Protocadherin 10 Expression Research Articles

PCDH8 is a novel prognostic biomarker in thyroid cancer and promotes cell proliferation and viability

Protocadherin 8 (PCDH8), a calcium-dependent transmembrane protein in the protocadherin family, regulates cell adhesion and signal transduction. While some studies have provided indirect evidence that PCDH8 has cancer-promoting properties, this association is controversial. In particular, its involvement in thyroid cancer (THCA) remains unclear. We aimed to elucidate the role of PCDH8 in THCA using bioinformatic analysis. Subsequently, the results were experimentally validated. The analysis conducted using the R programming language and online web tools explored PCDH8 expression levels, prognostic, and clinical implications, and its relationship with the tumor immune microenvironment in THCA. Furthermore, we examined the association between PCDH8 and co-expressed genes, highlighting their involvement in several biological processes relevant to THCA. The potential of PCDH8 as a therapeutic target for this pathology was also explored. Immunohistochemical (IHC) staining was performed on samples from 98 patients with THCA, and experimental validation was carried out. PCDH8 was significantly elevated in cancer tissues and associated with poor prognosis, several clinical factors, and immune cell and checkpoint abundance. Cox regression and survival analyses, together with Receiver Operating Curves (ROC) indicated that PCDH8 was an independent prognostic factor for THCA. Furthermore, PCDH8 impacts cell viability and proliferation, promoting tumorigenesis. Also, it influences tumor cell sensitivity to various drugs. Thus, PCDH8 might be a potential therapeutic target for THCA. IHC, cell culture, MTT, and colony formation experiments further confirmed our findings. This analysis provided insights into the potential carcinogenic role of PCDH8 in THCA, as it impacts cell viability and proliferation. Thus, PCDH8 might play an important role in its prognosis, immune infiltration, and diagnosis.

E3 ubiquitin ligase RNF180 prevents excessive PCDH10 methylation to suppress the proliferation and metastasis of gastric cancer cells by promoting ubiquitination of DNMT1

BackgroundDownregulation of certain tumor-suppressor genes (TSGs) by aberrant methylation of CpG islands in the promoter region contributes a great deal to the oncogenesis and progression of several cancers, including gastric cancer (GC). Protocadherin 10 (PCDH10) is a newly identified TSG in various cancers and is downregulated in GC; however, the specific mechanisms of PCDH10 in GC remain elusive. Here, we elucidated a novel epigenetic regulatory signaling pathway involving the E3 ubiquitin ligase RNF180 and DNA methyltransferase 1 (DNMT1), responsible for modulating PCDH10 expression by affecting its promoter methylation.ResultsWe revealed that PCDH10 was downregulated in GC cells and tissues, and low PCDH10 expression was correlated with lymph node metastasis and poor prognosis in patients with GC. Additionally, PCDH10 overexpression suppressed GC cell proliferation and metastasis. Mechanistically, DNMT1-mediated promoter hypermethylation resulted in decreased expression of PCDH10 in GC tissues and cells. Further analysis revealed that RNF180 can bind directly to DNMT1 and was involved in DNMT1 degradation via ubiquitination. Additionally, a positive correlation was found between RNF180 and PCDH10 expression and an inverse association between DNMT1 and PCDH10 expression showed considerable prognostic significance.ConclusionOur data showed that RNF180 overexpression upregulated PCDH10 expression via ubiquitin-dependent degradation of DNMT1, thus suppressing GC cell proliferation, indicating that the RNF180/DNMT1/PCDH10 axis could be a potential therapeutic target for GC treatment.

DNA Methylation and the Expression of the Tumor-Suppressor Genes Protocadherin-10 and Reprimo in Pediatric Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is the most prevalent hematologic malignancy among children. DNA methylation is well known to play a role in the initiation and progression of cancer. This research aimed to characterize the epigenetic inactivation and gene expression of Protocadherin-10 (PCDH10) and Reprimo (RPRM) in pediatric acute lymphoblastic leukemia, which is associated with epigenetic silencing in human cancers.
 Materials and Methods: This case-control study included 21 children (mean age, 4.5 years) with new cases of ALL. In addition, 15 patients with ALL were selected following treatment. Samples of bone marrow were extracted from each patient. Prior to and following treatment, 15 paired samples were analyzed. In addition, ALL patients were compared to 18 normal controls in a case study. MS-HRM and quantitative real-time PCR were utilized to examine the DNA-promoter methylation and gene expression of two TSGs from each group.
 Results: Expression of PCDH10 was significantly up regulated in treatment ALL group compared to the new cases of ALL patients (P= 0.0119), PCDH10 gene expression was higher in the normal control group than in the new cases of ALL (P <0.0001). RPRM gene, also had a slightly increase in gene expression in treatment ALL group compared to the new cases of ALL (P =0.0412). RPRM gene expression was higher in the normal control group than in the new cases of ALL) P = 0.0372). There was an increase in methylation in both PCDH10 and RPRM genes in new cases of ALL groups compared with treatment ALL group and normal group (P<0.0001 for both genes).
 Conclusions: New cases of ALL were associated with high DNA methylation level and low gene expression of PCDH10 and RPRM genes.

