The HOTAIR/miR-214/ST6GAL1 crosstalk modulates colorectal cancer procession through mediating sialylated c-Met via JAK2/STAT3 cascade
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The regulatory non-coding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), emerge as pivotal markers during tumor progression. Abnormal sialylated glycoprotein often leads to the malignancy of colorectal cancer (CRC).
Differential levels of HOTAIR and ST6GAL1 are analyzed by qRT-PCR. Functionally, CRC cell proliferation, aggressiveness and apoptosis are measured through relevant experiments, including CCK8 assay, colony formation assay, transwell assay, western blot and flow cytometry. Dual-luciferase reporter gene assay and RIP assay confirm the direct interaction between HOTAIR and miR-214. The lung metastasis, liver metatstasis and xenografts nude mice models are established to show the in vivo effect of HOATIR.
Here, differential levels of HOTAIR and ST6GAL1 are primarily observed in CRC samples and cells. Upregulated HOTAIR and ST6GAL1 are crucial predictors for poor CRC prognosis. Altered level of ST6GAL1 modulates CRC malignancy. Furthermore, ST6GAL1 and HOTAIR are confirmed as the direct targets of miR-214, and ST6GAL1 is regulated by HOTAIR via sponging miR-214. ST6GAL1 induces the elevated metabolic sialylation of c-Met, which is co-mediated by HOTAIR and miR-214. Sialylated c-Met affects the activity of JAK2/STAT3 pathway. The regulatory role of HOTAIR/miR-214/ST6GAL1 axis also impacts CRC procession. In addition, HOTAIR mediates lung metastasis, liver metastasis and tumorigenesis in vivo. ShHOTAIR and AMG-208 are combined to inhibit tumorigenesis for successful drug development.
The HOTAIR/miR-214/ST6GAL1 axis commands the CRC malignancy by modifying c-Met with sialylation and activating JAK2/STAT3 pathway. Our study presents novel insights into CRC progression and provided prospective therapeutic target for CRC.
KeywordsHOTAIR MiR-214 ST6GAL1 c-Met JAK2/STAT3 cascade
- 3’UTR 3′
Competing endogenous RNA
Long non-coding RNAs
α 2, 6-sialyltransferase 1
CRC is the third most common cancers, with a major burden of nearly 700,000 deaths each year worldwide . Patients with localized stage live a long survival period than the patients diagnosed with distant-stage . Additionally, drug resistance remains the major obstacle for CRC management. Therefore, it is urgent to identify effective markers for CRC diagnosis and clarify the potential molecular mechanism.
Glycosylation is a common type of post-translational modification of proteins. Sialylated glycoproteins are involved in cellular physiological activity and cancer development . Sialyltransferases (STs) are a family of anabolic enzymes, and correlate with cancer procession . Alpha 2, 6-sialyltransferases mediate the transfer of SA with an alpha 2, 6-linkage to it with terminal GAL or GALNAC residues. Abnormal ST6GAL1 is observed in prostate cancer  and hepatocellular cancer . ST6GAL1 restructures the sialylated glycoproteins on the cell surface, which enhances CRC malignancy . ST6GAL1 is a potential enzyme to modify sialylated mesenchymal-epithelial transition factor (c-Met), hyposialylated c-Met could attenuate cell motility . C-Met is a class of receptor tyrosine kinase, and exists on various epithelial cell surface . C-Met functions as proto-oncogene during cancer development . Specifically, CRC resistance is driven by abnormal c-Met via JAK2/STAT3 pathway . However, the potential mechanism is still unstated that ST6GAL1 modulates sialylated c-Met through JAK2/STAT3 pathway during CRC malignancy.
