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Anticancer Effect of Natural Product Sulforaphane by Targeting MAPK Signal through miRNA-1247-3p in Human Cervical Cancer Cells

The prognosis of cervical cancer remains poor. Sulforaphane, an active ingredient from cruciferous plants, has been identified as a potential anticancer agent in various cancers. However, there is little information about its effect on cervical cancer. Here, we conducted a present study to uncover the effect and the potential mechanisms of sulforaphane on cervical cancer. HeLa cells were treated with sulforaphane, and cell proliferation and apoptosis were assessed by Cell Counting Kit-8, Western blot, flow cytometry, and immunofluorescence. Then, next-generation sequencing (NGS) and bioinformatics tools were used to analyze mRNA-seq, miRNA-seq, and potential pathways. Finally, qRT-PCR, Cell Counting Kit-8, flow cytometry, small RNAs analysis, and Western blot were performed to evaluate the biological function of miR1247-3p and MAPK pathway in HeLa cell lines. Sulforaphane significantly suppressed the viability and induced apoptosis of HeLa cells. NGS and bioinformatics analysis showed sulforaphane exerted its anti-tumor activities through miR1247-3p and the MAPK signaling pathway. Further analysis suggested that sulforaphane could activate MAPK pathway via down-regulating the expression of miR-1247-3p. Sulforaphane inhibited proliferation and promoted apoptosis of HeLa cells via down-regulation of miR-1247-3p and activating the MAPK pathway. These findings provide preliminary experimental evidence for the treatment of cervical cancer with sulforaphane.


Introduction
Cervical cancer has become the fourth most common malignancy among women worldwide due to its high incidence and mortality rate [1] [2]. There are around 528,000 newlydiagnosed cervical cancer cases and 266,000 deaths every year, according to the latest global cancer statistics [3]. To date, effective treatments for patients with cervical cancer include surgery, chemotherapy, and radiotherapy [4]. Given many potential adverse side effects in these traditional treatments, so it is worthwhile to search for a novel effective treatment for cervical cancer [5].
Sulforaphane, an herbal isothiocyanate typically abundant in cruciferous vegetables such as broccoli, cauliflower, and cabbage, has many biological effects such as antiinflammation, anti-oxidation [6]. In particular, due to its anticarcinogenic role in pancreatic cancer, it has been noteworthy recently [7]. The antiproliferative and radiosensitizing properties for head and neck tumors also have been reported [8]. Although previous studies have identified the anti-tumor potential of sulforaphane in various kinds of cancers [9,10], the role of sulforaphane in cervical cancer has not been well recognized. MicroRNAs (miRNAs) are short, non-coding RNAs that play important roles in regulating many biological processes involving almost all aspects of cell physiology [11,12]. Typically, they regulate protein expression levels in physiological and pathophysiological processes by conjugating with complementary sequences of their target mRNAs [13,14]. As there are around 1000 miRNAs in the human genome, which modulate approximately one-third of the human genes [15], miRNAs are the most abundant regulators [16].
Accumulating evidence has shown that sulforaphane exerts its therapeutic effects through modulating the expression of cancer-related miRNAs on a variety of cancers, including colon cancer [17], lung cancer [18], gastric cancer [19], pancreatic cancer [20], skin cancer [21], acute myeloid leukemia [22], and nasopharyngeal cancer [23]. Besides, the previous study has demonstrated that miR-3156-3p, negatively associated with the incidence of HPV-positive cervical cancer, acted as a tumor-suppressive miRNA [24]. Furthermore, targeting MACC1, miR-338-3p could regulate the proliferation of cervical cancer cells via the MAPK pathway [25]. However, no elaborate research has found the association between miRNAs and sulforaphane in inhibiting cervical cancer until now. Therefore, given the anti-tumor capability of sulforaphane, we aimed at validating the anticancer activities and mechanisms of sulforaphane in cervical cancer, including downstream target miRNA and relevant signal pathway initiated by the miRNA.
In our study, we found that miR-1247-3p was highly related to both the apoptosis rate of cervical cancer cells cultured with sulforaphane and the activation of the MAPK pathway. Based on the high throughput sequencing and bioinformatics analysis, we ultimately disclosed the role of the miR-1247-3p-induced MAPK signal pathway in inhibition of cervical cancers treated with sulforaphane.
The sequence of mimic NC was: UUGUACUACACAAAAGUACUG.

