{"paper_id":"6c63f7ce-213c-4256-816c-be55eba1b3bd","body_text":"Colorectal cancer (CRC) is the fourth most deadly cancer in the world, accounting for 10 percent of diagnosed cancers and cancer-related deaths worldwide [ 1 ]. Nearly 900,000 people die of CRC each year [ 2 ]. Owing to aging population and dietary habits, the risk of CRC is high in high-income countries. Some other factors such as obesity, smoking, and lack of physical activity also increase the risk of CRC [ 3 ]. Much progress has been made in the treatment of CRC with surgical resection, adjuvant chemotherapy and radiotherapy. However, the recurrence and metastasis rate of CRC are still high [ 4 ]. Therefore, it is essential to elucidate the underlying mechanisms involving in the progression and development of this disease.\nP21 activated kinase 4 ( PAK4 ) is a member of Group 2 of the p21 activated kinases, which are downstream effectors of CdC42 and Rac1 and participate in biological behaviors of cancer [ 5 ]. It has been shown to suppress cell proliferation and its expression was upregulated in various cancers [ 6 ,  7 ]. A study on glioblastoma suggested that  PAK4  was down-regulated by miR-485 and inhibited the malignant biological behavior of glioblastoma cells [ 8 ]. In esophageal cancer, miR-199a-3p was reported to reduce cell proliferation by targeting  PAK4  [ 9 ]. Moreover, some studies have shown that certain miRNAs had suppressive effects on CRC cells through targeting  PAK4  [ 10 ,  11 ]. The breast cancer anti-estrogen resistance 3 ( BCAR3 ) is the member of novel Src homology 2 (SH2) containing protein (NSP) family [ 12 ]. Recent study showed that overexpression of  BCAR3  regulated cytoskeletal rearrangement [ 13 ]. It was also reported that  BCAR3  overexpression increased cell migration and invasion in endometriosis [ 14 ]. However, the role of  BCAR3  in CRC remains unclear.\nIn recent years, miRNAs have been shown to play a significant role in cancer occurrence, metastasis, and progression [ 15 ]. They are small noncoding RNAs of about 22 nucleotides in length and are crucial post-transcriptional and epigenetic regulators of gene expression in eukaryotes [ 16 ]. Increasing research have indicated that some miRNAs are important modulators on the progression of CRC [ 17 ,  18 ].\nIn this study, we aimed to explore the role of  PAK4  and  BCAR3  in CRC. We silenced the expression of  PAK4  and  BCAR3  separately and found that these two genes had different role on CRC cells. In view of the diversity of miRNA targets, we screened out miRNAs that inhibited both genes through the bioinformatics database. Surprisingly, we identified miR-199a/b-3p as the potential target miRNA of  PAK4  and  BCAR3 . Our results suggested that miR-199a/b-3p inhibited the proliferation, colony formation of CRC cells through targeting  PAK4 . Meanwhile, the inhibitory effects on migration and invasion were regulated by targeting  BCAR3 .\n\nHuman CRC cells (SW1116) were obtained from the Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). SW1116 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cells were cultured in humidified incubator at 37 °C with 5% CO2.\n3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich. SW1116 cells were seeded at 10,000 cells per well in 96-well plates. Cell proliferation was determined at 24, 48, and 72 h using MTT assay. MTT was dissolved in phosphate buffered saline solution at the concentration of 5 mg/ml. After incubated with 20 μl of MTT for 4 h at 37 °C, the medium was aspirated and dimethyl sulfoxide (DMSO) was added to dissolve formazan. The absorbance was measured at a wavelength of 570 nm using an automated Microplate Reader. The experiment was performed in triplicate.