LncRNAs are the transcripts longer than 200 nucleotides, and receive attention in cancer diagnosis and therapy . LncRNAs exhibit critical roles in mediating CRC development. As an oncogenic lncRNA, HOTAIR maintains various tumor malignancies, such as gastric cancer  and cervical cancer . HOTAIR also mediates 5-FU resistance in CRC cells . MiRNAs are a class of 19–25 nucleotides transcripts, without protein-coding ability. Mechanically, miRNAs suppresses mRNA transcription by directly binding to the 3′-untranslated region (3′-UTR) . Numerous studies expound that miRNAs involve in tumor progression. MiR-214 is remarkably downregulated in CRC  and hepatocellular carcinoma . Reversed interaction between HOTAIR and miR-214 has been illustrated in ovarian cancer . However, the mechanism that the HOTAIR/miR-214/ST6GAL1 crosstalk mediates c-Met sialylation is still unknown regarding CRC malignancy.
In the present study, the differential levels and regulatory roles of ST6GAL1 and HOTAIR are expounded. The regulatory network among HOTAIR, miR-214 and ST6GAL1 is established, which further mediates the altered sialylation of c-Met via JAK2/STAT3 pathway. We provide a novel mechanism that the HOTAIR/ miR-214/ST6GAL1 cross-talk modifies c-Met sialylation, and offers promising target for CRC diagnosis and therapy.
Materials and methods
Samples from CRC patients
The study and its informed consent have been examined and certified by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University (YJ-KY-FB-2016-16). Human primary CRC and adjacent tissues were extracted from 42 patients who underwent surgical resections from June 2015 to August 2017. The tumors were identified different stages (stage I, II, III and IV) based on the histopathological assessment. The samples were stored in liquid nitrogen for further analysis.
CRC cell culture
The CRC cell lines SW620, SW480, HCT-8, HCT-8/5-FU and LoVo were purchased from Keygen Biotech Co. Ltd. (Nanjing, China). SW620 and SW480 were cultured in Leibovitz’s L-15 (Gibco, Grand Island, NY) medium contained 10% inactivated fetal bovine serum (Gibco, Grand Island, NY). HCT-8, HCT-8/5-FU and LoVo cells were cultured in in RPMI 1640 (Gibco, Grand Island, NY) medium contained 10% inactivated fetal bovine serum. To develop the 5-FU resistant LoVo cells, 5-FU was added to the medium by stepwise increasing concentrations for over 6 months. HCT-8/5-FU and LoVo/5-FU were maintained in medium supplemented with 124.5 μM and 113.0 μM 5-FU. CRC cells were both incubated at 37 °C with a humidified atmosphere containing 5% CO2. All cells lines were routinely tested for mycoplasma, which were shown to be negative.
Real-time PCR analysis
The total RNA was extracted by Trizol (Invitrogen), and cDNA was synthesized by QuantiTect ReverseTranscription Kit (Qiagen, Valencia, CA). qRT-PCR was performed under an ABI Prism7500 fast real-time PCR system (Applied Biosystems, Foster City, CA) with mixing a QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA). Relative RNA expression was calculated with normalization to GAPDH.
Fluorescence in situ hybridization (FISH)
Cells were and fixed with 4% paraformaldehyde and dehydrated. Cells were hybridized with biotin-labeled HOTAIR and miR-214 probes (GenePharma) at 50 nM for at 73 °C for 5 min. The cells were degenerated and incubated at 37 °C overnight. Images were shown under a fluorescence microscope.
Western blot analysis
Proteins were electrophoresed with 10% SDS-PAGE gels and transferred to the polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Primary antibodies were incubated at 4 °C. The membrane was incubated with anti-rabbit IgG at 37 °C. All bands were determined and analyzed by LabWorks (TM ver4.6, UVP, BioImaging Systems, NY, USA). GAPDH was used as control.
Lectin pull-down assay
The CRC cells were lysed and treated with Sambucus nigra lectin (SNA, Vector Laboratories, Burlingame, CA) at 4 °C overnight. Glycoprotein eluting solution was used to wash the precipitated lysates. The relevant glycoproteins were collected and analyzed by western blot.
