MicroRNA-126 Inhibit Viability of Colorectal Cancer Cell by Repressing mTOR Induced Apoptosis and Autophagy
Received 12 November 2019
Accepted for publication 19 January 2020
Published 24 March 2020 Volume 2020:13 Pages 2459—2468
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Sanjeev Srivastava
Li Wei,1,* Zhanhong Chen,1,* Na Cheng,2,* Xing Li,1 Jie Chen,1 Donghao Wu,1 Min Dong,1 Xiangyuan Wu1
1Medical Oncology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, People’s Republic of China; 2Pathology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, People’s Republic of China
*These authors contributed equally to this work
Correspondence: Xiangyuan Wu; Min Dong
Medical Oncology, The Third Affiliated Hospital of Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, Guangdong, People’s Republic of China
Email [email protected]; [email protected]
Objective: Colorectal cancer (CRC) is a fatal disease, and tumor development is a complex cellular event involving a multistep cascade process involving proliferation, invasion, and migration. In recent years, it has been shown that microRNA-126 (miR-126) plays a key role in the tumorigenesis of CRC, but further studies are required to investigate the regulatory mechanisms through which this miRNA affects cell viability, autophagy, and apoptosis in CRC. We aimed to study the effect of miR-126 in gene regulation in CRC HCT116 cells.
Methods: CRC biopsy samples and normal colorectal tissue samples were used for miRNA profiling. Real-time quantitative PCR and WB were utilized to detect RNA and protein levels. MTT and colony formation assays were performed to examine cell viability. Furthermore, an immunofluorescence assay and Annexin V/PI flow cytometry were performed to detect autophagy and apoptosis, respectively.
Results: The expression of miR-126 was downregulated in CRC biopsies and cell lines compared with that in normal cells and tissues. The upregulation of miR-126 resulted in impaired viability and growth of CRC cells. Furthermore, with the overexpression of miR-126, cell autophagy was increased, as evidenced by LC3-I/II transformation and p62 degradation. Meanwhile, apoptosis induction was also observed because of the increased miR-126 levels. The autophagy inhibitor Bafilomycin A1 (BafA1) repressed both autophagy and apoptosis, indicating that miR-126 induced autophagy was responsible for the induction of apoptosis. A dual-luciferase reporter assay (DLRA) and bioinformatics prediction revealed that miR-126 silenced the mTOR gene by targeting the 3′-UTR. mTOR mRNA levels in CRC biopsy tissues and cell lines were upregulated to a greater extent than that in normal cells and tissues. Furthermore, HCT116 cells transfected with an miR-126 mimic showed a decreased expression of mTOR. In addition, the overexpression of mTOR counteracted miR-126 on autophagy and apoptosis.
Conclusion: Our study demonstrated that miR-126-induced can regulate the activity of CRC cells via autophagy and apoptosis and suggested a new mechanism of miR-126–mTOR interaction in CRC pathogenesis.
Keywords: CRC, miR-126, mTOR, apoptosis, autophagy
Colorectal cancer (CRC) is a fatal disease, and the incidence of synchronous liver metastases (SLM) is approximately 15% in all newly diagnosed CRC cases. SLM exhibit widespread resistance to traditional therapy and has a poor prognosis.1 Therefore, CRC is causes significant burden of cancer, with CRC proliferation and metastasis is most likely leading treatment failure. The extracellular matrix degradation, invasion, and adhesion of cancer cells, epithelial mesenchymal transformation, and abnormal expression of microRNA (miR) are the complex mechanisms involved in CRC.
miRNA is a short, conservative, and noncoding RNA that can bind to the 3′-untranslated region (3′-UTR) of target mRNAs and affect the posttranscriptional regulation of several genes.2,3 MiR-126 is encoded by intron 7 of the egfl7 gene.4 It has been shown that miR-126 is obviously related to vascular integrity, angiogenesis, and several human diseases.5,6 Studies have suggested that miR-126 could inhibit the development of tumors by targeting several genes such as IRS,7 VEGF,8 and PI3K.9 The downregulation of miR-126 plays a key role in tumorigenesis by regulating signaling pathways, indicating that miR-126 has a potential application in cancer treatment.9,10 In addition, miR-126 can be used as a new biomarker for acute myocardial infarction,11 liver metastasis of CRC,12 and type 2 diabetes.13
Recently, various studies have shown that miR-126 is related to the regulatory mechanism of CRC (abnormal activation or inactivation of signaling pathways). However, the effect of miR-126 on the proliferation and survival of CRC cells has not been fully elucidated. Therefore, this study aimed to prove the role of miR-126 in the autophagy and apoptosis of CRC cells.
