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mTOR signaling pathway
Biomedicine & Pharmacotherapy 105 (2018) 350–361
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MiR-214 promotes cell meastasis and inhibites apoptosis of esophageal squamous cell carcinoma via PI3K/AKT/mTOR signaling pathway
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Qiao Guanena, , Shi Junjieb, Wu Baolinc, Wang Chaoyangd, Yang Yajuana, Li Jinga, Li Junpengb, Ning Gailie, Wang Zhongpingf, Wang Juna a
Department of Gastroenterology, Handan City First Hospital, Handan, Hebei, 056002, China Department of Thoracic Surgery, Handan City First Hospital, Handan, Hebei, 056002, China c Department of Medical imaging, Handan City First Hospital, Handan, Hebei, 056002, China d Department of infectious disease, Handan City First Hospital, Handan, Hebei, 056002, China e Department of Gynaecology, Handan City First Hospital, Handan, Hebei, 056002, China f Department of Medicine, Handan City First Hospital, Handan, Hebei, 056002, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: MiR-214 Meastasis Apoptosis PI3K/AKT/mTOR pathway ESCC
There is growing evidence shown that microRNAs (miRNAs) are associated with cancer and can play a role in human cancers as oncogenes or tumor suppressor genes. MicroRNA-214 (miR-214) shows carcinogenesis in various tumor types, but little is known about biological functions of miR-214 in esophageal squamous cell carcinoma (ESCC). In this study, we observe that the expression of miR-214 is not only increased in human ESCC tissues, but also remarkably increased in cell lines correlates with LZTS1. In addition, the expression of miR-214 inhibited proliferation of ESCC cells in vitro and inhibit the growth of xenograft tumor in vivo. The results show miR-214 serve as a tumor promoter regulating cells migration, invasion and apoptosis in ESCC. Ferthermore, LZTS1 has been proved to as a functional target for miR-214 to regulate cells poliferation and apoptosis. In summary, these results suggest that miR-214 serves as tumor promoter to promote proliferation, migration, invasion and inhibit apoptosis of ESCC cells by targeting LZTS1 via PI3K/AKT/mTOR signaling pathway. The miR-214/LZTS1 pathway provides a new insight into the molecular mechanisms that the occurrence and development of ESCC and it provides a novel therapeutic target for ESCC.
1. Introduction Esophageal cancer is one of the leading causes of death in both developed and developing countries [1], especially in the East Asia, Africa and North America [2]. More than 90% of esophageal cancers are esophageal squamous cell carcinoma (ESCC) [3]. Although considerable progress has been made in diagnosis and treatment, esophageal cancer remains a devastating malignancy due to late diagnosis and rapidly aggressive progression. Patients with esophageal cancer are found to have high terminal metastasis and poor prognosis [4–6]. Therefore, exploration about the development mechanism of ESCC will be helpful for the detection, diagnosis and treatment of ESCC. MicroRNAs (miRNAs) are small non-coding RNA, usually 21–23 nucleotides long which serve as important regulators of gene expression by binding to the 3′ untranslated region (UTR) of target mRNAs [7,8], including in physiological and pathological processes, including cell proliferation, differentiation, migration and apoptosis [9–12]. In ESCC, many mirnas have been identified as effective tumor biomarkers for
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Corresponding author. E-mail address: [email protected] (Q. Guanen).
https://doi.org/10.1016/j.biopha.2018.05.149 Received 31 January 2018; Received in revised form 29 May 2018; Accepted 29 May 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS.
cancer diagnosis, or (and) key oncogenes (or tumor suppressor modulation [13–16]. However, few miRNAs are reported to be related to the occurrence and development of ESCC. Accumulating studies have show that miR-214 may induce a wide variety of human malignancies, including pancreatic, hepatoblastoma, hepatocellular, lung, breast, gastric, osteosarcoma, esophageal squamous cell carcinoma, prostate, ovarian, bladder melanoma and cervical cancers [17–21]. The phosphatidylinositol-3−OH kinase (PI3K)-Akt pathway is a major signaling cascade that is activated in a large variety of human cancers [22]. The phosphoinositide 3-kinase (PI3K)/AKT pathway enhances not only cell proliferation, but also cell invasion and migration [23]. PI3K regulates a large number of basic cellular responses and plays a key role in controlling cell survival and apoptosis. PI3K pathway may be the best characterized pathway to promote the growth or survival of cancer cells and inhibit apoptosis in cancer cells [24]. AKT, the key effect of cell survival, phosphorylation in many different growth factors and interleukin-activated responses via phosphatidylinol conversion through PI3K [25]. mTOR plays an important role in the
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described previously [26]. Microwave irradiation was performed on the sections which were incubated with polyclonal rabbit anti-human LZTS1 at room temperature of 2 h. Sections were washed with PBS and incubated with secondary antibodies at room temperature for 1 h. Staining intensity was evaluated. All images represent at least three separate experiments.
downstream signal transduction of AKT pathway and participates in cell proliferation, differentiation and apoptosis. In this study, our objective is to assess the molecular mechanism about miR-214 on ESCC cell proliferation and apoptosis and analyse the collective signaling pathways enriched by the predicted targets of the down-regulated miRNAs in ESCC carcinogenesis. In this study, we confirmed that miR-214 is negatively correlated with the expression level of LZTS1 inESCC. Our collective results indicated that miR-214 directly targets 3′-UTR of the LZTS1 transcript and inhibited its expression. The downregulation of miR-214 expression suppressed cancer cell viability, proliferation and tumorigenesis by PI3K/AKT/mTOR signaling pathway. This approach helps to clarify the role of miRNA in esophageal cancer and may be a new therapeutic target for ESCC.
2.6. Cell proliferation assay MTT assay and colony formation assay were used to explore the cell viability and cell proliferation ability. The cells were seeded in 96-well plate and cultured in medium including 10% FBS for 6 days. Then cells were detected by MTT assay at different time points. 10 ml of MTT solution was added to 100 ml medium, following incubated at 37 ℃ for 4 h. The absorbance was read on a spectrophotometer at 570 nm. Cell proliferation was detected by colony formation assay. After 12 days of inoculation, the cells were stained with crystal violet and the number of colonies was measured.
2. Materials and methods 2.1. Human tissue samples and cell line Thirty patients with esophageal squamous cell carcinoma and adjacent normal tissues were obtained from Handan First Hospital. The tissues were surgical removal and immediately stored at -80℃. The collection of human tissue samples was approved by the Ethics Committee of Handan First Hospital. The human esophageal squamous cell carcinoma cell line (KYSE150; ECA-109; KYSE450) and the normal cell line HET-1 A were provided by Institute of Biochemistry and Cell Biology of Chinese Academy of Science (China). These cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum under a humidified atmosphere of 95% atmosphere and 5% CO2 at 37 ℃.
