Tumor Suppressor MicroRNA-27a in Colorectal Carcinogenesis and Progression by Targeting SGPP1 and Smad2

The aberrant expression of microRNAs (miRNAs) is associated with colorectal carcinogenesis, but the underlying mechanisms are not clear. This study showed that the miRNA-27a (miR-27a) was significantly reduced in colorectal cancer tissues and colorectal cancer cell lines, and that the reduced miR-27a was associated with distant metastasis and colorectal cancer clinical pathological stages–miR-27a was lower at stages III/IV than that at stage II. Bioinformatic and systemic biological analysis predicted several targets of miR-27a, among them SGPP1 and Smad2 were significantly affected. SGPP1 and Smad2 at mRNA and protein levels were negatively correlated with miR-27a in human colorectal cancer tissues and cancer cell lines. Increased miR-27a significantly repressed SGPP1 and Smad2 at transcriptional and translational levels. Functional studies showed that increasing miR-27a inhibited colon cancer cell proliferation, promoted apoptosis and attenuated cell migration, which were also linked to downregulation of p-STAT3 and upregulation of cleaved caspase 3. In vivo, miR-27a inhibited colon cancer cell growth in tumor-bearing mice. Taken together, this study has revealed miR-27a as a tumor suppressor and has identified SGPP1 and Smad2 as novel targets of miR-27a, linking to STAT3 for regulating cancer cell proliferation, apoptosis and migration in colorectal cancer. Therefore, miR-27a could be a useful biomarker for monitoring colorectal cancer development and progression, and also could have a therapeutic potential by targeting SGPP1, Smad2 and STAT3 for colorectal cancer therapy.


Introduction
Colorectal cancer is one of the most common malignant diseases worldwide, but the causes of colorectal carcinogenesis and progression are largely unknown. Numerous studies have revealed that genetic and epigenetic changes and oncogenic signaling activation are the major causes of malignant transformation and progression. In recent years, the epigenetic alterations, in particular, the aberrant expression of microRNAs (miRNAs), have been shown critical roles in cancer formation, metastasis, and response to cancer therapy [1][2][3][4][5][6].
miRNAs are a novel class of small noncoding RNAs that typically inhibit the translation and stability of messenger RNAs (mRNAs) by binding to the 39-untranslated regions (39-UTR) of their target mRNAs [7]. miRNAs have 19-22 nucleotides and are found in all multi-cellular eukaryotic cells. miRNAs have important roles in various biological and pathological processes, such as development, cell proliferation, differentiation, apoptosis, inflammation, stress response and migration [1][2][3]. Increasing evidences have suggested that miRNAs are deregulated or upregulated in all types of cancers, acting either as tumor suppressors (e.g. miR-34, miR-15/16, let-7, miR 200 family) or as oncogenes (e.g. miR-155, miR-222/221, miR-17-5p, miR-21) [1,3,8], in which the miRNAs play key roles in important aspects of tumorigenesis, such as cancer initiation, differentiation, growth and progression [3,5,8], mainly by interfering with the expression of target genes involved in cell cycle, apoptosis, cell migration and invasion, angiogenesis. Using a miRNA array profile we have found that miRNA were differential expressed in colonic epithelial cells of a colorectal cancer mouse model, the Muc2 gene knockout mice [9]. One of the most changed miRNAs was miRNA-27a (miR-27a).
MiR-27a is located at chromosome 19 [10]. Its expression levels and biological functions in cancers are controversial. For instance, several studies have reported that miR-27a acts as an oncogene, whose expression is upregulated in breast cancers [11,12], colon cancer cell lines [13][14][15], and in hepatocellular adenocarcinoma cells [16], and that the increased expression of miR-27a is associated with breast cancer progression and poor outcomes [11,12]. Several studies have also observed that miR-27a exhibited oncogenic activity by directly suppressing ZBTB10/RINZF expression [10,17], resulting in upregulation of transcription factor specificity protein (Sp), vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). In another hand, miR-27a has also shown tumor suppressor roles, such as miR-27a is downregulated in esophageal cancers [18], oral squamous cell carcinoma [19], acute leukemia [20], and in non-small cell lung cancer (NSCLC) [21]. In NSCLC, miR-27a directly targets MET and EGFR 39 UTR, leading to reduced expression of MET and EGFR [21].
This study was to determine the expression of miR-27a and association with colorectal cancer formation, progression and the underlying mechanisms. We found that miR-27a was significantly reduced in human colorectal cancer tissues and in colorectal cancer cell lines. Using the approaches of miRNA array, systemic biology, in vitro manipulating expression of miR-27a and in vivo tumor-bearing mouse model, we found that miR-27a acted as a tumor suppressor in colorectal cancer, which was through targeting SGPP1 and Smad2.