PCDH8 participates in the growth process of colorectal cancer cells by regulating the AKT/GSK3β/β-catenin signaling pathway

Protocadherin 8 (PCDH8) is lower-expressed in many cancers (gastric cancer, breast cancer, bladder cancer and nasopharyngeal cancer), while the molecular mechanism underlying its effects in human colorectal cancer has not been reported. Therefore, this study aims to investigate the mechanism of PCDH8 in colorectal cancer. After colorectal cancer cells transfected with overexpressing or silencing the gene PCDH8, then the effects of PCDH8 on cell viability, migration, invasion and apoptosis were determined by a series of molecular biological experiments, respectively. The expressions of apoptosis-related factors, AKT/GSK3β/β-catenin signaling pathway- and epithelial-mesenchymal transition (EMT)-related proteins were quantified by western blotting. PCDH8 expression was significantly reduced in the colorectal cancer tissue and cell lines. Overexpressed PCDH8 significantly reduced the proliferation, migration and invasion of colorectal cancer cells, while silent PCDH8 had the opposite effects. Moreover, overexpressed PCDH8 could induce apoptosis, significantly down-regulate expressions of B-cell lymphoma-2 (Bcl-2), N-Cadherin and Vimentin but up-regulate those of Bcl-2 associated X (Bax), cleaved caspase-3 and E-Cadherin, whereas the negative regulations on the apoptosis and the expressions of apoptosis- and EMT-related proteins were observed in colorectal cancer cells following the silence of PCDH8. Furthermore, overexpressed PCDH8 significantly inhibited the phosphorylation of AKT and GSK3β, and repressed the expression of β-catenin, while silent PCDH8 had the opposite effects. In short, PCDH8 overexpression inhibits proliferation, invasion, EMT, and induces apoptosis of colorectal cancer cells by regulating AKT/GSK3β/β-catenin signaling pathway.

The effects of protocadherin 8 and WWOX in prostate adenocarcinoma

Objective: The protocadherin 8 (PCDH8) gene, located on chromosome 13q14.3 encodes an integral membrane protein. WWOX (fragile site FRA16D oxido-reductase) is a tumor suppressor gene located in region 16q23.324.1. The aim of this study was to investigate the staining pattern of protocadherin 8 and WWOX in adenomatous hyperplasia and prostate adenocarcinoma. Methods: Seventy adenomatous hyperplasia and 70 prostate adenocarcinoma preparations stored at the pathology department from 2013–2016 were retrospectively analyzed. Samples immunohistochemically stained with WWOX or protocadherin 8 were evaluated by two pathologists under a light microscope. After pathological investigation of the samples, the expression of WWOX and protocadherin 8 was scored. WWOX and PCDH8 expression was assessed semi-quantitatively according to staining intensity scored as none, mild, moderate, and strong (0 to 3+). For analysis of the data, the mild, moderate and strong staining scores were combined (as positive) and the data were classified as negative or positive. Differences in the WWOX or protocadherin 8 staining results between adenomatous hyperplasia and prostate adenocarcinoma cases were examined using a two-way chi-square test and binary logistic regression analysis. Result: Statistical analysis showed that WWOX expression was higher in adenomatous hyperplasia than in prostate adenocarcinomas. This difference was statistically significant (p = 0.035). There was no difference in PCDH8 expression between adenomatous hyperplasia and prostate adenocarcinoma samples (p = 0.217). Conclusion: In this study, the expression of WWOX decreased and the expression of PCDH8 remained unchanged in PCa cases. In terms of prognostic significance, it can be concluded that WWOX is a good prognostic parameter, while PCDH8 was an ineffective prognostic marker. In addition, the investigation of protocadherin 8 in larger series may provide more meaningful results.

HOTAIR Induces Methylation of PCDH10, a Tumor Suppressor Gene, by Regulating DNMT1 and Sponging with miR-148b in Gastric Adenocarcinoma.