The ampliations of ST6GAL1 and HOTAIR were cloned into pmirGLO vector (Promega). MiR-214 mimic, inhibitor, miR-NC, short hairpin RNA for HOTAIR and scramble shRNA were synthesized by GenePharma. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used for the transfection assay. qRT-PCR was used to detect the transfected efficiency.
Dual luciferase reporter gene assay
The pmirGLO Dual-Luciferase miRNA Target Expression Vector was obtained from GenePharma. The mRNA expression was shown by firefly luciferase, and the renilla luciferase intensity was used as normal control. Lipofectamine 3000 was utilized for the co-transfection. The dual luciferase reporter assay system (Promega) was used to conduct the experiment. Each experiment was performed in triplicate.
Cell viability assay
Cell proliferation assay was conducted by using cell counting kit-8 (CCK-8; Dojindo, Japan). 1 × 103 cells were plated into 96-well plate and 11 μL CCK8 was added into the plate for 4 h. The spectrometric absorbance was measured by microplate reader (Model 680; Bio-199 Rad, Hercules, CA, USA) at 450 nm. For chemoresistance to 5-FU, corresponding 5-FU was added into 96-well plate. Similarly, the absorbance was then measured to evaluate the chemoresistance to 5-FU. Each experiment was performed thrice.
Focus formation assay
2 × 103 single-cell suspension was obtained and seeded in cell culture dishes. The medium was renewed at regular intervals. The foci were formed obviously 12 days later. The colonies were fixed by 4% paraformaldehyde for 20 min, and stained with 0.2% crystal violet. The colonies were recorded and counted.
Cells were cultured in Boyden chambers containing a transwell membrane filter (Corning, New York, USA), and in serum-free medium overnight. The filter was coated with gelatin and matrigel. 5 × 104 cells were suspended on top, and complete medium was placed in the lower. The upper side cells were removed by a cotton swab. The invading cells were counted to estimate the invasive capacity. Five random fields were analyzed for each chamber.
The immunoprecipitation was performed by the protein A/G agarose beads (Thermo Fisher Scientific). Cell lysates incubated in immunoprecipitation lysis buffer (Beyotime) for 10 min. The lysates were conjugated with anti-c-Met (3 μg/ml, Abcam). The immunoprecipitates were washed with protein A/G agarose beads and used for further experiment.
Cells were incubated with FITC-SNA (Vector Laboratories, Burlingame, CA, USA) for 1 h. The FITC fluorescence intensity was detected by FACS Calibur (Becton-Dickinson, CA, USA). CRC cells were treated with 5-FU for 48 h and resuspended in 100 μL binding buffer. Annexin V and propidium iodide were used to stain cells for 10 min. With the usage of Annexin-V-FITC apoptosis detection kit (BD, Franklin Lakes, NJ, USA), the apoptotic cells were detected by FACS Calibur.
Immunohistochemistry (IHC) staining
The samples were collected and embedded with paraffin. The section was pre-treated with drying, deparaffining and rehydrating. The slides were immersed with 3% hydrogen peroxide for 10 min and labeled with the corresponding antibodies at 4 °C overnight. The secondary streptavidin-horseradish peroxidase conjugated antibody was used to label the primary antibody for 1 h. The slides were then counter stained with hematoxylin for 30s and cover slipped.
RNA immunoprecipitation (RIP) assay
The endogenous miR-214, combined with HOTAIR, was pulled down. The cell lysis was incubated in RIP immunoprecipitation buffer containing magnetic bead, conjugated with human anti-Ago2 antibody (Millipore). Mouse IgG (Millipore) was used as negative control. The protein was digested by proteinase K, and the immunoprecipitated RNA was obtained. The qRT-PCR assay was conducted to detect the isolated RNAs.
Tumor xenograft models
The experiments were approved by the Committee on the Ethics of Animal Experiments of the Dalian Medical University. The 4-week-old male nude mice were obtained from the Model Animal Research Institute of Nanjing University.
The lung metastasis model was built by injecting 5 × 106 cells into the tail vein. The procession was sustained at least 10 min. The mice were then sacrificed, and the lung was isolated.