Ethics Statement
The animal care and use were approved by the Institutional Animal Care and Use Committee of Xinxiang Medical University and University of Illinois at Chicago, and human samples collection and use were approved by the Institutional Review Board of Xinxiang Medical University. All patients gave informed consent in written.

Muc2 mouse colonic epithelia cells collection and miRNA array
As reported previously [9,22], the Muc22/2 and Muc2+/+ mice were generated by crossmating the Muc2+/2 mice, and the Muc22/2 mice spontaneously developed colorectal tumors at age about 3 months. Thus, mouse colonic epithelial cells were collected from 3-month aged Muc2+/+ and Muc22/2 mice, respectively. Four mice from each group were used. The total RNAs were extracted for miRNA array analysis. The miRNA array was performed in the Genomic Facility of University of Chicago (Chicago, Illinois). Affymetrix GeneChip miRNA Arrays version 3.0 was used for miRNA profile. The detailed experimental design, detailed protocol and data analysis could be accessed at Gene Expression Omnibus (GEO) (Access #GSE56577) and as published recently by us [23]. The animal use protocol was approved by both the University of Illinois at Chicago and Xinxiang Medical University Animal Care Committee.

Human samples collection
Forty-one paired human colorectal cancer tissues and their adjacent normal colonic mucosa were collected from November, 2012 through October, 2013, from the First Affiliated Hospital and the Affiliated Xinxiang Central Hospital, Xinxiang Medical University. Portion of the samples were snapped into liquid nitrogen and then stored at 280uC for RNA and protein extraction for quantitative RT-PCR (qRT-PCR) and western blotting analysis, respectively. Portion of the samples were fixed in 10% buffered formalin and embedded in paraffin. Patient clinical and histopathological information were summarized in Table 1. The sample collection was approved by the Institutional Review Board of Xinxiang Medical University. All patients gave informed consent in written.

Cell culture
Human colorectal cancer cell lines HCT116, Caco 2 and SW480 from ATCC were maintained in a complete MEM medium. Human normal colon mucosal epithelial cell line NCM460 was purchased from the Shanghai Bioleaf (Shanghai, China) and maintained in RPMI-1640 medium. The murine colon adenocarcinoma cell line MC38 (derived from C57BL6/J mouse) [27] was cultured in MEM (Invitrogen, Carlsbad, CA). All media were supplemented with 10% FBS and antibiotics (10,000 U/ml penicillin, 10 mg/ml streptomycin). Cells were cultured at 37uC in a humidified atmosphere containing 5% CO 2 .

RNA extraction and quantitative RT-PCR for miRNA and mRNA analysis
Trizol reagent (Invitrogen, Carlsbad, CA) was used for total RNA extraction from the frozen colorectal cancer and normal tissues following manufacturer's protocol. Quantitative RT-PCR (qRT-PCR) (Applied Biosystem Inc.) was used for miRNA and mRNA quantification analysis. The primers for miR-27a analysis were: reverse transcription (RT) primer: GTGCAGGGTCC-GAGGTCAGAGCCACCTGGGCAATTTTTTTTTTTGCG-GAA; forward primer: TTCGGTTCACAGTGGCT AAG;

Apoptosis analysis
To detect apoptosis, the HCT116 cells were transfected with miR-27a precursor or negative control miRNA precursor. After 48 hours, the cells were harvested and fixed with 70% ethanol followed by propidium iodide (P.I.) and Annexin V (Invitrogen, Carlsbad, CA) staining. Cells were then counted by flow cytometry (FACScan, BD Biosciences, San Jose, CA) for apoptosis analysis. Usually, about 10,000 cells were counted. The percentage of the MiRNA-27a in Colorectal Cancer PLOS ONE | www.plosone.org apoptosis was calculated by dividing the total cells by apoptotic cells.

