Background
Colorectal cancer (CRC) is one of the most common cancer types which shows high morbidity and mortality [
1]. The past decades have seen decreasing mortality of CRC as the early detection and treatment have advanced greatly, but the incidence of CRC increases worldwide and the onset age is becoming younger [
1‐
3]. Hence, it is still imminent to further clarify the exact pathogenesis of CRC. Multiple studies have been conducted to investigate the mutation of genes and their products [
4‐
7], which prove that the aberrant activation of signaling pathways [
8‐
11] and microsatellite instability (MSI) [
7,
11] are involved in oncogenesis and progression of CRC. Moreover, recent studies indicate that the regulation of micro-RNAs (miRNAs) is indispensable [
12‐
14].
Raf/MEK/ERK and PI3K/Akt are both signal transduction pathway that regulate intracellular processes in response to extracellular signals. ERK and AKT are, respectively, the key proteins of Raf/MEK/ERK pathway and PI3K/Akt pathway. The aberrant activation of Raf/MEK/ERK and PI3K/Akt signaling pathway is considered to be an essential issue in tumorigenesis and progression of CRC.
miRNAs are a class of small-regulatory RNA molecules, which are highly conserved across species. MiRNAs regulate gene expression through binding to the 3′-untranslated region (UTR) of their target mRNAs in a sequence-specific manner [
15]. In recent years, miRNAs are regarded as molecular biomarkers and therapeutic targets for CRC. A series of miRNAs have been proven to involve into CRC carcinogenesis, invasion and metastasis [
12,
16]. For example, MicroRNA-30b can function as a tumor suppressor in CRC by targeting KRAS, PIK3CD and BCL2 [
17], while MicroRNA-224, a tumor promoter, targets PHLPP1 and PHLPP2 [
18], sustains Wnt/β-catenin signaling and promotes aggressive phenotype of CRC [
19]. Besides, many other micro-RNAs such as miR-30a [
20], miR-140-5p [
21] and miR-153 [
22] are also known as important moderators in the progression of CRC. However, a large number of functional miRNAs remains to be investigated in CRC [
23].
Several studies have indicated the miR-422a takes part in many human diseases such as postmenopausal osteoporosis, osarcoma and colorectal adenocarcinoma [
24‐
26]. Moreover, miR-422a plays a positive role on head and neck squamous cell carcinoma by targeting NT5E/CD73 that promotes loco-regional recurrence, miR-422a were also found to significantly inhibit TMEM45B expression in squamous cell lung cancer [
27,
28]. Recent studies illuminate that miR-422a is associated with advanced stages of CRC, affects G1/S transition and potentially inhibits hTERT expression in CRC, which suggests miR-422a to be an independent prognostic factor of CRC [
29‐
31]. However, the more other target genes and underlying mechanism of miR-422a in the progression of CRC are largely unknown.
In this study, we report that miR-422a is down‑regulated in CRC tissues and cell lines; ectopic expression of miR-422a inhibits cell proliferation and tumor growth ability, inhibition of endogenous miR-422a, by contrast, promotes cell proliferation and tumor growth ability of CRC cells; miR-422a directly targets 3′-UTR of the AKT1 and MAPK1, down-regulation of miR-422a led to the activation of Raf/MEK/ERK and PI3K/AKT signaling pathways to promote cell proliferation in CRC.
Methods
Tissue specimens and cell cultures
The 30 freshly CRC specimens and their matched adjacent normal tissues frozen and stored in liquid nitrogen until further use were collected from operation room of Nanfang Hospital. Prior approval was obtained from the Southern Medical University Institutional Board (Guangzhou, China) before using these clinical materials for research. All samples were collected and analyzed with the prior written, informed consent of the patients. Four human CRC lines SW620, SW837, HCT15 and HCT116 were purchased from American Type Culture Collection Cell Biology Collection and were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; PAA Laboratories, Pasching, Austria) at 37 °C with 5% CO2.
