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MiR-422a targets MAPKK6 and regulates cell growth and apoptosis in colorectal cancer cells
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MiR-422a targets MAPKK6 and regulates cell growth and apoptosis in colorectal cancer cells ⁎
Peng Li, Qingmin Li, Yanqiang Zhang , Shaojun Sun, Shuntao Liu, Zhaoxi Lu Department of Clinical Laboratory, Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miRNA Colorectal cancer Cell growth Apoptosis p38
The important role of miR-422a in tumor has been reported in several studies. Recent research discovered that the expression of miR-422a was significantly decreased in colorectal cancer tissues, providing miR-422a as a tumor suppressor in CRC. However, the concrete mechanism of miR-422a regulating CRC cell is still unclear. In this study, we demonstrated that miR-422a could inhibit CRC cell growth and promote cell apoptosis via in vitro analyses. Moreover, computational methods were adopted to identify the targets of miR-422a. We found MAPKK6 was the direct target of miR-422a. Consequently, we further elucidated that miR-422a inhibited CRC cell growth and induced cell apoptosis by inhibiting p38/MAPK pathway. Besides that, we established the tumor xenograft model using nude mice and the inhibitory effects on tumor volumes and weights by miR-422a mimic transfection were also detected. Taken together, these findings demonstrated miR-422a exerted anti-cancer activities on CRC, which could be potentially used for CRC prognosis prediction and treatment.
1. Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer mortality [1,2]. Although lots of researches made in diagnostic methods and treatment strategies, the prognosis of CRC patients remains poor, especially in patients with advanced CRC [3,4]. The multiple alterations of tumor suppressor genes and oncogenes are related with CRC carcinogenesis. However, it is still limited to use these genes as markers for early diagnosis, clinical prognosis and prediction. Nowadays, a growing number of evidence showed that miRNAs may manipulate about 30% of human genes and play a crucial role in many cellular processes including differentiation, proliferation, migration and apoptosis [5–9]. However, the functional mechanisms of miRNAs still remain unclear during CRC carcinogenesis and progression. Therefore, several efforts have been made to find dysregulated expression of miRNAs, which could be served as potential biomarkers for colorectal cancer diagnosis and prognosis [10–12]. MicroRNAs, which are approximately 21-nucleotide-long noncoding RNA, anneal in the 3′-UTR of protein-coding mRNAs and lead to repression of translational efficiency and/or decreased mRNA levels [13,14] (Filipowicz, 2008 #452). Deregulation of miRNAs contributes to human pathogenesis including cancer [15]. For instance, the expression of let-7, miR-15, miR-16, miR-17 and miR-21 and were aberrantly altered in different cancer cells [16]. That is probably caused by
⁎
the amplification, deletion and rearrangement of miRNA genes located in the fragile sites in the genomic regions [17,18]. A theme is emerging that a miRNA could be considered either a tumor suppressor or oncogene depending on its targets in different cell types and tissues [19–21]. Identification of relevant targets or pathways controlled by miRNAs will ultimately provide insights into their biological functions. Altered expression of miR-422a has been reported in different types of cancer. For example, miR-422a was supposed to target TGF-β and influence Wnt signaling pathways, thus influencing osteosarcoma cell proliferation [22]; miR-422a inhibited glioma proliferation and invasion by targeting IGF1 and IGF1R [23]; Mao et al. [24] proved that miR422a suppressed MLH1 expression by targeting key mismatch repair protein (MutLα), leading to the instability of genome and tumorigenesis. According to the recent studies, miRNA arrays were adopted to identify new miRNAs that were related with CRC. MiR-422a was found to be deregulated in CRC patients and validated that had inhibitory effect on cell viability in colon cancer cell lines [25,26]. Besides that, miR-422a has been demonstrated to be a prognostic target and a potential tumor suppressor in CRC [27]. However, the underlying mechanisms of miR-422a regulating CRC progression remain unclear. In this study, we further confirmed the suppressive role of miR-422a in CRC via in vitro and in vivo experiments, and first found MAPKK6 was the target of miR-422a, indicating that p38/MAPK involved in the regulation of CRC cell apoptosis induced by miR-422a.
Corresponding author at: No. 67 Dongchang Road, Liaocheng, 252000, Shandong Province, China. E-mail address: [email protected] (Y. Zhang).
https://doi.org/10.1016/j.biopha.2018.03.013 Received 11 December 2017; Received in revised form 28 February 2018; Accepted 5 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Please cite this article as: Li, P., Biomedicine & Pharmacotherapy (2018), https://doi.org/10.1016/j.biopha.2018.03.013
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SW620 or HCT116 cells were plated in 96-well plate at a density of 3000 cells per well. Following cells were transfected with miR-422a mimic or scramble control for different time points. 1/10 vol of the medium cell counting kit solution was added, and the cells were further incubated for another three hours in cell incubator. The number of living cells was measured by using the microplate reader at 450 nm wavelength. Cell growth inhibition rate = (1 − A450, treated/A450, control) × 100%.
Table 1 The expression level of miR-422a in colorectal cancer patients. Variables
n
miRNA-422a (mean ± SD)
Age(years) < 60 ≧60
26 19
2.162 ± 1.39 2.561 ± 1.62
Tumor size(cm) ≧3.5 < 3.5
23 22
1.706 ± 1.35 2.417 ± 1.46
P value 0.151
0.048*
Gender Male Female
28 17
2.216 ± 1.13 2.607 ± 1.76
Invasion depth T1-T2 T3-T4
20 25
2.104 ± 1.19 2.325 ± 1.32
Lymph node metastasis N0 N1-N2
24 21
2.358 ± 1.15 2.153 ± 1.02
Distant metastasis M0 M1
27 18
3.583 ± 1.68 2.172 ± 1.41
TNM stage I, II III, IV
24 21
3.475 ± 1.57 2.126 ± 1.33
2.5. Flow cytometry analysis of apoptotic cells 0.165
CRC cells were plated in 60 mm dishes at a density of 1 × 106 cells per dish, and then transfected with miR-422a mimic or control. Seventy-two hours after transfection, the cells were trypsinized, and apoptosis was measured using Annexin V-FITC and propidium iodide [22] (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. This assay enabled the identification of early apoptotic (Annexin-V-FITC+/PI−) cells, late apoptotic/secondary necrotic (Annexin-V-FITC+/PI+) cells, and primary necrotic (Annexin-V-FITC−/ PI+) cells. The experiments were repeated three times for each sample.