\nAfter the treatments of SW1116 cells, cells were trypsinized to single-cell suspensions of 1000 cells and plated in 6-well plates. The plates were incubated at 37 °C in 5% CO 2 until sufficiently large clones were formed. The colonies were fixed with4% paraformaldehyde and stained with 0.5% crystal violet for 30 min.\nSW1116 cells were seeded into 6-well plates the day before transfection. The transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.\nTarget miRNAs for  PAK4  and  BCAR3  were predicted using the TargetScan database ( http://www.targetscan.org ) and DIANA database ( http://diana.imis.athena-innovation.gr ).\nTotal RNA from cells was extracted using Transzol reagent (Transgen) and then was converted into cDNA using the Transcript miRNA First-Strand cDNA Synthesis SuperMix (Transgen). In the 7500 Real-Time PCR System (ABI), quantitative reverse transcription was carried out in a 20 ul reaction system. qRT-PCR primers for  PAK4  were F: 5′-TCCCCCTGAGCCATTGTG-3′, R: 5′-TGACCTGTCTCCCCATCCA-3′.qRT-PCR primers for  BCAR3  were F: 5′-GCGGTGGAACTGAAGGATTC-3′, R: 5′ -TGGCAGTTTGGGTGTACTGG-3′. GAPDH was used as internal control: 5′-GGACCTGACCTGCCGTCTAG-3′ and 5′-GTAGCCCAGGATGCCCTTGA-3′. qRT-PCR primers for miR-199a/b-3p was F: 5′-GTCACAGTAGTCTGCACAT-3′. U6 was used as internal control: 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′. The 2 −ΔΔCt  method was used to analyze the expression levels of gene and miRNA.\nThe total cDNA from the SW1116 cells was used to amplify the 3′-UTR of  PAK4  and  BCAR3  by PCR. Then, the potential miR-199-3pbinding sites were inserted into the pmirGLO (Promega) vector to construct luciferase reporter vector. HEK293T cells were transfected withmiR-199a/b-3p mimics or Mir-negative controls (Gene pharma). At the same time, Mut or WT reporter vector was co-transfected into HEK293T cells. Cells were harvested and lysed with extraction buffer48 h after transfection. The results were assayed by Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The nucleotide sequences for  PAK4  3′UTR were: 50-GACCC TACTA CTGAA CTCCA GT-3′, and the mutagenesis sequences for  PAK4  30UTR-mut were: 5′-GACCC TACCGTGTAA CTCCA GT-3′. The nucleotide sequences for  BCAR3  3′UTR were: 5′-GACCC TACTA CTGAA CTCCA GT-3′, and the mutagenesis sequences for  BCAR3  3′UTR-mut were: 5′-GACCC TACCGTGTAA CTCCA GT-3′.\nCells were plated in 6-well plates and incubated overnight. The scratches were made using a sterilized 10 μl pipette tip. Then, culture medium was removed to eliminate dislodged cells. The scratch wound was observed at 0 and 24 h. All images were collected under the microscope. All experiments were repeated at least three times.\nMigration and invasion assays were performed in Transwell chambers (8 μm pore size, 24-well plate; Corning). 600 μl volume of 10% FBS-containing medium was added to the lower chamber and 1 × 10 4  cells were diluted in a 200 μl volume of serum-free medium were added to the upper chamber. For invasion assay, the upper chambers were coated with diluted Matrigel (BD Biosciences). After incubation for 48 h at 37 °C with 5% CO 2 , cells on the upper surface of the membranes were removed using a cotton swab. The remaining cells were fixed with 4% formaldehyde and stained with 1% crystal violet. The cells were counted and photographed under the inverted microscope in three fields of the view.\n20 μg of total proteins were loaded onto 8–10% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were subsequently blocked with 5% non-fat milk in TBST for 1 h at room temperature. The membranes were incubated with primary antibodies including  PAK4  (Cell Signaling Technology, dilution 1:1000) and  BCAR3  (Bethyl Laboratories, dilution 1:1000) at 4 °C overnight. GAPDH (Santa Cruz Biotechnology, dilution 1:1000) was used as the loading control. After washing 3 times, the membranes were incubated with anti-rabbit or anti-mouse IgG HRP-linked (Cell Signaling Technology, dilution 1:3000) secondary antibody at room temperature for 1 h and visualized by enhanced chemiluminescence detection kit (Thermo Fisher).