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Long non-coding RNA LINC01617 promotes proliferation and metastasis of esophageal cancer cells through AKT pathway
Accepted Manuscript Long non-coding RNA LINC01617 promotes proliferation and metastasis of esophageal cancer cells through AKT pathway
Dan Zhang, Wei Yang, Shuwei Wang, Fei Wang, Dayong Liu, Jingjing Dong, Nan Zhao, Yunye Wang, Haiyan Zhang PII: DOI: Reference:
S0378-1119(18)30916-8 doi:10.1016/j.gene.2018.08.054 GENE 43173
To appear in:
Gene
Received date: Revised date: Accepted date:
24 June 2018 12 August 2018 14 August 2018
Please cite this article as: Dan Zhang, Wei Yang, Shuwei Wang, Fei Wang, Dayong Liu, Jingjing Dong, Nan Zhao, Yunye Wang, Haiyan Zhang , Long non-coding RNA LINC01617 promotes proliferation and metastasis of esophageal cancer cells through AKT pathway. Gene (2018), doi:10.1016/j.gene.2018.08.054
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ACCEPTED MANUSCRIPT Long Non-Coding RNA LINC01617 Promotes Proliferation and Metastasis of Esophageal Cancer Cells Through AKT pathway
Dan Zhang#, Wei Yang#, Shuwei Wang, Fei Wang, Dayong Liu, Jingjing Dong, Nan Zhao, Yunye Wang* and Haiyan Zhang*
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Department of Surgery, Affiliated Hospital of Jilin Medical University, Jilin, China
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#: These two authors contributed the same in this paper
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*: Correspondence to: Yunye Wang, Department of Surgery, Affiliated Hospital of Jilin Medical University, E-mail: [email protected] Haiyan Zhang, Department of Surgery, Affiliated Hospital of Jilin Medical University, E-mail: [email protected]
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Abstract
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Objective To investigate the clinical significance of long non-coding RNA LINC01617 in esophageal cancer and explore the effect of LINC01617 on the proliferation and metastasis of esophageal cancer cells. Methods Real time fluorescence PCR was used to detect the expression of LINC01617 in 142 cases of esophageal cancer and adjacent tissues. The relationship between the expression level of LINC01617 and the survival rate of esophageal cancer patients was analyzed. The function of LINC01617 was detected in esophageal cancer cell lines. The tumor growth ability test was carried out in the nude mice. Results We found that LINC01617 was overexpressed in esophageal cancer, and its expression was associated with poor prognosis of esophageal cancer. In vitro experiments confirmed that knockout of LINC01617 significantly inhibited the proliferation, migration and invasion of esophageal cancer cells. Moreover, knockout of LNC01617 can inhibit the growth of esophageal cancer in nude mice. The Akt pathway may be involved in the regulation of cell activity in esophageal cancer. Conclusions LINC01617 may be involved in the occurrence and development of esophageal cancer, suggesting that LINC01617 can be used as a biomarker and potential therapeutic target for esophageal cancer. Key words
ACCEPTED MANUSCRIPT Esophageal cancer; LINC01617; AKT; Proliferation; Migration
Introduction
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Esophageal cancer is one of the most common malignant tumors in the world. It is listed as the sixth and third main cause of cancer death worldwide [1]. Although progress has been made in the diagnosis and treatment of esophageal cancer, the survival rate of 5 years after radical surgery is only 20 to 30%, mainly due to tumor metastasis, tumor recurrence and chemotherapeutic resistance [2]. Obviously, a better understanding of the complexity of esophageal cancer is essential for improving the treatment of this devastating disease. Studying the molecular mechanism of the occurrence and development of esophageal cancer helps to identify reliable diagnostic and prognostic markers for esophageal cancer. The human transcriptional group not only includes a large number of protein encoded messenger RNAs (mRNA), but also a large number of non protein encoded transcripts with structural, regulatory or unknown functions [3-8]. More and more evidence suggests that the expression of LncRNs may contribute to the diagnosis and prognosis of human cancer [5, 9-11]. Recently, researchers have discovered a new oncogene LncRNA molecule, LINC01617, which is highly expressed in blood samples of colon cancer patients relative to normal human blood samples [12]. However, the clinicopathological features of LINC01617 and its biological functions in esophageal cancer cells are not yet clear. This study is the first to study the clinical and experimental functions of LINC01617 in esophageal cancer. We found that the expression level of LINC01617 was associated with poor prognosis of esophageal cancer. Through in vivo and in vitro experiments, it is further confirmed that LINC01617 may regulate the proliferation and invasion of esophageal cancer cells by activating Akt signaling pathway. These findings can provide new clues for the role of LINC01617 in the development and progression of esophageal cancer.
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Material and Methods
Patients and tissue samples Tissue samples from 142 patients with esophageal cancer who underwent complete resection from March 2006 to January 2017 at the Affiliated Hospital of Jilin Medical University were studied. All patients did not receive preoperative radiotherapy or chemotherapy before operation. Demographic and clinicopathological data were collected from medical records and histopathological reports. The clinical stage was reassessed according to the seventh edition of the United States cancer TNM staging system joint committee. Follow up data were not until December 2017 or until death. The clinicopathological features of the patients with esophageal cancer are shown in Table 1. The project agreement was approved by the institutional ethics committee of
ACCEPTED MANUSCRIPT the Affiliated Hospital of Jilin Medical University. All patients were informed and agreed to use tumor tissue for clinical research. Cell lines and lentivirus transfection
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ECA109 and TE-1 cells (American Type Culture Collection, USA) were cultured in DMEM (Gibco, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, USA), 100 μ g/ml streptomycin (Gibco, USA), and 100 U/ml penicillin (Gibco, USA) at 37 °C with 5% CO2. Both cell lines were cytogenetically tested and authenticated before the cells were frozen. The lentivirus expressing shRNA specifically targeting LINC01617 (shLINC01617) or the scramble shRNA used as the negative control (shCtrl) was designed and constructed by Shanghai Genechem Company Ltd., China. Lentivirus particles were generated by co-transfecting recombined and packing vectors into 293T cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). ECA109 and TE-1 cells were then cultured in 6-well plates and transfected with shLINC01617 or shCtrl. The transfected cells were cultured for another 5 days. The knockdown efficiency of the target gene was further evaluated for both cell lines with quantitative real-time PCR (qRT-PCR).
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RNA extraction and quantitative RT-PCR.
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Total RNA was extracted from the corresponding tissues using a Total RNA isolation kit (Tiangen, Bejing, China) according to the manufacturer’s instructions. Reverse transcription was performed using the One Step Prime Script cDNA Synthesis Kit (Takara, Dalian, China) following the manufacturer’s instructions. Quantitative RT-PCR was performed using SYBR® Premix Ex Taq™ II (Takara, Dalian, China) according to the manufacturer’s instructions with the 7500 real-time RT-PCR system (Applied Biosystems, Foster City). The results was quantified by 2-△Ct. GAPDH were used as the normalization control. Each detection was performed in triplicate. Cell proliferation assays
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After the logarithmic growth period, ECA109 and TE-1 cells were digested by trypsin, and then suspended in the standard medium, then inoculated into 96 orifice plates with a density of 2000 cells/well. Cell proliferation was quantified by measurement of absorbance of water-soluble tetrazolium salt at 450 nm, using the Cell Counter Kit-8 (Dojindo, Kumamoto, Japan). Apoptosis assays Apoptosis was assessed using PI/annexin V-based flow cytometry using standard laboratory methodology. Briefly, cells were transfected as described above, and incubated for 5 days. The cells were then harvested, resuspended in binding buffer at a density of 1 × 106 cells/ml, and 100μl of this suspension was added to FACS tubes
ACCEPTED MANUSCRIPT and stained with PI and annexin V. Cells were mixed gently in a dark room for 15 min at room temperature, and then analyzed using flow cytometry. Caspase 3/7 activity assays
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Cell apoptosis were measured by Promega Caspase-Glo® 3/7Assay. Briefly, he cell suspension concentration was adjusted to 1 x 104 cells / holes at room temperature, and the non negative control cells of the target cells were added to the new 96 orifice with 100 micron l per pore, and a group of empty control groups containing cells were set up. 100 micron L Caspase-Glo reacting fluid is added to each hole. Cells were incubated for 18-22 hours at room temperature of 0.5-3 hours at room temperature, and then analyzed (Dojindo, Kumamoto, Japan).