0.125
0.158
0.038*
2.6. Predicted targets analysis of miR-422a
0.042*
The predicted targets of the miR-422a were analyzed by online bioinformatics methods, which including TargetScan (http://www. targetscan.org/vert_61/) and microRNA.org (http://www.microrna. org/microrna/). The way of TargetScan and microRNA.org predict biological targets of miRNAs are via searching for the presence of conserved 8 mer, 7 mer, and 6 mer sites that match the seed region of each miRNA [29]. The predicting target genes of miR-422a were analyzed through two algorithms from this database.
2. Materials and methods 2.1. Patients and tissue samples The colorectal carcinoma tissues and normal tissues were recruited from 45 patients diagnosed with CRC at Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University between 2012 and 2015. Informed consent was obtained from all patients, and all procedures were approved by the Ethics Committee of Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University. The CRC patients were not taken the radiotherapy, chemotherapy or other treatment prior to surgery. All tissues obtained from surgical resection were immediately stored at −80 °C until RNA extraction. The clinicopathological information of the patients was shown in Table 1, which was obtained from the hospital.
2.7. ′UTR-luciferase reporter gene assay The wild-type or mutant 3′UTR of MAPKK6 containing the predicted miR-422a binding sites was cloned into the pRL-TK vectors. 100 ng of pRL-TK-3′UTR-wt or mutant and 20 ng of pGL3.0 control vector were co-transfected in CRC cells planted in 24 well plate. Four hours after the transfection, 50 nM of miR-422a mimic or control was transfected into the cells respectively. After another 48 h’ incubation, cells were lysed, and the luciferase activities in each group were analyzed by dual luciferase assay kit (Promega, Madison, USA).
2.2. Cell culture
2.8. Fluorescence analysis
The colorectal carcinoma SW620 and HCT116 cells were purchased from American type culture collection (ATCC, Manassas, VA, USA). All cells were cultured in DMEM medium (Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) at 37 °C with 5% CO2 in a tissue culture incubator.
SW620 and HCT116 cells were seeded on 24-mm sterilized coverslips, and then were co-transfected with MAPKK6-3′UTR-wt or mutant plasmids containing RFP expressing gene, and miR-422a mimic or control respectively. After 72 h’ incubation, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with DAPI reagent. Images were captured by fluorescence microscopy (NIKON Instruments Inc., Tokyo, Japan) and analyzed with ImageJ software.
2.3. Cell transfection and vectors construction 2 × 105 CRC cells were plated in six well plates until 40–60% confluence and then were transfected with 50 pmol miR-422a mimic or scrambled sequence control (GenePharma, Shanghai, China) by using lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cells were then lysed and normalized for the following experiments. MAPKK6 overexpression plasmid (pcDNA3.1-MAPKK6) was constructed as previously described [28]. Lipofectamine 2000 reagent (Invitrogen) was used for cell transfection.
2.9. Real-time PCR analysis Total RNA was isolated from cells with Trizol (Invitrogen) reagent, according to the manufacturer’s instructions. The concentration and purity of isolated RNA was estimated using the ND-1000 microspectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was assessed on BioAnalyzer 2100 using BioAnalyzer RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Palo Alto, CA, USA). Then, 2 ug of RNA was utilized for cDNA synthesis using SuperScript II RNase H Reverse Transcriptase (Life Technologies). Following the reverse transcription reactions, real-time PCR was performed with SYBR Green (Thermo Fisher) detecting by Bio-Rad CFX
2.4. Cell proliferation assay Cell proliferation was determined by cell counting kit 8 (#96992, Sigma Aldrich, USA) according to the protocol of manufacture. Briefly, 2
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Fig. 1. miR-422a inhibited colorectal cancer cell growth. (A) Total RNA was extracted from both tumor tissues and normal tissues of 45 colorectal cancer patients. The levels of miR-422a were analyzed by RT-PCR (* P < 0.05, n = 45). (B) The cell growth of both CRC SW620 and HCT116 cells were inhibited by miR-422a mimic transfection in a time-dependent manner (n = 3). (C) Representative photographs of cell number and morphology for each group were taken under microscope.
hour with horseradish peroxidase-coupled secondary antibodies at room temperature. Signals were detected using the ECL kit (Amersham Pharmacia Biotech).
connect Real-time System (Bio-Rad, USA). The qPCR procedure was performed as follows: initial denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 15 s; and then an annealing extension at 60 °C for 30 s. The absorbance values were measured at the extension stage. Real-time PCR results were analyzed and the expression of mRNA levels was calculated using the 2−ΔΔCt method and normalized to GAPDH, an internal reference control. Primer for MAPKK6: 5′-AGCGG ATCCGAGCCACAGTAAATA-3′ (sense) and 5′-CCCGAAACAGTG CGCC ATAAAAG-3′ (anti-sense).
2.11. Xenografts in nude mice Six weeks old male BALB/c nu/nu mice were purchased from Beijing Vital River Laboratory Animal Technology Company and maintained under specific pathogen-free conditions. 1 × 107 SW620 or HCT116 cells were subcutaneously injected at the back of each mouse. The tumor volume was measured by micrometer calipers every three days. When tumor size reached to the mean volume of 100 mm3, the mice were intratumor injection with 50 μg of miR-422a mimic dissolved in 100 μL of DMEM mixed with 5 μL of lipofectamine 2000 (Invitrogen) or scramble three times a week for five weeks. The mice were sacrificed at the end of five weeks for tumor weight and volume analysis. All mice experiments were performed under the strict protocols approved by the Committee on Animal Experimentation of Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University.
2.10. Western blot After transfection with miR-422a mimic for 72 h, CRC cells were lysed, and total proteins were extracted. Protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher, USA). 30 μg of protein was loaded for each group, and electrophoresis was performed in 10% polyacrylamide SDS gel. Later on, the gel was blotted to polyvinylidene fluoride membranes (Millipore, MA, USA) following with a blocking in a 5% non-fat dry milk solution in the washing buffer. The membranes were then incubated at 4 °C overnight with the primary antibodies. MAPKK6, p-p38, p38, cleaved caspase-3, TAO2 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); p-ASK1 and ASK1 were obtained from Cell Signaling Technology (Danvers, MA, USA). After washing three times with washing buffer containing 0.05% Tween-20, the membranes were incubated for one
2.12. TUNEL assay Tumors were fixed in formalin and pieced at 6 mm and then dewaxed in alcohol. Apoptosis in tumor tissues were detected in by TUNEL assay using the in situ TUNEL kit (Thermo Fisher Scientific, 3
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reporter plasmids containing a red fluorescence protein (RFP) gene, we also observed the effect of miR-422a mimic on MAPKK6-3′-UTR reporter plasmids under the fluorescence microscope. Consistent with the dual luciferase reporter assay, miR-422a mimic reduced the RFP expression of wild-type, but not mutant reporter plasmid in CRC cells (Fig. 3C). In addition, overexpression of MAPKK6 enhanced CRC cell proliferation, which was reversed by miR-422a mimic (Fig. 3D). Meanwhile, the protein level of MAPKK6 was down regulated by miR422a as well (Fig. 3E, F). Thus, these findings suggested that miR-422a could directly target MAPKK6.