\nAll statistical analysis was performed using SPSS 22.0 software. Data were presented as the mean ± SD and differences between groups were analyzed using Student’s  t  test.  P  < 0.05 was considered statistically significant.\n\nTo investigate the role of  PAK4  in CRC cells, we silenced  PAK4  expression by transfecting  PAK4  siRNA expression vectors in SW1116 cells. The results from western blot showed that  PAK4  was significantly decreased in the cells transfected with  PAK4  siRNA vectors (Fig.  1 A). MTT and colony formation assay indicated that the knockdown of  PAK4  significantly inhibited the proliferation and decreased the number of colonies in SW1116 cells (Fig.  1 B–D). However, scratch wound and transwell assays showed that the knockdown of  PAK4  had no significant effect on migration and invasion of SW1116 cells (Fig.  1 E–H). These results indicated the role of  PAK4  in colorectal cell proliferation and colony formation. Fig. 1 Silence of PAK4 inhibited proliferation and colony formation in CRC.  A  Western blot was used to measure the protein levels of the PAK4 and GAPDH protein in SW1116 cells transfected with PAK4 siRNA expression plasmid. GAPDH was used as the loading control.  B  MTT assay was performed on SW1116 cells transfected with PAK4 siRNA expression plasmid; ** p  < 0.01.  C ,  D  Colony formation assay was performed on SW1116 cells transfected with PAK4 siRNA expression plasmid; ** p  < 0.01.  E ,  F  Cell migration was analyzed using scratch wound assay. (bar, 200 μm).  G ,  H  Cell invasion was analyzed using transwell assay. (bar, 100 μm)\nSilence of PAK4 inhibited proliferation and colony formation in CRC.  A  Western blot was used to measure the protein levels of the PAK4 and GAPDH protein in SW1116 cells transfected with PAK4 siRNA expression plasmid. GAPDH was used as the loading control.  B  MTT assay was performed on SW1116 cells transfected with PAK4 siRNA expression plasmid; ** p  < 0.01.  C ,  D  Colony formation assay was performed on SW1116 cells transfected with PAK4 siRNA expression plasmid; ** p  < 0.01.  E ,  F  Cell migration was analyzed using scratch wound assay. (bar, 200 μm).  G ,  H  Cell invasion was analyzed using transwell assay. (bar, 100 μm)\nTo investigate the role of  BCAR3  in CRC cells, we silenced  BCAR3  expression by transfecting  BCAR3  siRNA expression vectors in SW1116 cells. The results from western blot showed that  BCAR3  was significantly decreased in the cells transfected with  BCAR3  siRNA vectors (Fig.  2 A). MTT and colony formation assay indicated that the knockdown of  BCAR3 had no significant effect on proliferation and colony formation in SW1116 cells (Fig.  2 B–D). Scratch wound and transwell assays showed that the knockdown of  BCAR3  significantly inhibited cell migration and invasion of SW1116 cells (Fig.  2 E–H). These results indicated the role of  BCAR3  in colorectal cell migration and invasion. Fig. 2 Silence of BCAR3inhibited cell migration and invasion in CRC.  A  Western blot was used to measure the protein levels of the BCAR3 and GAPDH protein in SW1116 cells transfected with BCAR3 siRNA expression plasmid. GAPDH was used as the loading control.  B  MTT assay was performed on SW1116 cells transfected with BCAR3 siRNA expression plasmid.  C ,  D  Colony formation assay was performed on SW1116 cells transfected with BCAR3 siRNA expression plasmid.  E ,  F  Cell migration was analyzed using scratch wound assay (bar, 200 μm); ** p  < 0.01. (G, H) Cell invasion was analyzed using transwell assay (bar, 100 μm); ** p  < 0.01\nSilence of BCAR3inhibited cell migration and invasion in CRC.  A  Western blot was used to measure the protein levels of the BCAR3 and GAPDH protein in SW1116 cells transfected with BCAR3 siRNA expression plasmid. GAPDH was used as the loading control.  