miR-144-3p inhibited the growth, metastasis and epithelial-mesenchymal transition of colorectal adenocarcinoma by targeting ZEB1/2

miR-144-3p is aberrantly expressed in several types of human cancer and functions as a tumor suppressor by inhibiting metastasis. However, the clinical significance and biological function of miR-144-3p in colorectal adenocarcinoma (CRA) have yet to be elucidated. Here we reported that miR-144-3p expression level was significantly down-regulated in CRA tissues compared with matched noncancerous colorectal mucosae tissues. Low miR-144-3p expression was correlated with adverse clinicopathologic characteristics and poor prognosis of CRA patients. Cox regression analysis showed that low miR-144-3p expression was an independent risk factor for DFS and OS in CRA. In vitro and in vivo assays showed that miR-144-3p significantly inhibited proliferation, migration and invasion of CRA cells. In particular, miR-144-3p could suppress EMT process of CRA cells by regulating the cytoskeleton and EMT markers. Bioinformatics analysis indicated that EMT associated transcription factors ZEB1 and ZEB2 were potential targets of miR-144-3p, and miR-144-3p inhibited ZEB1 and ZEB2 expression and was negatively correlated with their expression in CRA. Finally, we confirmed that ZEB1 and ZEB2 down-regulation collaboratively mediated the inhibitory effect of miR-144-3p on proliferation, invasion and EMT of CRA cells. In conclusion, our study provided evidence that miR-144-3p could inhibit CRA cell proliferation, invasion and EMT by targeting ZEB1/2.


INTRODUCTION
Colorectal cancer (CRC) has become the second leading cause of cancer related death, accounting for about 1 in 10 cancer death [1]. About 98% of CRC histopathologic type is colorectal adenocarcinoma (CRA) [2]. Despite improved survival rate of CRA patients due to the development of chemotherapeutic treatments and surgical technology, about 30% CRA patients present with metastasis at the time of diagnosis, which is the major obstacle to improving the prognosis of CRA patients [3]. The liver is the major organ for CRA metastasis, and liver metastasis would develop in about 15% of operable CRA patients and over 50% of inoperable CRA patients [4].

AGING
Tumor metastasis involves complex cascade, which is regulated by molecular mechanisms [5]. Therefore, understanding the molecular mechanisms of CRA metastasis cascade will help develop new targeted drugs and improve the survival rate of CRA patients.
In this study, we investigated the expression profile and clinical significance of miR-144-3p in CRA patients and explored molecular mechanisms of miR-144-3p in CRA.

Colorectal adenocarcinoma samples
Fresh frozen colorectal adenocarcinoma tissues (CRAT) and corresponding noncancerous colorectal mucosae tissues (NCMT) harvested from 160 CRA patients at the First Affiliated Hospital of Nanchang University from January 2009 to December 2011 were set as training cohort. In addition, matched NCMTs, CRATs, liver metastatic nodule (LMNs) were obtained from 8 patients with liver-only metastases. Another 140 matched fresh frozen CRATs and NCMTs from CRA patients undergoing radical resection between July 2011 and July 2013 at People's Hospital of Pingxiang were set as validation cohort. None of CRA patients had chemotherapy or radiotherapy before the surgery. This study was approved by Ethics Committee of First Affiliated Hospital of Nanchang University. All participants provided informed consent.

Follow-up study
Overall survival (OS) indicated the time from the surgery to tumor related death or the last follow-up if the patients survived [2]. Disease-free survival (DFS) indicated the time from the surgery till the patient survived without recurrence or metastasis [2]. All follow-up data were collected for further analysis.

Cell culture and transfection
Cancer cell lines HCT116, LoVo, SW480, HT-29, SW620 and colorectal mucosal cell line FHC were provided by American Type Culture Collection. Cell were cultured and cell transfection was performed with miR-144-3p mimic, inhibitor and their corresponding control sequences (RiboBio, Guangzhou, China) by using Lipofectamine (Invitrogen, Carlsbad, CA, USA).