2.7. Cell cycle analysis Cells were collected and fixed in 70% ethanol at 4℃ overnight. The cells were incubated with RNase A in PBS for 30 min after the ethanol was removed. Whereafter the cells were incubated in 0.5 ml of 50 mg/ ml propidium iodide (PI) for 30 min in the dark. The cells throughout the cell cycle was analyzed by Cell Lab Quanta SC flow cytometry (Beckman Coulter, Fullerton, CA) according to the established method as described previously [27] after washed with cold PBS twice. 2.8. TUNEL assays
2.2. Cell transfections
4×103 cells were seeded on a 14-well slide in triplicate for 24 h, then fresh medium containing 10% FBS were replaced and cultured for 48 h. We use an In situ Cell Death Detection Kit with Fluorescein (Roche Applied Science, IN, USA) to detecte cell apoptosis. DNA was stained with DAPI which was used to visualize the nuclei. The TUNEL assays were quantified by counting the DAPI. The apoptotic rate was calculated by dividing the average number of TUNEL-positive cells by that of the DAPI-positive cells in one field. Experiments were performed at least three separate experiments.
The lentivirus expressing human miR-214 inhibitor and an empty lentivirus were purchased from RiboBio (RiboBio Biotech, China). Transfections were performed using Lipofectamine 2000 according to the manufacturer's protocol. Cells were incubated for 72 h. Lentivirusinduced miR-214 downregulation was assessed by RT-PCR, western blot and immunohistochemical. 2.3. RNA extraction and quantitative real-time PCR Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). TaqMan miRNA assays were performed to assess the expression level of miR-214. Briefly, 3 mg of small RNA extracted from tissue samples or cells was reverse transcribed to cDNA. The cDNA was subsequently used for the amplification of miR-214, U6 was used as an internal control. Real-time PCR was performed using SYBR Premix Ex Taq II. The PCR conditions were performed at 94℃ for 4 min, followed by 40 cycles of amplification: 94℃ for 40 s, 52℃ for 40 s, 72℃ for 40 s. All samples were normalized to control and calculated using the 2−ΔΔCT analysis method.
2.9. Cell migration and invasion assays Wound healing assay was used to assessed cell migration ability. Cells were inoculated into 24 well culture plates and cultured until confluent. A P200 pipette tip was used to make a straight line simulation “wound”. In addition, cells were washed with appropriate culture medium for 3 times to remove cell debris. The degree of wound closure was monitored at different times. Futhermore, migration and invasion ability were detected by transwell, a total of 1 × 104 cells were inoculated into the upper part of a transwell chamber (transwell filter inserts with a pore size of 8 μm), which was precoated with or without 30 μl of 1 mg/ml matrigel for 2 h. The number of migrated cells on the lower surface of the membrane was counted under a light microscope with a magnification of ×400 in 10 fields. All assays were performed three times.
2.4. Western blotting Cells were lysed with RIPA lysis buffer and proteins were harvested. Total cell protein extracts were separated by 10% SDS polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane which block with 5% non-fat milk. Whereafter the blots were incubated with primary antibodies at 4℃ overnight, followed by incubation with secondary antibody at room temperature for 1 h. Immunoblots were visualized using the ECL detection system and the protein levels were quantified using ImageJ software.
2.10. In vivo tumorigenesis assay Male BALB/c nude mice (6–7 weeks old) were randomly divided into three groups (n = 10). 5 × 106 si-miR-214, Lv-NC or control cells were injected subcutaneously into the right flank of the nude mice axillary. The mice were sacrificed and evaluated after 2 weeks. Tumor growth was analyzed by measuring tumor length and width, and tumor volume was calculated according to 1/2×length × width2.
2.5. Immunohistochemistry Immunohistochemistry was performed according to the methods 351
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2.11. ′UTR luciferase reporter assay
expression of miR-214 in ESCC tissues was noteworthy higher than that of the adjacent non-tumor tissues (P < 0.01). In addition, we measured the expression of miR-214 in ESCC cell lines and normal epithelial cell line. The results show that miR-214 expression was significantly increased in ESCC cell lines (Fig. 1B). As LZTS1 was demonstrated as a potential target of miR-214 using the targetscan, we also studied the expression of LZTS1 protein in ESCC cell lines and normal epithelial cell line, we discovered that the expression of LZTS1 protein was decreased in ESCC cell lines (Fig. 1C), thus miR-214 was inversely correlated with LZTS1 expression. The ability of cells migratory and invasive were carried out by transwell assay (Fig. 1D, E). These results indicated that the expression levels of miR-214 was up-expression in ESCC tissues and cell lines.
The human LZTS1 3′UTR luciferase reporter construct (LZTS13′UTR WT) was generated by cloning LZTS1 mRNA 3′UTR sequence into downstream of pMIR-report construct. We used PCR to generated the LZTS1 3′UTR sequence. Dual-Luciferase Reporter Assay System was used to assessed luciferase activity. Data were normalized by dividing firefly luciferase activity and each experiment was repeated three times. 2.12. Statistical analysis The difference between different two groups of three independent experiments was analyzed by Student’s t-test. one-way analysis of variance (ANOVA) was used to analyzed the difference among more than two groups. The data are presented as means ± S.D. P < 0.05 was considered to be statistically significant.
3.2. Effects of miR-214 on ESCC cell proliferation in vitro We induced down-regulation of miR-214 expression by transfected with lentivirus in ECA-109 cells to explore the biological functions of miR-214 in esophageal cancer cell lines. These efficiencies were confirmed by RT-PCR, western blot and immunohistochemical (Fig. 2A–D). To determine whether miR-214 could obviously regulating ESCC proliferation, MTT and colony formation assays are used to explore the role of miR-214 in the progression of ESCC. The MTT assay showed that cell viability was noteworthy suppressed which in the cells transfected with lentivirus compared with the control group (Fig. 3A). In addition, colony formation experiments showed that the cells transfected with lentivirus formed considerably smaller and fewer colonies than those
3. Results 3.1. MiR-214 is overexpressed in esophageal squamous cell carcinoma tissues and cell lines To identify miRNAs that regulate the metastatic cascade in ESCC, we performed miRNAs array on ESCC tissues and corresponding normal tissues. In this study, we firstly carried out RT-PCR to detect the expression of miR-214 in 30 pairs of fresh ESCC tissues and its adjacent non-tumor tissues. As shown in Fig. 1A, the results showed that the
Fig. 1. miR-214 expression in ESCC tissues and cell lines. (A) expression of miR-214 is elevated in ESCC tissues compared with adjacent non-tumor tissues. (B) quantitative RT-PCR analysis of miR-214 expression levels in ESCC cell lines (KYSE150; ECA-109; KYSE450) compared with normal epithelial cell line (HET-1 A). (C) western blotting analysis of LZTS1 protein expression in ESCC cell lines and normal epithelial cell line. (D, E) the migratory and invasive ability of cell lines were performed by transwell assay. The results were presented as values of means ± SD of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001. 352
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Fig. 2. We induced down-regulation of miR-214 expression by transfected with lentivirus in ECA-109 cells. (A–C) cells transfected with lentivirus showed a decrease in miR-214 expression and increase in LZTS1 expression. (D) the expression of LZTS1 was detected by immunohistochemical. The test was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001.