Materials and Methods
The clinical data of 30 patients undergoing CRC radical resection in the Third Affiliated Hospital of the Sun Yat-sen University were collected between September 2016 and June 2019. All subjects (15 men and 15 women; age range, 22–76 years; mean age, 57.3 ± 8.36 years) were diagnosed with colorectal adenocarcinoma; no other tumors were detected. The patients underwent nonemergency surgery and did not receive any preoperative chemotherapy. Among the 30 patients, there were 4 cases of left-side colon cancer, 4 cases of right-side colon cancer, and 22 cases of rectal cancer. Tumor diameters were < 4 cm (n = 5), 4–6 cm (n = 19), and >6 cm (n = 6). Histopathological examination of specimens obtained after radical operation showed that CRC was malignant In accordance with the NCCN Guidelines, patients were divided based on the cancer stage into stage II and above categories, following which they received postoperative chemotherapy. Samples of the adjacent normal colorectal tissues were obtained 5 cm from the distal end of the tumor. The patients had no history of preoperative radiotherapy or chemotherapy. This study has been approved by the Ethics Committee of the Third Affiliated Hospital of the Sun Yat-sen University. All study participants provided written informed consent before participating in the study.
Cell Culture and Transfection
HCT116 (Colorectal carcinoma, Human, Dukes’ type A) and HEK293 (Permanent line established from primary embryonic human kidney, Human) cells were cultured in DMEM with 10% fetal bovine serum (FBS, Invitrogen). NCM460 (Primary ductal carcinoma, Human, Epithelial, Lymphoblast. Grade III), HT29 (Colorectal adenocarcinoma. Human, Grade I), Caco-2 (Colorectal adenocarcinoma. Human, Grade II), SW620 (Colorectal adenocarcinoma. Human. Dukes’ type C), and SW480 (Colorectal Adenocarcinoma. Human. Grade IV. Dukes’ type B) cells were cultured in RPMI-1640 (Thermo) with 10% FBS. All cell lines were obtained from ATCC.
pcDNA-mTOR plasmids were obtained from Shanghai Gene Pharmaceutical Co., Ltd., and the miRNA mimics and controls were obtained from Ribobio. Lipofectamine 3000 (Thermo) was used as a transfection reagent for transfection based on manufacturer’s instructions.
Total RNA was separated using the phenol–chloroform method, and the quality of RNA was assessed via capillary electrophoresis. Following small RNA sequencing, libraries were generated and the high sensitivity DNA chip in the Agilent Bioanalyzer 2100 system was used for the quantification of these libraries. The quality control of raw sequence files was evaluated using a FastQC quality control instrument. The elimination of adapters using Cutadapt (Ver. 1.2.1) and the cutting of low-quality sequences was performed to exclude low-quality data. The miRDeep2 software was used for the assessing miRNA activity (Ver. 184.108.40.206). The differential expression and change of the samples and controls was detected using differential expression sequencing.
Soft Agar Colony Formation Assay
The cells were transfected with either an NC mimic or an miR-126 mimic and were resuspended in DMEM 2 days after transfection; following this, they were transferred to a 12-well plate with 0.5% bottom agar. After 14 days, the colonies were counted in three random areas of each plate.
Cell viability was evaluated using an MTT assay; 20 µL of MTT and 100µL of DMSO were added to each well. After adding MTT, formazan dye formed; the sample was further mixed for 10 min to dissolve it. Sample absorbance at 490 nm was determined using an Infinite M200 microplate reader.
Cell Death Assessment
An annexin V-FITC and PI apoptosis detection kit was used for cell death assessment. After transfection, the cells were resuspended in binding buffer (20 µL) with annexin V-FITC (10 µL) and PI (5 µL). The samples were incubated in darkness for 20 min before evaluating cell death via flow cytometry (FC).