PCR
Total RNA was extracted by using TRIzol (Invitrogen) and cDNA was synthesized by using cDNA kit (Toyobo, Japan). PCR analysis was performed by using SYBR ® -Green Master kit (Toyobo) (details in Supplementary Materials).

Western blot analysis
Total proteins were extracted by using RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China), and the details for Western blotting were shown in Supplementary Materials.

Cell proliferation assay
The proliferation ability of CRA cells was analyzed by methyl thiazolyl tetrazolium (MTT), EdU proliferation and colony formation analysis (details in Supplementary Materials).

Transwell and cell adhesion assays
Cell motility and invasive ability and cell adhesion were examined by transwell migration and invasion assay, cellcell adhesion and cell-extracellular matrix (ECM) assay, respectively (details in Supplementary Materials).

Immunofluorescence (IF) and flow cytometry
The details of IF staining and flow cytometry of vimentin and E-cadherin were described in Supplementary Materials. Briefly, cells were fixed and incubated with rhodamine conjugated phalloidin (Solarbio, Beijing, China). The nuclei were stained with DAPI and cells were observed under fluorescence microscope (Nikon Corporation, Japan).

Animal experiments
CRA cells (5 × 10 6 ) were subcutaneously injected into left upper flank of nude male BALB/c mice (4-5 weeks old). Tumor volume was calculated with formula: tumor volume (cm 3 ) = (long axis × short axis 2 )/2 [22]. After 6 weeks, tumor tissues were dissected. For in vivo metastatic assay, the spleen was exposed after anesthesia and the incision on left lateral flank in nude mice. One month after intrasplenic injection of 1×106 CRA cells, the mice were euthanized and liver specimens were collected to examine metastatic nodules.

Statistical analysis
All data were analyzed with software SPSS 18 (SPSS, Chicago, IL, USA). The differences between two groups were analyzed by t test or χ 2 test. Survival curve was calculated by Kaplan-Meier method. Factors associated with OS and DFS were identified by Cox proportional hazard regression analysis. P < 0.05 indicated significance.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Institutional Review Boards of the First Affiliated Hospital of Nanchang University and Jiangxi Pingxiang People's Hospital, and was performed in accordance with the Declaration of Helsinki and current ethical guidelines. Prior informed consent was obtained from all participants.

Consent for publication
Patients provided written informed consent for publication. All authors have read and approved of publication of this manuscript.

Availability of data and materials
The datasets used and/or analyzed during the current study are openly available.

miR-144-3p was significantly downregulated in CRA
The analysis of 160 pairs of fresh frozen CRATs and corresponding NCMTs from training cohort showed that miR-144-3p level was lower in CRATs than in NCMTs ( Figure 1A). Furthermore, miR-144-3p level was lower in CRATs with TNM stage III than in those with TNM stage I/II ( Figure 1B). Moreover, analysis of 8 matched NCMTs, CRATs and LMNs demonstrated that miR-144-3p level gradually decreased from NCMTs, CRATs to LMNs ( Figure 1C). Analysis of miR-144-3p level in CRATs and LMNs from the GEO dataset (GSE44121) showed that miR-144-3p level was lower in LMNs than in CRATs ( Figure 1D).

miR-144-3p inhibited CRA cell proliferation
Next we explored biological function of miR-144-3p in CRAPCR analysis of miR-144-3p expression in AGING CRA cell lines and colorectal mucosal cell line FHC showed lower expression of miR-144-3p in CRA cell lines than in FHC cell line (Figure 2A). In particular, HCT116 had the highest and Lovo had the lowest level of miR-144-3p, and they were selected for subsequent assays. Transfection of miR-144-3p mimic effectively increased miR-144-3p level in Lovo cells and transfection of mR-144-3p inhibitor reduced miR-144-3p level in HCT116 cells ( Figure 2B).

miR-144-3p inhibited CRA metastasis
Transwell migration assays showed that miR-144-3p mimic significantly inhibited Lovo cell migration, while miR-144-3p downregulation in HCT116 significantly increased cell migration ( Figure 3A). Transwell invasion assays showed similar results ( Figure 3B). Adhesion assays showed that miR-144-3p mimic significantly enhanced cell-cell adhesion but decreased cell-ECM adhesion in Lovo cells. However, miR-144-3p inhibitor significantly decreased cell-cell adhesion but increased cell-ECM adhesion in HCT116 cells ( Figure 3C, 3D). Furthermore, we found lower number of liver metastatic nodules from mice with the injection of Lovo cells transfected with miR-144-3p mimic and higher number of liver metastatic nodules from mice with the injection of HCT116 cells transfected with miR-144-3p inhibitor, compared to control mice ( Figure 3E).