Waltham, MA, USA) according to the manufacturer’s instructions. 2.13. Statistical analysis All values are presented as the mean ± SD. Statistics were performed with two-tailed Student’s t-test available in GraphPad Prism (version 5.0, La Jolla, CA, USA). All experiments were independently repeated three times, unless otherwise stated. 3. Results
3.4. MiR-422a induces apoptosis via inhibiting p38 expression
3.1. Expression of miR-422a in colorectal cancer tissues and the effect of miR-422a on CRC cell growth
Since MAPKK6 has been considered to be the target of miR-422a, the mRNA level of MAPPK6 was down-regulated by miR-422a mimic treatment assessed by real-time PCR (Fig. 4A). Among these molecules, we found that the expression of p-p38 was down-regulated in CRC cells transfected with miR-422a mimic. However, the level of cleaved caspase-3 increased (Fig. 4B, C), indicating that miR-422a could promote CRC cell apoptosis through inhibiting p38/MAPK pathway. Moreover, miR-422a has been demonstrated that could induce CRC cell apoptosis, in order to further investigate the possible mechanism involved in this process, we determined several molecules expression supposed to be the downstream of MAPKK6 signaling. ASK1 and TAO2 are MAP kinase kinase kinases, activated in response to a number of stress-related stimuli, which in turn activate JNK and p38 and play important role during cancer development [30,31]. In the current study, miR-422a had no effect on the expressions of TAO2 and p-ASK1 (Fig. 5A–C).
The expression of miR-422a in 45 pairs of colorectal cancer tissues and normal adjacent tissues was determined by RT-PCR. The results indicated that the level of miR-422a was extremely higher in adjacent tissues compared with tumor tissues (P < 0.05; Fig. 1A). Meanwhile, the correlation between miR-422a levels and clinical information of CRC patients was investigated. The data shown in Table 1 suggested that the level of miR-422a significantly correlated with tumor size (P = 0.048), distant metastasis (P = 0.038), and TNM stage (P = 0.042). However, there was no significant correlation between miR-422a expression and age, gender, invasion depth and lymph node metastasis (P > 0.05). Based on the above analysis, we assumed that miR-422a may exert an anti-tumor effect on colorectal cancer. In order to explore the biological role of miR-422a in CRC cells, miR-422a mimic were transiently transfected into SW620 and HCT116 cells, respectively. Subsequently, CRC cell viability was determined by cell counting kit 8 (CCK8) at different time after transfection. As shown in Fig. 1B, miR-422a inhibited the growth of both SW620 and HCT116 cells in a time-dependent manner. Cell morphology and density were also observed under the microscope after 72 h transfection. Consistent with the cell growth results, there was a significant reduced cell density in miR422a mimic treated cells compared with vehicle control or scramble sequence treated cells (Fig. 1C). Moreover, cell morphology turned into round instead of spreading well, indicating that miR-422a may not only inhibit cell growth, but also induce cell apoptosis.
3.5. MiR-422a inhibits tumor growth in CRC cell xenografts in vivo The effect of miR-422a on tumor cell growth was also detected in primary tumor xenograft nude mice model. The volumes of tumors with miR-422a mimic treatment were significantly inhibited in a time dependent manner compared with those in the control group (Fig. 6A). The average tumor weight at the end of the experiment was calculated from 6 mice in each group. Tumor weight was also dramatically decreased after miR-422a mimic treatment (Fig. 6B). Representative figures of tumor dimensions in nude mice at the end of the experiment were shown in Fig. 6C. In addition, the body weights of mice were stable which indicated the miR-422a mimic injections were tolerable (Fig. 6D).
3.2. MiR-422a promotes CRC cell apoptosis As observed in Fig. 1C, CRC cells appeared apoptosis characteristics after transfection with miR-422a mimic. Thus, flow cytometry analysis by Annexin Ⅴ and PI staining was performed to examine the proportion of apoptotic cells. The data showed that miR-422a mimic dramatically induced early apoptotic cell number (Annexin V-positive and PI-negative) and late apoptotic cell number (Annexin V-positive and PI-positive) (Fig. 2A). The apoptosis rate was calculated according to the ratio of cells, which were in early and late stage of apoptosis, and total cells (Fig. 2B). These results indicating that miR-422a could induce CRC cell apoptosis.
3.6. Mir-422a induced apoptosis in tumor tissues in CRC xenografts TUNEL assay was conducted in order to further determine the role of miR-422a against CRC in vivo. As shown in Fig. 7A and B, TUNEL positive cells were dramatically increased in both miR-422a mimic groups compared to the control mice, which was consistent with the results of in vitro. 4. Discussion
3.3. MiR-422a directly targets MAPKK6 This is the first study to show that miR-422a could negatively regulate MAPKK6 at the post-transcriptional level, via targeting its 3′UTR (nt518-524). It is also firstly demonstrated that miR-422a could downregulate cell growth and promotes apoptosis via p38/MAPK signaling pathway in CRC cells. The in vivo relevance of these findings is supported by correlation studies in resected tissues of colorectal cancer patients, and in xenograft tumor models. Moreover, it is noteworthy that in clinical samples the expression of miR-422a is not only negatively correlated with tumor size, but also shows the inverse correlation of the distant metastasis and TNM stage (Table 1), which is further provides miR-422a as a tumor suppressor in colorectal cancer. According to the present literatures, the functional role of miR-422a in cancer has been well investigated. MiR-422a is considered as a tumor
To understand the mechanisms by which miR-422a inhibits CRC cell growth and promotes apoptosis, several computational analysis were performed to identify the targets of miR-422a in humans. Among all the potential targets, MAPKK6 is of particular interest because its high predicted score in both TargetScan and microRNA.org (http://www. microrna.org/microrna/) online bioinformatics methods. Therefore, the following experiments were carried out to confirm whether MAPPK6 is a direct target of miR-422a. We constructed wild-type and mutant MAPKK6-3′-UTR expression plasmids, which were fused with a luciferase reporter gene (Fig. 3A). According to the results, miR-422a mimic significantly inhibited the luciferase activity of wild-type but not mutant reporter genes in SW620 and HCT116 cells (Fig. 3B). Since the 4
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Fig. 2. MiR-422a promoted apoptosis in CRC cells. (A) CRC SW620 and HCT116 cells were transfected with miR-422a mimic for 72 h. The apoptosis was determined by Annexin V/PI staining, and then was analyzed by flow cytometry (X axis, Annexin V; Y axis, PI). (B) Calculated cell apoptotic rate was shown (** P ＜ 0.01 compared with control, n = 3).