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Colony formation assays
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After reaching the logarithmic growth phase ECA109 and TE-1 cells were trypsinized, counted, and seeded at a density of 800 cells/well into six-well plates containing regular culture medium. After 14 days the cells were washed twice with PBS, fixed with methanol, and stained with Giemsa. The colonies were then photographed and scored. Cell migration and invasion assay
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Migration and invasion assays were performed using transwell chambers. For migration assay, 5×104 cells were seeded into the upper chamber of transwells (BD Bioscience). For invasion assay, 1×105 cells were added into the upper chamber precoated with matrigel (BD Bioscience). In both assays, cells were maintained in medium without serum in the upper chamber, and medium containing 10% FBS was added to the lower chamber as chemoattractant. After 24 hours incubation, cells that did not migrate or invade through the membrane were wiped out. Then the membranes were fixed and stained with 0.5% crystal violet. Three random fields were counted per chamber using an inverted microscope (Olympus), and each experiment was repeated three times. Microarray gene expression analysis After infection with LINC01617-KD total RNA was extracted from TE-1 cells, and 50–500 ng of RNA was used to generate biotin-modified amplified RNA (aRNA) using a GeneChip 3’ IVT Express Kit (Affymetrix, USA). Reverse transcription was performed using a T7 oligo (dT) primer and a first-strand IVT Labelling Master Mix was used to produce multiple copies of biotin-modified aRNA. The aRNA was then purified and quantified. After fragmentation the aRNA was hybridized to the GeneChip Human Genome U133 plus 2.0 Array (Affymetrix, USA). After hybridization the chips were stained with phycoerythrin and washed in a Genechip Fluidics Station 450. The microarray signals were scanned and analysed using a
ACCEPTED MANUSCRIPT Genechip Array Scanner 3000 7G. Bioinformatics
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Differentially expressed genes (DEGs) between the LINC01617-KD and NC groups with corrected p-values of < 0.05 and an absolute fold change of > 1.5 were considered to be significantly differentially expressed. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes and BioCarta pathway databases were performed on significant DEGs.
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Western blotting
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Whole cells were directly lysed in 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton containing aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Proteins were separated using SDS–PAGE and then transferred to PVDF membranes at 4 °C (300 mA for 150 min). Membranes were blocked in 5% skim milk in TBST overnight at 4 °C, and then incubated with primary antibodies overnight at 4 °C. They were then incubated with secondary antibodies. After washing three times in TBST the membranes were incubated in Amersham™ ECL Plus Western Blotting Detection System (GE Healthcare, UK) before visualization. Statistical analysis
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Statistical analyses were performed using GraphPad Prism and GraphPad InStat software (GraphPad Software, La Jolla, CA, USA). Overall survival (OS) curves were plotted using the Kaplan-Meier method measured from the day of surgery, while the log-rank test was applied for comparison. According to LINC01617 expression level, cases were divided into two groups by the minimum p-value approach, which is a comprehensive method to find the optimal risk separation cut-off point in continuous gene expression measurements T-tests were used to determine significance, with a p-value of < 0.05 to indicate a significant difference.
Results
Overexpression of LINC01617 in esophageal carcinoma For the first time, we detected the expression of LINC01617 in 142 pairs of esophageal cancer tissues and their corresponding esophageal tissues by qRT-PCR. As shown in Figure 1A, the expression level of LINC01617 in esophageal cancer tissues is significantly higher than that in esophageal cancer tissues (P < 0.001, Fig. 1A). Table 1 summarizes the relationship between upregulation of LINC01617 and clinicopathological features of esophageal cancer. The results showed that the expression of LINC01617 in esophageal cancer tissues was closely related to lymph
ACCEPTED MANUSCRIPT node metastasis (P < 0.001) and TNM staging (P = 0.003). In addition, we found that patients with higher LINC01617 expression had poorer prognosis. Compared with patients with low LINC01617 expression, the overall survival rate of patients with LINC01617 expression was significantly reduced (P < 0.001, Fig. 1B). Knockdown of LINC01617 inhibits proliferation of esophageal cancer cells in vivo and in vitro
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In order to further study the biological role of LINC01617 in esophageal cancer, we used lentivirus to express shLINC01617, LINC01617 or shCtrl (Fig. 2A) in ECA109 and TE-1 cells. More than 70% knockdown efficiency was confirmed by qRT-PCR (Fig. 2B). The overexpression fold change were 26 and 19 in ECA109 and TE-1 cells (Fig. 2B). The proliferation rate of shLINC01617 treated cells was significantly lower than that of the control group and vise versa (P < 0.001) (Fig. 2C and 2D). In addition, compared with the control group, LINC01617 knockout cells produced significantly smaller xenografts in nude mice (Fig. 2E-2G).
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Knockdown of LINC01617 reduces cell clone formation and promotes cell apoptosis In order to understand the mechanism of LINC01617 on the proliferation of esophageal cancer cells, the ability of clone formation was analyzed. After silencing LINC01617 expression, we observed that the clones of ECA109 and TE-1 cells were significantly reduced and vise versa in LINC01617 overexpression group (Fig. 3A). In addition, Annexin V staining showed that the rate of apoptosis increased, and the apoptotic marker Caspase-3/7 activity was also detected after knockdown of LINC01617 (Fig. 3B and 3C).
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Knockdown of LINC01617 inhibits migration and invasion of esophageal cancer cells
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The results of Table 1 indicate that the over expression of LINC01617 is related to lymph node metastasis. Next we studied whether LINC01617 could regulate the migration of esophageal cancer cells. Compared with shCtrl group, there were fewer ECA109 and TE-1 cell migration cells in LINC01617 inhibition group (P < 0.001, Fig. 4A). In addition, Figure 4B showed that in ECA109 and TE-1 cells, the invasive cells in LINC01617 inhibition group were also less than those in shCtrl group (P < 0.001) and vise versa in LINC01617 overexpression group. Wound healing test can not simulate the nutritional gap in tumor microenvironment. Therefore, we did not carry out wound healing test to evaluate the effect of LINC01617 on esophageal cancer cells. LINC01617 may mediate the occurrence and development of esophageal cancer through Akt signaling pathway.
