization in HCT116 cells. Conversely, overexpression of ZNF746 masked the antitumor effect of Morusin to reduce the expression of c-Myc and increase the number of TUNEL positive cells in HCT116 cells, demonstrating the pivotal role of C-Myc and ZNF746 in Morusin induced apoptosis.
Accumulating evidence reveals that miRNAs are critically involved in the development, cell differentiation, cell cycle, apoptosis, metastasis, and angiogenesis in several cancers as an oncogene or tumor suppressor [39,40]. Among several miRNAs, miR193a-5p is reported to inhibit liver carcinogenesis [41] and induce G1 arrest in CRCs [42]. RT-PCR reveals that Morusin upregulated miR193a-5p in HCT116 cells. As shown in bioinformatics data, miR-193a-5p directly binds to the 3 -Untranslated region of ZNF746 and c-Myc; miR193a-5p mimic reduced the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells, while miR193a-5p inhibitor reduced the ability of Morusin to suppress the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells, indicating miR-193a-5p suppresses the expression of ZNF746, c-Myc, and pro-PARP during apoptotic effect of Morusin.

Conclusions
Taken together, Morusin increased cytotoxicity and sub G1 population, cleaved PARP, and attenuated the expression of pro-caspase-3 and pro-PARP in HCT116 and SW480 cells. Additionally, Morusin suppressed the expression of C-Myc and ZNF746, disturbed the binding of ZNF746 and c-Myc, and upregulated miR193a-5p in HCT116 cells. Conversely, the miR193a-5p inhibitor masked the ability of Morusin to attenuate the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells. Overall, these findings suggest that miR193a-5p mediated inhibition of C-Myc and ZNF746 signaling plays a pivotal role in Morusin induced apoptosis in CRCs (Figure 8).

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