B  MTT assay was performed on SW1116 cells transfected with BCAR3 siRNA expression plasmid.  C ,  D  Colony formation assay was performed on SW1116 cells transfected with BCAR3 siRNA expression plasmid.  E ,  F  Cell migration was analyzed using scratch wound assay (bar, 200 μm); ** p  < 0.01. (G, H) Cell invasion was analyzed using transwell assay (bar, 100 μm); ** p  < 0.01\nGiven that  PAK4  and  BCAR3  had different roles on CRC cells and the diversity of miRNA targets, we screened out potential target miRNAs of these two genes by bioinformatics analysis. According to the results from TargetScan and DIANA database, we chose miR-199a/b-3p as the target miRNA (Fig.  3 A). There are two potential target sites for miR-199a/b-3p at position 128–153 and 583–606 of  PAK4  mRNA 3’UTR (Fig.  3 B). There are four potential target sites for miR-199a/b-3p at position 224–239, 262–276,381–401, and 1188–1208 of  BCAR3  mRNA 3’UTR (Fig.  3 C). Luciferase reporter assay was conducted to examine the hypothesis that miR-199a/b-3p targeted the 3'-UTR of  PAK4  and  BCAR3 . The results indicated that miR-199a/b-3p significantly decreased the luciferase activity of the reporter with wild type 3’-UTR of  PAK4  and  BCAR3  (Fig.  3 D, E). qRT-PCR and western blot analyses verified that miR-199a/b-3p decreased  PAK4  and  BCAR3  expression at mRNA and protein levels in SW1116 cells (Fig.  3 F–H). Results showed that miR-199a/b-3p could directly target the 3′-UTR of  PAK4  and  BCAR3 . Fig. 3 miR‑199a/b-3p was the target of PAK4 and BCAR3.  A  TargetScan and DIANA database were used to predict the target miRNAs of PAK4 and BCAR3.  B  Sequences of predicted miR-199a/b-3p binding sites within PAK4 3’-UTR in DIANA database.  C  Sequences of predicted miR-199a/b-3p binding sites within BCAR3 3’-UTR in DIANA database.  D ,  E  Effect of miR-199a/b-3p on luciferase intensity controlled by the wild type (WT) or mutant (MUT) 3’UTR of PAK4/BCAR3 was determined using the luciferase assay; ** p  < 0.01.  F ,  G  mRNA levels of PAK4/BCAR3 were detected using quantitative RT-PCR in SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01.  H  Protein levels of PAK4 and BCAR3 were detected using western blot in SW1116 cells transfected with miR-199a/b-3p mimics\nmiR‑199a/b-3p was the target of PAK4 and BCAR3.  A  TargetScan and DIANA database were used to predict the target miRNAs of PAK4 and BCAR3.  B  Sequences of predicted miR-199a/b-3p binding sites within PAK4 3’-UTR in DIANA database.  C  Sequences of predicted miR-199a/b-3p binding sites within BCAR3 3’-UTR in DIANA database.  D ,  E  Effect of miR-199a/b-3p on luciferase intensity controlled by the wild type (WT) or mutant (MUT) 3’UTR of PAK4/BCAR3 was determined using the luciferase assay; ** p  < 0.01.  F ,  G  mRNA levels of PAK4/BCAR3 were detected using quantitative RT-PCR in SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01.  H  Protein levels of PAK4 and BCAR3 were detected using western blot in SW1116 cells transfected with miR-199a/b-3p mimics\nTo further explore the role of miR-199a/b-3p in colorectal cells, we transfected the miR-199a/b-3p mimics or negative controls into SW1116 cells. qRT-PCR was used to detect the relative expression of miR-199a/b-3p. The level of miR-199a/b-3p was significantly increased in mimics transfected cells (Fig.  4 A). MTT and colony formation assays indicated that miR-199a/b-3p mimics inhibited the proliferation and decreased the number of colonies in SW1116 cells (Fig.  4 B–D). Fig. 4 miR-199a/b-3p inhibited cell proliferation and colony formation in CRC.  A  Levels of miR-199a/b-3p were detected using quantitative RT-PCR in SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01.  B  MTT assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01.  