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Identification of Coiled-Coil Domain–Containing Protein 180 and Leucine-Rich Repeat–Containing Protein 4 as Potential Immunohistochemical Markers for Liposarcoma Based on Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded Tissue

2019; Elsevier BV; Volume: 189; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2019.01.013

ISSN

1525-2191

Autores

Tomoyuki Aoyama, Akira Takasawa, Kumi Takasawa, Yusuke Ono, Makoto Emori, Masaki Murata, Takahiro Hayasaka, Naoki Fujitani, Makoto Osanai, Toshihiko Yamashita, Tadashi Hasegawa, Norimasa Sawada,

Tópico(s)

Lipid metabolism and biosynthesis

Resumo

Recent technical improvements in both mass spectrometry and protein extraction have made it possible to use formalin-fixed, paraffin-embedded (FFPE) tissues for proteome analysis. In this study, comparable proteome analysis of FFPE tissues revealed multiple candidate marker molecules for differentiating atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) from lipoma. A total of 181 unique proteins were identified for ALT/WDL. Of the identified proteins, coiled-coil domain–containing protein 180 (CCDC180) and leucine-rich repeat–containing protein 4 (LRRC4) were studied as candidate markers of ALT/WDL. CCDC180 and LRRC4 immunohistochemistry clearly stained tumor cells of ALT/WDL and dedifferentiated liposarcoma and could differentiate them from lipoma with high accuracy. Cell biological methods were used to further examine the expression of the candidate marker molecules in liposarcoma cells. In liposarcoma cells, knockdown of CCDC180 and LRRC4 inhibited cell proliferation. CCDC180 inhibited cell migration, invasion, and apoptosis resistance in WDL cells. Adipogenic differentiation suppressed the expression of CCDC180 and LRRC4 in WDL cells. These results indicated that LRRC4 and CCDC180 are novel immunohistochemical markers for differentiating ALT/WDLs. Their expression was associated with adipocyte differentiation and contributed to malignant potentials of WDL cells. Proteome analysis using a standard stock of FFPE tissues can reveal novel biomarkers for various diseases, which contributes to the progress of molecular pathology. Recent technical improvements in both mass spectrometry and protein extraction have made it possible to use formalin-fixed, paraffin-embedded (FFPE) tissues for proteome analysis. In this study, comparable proteome analysis of FFPE tissues revealed multiple candidate marker molecules for differentiating atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) from lipoma. A total of 181 unique proteins were identified for ALT/WDL. Of the identified proteins, coiled-coil domain–containing protein 180 (CCDC180) and leucine-rich repeat–containing protein 4 (LRRC4) were studied as candidate markers of ALT/WDL. CCDC180 and LRRC4 immunohistochemistry clearly stained tumor cells of ALT/WDL and dedifferentiated liposarcoma and could differentiate them from lipoma with high accuracy. Cell biological methods were used to further examine the expression of the candidate marker molecules in liposarcoma cells. In liposarcoma cells, knockdown of CCDC180 and LRRC4 inhibited cell proliferation. CCDC180 inhibited cell migration, invasion, and apoptosis resistance in WDL cells. Adipogenic differentiation suppressed the expression of CCDC180 and LRRC4 in WDL cells. These results indicated that LRRC4 and CCDC180 are novel immunohistochemical markers for differentiating ALT/WDLs. Their expression was associated with adipocyte differentiation and contributed to malignant potentials of WDL cells. Proteome analysis using a standard stock of FFPE tissues can reveal novel biomarkers for various diseases, which contributes to the progress of molecular pathology. Proteomics is the large-scale analysis of a proteome, a data set of total proteins in a biological system including their amount, cellular structure, and function. Every alteration of proteins directly reflects some biological or clinical events and can become an important factor of pathologic abnormality. Recently, mass spectrometry (MS)–based proteomics has been used to discover biomarkers for a variety of diseases as well as to elucidate molecular mechanisms.1Voshol H. Brendlen N. Müller D. Inverardi B. Augustin A. Pally C. Wieczorek G. Morris R.E. Raulf F. van Oostrum J. 