Immunofluorescence Assay (IFA) for LC3 Detection
In the LC3 experiment, GFP-LC3B was used to transfect the cells. A fluorescence microscope was used to record the images, and the GFP puncta were observed in 10 different fields.
Western Blotting (WB)
A mixture of protease inhibitors (Roche) in RIPA buffer (pH 8.0) was used for cell lysis. Protein levels were detected, separated on 10% SDS-PAGE, and transferred to 0.45 µm PVDF membrane. Further, at room temperature, PBST with 5% BSA was used to block the membrane for 60 min. Subsequently, the membranes were incubated with primary antibodies: Bcl-2 ab (ab59348, Abcam, 1:400), Bax ab (ab32503, Abcam, 1:1000), LC3B-I ab (ab51520, Abcam, 1:1000), p62 ab (ab56416, Abcam, 1:100), mTOR ab (ab2732, Abcam, 1:2000), and GAPDH (ab8245, Abcam, 1:5000) at 4°C for 60 min. The membrane and the secondary antibody were incubated at room temperature for 60 min with Amersham ECL peroxidase. A Super Signal West Femto Maximum Sensitivity Substrate Kit was used to determine the immunoreactivity.
RNA Separation and Quantitative Real-Time PCR (Q-PCR)
The total RNA was separated using Trizol, and a Roche Light Cycler 480 Q-PCR system was used to detect the transcription level. The internal reference was GAPDH. A SYBR Green PCR Master Mix was used for Q-PCR (20 μL). The 2-ΔΔCT method was used for quantification; gene expression was normalized to GAPDH, whereas miR-126 expression was normalized to U6 controls. The primer sequences were as follows: miR-126 F: 5ʹ-GTC CGC TCG TAC CGT GAG TAA TA-3ʹ; U6 F: 5ʹ-CTC GCT TCG GCA GCA CA-3ʹ, U6 R: 5ʹ-AAC GCT TCA CGA ATT TGC GT-3ʹ; mTOR F: 5ʹ-CGC TGT CAT CCC TTT ATC G-3ʹ, mTOR R: 5ʹ-ATG CTC AAA CAC CTC CAC C-3ʹ; GAPDH F: 5ʹ-GCA CCG TCA AGG CTG AGA A-3ʹ, GAPDH R: 5ʹ-AGG TCC ACC ACT GAC ACG TT-3ʹ.
Dual-Luciferase Reporter Assay(DLRA)
The mTOR 3′-UTR sequence was amplified using the GV126 luciferase gene. In addition to miR-126, the binding site of the mTOR gene was ablated via a site-directed mutation; these samples were used as controls. The transfection efficiency was normalized using a thymidine kinase promoter and plasmid with Renilla luciferase. HEK293T cells were co-transfected with NC as well as miR-126 mimic with luciferase reporter vectors and the luciferase assay was conducted.
The results were presented as means ± SDs. The differences between the groups were assessed using a two-tailed Student’s t-test or ANOVA with Tukey’s post hoc test. A P-value of < 0.05 indicated statistical significance.
CRC Samples and Cell Lines Showed Reduced miR-126 Expression
miRNA microarray analysis was performed in CRC and normal cells in order to prove the influence of miRs on the occurrence and development of CRC. The results indicated that miR-126 expression was markedly reduced in CRC samples compared with that in healthy controls (Figure 1A and B). The miR-126 expression in CRC cells was obviously downregulated compared with that in the adjacent normal tissues (Figure 1C). Additionally, CRC cell lines (HCT-116, NCM460, SW620, and SW480) showed lower miR-126 expression than the HEK293T cells (Figure 1D). These data confirmed a low expression of miR-126 in CRC cells.
Inhibition of Proliferation and Induction of Autophagy and Apoptosis in HCT-116 Cells by miR-126
Next, we explored the role of miR-126 in the proliferation of HCT-116 cells. An increase in miR-126 expression was found after transfecting HCT-116 cells with an miR-126 mimic (Figure 2A). Compared with the NC mimic group, MTT data collected at 24, 48, and 72 h after transfection revealed that the proliferation rates of HCT-116 cells were greatly decreased after the transfection of miR-498 mimic (Figure 2B). Moreover, the colony formation assay indicated that miR-126 could decrease the number of colonies formed by HCT-116 cells (Figure 2C).