PurposeHOX transcript antisense intergenic RNA (HOTAIR), as a long non-coding RNA, has been reported to regulate carcinogenesis by epigenetic mechanism in various cancers. Protocadherin 10 (PCDH10) is one of the well-known tumor suppressor genes, and is frequently methylated in gastric cancers (GC). We aimed to investigate the detailed pathway of how HOTAIR contributes to the target gene in gastric carcinogenesis.Materials and MethodsWe investigated the mechanism of HOTAIR on carcinogenesis and metastasis of GC. Methylation-specific PCR was performed to identify the interaction between HOTAIR and PCDH10. In addition, we investigated the interaction between miR-148b and HOTAIR by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay.ResultsThe expression of HOTAIR was significantly upregulated in GC tissues (p<0.05) and GC cell lines (p<0.01), while PCDH10 was downregulated in GC tissues (p<0.05). The knockdown of HOTAIR (si-HOTAIR1 and 2) significantly upregulated the mRNA/protein expression of PCDH10 and reduced the methylation of PCDH10 compared to the control in MKN 28 and MKN 74. Si-HOTAIR1 and 2 significantly reduced DNA methyltransferase 1 (DNMT1) expression, and overexpression of HOTAIR increased DNMT1 expression. In RIP, we found that miR-148b interacted with HOTAIR. Si-HOTAIRs increased miR-148b expression, and miR-148b mimic inversely reduced HOTAIR expression. Si-HOTAIRs and miR-148b mimic reduced DNMT1 expression and increased PCDH10 expression compared to the control.ConclusionThis study demonstrated that HOTAIR interacts with miR-148b and DNMT1, eventually leading to PCDH10 methylation, which contributes to the progression of GC. Our findings provide a better understanding for detailed pathway of HOTAIR in epigenetic mechanism of GC.

PCDH10 exerts tumor-suppressor functions through modulation of EGFR/AKT axis in colorectal cancer.

Protocadherin 10 (PCDH10) is identified as a tumor suppressor in multiple cancers. The molecular mechanisms that mediate the functions of PCDH10 have yet to be fully elucidated. Here, we demonstrated that ectopic expression of PCDH10 in colorectal cancer (CRC) cells induced cell cycle retardation and increased apoptosis through regulation of the p53/p21/Rb axis and Bcl-2 expression. Overexpression of PCDH10 reversed the epithelial-mesenchymal transition (EMT) process with morphological changes and EMT marker alterations. Mechanistic study revealed that PCDH10 inhibited AKT/GSK3β signaling pathway which in turn reduced β-catenin activity and thus attenuated Snail and Twist1 expression. Furthermore, PCDH10 inhibited the stemness of CRC cells, including spheroid formation and stem cell markers. A proteomics approach revealed that PCDH10 could interact with EGFR, which was further verified by co-immunoprecipitation. Moreover, restoration of PCDH10 expression reduced EGFR phosphorylation. Accordingly, our work proposes a novel pathway by which PCDH10 directly engages in the negative regulation of EGFR/AKT/β-catenin signaling pathway, resulting in tumor suppression.

Quantitation Analysis of PCDH10 Methylation in Adolescent Idiopathic Scoliosis Using Pyrosequencing Study.

A retrospective and comparative study. To evaluate the difference of DNA methylation in protocadherin10 (PCDH10) genes between adolescent idiopathic scoliosis (AIS) and normal controls, and to assess the association between DNA methylation and the etiology of AIS. The PCDH10 gene showed abnormal expression in AIS. However, the mechanism was still unclear. DNA methylation was an important epigenetic mechanism at the interface between genetics and environmental phenotype, seeming to be a suitable epigenetic mark for the abnormal expression of PCDH10 in AIS. There were 50 AIS patients and 50 healthy controls included in the study. The peripheral blood sample of each participant was taken. The pyrosequencing assay was used to assess the methylation status of PCDH10 promoter and real time PCR (RT-PCR) was used to detect the PCDH10 gene expression. The comparison analysis was performed using independent t test and 2-tailed Pearson coefficients was calculated for the correlation analysis. The average methylation level was 4.32 ± 0.73 in AIS patients and 3.14 ± 0.97 in healthy controls (P < 0.001). The PCDH10 gene expression was 0.23 ± 0.04 in AIS patients and 0.36 ± 0.08 in normal controls (P < 0.0001). Statistically significant linear correlation was found between PCDH10 gene methylation level and Cobb angle of major curve (P < 0.001). Besides, a significant negative correlation between PCDH10 methylation and PCDH10 gene expression was found (P < 0.001). AIS patients were associated with high DNA methylation level and low gene expression of PCDH10 gene rather than normal controls. The high methylation level indicated high Cobb angle of major curves in AIS. The abnormal DNA methylation may widely exist and serve as a potential mechanism for AIS. 3.