Wound healing assay
As reported previously [26], the HCT116 cells transiently transfected with miR-27a precursor or negative control miRNA precursor were seeded in a 100-mm Petri dish. A wound was made by scratching on the Petri dish bottom, followed by another 48 hours growth.

Immunoblotting
For immunoblotting, the human colon cancer cells HCT116 and SW480 were collected 72 hours after transfection with miR-27a precursor or negative control miRNA precursor. Cells were lysed using 1x RIPA buffer (Upstate Biotechnology, Lake Placid, NY) containing a protease inhibitor cocktail (Sigma, St. Louis, MO). After cell lysis, 45 mg of protein was loaded on a 10% SDS gel followed by transfer to PVDF membrane. Antibodies against SGPP1 (Abcam, Cambridge, MA), Smad2, Stat3, phosphorylated

Tumor-bearing (Xenografts) study
As reported recently [26], 1.5610 5 murine colon adenocarcinoma cells MC38 re-suspended in 150 ml PBS were injected subcutaneously into the flank of the normal C57B/l6 mice at age about 8 weeks (5 mice per group). Both MC38 cell line and the mice were in C57BL/6 background and no rejection occurred. The animals were maintained in a pathogen-free barrier facility and closely monitored by animal facility staff. The grown tumors (xenografts) were measured every 3 day starting 21 days post inoculation of MC38 cells using caliper as length x width x width/ 2 (mm 3 ). 6.26 mg of miR-27a precursor or negative miRNA (GenePharma, Shanghai, China) mixed with 1.6 ml transfection reagent Lipofectamine 2000 (Invitrogen) in 50 ml PBS were injected into the tumors every 3 days, for total of 3 times. 30 days after inoculation, the animals were sacrificed and the xenografts were isolated, the weight (gram) and volume (mm 3 ) of the xenografts were determined. All procedures were conducted according to the Animal Care and Use guideline approved by Xinxiang Medical University Animal Care Committee.

MiR-27a expression was reduced in Muc22/2 mouse colonic epithelial cells, in human colorectal cancer tissues and colorectal cancer cells
Our previous studies have demonstrated that Muc22/2 mice spontaneously developed colorectal cancers and the carcinogenesis is linked to chronic inflammation [9,22,28,29]. Interestingly, chronic colitis is frequently seen at the early age of Muc22/2 mice, and colorectal tumors are frequently seen after 3 months of age. Thus, the 3 months could be a borderline of the transition from chronic inflammation to tumor formation. To indentify the potent miRNAs involving in the malignant transformation, we isolated colonic epithelial cells from the age of 3 months mice, extracted RNA and performed miRNA profile. As shown in Figure 1A, miRNA profile showed differential expression of miRNAs in both Muc22/2 and Muc2+/+ mouse colonic epithelial cells. Based on potential biology roles in proliferation and inflammation, miRNA profile and published literatures, miR-27a was chosen for further studies. Since genetic deficiency of the Muc2 gene in mice causes colorectal cancer formation, the decreased expression of miR-27a in colonic epithelial cells could be involved in the carcinogenesis.
To determine the role of miR-27a in colonic epithelial cell malignant transformation, miR-27a expression levels were determined in human colorectal cancer and their adjacent tissues. In comparison with the adjacent normal colon mucosa, 73% (30/41) of colorectal cancer tissues shown reduced miR-27a expression, and only 27% (11/41) colorectal cancers exerted upregulated expression of miR-27a, the difference was significant ( Figure 1B, p,0.01). More importantly, the reduced expression of miR-27a was also associated with colorectal cancer pathological stages -miR-27a levels were more downregulated at stages III/IV than those at stage II ( Figure 1C, p,0.0001). In addition, reduced miR-27a was correlated with distant metastasis ( Table 1).
The expression levels of miR-27a were further determined in human colorectal cancer cell lines. As shown in Figure 1D, miR-27a expression was downregulated 95%, 90% and 52% in HCT116, Caco-2 and SW480 cells, respectively, compared to the immortalized normal human colon epithelial cell NCM460.