ZEB1 and ZEB2 mediated the inhibition of EMT by miR-144-3p
Finally, we examined whether miR-144-3p may inhibit EMT of CRA cells by targeting ZEB1 and ZEB2. In Lovo miR-144-3p mimic cells, the upregulation of ZEB1 or ZEB2 alone could partly upregulate vimentin expression and partly inhibit E-cadherin expression ( Figure 7A, 7B). When both ZEB1 and ZEB2 were upregulated, vimentin expression increased while Ecadherin expression decreased significantly ( Figure 7A, 7B). In HCT116 miR-144-3p inhibitor , knockdown of ZEB1 or ZEB2 alone could partly downregulate vimentin expression and upregulate E-cadherin expression, but the effects were better after knockdown of both ZEB1 and ZEB2 ( Figure 7A, 7B). Immunofluorescence and flow cytometry confirmed that ZEB1 and ZEB2 mediated the effect of miR-144-3p on EMT in CRA cells ( Figure 7C, 7D).
Next we performed IHC to detect E-cadherin, vimentin, ZEB1 and ZEB2 in serial sections of NCMT and CRAT with high or low miR-144-3p expression level, and found that NCMT and CRAT with high miR-144-3p level had low levels of ZEB1, ZEB2 and vimentin and high level of E-cadherin, while CRAT with low miR-144-3p level exhibited opposite levels of these proteins ( Figure 7E).

DISCUSSION
This study showed that miR-144-3p level in CRATs was lower compared to matched NCMTs, and was lower in CRATs with advanced TNM stage than in CRATs with early TNM stage. In addition, miR-144-3p level progressively decreased from matched NCMTs, CRATs to LMNs. These results suggest that miR-144-3p may modulate CRA metastasis.
In this study, miR-144-3p level in CRA was significantly correlated to aggressive clinicopathological features and poor OS and DFS. In addition, low miR-144-3p level was a risk factor for OS and DFS in CRA. Therefore, miR-144-3p can be a prognostic biomarker for CRA patients after radical surgery.
Studies have shown that miR-144-3p could inhibit tumor proliferation, invasion and metastasis [36][37][38]. However, its biological function in CRA remains unclear. We demonstrated that miR-144-3p inhibited CRA cell proliferation, invasion and metastasis, indicating its pivotal role in CRA progression. miRNAs play critical role in regulating EMT during CRA development [39]. miR-144-3p could inhibit EMT and AGING AGING metastasis of renal cell carcinoma, gastric cancer, and breast cancer [18][19][20]. In this study we found that miR-144-3p regulated cell cytoskeleton, decreased vimentin expression while increased E-cadherin expression in CRA cells. These results indicate that miR-144-3p may suppress CRA metastasis by inhibiting EMT process.
ZEB1 and ZEB2 are EMT-associated transcription factors involved in tumor metastasis [40][41][42]. Based on miRNA target prediction algorithms, both ZEB1 and ZEB2 were predicted to contain binding site of miR-144-3p. We demonstrated that miR-144-3p bound 3'-UTR of ZEB1 and ZEB2, and inhibited their expression in CRA. In addition, ZEB1 and ZEB2 are overexpressed in CRA and are related with poor prognosis for CRA patients [43,44].
In this study, the levels of miR-144-3p and ZEB1/2 were negatively correlated in CRATs, indicating that miR-144-3p may downregulate ZEB1 and ZEB2 in CRA. We further confirmed that overexpression of ZEB1 or ZEB2 alone could only partly recover inhibitory effects of miR-144-3p on CRA cell proliferation, migration and invasion, while overexpression of both ZEB1 and ZEB2 abrogated inhibitory effects of miR-144-3p on CRA cell proliferation, migration and invasion. Although miR-144 downregulated ZEB1 and ZEB2 in various types of cancers [44,45], this is the first study to investigate the interaction of ZEB1/2 and miR-144 in CRA. By rescue experiments, we confirmed that ZEB1 and ZEB2 downregulation collaboratively mediated inhibitory effects of miR-144-3p on EMT in CRA. Therefore, our study systematically explored the role of miR-144-3p in CRA.