Plasmids and transfection
The miR-422a mimic, miR-422a inhibitor and the negative control were obtained from RiboBio (Guangzhou, China) and transfected into CRC cells using Lipofectamine 2000 reagent (Invitrogen). The full length of MAPK1 3′-UTR contains 4593 bps and the AKT1 3′-UTR has 1011 bps. The miR-422a binding site in the MAPK1 3′-UTR is located at 176–188 bps, while 411–425 bps in the AKT1 3′-UTR. The region of the human MAPK1 3′-UTR from 172 to 191 bps and AKT1 3′-UTR from 407 to 429 bp were generated by PCR amplification and subcloned into the MluI/NheI sites of the pGL3-basic luciferase reporter plasmid (Promega). The primers are listed in Additional file
1: Table S1.
RNA isolation, reverse transcription (RT) and real-time PCR
Total RNA from cell lines, CRC tissues and normal mucosa was extracted using Trizol reagent (Invitrogen). The cDNA was reverse-transcribed at 37 °C for an hour and 85 °C for 5 min in a 10 µl reaction system including 3.75 μl (0.25–8 μg) RNA sample, 5 μl mRQ Buffer (2×), 1.25 μl mRQ Enzyme (takara, Guangzhou, China), then add 90 μl H
2O to bring the total volume to 100 μl. These cDNAs were ready for the miRNA quantification according to the protocol (takara Guangzhou, China). The PCR reaction was performed as follows: 95 °C for 10 s, 40 cycles of 95 °C for 5 s and 60 °C for 20 s and 95 °C for 60 s, 55 °C for 30 s, and 95 °C for 30 s. Sequences of the primers are summarized in Additional file
2: Table S2.
Western blotting
We carried out western blotting according to standard methods, which protein lysates were prepared, subjected to SDS–PAGE and transferred onto PVDF membranes, using anti-AKT (Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (CST, USA), anti-GSK-3β (CST, USA), anti-p21, anti-p27, anti-CyclinD1 (Bioworld Technology, St. Louis Park, MN, USA), anti-p-AKT (CST, USA), anti-p-GSK-3βand anti-p-ERK1/2 (CST, USA). Anti-α-Tubulin (Sigma, St. Louis, MO, USA) monoclonal antibody served as a internal reference.
The miR-422a mimics, anti- miR-422a inhibitors and negative control oligos were transiently transfected into CRC cells for the MTT assay, colony-formation assay, soft agar colony-formation assay, flow-cytometry and luciferase assays, as previously described [
32]. Further details are described in Additional file
3: Additional materials and methods. All experiments were repeated at least three times and the data are presented as mean ± SD.
Tumorigenesis assay
200 μl cell suspension (2 × 10
6 cells) prepared with 0.9% normal saline were subcutaneously injected into 4-week-old Balb/Cathymic nude mice (nu/nu) bought from the Animal Center of Southern Medical University, Guangzhou, China. Tumor volumes were measured on the indicated days. Details refer to studies described previously [
18,
33].
Statistical analysis
All statistical analyses were analyzed using SPSS21.0 for Windows. The independent-samples t test and paired-samples t-test were used to analyze two groups. The correlation analysis was used to explain the relationship between miR-422a expression and its target gene. p < 0.05 was considered statistically significant.
Discussion
It is known that miRNAs function as suppressive or carcinogenic factors in a variety of cancers [
34]. For example, miR-638 is an important tumour suppressor [
35], while miR-153 represents an onco-microRNA in CRC [
22]. Previous findings suggested that miR-422a might work as a tumor suppressor [
36] in glioblastoma and hepatocellular carcinoma involving in a feedback loop with family of forkhead box (FOX) [
37]. Recently, miR-422a have been found to function as a protector against CRC because the expression level of miR-422a is lower in CRC tissue compared with normal tissue, and it is revealed that miR-422a may be relevant with cell proliferation [
31]. But the mechanism of how miR-422a inhibit cell proliferation and in the progression of CRC remain to be elucidated. Our results indicated that miR-422a was down-regulated in human CRC, induced G1 arrest and inhibited cell proliferation and tumour growth thought regulating the activity of Raf–MEK–ERK and PI3K–AKT signaling pathways.