3.4. Effect of miR-214 on meastasis and apoptosis in ESCC cell
transfected with controls (Fig. 3B). Our results show that the downregulation of miR-214 expression significantly suppressed the growth of esophageal cancer cells. Next, we study the potential mechanism underlying the effect of miR-214 on ESCC cell growth. We use flow cytometry to analyse cell cycle distribution to determine the number of cells at different stages (Fig. 3D). Western blot analysis was used to analyzed the levels of three factors involved in cell cycle regulation. The expression of p27 and Cyclin D1 protein were discovered to be reduced by miR-214 inhibitor compared to control group (Fig. 3E). But there was no significant difference in p21 protein expression in the three cell groups. These results suggest that miR-214 might regulate p27 and Cyclin D1 rather than p21 in cells, which is possibly affiliated to its effect on cell proliferation.
In cell migration experiment, down-regulation of miR-214 expression significantly supressed the migration ability of ECA-109 cells (Fig. 5A, B). Similar results in invasion assays, the number of invasive cells in down-regulated miR-214 groups were significantly decreased compared with the control group. Western blot was used to test the expression of meastasis relatived proteins (Fig. 5D). In order to further explore the influence of miR-214 expression on cell apoptosis. The raised percentage of apoptotic cells in ECA-109 cells as measured by flow cytometry. The number of apoptotic cells was noteworthy raised in si-miR-214 cells compared with control group (Fig. 5E). Western blot was used to test several apoptotic related factors, including Bcl-2, Bax, Caspase-3 and Caspase-9 levels. As show in Fig. 5F, we found that down-regulation of miR-214 significantly supressed the levels of Bcl-2. Fuethermore, knocking down of miR-214 in ECA-109 cells increased Bax, Caspase-3 as well as Caspase-9 levels.
3.3. Effects of miR-214 on ESCC xenograft tumour growth in vivo In order to further study the effects of miR-214 the expression of tumor growth in vivo, we conducted xenograft tumor model test with subcutaneous injection of ECA-109 cells stable downexpressing miR214 or scrambled miRNA into the nude mice. The tumour sizes and weight were measured every three days. The tumor growth rate and volume were smaller which injected with ECA-109 cells stably downexpressing miR-214 than that of the control group (Fig. 4A, B). As shown in Fig. 4C, The mice were executed on the 30th day and the tumours were weighed. The tumour weight between the three groups differed notably. Furthermore, the subcutaneous tumor tissues were examined by H&E (Fig. 4D). TUNEL assay was used to detect the effects of miR-214 on the ESCC apoptosis. The tunel assay showed that the number of tunel positive cells in miR-214 inhibitor group induced compaired with the control group (Fig. 4E).
3.5. LZTS1 is a direct target of miR-214 To illustrate the molecular mechanism that miR-214 promoted cell proliferation and inhibited apoptosis, we used mRNA target-predicting algorithms (TargetScan) based on the presence of binding sites in the 3′UTR (Fig. 6A, B). To establish a direct relationship between miR-214 and LZTS1, we cloned the WT-LZTS1 3′-UTR or Mut-LZTS1 3′-UTR into a dual-luciferase UTR vector. In addition, we conducted luciferase reporter assay to test whether LZTS1 was a direct target of miR-214. We discovered that the luciferase activity was noteworthy increased by cotransfection with miR-214 mimics and the vector carrying the WTLZTS1 3′-UTR (Fig. 6C) in esophageal cancer cell lines. Furthermore, we 353
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Fig. 3. Influences of miR-214 on ESCC cells growth. (A) down-regulated miR-214 expression has remarkably inhibited on ESCC cell proliferative ability of ECA-109 cells respectively through colony formation assay. (B, C) in vitro viability of cells significantly suppressed by miR-214 inhibitor through MTT assay and cell adhesion assay. (D) cell cycle progression was assayed by flow cytometry in 3 different groups. (E) western blot analysis for the protein levels of p27, Cyclin D1 and p21 in cells transfected with miR-214 inhibitor and cultured in serum free medium for 48 h. β-actin was used as an internal control. * P < 0.05; ** P < 0.01 compared with control.
immunofluorescence. We found that Cyclin D1, p-PI3K, p-AKT and pmTOR expression in miR-214 lentivirus group was noteworthy lower than that in the control and empty vector group. Moreover, PI3K, AKT and mTOR expression was basically the same in miR-214 lentivirus group and all significantly lower than that in control group (Figs. 7 and 8). Taken together, these results indicate that miR-214 regulates the PI3K/AKT/mTOR pathway, which contributes to the effect of LZTS1on cell proliferation and apoptosis in ESCC cells.
found that although ectopically expressing miR-214 repress the expression of LZTS1 (Fig. 6D). These results demonstrated that miR-214 negatively regulates LZTS1 expression by directly targeting its 3′-UTR.
3.6. MiR-214 regulates cell proliferation through the PI3K/AKT/mTOR signaling pathway in ESCC cells To investigate the molecular mechanism about the inhibition of miR-214 on cell proliferation, the expression of LZTS1, Cyclin D1, phosphorylated-PI3K (p-PI3K), PI3K, p-AKT, AKT, p-mTOR and mTOR were determined by western blot, immunohistochemical and 354
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Fig. 4. Down-expression miR-214 suppresses ESCC xenograft tumor growth in vivo. (A) ECA-109 cells (2 × 106) were injected in the hind limbs of nude mice. After thirty days, the cells stably downexpressing miR-214 formed smaller tumours than those expressing scrambled controls. (B, C) tumor volumes and weight was measured. (D) H&E analysis of the pathological features of the tumors from the three groups. (E) TUNEL assay of apoptosis in ESCC. The data are presented as the means ± SD, and p-values were calculated using Student’st-test. **P < 0.01 and ***P < 0.001 vs. NC and control.