Cell culture.
Human cervical cancer cell lines HeLa was purchased from the American Model Culture Collection (ATCC), which was preserved by the State Key Laboratory of Biotherapy of Sichuan University. The cell was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 unit/mL penicillin( Beyotime, SH, China), and 100 mg/mL streptomycin (Beyotime). These cells were cultured in a humid chamber at 37℃ under 5% CO2 in the atmosphere.

Cell viability assay.
CCK8 kit was used to detect cell proliferation. Briefly, 5x10 3 cells were plated in96 well plates. After attachment overnight, cells were treated with sulforaphane (0, 10, 20, 40µM) for24h, and then incubated in 10% CCK-8 for another 2h. The cells in the 0 µM-treated group were cultured with the same volume of DMSO as the 40µM-treated group. The cell proliferation rate was measured at 450nm absorbance. The experiment was carried out three times.

Transcriptome sequencing and analysis.
HeLa cells were harvested for RNA extraction after 8 h of sulforaphane treatment at a concentration of 40 µM. The cells incubated with the same volume of DMSO as the sulforaphane (40µM)-treated group were set as the control. Each group included three samples. Trizol Reagent (Invitrogen, San Diego, CA, USA) was used to isolate total RNA. Illumina HiSeq 2500 platform was utilized to perform transcriptome sequencing, and 150bp paired-end raw reads were generated. Illumina sequencing was carried out at Novogene, Beijing, China. The expression level of each transcript was measured as the number of clean reads mapped to its reference sequence (GRCh 38). Kallisto [26] was adopted to align and quantify clean reads, genes expressed in at least 1 sample were defined as detected genes. Differential expression analysis was performed through the DEseq2(v 1.24.0) R package [27], DEGs were selected out based on the interval of p < 0.05 & |log2 (fold change)| > 1. Enrichment of pathways was performed by KOBAS 3.0 [28] with the corresponding groups of DEGs. The hypergeometric test method was Fisher's exact test, and the FDR correction method was Benjamini and Hochberg.

Small RNA Sequencing and Analysis.
Small RNAs were sequenced by Illumina Hiseq 2500/2000 platform, and 50 bp singleend reads were generated. Clean data were obtained by removing adaptor sequences, collapsing reads with the same sequence, and reads with less than 15 bases in length from raw data. Then, clean data were analyzed based on the miRDeep2 (2.0.0.8) [29]. Small RNA reads were mapped to miRNA precursors from miRBASE release 22.1 [30] by miRDeep2, which allowed zero mismatches. Mapped reads were quantified by miRDeep2. Reads were matched to human miRNA precursors using bowtie2 [31], and 1520 miRNAs were detected. Analysis of the differential expressed miRNAs(DEMs) between sulforaphane-untreated and sulforaphane-treated groups was performed by the DEseq2 R package. P< 0.05 & |log2 (fold change)| > 2 was set to as the significance threshold in this test. The target genes of DEMs were predicted by TargetScanHuman(v 7.2)[32].The Enrichment of KEGG pathways was performed by KOBAS 3.0 with the target genes.

Flowcytometry.
FITC-Annexin V/PI Detection Kit I (BD Biosciences, San Jose, CA) was used for quantifying the apoptotic cell rate after sulforaphane treatment. The HeLa cells (5×10 5 cells/well) were plated in 6-well plates. After 24 h incubation, the cells were cultured with different concentrations of sulforaphane for 24 h. Then the cells were collected and incubated with Annexin V-FITC for 5 min in the dark and then stained with PI for 5 min. The sample was sorted by NovoCyte flow cytometry. Data were analyzed by NovoExpress 1.1.2 software.

miRNA transfection.
MiR-1247-3p mimic and its scrambled control (NC) were both purchased from GenePharma (Shanghai, China). For transfection, the cells were seeded at a density of 4×10 4 cells per well into 6-well culture plates. As the cells reached 50-80 % confluence, transient transfection was performed by using Lipofectamine™ 3000 (Invitrogen) according to the manufactures' protocol. The transfection was stopped after 24 hours, and cells were collected for subsequent experiments, including cell viability and apoptosis assays. In cell viability assay, the cells transfected with different miRNA mimics were administrated with sulforaphane (0, 10, 20, and 40µM) for 24h. In apoptosis assay, the flow cytometry detected the apoptotic cell rate of sulforaphane-treated cells (0, 40µM) for 24h. The cells in the sulforaphane (0 µM)treated group(control group) were incubated with the same volume of DMSO as the sulforaphane (40 µM)-treated group.