CONCLUSIONS
In summary, miR-144-3p was downregulated in CRA and its downregulation was significantly correlated to poor prognosis of CRA patients. Moreover, miR-144-3p inhibited CRA cell proliferation, invasion, and EMT process in vitro and suppressed CRA metastasis in vivo. Furthermore, we revealed the mechanism that miRNA-144-3p inhibited ZEB1 and ZEB2 expression to suppress CRA growth and metastasis. Therefore, our study suggested that miR-144-3p could be a therapeutic target and prognostic marker for CRA.

Cell lines
The normal colorectal mucosal cell line FHC, and colorectal adenocarcinoma (CRA) cell line SW480, SW620, HCT116, LoVo, HT-29 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Short tandem repeat (STR) DNA fingerprinting was used to authenticate all cell lines prior to commencement of the study. All cell lines were routinely cultured with RPMI-1640 (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), and maintained in a 5% CO2 humidified incubator at 37° C.

qRT-PCR analysis
Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc) according to the manufacturer's protocol. After determining the concentration, the RNA was then reverse transcribed to obtain cDNA using the universal cDNA synthesis kit (Toyobo, Osaka, Japan). qRT-PCR analysis was performed using the SYBR ® -Green Realtime PCR Master Mix assay kit (Toyobo, Osaka, Japan). GAPDH and U6 were used as internal controls for gene mRNA and miRNA respectively. The primer sequences were as follows: Cell transfection miR-144-3p mimic or inhibitor and their corresponding control sequences (RiboBio, Guangzhou, China) were transfected into the indicated CRA cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific Inc) following the manufacturer's protocol. After 48h transfection, the transfected cells were harvested and verified their efficiency for subsequent experiments. For transfection of ZEB1 and ZEB2 ORF for ZEB1 and ZEB2 overexpression or short hairpin RNA (shRNA) for ZEB1 and ZEB2 knockdown, the lentiviral vectors encoding ZEB1 and ZEB2 ORF or shRNA-ZEB1/2 were synthesized and purchased from GenePharma Co., Ltd (Shanghai, China). The empty vector was used as the negative control. The sequences of shRNAs for ZEB1 and ZEB2 knockdown were as follows: shRNA-ZEB1: 5'-CCUAGUCAGCCACCUUUAATT -3'; shRNA-ZEB2: 5'-GUAAUGACUAGGGCUA UUA-3'. Lentiviral vectors were transfected into cells in 6-well plates using Lipofectamine ® 3000 (Invitrogen, Thermo Fisher Scientific Inc.) according the manufacturer's protocol. The efficiency of knockdown or overexpression of ZEB1 and ZEB2 was confirmed by qRT-PCR and western blot. The stably transfected cells were subjected to subsequent experiments.

Cell proliferation and colony formation assays
Methyl thiazolyl tetrazolium (MTT) assays and colony formation assays were used to determine CRA cell proliferation ability. For the MTT assays, 5 × 10 3 cells were seeded into each well of 96-well plates. 100 µl fresh medium containing 0.5 mg/ml MTT (MilliporeSigma) was added into each well and incubated at 37° C for 4 h. The absorbance was measured at 570 nm. For the colony formation assay, about 500 cells were seeded into 35-mm cell-culture dishes (Corning Incorporated; Corning, NY, USA) and cultured for 2 weeks. The number of colonies per dish was calculated following being stained with crystal violet (Beyotime Institute of Biotechnology, Jiangsu, China). Only positive colonies (diameter > 40µm) were calculated and compared. The experiments were repeated three times.