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Fig. 5. Effect of miR-214 on cell meastasis and apoptosis in ESCC cells. (A, B) cell migration ability as determined using wound healing assay and transwell assay. (C) cell invasion ability as determined using wound healing assay and transwell assay. (D) the meastasis relatived proteins was detected by western blot. (E) the apoptosis of ECA-109 cells measured by flow cytometry after transfection 48 h. (F) the cell apoptosis related factors was detected by western blotting. The average values ± SEM were calculated from three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 6. LZTS1 is a direct target of miR-214. (A) wild-type (Wt) and mutant (Mt) miR-214 target sequences in the LZTS1 3′-UTR. (B) relative luciferase activity of ECA109 cells after cotransfection with wild type (wt) or mutant (mut) LZTS1 3′-UTR reporter genes and miR-214 mimics. The relative firefly luciferase activity was measured after transfection for 48 h. (C, D) western blot analysis was performed for LZTS1 expression in ECA-109 cells. The data are presented as the means ± SD, and p-values were calculated using Student’s t-test.*P < 0.05, **P < 0.01, ***P < 0.001.
4. Discussion
various cancers, and changes in miRNA expression may cause cancer in humans by negatively regulating multiple target genes [29]. The identification of cancer-specific miRNAs and their targets is of the utmost importance for understanding the role of tumor metastasis and is crucial for determining new therapeutic targets [30,31]. MiR-214 has been found that abnormal expression in human tumor and plays a
MicroRNAs (miRNAs) are small (17–25 nucleotides), non-coding RNAs that negatively control the target gene expression by inducing translational inhibition or target mRNAs degradation [28]. There is increasing evidence has shown some miRNAs express abnormalities in 357
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Fig. 7. miR-214 regulates the cell proliferation factors though the PI3K/AKT/mTOR pathway in ESCC cells. (A) miR-214 regulates the PI3K/AKT/mTOR pathway activity and expression of some related proteins (LZTS1, Cyclin D1, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR) in ESCC cells. (B) the mRNA expression of LZTS1, Cyclin D1, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR in the control, empty vector and miR-214 lentivirus group by RT-PCR. (C) the expression of PI3K/AKT/ mTOR related proteins was detectd by immunohistochemical.
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Fig. 8. The expression of PI3K/AKT/mTOR related proteins was detectd by immunofluorescence. (A–F) LZTS1, Cyclin D1, p-PI3K, PI3K, p-mTOR and mTOR was detectd by immunofluorescence.
However, the molecular mechanism of miR-214 in esophageal squamous cell progression has not been elucidated. In our study, we first demonstrated that miR-214 promotes metastasis and inhibites apoptosis by targeting LZTS1 through PI3K/AKT/mTOR signaling pathway in ESCC. We preliminarily examined the changes of miRNAs in
different role in cancer development. Previous studies have shown that miR-214 has been associated with increased proliferation, metastasis, invasion and functions as an oncogene for specific types of cancer in humans [32–35]. However, miR-214 expression was down-regulated to inhibit cell growth in cervical cancer and breast cancer [36–38]. 359
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Conflicts of interest
human primary ESCC tissues with or without metastasis by RT-PCR. Moreover, microarray analysis was used to determine miR-214 as one of the most significantly upregulated miRNAs in ESCC tissues. Considering that miR-214 play an important role in regulation of cell proliferation and apoptosis, the effect of miR-214 on these phenotypes of ESCC cells was further investigated. It is worth noting that silence of miR-214 evidently supressed cell proliferation, tumor growth and promoted apoptosis in vitro and in vivo. Based on these results, we believe that miR-214 may play a role as tumor promoter in ESCC, which is potential to be a new predictor of ESCC progression. The leucine zipper putative tumor suppressor 1 (LZTS1) is a tumor suppressor gene located at chromosome 8p22 [39], which is often reduced or absent in human breast cancers and bladder cancers [40,41]. LZTS1 defcient mice develop cancers with diverse histogenetic backgrounds [42,43], suggesting that LZTS1 acts as a major tumor suppressor gene in multiple cell types. Our data also indicate that LZTS1 is a direct functional target of miR-214 in ESCC cells. There are several pieces of evidence to support this. Firstly, the complementary sequence of miR-214 is confirmed in the 3′-UTR of LZTS1 mRNA. The activity of 3′-UTR luciferase activity was decreased in area of miR-214 overexpressed, which was eliminated by mutation of the miR-214 seed binding site. Secondly, miR-214 overexpression decreased LZTS1 protein and this effect was reduced by miR-214 knockdown. These results strongly suggest that LZTS1 was a direct target of miR-214 in ESCC cells. The relationship between miR-214 and LZTS1 was established and confirm that miR-214 acts as an onco-miRNA by targeting LZTS1 in ESCC cells. We went onto detail the apoptosis-related mechanisms participate in miR-214′s action. Apoptosis induced by serum starvation is generally considered to be caspase-dependent. Caspase-3 activity is essential for apoptosis after serum withdrawal, but whether apoptosis occurs is still controversial through the internal pathway (via caspase-9 activation). In this case, we found that the heterotopic expression of miR-214 in human esophageal cancer ECA-109 cells increased the expressions of caspase-3, Bax and caspase-9, which were partly conversed by downexpression of miR-214. In contrast, the expression of Bcl-2 was decreased. These data demonstate that the intrinsic pathway is participate in the apoptosis induced by enforced expression of miR-214. Although miR-214 has been related to regulation of tumor growth and proliferation, the molecular mechanisms remains unclear. mTOR plays an important role in downstream signaling of the AKT pathway and is participates in cell proliferation, differentiation, apoptosis and other cellular processes. In addition, the PI3K/AKT/mTOR pathway is frequently activated and plays an important role in metastasis and tumor growth in various cancers. Therefore, we speculate that miR-214 affects the proliferation and tumor growth of ESCC by controlling the PI3K/AKT/mTOR signaling pathway. We studied the expression levels of some PI3K/AKT/mTOR signaling pathway related genes, such as p-AKT, AKT, Cyclin D1, p-PI3K, PI3K, p-mTOR and mTOR. These results demonstrate that these molecular protein levels were significantly inhibited, suggesting that miR-214 promote proliferation and growth of ESCC cells via PI3K/AKT/mTOR signaling pathway. In conclusion, we have reported the tumor enhancement role of miR-214 in human ESCC. The expression levels of miR-214 is noteworthy increased in ESCC cells and tissues, down-expression of miR214 can inhibite cell growth in vitro and tumorigenesis in vivo. In addition, it promotes cell migration, invasion and suppresses apoptosis. Moreover, LZTS1 is a direct and functional target of miR-214 and this function of miR-214 in ESCC is mainly mediated by the PI3K/AKT/ mTOR signaling pathway. Given these, miR-214 may be a useful prognostic marker and a potential therapeutic strategy for future treatment of esophageal cancer.
