MicroRNA-491-5p inhibit gastric cancer development by targeting EMT, cell adhesion genes and IFITM2

Background: Gastric cancer (GC) is one of the deadliest cancers in China. And, it can be regulated by MicroRNAs (miRNAs) generally. miR-491-5p function as a tumor suppressor in different types of cancer, but we still don’t know the role of miR-491-5p in gastric cancer. Methods: Functional experiments including CCK-8 assay and transwell assay were performed. Furthermore, the underlying mechanism was explored through qRT-PCR and western blot assay. In addition, the function of miR-491-5p was also identied in vivo. Results: In this study, we found that high level of miR-491-5p caused a weak cell proliferation, migration and invasion abilities. In order to explore the role of miR-491-5p in vivo, we set a xenograft mouse model, and also found that high level of miR-491-5p suppressed tumor growth. Moreover, we found that miR-491-5p regulate the tumor development thought regulate the expression of EMT(Epithelial-mesenchymal transition), cell adhesion genes and IFITM2. Conclusions: These data show that miR-491-5p function as a tumor suppressor in GC both in vitro and in vivo.


Background
Gastric cancer (GC) is an aggressive and deadly malignancy. Surgery and endoscopic mucosal resection (EMR) with or without adjuvant systemic chemotherapy are the primary treatments for gastric cancer [1] .
Currently, there are no effective drugs for gastric cancer. Despite a steady decline in overall morbidity and mortality over the past few decades, most of patients diagnosed with GC are already in an advancedstage so that the 5-year survival rate is very low [2] . Due to its high mortality and geographical distribution, gastric cancer has become an important public health problem in China [3] . Therefore, it is urgent to further explore the gastric cancer progress and develop mechanisms.
MicroRNAs (miRNAs) have been shown to be oncogenes in kind of cancer. To the contrary, it also had been reported as a tumor suppressor in some other kind of cancer [4,5] . In GC, miRNAs have their own unique expression pro le, abnormal expression of miRNAs are associated with abnormal histological subtypes and cause a progression of cancer. miR-491-5p function as a tumor suppressor in kind of cancer that regulate the abilities of cell proliferation, migration, invasion and associated with apoptosis even chemoresistance activities. However, the function of miR-491-5p in GC is not very clearly. Some research reported that miR-491-5p is low expressed in GC, and cause a progression of gastric cancer through regulated Wnt3a/beta-catenin signaling. However, the role of miR-491-5p in GC, particularly metastatic gastric cancer is still not very clearly [6] .
In this research, we are going to nd out the function of miR-491-5p in gastric cancer and the mechanism of miR-491-5p function as a tumor suppresser in gastric cancer.

cell transfection
Human GC cell line AGS was maintained in DMEM-H containing 10% FBS and grown in 5% CO 2 at 37℃.
A total of 1.0×10^6 cells were seeded into 10 cm cell culture dish in 24H before transfection, so that the cells can grow into 70-80% cell density when transfection. First, prepare A and B transfer solution in a sterile 1.5 ML centrifugal tube (A solution: miRNA mimics 20 pmol 30 μL in 100 μL Opti-MEM; B solution: Lipofectamine 2000 Reagent in 100 μL Opti-MEM.), gently mixed and let stand at room temperature for 5 min. Add liquid droplets B to liquid A and mix it gently. Stand at room temperature for 20 min. Second, add the mix transfer solution into the dish, make total volume to 5 ML with Opti-MEM, shake the cell culture dish to make it evenly distributed, and then culture it in a cell incubator at 37℃ and 5% CO 2 . After 4 hours, each dish was changed into 5 ML of DMEM-H culture medium with 10% FBS, and the culture was continued in a cell incubator containing 5% CO 2 at 37℃.

MTT assay
AGS cells were seeded into a 96-well plate in 24 h after transfection, 3000 cells/well, MTT was used to test the cell viability at 24 h, 48 h, 72h. We added 10 μl MTT solution with a concentration of 10 mg/mL into each well, continue to culture for 4 h. Carefully suck away the original culture solution, add 100 μl DMSO to each well, and then oscillate in the shaking bed for 10 min to dissolve the blue and purple crystalline formazan. On the enzyme-linked immune detector measured at the wavelength of 490nm absorbance value of each hole.