For liver metastasis, 4-week-old male nude mice were randomly used to build the liver metastasis model under anesthetizing. The spleen was exposed and injected with 5 × 106 CRC cell, and this procession sustained at least 5 min. The endpoints of in vivo metastasis experiments were based on the presence of clinical signs of liver metastasis, weight loss, the appearance of ascites and energielos with reduced action. The nude mice were sacrificed 28 days later, and the liver and the spleen were collected and photographed.
For xenografts model, 1 × 107 GFP labeled CRC cells were injected subcutaneously into the right flank of each nude mouse. The mice were randomly divided into control and treatment groups when the mice bearing palpable tumors. The groups received DMSO or AMG-208, respectively. The endpoints of in vivo xenografts experiments were based on the presence of weight loss, tumor volume higher than 4cm3 and energielos with reduced action. The representative X-Ray photos and fluorescent photos were captured to reveal the tumor volume in vivo. The photos were taken by in-vivo Imaging System of Dalian Medical University. Then the mice were humanely sacrificed and the tumors were isolated for further analysis.
SPSS 17.0 software was used to analyze the experimental data. Data were presented as means ± standard deviation (SD), and each experiment was carried out thrice at least. Student’s t-test was used to compare the significant difference of two groups. SD represented the variation of data values. The data variance of each group was similar. The one-way analysis of variance (ANOVA) was used to determine the significant difference of multiple groups. The survival curves were calculated by Kaplan-Meier method, and the difference was assessed by a log-rank test. Spearman’s correlation analysis was used to identify the association between miRNAs and mRNA expression. Statistical significance was defined as P value < 0.05.
HOTAIR and ST6GAL1 are upregulated during CRC progression
ST6GAL1 mediates the malignancy of CRC cell lines
ST6GAL1 and HOTAIR function as direct target of miR-214
HOTAIR/miR-214/ST6GAL1 axis mediates α 2, 6-sialylation of c-met and activates JAK2/STAT3 pathway
Modulation of HOTAIR and miR-214 influences CRC procession in vitro
HOTAIR mediates CRC malignancy in vivo
The occurrence of CRC remains a multilevel event, and unrestrained metastasis often led to the advanced CRC progression. Moreover, drug resistance is the main cause of the unsatisfactory therapy. It is imperative to clarify the involvement of modulatory ncRNAs in regulating sialylation during CRC evolution. This study provides in-depth understanding of HOTAIR/miR-214/ST6GAL1 cross-talk regulating c-Met sialylation via JAK2/STAT3 pathway during CRC progression.
Sialylation exists as a key modification of proteins in cancer. Abnormal sialylation is regarded as the hallmark of tumor development. With the usage of Mass Spectrometry, higher ST6GAL1 is confirmed in CRC cells . Moreover, differential ST6GAL1 is also closely correlated with CRC progression . Similar observations are shown in our study, overexpressed ST6GAL1 is verified in CRC tissues, and exhibited high association with CRC advancement. Highly ST6GAL1 level is also verified in CRC cells. Related research depicts the effect of ST6GAL1 on CRC malignancy , and the potential role of ST6GAL1 in promoting tumor development is also clarified in breast cancer . The critical function of ST6GAL1 involved into CRC progression is further corroborated and expounded both in vitro and in vivo in this study. ST6GAL1 regulation is found to be the essential factor for CRC malignancy. Therefore, ST6GAL1 emerges as a promising marker for assessing CRC progression.