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To systematically explore the downstream pathways, the microarray data were analyzed by IPA “canonical pathway” module. The exported data showed that several critical pathways involved in oncogenic function such as “PI3K/AKT Signaling” and “JAK/Stat Signaling” were inhibited by LINC01617 knockdown (Figure 4C ). All gene list changed were in Supplemtary Table 2. The major changed genes (top 2 up-regulated and top 2 down-regulated) genes were confirmed by qPCR and the data were shown in Supplementary Figure 1. The gene association diagram was shown in Supplementary Figure 2. The Akt pathway is a signal transduction pathway that promotes the survival and growth of almost all types of cancers. The expression of member genes of Akt pathway was detected by Western blot. As shown in Fig. 4D, when LINC01617 was inhibited in ECA109 and TE-1, the expression of phosphorylated Akt decreased, and the expression of c-myc, CCND1 and FN1 decreased significantly. On the other hand, when LINC01617 was inhibited, the expression of p27 and p53 increased. Furthermore, AKT inhibitor “AZD5363” reduced cancer cell proliferation and migration and AKT activator “crosstide” rescued cell proliferation and migration inhibition induced by LINC01617 knockdown in ECA109 and TE-1 cells (Figure 5B-5E). These results indicated that LINC01617 may regulate esophageal cancer cells through Akt pathway. Discussion
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LncRNAs was previously considered to be "transcriptional noise" or background transcription [13, 14]. However, in recent years, more and more regulatory LncRNAs has contributed to the development and progression of cancer in many types of cancer, which can be used as a biomarker and prognostic factor [14, 15]. Previous studies have confirmed that LINC01617 is a LncRNA associated with colorectal cancer. A previous study showed that LINC01617 was highly expressed in blood samples of colorectal cancer patients compared with normal human blood samples. [12]. The purpose of this study was to investigate the clinical significance and role of LncRNA LINC01617 in esophageal cancer. Our results indicate that LINC01617 is significantly increased in esophageal cancer tissues compared with adjacent non neoplastic tissues and is associated with overall survival. Inhibition of LINC01617 results in apoptosis and cell proliferation inhibition of ECA109 and TE-1 cells. Compared with the control group, LINC01617 knockout cells produced smaller xenografts in nude mice. In addition, LINC01617 inhibition also reduced the migration and invasion of esophageal cancer cells and LINC01617 overexpression significantly promoted the cancer cell migration and invasion. It is suggested that LINC01617 plays a carcinogenic role in the occurrence and development of esophageal cancer. Previous studies have shown that the Akt pathway is involved in cell proliferation and apoptosis affecting the progression of esophageal cancer [16, 17]. Microarray analysis data indicated that PI3K/AKT sgnaling were enriched when LINC01617 were
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inhibited. In addition, as a downstream factor of AKT signaling pathway, P21, p53, FN1, c-myc and CCND1 have been reported to play an important role in the development and development of many different cancers [18-20]. Our findings suggest that Akt signal transduction may be an important reason for LINC01617 mediated proliferation of esophageal cancer cells. This study found that after LINC01617 knockdown, the migration and invasion of esophageal cancer cells were significantly inhibited. Interestingly, the expression of FN1 decreased in shLINC01617 transfected esophageal cancer cells. Through qPCR and WB, we further confirmed that FN1 was overexpressed in esophageal cancer patients, and its expression level was significantly correlated with the expression level of LINC01617. As a regulatory gene for cell migration and metastasis, FN1 expression was up-regulated in various types of cancer cells [21, 22]. It is reported that high FN1 expression indicates the activation of epithelial mesenchymal transition (EMT) in cancer tissues [22, 23]. More and more evidences show that EMT may play a key role in tumor metastasis [24, 25]. In addition, the AKT signal has recently been identified as a key regulatory factor induced by EMT [26]. The diagram of signal casdaed by LINC01617 was shown in Figure 6. It is speculated that LINC01617 may promote the migration and invasion of esophageal cancer cells by activating EMT, and its mechanism needs further study. The mechanism linking LINC01617 and Akt is worth further exploring. There are some limitations in our research. Why LINC01617 is frequently upregulated in esophageal cancer cells, and how LINC01617 regulates the Akt pathway remains elusive. In conclusion, this study first discussed the role of LINC01617 in the occurrence and development of esophageal cancer. It is suggested that LINC01617 may be a potential diagnostic marker for esophageal cancer. LINC01617 is involved in many areas of tumor progression, including cell proliferation, migration and invasion. Drug intervention or knockout of LINC01617 function may have potential therapeutic value in the prevention of esophageal cancer. Therefore, our results not only provide a molecular basis for the role of LINC01617 in esophageal cancer, but also provide new therapeutic targets for the treatment of esophageal cancer.
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Acknowledgements
This work was supported by Jilin Research Fund 201737145.
Figure Legend Figure 1: LINC01617 is overexpressed in esophageal cancer tissues. A. qPCR analysis of LINC01617 level in 142 pairs of esophageal cancer tissues and nontumorous tissues. B. Kaplan–Meier curves with univariate analyses (log-rank) for patients with esophageal cancer with low LINC01617 expressing (n=72) versus high LINC01617 expressing tumors (n=70). ***p<0.001.
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Figure 2: Knockdown of LINC01617 inhibits proliferation of esophageal cancer cells in vivo and in vitro. A. Microscopic images of esophageal cancer cells infected with lentivirus at MOI of 20. B. Knockdown efficiency and overexpression fold change of LINC01617 in two esophageal cancer cell lines. ***p<0.001. LINC01617 knockdown and overexpression significantly inhibited and promoted the growth tare of C. ECA-109 and D. TE-1, as shown by cell count for 5 days MTT assay, respectively. E. Images of tumors derived from ECA-109 cells in nude mice. F. The changes in volume of the tumors of different groups. G. Tumor weight at time of sacrifice. *p<0.05.
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Figure 3: Knockdown an overexpression of LINC01617 reduces cell clone formation and promotes cell apoptosis. A. Photomicrographs of Giemsa-stained colonies of ECA-109 and TE-1 cells growing in 6-well plates for 14 days after infection. The number of cells in each colony was counted. B. FCM showed that knockout of LINC01617 expression increased ECA-109 and TE-1 cell apoptosis compared with the control ***P < 0.001. C. Caspase 3/7 activity in ECA-109 and TE-1 cells after LINC01617 inhibition ***p<0.001.
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Figure 4: Knockdown of LINC01617 inhibits esophageal cancer cell migration and invasion in vitro. A. Cell migration and B. invasiveness were significantly reduced and promoted after silencing and overexpressing LINC01617 in ECA-109 and TE-1 cells respectively ***p < 0.001. C. Heatmap representation of genes significant differential expressions in JVM-2 cells infected with lentivirus expressing either NC-siRNA or LINC01617-siRNA under the criteria p<0.05 and | fold change | >1.5. Genes and samples were listed in rows and columns, respectively. A color scale for the normalized expression data was shown at the bottom of the microarray heatmap (green represents downregulated genes while red represents upregulated genes). LINC01617 related disease and function enrichment was analyzed based on IPA databases. D. Western blot analysis showed that knockdown of LINC01617 resulted in down-regulation of phosphorylated AKT, c-Myc, CCND1 and FN1 and up-regulation of p27 and p53 in ECA-109 and TE-1 cells. Figure 5. A. Western blot analysis showed that AZD5363 treatment resulted in down-regulation of p-AKT in ECA-109 and TE-1 cells. Crosstide rescued the AKT inactivation in ECA-109 and TE-1 cells. B. Clony formation analysis showed that AZD5363 reduced the proliferation of ECA-109 and TE-1 cells. C. Crosstide rescud the anti-proliferative effect of LINC01617 knockdown in ECA-109 and TE-1 cells (**p<0.01, ***p<0.001, ##p<0.01, t-test). D. Transwell analysis showed that AZD5363 reduced the migration of ECA-109 and TE-1 cells. E. Crosstide rescud the anti-migrative effect of LINC01617 knockdown in ECA-109 and TE-1 cells (*p<0.05, **p<0.01, ***p<0.001, ##p<0.01, t-test). Figure 6. Diagram of signal casdaed by LINC01617.
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Supplemantary Figure 1. The 4 major changed genes ABCA1, ALDOC, KDSR and MED21 were tested by qPCR to validate the microarray data. Supplementary Figure 2. LINC01617 promoted the AKT signal transduction to regulate cancer cell proliferation and migration. The gene association diagram was shown.