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Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue.Tumor Biol. 2016; 37: 13595-13606Crossref PubMed Scopus (29) Google Scholar Lipomatous tumors represent the largest single group of mesenchymal tumors, including benign-type lipoma and malignant liposarcoma.16Fletcher C.D. Unni K.K. Mertens F. WHO Classification of Tumors of Soft Tissue and Bone.ed 4. IARC Press, Lyon, France2013Google Scholar Among the liposarcomas, atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) represents the largest subgroup and is similar to benign lipoma in histologic appearance. In clinical pathology, it is important to differentiate ALT/WDL from lipoma because ALT/WDL can proceed on to dedifferentiation, a critical risk factor for distant metastasis. Genomic PCR and fluorescence in situ hybridization, for detecting amplification of chromosome 12q13-q15 in ALT/WDL, are helpful for differential diagnosis, although those analyses are not appropriate for clinical practice everywhere. Overexpressions of E3-ubiquitin–protein ligase Mdm2 (MDM2), cyclin-dependent kinase 4 (CDK4), and high-mobility group protein, which result from 12q13-q15 amplification, are also used as ALT/WDL markers in immunohistochemistry.16Fletcher C.D. Unni K.K. Mertens F. WHO Classification of Tumors of Soft Tissue and Bone.ed 4. IARC Press, Lyon, France2013Google Scholar, 17Dei Tos A.P. Doglioni C. Piccinin S. Sciot R. Furlanetto A. Boiocchi M. Dal Cin P. Maestro R. Fletcher C.D. Tallini G. Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours.J Pathol. 2000; 190: 531-536Crossref PubMed Google Scholar, 18Hostein I. Pelmus M. Aurias A. Pedeutour F. Mathoulin-Pélissier S. Coindre J.M. 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Coindre J.M. Pedeutour F. Bianchini L. Prognostic value of HMGA2, CDK4, and JUN amplification in well-differentiated and dedifferentiated liposarcomas.Mod Pathol. 2015; 28: 1404-1414Crossref PubMed Scopus (47) Google Scholar, 22Clay M.R. Martinez A.P. Weiss S.W. Edgar M.A. MDM2 and CDK4 immunohistochemistry: should it be used in problematic differentiated lipomatous tumors? a new perspective.Am J Surg Pathol. 2016; 40: 1647-1652Crossref PubMed Scopus (47) Google Scholar However, immunohistochemistry of the molecules has limited applicability to practical use because of the small number of immunopositive cells in specimens. Therefore, useful immunohistochemical markers are required to distinguish ALT/WDL from lipoma. In this study, comparable proteome analysis of FFPE tissues revealed multiple candidate marker molecules for differentiating ALT/WDL from lipoma. It was also confirmed that identified molecules, coiled-coil domain–containing protein 180 (CCDC180) and leucine-rich repeat–containing protein 4 (LRRC4), were useful in immunohistochemistry. In addition, it was demonstrated that they contributed to malignant roles of WDL cells and that they were up-regulated in association with dedifferentiation. Dithiothreitol, iodoacetamide, ammonium bicarbonate, acetonitrile, ethanol, guanidine hydrochloride (guanidine-HCl), hydrochloric acid, formic acid, dexamethasone, and cisplatin were purchased from Wako (Osaka, Japan). Heptane, methanol, acetic acid, and chloroform were purchased from Kanto Chemical (Tokyo, Japan). 