Further, we examined whether miR-126 upregulation mediated autophagy of HCT-116 cells. IFA was first utilized to trace the location of LC3B protein following the increase in miR-126 expression. LC3B (green) accumulated as a result of miR-126 mimic transfection (Figure 3A). Furthermore, miR-126 upregulation increased the autophagy rate of HCT-116 cells, as indicated by decreased LC3 transformation and processing (increased LC3-II levels), together with p62 degradation (Figure 3B). These data demonstrated that miR-126 mediates autophagy of HCT-116 cells.
It is assumed that miR-126 can regulate the apoptosis of HCT-116 cells. FC using annexin V-FITC/PI suggested that there was an increase in apoptosis after transfection with the miR-126 mimic compared with transfection with the NC mimic group (Figure 4A). We also explored the role of miR-126 in Bcl-2 and Bax expression. The expression of the antiapoptotic Bcl-2 was decreased as a result of transfection with the miR-126 mimic, whereas that of the proapoptotic Bax was increased (Figure 4B); these findings suggest that miR-126 also triggered apoptosis in HCT-116 cells.
Next, we tested whether the apoptosis induced by miR-126 was dependent on autophagy. miR-126 was further used to simulate the presence and absence of Baimelimin A1 (BafA1), and the apoptotic cells were detected by annexin V-FITC/PI staining and FC. BafA1 treatment in miR-126-upregulated HCT-116 cells resulted in the downregulation of LC3-II levels and upregulation of p62 levels, indicating that autophagy was successfully inhibited (Figure 5A). On the other hand, BafA1 treatment also led to a significant reduction in annexin-V-positive cells (Figure 5B). In total, these data suggested that miR-126 can induce apoptosis in an autophagic-dependent manner.
MiR-126 Aimed at 3′-UTRs of mTOR
Bioinformatics prediction revealed that miR-126 may target the 3′-UTRs of mTOR (Figure 6A).14 DLRA was used to study the direct relationship between miR-126 and the 3′-UTRs of mTOR (Figure 6B). The data revealed that, compared with the other control groups, luciferase function was inhibited by 55% by the fusion of the miR-126 mimic with the 3′-UTRs of mTOR after transfection with the miR-126 mimic. Our study indicated that there was an increase in mTOR expression in CRC cells compared with that in the normal adjacent tissue and HEK293T cells (Figure 6C and D). Furthermore, we determined the effect of miR-126 on mTOR expression in HCT-116 cells. The protein and mRNA expressions of mTOR were obviously decreased after transfection with the miR-126 mimic (Figure 6E and F). The results demonstrated that the mTOR expression was decreased after the miR-126 expression was upregulated, and the 3′-UTRs of mTOR were targeted by miR-126.
mTOR Overexpression Diminished the Effect of miR-126 on the Viability of HCT-116 Cells
To explore whether mTOR could reverse the effects of miR-126 on CRC cell viability (autophagy and apoptosis), mTOR was overexpressed in HCT-116 cells and co-transfected with miR-126 mimic. WB and Q-PCR were used to detect the change in mTOR expression (Figure 7A and B). Further, we showed that the overexpression of mTOR rescued the proliferation of HCT-116 cells, was inhibited inhibited by the miR-126 mimic, based on data from the MTT assay (Figure 7C). The overexpression of mTOR led to an obvious decrease in apoptotic cells transfected with the miR-126 mimic (Figure 7D and E). Additionally, our data showed that mTOR overexpression resulted in a significant reduction in the level of LC3-II, as well as in p62 stability (Figure 7F). Therefore, mTOR expression can restore cell proliferation by reducing miR-126-induced autophagy and apoptosis.
CRC is one of the most common malignant tumors; thus, an effective treatment is essential for patients with CRC.15 The present study revealed that the expression of mTOR was downregulated by miR-126, showing an anticancer effect via autophagy and apoptosis mediation in CRC cells. Further validation revealed that mTOR overexpression can promote the proliferation of CRC cells. Moreover, miR-126-tirggered autophagy was responsible for BafA1-induced apoptosis.