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Reference [1] X.-Y. Ming, L. Fu, L.-Y. Zhang, Y.-R. Qin, T.-T. Cao, K.W. Chan, S. Ma, D. Xie, X.-Y. Guan, Integrin α7 is a
RI
functional cancer stem cell surface marker in oesophageal squamous cell carcinoma, Nature Communications, 7 (2016) 13568.
SC
[2] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, International Journal of Cancer, 127 (2010) 2893-2917. [3] J.E. Wilusz, H. Sunwoo, D.L. Spector, Long noncoding RNAs: functional surprises from the RNA
NU
world, Genes & Development, 23 (2009) 1494-1504.
[4] L. Hua, C.-Y. Wang, K.-H. Yao, J.-T. Chen, J.-J. Zhang, W.-L. Ma, High expression of long non-coding RNA ANRIL is associated with poor prognosis in hepatocellular carcinoma, International Journal of
MA
Clinical and Experimental Pathology, 8 (2015) 3076-3082.
[5] L. Li, L. Zhang, Y. Zhang, F. Zhou, Increased expression of LncRNA BANCR is associated with clinical progression and poor prognosis in gastric cancer, Biomedicine & Pharmacotherapy, 72 (2015) 109-112. [6] G. Loewen, J. Jayawickramarajah, Y. Zhuo, B. Shan, Functions of lncRNA HOTAIR in lung cancer,
D
Journal of Hematology & Oncology, 7 (2014) 90.
[7] M. NAEMURA, C. MURASAKI, Y. INOUE, H. OKAMOTO, Y. KOTAKE, Long Noncoding RNA ANRIL 35 (2015) 5377-5382.
PT E
Regulates Proliferation of Non-small Cell Lung Cancer and Cervical Cancer Cells, Anticancer Research, [8] K. Zhang, H. Shi, H. Xi, X. Wu, J. Cui, Y. Gao, W. Liang, C. Hu, Y. Liu, J. Li, N. Wang, B. Wei, L. Chen, Genome-Wide lncRNA Microarray Profiling Identifies Novel Circulating lncRNAs for Detection of
CE
Gastric Cancer, Theranostics, 7 (2017) 213-227. [9] K. Qian, G. Liu, Z. Tang, Y. Hu, Y. Fang, Z. Chen, X. Xu, The long non-coding RNA NEAT1 interacted with miR-101 modulates breast cancer growth by targeting EZH2, Archives of Biochemistry and
AC
Biophysics, 615 (2017) 1-9.
[10] T. Alam, Y.A. Medvedeva, H. Jia, J.B. Brown, L. Lipovich, V.B. Bajic, Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes, PLoS ONE, 9 (2014) e109443.
[11] J.S. Mattick, Non‐coding RNAs: the architects of eukaryotic complexity, EMBO reports, 2 (2001) 986-991. [12] J. Shi, X. Li, F. Zhang, C. Zhang, Q. Guan, X. Cao, W. Zhu, X. Zhang, Y. Cheng, K. Ou, Q. Chen, S. Hu, Circulating lncRNAs associated with occurrence of colorectal cancer progression, American Journal of Cancer Research, 5 (2015) 2258-2265. [13] J. Ponjavic, C.P. Ponting, G. Lunter, Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs, Genome Research, 17 (2007) 556-565. [14] T. Derrien, R. Guigó, R. Johnson, The Long Non-Coding RNAs: A New (P)layer in the “Dark Matter”,
ACCEPTED MANUSCRIPT Frontiers in Genetics, 2 (2011) 107. [15] K. Struhl, Transcriptional noise and the fidelity of initiation by RNA polymerase II, Nat Struct Mol Biol, 14 (2007) 103-105. [16] G. Xu, Y. Guo, D. Xu, Y. Wang, Y. Shen, F. Wang, Y. Lv, F. Song, D. Jiang, Y. Zhang, Y. Lou, Y. Meng, Y. Yang, Y. Kang, TRIM14 regulates cell proliferation and invasion in osteosarcoma via promotion of the AKT signaling pathway, Scientific reports, 7 (2017) 42411. [17] G. Wang, T. Zhang, W. Sun, H. Wang, F. Yin, Z. Wang, D. Zuo, M. Sun, Z. Zhou, B. Lin, J. Xu, Y. Hua, H. Li, Z. Cai, Arsenic sulfide induces apoptosis and autophagy through the activation of ROS/JNK and suppression of Akt/mTOR signaling pathways in osteosarcoma, Free radical biology & medicine, 106
PT
(2017) 24-37.
[18] J. Dai, R.J. Wei, R. Li, J.B. Feng, Y.L. Yu, P.S. Liu, A study of CCND1 with epithelial ovarian cancer cell
RI
proliferation and apoptosis, European review for medical and pharmacological sciences, 20 (2016) 4230-4235.
SC
[19] J. Long, C. Ou, H. Xia, Y. Zhu, D. Liu, MiR-503 inhibited cell proliferation of human breast cancer cells by suppressing CCND1 expression, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 36 (2015) 8697-8702.
NU
[20] H. Gu, T. Yang, S. Fu, X. Chen, L. Guo, Y. Ni, MicroRNA-490-3p inhibits proliferation of A549 lung cancer cells by targeting CCND1, Biochemical and biophysical research communications, 444 (2014) 104-108.
MA
[21] J. Wu, Y. Wang, X. Xu, H. Cao, S. Sahengbieke, H. Sheng, Q. Huang, M. Lai, Transcriptional activation of FN1 and IL11 by HMGA2 promotes the malignant behavior of colorectal cancer, Carcinogenesis, 37 (2016) 511-521.
[22] S. Viana Lde, R.J. Affonso, Jr., S.R. Silva, M.V. Denadai, D. Matos, C. Salinas de Souza, J. Waisberg,
D
Relationship between the expression of the extracellular matrix genes SPARC, SPP1, FN1, ITGA5 and ITGAV and clinicopathological parameters of tumor progression and colorectal cancer dissemination,
PT E
Oncology, 84 (2013) 81-91.
[23] H. Zhang, Z. Sun, Y. Li, D. Fan, H. Jiang, MicroRNA-200c binding to FN1 suppresses the proliferation, migration and invasion of gastric cancer cells, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 88 (2017) 285-292. 2387-2388.
CE
[24] S.C. Wei, L. Fattet, J. Yang, The forces behind EMT and tumor metastasis, Cell cycle, 14 (2015) [25] S. Heerboth, G. Housman, M. Leary, M. Longacre, S. Byler, K. Lapinska, A. Willbanks, S. Sarkar,
AC
EMT and tumor metastasis, Clinical and translational medicine, 4 (2015) 6. [26] S.J. Grille, A. Bellacosa, J. Upson, A.J. Klein-Szanto, F. van Roy, W. Lee-Kwon, M. Donowitz, P.N. Tsichlis, L. Larue, The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines, Cancer research, 63 (2003) 2172-2178.
ACCEPTED MANUSCRIPT Table 1. Relationship between the expression level of lncRNA LINC01617 and clinical characteristics of esophageal cancer. Clinical Characteristics
Lymphnode metastasis
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TNM stage
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Gender
>=60 <60 Male Female Yes No I~II III~IV
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LINC01617 High Low expression expression 43 31 37 41 58 45 22 28 58 31 14 49 20 42 52 38
P value
0.198 0.17 <0.001 0.003
ACCEPTED MANUSCRIPT Highlights
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To our knowledge, this study reported for the first time that: 1. LINC01617 is frequently up-regulated in esophageal cancer tissues. The over-expression of LINC01617 is associated with poor prognosis. 2. LINC01617 is a pro-proliferatiive lnRNA for esophageal cancer 3. LINC01617 regulates esophageal cancer cell proliferation and migration through AKT regulation.