EdU proliferation assays
Cell proliferation was detected using the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) with the EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China). Briefly, the indicated cells were seeded in 96-well plates and cultured in complete media under the condition of 5% CO2, at 37° C. After incubation with 50 µM EdU for 4h, the cells were fixed, permeabilized and stained with EdU in order according to the manufacturer's protocol. Then the cell nuclei were stained with DAPI at a concentration of 1 µg/ml for 30 second. The typical images of the plates were taken using an inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan). The experiments were repeated with three wells.

Transwell assay
Transwell migration and invasion assays were separately used to test CRA cell motility and invasion ability. Briefly, after pre-incubation with 10 µg/ml Mitomycin-C for 1 h to inhibit cell proliferation, about 1x10 5 cells in serum free medium were placed into the upper chamber of the insert. The upper chamber of the insert was coated with Matrigel (BD Biosciences; Franklin Lakes, NJ, USA) used for Transwell invasion assays, while the upper chamber of the insert without Matrigel used for Transwell migration assays. Following incubation for 24 h, the number of cells that adhered to the lower membrane of the inserts was calculated after stained with 0.1% crystal violet (Beyotime Institute of Biotechnology). For each group, the assays were repeated three times, and five random fields of view were selected for analysis.

Adhesion assay
The adhesive ability of the CRA cells was analyzed by cell-extracellular matrix (ECM) and cell-cell adhesion assays. For the cell-ECM adhesion assay, a 96-well plate was plated with fibronectin at 37° C for 60 min and washed twice with DMEM buffer supplied with 0.1% BSA (HyClone, GE Healthcare Life Sciences; Logan, UT, USA). The plates were blocked with DMEM buffer supplied with 0.5% BSA at 37° C in a 5% CO2 incubator for 60 min. About 1 × 10 4 cells (100 µl cells at a density of about 1x10 5 /ml) were added into each well of a 96-well plate (Costar; Corning Incorporated, Corning, NY, USA) and cultured at 37° C. Five wells for each group were determined at 60, 90 or 120 min. After remove of the nonadhesion cells, then 100 µl fresh medium containing 0.5 mg/ml MTT (MilliporeSigma) was added into each well and incubated at 37° C for 4 hours. The wells were then added 100 µl DMSO following remove of the medium and shaken at room temperature for 10 min. Finally, the absorbance was measured at 570 nm. For the cell-cell adhesion assay, sub-confluent (70-80%) cell layers were rinsed twice with Ca2-and Mg2-free PBS and separated by incubation in HBSS containing 1 mmol/L EDTA at 37° C for 20 min. And then, 100 µl single cells at density of about 1 × 10 5 /ml were added into a 96-well plate (Corning Costar Incorporated) with a fully confluent single cell layer, and cultured at 37° C for 0-120 min. The non-adhesion cells were collected from the wells, and counted using an inverted TE-2000S microscope (Nikon Corporation). The adhesion rate was determined by calculating representative aliquots from each well on a hematocytometer. The adhesion rate was calculated at 60, 90 or 120 min as follow: N0-Nt/N0 ×100%, where Nt is the number of non-adhesion cells at the incubation time t, and N0 is the total number of cells added.

Immunofluorescence (IF)
The CRA cells were grown on the glass coverslips, and then fixed with 4% paraformaldehyde. After permeated in phosphate-buffered saline (PBS) with 0.2% Trion X-100, CRA cells were blocked for an hour with 1% bovine serum albumin (BSA), and then incubated with primary antibody overnight at 4° C. In the following day, the cells incubated with appropriate concentration of secondary antibody (Beyotime Institute of Biotechnology) and DAPI (Beyotime Institute of Biotechnology) separately. Images of the slides were captured using an inverted fluorescence microscope. Primary antibodies for E-cadherin, vimentin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Flow cytometry
CRA cells were seeded in 6-well plates and incubated for 24 h. Then monocytes were washed and incubated with E-cadherin, vimentin antibody (CST; Danvers, MA, USA) at a dilution of 1:10 in phosphate buffered saline (PBS)-bovine serum albumin (BSA) for 30 min at 4° C.