Migration assays
For cell migration assays, completely discard the cell culture solution of each group, wash the cells with PBS, add digestive juice (0.02%EDTA, 0.25% trypsin) for digestion after each well, and conduct the Transwell experiment. 4×10^5 AGS cells were plated in chamber insert, serum-free medium is put on the top of the insert, the bottom of the plate to join DMEM medium containing 20% FBS. Cells were incubated for 72 h at 37℃. Remove the Transwell chamber, wipe off the cells on the top of the insert with cotton swabs, and gently wash off the remaining cells with PBS. The mixed solution was prepared with methanol and glacial acetic acid according to 3:1, and the cells on the opposite side of the Transwell chamber were xed for 30 min. Cells with crystal violet blue staining for 15 minutes, and then washed it with double-distilled water (ddH2O). Data are shown as mean± s.d. from three independent experiments.

Invasion assays
For cell invasion assays, completely discard the cell culture solution of each group, wash the cells with PBS, add digestive juice (0.02%EDTA, 0.25% trypsin) for digestion after each well, and conduct the Transwell experiment. Dissolve matrigel gel, add 50μl of gel to each chamber, 4×10^5 AGS cells were plated in chamber insert, serum-free medium is put on the top of the insert, the bottom of the plate to join DMEM medium containing 20% FBS. Cells were incubated for 72 h at 37℃. Remove the Transwell chamber, wipe off the cells upside of the insert with cotton swabs, and gently wash off the remaining cells with PBS. The mixed solution was prepared with methanol and glacial acetic acid according to 3:1, and

HE staining
Clinical samples were soaked in xylene for 10 min, and then soaked for another 10 min after replacing xylene. And then soak in gradient ethanol (100%, 95%, 85%, 70% and 50%) for 30 second to 1 min respectively. Wash twice with PBS, each time for 5 min. stained with heamatoxylin for 2 min, wash with tap water. Hydrochloric acid alcohol was differentiated three times, each time for 2 seconds, and wash with tap water. Soaked with gradient ethanol (50%, 70%, 85%, 95%, 100%) to dehydration. Soak the tissue slices in xylene for 10 min, and soak them for another 10 min after replacing xylene. Add 1-2 drops of neutral gum and tilt the cover glass from one end to prevent bubbles. Observation with microscope, take photos with 3 visual elds, 200×.

In vivo xenograft study
The animals used in this study were obtained from the company of Beijing Hua kang. To the side of the subcutaneous implanted into nude mice AGS cells, We measure the nude mice weight once a week. And after experiment, these animals were killed by the injection of Barbiturate. Then the tumors were dissected, weighed and photographed.

Statistical analyses.
The differences between two groups were tested by student's t-test, When * p 0.05, we think it has statistical signi cance. Student's test was used for all statistical analyses with the SPSS 17.0 software.

miR-491-5p inhibits gastric cancer cells proliferation and metastasis
It has been reported that miR-491-5p is low expressed in gastric cancer, and perhaps function as a tumor suppressor. In order to nd out the function of miR-491-5p in gastric cancer, we transfect miR-491-5p into AGS cells and then observed the change of cells proliferation and metastasis abilities. The results of MTT assay shows that the cells transfected with miR-491-5p mimics have a low cell activity than control. The inhibitor of miR-491-5p we named it ASO-miR-491-5p treated can increase the cell activity. The same result can be got in 24 h, 48 h and 72h (Fig.1A). Transwell assay was used to test the invasion and metastasis changes of AGS cells. As expected, transfection with the miR-491-5p mimics decrease the invasion and metastasis abilities of AGS cells, and the miR-491-5p inhibitor signi cantly increase the invasion and metastasis abilities ( Fig.1B-1C). To further test the effect of miR-491-5p on AGC cells growth, we performed colony formation assay. Cell transfected miR-491-5p showed decreased colony formation, and the simultaneous miR-491-5p inhibitor increased colony formation compared with the control cells (Fig. 1D).

miR-491-5p effectively downregulates genes associated with EMT, cell adhesion and IFITM2
EMT plays an important role in cancer metastasis. At rst, it can cause the downregulation of E-cadherin, so that leading to loss of cell-cell contact. Secondly, it could upregulate N-cadherin, Vimentin, which mediate the mesenchymal phenotype of cells. It has been reported that β-catenin can be mediate by Ecadherin. When E-cadherin is downregulated, β-catenin become more active, so that it translocated to the nucleus and activated the expression of target genes including IFITM family members that promote tumor initiation and progression [7] .
To test the association between miR-491-5p and EMT associated genes expression, we performed a qRT-PCR assay. The result shows that high miR-491-5p level related to low expression of N-cadherin, βcatenin, Vimentin and IFITM2 and high expression of E-cadherin. When we used the miR-491-5p inhibitor ASO-miR-491-5p, the expression of these genes can be recovery. The result shows that miR-491-5p can directly downregulated the expression of the EMT associated and cell adhesion gene in transcription level ( Fig.2A). Moreover, the western blot results were consistent with the RT-PCR result (Fig.2B).