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
MiR-214 promotes cell meastasis and inhibites apoptosis of esophageal squamous cell carcinoma via PI3K/AKT/mTOR signaling pathway
T
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Qiao Guanena, , Shi Junjieb, Wu Baolinc, Wang Chaoyangd, Yang Yajuana, Li Jinga, Li Junpengb, Ning Gailie, Wang Zhongpingf, Wang Juna a
Department of Gastroenterology, Handan City First Hospital, Handan, Hebei, 056002, China Department of Thoracic Surgery, Handan City First Hospital, Handan, Hebei, 056002, China c Department of Medical imaging, Handan City First Hospital, Handan, Hebei, 056002, China d Department of infectious disease, Handan City First Hospital, Handan, Hebei, 056002, China e Department of Gynaecology, Handan City First Hospital, Handan, Hebei, 056002, China f Department of Medicine, Handan City First Hospital, Handan, Hebei, 056002, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: MiR-214 Meastasis Apoptosis PI3K/AKT/mTOR pathway ESCC
There is growing evidence shown that microRNAs (miRNAs) are associated with cancer and can play a role in human cancers as oncogenes or tumor suppressor genes. MicroRNA-214 (miR-214) shows carcinogenesis in various tumor types, but little is known about biological functions of miR-214 in esophageal squamous cell carcinoma (ESCC). In this study, we observe that the expression of miR-214 is not only increased in human ESCC tissues, but also remarkably increased in cell lines correlates with LZTS1. In addition, the expression of miR-214 inhibited proliferation of ESCC cells in vitro and inhibit the growth of xenograft tumor in vivo. The results show miR-214 serve as a tumor promoter regulating cells migration, invasion and apoptosis in ESCC. Ferthermore, LZTS1 has been proved to as a functional target for miR-214 to regulate cells poliferation and apoptosis. In summary, these results suggest that miR-214 serves as tumor promoter to promote proliferation, migration, invasion and inhibit apoptosis of ESCC cells by targeting LZTS1 via PI3K/AKT/mTOR signaling pathway. The miR-214/LZTS1 pathway provides a new insight into the molecular mechanisms that the occurrence and development of ESCC and it provides a novel therapeutic target for ESCC.
1. Introduction Esophageal cancer is one of the leading causes of death in both developed and developing countries [1], especially in the East Asia, Africa and North America [2]. More than 90% of esophageal cancers are esophageal squamous cell carcinoma (ESCC) [3]. Although considerable progress has been made in diagnosis and treatment, esophageal cancer remains a devastating malignancy due to late diagnosis and rapidly aggressive progression. Patients with esophageal cancer are found to have high terminal metastasis and poor prognosis [4–6]. Therefore, exploration about the development mechanism of ESCC will be helpful for the detection, diagnosis and treatment of ESCC. MicroRNAs (miRNAs) are small non-coding RNA, usually 21–23 nucleotides long which serve as important regulators of gene expression by binding to the 3′ untranslated region (UTR) of target mRNAs [7,8], including in physiological and pathological processes, including cell proliferation, differentiation, migration and apoptosis [9–12]. In ESCC, many mirnas have been identified as effective tumor biomarkers for
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Corresponding author. E-mail address: [email protected] (Q. Guanen).
https://doi.org/10.1016/j.biopha.2018.05.149 Received 31 January 2018; Received in revised form 29 May 2018; Accepted 29 May 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS.
cancer diagnosis, or (and) key oncogenes (or tumor suppressor modulation [13–16]. However, few miRNAs are reported to be related to the occurrence and development of ESCC. Accumulating studies have show that miR-214 may induce a wide variety of human malignancies, including pancreatic, hepatoblastoma, hepatocellular, lung, breast, gastric, osteosarcoma, esophageal squamous cell carcinoma, prostate, ovarian, bladder melanoma and cervical cancers [17–21]. The phosphatidylinositol-3−OH kinase (PI3K)-Akt pathway is a major signaling cascade that is activated in a large variety of human cancers [22]. The phosphoinositide 3-kinase (PI3K)/AKT pathway enhances not only cell proliferation, but also cell invasion and migration [23]. PI3K regulates a large number of basic cellular responses and plays a key role in controlling cell survival and apoptosis. PI3K pathway may be the best characterized pathway to promote the growth or survival of cancer cells and inhibit apoptosis in cancer cells [24]. AKT, the key effect of cell survival, phosphorylation in many different growth factors and interleukin-activated responses via phosphatidylinol conversion through PI3K [25]. mTOR plays an important role in the
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described previously [26]. Microwave irradiation was performed on the sections which were incubated with polyclonal rabbit anti-human LZTS1 at room temperature of 2 h. Sections were washed with PBS and incubated with secondary antibodies at room temperature for 1 h. Staining intensity was evaluated. All images represent at least three separate experiments.
downstream signal transduction of AKT pathway and participates in cell proliferation, differentiation and apoptosis. In this study, our objective is to assess the molecular mechanism about miR-214 on ESCC cell proliferation and apoptosis and analyse the collective signaling pathways enriched by the predicted targets of the down-regulated miRNAs in ESCC carcinogenesis. In this study, we confirmed that miR-214 is negatively correlated with the expression level of LZTS1 inESCC. Our collective results indicated that miR-214 directly targets 3′-UTR of the LZTS1 transcript and inhibited its expression. The downregulation of miR-214 expression suppressed cancer cell viability, proliferation and tumorigenesis by PI3K/AKT/mTOR signaling pathway. This approach helps to clarify the role of miRNA in esophageal cancer and may be a new therapeutic target for ESCC.
2.6. Cell proliferation assay MTT assay and colony formation assay were used to explore the cell viability and cell proliferation ability. The cells were seeded in 96-well plate and cultured in medium including 10% FBS for 6 days. Then cells were detected by MTT assay at different time points. 10 ml of MTT solution was added to 100 ml medium, following incubated at 37 ℃ for 4 h. The absorbance was read on a spectrophotometer at 570 nm. Cell proliferation was detected by colony formation assay. After 12 days of inoculation, the cells were stained with crystal violet and the number of colonies was measured.
2. Materials and methods 2.1. Human tissue samples and cell line Thirty patients with esophageal squamous cell carcinoma and adjacent normal tissues were obtained from Handan First Hospital. The tissues were surgical removal and immediately stored at -80℃. The collection of human tissue samples was approved by the Ethics Committee of Handan First Hospital. The human esophageal squamous cell carcinoma cell line (KYSE150; ECA-109; KYSE450) and the normal cell line HET-1 A were provided by Institute of Biochemistry and Cell Biology of Chinese Academy of Science (China). These cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum under a humidified atmosphere of 95% atmosphere and 5% CO2 at 37 ℃.