Activation of MAPKK6-p38 pathway is involved in multiple signaling regulations, including auto-inhibition, dimerization and ubiquitination. However, the mechanisms of MAPKK6-p38 pathway deactivation, which is closely related to signaling transduction, are not well investigated. MAPPK6 has been shown to interact with TAO2 and ASK1, then activate p38 or/and JNK pathway in many tumor types [30,31]. However, in the present study the proteins expressions of TAO2 and pASK1 were not affected by miR-422a, which illustrated that miR-422a
suppressor in glioma [23], non-small cell lung cancer [32], osteosarcoma [22], bladder cancer [33], and et al. A limited number of genes, including PIK3CA [33], CD73 [34], hTERT [26], forkhead box [35], have been reported to be the targets of miR-422a, suggesting potential function in regulating cell proliferation, apoptosis, and invasion. However, the precise role of miR-422a in CRC cancer remains to be defined. In this study, we identified MAPKK6 as a new target of miR-422a. The p38 activator MAPPK6, which is also a dual specific protein kinase.
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Fig. 3. MAPKK6 was a directly target of miR-422a. (A) Gene structure of MAPKK6 showing the predicted target site of miR-422a in its 3′UTR. (B) SW620 or HCT116 cells were transfected with reporter gene containing wild-type (3′UTR-WT) or mutant (3′UTR-MT) MAPKK6 3′-UTR along with miR-422a mimic or control as indicated (** P ＜ 0.01, n = 3). (C) MiR-422a targeting MAPKK6 3′UTR was explored under fluorescence microscope by using the reporter gene fused with a RFP containing 3′UTR-WT or 3′UTR-MT of MAPKK6 (n = 3). (D) SW620 cells were infected with pcDNA3.1 (control vector) or pcDNA3.1-MAPPK6 and the cell growth was detected with CCK-8 (* P ＜ 0.05, ** P ＜ 0.01, n = 3). (E) Western blotting analysis of MAPKK6 proteins following exposure to miR-422a mimic in SW620 cells. (F) Relative protein levels were quantified by using β-actin as control (** P ＜ 0.01, n = 3).
or pharmacologic inhibition using PH797804 reduces tumor burden in mice [37]. Treatment with the p38 MAPK inhibitor SB202190 also reduces colon tumor growth either in xenografts of human colon cancer cell lines or in mice that express APCmin [38]. These results support that p38 facilitates colon tumor progression.
induced CRC cell apoptosis mainly via MAPKK6-p38 direct action. It is possible that proteins TAO2 and ASK1 do not play a leading role in CRC. P38 has participated in multiple cellular processes, which is usually activated by inflammatory cytokines, stress signals, and other signals. Since p38 pathway could balance the cell growth and differentiation, and induce apoptosis by integrating signals, it is mainly recognized as an important tumor suppressor. However, recent reports have also illustrated pro-tumorigenic functions for p38. Thus, p38 signaling may facilitate the survival and proliferation of tumor cells contributing to the progression of some tumor types. In addition, p38 activation helps tumor cells to survive chemotherapeutic treatments. In CRC, p38 regulates intestinal homeostasis and the integrity of the colon epithelia. Down-regulation of p38 in intestinal epithelial cells increases proliferation, reduces the number of mucus-producing goblet cells and affects epithelial barrier function by altering tight junction assembly [36,37]. As a consequence, mice with p38 deficient intestinal epithelial cells are more susceptible to colitis-associated colon tumorigenesis [37]. In contrast, down-regulation of p38 in colon tumor cells
5. Conclusion In the current study, we supposed that miR-422a targets MAPKK6, resulting in a decrease of MAPKK6 and P38, which inhibited CRC cell growth. Meanwhile, cell apoptosis was also detected by miR-422a treatment. Since, p38 MAPK pathway was reported to induce apoptosis, this induction of apoptosis may be caused by other effects of the targets of miR-422a. These findings shed new lights on the future CRC treatment using miR-422a. Conflicts of interest The authors declare no competing financial interests.
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Fig. 4. MiR-422a induced CRC cell apoptosis by inhibiting MAPKK6 expression. (A) MAPKK6 mRNA expression was determined by RT-PCR in SW620 and HCT116 cells after miR-422a mimic transfection (** P ＜ 0.01, n = 3). (B) Western blotting analysis of MAPKK6, p-p38, p38 and cleaved caspase-3 proteins following exposure to miR-422a mimic in CRC cells. (C), (D) and (E) Relative protein levels were quantified by using β-actin as control (** P ＜ 0.01, n = 3).
Fig. 5. MiR-422a had no effect on the expression of p-ASK1, ASK1 and TAO2 proteins (A) Western blotting analysis of p-Ask1, Ask1 and TAO2 proteins following exposure to miR-422a mimic. (B) and (C) Relative protein levels were quantified by using β-actin as control (** P ＜ 0.01, n = 3).
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Fig. 6. MiR-422a inhibited tumor growth in nude mice xenograft. (A)Tumor volumes in xenograft tumor growth of SW620 and HCT116 cells nude mice were measured every week after treatment of miR-422a mimic. Points indicate mean values (n = 6); bars indicate SD (standard deviation). (B) Tumor weights at the end of the experiment (* P ＜ 0.05 compared with vehicle control, n = 6). (C) Representative photographs of tumor dimensions at the end of the experiment. (D) Body weights of mice were monitored weekly (n = 6).
Fig. 7. Figure 7. Mir-422a induced apoptosis in tumor tissues in CRC xenografts. (A) SW620 and HCT116 tumor bearing nude mice were treated with miR-422a mimic or scramble for 5 weeks. Then the tumor tissues were isolated and subjected to TUNEL detection. (B) TUNEL positive cells in each group were quantified per field (** P ＜ 0.01, n = 3).