Epigenetic Inactivation of Protocadherin 10 by Methylation in Colorectal Cancer

Aberrant promoter methylation of CpG islands of tumor-suppressor genes has been recognized as one of the important tumor markers for cancer detection. The aim of this study was to investigate the promoter methylation status of protocadherin 10 (PCDH10), a tumor suppressor gene, in Iranian colorectal cancer (CRC) patients. Cancerous and the adjacent normal tissues obtained from 38 CRC patients were used to assess the methylation status of PCDH10 with Methylation Specific PCR, in addition, to study the expression level of this gene by quantitative PCR. The relationship between hypermethylation and the demographic characteristics of these patients was analyzed. The promoter methylation level of PCDH10 was statistically different between tumoral and normal tissues in CRC patients. Twenty-seven out of 38 patients showed hypermethylation with a sensitivity of 73% and a specificity of 97%. PCDH10 expression decreased in 15 cases (46%) as 16 cases (50 %) showed overexpression and 1 case (4%) had no changes. Not a significant association was reported between PCDH10 hypermethylation and the clinicopathological characteristics (P&gt;0.05). Our results indicated that PCDH10 methylation has a critical function in CRC, with a nearly elevated sensitivity and a high specificity in the Iranian population, qualify it as a potential candidate biomarker. © 2019 Tehran University of Medical Sciences. All rights reserved. Acta Med Iran 2019;57(8):472-477.

PCDH10 inhibits invasion of lymphoma cells by regulating β-catenin.

The aim of this study was to investigate the effect of protocadherin 10 (PCDH10) on the invasive potential of lymphoma cells by regulating matrix metalloproteinase-7 (MMP7) and MMP9 via targeting β-catenin. The mRNA and protein expressions of PCDH10, β-catenin, MMP7 and MMP9 in lymphoma cell lines were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Raji cells with low expression of PCDH10 were transiently transfected with pCMV5-HA-PCDH10 or pcDNA5-His-β-catenin. Meanwhile, HUT-78 cells with high expression of PCDH10 were transfected with PCDH10-shRNA or β-catenin-shRNA. Subsequently, the expression levels of β-catenin, MMP7 and MMP9 in transfected lymphoma cells were determined as well. In addition, the regulatory effects of PCDH10 on the invasive potential of lymphoma cells were explored by transwell assay. PCDH10 expression was negatively correlated with the expressions of β-catenin, MMP7 and MMP9 in several lymphoma cell lines. Transfection of HA-PCDH10 in human-derived malignant B lymphoma cell line Raji markedly down-regulated the protein levels of β-catenin, MMP7 and MMP9. However, the mRNA level of β-catenin was not influenced by PCDH10. Interference with PCDH10 in HUT-78 cells with high expression of PCDH10 significantly increased the protein expressions of β-catenin, MMP7, and MMP9. However, no significant changes were observed in the mRNA expression of β-catenin. In addition, knockdown of β-catenin in cells with high expression of PCDH10 remarkably down-regulated the expression levels of MMP7 and MMP9. PCDH10 overexpression in lymphoma cells downregulates β-catenin expression, as well as inhibits the expressions of MMP7 and MMP9, eventually inhibiting the invasive potential of lymphoma cells.

Estrogen receptor‑α is a determinant of protocadherin‑8 expression in breast cancer cells

Protocadherin‑8 (PCDH8) is a member of the protocadherin subfamily within the cadherin superfamily of cell adhesion molecules. PCDH8 is silenced in a variety of solid tumors including those of the breast, and believed to be a tumor suppressor. Here we show that PCDH8 is suppressed by estrogen in MCF-7 and T47D breast cancer cells. The suppression is dependent on estrogen receptor‑α (ERα) expression. Knockdown of ERα alone induces the expression of PCDH8. The inverse correlation between PCDH8 and ERα expression is also apparent from the analysis of PCDH8 expression in breast tumors from the TCGA-BRCA dataset. Since estrogen produces mitogenic effects in ERα positive breast cancer cells, estrogen-mediated suppression of PCDH8 is consistent with its purported tumor suppressor function. Functional studies on PCDH8 in breast cancer cells may yield valuable insights into the mechanisms of estrogen-mediated epithelial-mesenchymal transition.