3-Isobutyl-1-methylxanthine, oleic acid, trifluoroacetic acid (TFA), and trypsin were purchased from Sigma-Aldrich (St. Louis, MO). The Qproteome FFPE tissue kit was purchased from Qiagen (Hilden, Germany). Antibodies used in this study are listed in Table 1.Table 1List of Antibodies Used in This StudyAntibodyClonalitySpeciesSupplierDilutionApplicationAnti-C9orf174 (CCDC180)PolyclonalRabbitAbcam (Cambridge, UK)1:1000WBAnti–netrin G2 ligand (LRRC4)MonoclonalMouseAbcam1:1000WBAnti-FABP4PolyclonalRabbitAbcam1:1000WBAnti-actinPolyclonalRabbitSigma-Aldrich1:3000WBAnti-CCDC180PolyclonalRabbitAtlas Antibodies (Stockholm, Sweden)1:50IHCAnti-LRRC4PolyclonalRabbitAtlas Antibodies1:200IHCAnti-chymaseMonoclonalMouseThermo Fisher Scientific (Waltham, MA)1:4000IHCAnti-MDM2MonoclonalMouseCalbiochem (La Jolla, CA)1:50IHCAnti-CDK4MonoclonalMouseInvitrogen (Carlsbad, CA)1:50IHCAnti–Ki-67PolyclonalRabbitCell Signaling Technology, Inc. (Beverly, MA)1:100IHCAnti–cleaved caspase-3PolyclonalRabbitCell Signaling Technology, Inc.1:100IHCAnti–cleaved PARPMonoclonalRabbitCell Signaling Technology, Inc.1:200IHCCDK4, cyclin-dependent kinase 4; FABP4, fatty acid–binding protein 4; IHC, immunohistochemistry; MDM2, E3-ubiquitin–protein ligase Mdm2; PARP, poly (ADP-ribose) polymerase; WB, Western blot analysis. Open table in a new tab CDK4, cyclin-dependent kinase 4; FABP4, fatty acid–binding protein 4; IHC, immunohistochemistry; MDM2, E3-ubiquitin–protein ligase Mdm2; PARP, poly (ADP-ribose) polymerase; WB, Western blot analysis. The present research protocol was approved by the Sapporo Medical University Ethics Committee (Sapporo, Japan), and informed consent was obtained from all participating patients. The tissues used in this study were from 47 patients who underwent surgical resection for ALT/WDL, dedifferentiated liposarcoma (DDL), or lipoma at Sapporo Medical University Hospital (Sapporo, Japan) during the period from 2006 to 2017. FFPE tissue blocks from 22 cases of ALT/WDL, 18 cases of DDL, and 25 cases of lipoma were obtained from the pathology file of Sapporo Medical University Hospital. Clinicopathological features of the patients are summarized in Supplemental Table S1. After detailed histopathological analysis, 4 of 22 ALT/WDL cases and 4 of 25 lipoma cases were selected for downstream peptide sample preparation. Peptide sample preparation was performed, as described before, with some modifications.15Yamamoto T. Kudo M. Peng W.X. Takata H. Takakura H. Teduka K. Fujii T. Mitamura K. Taga A. Uchida E. Naito Z. Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue.Tumor Biol. 2016; 37: 13595-13606Crossref PubMed Scopus (29) Google Scholar, 23Paulo J.A. Lee L.S. Banks P.A. Steen H. Conwell D.L. Proteomic analysis of formalin-fixed paraffin-embedded pancreatic tissue using liquid chromatography tandem mass spectrometry.Pancreas. 2012; 41: 175-185Crossref PubMed Scopus (27) Google Scholar, 24Shah S.J. Yu K.H. Sangar V. Parry S.I. Blair I.A. Identification and quantification of preterm birth biomarkers in human cervicovaginal fluid by liquid chromatography/tandem mass spectrometry.J Proteome Res. 2009; 8: 2407-2417Crossref PubMed Scopus (61) Google Scholar, 25Oki G. Wada T. Iba K. Aiki H. Sasaki K. Imai S. Sohma H. Matsumoto K. Yamaguchi M. Fujimiya M. Yamashita T. Kokai Y. Metallothionein deficiency in the injured peripheral nerves of complex regional pain syndrome as revealed by proteomics.Pain. 2012; 153: 532-539Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar FFPE tissue blocks were divided into sections on a microtome to eight serial sections (10 to 15 μm thick and approximately 100 mm2 in size). Each of the FFPE tissue sections was deparaffinized by 0.5 mL heptane in a 2.0-mL microtube at room temperature for 90 minutes. After adding 25 μL of methanol, the tissue was collected by centrifugation. The supernatant was removed, and the pellet was left to dry in air at room temperature. Subsequent protein extraction was performed by using a Qproteome FFPE tissue kit or the guanidine-HCl method (Figure 1). In cases in which the Qproteome FFPE tissue kit was used, the air-dried tissue pellet was resuspended in 100 μL of