An increasing number of studies have shown that miR-126 is a tumor suppressor and that its expression is downregulated in multiple tumors. Various molecular signal cascades involved in the regulation of cancer cells have been described to be modulated by miR-126 and vice versa in a cell type–specific and context-dependent manner (Figure 8). Furthermore, our previous related studies16–19 have shown that miR-126 can inhibit the proliferation and invasion of CRC cells in vitro. Liu et al demonstrated VEGF-A to be a target of miR-126, as the infection of lung carcinoma cell lines with lentiviral miR-126 caused a decrease in VEGF-A. This led to a cell cycle arrest in lung cancer cells and to the inhibition of tumor cell growth in vitro and in vivo.8 Li et al demonstrated that the transformation of mouse embryonic Cx43KO brain cells with the oncogene v-Src caused a decrease in miR-126 expression. Additionally, the 3´ UTR of Crk, a component of the focal adhesion network involved in integrin signaling, has been reported as a target of miR-126.20 Chen et al suggested that reduced miR-126 expression facilitates angiogenesis of gastric cancer through its regulation of the VEGF-A-mediated Akt/mTOR pathway.21 Downregulation of miR-126 expression increases the expression of VEGF-A and its downstream mTOR signaling. In contrast, restoration of miR-126 expression reduces the expression of VEGF-A and suppresses the activation of its downstream AKT and mTOR. Banerjee et al and Guo et al have shown that miR-126 targets sites in the promoter of p85β, a PI3K subunit that stabilizes and propagates PI3K signal in CRC cells, which is involved in the carcinogenesis of CRC.9,22 Thus, the information about the role of miR-126 in the regulation of mTOR signaling is not new, but authors have shown further proof of its importance in CRC. Although the potential role and mechanism of action of miR-126 in CRC cell survival have been reported previously. In the present study, we have mainly studied the effect of miR-126 on the autophagy and apoptosis of CRC cells in vitro and in vivo. After overexpression of miR-126 in CRC cells, mTOR expression was decreased, and apoptosis and autophagy were induced. These results suggest that miR-126 is a tumor suppressor and that it considerably inhibits CRC cell proliferation by modulating the mTOR pathway.
The mTOR sensor has become a target for drug development; because it plays a vital role in cancer biology. In addition, the advent of the next generation mTOR inhibitors marks an exciting new stage in mTOR-related therapy. Indeed, the Pi3k/Akt/mTOR signal transduction axis is one of the main mechanisms for maintaining the growth and metastasis of tumors.23 The PI3K/Akt/mTOR axis is involved in many biological events, such as apoptosis,24,25 inflammation,26,27 and autophagy.28 Wu et al proved that the positive expression rate of Glut1 was 75% in CRC tissues and 0% in the adjacent normal tissues. Compared with the adjacent normal tissues, in CRC tissues, the expressions of Glut1, TGF-β1, PI3K, AKT, and mTOR were increased, whereas those of PTEN, Bax, cleaved caspase-3, and cleaved-PARP were decreased. These results revealed that Glut1 gene silencing can inhibit proliferation and promote apoptosis of CRC cells by inhibiting the TGF-β/PI3K-AKT-mTOR signaling pathway.29 mTOR also plays an important role in the autophagy of CRC cells. Aspirin can inhibit mTOR effectors (S6K1 and 4E-BP1) by decreasing the mTOR signal transduction in CRC cells and can also induce autophagy, which is a characteristic of mTOR inhibition.30 In the present study, after overexpression of mTOR in CRC cells transfected with miR-126 mimic, we found that mTOR played an oncogenic role in CRC cells, as evidenced by the restored proliferation and reduced apoptosis and autophagy, which was also contrary to the function of miR-126.
In conclusion, we explored the role of miR-126 in the occurrence and development of CRC, and revealed the antitumor effect of miR-126 on CRC. A decrease in miR-126 expression in CRC tumors is related to impaired growth of cancer cells. Therefore, miR-126 can be further applied as a diagnostic and prognostic biomarker of CRC.
Ethics Approval and Informed Consent
This study has been approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. All study participants provided written informed consent before participating in the study.
All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
The authors report no conflicts of interest in this work.
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