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Overexpression of Agrin Promotes Tumor Proliferation and Correlates with Poor Prognosis in Cholangiocarcinoma

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Overexpression of Agrin Promotes Tumor Proliferation and Correlates with Poor Prognosis in Cholangiocarcinoma Overexpression of Agrin promotes tumor proliferation and correlates with poor prognosis in cholangiocarcinoma Meimei He wenzhou university Shasha Ji wenzhou university Junxue Tu wenzhou university Dan Lou ( [email protected] ) Wenzhou University https://orcid.org/0000-0002-9269-4737 Research Keywords: cholangiocarcinoma, Agrin, YAP, metastasis, proliferation Posted Date: August 4th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-50004/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract Background Agrin exists as a shorter Type II Transmembrane form with an internal signal peptide, and is closely correlated with the activation of multiple intercellular signaling pathways. The aim of the present study was to investigate the role of Agrin in the development of cholangiocarcinoma (CCA). Methods RT-qPCR and western blotting were performed to detect the expressional level of target genes, including Agrin, in CCA tissues or cell lines. The correlation between Agrin, and tumor characteristics and prognosis, was analyzed using independent sample t-test, the Kaplan-Meier method and Cox proportional hazard model, respectively. Proliferation, migration, invasion and tumorigenesis in CCA cells was determined by CCK8 assay, cell cycle detection, Transwell assay and nude mouse tumorigenicity assay, respectively. Results Agrin was signicantly upregulated in CCA tissues, as compared to the adjacent non-tumor tissues, and was correlated with poorer tumor characteristics such as portal vein tumor thrombus, intrahepatic metastasis and poor survival. The Agrin overexpression in CCA cell lines clearly promoted proliferation, colony formation, migration, invasion and cell cycle progression, but Agrin knockdown had the opposite effect. Furthermore, CCA cells with inhibitory Agrin expression presented with less and smaller tumors, as compared with the control group in vivo. Mechanistic analysis indicated that Agrin was able to activate the Hippo signaling pathway and induce yap to enter the cell nucleus. Conclusions We found that Agrin promotes the CCA progression via activating the Hippo signaling pathway, which could be a potentially promising target for CCA treatment. Introduction Cholangiocarcinoma (CCA) is the most frequent biliary adenocarcinoma associated with high malignancy, accounting for 10–15% of primary liver cancer cases[1]. According to where the tumor originates from, CCA is classied into intrahepatic and extrahepatic CCA, the latter including perihilar and distal CCA. Most of CCA patients are diagnosed at a median age of 65 years, but patients with primary sclerosing cholangitis commonly present with CCA at the age of ≤ 40 years[2]. The high incidence rates of hepatolithiasis and high prevalence of hepatitis B virus may be the main cause of CCA initiation in China[3]. In recent decades, there has been increasing concern over the rising incidence and high tumor- Page 2/20 related death rates of CCA worldwide. For CCA patients, surgery is the only potentially curative option. Unfortunately, rapid preoperative tumor progression and high postoperative tumor recurrence rates signicantly limit the R0 resection rates and long-term tumor-free survival (TFS) time[4]. Recently, great advances have been made in tumor management[5], and related immunotherapy has been achieved, which strongly suggests that further understanding of the molecular mechanism underlying tumor progression and recurrence would be useful for improving prognosis. The present study aimed at determining the role of Agrin in regulating cell biofunction in CCA. Agrin is a 210-kDa basal lamina-related heparan sulfate proteoglycan that exists as either a shorter Type II Transmembrane protein or a secreted protein in the extracellular matrix[6]. Its two parts have been shown to have a completely different function, with the carboxy-terminal part exhibiting a synaptogenic activity and triggering the acetylcholine receptors (AChRs) by activating the low-density lipoprotein receptor-related protein 4 (Lrp4) receptor and the muscle-specic kinase (MuSK), and the aminoterminal end consisting of a signal sequence, which is needed for the secretory pathway, and an aminoterminal Agrin domain, which is required for binding with basal lamina-associated laminins[7]. The majority of studies have focused on investigating the essential role of Agrin in the formation of the vertebrate neuromuscular junction during embryogenesis. For example, Nitkin et al[8] and Jones et al[9] conrmed that ectopic Agrin expression could promote the formation of postsynaptic specializations, such as the induction of