2.7. Cell cycle analysis Cells were collected and fixed in 70% ethanol at 4℃ overnight. The cells were incubated with RNase A in PBS for 30 min after the ethanol was removed. Whereafter the cells were incubated in 0.5 ml of 50 mg/ ml propidium iodide (PI) for 30 min in the dark. The cells throughout the cell cycle was analyzed by Cell Lab Quanta SC flow cytometry (Beckman Coulter, Fullerton, CA) according to the established method as described previously [27] after washed with cold PBS twice. 2.8. TUNEL assays
2.2. Cell transfections
4×103 cells were seeded on a 14-well slide in triplicate for 24 h, then fresh medium containing 10% FBS were replaced and cultured for 48 h. We use an In situ Cell Death Detection Kit with Fluorescein (Roche Applied Science, IN, USA) to detecte cell apoptosis. DNA was stained with DAPI which was used to visualize the nuclei. The TUNEL assays were quantified by counting the DAPI. The apoptotic rate was calculated by dividing the average number of TUNEL-positive cells by that of the DAPI-positive cells in one field. Experiments were performed at least three separate experiments.
The lentivirus expressing human miR-214 inhibitor and an empty lentivirus were purchased from RiboBio (RiboBio Biotech, China). Transfections were performed using Lipofectamine 2000 according to the manufacturer's protocol. Cells were incubated for 72 h. Lentivirusinduced miR-214 downregulation was assessed by RT-PCR, western blot and immunohistochemical. 2.3. RNA extraction and quantitative real-time PCR Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). TaqMan miRNA assays were performed to assess the expression level of miR-214. Briefly, 3 mg of small RNA extracted from tissue samples or cells was reverse transcribed to cDNA. The cDNA was subsequently used for the amplification of miR-214, U6 was used as an internal control. Real-time PCR was performed using SYBR Premix Ex Taq II. The PCR conditions were performed at 94℃ for 4 min, followed by 40 cycles of amplification: 94℃ for 40 s, 52℃ for 40 s, 72℃ for 40 s. All samples were normalized to control and calculated using the 2−ΔΔCT analysis method.
2.9. Cell migration and invasion assays Wound healing assay was used to assessed cell migration ability. Cells were inoculated into 24 well culture plates and cultured until confluent. A P200 pipette tip was used to make a straight line simulation “wound”. In addition, cells were washed with appropriate culture medium for 3 times to remove cell debris. The degree of wound closure was monitored at different times. Futhermore, migration and invasion ability were detected by transwell, a total of 1 × 104 cells were inoculated into the upper part of a transwell chamber (transwell filter inserts with a pore size of 8 μm), which was precoated with or without 30 μl of 1 mg/ml matrigel for 2 h. The number of migrated cells on the lower surface of the membrane was counted under a light microscope with a magnification of ×400 in 10 fields. All assays were performed three times.
2.4. Western blotting Cells were lysed with RIPA lysis buffer and proteins were harvested. Total cell protein extracts were separated by 10% SDS polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane which block with 5% non-fat milk. Whereafter the blots were incubated with primary antibodies at 4℃ overnight, followed by incubation with secondary antibody at room temperature for 1 h. Immunoblots were visualized using the ECL detection system and the protein levels were quantified using ImageJ software.
2.10. In vivo tumorigenesis assay Male BALB/c nude mice (6–7 weeks old) were randomly divided into three groups (n = 10). 5 × 106 si-miR-214, Lv-NC or control cells were injected subcutaneously into the right flank of the nude mice axillary. The mice were sacrificed and evaluated after 2 weeks. Tumor growth was analyzed by measuring tumor length and width, and tumor volume was calculated according to 1/2×length × width2.
2.5. Immunohistochemistry Immunohistochemistry was performed according to the methods 351
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2.11. ′UTR luciferase reporter assay
expression of miR-214 in ESCC tissues was noteworthy higher than that of the adjacent non-tumor tissues (P < 0.01). In addition, we measured the expression of miR-214 in ESCC cell lines and normal epithelial cell line. The results show that miR-214 expression was significantly increased in ESCC cell lines (Fig. 1B). As LZTS1 was demonstrated as a potential target of miR-214 using the targetscan, we also studied the expression of LZTS1 protein in ESCC cell lines and normal epithelial cell line, we discovered that the expression of LZTS1 protein was decreased in ESCC cell lines (Fig. 1C), thus miR-214 was inversely correlated with LZTS1 expression. The ability of cells migratory and invasive were carried out by transwell assay (Fig. 1D, E). These results indicated that the expression levels of miR-214 was up-expression in ESCC tissues and cell lines.
The human LZTS1 3′UTR luciferase reporter construct (LZTS13′UTR WT) was generated by cloning LZTS1 mRNA 3′UTR sequence into downstream of pMIR-report construct. We used PCR to generated the LZTS1 3′UTR sequence. Dual-Luciferase Reporter Assay System was used to assessed luciferase activity. Data were normalized by dividing firefly luciferase activity and each experiment was repeated three times. 2.12. Statistical analysis The difference between different two groups of three independent experiments was analyzed by Student’s t-test. one-way analysis of variance (ANOVA) was used to analyzed the difference among more than two groups. The data are presented as means ± S.D. P < 0.05 was considered to be statistically significant.
3.2. Effects of miR-214 on ESCC cell proliferation in vitro We induced down-regulation of miR-214 expression by transfected with lentivirus in ECA-109 cells to explore the biological functions of miR-214 in esophageal cancer cell lines. These efficiencies were confirmed by RT-PCR, western blot and immunohistochemical (Fig. 2A–D). To determine whether miR-214 could obviously regulating ESCC proliferation, MTT and colony formation assays are used to explore the role of miR-214 in the progression of ESCC. The MTT assay showed that cell viability was noteworthy suppressed which in the cells transfected with lentivirus compared with the control group (Fig. 3A). In addition, colony formation experiments showed that the cells transfected with lentivirus formed considerably smaller and fewer colonies than those
3. Results 3.1. MiR-214 is overexpressed in esophageal squamous cell carcinoma tissues and cell lines To identify miRNAs that regulate the metastatic cascade in ESCC, we performed miRNAs array on ESCC tissues and corresponding normal tissues. In this study, we firstly carried out RT-PCR to detect the expression of miR-214 in 30 pairs of fresh ESCC tissues and its adjacent non-tumor tissues. As shown in Fig. 1A, the results showed that the
Fig. 1. miR-214 expression in ESCC tissues and cell lines. (A) expression of miR-214 is elevated in ESCC tissues compared with adjacent non-tumor tissues. (B) quantitative RT-PCR analysis of miR-214 expression levels in ESCC cell lines (KYSE150; ECA-109; KYSE450) compared with normal epithelial cell line (HET-1 A). (C) western blotting analysis of LZTS1 protein expression in ESCC cell lines and normal epithelial cell line. (D, E) the migratory and invasive ability of cell lines were performed by transwell assay. The results were presented as values of means ± SD of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001. 352
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Fig. 2. We induced down-regulation of miR-214 expression by transfected with lentivirus in ECA-109 cells. (A–C) cells transfected with lentivirus showed a decrease in miR-214 expression and increase in LZTS1 expression. (D) the expression of LZTS1 was detected by immunohistochemical. The test was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001.