References
Acknowledgements Thanks Ke Wu for carefully proof-reading and the comments to the experiments.
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MiR-422a targets MAPKK6 and regulates cell growth and apoptosis in colorectal cancer cells ⁎
Peng Li, Qingmin Li, Yanqiang Zhang , Shaojun Sun, Shuntao Liu, Zhaoxi Lu Department of Clinical Laboratory, Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miRNA Colorectal cancer Cell growth Apoptosis p38
The important role of miR-422a in tumor has been reported in several studies. Recent research discovered that the expression of miR-422a was significantly decreased in colorectal cancer tissues, providing miR-422a as a tumor suppressor in CRC. However, the concrete mechanism of miR-422a regulating CRC cell is still unclear. In this study, we demonstrated that miR-422a could inhibit CRC cell growth and promote cell apoptosis via in vitro analyses. Moreover, computational methods were adopted to identify the targets of miR-422a. We found MAPKK6 was the direct target of miR-422a. Consequently, we further elucidated that miR-422a inhibited CRC cell growth and induced cell apoptosis by inhibiting p38/MAPK pathway. Besides that, we established the tumor xenograft model using nude mice and the inhibitory effects on tumor volumes and weights by miR-422a mimic transfection were also detected. Taken together, these findings demonstrated miR-422a exerted anti-cancer activities on CRC, which could be potentially used for CRC prognosis prediction and treatment.
1. Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer mortality [1,2]. Although lots of researches made in diagnostic methods and treatment strategies, the prognosis of CRC patients remains poor, especially in patients with advanced CRC [3,4]. The multiple alterations of tumor suppressor genes and oncogenes are related with CRC carcinogenesis. However, it is still limited to use these genes as markers for early diagnosis, clinical prognosis and prediction. Nowadays, a growing number of evidence showed that miRNAs may manipulate about 30% of human genes and play a crucial role in many cellular processes including differentiation, proliferation, migration and apoptosis [5–9]. However, the functional mechanisms of miRNAs still remain unclear during CRC carcinogenesis and progression. Therefore, several efforts have been made to find dysregulated expression of miRNAs, which could be served as potential biomarkers for colorectal cancer diagnosis and prognosis [10–12]. MicroRNAs, which are approximately 21-nucleotide-long noncoding RNA, anneal in the 3′-UTR of protein-coding mRNAs and lead to repression of translational efficiency and/or decreased mRNA levels [13,14] (Filipowicz, 2008 #452). Deregulation of miRNAs contributes to human pathogenesis including cancer [15]. For instance, the expression of let-7, miR-15, miR-16, miR-17 and miR-21 and were aberrantly altered in different cancer cells [16]. That is probably caused by
⁎
the amplification, deletion and rearrangement of miRNA genes located in the fragile sites in the genomic regions [17,18]. A theme is emerging that a miRNA could be considered either a tumor suppressor or oncogene depending on its targets in different cell types and tissues [19–21]. Identification of relevant targets or pathways controlled by miRNAs will ultimately provide insights into their biological functions. Altered expression of miR-422a has been reported in different types of cancer. For example, miR-422a was supposed to target TGF-β and influence Wnt signaling pathways, thus influencing osteosarcoma cell proliferation [22]; miR-422a inhibited glioma proliferation and invasion by targeting IGF1 and IGF1R [23]; Mao et al. [24] proved that miR422a suppressed MLH1 expression by targeting key mismatch repair protein (MutLα), leading to the instability of genome and tumorigenesis. According to the recent studies, miRNA arrays were adopted to identify new miRNAs that were related with CRC. MiR-422a was found to be deregulated in CRC patients and validated that had inhibitory effect on cell viability in colon cancer cell lines [25,26]. Besides that, miR-422a has been demonstrated to be a prognostic target and a potential tumor suppressor in CRC [27]. However, the underlying mechanisms of miR-422a regulating CRC progression remain unclear. In this study, we further confirmed the suppressive role of miR-422a in CRC via in vitro and in vivo experiments, and first found MAPKK6 was the target of miR-422a, indicating that p38/MAPK involved in the regulation of CRC cell apoptosis induced by miR-422a.
Corresponding author at: No. 67 Dongchang Road, Liaocheng, 252000, Shandong Province, China. E-mail address: [email protected] (Y. Zhang).
https://doi.org/10.1016/j.biopha.2018.03.013 Received 11 December 2017; Received in revised form 28 February 2018; Accepted 5 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Please cite this article as: Li, P., Biomedicine & Pharmacotherapy (2018), https://doi.org/10.1016/j.biopha.2018.03.013
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SW620 or HCT116 cells were plated in 96-well plate at a density of 3000 cells per well. Following cells were transfected with miR-422a mimic or scramble control for different time points. 1/10 vol of the medium cell counting kit solution was added, and the cells were further incubated for another three hours in cell incubator. The number of living cells was measured by using the microplate reader at 450 nm wavelength. Cell growth inhibition rate = (1 − A450, treated/A450, control) × 100%.
Table 1 The expression level of miR-422a in colorectal cancer patients. Variables
n
miRNA-422a (mean ± SD)
Age(years) < 60 ≧60
26 19
2.162 ± 1.39 2.561 ± 1.62
Tumor size(cm) ≧3.5 < 3.5
23 22
1.706 ± 1.35 2.417 ± 1.46
P value 0.151
0.048*
Gender Male Female
28 17
2.216 ± 1.13 2.607 ± 1.76
Invasion depth T1-T2 T3-T4
20 25
2.104 ± 1.19 2.325 ± 1.32
Lymph node metastasis N0 N1-N2
24 21
2.358 ± 1.15 2.153 ± 1.02
Distant metastasis M0 M1
27 18
3.583 ± 1.68 2.172 ± 1.41
TNM stage I, II III, IV
24 21
3.475 ± 1.57 2.126 ± 1.33
2.5. Flow cytometry analysis of apoptotic cells 0.165
CRC cells were plated in 60 mm dishes at a density of 1 × 106 cells per dish, and then transfected with miR-422a mimic or control. Seventy-two hours after transfection, the cells were trypsinized, and apoptosis was measured using Annexin V-FITC and propidium iodide [22] (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. This assay enabled the identification of early apoptotic (Annexin-V-FITC+/PI−) cells, late apoptotic/secondary necrotic (Annexin-V-FITC+/PI+) cells, and primary necrotic (Annexin-V-FITC−/ PI+) cells. The experiments were repeated three times for each sample.