C ,  D  Colony formation assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01\nmiR-199a/b-3p inhibited cell proliferation and colony formation in CRC.  A  Levels of miR-199a/b-3p were detected using quantitative RT-PCR in SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01.  B  MTT assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01.  C ,  D  Colony formation assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics; ** p  < 0.01\nWe then determined the effect of miR-199a/b-3p on migration and invasion. Scratch wound assay results showed that themiR-199a/b-3p mimics significantly inhibited the migratory ability of SW1116 cells (Fig.  5 A, B). Transwell assay results showed that miR-199a/b-3p mimics also significantly inhibited the invasive ability of SW1116 cells (Fig.  5 C, D). These results indicated that miR-199a/b-3p could suppress cell migration and invasion in CRC cells. Fig. 5 miR-199a/b-3p inhibited cell migration and invasion in CRC.  A ,  B  SW1116 cells were transfected with miR-199a/b-3p mimics. Cell migration was analyzed using scratch wound assay. (bar, 200 μm).  C ,  D  SW1116 cells were transfected with miR-199a/b-3p mimics. Cell invasion was analyzed using transwell assay. (bar, 100 μm)\nmiR-199a/b-3p inhibited cell migration and invasion in CRC.  A ,  B  SW1116 cells were transfected with miR-199a/b-3p mimics. Cell migration was analyzed using scratch wound assay. (bar, 200 μm).  C ,  D  SW1116 cells were transfected with miR-199a/b-3p mimics. Cell invasion was analyzed using transwell assay. (bar, 100 μm)\nSince the inhibitory effects of miR-199a/b-3p included the effects of silencing  PAK4  and  BCAR3 , we examined that whether the function could be reversed by overexpressing  PAK4  or  BCAR3 . We transfected negative controls, miR-199a/b-3p mimics, miR-199a/b-3p+ BCAR3 , or miR-199a/b-3p+ PAK4  in SW1116 cells. MTT and colony formation assays showed that the restoration of  PAK4  expression reversed the effect of miR-199a/b-3p on cell proliferation and colony formation (Fig.  6 A–C). The results from scratch wound and transwell assays showed that ectopic  BCAR3  expression reversed the inhibitory effect of miR-199a/b-3p on cell migration and invasion (Fig.  6 D–G). The above results indicated that miR-199a/b-3p inhibited viability and mobility of CRC cells by targeting  PAK4  and  BCAR3,  respectively. Fig. 6 Effects of miR-199a/b-3p could be partially reversed by PAK4 and BCAR3 overexpression.  A  MTT assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics and/or PAK4/BCAR3 overexpression plasmid; ** p  < 0.01.  B ,  C  Colony formation assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics and/or PAK4/BCAR3 overexpression plasmid; ** p  < 0.01.  D ,  F  Cell migration was analyzed using scratch wound assay. (bar, 200 μm);** p  < 0.01.  E ,  G  Cell invasion was analyzed using transwell assay. (bar, 100 μm);** p  < 0.01\nEffects of miR-199a/b-3p could be partially reversed by PAK4 and BCAR3 overexpression.  A  MTT assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics and/or PAK4/BCAR3 overexpression plasmid; ** p  < 0.01.  B ,  C  Colony formation assay was performed on SW1116 cells transfected with miR-199a/b-3p mimics and/or PAK4/BCAR3 overexpression plasmid; ** p  < 0.01.  D ,  F  Cell migration was analyzed using scratch wound assay. (bar, 200 μm);** p  < 0.01.  E ,  G  Cell invasion was analyzed using transwell assay. (bar, 100 μm);** p  < 0.01\n\nCRC is a complex disease and one of the main causes of death from cancer worldwide [ 19 ,  20 ]. The morbidity of CRC is increasing every year. Meanwhile the global incidence of CRC is expected to increase to 2.5 million by 2035 [ 21 ]. Although the life expectancy for CRC patients has increased as a result of advances in screening and treatment, the prognosis of these patients remains poor [ 22 ]. Therefore, it is of great significance to understand the underlying mechanism of its occurrence and development and to find out the approaches to overcome the poor prognosis.