Down-regulated of PCDH10 predicts poor prognosis in hepatocellular carcinoma patients.

Protocadherin10 (PCDH10), a member of the nonclustered protocadherin family, functions as a tumor suppressor in many cancers. The aim of this study was to evaluate the expression level and prognostic value of PCDH10 in hepatocellular carcinoma (HCC) patients.Quantitative real-time polymerase chain reaction was used to analyze the expression level of PCDH10 in HCC tissues and adjacent nontumor tissues. The association of PCDH10 expression with clinicopathological features of patients was evaluated by chi-squared test. Overall survival was estimated using the Kaplan–Meier method. Besides, the patient prognosis was also evaluated by Cox regression analysis.PCDH10 expression was significantly lower in HCC tissues than that in adjacent nontumor tissues (P = .000). Kaplan–Meier curves showed that patients with lower PCDH10 expression had a worse overall survival. Moreover, PCDH10 expression level was associated tumor size (P = .005), tumor node metastasis stage (P = .002), smoking status (P = .000), and drinking status (P = .005). Multivariate analysis showed that the expression of PCDH10 (P = .000; hazard ratio = 4.784; 95% confidence interval: 2.550–8.977) was an independently associated with poor overall survival rates, as well as smoking status and drinking status.Our findings indicated that the decreased expression of PCDH10 was closely associated with poor prognosis of HCC patients. It might be considered as a valuable biomarker for HCC.

Expression of protocadherin8: Function as a tumor suppressor in hypopharyngeal carcinoma.

Protocadherin8 (PCDH8), an integral membrane protein, was reported to be a tumor suppressor involved in tumorigenesis in cancers. We aimed to investigate the expression of PCDH8 and its clinicopathological significance in hypopharyngeal carcinoma. We also examined the possible inactivation mechanism of PCDH8. A total of 80 pairs of hypopharyngeal carcinoma tumor tissues and non-tumor tissues were investigated to examine the immunohistochemical expression of PCDH8. Prognostic value and clinicopathological significance of PCDH8 expression were examined by Kaplan-Meier and log-rank test and Cox's ones. Ten pairs of tumor tissues and non-tumor tissues were analyzed by RT-PCR and 4 pairs by Western blot respectively. Promoter methylation of PCDH8 was observed in 14 pairs of tumor tissues and non-tumor tissues by methylation-specific PCR (MSP). The expression of PCDH8 in tumor tissues was depressed immunohistochemically when compared with non-tumor tissues and was significantly lower in the advanced pathological stage. Meanwhile, the expression of PCDH8 served as an independent prognostic risk factor for overall survival of hypopharyngeal carcinoma. The mRNA and protein levels of PCDH8 in tumor tissues was also down-regulated than in non-tumor tissues. Moreover, aberrant promoter methylation of PCDH8 occurred frequently in tumor tissues, rather than in non-tumor tissues. For the first time, our study demonstrates the tumor-suppressive function of PCDH8 and its epigenetic inactivation by promoter methylation in hypopharyngeal carcinoma. It suggests that PCDH8 may serve as a useful prognostic biomarker and a potential therapeutic target for hypopharyngeal carcinoma patients.

Protocadherin-8 promotes invasion and metastasis via laminin subunit γ2 in gastric cancer.

Growing evidence suggests that protocadherins (PCDH) play crucial roles in pathogenesis and progression of cancers, including gastric cancer (GC). Protocadherin‐8 (PCDH8) was previously reported to be involved in metastasis of GC, but functional studies yielded inconsistent results and the molecular mechanism remained unknown. The present study aimed to explore the clinical relevance, function and molecular mechanism of PCDH8 in GC. Data from the GEPIA and Kaplan–Meier plotter databases showed that high expression of PCDH8 was significantly correlated with poorer prognosis in GC. Ectopic expression of PCDH8 in GC cells promoted invasion and migration in vitro and metastasis in vivo, and knockdown of PCDH8 inhibited invasion and migration in vitro. RNA sequencing followed by gene set enrichment analysis found a remarkable enrichment in the extracellular matrix receptor interaction pathway, with the expression of laminin subunit γ2 (LAMC2) being significantly increased in the PCDH8‐overexpressing group. High expression of LAMC2 was significantly correlated to poor prognosis in GC in GEPIA database. Upregulation of LAMC2 following PCDH8 overexpression was further confirmed by immunohistochemistry in liver metastatic lesions of nude mice. To our knowledge, this is the first report of the metastasis‐enhancing property and molecular mechanism through upregulation of LAMC2 of PCDH8 in cancer. High expression of PCDH8 could be used as a biomarker for poor prognosis in clinical practice.