0.125
0.158
0.038*
2.6. Predicted targets analysis of miR-422a
0.042*
The predicted targets of the miR-422a were analyzed by online bioinformatics methods, which including TargetScan (http://www. targetscan.org/vert_61/) and microRNA.org (http://www.microrna. org/microrna/). The way of TargetScan and microRNA.org predict biological targets of miRNAs are via searching for the presence of conserved 8 mer, 7 mer, and 6 mer sites that match the seed region of each miRNA [29]. The predicting target genes of miR-422a were analyzed through two algorithms from this database.
2. Materials and methods 2.1. Patients and tissue samples The colorectal carcinoma tissues and normal tissues were recruited from 45 patients diagnosed with CRC at Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University between 2012 and 2015. Informed consent was obtained from all patients, and all procedures were approved by the Ethics Committee of Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University. The CRC patients were not taken the radiotherapy, chemotherapy or other treatment prior to surgery. All tissues obtained from surgical resection were immediately stored at −80 °C until RNA extraction. The clinicopathological information of the patients was shown in Table 1, which was obtained from the hospital.
2.7. ′UTR-luciferase reporter gene assay The wild-type or mutant 3′UTR of MAPKK6 containing the predicted miR-422a binding sites was cloned into the pRL-TK vectors. 100 ng of pRL-TK-3′UTR-wt or mutant and 20 ng of pGL3.0 control vector were co-transfected in CRC cells planted in 24 well plate. Four hours after the transfection, 50 nM of miR-422a mimic or control was transfected into the cells respectively. After another 48 h’ incubation, cells were lysed, and the luciferase activities in each group were analyzed by dual luciferase assay kit (Promega, Madison, USA).
2.2. Cell culture
2.8. Fluorescence analysis
The colorectal carcinoma SW620 and HCT116 cells were purchased from American type culture collection (ATCC, Manassas, VA, USA). All cells were cultured in DMEM medium (Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) at 37 °C with 5% CO2 in a tissue culture incubator.
SW620 and HCT116 cells were seeded on 24-mm sterilized coverslips, and then were co-transfected with MAPKK6-3′UTR-wt or mutant plasmids containing RFP expressing gene, and miR-422a mimic or control respectively. After 72 h’ incubation, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with DAPI reagent. Images were captured by fluorescence microscopy (NIKON Instruments Inc., Tokyo, Japan) and analyzed with ImageJ software.
2.3. Cell transfection and vectors construction 2 × 105 CRC cells were plated in six well plates until 40–60% confluence and then were transfected with 50 pmol miR-422a mimic or scrambled sequence control (GenePharma, Shanghai, China) by using lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cells were then lysed and normalized for the following experiments. MAPKK6 overexpression plasmid (pcDNA3.1-MAPKK6) was constructed as previously described [28]. Lipofectamine 2000 reagent (Invitrogen) was used for cell transfection.
2.9. Real-time PCR analysis Total RNA was isolated from cells with Trizol (Invitrogen) reagent, according to the manufacturer’s instructions. The concentration and purity of isolated RNA was estimated using the ND-1000 microspectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was assessed on BioAnalyzer 2100 using BioAnalyzer RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Palo Alto, CA, USA). Then, 2 ug of RNA was utilized for cDNA synthesis using SuperScript II RNase H Reverse Transcriptase (Life Technologies). Following the reverse transcription reactions, real-time PCR was performed with SYBR Green (Thermo Fisher) detecting by Bio-Rad CFX
2.4. Cell proliferation assay Cell proliferation was determined by cell counting kit 8 (#96992, Sigma Aldrich, USA) according to the protocol of manufacture. Briefly, 2
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Fig. 1. miR-422a inhibited colorectal cancer cell growth. (A) Total RNA was extracted from both tumor tissues and normal tissues of 45 colorectal cancer patients. The levels of miR-422a were analyzed by RT-PCR (* P < 0.05, n = 45). (B) The cell growth of both CRC SW620 and HCT116 cells were inhibited by miR-422a mimic transfection in a time-dependent manner (n = 3). (C) Representative photographs of cell number and morphology for each group were taken under microscope.
hour with horseradish peroxidase-coupled secondary antibodies at room temperature. Signals were detected using the ECL kit (Amersham Pharmacia Biotech).
connect Real-time System (Bio-Rad, USA). The qPCR procedure was performed as follows: initial denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 15 s; and then an annealing extension at 60 °C for 30 s. The absorbance values were measured at the extension stage. Real-time PCR results were analyzed and the expression of mRNA levels was calculated using the 2−ΔΔCt method and normalized to GAPDH, an internal reference control. Primer for MAPKK6: 5′-AGCGG ATCCGAGCCACAGTAAATA-3′ (sense) and 5′-CCCGAAACAGTG CGCC ATAAAAG-3′ (anti-sense).
2.11. Xenografts in nude mice Six weeks old male BALB/c nu/nu mice were purchased from Beijing Vital River Laboratory Animal Technology Company and maintained under specific pathogen-free conditions. 1 × 107 SW620 or HCT116 cells were subcutaneously injected at the back of each mouse. The tumor volume was measured by micrometer calipers every three days. When tumor size reached to the mean volume of 100 mm3, the mice were intratumor injection with 50 μg of miR-422a mimic dissolved in 100 μL of DMEM mixed with 5 μL of lipofectamine 2000 (Invitrogen) or scramble three times a week for five weeks. The mice were sacrificed at the end of five weeks for tumor weight and volume analysis. All mice experiments were performed under the strict protocols approved by the Committee on Animal Experimentation of Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University.
2.10. Western blot After transfection with miR-422a mimic for 72 h, CRC cells were lysed, and total proteins were extracted. Protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher, USA). 30 μg of protein was loaded for each group, and electrophoresis was performed in 10% polyacrylamide SDS gel. Later on, the gel was blotted to polyvinylidene fluoride membranes (Millipore, MA, USA) following with a blocking in a 5% non-fat dry milk solution in the washing buffer. The membranes were then incubated at 4 °C overnight with the primary antibodies. MAPKK6, p-p38, p38, cleaved caspase-3, TAO2 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); p-ASK1 and ASK1 were obtained from Cell Signaling Technology (Danvers, MA, USA). After washing three times with washing buffer containing 0.05% Tween-20, the membranes were incubated for one
2.12. TUNEL assay Tumors were fixed in formalin and pieced at 6 mm and then dewaxed in alcohol. Apoptosis in tumor tissues were detected in by TUNEL assay using the in situ TUNEL kit (Thermo Fisher Scientific, 3
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reporter plasmids containing a red fluorescence protein (RFP) gene, we also observed the effect of miR-422a mimic on MAPKK6-3′-UTR reporter plasmids under the fluorescence microscope. Consistent with the dual luciferase reporter assay, miR-422a mimic reduced the RFP expression of wild-type, but not mutant reporter plasmid in CRC cells (Fig. 3C). In addition, overexpression of MAPKK6 enhanced CRC cell proliferation, which was reversed by miR-422a mimic (Fig. 3D). Meanwhile, the protein level of MAPKK6 was down regulated by miR422a as well (Fig. 3E, F). Thus, these findings suggested that miR-422a could directly target MAPKK6.