3.4. Effect of miR-214 on meastasis and apoptosis in ESCC cell
transfected with controls (Fig. 3B). Our results show that the downregulation of miR-214 expression significantly suppressed the growth of esophageal cancer cells. Next, we study the potential mechanism underlying the effect of miR-214 on ESCC cell growth. We use flow cytometry to analyse cell cycle distribution to determine the number of cells at different stages (Fig. 3D). Western blot analysis was used to analyzed the levels of three factors involved in cell cycle regulation. The expression of p27 and Cyclin D1 protein were discovered to be reduced by miR-214 inhibitor compared to control group (Fig. 3E). But there was no significant difference in p21 protein expression in the three cell groups. These results suggest that miR-214 might regulate p27 and Cyclin D1 rather than p21 in cells, which is possibly affiliated to its effect on cell proliferation.
In cell migration experiment, down-regulation of miR-214 expression significantly supressed the migration ability of ECA-109 cells (Fig. 5A, B). Similar results in invasion assays, the number of invasive cells in down-regulated miR-214 groups were significantly decreased compared with the control group. Western blot was used to test the expression of meastasis relatived proteins (Fig. 5D). In order to further explore the influence of miR-214 expression on cell apoptosis. The raised percentage of apoptotic cells in ECA-109 cells as measured by flow cytometry. The number of apoptotic cells was noteworthy raised in si-miR-214 cells compared with control group (Fig. 5E). Western blot was used to test several apoptotic related factors, including Bcl-2, Bax, Caspase-3 and Caspase-9 levels. As show in Fig. 5F, we found that down-regulation of miR-214 significantly supressed the levels of Bcl-2. Fuethermore, knocking down of miR-214 in ECA-109 cells increased Bax, Caspase-3 as well as Caspase-9 levels.
3.3. Effects of miR-214 on ESCC xenograft tumour growth in vivo In order to further study the effects of miR-214 the expression of tumor growth in vivo, we conducted xenograft tumor model test with subcutaneous injection of ECA-109 cells stable downexpressing miR214 or scrambled miRNA into the nude mice. The tumour sizes and weight were measured every three days. The tumor growth rate and volume were smaller which injected with ECA-109 cells stably downexpressing miR-214 than that of the control group (Fig. 4A, B). As shown in Fig. 4C, The mice were executed on the 30th day and the tumours were weighed. The tumour weight between the three groups differed notably. Furthermore, the subcutaneous tumor tissues were examined by H&E (Fig. 4D). TUNEL assay was used to detect the effects of miR-214 on the ESCC apoptosis. The tunel assay showed that the number of tunel positive cells in miR-214 inhibitor group induced compaired with the control group (Fig. 4E).
3.5. LZTS1 is a direct target of miR-214 To illustrate the molecular mechanism that miR-214 promoted cell proliferation and inhibited apoptosis, we used mRNA target-predicting algorithms (TargetScan) based on the presence of binding sites in the 3′UTR (Fig. 6A, B). To establish a direct relationship between miR-214 and LZTS1, we cloned the WT-LZTS1 3′-UTR or Mut-LZTS1 3′-UTR into a dual-luciferase UTR vector. In addition, we conducted luciferase reporter assay to test whether LZTS1 was a direct target of miR-214. We discovered that the luciferase activity was noteworthy increased by cotransfection with miR-214 mimics and the vector carrying the WTLZTS1 3′-UTR (Fig. 6C) in esophageal cancer cell lines. Furthermore, we 353
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Fig. 3. Influences of miR-214 on ESCC cells growth. (A) down-regulated miR-214 expression has remarkably inhibited on ESCC cell proliferative ability of ECA-109 cells respectively through colony formation assay. (B, C) in vitro viability of cells significantly suppressed by miR-214 inhibitor through MTT assay and cell adhesion assay. (D) cell cycle progression was assayed by flow cytometry in 3 different groups. (E) western blot analysis for the protein levels of p27, Cyclin D1 and p21 in cells transfected with miR-214 inhibitor and cultured in serum free medium for 48 h. β-actin was used as an internal control. * P < 0.05; ** P < 0.01 compared with control.
immunofluorescence. We found that Cyclin D1, p-PI3K, p-AKT and pmTOR expression in miR-214 lentivirus group was noteworthy lower than that in the control and empty vector group. Moreover, PI3K, AKT and mTOR expression was basically the same in miR-214 lentivirus group and all significantly lower than that in control group (Figs. 7 and 8). Taken together, these results indicate that miR-214 regulates the PI3K/AKT/mTOR pathway, which contributes to the effect of LZTS1on cell proliferation and apoptosis in ESCC cells.
found that although ectopically expressing miR-214 repress the expression of LZTS1 (Fig. 6D). These results demonstrated that miR-214 negatively regulates LZTS1 expression by directly targeting its 3′-UTR.
3.6. MiR-214 regulates cell proliferation through the PI3K/AKT/mTOR signaling pathway in ESCC cells To investigate the molecular mechanism about the inhibition of miR-214 on cell proliferation, the expression of LZTS1, Cyclin D1, phosphorylated-PI3K (p-PI3K), PI3K, p-AKT, AKT, p-mTOR and mTOR were determined by western blot, immunohistochemical and 354
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Fig. 4. Down-expression miR-214 suppresses ESCC xenograft tumor growth in vivo. (A) ECA-109 cells (2 × 106) were injected in the hind limbs of nude mice. After thirty days, the cells stably downexpressing miR-214 formed smaller tumours than those expressing scrambled controls. (B, C) tumor volumes and weight was measured. (D) H&E analysis of the pathological features of the tumors from the three groups. (E) TUNEL assay of apoptosis in ESCC. The data are presented as the means ± SD, and p-values were calculated using Student’st-test. **P < 0.01 and ***P < 0.001 vs. NC and control.