endplate-specic gene transcription of the e-subunit of the AChR in vivo and aggregates of several important molecules on cultured myotubes in vitro. Conversely, the inhibition of Agrin resulted in very few pre- and postsynaptic specializations, and led to mice dying at birth due to nonfunctional respiratory musculature[10]. In addition, several recent studies have veried that Agrin is necessary for tumor initiation and progression. Sayan et al demonstrated that Agrin promoted cellular proliferation, migration and invasion in hepatocellular carcinoma, and activated the FAK signaling pathway through the formation of the Agrin-Lrp4-MuSK signaling complex[11]. Rivera C showed that Agrin silencing decreased the malignant ability, and inhibited the phosphorylation of FAK, ERK and cyclin D1 proteins in oral squamous cell carcinoma cells[12]. However, the relationship between Agrin and CCA development had not yet been explored. Herein, it was revealed that Agrin promotes CCA malignant characteristics as an activator of the Hippo signaling pathway regulating cell proliferation, migration and invasiveness. Furthermore, shRNA- mediated Agrin knockdown signicantly suppressed tumor spheroid formation in vivo. Collectively, the present results showed that Agrin upregulation was required for CCA development, and that Agrin could potentially serve as a therapeutic target. Material And Methods Cell lines and cell transfection Two human CCA cell lines (HUCCT-1 and CCLP-1) were used in the study, which were purchased from the Institutes of Biological Sciences (Shanghai, China) and cultured following the manufacturer’s Page 3/20 instructions. HUCCT-1 and CCLP-1 were incubated using 1640 complete medium (Thermo Fisher Scientic, Inc.) supplemented with 10% fetal bovine serum (FBS) and antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin) in a 5% CO2 incubator at 37˚C. Lentivirus encoding both an shRNA hairpin (5’- CAGGAGAAUGUCUUCAAGATT-3’) specically targeting human Agrin and green uorescent protein, as well as the vector pcDNA3.1 embracing the full-length cDNA of Agrin, were used to infect the cells, according to the manufacturer’s instructions. Lentivirus encoding a scramble sequence was used as a control. RNA extraction and RT-qPCR The extraction of total RNA from tissues and cells was performed using TRIZOL reagent (Thermo Fisher Scientic, Inc.), following the manufacturer’s instructions, and a ReverTra Ace® kit (Thermo Fisher Scientic, Inc.) was used to reverse-transcribe the RNA into cDNA. RT-qPCR was performed using SYBR Premix Ex Taq™ (Takara Bio, Inc.) and the Thermal Cycler Dice Detection System. The following primers were used to specially amplify the Agrin gene and β-actin. Agrin: forward, 5’-ACACCGTCCTCAACCTGAAG-3’ reverse, 5’-CCAGGTTGTAGCTCAGTTGC-3’; β-actin: forward, 5’-AGAGCCTCGCCTTTGCCGATCC-3’ reverse, 5’-CTGGGCCTCGTCGCCCACATA-3’. Cell cycle analysis Cells with Agrin plasmid or shRNA transfection received serum starvation to induce cell cycle synchronization for 48 h, then seeded at a density of 10,000 cells per 6-cm plate. The monolayer cells with ~ 70% conuency were harvested as single cell suspensions and xed in 90% ethanol overnight. Following washing twice with PBS, cells were incubated with 0.5 ml DNA Prep Stain in the dark for 30 min at room temperature, followed by ow cytometric analysis. The percentage of the cell population in each phase was calculated using ModFit LT software. Colony formation Colony formation assay was performed through seeding CCA cells in 6 wells plates. A total of 10,00 cells was seeded, and cultured in fresh medium for 2 weeks. Subsequently, the colonies stained with crystal violet were photographed and counted. Counting kit-8 assay Page 4/20 The effect of Agrin on CCA cell viability was detected by cell counting kit-8 (Dojindo Molecular Technologies, Inc.) assay. Briey, CCA cells were plated in a 96-well plate at a density of 1,000 cells per well. Following culture for 24, 48, 72, 96 and 120 h, fresh medium containing 10% Cell Counting Kit-8 was added to the well, and the absorbance value at a wavelength of 450 nm was detected as an indicator of cell viability. Cell migration and invasion assays Migration and invasion assays were conducted using the Transwell system (24-well insert, 8.0-µm pores). CCA cells of 1 × 104 per well were suspended in 100 µl non-FBS culture medium and plated into the upper chamber. The upper chamber, which was coated with Matrigel, was used for the invasion assay, and the one without Matrigel for the migration assay. Medium containing 10% FBS was added into the bottom chamber to drive cell translocation at 500 µl per well. After 24, 48 and 72 h, cells on the upper surface of the chamber were cleaned with cotton swabs, and those on the bottom surface were reserved and xed in 95% methanol for 20 min, then stained with 0.4% crystal violet and photographed.
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