Waltham, MA, USA) according to the manufacturer’s instructions. 2.13. Statistical analysis All values are presented as the mean ± SD. Statistics were performed with two-tailed Student’s t-test available in GraphPad Prism (version 5.0, La Jolla, CA, USA). All experiments were independently repeated three times, unless otherwise stated. 3. Results
3.4. MiR-422a induces apoptosis via inhibiting p38 expression
3.1. Expression of miR-422a in colorectal cancer tissues and the effect of miR-422a on CRC cell growth
Since MAPKK6 has been considered to be the target of miR-422a, the mRNA level of MAPPK6 was down-regulated by miR-422a mimic treatment assessed by real-time PCR (Fig. 4A). Among these molecules, we found that the expression of p-p38 was down-regulated in CRC cells transfected with miR-422a mimic. However, the level of cleaved caspase-3 increased (Fig. 4B, C), indicating that miR-422a could promote CRC cell apoptosis through inhibiting p38/MAPK pathway. Moreover, miR-422a has been demonstrated that could induce CRC cell apoptosis, in order to further investigate the possible mechanism involved in this process, we determined several molecules expression supposed to be the downstream of MAPKK6 signaling. ASK1 and TAO2 are MAP kinase kinase kinases, activated in response to a number of stress-related stimuli, which in turn activate JNK and p38 and play important role during cancer development [30,31]. In the current study, miR-422a had no effect on the expressions of TAO2 and p-ASK1 (Fig. 5A–C).
The expression of miR-422a in 45 pairs of colorectal cancer tissues and normal adjacent tissues was determined by RT-PCR. The results indicated that the level of miR-422a was extremely higher in adjacent tissues compared with tumor tissues (P < 0.05; Fig. 1A). Meanwhile, the correlation between miR-422a levels and clinical information of CRC patients was investigated. The data shown in Table 1 suggested that the level of miR-422a significantly correlated with tumor size (P = 0.048), distant metastasis (P = 0.038), and TNM stage (P = 0.042). However, there was no significant correlation between miR-422a expression and age, gender, invasion depth and lymph node metastasis (P > 0.05). Based on the above analysis, we assumed that miR-422a may exert an anti-tumor effect on colorectal cancer. In order to explore the biological role of miR-422a in CRC cells, miR-422a mimic were transiently transfected into SW620 and HCT116 cells, respectively. Subsequently, CRC cell viability was determined by cell counting kit 8 (CCK8) at different time after transfection. As shown in Fig. 1B, miR-422a inhibited the growth of both SW620 and HCT116 cells in a time-dependent manner. Cell morphology and density were also observed under the microscope after 72 h transfection. Consistent with the cell growth results, there was a significant reduced cell density in miR422a mimic treated cells compared with vehicle control or scramble sequence treated cells (Fig. 1C). Moreover, cell morphology turned into round instead of spreading well, indicating that miR-422a may not only inhibit cell growth, but also induce cell apoptosis.
3.5. MiR-422a inhibits tumor growth in CRC cell xenografts in vivo The effect of miR-422a on tumor cell growth was also detected in primary tumor xenograft nude mice model. The volumes of tumors with miR-422a mimic treatment were significantly inhibited in a time dependent manner compared with those in the control group (Fig. 6A). The average tumor weight at the end of the experiment was calculated from 6 mice in each group. Tumor weight was also dramatically decreased after miR-422a mimic treatment (Fig. 6B). Representative figures of tumor dimensions in nude mice at the end of the experiment were shown in Fig. 6C. In addition, the body weights of mice were stable which indicated the miR-422a mimic injections were tolerable (Fig. 6D).
3.2. MiR-422a promotes CRC cell apoptosis As observed in Fig. 1C, CRC cells appeared apoptosis characteristics after transfection with miR-422a mimic. Thus, flow cytometry analysis by Annexin Ⅴ and PI staining was performed to examine the proportion of apoptotic cells. The data showed that miR-422a mimic dramatically induced early apoptotic cell number (Annexin V-positive and PI-negative) and late apoptotic cell number (Annexin V-positive and PI-positive) (Fig. 2A). The apoptosis rate was calculated according to the ratio of cells, which were in early and late stage of apoptosis, and total cells (Fig. 2B). These results indicating that miR-422a could induce CRC cell apoptosis.
3.6. Mir-422a induced apoptosis in tumor tissues in CRC xenografts TUNEL assay was conducted in order to further determine the role of miR-422a against CRC in vivo. As shown in Fig. 7A and B, TUNEL positive cells were dramatically increased in both miR-422a mimic groups compared to the control mice, which was consistent with the results of in vitro. 4. Discussion
3.3. MiR-422a directly targets MAPKK6 This is the first study to show that miR-422a could negatively regulate MAPKK6 at the post-transcriptional level, via targeting its 3′UTR (nt518-524). It is also firstly demonstrated that miR-422a could downregulate cell growth and promotes apoptosis via p38/MAPK signaling pathway in CRC cells. The in vivo relevance of these findings is supported by correlation studies in resected tissues of colorectal cancer patients, and in xenograft tumor models. Moreover, it is noteworthy that in clinical samples the expression of miR-422a is not only negatively correlated with tumor size, but also shows the inverse correlation of the distant metastasis and TNM stage (Table 1), which is further provides miR-422a as a tumor suppressor in colorectal cancer. According to the present literatures, the functional role of miR-422a in cancer has been well investigated. MiR-422a is considered as a tumor
To understand the mechanisms by which miR-422a inhibits CRC cell growth and promotes apoptosis, several computational analysis were performed to identify the targets of miR-422a in humans. Among all the potential targets, MAPKK6 is of particular interest because its high predicted score in both TargetScan and microRNA.org (http://www. microrna.org/microrna/) online bioinformatics methods. Therefore, the following experiments were carried out to confirm whether MAPPK6 is a direct target of miR-422a. We constructed wild-type and mutant MAPKK6-3′-UTR expression plasmids, which were fused with a luciferase reporter gene (Fig. 3A). According to the results, miR-422a mimic significantly inhibited the luciferase activity of wild-type but not mutant reporter genes in SW620 and HCT116 cells (Fig. 3B). Since the 4
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Fig. 2. MiR-422a promoted apoptosis in CRC cells. (A) CRC SW620 and HCT116 cells were transfected with miR-422a mimic for 72 h. The apoptosis was determined by Annexin V/PI staining, and then was analyzed by flow cytometry (X axis, Annexin V; Y axis, PI). (B) Calculated cell apoptotic rate was shown (** P ＜ 0.01 compared with control, n = 3).