miR-491-5p suppresses the growth of AGS xenograft tumors
Based on our in vitro results, we further investigated the inhibitory potential of miR-491-5p on GC growth in vivo. To the side of the subcutaneous implanted into nude mice AGS cells. We measure the nude mice weight once a week, and there were no big difference between mimics NC and miR-491-5p mimics group.
It means that the tumor growth don't have effect on mice growth (Fig.3A). At the endpoint of the assay, we remove the tumor and weight it. As expected, the tumors in miR-491-5p mimics group are not as big as mimics NC group (Fig.3B-3D). The miR-491-5p mimics transfect decrease the growth of GC cells in vivo.

miR-491-5p regulates genes associated with EMT, cell adhesion and IFITM2 in vivo
H&E staining were performed to observe the tumor histomorphology in mice. In miR-491-5p mimics group, the cells show less nuclear heterogeneity than control group (Fig.4A). To further con rm the result that miR-491-5p can downregulate the genes associated with EMT, cell adhesion and IFITM2 in vivo, we used western blot to test the level of these protein expression. N-cadherin, β-catenin, Vimentin and IFITM2 were low expressed in miR-491-5p mimics group, E-cadherin was high expressed in miR-491-5p mimics group (Fig.4B). Moreover, the immunohistochemical results con rm the result (Fig.4C). In total, miR-491-5p could downregulate the genes associated with EMT, cell adhesion and IFITM2 in vivo.

Discussion
There is increasing evidence that miRNA can play a proliferating or anti-proliferating role in physiological conditions, while abnormal expression of miRNA can cause a lot of healthy problems, including cancer [2,[8][9][10] . miRNAs dysregulation has been identi ed as a marker of caners. And also found the miRNAs expression changes in gastric cancer [11][12][13] . Recently discovered miRNA miR-491, is downregulated in several types of cancer, including breast cancer, oral squamous cell carcinoma, hepatocellular carcinoma, pancreatic, glioblastoma, ovarian cancer and hepatocellular carcinoma [13][14][15] . miR-491-5p has been shown to be a mature form of miR-491 that induce apoptosis and inhibit the proliferation of pancreatic, ovarian, breast cancer and colorectal cells, as well as the migration and invasion of glioma and breast, oral squamous cell. It means that miR-491-5p play as a tumor suppressor [1,16,17] .
It has been reported that miR-491-5p not only inhibit the proliferation, invasion and migration but also induced cell apoptosis and suppressed tumor growth [18] ; In estrogen-receptor-α-positive breast cancer, miR-491-5p functions as a tumor suppressor through mediate JMJD2B [11] ; In oral squamous cell carcinoma, miR-491-5p and GIT1 function as modulators and biomarkers of invasion and metastasis. Our data shown that increasing expression of miR-491-5p cause a low cell proliferation, migration and invasion abilities. In order to con rm the conclusion, we established a xenograft mouse model, and the result shows that miR-491-5p overexpression suppressed tumor growth. In summary, miR-491-5p functions as a tumor-inhibitory in gastric cancer in vitro and in vivo.
EMT has to obvious characteristics, one is that acquisition of a mesenchymal phenotype and the other is loss of epithelial characteristics. These two characteristics have closely related to tumor metastasis and tumor recurrence. There are some EMT molecular markers, the level of N-cadherin, E-catenin and Vimentin suggest the state of EMT [19] . Cell-cell adhesion de nes cellular and tissue morphogenesis so that it is a fundamental biological process in multicellular organisms. During normal tissue homeostasis, signals transmitted by adhesion between cells and to the underlying extracellular matrix (ECM) are closely related to gene regulation. WNT signaling activation and EMT-associated changes have closely relate with the displacement of β-catenin from adherens junctions in normal and cancer cells. Interferon (IFN)-induced transmembrane protein (IFITM) family has at least three members, IFITM1, IFITM2 and IFITM3. IFITMs have been reported could control the cell cycle, and then function as tumor suppressors, while the conserved cluster of differentiation 225 domains is important for the function.It has been reported that IFITM2 acted as a pro-apoptotic gene in a p53-independent manner in colon cancer [20] . In this study, we found out that miR-491-5p function as a tumor-inhibitory in gastric cancer, and the molecular mechanism is that it direct targeting EMT, cell adhesion genes, including N-cadherin, β-catenin, E-cadherin, Vimentin and IFITM2.

Conclusion
In conclusion, we demonstrated that miR-491-5p can downregulate the cell viability, invasion, metastasis and clone formation abilities of GC cells; miR-491-5p can also downregulate the GC associated protein expression and inhibit the tumor grow in mouse model.

Supplementary Files
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