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Fig. 5. Effect of miR-214 on cell meastasis and apoptosis in ESCC cells. (A, B) cell migration ability as determined using wound healing assay and transwell assay. (C) cell invasion ability as determined using wound healing assay and transwell assay. (D) the meastasis relatived proteins was detected by western blot. (E) the apoptosis of ECA-109 cells measured by flow cytometry after transfection 48 h. (F) the cell apoptosis related factors was detected by western blotting. The average values ± SEM were calculated from three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 6. LZTS1 is a direct target of miR-214. (A) wild-type (Wt) and mutant (Mt) miR-214 target sequences in the LZTS1 3′-UTR. (B) relative luciferase activity of ECA109 cells after cotransfection with wild type (wt) or mutant (mut) LZTS1 3′-UTR reporter genes and miR-214 mimics. The relative firefly luciferase activity was measured after transfection for 48 h. (C, D) western blot analysis was performed for LZTS1 expression in ECA-109 cells. The data are presented as the means ± SD, and p-values were calculated using Student’s t-test.*P < 0.05, **P < 0.01, ***P < 0.001.
4. Discussion
various cancers, and changes in miRNA expression may cause cancer in humans by negatively regulating multiple target genes [29]. The identification of cancer-specific miRNAs and their targets is of the utmost importance for understanding the role of tumor metastasis and is crucial for determining new therapeutic targets [30,31]. MiR-214 has been found that abnormal expression in human tumor and plays a
MicroRNAs (miRNAs) are small (17–25 nucleotides), non-coding RNAs that negatively control the target gene expression by inducing translational inhibition or target mRNAs degradation [28]. There is increasing evidence has shown some miRNAs express abnormalities in 357
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Fig. 7. miR-214 regulates the cell proliferation factors though the PI3K/AKT/mTOR pathway in ESCC cells. (A) miR-214 regulates the PI3K/AKT/mTOR pathway activity and expression of some related proteins (LZTS1, Cyclin D1, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR) in ESCC cells. (B) the mRNA expression of LZTS1, Cyclin D1, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR in the control, empty vector and miR-214 lentivirus group by RT-PCR. (C) the expression of PI3K/AKT/ mTOR related proteins was detectd by immunohistochemical.
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Fig. 8. The expression of PI3K/AKT/mTOR related proteins was detectd by immunofluorescence. (A–F) LZTS1, Cyclin D1, p-PI3K, PI3K, p-mTOR and mTOR was detectd by immunofluorescence.
However, the molecular mechanism of miR-214 in esophageal squamous cell progression has not been elucidated. In our study, we first demonstrated that miR-214 promotes metastasis and inhibites apoptosis by targeting LZTS1 through PI3K/AKT/mTOR signaling pathway in ESCC. We preliminarily examined the changes of miRNAs in
different role in cancer development. Previous studies have shown that miR-214 has been associated with increased proliferation, metastasis, invasion and functions as an oncogene for specific types of cancer in humans [32–35]. However, miR-214 expression was down-regulated to inhibit cell growth in cervical cancer and breast cancer [36–38]. 359
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Conflicts of interest
human primary ESCC tissues with or without metastasis by RT-PCR. Moreover, microarray analysis was used to determine miR-214 as one of the most significantly upregulated miRNAs in ESCC tissues. Considering that miR-214 play an important role in regulation of cell proliferation and apoptosis, the effect of miR-214 on these phenotypes of ESCC cells was further investigated. It is worth noting that silence of miR-214 evidently supressed cell proliferation, tumor growth and promoted apoptosis in vitro and in vivo. Based on these results, we believe that miR-214 may play a role as tumor promoter in ESCC, which is potential to be a new predictor of ESCC progression. The leucine zipper putative tumor suppressor 1 (LZTS1) is a tumor suppressor gene located at chromosome 8p22 [39], which is often reduced or absent in human breast cancers and bladder cancers [40,41]. LZTS1 defcient mice develop cancers with diverse histogenetic backgrounds [42,43], suggesting that LZTS1 acts as a major tumor suppressor gene in multiple cell types. Our data also indicate that LZTS1 is a direct functional target of miR-214 in ESCC cells. There are several pieces of evidence to support this. Firstly, the complementary sequence of miR-214 is confirmed in the 3′-UTR of LZTS1 mRNA. The activity of 3′-UTR luciferase activity was decreased in area of miR-214 overexpressed, which was eliminated by mutation of the miR-214 seed binding site. Secondly, miR-214 overexpression decreased LZTS1 protein and this effect was reduced by miR-214 knockdown. These results strongly suggest that LZTS1 was a direct target of miR-214 in ESCC cells. The relationship between miR-214 and LZTS1 was established and confirm that miR-214 acts as an onco-miRNA by targeting LZTS1 in ESCC cells. We went onto detail the apoptosis-related mechanisms participate in miR-214′s action. Apoptosis induced by serum starvation is generally considered to be caspase-dependent. Caspase-3 activity is essential for apoptosis after serum withdrawal, but whether apoptosis occurs is still controversial through the internal pathway (via caspase-9 activation). In this case, we found that the heterotopic expression of miR-214 in human esophageal cancer ECA-109 cells increased the expressions of caspase-3, Bax and caspase-9, which were partly conversed by downexpression of miR-214. In contrast, the expression of Bcl-2 was decreased. These data demonstate that the intrinsic pathway is participate in the apoptosis induced by enforced expression of miR-214. Although miR-214 has been related to regulation of tumor growth and proliferation, the molecular mechanisms remains unclear. mTOR plays an important role in downstream signaling of the AKT pathway and is participates in cell proliferation, differentiation, apoptosis and other cellular processes. In addition, the PI3K/AKT/mTOR pathway is frequently activated and plays an important role in metastasis and tumor growth in various cancers. Therefore, we speculate that miR-214 affects the proliferation and tumor growth of ESCC by controlling the PI3K/AKT/mTOR signaling pathway. We studied the expression levels of some PI3K/AKT/mTOR signaling pathway related genes, such as p-AKT, AKT, Cyclin D1, p-PI3K, PI3K, p-mTOR and mTOR. These results demonstrate that these molecular protein levels were significantly inhibited, suggesting that miR-214 promote proliferation and growth of ESCC cells via PI3K/AKT/mTOR signaling pathway. In conclusion, we have reported the tumor enhancement role of miR-214 in human ESCC. The expression levels of miR-214 is noteworthy increased in ESCC cells and tissues, down-expression of miR214 can inhibite cell growth in vitro and tumorigenesis in vivo. In addition, it promotes cell migration, invasion and suppresses apoptosis. Moreover, LZTS1 is a direct and functional target of miR-214 and this function of miR-214 in ESCC is mainly mediated by the PI3K/AKT/ mTOR signaling pathway. Given these, miR-214 may be a useful prognostic marker and a potential therapeutic strategy for future treatment of esophageal cancer.
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