extraction buffer, briefly sonicated, and incubated at 100°C for 20 minutes. It was then incubated in the tube at 80°C for 2 hours with agitation and centrifugation (14,000 × g for 15 minutes at 4°C). After cooling, the supernatant was collected in a new microtube. To purify the protein sample, 400 μL of methanol, 100 μL of chloroform, and 300 μL of water were added to each tube in turn, followed by vortex mixing for 10 seconds and centrifugation. After removal of the upper layer, the protein was formed as a white gel-like pellet on the bottom of the tube when centrifuged after adding 300 μL of methanol. After washing the pellet with ethanol and centrifugation, the pellet was dissolved in 20 μL of β-ME Sample Treatment (Cosmo Bio, Tokyo, Japan). The protein samples were applied to 4% to 20% gradient SDS polyacrylamide gel (Cosmo Bio) and electrophoresed to stack the protein into the gel until the dye front reached to 5 cm below the bottom of the sample-loaded well. The protein-stacked gels were excised, and the gels were further divided into approximately 1-mm square cubes. After reduction and alkylation, in-gel digestion was performed with trypsin overnight at 37°C, as described by Shevchenko et al.26Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.Proc Natl Acad Sci U S A. 1996; 93: 14440-14445Crossref PubMed Scopus (1298) Google Scholar In cases in which the guanidine-HCl method was used to extract proteins from the pellet, the dried material was resuspended in 250 μL of 6 mol/L guanidine-HCl/50 mmol/L ammonium bicarbonate/20 mmol/L dithiothreitol (pH 8.5), briefly sonicated, and incubated at 100°C for 20 minutes and then incubated in the tube at 80°C for 2 hours with agitation. After cooling, the recovered proteins were quantified with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA). Iodoacetamide was added to a final concentration of 40 mmol/L, and the sample was incubated at room temperature in the dark for 1 hour. The alkylation reaction was quenched by adding 3 μL of 2 mol/L dithiothreitol. For tryptic digestion, the sample was diluted (1:6) with 50 mmol/L ammonium bicarbonate (pH 8.1) to reduce the concentration of guanidine-HCl buffer (final concentration of 1 mol/L) and digested with trypsin overnight. After digestion, the mixture was acidified with TFA (final concentration of 0.1%). To remove interfering substances before mass spectrometry, the mixture was desalted with MonoSpin C18 (GL Sciences, Tokyo, Japan), according to the manufacturer's instructions. Finally, peptides were eluted with 0.1% formic acid in 60% acetonitrile and stored at −20°C until analysis. Peptides were subsequently separated on a HiQsilC18W-3 column (100 μm inner diameter × 100; KYA Technology, Tokyo, Japan), according to a previous report.25Oki G. Wada T. Iba K. Aiki H. Sasaki K. Imai S. Sohma H. Matsumoto K. Yamaguchi M. Fujimiya M. Yamashita T. Kokai Y. Metallothionein deficiency in the injured peripheral nerves of complex regional pain syndrome as revealed by proteomics.Pain. 2012; 153: 532-539Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Elution solvent A and solvent B were 0.1% TFA and 0.1% TFA, respectively, in 70% acetonitrile. The gradient was 5% to 50% for solvent B over 50 minutes at a flow rate of 300 nL/minute. Separated peptides were spotted onto 384-well AB OptiTOF MALDI Plate Inserts (AB Sciex, Foster City, CA). The 150-nL peptide fractions were overlaid with 700 nL aCHCA (Sigma, Tokyo, Japan) in 80 mg/mL ammonium citrate, 70% acetonitrile, and 0.1% TFA. A 4800 Plus MALDI-TOF/TOF Analyzer (AB Sciex), with 4000 Series Explorer version 3.5 software (AB Sciex), was used for mass spectrometric analyses. The mass accuracy was calibrated by using tryptic bovine serum albumin standard (KYA Technology) and a six-peptide mixture (AB Sciex). MS/MS acquisitions of MS spectra with signal to noise ratio >100 were performed using air as the collision gas with a collision energy of 1 kV. Peptide tolerance was 100 ppm, and MS/MS tolerance was 1 Da. Data were processed by Protein Pilot version 3.0 (AB Sciex, Framingham, MA) using the Paragon search algorithm.27Shilov I.V. Seymour S.L. Patel A.A. Loboda A. Tang W.H. Keating S.P. Hunter C.L. Nuwaysir L.M. Schaeffer D.A. The Paragon algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.Mol Cell Proteomics. 