ACCEPTED MANUSCRIPT Abbreviations
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qRT-PCR: quantitative real-time PCR DMSO: dimethyl sulfoxide FCM: Flow cytometry PBS: phosphate-buffered saline
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Dan Zhang, Wei Yang, Shuwei Wang, Fei Wang, Dayong Liu, Jingjing Dong, Nan Zhao, Yunye Wang, Haiyan Zhang PII: DOI: Reference:
S0378-1119(18)30916-8 doi:10.1016/j.gene.2018.08.054 GENE 43173
To appear in:
Gene
Received date: Revised date: Accepted date:
24 June 2018 12 August 2018 14 August 2018
Please cite this article as: Dan Zhang, Wei Yang, Shuwei Wang, Fei Wang, Dayong Liu, Jingjing Dong, Nan Zhao, Yunye Wang, Haiyan Zhang , Long non-coding RNA LINC01617 promotes proliferation and metastasis of esophageal cancer cells through AKT pathway. Gene (2018), doi:10.1016/j.gene.2018.08.054
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ACCEPTED MANUSCRIPT Long Non-Coding RNA LINC01617 Promotes Proliferation and Metastasis of Esophageal Cancer Cells Through AKT pathway
Dan Zhang#, Wei Yang#, Shuwei Wang, Fei Wang, Dayong Liu, Jingjing Dong, Nan Zhao, Yunye Wang* and Haiyan Zhang*
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Department of Surgery, Affiliated Hospital of Jilin Medical University, Jilin, China
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#: These two authors contributed the same in this paper
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*: Correspondence to: Yunye Wang, Department of Surgery, Affiliated Hospital of Jilin Medical University, E-mail: [email protected] Haiyan Zhang, Department of Surgery, Affiliated Hospital of Jilin Medical University, E-mail: [email protected]
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Abstract
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Objective To investigate the clinical significance of long non-coding RNA LINC01617 in esophageal cancer and explore the effect of LINC01617 on the proliferation and metastasis of esophageal cancer cells. Methods Real time fluorescence PCR was used to detect the expression of LINC01617 in 142 cases of esophageal cancer and adjacent tissues. The relationship between the expression level of LINC01617 and the survival rate of esophageal cancer patients was analyzed. The function of LINC01617 was detected in esophageal cancer cell lines. The tumor growth ability test was carried out in the nude mice. Results We found that LINC01617 was overexpressed in esophageal cancer, and its expression was associated with poor prognosis of esophageal cancer. In vitro experiments confirmed that knockout of LINC01617 significantly inhibited the proliferation, migration and invasion of esophageal cancer cells. Moreover, knockout of LNC01617 can inhibit the growth of esophageal cancer in nude mice. The Akt pathway may be involved in the regulation of cell activity in esophageal cancer. Conclusions LINC01617 may be involved in the occurrence and development of esophageal cancer, suggesting that LINC01617 can be used as a biomarker and potential therapeutic target for esophageal cancer. Key words
ACCEPTED MANUSCRIPT Esophageal cancer; LINC01617; AKT; Proliferation; Migration
Introduction
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Esophageal cancer is one of the most common malignant tumors in the world. It is listed as the sixth and third main cause of cancer death worldwide [1]. Although progress has been made in the diagnosis and treatment of esophageal cancer, the survival rate of 5 years after radical surgery is only 20 to 30%, mainly due to tumor metastasis, tumor recurrence and chemotherapeutic resistance [2]. Obviously, a better understanding of the complexity of esophageal cancer is essential for improving the treatment of this devastating disease. Studying the molecular mechanism of the occurrence and development of esophageal cancer helps to identify reliable diagnostic and prognostic markers for esophageal cancer. The human transcriptional group not only includes a large number of protein encoded messenger RNAs (mRNA), but also a large number of non protein encoded transcripts with structural, regulatory or unknown functions [3-8]. More and more evidence suggests that the expression of LncRNs may contribute to the diagnosis and prognosis of human cancer [5, 9-11]. Recently, researchers have discovered a new oncogene LncRNA molecule, LINC01617, which is highly expressed in blood samples of colon cancer patients relative to normal human blood samples [12]. However, the clinicopathological features of LINC01617 and its biological functions in esophageal cancer cells are not yet clear. This study is the first to study the clinical and experimental functions of LINC01617 in esophageal cancer. We found that the expression level of LINC01617 was associated with poor prognosis of esophageal cancer. Through in vivo and in vitro experiments, it is further confirmed that LINC01617 may regulate the proliferation and invasion of esophageal cancer cells by activating Akt signaling pathway. These findings can provide new clues for the role of LINC01617 in the development and progression of esophageal cancer.
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Material and Methods
Patients and tissue samples Tissue samples from 142 patients with esophageal cancer who underwent complete resection from March 2006 to January 2017 at the Affiliated Hospital of Jilin Medical University were studied. All patients did not receive preoperative radiotherapy or chemotherapy before operation. Demographic and clinicopathological data were collected from medical records and histopathological reports. The clinical stage was reassessed according to the seventh edition of the United States cancer TNM staging system joint committee. Follow up data were not until December 2017 or until death. The clinicopathological features of the patients with esophageal cancer are shown in Table 1. The project agreement was approved by the institutional ethics committee of
ACCEPTED MANUSCRIPT the Affiliated Hospital of Jilin Medical University. All patients were informed and agreed to use tumor tissue for clinical research. Cell lines and lentivirus transfection
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ECA109 and TE-1 cells (American Type Culture Collection, USA) were cultured in DMEM (Gibco, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, USA), 100 μ g/ml streptomycin (Gibco, USA), and 100 U/ml penicillin (Gibco, USA) at 37 °C with 5% CO2. Both cell lines were cytogenetically tested and authenticated before the cells were frozen. The lentivirus expressing shRNA specifically targeting LINC01617 (shLINC01617) or the scramble shRNA used as the negative control (shCtrl) was designed and constructed by Shanghai Genechem Company Ltd., China. Lentivirus particles were generated by co-transfecting recombined and packing vectors into 293T cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). ECA109 and TE-1 cells were then cultured in 6-well plates and transfected with shLINC01617 or shCtrl. The transfected cells were cultured for another 5 days. The knockdown efficiency of the target gene was further evaluated for both cell lines with quantitative real-time PCR (qRT-PCR).
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RNA extraction and quantitative RT-PCR.
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Total RNA was extracted from the corresponding tissues using a Total RNA isolation kit (Tiangen, Bejing, China) according to the manufacturer’s instructions. Reverse transcription was performed using the One Step Prime Script cDNA Synthesis Kit (Takara, Dalian, China) following the manufacturer’s instructions. Quantitative RT-PCR was performed using SYBR® Premix Ex Taq™ II (Takara, Dalian, China) according to the manufacturer’s instructions with the 7500 real-time RT-PCR system (Applied Biosystems, Foster City). The results was quantified by 2-△Ct. GAPDH were used as the normalization control. Each detection was performed in triplicate. Cell proliferation assays
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After the logarithmic growth period, ECA109 and TE-1 cells were digested by trypsin, and then suspended in the standard medium, then inoculated into 96 orifice plates with a density of 2000 cells/well. Cell proliferation was quantified by measurement of absorbance of water-soluble tetrazolium salt at 450 nm, using the Cell Counter Kit-8 (Dojindo, Kumamoto, Japan). Apoptosis assays Apoptosis was assessed using PI/annexin V-based flow cytometry using standard laboratory methodology. Briefly, cells were transfected as described above, and incubated for 5 days. The cells were then harvested, resuspended in binding buffer at a density of 1 × 106 cells/ml, and 100μl of this suspension was added to FACS tubes
ACCEPTED MANUSCRIPT and stained with PI and annexin V. Cells were mixed gently in a dark room for 15 min at room temperature, and then analyzed using flow cytometry. Caspase 3/7 activity assays
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Cell apoptosis were measured by Promega Caspase-Glo® 3/7Assay. Briefly, he cell suspension concentration was adjusted to 1 x 104 cells / holes at room temperature, and the non negative control cells of the target cells were added to the new 96 orifice with 100 micron l per pore, and a group of empty control groups containing cells were set up. 100 micron L Caspase-Glo reacting fluid is added to each hole. Cells were incubated for 18-22 hours at room temperature of 0.5-3 hours at room temperature, and then analyzed (Dojindo, Kumamoto, Japan).