miR-27a targets prediction
Above findings strongly suggested miR-27a was frequently downregulated in colorectal cancers. Therefore, it is essential to determine whether the reduction of miR-27a is involved in colorectal cancer formation and progression, and it is warranted to identify miR-27a targets and reveal the underlying molecular mechanisms. Previous studies have demonstrated that ZBTB10/ RINZF is a direct target of oncogenic miR-27a in breast cancers [10,17] and colon cancer cell lines [15], but this could not be the case in the colorectal cancer tissues because herein miR-27a seemed to be a tumor suppressor in colorectal cancers. We employed multiple tools to predict novel targets of miR-27a. Supplemental Figure S1A (Supplemental materials) showed 346 potent targets at different signaling pathways, among them, the categories of cell cycle, cell death, cell differentiation, cell growth and proliferation and cellular homeostasis, etc, could be more relevant to cancer development and progression. We further narrowed down the targets using GO program, and the targets at the categories of cellular process, single organism process, biological regulation and metabolic process, etc, exhibited stronger scores, meaning more relevant to miR-27a-associated functions in cancer formation and progression (Supplemental Materials Figure  S1B).

SGPP1 and Smad2 were the targets of miR-27a
Among the hundreds of targets, the two novel targets of miR-27a, Sphingosine-1-phosphate phosphatase 1 (SGPP1) and Smad2, were chosen for further studies. SGPP1 is a catalyze of Sphingosine-1-phosphate (S1P), the later is a bioactive sphingolipid metabolite that regulates diverse biologic processes [30,31], and recent report showed that SIP is linked to signal transducer and activator of transcription 3 (STAT3) activation and the development of colitis-associated colorectal cancer [31]. But the biological role of SGPP1 on carcinogenesis is not clear. Smad 2 is a member of Smad family and is associated with cell growth and proliferation through transforming growth factor b (TGF-b) signaling pathway [32,33].
Genomic alignment showed that 39-UTR of SGPP1 and Smad2 have one or two miR-27a binding sites (Figure 2A). To determine whether miR-27a could repress SGPP1 and Smad2 expression by targeting its binding site at 39-UTR in SGPP1 and Smad2, the PCR products containing full length of 39-UTR with intact target site of miR-27a recognition sequences were inserted into the luciferase reporter vector. The plasmids were transfected into HCT116 cells that had been transfected with control miRNA mimics or miR-27a mimics, and the luciferase reporter activities were measured. As shown in Figure 2B, SGPP1 reporter activities were reduced about 65% and both of the Smad2 reporter activities (Smad2-a and Smad2-b) were reduced about 90%, respectively. These finding confirmed that SGPP1 and Smad2 were the targets of miR-27a.
The repression of SGPP1 and Smad2 expression by miR-27a were determined by qRT-PCR and immunoblotting. As shown in Figure 2C, SGPP1 and Smad2 mRNA levels were repressed about 60% and 50% by miR-27a in HCT116 cells, respectively, consistent with the repression on their reporter luciferase activities. SGPP1 and Smad2 protein levels were also downregulated by miR-27a on both HCT116 and SW480 colorectal cancer cells ( Figure 2D).

SGPP1 and Smad2 were inversely correlated with miR-27a in colorectal cancer
As SGPP1 and Smad2 were likely the targets of miR-27a, we then determined the expression levels of SGPP1 and Smad2 in human colorectal cancers and cancer cell lines. As shown in Figure 3A, SGPP1 mRNA levels were 1.6-, 3.2-and 2.4-fold in HCT116, Caco-2 and SW480 cells, respectively, than that in the normal colonic epithelial cell NCM460 cells. Similar as the expression levels of SGPP1, Smad2 mRNA levels were also upregulated in HCT116, Caco-2 and SW480 cells, showing 1.4-, 2.3-and 1.8-fold higher than that in the normal colonic epithelia NCM460 cells ( Figure 3A). Both SGPP1 and Smad2 mRNA levels were inversely correlated with the miR-27a levels at these human colorectal cancer cells ( Figure 1D). The expression of SGPP1 and Smad2 were then evaluated in human colorectal cancers by immunohistochemical staining. Compared to the adjacent normal colon mucosa, both SGPP1 and Smad2 expression were much higher at colorectal adenocarcinomas ( Figure 3B), negatively correlated with miR-27a levels in colorectal cancers in which miR-27a was frequently reduced ( Figure 1B).