Activation of MAPKK6-p38 pathway is involved in multiple signaling regulations, including auto-inhibition, dimerization and ubiquitination. However, the mechanisms of MAPKK6-p38 pathway deactivation, which is closely related to signaling transduction, are not well investigated. MAPPK6 has been shown to interact with TAO2 and ASK1, then activate p38 or/and JNK pathway in many tumor types [30,31]. However, in the present study the proteins expressions of TAO2 and pASK1 were not affected by miR-422a, which illustrated that miR-422a
suppressor in glioma [23], non-small cell lung cancer [32], osteosarcoma [22], bladder cancer [33], and et al. A limited number of genes, including PIK3CA [33], CD73 [34], hTERT [26], forkhead box [35], have been reported to be the targets of miR-422a, suggesting potential function in regulating cell proliferation, apoptosis, and invasion. However, the precise role of miR-422a in CRC cancer remains to be defined. In this study, we identified MAPKK6 as a new target of miR-422a. The p38 activator MAPPK6, which is also a dual specific protein kinase.
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Fig. 3. MAPKK6 was a directly target of miR-422a. (A) Gene structure of MAPKK6 showing the predicted target site of miR-422a in its 3′UTR. (B) SW620 or HCT116 cells were transfected with reporter gene containing wild-type (3′UTR-WT) or mutant (3′UTR-MT) MAPKK6 3′-UTR along with miR-422a mimic or control as indicated (** P ＜ 0.01, n = 3). (C) MiR-422a targeting MAPKK6 3′UTR was explored under fluorescence microscope by using the reporter gene fused with a RFP containing 3′UTR-WT or 3′UTR-MT of MAPKK6 (n = 3). (D) SW620 cells were infected with pcDNA3.1 (control vector) or pcDNA3.1-MAPPK6 and the cell growth was detected with CCK-8 (* P ＜ 0.05, ** P ＜ 0.01, n = 3). (E) Western blotting analysis of MAPKK6 proteins following exposure to miR-422a mimic in SW620 cells. (F) Relative protein levels were quantified by using β-actin as control (** P ＜ 0.01, n = 3).
or pharmacologic inhibition using PH797804 reduces tumor burden in mice [37]. Treatment with the p38 MAPK inhibitor SB202190 also reduces colon tumor growth either in xenografts of human colon cancer cell lines or in mice that express APCmin [38]. These results support that p38 facilitates colon tumor progression.
induced CRC cell apoptosis mainly via MAPKK6-p38 direct action. It is possible that proteins TAO2 and ASK1 do not play a leading role in CRC. P38 has participated in multiple cellular processes, which is usually activated by inflammatory cytokines, stress signals, and other signals. Since p38 pathway could balance the cell growth and differentiation, and induce apoptosis by integrating signals, it is mainly recognized as an important tumor suppressor. However, recent reports have also illustrated pro-tumorigenic functions for p38. Thus, p38 signaling may facilitate the survival and proliferation of tumor cells contributing to the progression of some tumor types. In addition, p38 activation helps tumor cells to survive chemotherapeutic treatments. In CRC, p38 regulates intestinal homeostasis and the integrity of the colon epithelia. Down-regulation of p38 in intestinal epithelial cells increases proliferation, reduces the number of mucus-producing goblet cells and affects epithelial barrier function by altering tight junction assembly [36,37]. As a consequence, mice with p38 deficient intestinal epithelial cells are more susceptible to colitis-associated colon tumorigenesis [37]. In contrast, down-regulation of p38 in colon tumor cells
5. Conclusion In the current study, we supposed that miR-422a targets MAPKK6, resulting in a decrease of MAPKK6 and P38, which inhibited CRC cell growth. Meanwhile, cell apoptosis was also detected by miR-422a treatment. Since, p38 MAPK pathway was reported to induce apoptosis, this induction of apoptosis may be caused by other effects of the targets of miR-422a. These findings shed new lights on the future CRC treatment using miR-422a. Conflicts of interest The authors declare no competing financial interests.
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Fig. 4. MiR-422a induced CRC cell apoptosis by inhibiting MAPKK6 expression. (A) MAPKK6 mRNA expression was determined by RT-PCR in SW620 and HCT116 cells after miR-422a mimic transfection (** P ＜ 0.01, n = 3). (B) Western blotting analysis of MAPKK6, p-p38, p38 and cleaved caspase-3 proteins following exposure to miR-422a mimic in CRC cells. (C), (D) and (E) Relative protein levels were quantified by using β-actin as control (** P ＜ 0.01, n = 3).
Fig. 5. MiR-422a had no effect on the expression of p-ASK1, ASK1 and TAO2 proteins (A) Western blotting analysis of p-Ask1, Ask1 and TAO2 proteins following exposure to miR-422a mimic. (B) and (C) Relative protein levels were quantified by using β-actin as control (** P ＜ 0.01, n = 3).
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Fig. 6. MiR-422a inhibited tumor growth in nude mice xenograft. (A)Tumor volumes in xenograft tumor growth of SW620 and HCT116 cells nude mice were measured every week after treatment of miR-422a mimic. Points indicate mean values (n = 6); bars indicate SD (standard deviation). (B) Tumor weights at the end of the experiment (* P ＜ 0.05 compared with vehicle control, n = 6). (C) Representative photographs of tumor dimensions at the end of the experiment. (D) Body weights of mice were monitored weekly (n = 6).
Fig. 7. Figure 7. Mir-422a induced apoptosis in tumor tissues in CRC xenografts. (A) SW620 and HCT116 tumor bearing nude mice were treated with miR-422a mimic or scramble for 5 weeks. Then the tumor tissues were isolated and subjected to TUNEL detection. (B) TUNEL positive cells in each group were quantified per field (** P ＜ 0.01, n = 3).
References
Acknowledgements Thanks Ke Wu for carefully proof-reading and the comments to the experiments.
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