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Colony formation assays
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After reaching the logarithmic growth phase ECA109 and TE-1 cells were trypsinized, counted, and seeded at a density of 800 cells/well into six-well plates containing regular culture medium. After 14 days the cells were washed twice with PBS, fixed with methanol, and stained with Giemsa. The colonies were then photographed and scored. Cell migration and invasion assay
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Migration and invasion assays were performed using transwell chambers. For migration assay, 5×104 cells were seeded into the upper chamber of transwells (BD Bioscience). For invasion assay, 1×105 cells were added into the upper chamber precoated with matrigel (BD Bioscience). In both assays, cells were maintained in medium without serum in the upper chamber, and medium containing 10% FBS was added to the lower chamber as chemoattractant. After 24 hours incubation, cells that did not migrate or invade through the membrane were wiped out. Then the membranes were fixed and stained with 0.5% crystal violet. Three random fields were counted per chamber using an inverted microscope (Olympus), and each experiment was repeated three times. Microarray gene expression analysis After infection with LINC01617-KD total RNA was extracted from TE-1 cells, and 50–500 ng of RNA was used to generate biotin-modified amplified RNA (aRNA) using a GeneChip 3’ IVT Express Kit (Affymetrix, USA). Reverse transcription was performed using a T7 oligo (dT) primer and a first-strand IVT Labelling Master Mix was used to produce multiple copies of biotin-modified aRNA. The aRNA was then purified and quantified. After fragmentation the aRNA was hybridized to the GeneChip Human Genome U133 plus 2.0 Array (Affymetrix, USA). After hybridization the chips were stained with phycoerythrin and washed in a Genechip Fluidics Station 450. The microarray signals were scanned and analysed using a
ACCEPTED MANUSCRIPT Genechip Array Scanner 3000 7G. Bioinformatics
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Differentially expressed genes (DEGs) between the LINC01617-KD and NC groups with corrected p-values of < 0.05 and an absolute fold change of > 1.5 were considered to be significantly differentially expressed. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes and BioCarta pathway databases were performed on significant DEGs.
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Western blotting
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Whole cells were directly lysed in 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton containing aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Proteins were separated using SDS–PAGE and then transferred to PVDF membranes at 4 °C (300 mA for 150 min). Membranes were blocked in 5% skim milk in TBST overnight at 4 °C, and then incubated with primary antibodies overnight at 4 °C. They were then incubated with secondary antibodies. After washing three times in TBST the membranes were incubated in Amersham™ ECL Plus Western Blotting Detection System (GE Healthcare, UK) before visualization. Statistical analysis
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Statistical analyses were performed using GraphPad Prism and GraphPad InStat software (GraphPad Software, La Jolla, CA, USA). Overall survival (OS) curves were plotted using the Kaplan-Meier method measured from the day of surgery, while the log-rank test was applied for comparison. According to LINC01617 expression level, cases were divided into two groups by the minimum p-value approach, which is a comprehensive method to find the optimal risk separation cut-off point in continuous gene expression measurements T-tests were used to determine significance, with a p-value of < 0.05 to indicate a significant difference.
Results
Overexpression of LINC01617 in esophageal carcinoma For the first time, we detected the expression of LINC01617 in 142 pairs of esophageal cancer tissues and their corresponding esophageal tissues by qRT-PCR. As shown in Figure 1A, the expression level of LINC01617 in esophageal cancer tissues is significantly higher than that in esophageal cancer tissues (P < 0.001, Fig. 1A). Table 1 summarizes the relationship between upregulation of LINC01617 and clinicopathological features of esophageal cancer. The results showed that the expression of LINC01617 in esophageal cancer tissues was closely related to lymph
ACCEPTED MANUSCRIPT node metastasis (P < 0.001) and TNM staging (P = 0.003). In addition, we found that patients with higher LINC01617 expression had poorer prognosis. Compared with patients with low LINC01617 expression, the overall survival rate of patients with LINC01617 expression was significantly reduced (P < 0.001, Fig. 1B). Knockdown of LINC01617 inhibits proliferation of esophageal cancer cells in vivo and in vitro
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In order to further study the biological role of LINC01617 in esophageal cancer, we used lentivirus to express shLINC01617, LINC01617 or shCtrl (Fig. 2A) in ECA109 and TE-1 cells. More than 70% knockdown efficiency was confirmed by qRT-PCR (Fig. 2B). The overexpression fold change were 26 and 19 in ECA109 and TE-1 cells (Fig. 2B). The proliferation rate of shLINC01617 treated cells was significantly lower than that of the control group and vise versa (P < 0.001) (Fig. 2C and 2D). In addition, compared with the control group, LINC01617 knockout cells produced significantly smaller xenografts in nude mice (Fig. 2E-2G).
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Knockdown of LINC01617 reduces cell clone formation and promotes cell apoptosis In order to understand the mechanism of LINC01617 on the proliferation of esophageal cancer cells, the ability of clone formation was analyzed. After silencing LINC01617 expression, we observed that the clones of ECA109 and TE-1 cells were significantly reduced and vise versa in LINC01617 overexpression group (Fig. 3A). In addition, Annexin V staining showed that the rate of apoptosis increased, and the apoptotic marker Caspase-3/7 activity was also detected after knockdown of LINC01617 (Fig. 3B and 3C).
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Knockdown of LINC01617 inhibits migration and invasion of esophageal cancer cells
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The results of Table 1 indicate that the over expression of LINC01617 is related to lymph node metastasis. Next we studied whether LINC01617 could regulate the migration of esophageal cancer cells. Compared with shCtrl group, there were fewer ECA109 and TE-1 cell migration cells in LINC01617 inhibition group (P < 0.001, Fig. 4A). In addition, Figure 4B showed that in ECA109 and TE-1 cells, the invasive cells in LINC01617 inhibition group were also less than those in shCtrl group (P < 0.001) and vise versa in LINC01617 overexpression group. Wound healing test can not simulate the nutritional gap in tumor microenvironment. Therefore, we did not carry out wound healing test to evaluate the effect of LINC01617 on esophageal cancer cells. LINC01617 may mediate the occurrence and development of esophageal cancer through Akt signaling pathway.