Immunofluorescence.
HeLa cells were seed onto a 14-mm cover glass in 24-well plate and incubated overnight. Cells were treated by different concentrations of sulforaphane for 48h. Then the culture medium was removed, and cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10min and washed three times. The unspecific binding sites were blocked with PBST containing 1% BSA and 0.05% Triton X-100. Subsequently, cells were incubated with primary antibody overnight at 4°C. The secondary antibodies combined with FITC were used. DAPI was performed to stained cell nuclei, and the samples were examined with fluorescence microscopy.

RT-PCR.
To confirm the bioinformatics analysis, total RNA was extracted by using RNA Extraction Kit (TIANGEN BIOTECH, DP419) after treatment with or without sulforaphane (40 µM) for 8 h, and reverse transcribed to cDNA by using Prime Script RT Kit (Takara, RR036) according to the manufacturers' instructions. The qPCR array was performed by using SYBR Select Master Mix (Invitrogen, 4472908) with specific gene primers in StepOnePlus PCR System (Thermo). RT-qPCR was conducted by the following conditions: 95°C for 15 min, followed by 40 cycles of 94°C for 20 s and 60°C for 34 s. RNU6 was used as an internal reference. The target genes expression levels were measured by using the 2 − ⊿ Ct method. The harvested cells were washed twice with a pre-cooled PBS buffer. Cellular proteins were extracted in RIPA lysis buffer (Biosharp, Guangzhou, GD, China), including protease inhibitor cocktail (Millipore) over ice for 30 minutes. The supernatant was gathered by centrifuging at 13000rpm for 15 min at 4°C. The protein concentrations of samples were evaluated by BCA protein assay kit (Beyotime, China). Afterwards, the protein extracts were separated in SDS-PAGE gel and transferred onto nitrocellulose (NC) membranes (Merck Millipore, MS, USA). Then, the membranes were blocked at room temperature for 1.5h and incubated with proper primary antibody and the corresponding secondary antibody. These primary antibodies were purchased from HuaBio (Hangzhou, ZJ, China), including anti-Cytochrome C antibody (#ET1610-16), anti-Bax antibody (#ET1603-34), anti-β-Actin antibody (#R1207-1). The other primary antibodies were purchased from Cell Signaling Technology, including anti-p-ERK (#4370), anti-ERK(#4695), (anti-p-JNK(#9255), anti -JNK(#9252), anti-p-P38(#4511) and anti -P38(#8690). The specific protein bands were detected via chemiluminescence detection.

Statistical analysis.
All the statistics were carried out in GraphPad Prism 7 and 8. All data are shown as mean ± SD. A two-tailed Student's t-test was used to analyze statistical significance. The statistic differences are shown as * p < 0.05, ** p b< 0.01, *** p < 0.001, ns, no significance.

Sulforaphane inhibited the proliferation and induced apoptosis of HeLa cells.
To evaluate the effect of sulforaphane on cell viability, the HeLa cell was cultured with sulforaphane of different concentrations (0, 10, 20, 40, and 60µM) for 24 h. According to the results of our CCK-8 array, sulforaphane suppressed the viability of HeLa cells in a concentration-dependent manner (Fig. 1A). As shown in Fig. 1B, the flow cytometry analysis showed the apoptotic rate of HeLa cells of 40µM treatment after 24h remarkably increased compared with the rate of 0µM after 24h. Moreover, the apoptosis rate determined by the TUNEL assay increased (Fig. 1C). To further investigate apoptosis induced by sulforaphane, we analyzed the expression of Bcl-2, Bax, and Cytochrome C. As demonstrated in Fig. 1D, the expression level of anti-apoptotic Bcl-2 decreased. At the same time, pro-apoptotic Bax and Cytochrome C were up-regulated in a dose-dependent manner, which contributed to an increase in the Bax/Bcl-2 expression ratio. All of these proteins were crucial molecular regulators in the mitochondrial-mediated apoptotic pathway [33]. These data indicated that sulforaphane could inhibit the proliferation and induce the apoptosis of HeLa cells.