The disorder of the cell cycle is largely responsible for uncontrolled cell proliferation, which is the basic feature of cancer. There are several different cyclins that are activated in different parts of the cell cycle and that cause the CDKs, the key regulatory proteins transforming cell cycle phases, to phosphorylate different substrates. These cyclins are divided into two main groups: G1/S cyclins including cyclin A, Cyclin D and Cyclin E, and G2/M cyclins. The three D type cyclins (cyclin D1, cyclin D2, cyclin D3) bind to CDK4 and to CDK6 and regulate transition from G1 to S phase [
38,
39]. Aberrant cyclin D1 expression has been reported in many human cancers [
38]. Cyclin D1 is firstly induced by Ras, a small GTP-binding protein, and CDK-cyclin D1 complexes are essential for entry into S phase from G1 phase [
40,
41]. CDK activity can be counteracted by CDK inhibitors (CKI) that has distinct two families. Cip/Kip family belongs to CKI, including p21 (Waf1, Cip1), p27 (Cip2), p57 (Kip2) [
42,
43]. Our study found that miR-422a up-regulated expression of p27 and p21 and inhibited expression of cyclin D1, arresting the CRC cells at G1/G0 phase and suppressing CRC proliferation.
Cell cycle is regulated by the ubiquitin pathway [
44], p53-p21-DREAM-CDE/CHR pathway [
45], WNT signaling pathway [
46,
47] and so on. The Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinases (ERK) pathway [
48] and Ras/PI3K/AKT pathways [
49] are also involved in cell cycle regulation, when Ras actives both Raf and PI3K which further respectively active ERK and AKT [
50]. Activation of AKT and ERK can phosphorylate GSK3β, preventing phosphorylation of cyclin D1 which leads to cell cycle progression [
51‐
54]. Mitogen-activated protein kinase (MAPK) is protein kinases and three MAPK families have been clearly characterized, namely classical MAPK (also known as ERK), C-Jun N-terminal kinse/stress-activated protein kinase (JNK/SAPK) and p38 kinase [
55]. Since ERK1(MAPK3) and its close relative ERK2 (MAPK1) are both involved in growth factor signaling, the family was termed “mitogen-activated”. We have confirmed that miR-422a reduced total and phosphorylation level of ERK1/2 and AKT1. Numbers of researches have shown that microRNA participates in regulation of these signal pathways. For instance, miR-224 directly targets GSK3β and SFRP2 and activates the Wnt/β-catenin signaling in CRC cells [
19]. And miR-1 was reported as a tumor suppressor that restrained epithelial-mesenchymal transition and metastasis of colorectal carcinoma via the MAPK and PI3K/AKT pathway [
56].
Indeed, KRAS gene is mutated in nearly 50% of CRCs [
57], which is activated after the extracellular mitogen binds to the membrane receptor and leads to aberrant activations of downstream pro-survival signaling cascades including Raf/MEK/ERK, PI3K/PKB(AKT), and Ral-GTPase active MAPK/GSK pathway [
58]. Importantly, we found that expression of KRAS was not significantly changed by miR-422, but a dual inhibition of the Raf/MEK/ERK and PI3K/AKT signaling pathways was mediated by miR-422a, binding to the 3′UTR of MAPK1(ERK2) and AKT1 mRNAs. The anti-proliferative molecule GSK3β is an essential regulator of the intrinsic apoptotic pathway, and has been implicated in the development and progression of CRC [
51]. GSK3β is also a downstream effector of ERK2 or AKT1 [
50]. The present study reveals that miR-422a increased GSK3β expression through regulation of RAS/Raf/MEK–/RK and RAS/PI3K/AKT and finally inhibited cells proliferation in CRC. It has been documented that the expression of p27
Kip1, p21
Cip1 and cyclinD1 can be transcriptionally regulated by GSK3β and, in turn, the transcriptional activity of GSK3β is modulated by AKT and ERK phosphorylation [
9,
42,
43,
59‐
61].
Authors’ contributions
WTL, YPY and YQD designed the experiments; WTW, XXN, ZYX conducted experiments; SYW, HLJ, YXW provided research materials and methods; WTW and XXN analyzed data; and YPY and WTW wrote the manuscript. WTW, XXN, ZYX, SYW, HLJ, YXW, WTL, YPY and YQD contributed towards the article. All authors read and approved the final manuscript.