MiR-27a inhibited cell proliferation, enhanced apoptosis and attenuated cancer cell migration
To determine miR-27a tumor suppressing functions, miR-27a precursor were transfected into the HCT116 cell, and the effects on cell proliferation, apoptosis and migration were analyzed. As shown in Figure 4A and 4B, increasing miR-27a significantly inhibited cancer cell proliferations after 48 and 72 h at HCT116, Caco-2 and SW480 cells, although the effects at 24 h post transfection were not changed. In addition, increasing miR-27a significantly enhanced cancer cell apoptosis (45% at miR-27a groups versus 15% at vector control group, p,0.01). The examples of apoptosis changes were shown at Figure 4C.
To determine the mechanisms of miR-27a mediated inhibition of cell proliferation and migration and enhancement of apoptosis, we determined the changes of STAT3 and Caspase3. We found that besides the downregulation of total STAT3 by miR-27a, the phosphorylated STAT-3 (p-STAT3) was also dramatically repressed by miR-27a ( Figure 4E). Since miR-27a was reduced in human colorectal cancers, we determined whether p-STAT3 was increased in colorectal cancers. P-STAT was indeed increased in colorectal cancers ( Figure 4F).

MiR-27a inhibited cancer growth in mice
To determine miR-27a tumor suppressing effects in vivo, we transplanted murine colon cancer cells MC38 into wild-type C57Bl/6 mice and injected a mixture of miR-27a precursor and Lipofectamine 2000 into the tumors when the tumors were palmable at day 21 post inoculation. Compared to vector control, miR-27a treatment significantly inhibited cancer cell growth in mice, in terms of significant reduction of tumor sizes and weight ( Figure 5A, 5B). qRT-PCR showed that miR-27a level was still at a higher level in the tumors isolated from mouse xenografts ( Figure 5C).