2007; 6: 1638-1655Crossref PubMed Scopus (1059) Google Scholar MS/MS data were searched against the human International Protein Index database version 3.63 (European Bioinformatics Institute, Cambridgeshire, UK). The Unused ProtScore reflects the total amount of unique peptide evidence related to a given protein. Specimens of 22 cases of ALT/WDL, 18 cases of DDL, and 25 cases of lipoma, obtained by surgical resections during the period from 2006 to 2017, were retrieved from the pathology file of Sapporo Medical University Hospital. The hematoxylin and eosin slides of all cases were reviewed to select representative sections. New sections from paraffin blocks were examined by the labeled polymer method. Immunohistochemical labeling was visualized using Dako REAL EnVision Detection System (Agilent Technologies, Inc., Santa Clara, CA), as per the manufacturer's protocol.28Akimoto T. Takasawa A. Murata M. Kojima Y. Takasawa K. Nojima M. Aoyama T. Hiratsuka Y. Ono Y. Tanaka S. Osanai M. Hasegawa T. Saito T. Sawada N. Analysis of the expression and localization of tight junction transmembrane proteins, claudin-1, -4, -7, occludin and JAM-A, in human cervical adenocarcinoma.Histol Histopathol. 2016; 31: 921-931PubMed Google Scholar, 29Ueda A. Takasawa A. Akimoto T. Takasawa K. Aoyama T. Ino Y. Nojima M. Ono Y. Murata M. Osanai M. Hasegawa T. Saito T. Sawada N. Prognostic significance of the co-expression of EGFR and HER2 in adenocarcinoma of the uterine cervix.PLoS One. 2017; 12: e0184123Crossref PubMed Scopus (13) Google Scholar, 30Keira Y. Takasawa A. Murata M. Nojima M. Takasawa K. Ogino J. Higashiura Y. Sasaki A. Kimura Y. Mizuguchi T. Tanaka S. Hirata K. Sawada N. Hasegawa T. An immunohistochemical marker panel including claudin-18, maspin, and p53 improves diagnostic accuracy of bile duct neoplasms in surgical and presurgical biopsy specimens.Virchows Arch. 2015; 466: 265-277Crossref PubMed Scopus (27) Google Scholar Antigen of CCDC180, LRRC4, chymase, MDM2, and CDK4 retrieval was performed by immersing sections in 10 mmol/L tris/1 mmol/L EDTA buffer (pH 9.0) or 10 mmol/L citrate buffer (pH 6.0) and boiling them in a microwave (95°C, 30 minutes), followed by overnight incubation at 4°C with antibodies to the following antigens: CCDC180, LRRC4, chymase, MDM2, and CDK4. The intensity of staining was assessed as strong (3+), moderate (2+), weak (1+), or negative (0). All slides were independently evaluated by two pathologists (A.T. and M.M.). Discordant cases were discussed, and a consensus was reached.31Aoyama T. Takasawa A. Murata M. Osanai M. Takano K. Hasagawa T. Sawada N. Immunoreactivity patterns of tight junction proteins are useful for differential diagnosis of human salivary gland tumors.Med Mol Morphol. 2019; 52: 23-35Google Scholar The human liposarcoma cell lines 93T449 and SW872 were purchased from ATCC (Manassas, VA). 93T449 cells (derived from well-differentiated liposarcoma) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). SW872 cells were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum. The medium for the cell lines contained 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were plated on 60-mm culture dishes (Corning Glass Works, Corning, NY) and incubated in a humidified 5% CO2 incubator at 37°C. 93T449 cells were treated with 40 mmol/L cisplatin for 24 hours. One-day post-confluent 93T449 cells were treated with an adipogenic cocktail containing 0.5 mmol/L 3-isobutyl-1-methylxanthine and 0.25 μmol/L dexamethasone. After 4 days, the medium was replaced with fresh Dulbecco's