Aberrant lncRNA expression is crucial issue for cancer development. High expression of HOTAIR reveals closely association with cancers, such as gastric cancer , lung cancer  and CRC . Our results provide comprehensive evidence that HOTAIR is extremely associated with poor CRC advancement. For clinical investigation, decreased HOTAIR indicates longer 5-year survival rate. HOTAIR exhibits closely association with CRC malignancy, while the precise mechanism of HOTAIR involved in CRC progression is still unclear. CeRNA theory seems as a reasonable hypothesis to verify the potential mechanism. Reasonably, HOTAIR contributes to CRC liver metastasis by sponging miR-218 . The correlation of HOTAIR and miR-214 is fully expounded in ovarian cancer . By directly binding with FGFR1, miR-214 efficiently mediates the procession of CRC liver metastasis . MiR-214 is verified as tumor suppressor of colon cancer . Interestingly, miR-214 also impacts monocyte apoptosis by targeting PTEN . In this research, miR-214 is identified as the target of ST6GAL1 and HOTAIR. HOTAIR and miR-214 co-locate in the cytoplasm of CRC cells, which provide the possible interaction between the two molecules. We also affirm the endogenous interaction between HOTAIR and miR-214 as the potential reason for CRC procession. Therefore, HOTAIR/miR-214/ST6GAL1 crosstalk might efficiently contribute to CRC progression.
Glycoprotein functions as the actual executor of cell physiological and pathological processes. ST6GAL1 is regarded as the essential enzyme to form α 2, 6-linked sialic acids, the major structure of sialylated glycoprotein. Unusual activation of c-Met might be considered as the initiation of cancer malignancy . Aberrant modification of c-Met, with α 2, 6-sialic terminal structure, participates in CRC progression, and often leads to the long-term changes in neoplastic cell motility . In accordance with our study, regulation of miR-214 and HOTAIR results in the altered ST6GAL1 level. Additionally, ST6GAL1 induces continuous sialylated c-Met activation, which is the potential mechanism accounting for CRC evolution. We hypothesize that HOTAIR and miR-214 could efficiently regulate ST6GAL1 mRNA level, which induced the alteration of ST6GAL1 protein. The ST6GAL1 enzyme catalyzes the sialylation of c-Met, forms a regulatory HOTAIR/miR-214/ST6GAL1 crosstalk in commanding α 2, 6 sialylation of c-Met during CRC procession. Recent researches show that c-Met influences the STAT3 dependent pathway . C-Met blocking assay offers us the notion that the directly activation of JAK2/STAT3 pathway is attributed to α 2, 6 sialylated c-Met expression. Moreover, miR-214 also involves in the activation of NF-κB, Wnt, JAK/STAT and TP53 pathways . Co-transfection of HOTAIR and miR-214 also regulates the main molecules of JAK2/STAT3 pathway. HOTAIR and miR-214 influences CRC malignancy. In accordance with the in vitro experimental data, alteration of HOTAIR affects CRC progression, especially the lung and liver metastasis in the nude mouse model. The inhibitory effect of HOTAIR on CRC tumorigenesis is also demonstrated in vivo.
The comprehensive research data provide strengthen evidence that HOTAIR/miR-214/ST6GAL1 axis commands the sialylated c-Met, and further activates JAK2/STAT3 pathway in CRC progression. The HOTAIR/miR-214/ST6GAL1 crosstalk could be regarded as a potential strategy for CRC. However, CRC is still multi-factorial disease and should be urgent captured. Further investigation is still urgent required.
The co-mediation of HOTAIR and miR-214 involves in regulation the sialylated c-Met via ST6GAL1, and is clarified in CRC progression. The regulatory network might be inspired for postponing CRC malignancy. The current study presents valuable information regarding the early diagnosis and therapeutic target for CRC.
Thank you for the editors and reviewers for their helpful comments on this paper.
BL is responsible for conducting experiments, acquisition of data, analysis and drafted the manuscript. QL, YH and YQ provided technical and material support. SL, SP and YX collected the clinical data. LJ was responsible for designing the experiments and research supervision. All authors read and approve the final manuscript.
This work was supported by the National Natural Science Foundation of China (81772277).
Ethics approval and consent to participate
The study has been examined and certified by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University (Ethics Reference NO: YJKY-FB-2016-16), and informed consent was obtained from all participants included in the study, in agreement with institutional guidelines.
Consent for publication
The authors declare that they have no competing interests.
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