\nPAK4  has been reported to be involved in the regulation of cell cycle regulatory proteins p21, p16, and CDK6 [ 23 ]. Studies showed that the down-regulation of  PAK4  had inhibitory effects on CRC cells [ 10 ,  11 ].Consistent with these previous reports, our study demonstrated that the knockdown of  PAK4  significantly inhibited the proliferation and colony formation of SW1116 cells. However, there was no significant effect on migration and invasion.\nRecently, reports have shown that the overexpression of  BCAR3  could regulate cytoskeletal rearrangement and improved the cell migration and invasion in endometriosis [ 13 ,  14 ]. However, there no relevant studies conducted on the role of  BCAR3  in CRC. Therefore, we knocked down the expression of  BCAR3  to explore the role of  BCAR3  in CRC. Results showed that the knockdown of  BCAR3  significantly inhibited cell migration and invasion of SW1116 cells, but there was no significant effect on proliferation and colony formation.\nMiRNAs are endogenous non-coding small RNAs, which play important roles in plants and animals [ 24 ]. They inhibit the gene translation or degrading the target mRNA by binding to the 3ʹ-untranslated region (3ʹ-UTR) of target genes [ 25 ]. A single miRNA can target numerous mRNA and regulate the expression of multiple genes, which involved in cancer progression in multiple ways. Much more studies have shown that miRNAs play essential roles in promoting or inhibiting the proliferation, invasion, apoptosis and drug resistance of tumor cells by regulating some key genes [ 26 – 28 ]. Some studies revealed that miRNAs are involved in progression of CRC [ 29 ,  30 ].\nConsidering the different roles of  PAK4  and  BCAR3  on CRC cells and the multiple targets of miRNA, we decided to predict the potential target miRNAs of  PAK4  and  BCAR3 . From the results in TargetScan and DIANA database, we surprisingly found miR-199a/b-3p could be the target miRNA of both genes. Our luciferase reporter assay results showed that  PAK4  and  BCAR3  were potential targets of miR-199a/b-3p in SW1116 cells. Moreover, qRT-PCR and western blot analyses verified that miR-199a/b-3p decreased  PAK4  and  BCAR3  expression at mRNA and protein levels in SW1116 cells, which meant that miR-199a/b-3p could directly target the 3'-UTR of  PAK4  and  BCAR3 .\nAs for miR-199a/b-3p, there are several studies of its suppressive role in tumor progression. MiR-199a/b-3p could suppress hepatocellular carcinoma through inhibiting  PAK4/Raf/MEK/ERK  pathway [ 31 ]. In this study, our results indicated that miR-199a/b-3p significantly inhibited the proliferation, colony formation, migration, and invasion of SW1116 cells.\nWe then performed rescue experiments to explore whether  PAK4  and  BCAR3  were downstream functional regulators involved in miR-199a/b-3p regulation in SW1116 cells. Overexpression of  PAK4  significantly alleviated the effects of miR-199a/b-3p in SW1116 cells on cell proliferation and colony formation. Meanwhile, the overexpression of  BCAR3  significantly reversed the effects of miR-199a/b-3p in SW1116 cells on cell migration and invasion. The above results indicated that miR-199a/b-3p inhibited the proliferation, colony formation of SW116 cells through targeting  PAK4 . Meanwhile, the inhibitory effects on migration and invasion were regulated by targeting  BCAR3 .\nIn summary, our research demonstrated that miR-199a/b-3p inhibited viability and mobility of CRC cells by regulating  PAK4  and  BCAR3,  respectively. This study proposed a new miRNA for filtrating multi-targeted neoplastic suppressant to improve the prognosis of CRC in patients.","source_license":"CC-BY-4.0","license_restricted":false}