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To systematically explore the downstream pathways, the microarray data were analyzed by IPA “canonical pathway” module. The exported data showed that several critical pathways involved in oncogenic function such as “PI3K/AKT Signaling” and “JAK/Stat Signaling” were inhibited by LINC01617 knockdown (Figure 4C ). All gene list changed were in Supplemtary Table 2. The major changed genes (top 2 up-regulated and top 2 down-regulated) genes were confirmed by qPCR and the data were shown in Supplementary Figure 1. The gene association diagram was shown in Supplementary Figure 2. The Akt pathway is a signal transduction pathway that promotes the survival and growth of almost all types of cancers. The expression of member genes of Akt pathway was detected by Western blot. As shown in Fig. 4D, when LINC01617 was inhibited in ECA109 and TE-1, the expression of phosphorylated Akt decreased, and the expression of c-myc, CCND1 and FN1 decreased significantly. On the other hand, when LINC01617 was inhibited, the expression of p27 and p53 increased. Furthermore, AKT inhibitor “AZD5363” reduced cancer cell proliferation and migration and AKT activator “crosstide” rescued cell proliferation and migration inhibition induced by LINC01617 knockdown in ECA109 and TE-1 cells (Figure 5B-5E). These results indicated that LINC01617 may regulate esophageal cancer cells through Akt pathway. Discussion
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LncRNAs was previously considered to be "transcriptional noise" or background transcription [13, 14]. However, in recent years, more and more regulatory LncRNAs has contributed to the development and progression of cancer in many types of cancer, which can be used as a biomarker and prognostic factor [14, 15]. Previous studies have confirmed that LINC01617 is a LncRNA associated with colorectal cancer. A previous study showed that LINC01617 was highly expressed in blood samples of colorectal cancer patients compared with normal human blood samples. [12]. The purpose of this study was to investigate the clinical significance and role of LncRNA LINC01617 in esophageal cancer. Our results indicate that LINC01617 is significantly increased in esophageal cancer tissues compared with adjacent non neoplastic tissues and is associated with overall survival. Inhibition of LINC01617 results in apoptosis and cell proliferation inhibition of ECA109 and TE-1 cells. Compared with the control group, LINC01617 knockout cells produced smaller xenografts in nude mice. In addition, LINC01617 inhibition also reduced the migration and invasion of esophageal cancer cells and LINC01617 overexpression significantly promoted the cancer cell migration and invasion. It is suggested that LINC01617 plays a carcinogenic role in the occurrence and development of esophageal cancer. Previous studies have shown that the Akt pathway is involved in cell proliferation and apoptosis affecting the progression of esophageal cancer [16, 17]. Microarray analysis data indicated that PI3K/AKT sgnaling were enriched when LINC01617 were
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inhibited. In addition, as a downstream factor of AKT signaling pathway, P21, p53, FN1, c-myc and CCND1 have been reported to play an important role in the development and development of many different cancers [18-20]. Our findings suggest that Akt signal transduction may be an important reason for LINC01617 mediated proliferation of esophageal cancer cells. This study found that after LINC01617 knockdown, the migration and invasion of esophageal cancer cells were significantly inhibited. Interestingly, the expression of FN1 decreased in shLINC01617 transfected esophageal cancer cells. Through qPCR and WB, we further confirmed that FN1 was overexpressed in esophageal cancer patients, and its expression level was significantly correlated with the expression level of LINC01617. As a regulatory gene for cell migration and metastasis, FN1 expression was up-regulated in various types of cancer cells [21, 22]. It is reported that high FN1 expression indicates the activation of epithelial mesenchymal transition (EMT) in cancer tissues [22, 23]. More and more evidences show that EMT may play a key role in tumor metastasis [24, 25]. In addition, the AKT signal has recently been identified as a key regulatory factor induced by EMT [26]. The diagram of signal casdaed by LINC01617 was shown in Figure 6. It is speculated that LINC01617 may promote the migration and invasion of esophageal cancer cells by activating EMT, and its mechanism needs further study. The mechanism linking LINC01617 and Akt is worth further exploring. There are some limitations in our research. Why LINC01617 is frequently upregulated in esophageal cancer cells, and how LINC01617 regulates the Akt pathway remains elusive. In conclusion, this study first discussed the role of LINC01617 in the occurrence and development of esophageal cancer. It is suggested that LINC01617 may be a potential diagnostic marker for esophageal cancer. LINC01617 is involved in many areas of tumor progression, including cell proliferation, migration and invasion. Drug intervention or knockout of LINC01617 function may have potential therapeutic value in the prevention of esophageal cancer. Therefore, our results not only provide a molecular basis for the role of LINC01617 in esophageal cancer, but also provide new therapeutic targets for the treatment of esophageal cancer.
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Acknowledgements
This work was supported by Jilin Research Fund 201737145.
Figure Legend Figure 1: LINC01617 is overexpressed in esophageal cancer tissues. A. qPCR analysis of LINC01617 level in 142 pairs of esophageal cancer tissues and nontumorous tissues. B. Kaplan–Meier curves with univariate analyses (log-rank) for patients with esophageal cancer with low LINC01617 expressing (n=72) versus high LINC01617 expressing tumors (n=70). ***p<0.001.
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Figure 2: Knockdown of LINC01617 inhibits proliferation of esophageal cancer cells in vivo and in vitro. A. Microscopic images of esophageal cancer cells infected with lentivirus at MOI of 20. B. Knockdown efficiency and overexpression fold change of LINC01617 in two esophageal cancer cell lines. ***p<0.001. LINC01617 knockdown and overexpression significantly inhibited and promoted the growth tare of C. ECA-109 and D. TE-1, as shown by cell count for 5 days MTT assay, respectively. E. Images of tumors derived from ECA-109 cells in nude mice. F. The changes in volume of the tumors of different groups. G. Tumor weight at time of sacrifice. *p<0.05.
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Figure 3: Knockdown an overexpression of LINC01617 reduces cell clone formation and promotes cell apoptosis. A. Photomicrographs of Giemsa-stained colonies of ECA-109 and TE-1 cells growing in 6-well plates for 14 days after infection. The number of cells in each colony was counted. B. FCM showed that knockout of LINC01617 expression increased ECA-109 and TE-1 cell apoptosis compared with the control ***P < 0.001. C. Caspase 3/7 activity in ECA-109 and TE-1 cells after LINC01617 inhibition ***p<0.001.
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Figure 4: Knockdown of LINC01617 inhibits esophageal cancer cell migration and invasion in vitro. A. Cell migration and B. invasiveness were significantly reduced and promoted after silencing and overexpressing LINC01617 in ECA-109 and TE-1 cells respectively ***p < 0.001. C. Heatmap representation of genes significant differential expressions in JVM-2 cells infected with lentivirus expressing either NC-siRNA or LINC01617-siRNA under the criteria p<0.05 and | fold change | >1.5. Genes and samples were listed in rows and columns, respectively. A color scale for the normalized expression data was shown at the bottom of the microarray heatmap (green represents downregulated genes while red represents upregulated genes). LINC01617 related disease and function enrichment was analyzed based on IPA databases. D. Western blot analysis showed that knockdown of LINC01617 resulted in down-regulation of phosphorylated AKT, c-Myc, CCND1 and FN1 and up-regulation of p27 and p53 in ECA-109 and TE-1 cells. Figure 5. A. Western blot analysis showed that AZD5363 treatment resulted in down-regulation of p-AKT in ECA-109 and TE-1 cells. Crosstide rescued the AKT inactivation in ECA-109 and TE-1 cells. B. Clony formation analysis showed that AZD5363 reduced the proliferation of ECA-109 and TE-1 cells. C. Crosstide rescud the anti-proliferative effect of LINC01617 knockdown in ECA-109 and TE-1 cells (**p<0.01, ***p<0.001, ##p<0.01, t-test). D. Transwell analysis showed that AZD5363 reduced the migration of ECA-109 and TE-1 cells. E. Crosstide rescud the anti-migrative effect of LINC01617 knockdown in ECA-109 and TE-1 cells (*p<0.05, **p<0.01, ***p<0.001, ##p<0.01, t-test). Figure 6. Diagram of signal casdaed by LINC01617.
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Supplemantary Figure 1. The 4 major changed genes ABCA1, ALDOC, KDSR and MED21 were tested by qPCR to validate the microarray data. Supplementary Figure 2. LINC01617 promoted the AKT signal transduction to regulate cancer cell proliferation and migration. The gene association diagram was shown.
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Reference [1] X.-Y. Ming, L. Fu, L.-Y. Zhang, Y.-R. Qin, T.-T. Cao, K.W. Chan, S. Ma, D. Xie, X.-Y. Guan, Integrin α7 is a
RI
functional cancer stem cell surface marker in oesophageal squamous cell carcinoma, Nature Communications, 7 (2016) 13568.