Discussion
It is well known that the functions and target genes of a miRNA are tissue-and cancer-type specific. Functional studies have shown that miR-27a has shown both oncogenic and tumor suppressive functions in different cell lines and human cancer tissues. For example, miR-27a directly suppresses ZBTB10/RINZF expression [10,17]. and in turn upregulates VEGF and VEGF receptor in the cancers of breast [11,12] and colon [13][14][15], and overexpression of miR-27a is associated with poor outcomes [11,12]. Whereas, in the cancers of esophagus [18], oral cavity [19], lung [21], and head and neck [34], miR-27a is downregulated, and miR-27a directly targets MET and EGFR and suppresses their expression in lung cancer [21]. In this study, we found that miR-27a was significantly downregulated in colorectal cancers and colorectal cancer cells. Importantly, the downregulated miR-27a was also associated with colorectal cancer pathological stages and distant metastasis, showing tumor suppressor roles in colorectal cancer. First, in vitro studies showed that increased expression of miR-27a inhibited colorectal cancer cell proliferation, promoted apoptosis and attenuated cancer cell migration. Second, in a tumor-bearing mouse model, a direct injection of miR-27a to tumor suppressed tumor growth. These findings strongly suggested that miR-27a could be used as a biomarker to monitor cancer development and progression, and could be used as a potential therapeutic target and even a potential therapeutic agent for colorectal cancer.
The mechanistic studies further showed that miR-27a-mediated tumor suppressor could be through targeting SGPP1, Smad2 and STAT3. Previous studies have demonstrated that Apc mutation and Wnt/b-catenin signaling activation, and chronic inflammation in colon, are the two major causes for colorectal cancer formation [35][36][37]. The studies from us have shown that genetic deletion of the Muc2 gene is sufficient to cause chronic colitis and rectal inflammation at early stage, and leads to colorectal cancer formation at late stage [9,22,29,38]. The malignant transformation is linked to the activation of inflammatory signaling and upregulation of cytokines [22,38]. And the chronic inflammation facilitated Apc-mutation-caused gastrointestinal tumor formation in the Apc/Muc2 double gene knockout mouse model of colorectal cancer [22]. In fact, other studies have demonstrated that the transcription factor STAT3 also plays a critical role in inflammation-associated colorectal cancer formation [31,[39][40][41][42]. During the transition of chronic intestinal inflammation to colitisassociated cancer, STAT3 could be persistently activated by sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1), which was linked to production of the multifunctional NF-kB-regulated cytokine IL-6, and consequently upregulating of the S1P receptor, S1PR1 [31]. While, the balance of S1P levels are regulated by sphingosine-1-phosphate phosphatase 1 (SGPP1) [30].
Using comprehensive approaches, including qRT-PCR, immublotting and in situ immunohistochemical staining, we showed that the expression of SGPP1 at mRNA and protein levels were upregulated in colorectal cancers and colorectal cancer cell lines, which were inversely correlated with the expression of miR-27a. Both dual luciferase assay and increasing expression of miR-27a further showed the inhibitory effects of miR-27a on SGPP1. Thus, this is the first to reveal that SGPP1 is a potent direct target of miR-27a, although the evidence of direction regulatory interaction is needed for further investigation. Limited studies have demonstrated that SGPP1 catalyzes the degradation of S1P via salvage and recycling of sphingosine into long-chain ceramides, and that aberrant expression of SGPP1 is associated with idiopathic pulmonary fibrosis, and pulmonary fibrosis [30]. However, the biological functions for SGPP1 in carcinogenesis are largely unknown and worthy further elucidative.
Smad2 is a member of the Smad family and is a key element in TGF-b signaling. Therefore, accompanying actions of TGF-b in different circumstances, e.g., in regulating development and differentiation in physiological cell process, and in facilitating cell growth and migration and angiogenesis in cancers, Smad2 exerts dual functions as tumor suppressor or oncogene [32,33,43,44]. In the current study, we found that, similar as SGPP1, Samd2 expression at mRNA and protein levels was upregulated in colorectal cancers and cell lines, exhibiting oncogenic phenotypes; moreover, Smad2 was repressed at transcriptional and translational levels by miR-27a, suggesting the direct target of miR-27a, a novel finding that has not been reported previously. A few recent studies have reported that Smad2, similar as Smad3 and Smad4, is mutant in cancers [43], but Smad2 plays differential roles from Smad3 and Samd4 in TGF-b signaling [33]. The mutation frequency in colorectal cancer and whether the mutation of Smad2 leads to stability of SMAD2 protein in the colorectal cancer is not clear and warrants further investigation. Previous studies have also shown therapeutic role of Smad2, that blocking Smad2 could suppress TGF-b-induced tumorigenesis, epithelialmesenchymal transition (EMT), cell motility, and invasion [45], indicating that targeting miR-27a/Smad2 could have a great impact on developing a novel strategy for colorectal cancer therapy. The new signal pathway of miR-27a-Smad2-TGF-b could also contribute to the inhibitory role of miR-27a on cancer cell migration ( Figure 4D), invasion and metastasis, detailed mechanism is under investigation.
In summary, we have demonstrated that miR-27a is frequently downregulated in colorectal cancer, and the reduced miR-27a is correlated with cancer distant metastasis and histopathological stages, and thus, miR-27a acts as a tumor suppressor. Both in vivo and in vitro studies have identified SGPP1 and Smad2 as two novel targets of miR-27a, which is linked to STAT3 to regulate cancer cell proliferation, apoptosis and migration. Therefore, miR-27a could be a useful biomarker for colorectal cancer development and progression, and also could have a therapeutic potential targeting SGPP1, Smad2 and Stat3 for colorectal cancer therapy. Figure S1 Prediction of miR-27a targets. A. The targets were categorized by biological process (filtered by sequences numbers, cutoff = 5.0). B, The targets were clustered into multiple categories by biological process level 2 using Go Oncology tool. (TIF)