An integrated analysis of altered mRNAs and miRNAs via high-throughput sequencing.
According to the previous results, transcriptome sequencing and bioinformatics analysis were performed to the HeLa cells treated with sulforaphane at a concentration of 40 µM for 8h. Next-generation sequencing was applied to identify a large number of altered mRNAs and miRNAs after treatment. The analysis of transcription sequencing data described that a total of 21,582 expressed genes were detected. The expression level of 7838 genes changed significantly (P < 0.05) in the sulforaphane-treated group compared with the control group (Fig 2A). Ultimately, 2041 DEGs were identified by the DEseq2(v 1.24.0) R package. After the analysis of miRNA sequencing data, a total of 1588 miRNAs were spotted. Then the differential expression analysis of these detected miRNAs was performed between the sulforaphane-treated group and the control group ( Fig 2B). Finally, 101 significantly changed miRNAs were found (P < 0.05), which contained 39 up-regulated miRNAs and 62 downregulated miRNAs. To better determine the function of DEGs after sulforaphane expose, gene ontology (GO) enrichment analysis was used to find significantly enriched terms, leading to a more comprehensive understanding of the effects sulforaphane exerting on HeLa cells (Fig 2C). The results displayed that many biological processes were significantly altered after sulforaphane treatment, including response to unfolding protein (GO: 0006986), reproductive structure development (GO: 0048608), reproductive system development (G0:0061458), embryonic organ development(GO: 0048568).

Upregulation of miR-1247-3p promoted cell proliferation and induced anti-apoptosis in HeLa cells.
Several studies suggested that sulforaphane initiated its anti-tumor and antiinflammatory activities by regulating several miRNAs [23,[34][35][36][37]. To further explore the role of sulforaphane, we evaluated miRNAs expression via high throughput RNA sequencing. Further, we identified the differential expression level of the miRNAs by the volcano plot in both the control group and the experiment group, as shown in Fig. 3A. Among the four significantly differentially expressed miRNAs, two miRNAs were up-regulated (miR-320a-5p, miR-12135), and the other two miRNAs were downregulated (miR-1247-3p, miR-33b-3p). qPCR was used to confirm the differential miRNA expression. The expression of these miRNAs, except for miR-320a-5p, was consistent with RNAsequencing results (Fig.3B). Next, we tried to examine whether the four miRNAs would impact tumor cell proliferation. The result of the CCK-8 array exhibited that miR-1247-3p mimic could significantly increase HeLa cell proliferation (Fig.3C). Additionally, flow cytometry results confirmed that miR-1247-3p mimic dramatically inhibited cell apoptosis induced by sulforaphane (Fig. 3D). Thus, these results suggested miR-1247-3p could contribute to the antiapoptotic effect of sulforaphane on HeLa cells.

Sulforaphane activated MAPK signaling pathway through downregulating miR-1247-3p.
To find the mechanism of sulforaphane-induced apoptosis and further investigate the functions of miR-1247-3p, 2725 target genes were obtained (SupplyTable1) to predict the potential target pathways of miR-1247-3p ( Fig 4A). Then KEGG pathway analysis was also carried out to explore the signaling pathways of the DEGs between sulforaphane-untreated and treated groups. The top 10 significantly enriched pathways are shown in Fig 4B,   So we tried to examine whether miR-1247-3p was implicated in the regulatory effect of sulforaphane via MAPK pathway on HeLa cells. The results showed that sulforaphane remarkably elevated the phosphorylation levels of JNK, P38, ERK (Fig. 4C). To further confirm these results, we detected JNK, P38, ERK protein levels between the mimic control group and miR-1247-3p mimic group. Results in Fig. 4D showed that the expression of p-JNK, p-P38, and p-ERK in cells transfected with miR-1247-3P mimic was much lower than that in mimic control-transfected cells. Therefore, these results implied that sulforaphane might downregulate miR-1247-3p to activate MAPK signaling pathway.

Conclusions
In summary, high throughput sequencing was applied in sulforaphane-treated HeLa cells, and we identified miR1247-3p downregulation was implicated in the pro-apoptosis effect of sulforaphane. Additionally, miR1247-3p-driven MAPK signaling pathway might be attributable to the apoptosis of HeLa cells. Based on the aforementioned mechanism, sulforaphane could serve as a promising anticancer agent in the treatment of cervical cancer.