SC
[2] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, International Journal of Cancer, 127 (2010) 2893-2917. [3] J.E. Wilusz, H. Sunwoo, D.L. Spector, Long noncoding RNAs: functional surprises from the RNA
NU
world, Genes & Development, 23 (2009) 1494-1504.
[4] L. Hua, C.-Y. Wang, K.-H. Yao, J.-T. Chen, J.-J. Zhang, W.-L. Ma, High expression of long non-coding RNA ANRIL is associated with poor prognosis in hepatocellular carcinoma, International Journal of
MA
Clinical and Experimental Pathology, 8 (2015) 3076-3082.
[5] L. Li, L. Zhang, Y. Zhang, F. Zhou, Increased expression of LncRNA BANCR is associated with clinical progression and poor prognosis in gastric cancer, Biomedicine & Pharmacotherapy, 72 (2015) 109-112. [6] G. Loewen, J. Jayawickramarajah, Y. Zhuo, B. Shan, Functions of lncRNA HOTAIR in lung cancer,
D
Journal of Hematology & Oncology, 7 (2014) 90.
[7] M. NAEMURA, C. MURASAKI, Y. INOUE, H. OKAMOTO, Y. KOTAKE, Long Noncoding RNA ANRIL 35 (2015) 5377-5382.
PT E
Regulates Proliferation of Non-small Cell Lung Cancer and Cervical Cancer Cells, Anticancer Research, [8] K. Zhang, H. Shi, H. Xi, X. Wu, J. Cui, Y. Gao, W. Liang, C. Hu, Y. Liu, J. Li, N. Wang, B. Wei, L. Chen, Genome-Wide lncRNA Microarray Profiling Identifies Novel Circulating lncRNAs for Detection of
CE
Gastric Cancer, Theranostics, 7 (2017) 213-227. [9] K. Qian, G. Liu, Z. Tang, Y. Hu, Y. Fang, Z. Chen, X. Xu, The long non-coding RNA NEAT1 interacted with miR-101 modulates breast cancer growth by targeting EZH2, Archives of Biochemistry and
AC
Biophysics, 615 (2017) 1-9.
[10] T. Alam, Y.A. Medvedeva, H. Jia, J.B. Brown, L. Lipovich, V.B. Bajic, Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes, PLoS ONE, 9 (2014) e109443.
[11] J.S. Mattick, Non‐coding RNAs: the architects of eukaryotic complexity, EMBO reports, 2 (2001) 986-991. [12] J. Shi, X. Li, F. Zhang, C. Zhang, Q. Guan, X. Cao, W. Zhu, X. Zhang, Y. Cheng, K. Ou, Q. Chen, S. Hu, Circulating lncRNAs associated with occurrence of colorectal cancer progression, American Journal of Cancer Research, 5 (2015) 2258-2265. [13] J. Ponjavic, C.P. Ponting, G. Lunter, Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs, Genome Research, 17 (2007) 556-565. [14] T. Derrien, R. Guigó, R. Johnson, The Long Non-Coding RNAs: A New (P)layer in the “Dark Matter”,
ACCEPTED MANUSCRIPT Frontiers in Genetics, 2 (2011) 107. [15] K. Struhl, Transcriptional noise and the fidelity of initiation by RNA polymerase II, Nat Struct Mol Biol, 14 (2007) 103-105. [16] G. Xu, Y. Guo, D. Xu, Y. Wang, Y. Shen, F. Wang, Y. Lv, F. Song, D. Jiang, Y. Zhang, Y. Lou, Y. Meng, Y. Yang, Y. Kang, TRIM14 regulates cell proliferation and invasion in osteosarcoma via promotion of the AKT signaling pathway, Scientific reports, 7 (2017) 42411. [17] G. Wang, T. Zhang, W. Sun, H. Wang, F. Yin, Z. Wang, D. Zuo, M. Sun, Z. Zhou, B. Lin, J. Xu, Y. Hua, H. Li, Z. Cai, Arsenic sulfide induces apoptosis and autophagy through the activation of ROS/JNK and suppression of Akt/mTOR signaling pathways in osteosarcoma, Free radical biology & medicine, 106
PT
(2017) 24-37.
[18] J. Dai, R.J. Wei, R. Li, J.B. Feng, Y.L. Yu, P.S. Liu, A study of CCND1 with epithelial ovarian cancer cell
RI
proliferation and apoptosis, European review for medical and pharmacological sciences, 20 (2016) 4230-4235.
SC
[19] J. Long, C. Ou, H. Xia, Y. Zhu, D. Liu, MiR-503 inhibited cell proliferation of human breast cancer cells by suppressing CCND1 expression, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 36 (2015) 8697-8702.
NU
[20] H. Gu, T. Yang, S. Fu, X. Chen, L. Guo, Y. Ni, MicroRNA-490-3p inhibits proliferation of A549 lung cancer cells by targeting CCND1, Biochemical and biophysical research communications, 444 (2014) 104-108.
MA
[21] J. Wu, Y. Wang, X. Xu, H. Cao, S. Sahengbieke, H. Sheng, Q. Huang, M. Lai, Transcriptional activation of FN1 and IL11 by HMGA2 promotes the malignant behavior of colorectal cancer, Carcinogenesis, 37 (2016) 511-521.
[22] S. Viana Lde, R.J. Affonso, Jr., S.R. Silva, M.V. Denadai, D. Matos, C. Salinas de Souza, J. Waisberg,
D
Relationship between the expression of the extracellular matrix genes SPARC, SPP1, FN1, ITGA5 and ITGAV and clinicopathological parameters of tumor progression and colorectal cancer dissemination,
PT E
Oncology, 84 (2013) 81-91.
[23] H. Zhang, Z. Sun, Y. Li, D. Fan, H. Jiang, MicroRNA-200c binding to FN1 suppresses the proliferation, migration and invasion of gastric cancer cells, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 88 (2017) 285-292. 2387-2388.
CE
[24] S.C. Wei, L. Fattet, J. Yang, The forces behind EMT and tumor metastasis, Cell cycle, 14 (2015) [25] S. Heerboth, G. Housman, M. Leary, M. Longacre, S. Byler, K. Lapinska, A. Willbanks, S. Sarkar,
AC
EMT and tumor metastasis, Clinical and translational medicine, 4 (2015) 6. [26] S.J. Grille, A. Bellacosa, J. Upson, A.J. Klein-Szanto, F. van Roy, W. Lee-Kwon, M. Donowitz, P.N. Tsichlis, L. Larue, The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines, Cancer research, 63 (2003) 2172-2178.
ACCEPTED MANUSCRIPT Table 1. Relationship between the expression level of lncRNA LINC01617 and clinical characteristics of esophageal cancer. Clinical Characteristics
Lymphnode metastasis
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TNM stage
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Gender
>=60 <60 Male Female Yes No I~II III~IV
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LINC01617 High Low expression expression 43 31 37 41 58 45 22 28 58 31 14 49 20 42 52 38
P value
0.198 0.17 <0.001 0.003
ACCEPTED MANUSCRIPT Highlights
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To our knowledge, this study reported for the first time that: 1. LINC01617 is frequently up-regulated in esophageal cancer tissues. The over-expression of LINC01617 is associated with poor prognosis. 2. LINC01617 is a pro-proliferatiive lnRNA for esophageal cancer 3. LINC01617 regulates esophageal cancer cell proliferation and migration through AKT regulation.
ACCEPTED MANUSCRIPT Abbreviations
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qRT-PCR: quantitative real-time PCR DMSO: dimethyl sulfoxide FCM: Flow cytometry PBS: phosphate-buffered saline
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