modified Eagle's medium supplemented with 8% fetal bovine serum containing 100 μmol/L oleic acid, and the medium was replaced every 2 to 3 days for 21 days. Cells were harvested for Western blot analysis or examined by using Oil Red O staining (Wako Pure Chemical Industries, Ltd, Osaka, Japan). Cells in 60-mm culture dishes were washed with phosphate-buffered saline, fixed with 10% formalin neutral buffer solution for 5 minutes, incubated with 0.3% (w/v) Oil Red O in 60% (v/v) isopropanol for 30 minutes at room temperature, and washed with distilled water. Fat droplets in the adipogenesis-induced cells were evaluated with a phase-contrast microscope.32Serlachius M. Andersson L.C. Upregulated expression of stanniocalcin-1 during adipogenesis.Exp Cell Res. 2004; 296: 256-264Crossref PubMed Scopus (33) Google Scholar siRNA targeting the mRNA sequences of human CCDC180 or LRRC4 and siRNA universal negative control were purchased from Sigma-Aldrich. Transfection of siRNA was performed using RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer's instructions. siRNAs used in this study are listed in Table 2.Table 2List of siRNAs Used in This StudyName of siRNAGeneSequence of siRNAsiCCDC#1CCDC1805′-GCCAAUACUUCUUUGUGCGUGAAAU-3′siCCDC#2CCDC1805′-GGUUGCUGACGAAUCUGCAAGUGAA-3′siLRRC#1LRRC45′-CUGGUUACCAGCCGGCAUA-3′siLRRC#2LRRC45′-GUAUAUCUCUGAGGGAGCU-3′ Open table in a new tab Cell blocks were prepared by using the sodium alginate method.28Akimoto T. Takasawa A. Murata M. Kojima Y. Takasawa K. Nojima M. Aoyama T. Hiratsuka Y. Ono Y. Tanaka S. Osanai M. Hasegawa T. Saito T. Sawada N. Analysis of the expression and localization of tight junction transmembrane proteins, claudin-1, -4, -7, occludin and JAM-A, in human cervical adenocarcinoma.Histol Histopathol. 2016; 31: 921-931PubMed Google Scholar Briefly, cells were collected in a 2.0-mL tube and washed with phosphate-buffered saline. Supernatant was discarded after centrifugation, and cells were fixed in 10% formalin solution for 12 to 24 hours at 4°C. Fixative was discarded after centrifugation, and aggregated cells were gently suspended in 1% sodium alginate solution (Wako Pure Chemical Industries, Ltd). Then, 1 mol/L CaCl2 was added to form gel, and the fixed cell–containing gel was embedded in paraffin. Thin sections (4 μm thick) were cut from paraffin-embedded cell blocks and stained with hematoxylin and eosin. Deparaffinized sections were immersed in 10 mmol/L sodium citrate buffer (pH 6.0) and autoclaved for antigen retrieval. Primary antibodies were anti–Ki-67 or cleaved caspase-3. Immunohistochemical labeling was visualized using the EnVision system, as per the manufacturer's protocol.28Akimoto T. Takasawa A. Murata M. Kojima Y. Takasawa K. Nojima M. Aoyama T. Hiratsuka Y. Ono Y. Tanaka S. Osanai M. Hasegawa T. Saito T. Sawada N. Analysis of the expression and localization of tight junction transmembrane proteins, claudin-1, -4, -7, occludin and JAM-A, in human cervical adenocarcinoma.Histol Histopathol. 2016; 31: 921-931PubMed Google Scholar, 29Ueda A. Takasawa A. Akimoto T. Takasawa K. Aoyama T. Ino Y. Nojima M. Ono Y. Murata M. Osanai M. Hasegawa T. Saito T. Sawada N. Prognostic significance of the co-expression of EGFR and HER2 in adenocarcinoma of the uterine cervix.PLoS One. 2017; 12: e0184123Crossref PubMed Scopus (13) Google Scholar RNA isolation and RT-PCR were performed, as described previously.33Takasawa A. Murata M. Takasawa K. Ono Y. Osanai M. Tanaka S. Nojima M. Kono T. Hirata K. Kojima T. Sawada N. Nuclear localization of tricellulin promotes the oncogenic property of pancreatic cancer.Sci Rep. 2016; 6: 33582Crossref PubMed Scopus (16) Google Scholar Primers used for RT-PCR of CCDC180 and LRRC4 are listed in Table 3.Table 3List of Primers Used for RT-PCRGeneForward primerReverse primerProduct size, bpCCDC1805′-AAGAAGGCTGTACCCAGTGC-3′5′-TCTCCCAGAGGAGGGTCAAG-3′192LRRC

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