Low Expression of Protocadherin-8 Promotes the Progression of Ovarian Cancer.

ObjectiveOvarian cancer (OC) is the second most lethal gynecological cancer among women throughout the world. Protocadherin-8 (PCDH8) could function as a candidate tumor suppressor. However, the link between PCDH8 and OC development is poorly understood.Materials and MethodsA total of 68 OC patients were retrospectively enrolled. Clinical information was collected and cancer tissues were used for tissue microarray. The PCDH8 expression was determined on tissue microarray by immunohistochemical staining, and PCDH8 protein was detected in cancer tissues and adjacent tissue by western blotting. Human OC cell lines (SKOV-3 and OVCAR-3) were used to assess the effects of PCDH8 overexpression by western blot and real-time PCR analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay, wound healing migration assay, colony formation assay and invasion assays were performed to assess the influence of PCDH8 on cell function. Cells with Luc-nonspecific Lentiviral or Luc-Lentiviral with PCDH8 gene were subcutaneously injected into nude mice to observe the effect of PCDH8 gene on tumor growth. Bioluminescence imaging was used to observe tumor volume.ResultsWe found a low expression of PCDH8 in OC tissues versus the corresponding adjacent tissue. The PCDH8 expression, International Federation of Gynecology and Obstetrics stage, metastasis and recurrence were the independent prognostic factors for over-all survival by multivariate analyses. Furthermore, the patients with recurrence presented a low level of PCDH8 in OC tissues, and patients with advanced tumor stage also had a low PCDH8 expression. Importantly, the low expression of PCDH8 in OC tissues had a poor prognosis with a low overall survival rate. Overexpression of PCDH8 could inhibit OC cell growth/proliferation, migration, invasion, and colony formation in vitro. In vivo experiments also proved that overexpression of PCDH8 could inhibit OC cell growth/proliferation.ConclusionsProtocadherin-8 might be considered as a candidate tumor suppressor and play a crucial role in the progression of OC.

Retracted Article: Overexpression of PCDH8 inhibits proliferation and invasion, and induces apoptosis in papillary thyroid cancer cells.

Protocadherin8 (PCDH8), a member of the protocadherin (PCDH) family, is involved in the progression of several types of cancers. However, the expression and biological roles of PCDH8 in papillary thyroid carcinoma (PTC) remain largely unknown. Therefore, in the present study, we detected the expression of PCDH8 in human PTC tissues and cell lines, and evaluated its role in PTC cells. Our results demonstrated that PCDH8 was lowly expressed in human PTC tissues and cell lines. In addition, ectopic expression of PCDH8 efficiently inhibited the proliferation, migration and invasion of PTC cells, as well as prevented the epithelial-mesenchymal transition (EMT) phenotype in PTC cells. Furthermore, PCDH8 efficiently induced apoptosis and autophagy in PTC cells. Mechanistically, overexpression of PCDH8 significantly prevented the activation of the Wnt/β-catenin pathway in PTC cells. Taken together, these findings showed that overexpression of PCDH8 inhibits proliferation and migration/invasion, and induces apoptosis of PTC cells through the Wnt/β-catenin signaling pathway.

108 Single-Cell RNA Sequencing Reveals Blastomere Heterogeneity and Early Lineage Specification Events in Bovine Embryos During Major Embryonic Genome Activation

During early embryonic stages, gene products generated by the embryo acquire control over embryonic development. At the 8- to 16-cell stage, major embryonic genome activation (EGA) occurs in bovine embryos. Morphological observations, such as size of blastomeres and distribution of microvilli, suggest heterogeneity of individual cells already at this developmental stage. To study this heterogeneity on the transcriptome level, we performed single-cell RNA sequencing (scRNA-seq) of 161 blastomeres from 14 in vitro-produced bovine embryos at Day 2 and Day 3 post-fertilization. After removing the zona pellucida, blastomeres were mechanically separated in Ca2+- and Mg2+-free PBS, individually collected, and lysed. Complementary DNA libraries were prepared by the single cell RNA-barcoding and sequencing (SCRB-Seq) protocol. Exogenous RNA was added for quality control and cell specific barcodes and unique molecular identifiers (UMI) were used to enable pooling of libraries and to exclude PCR duplicates, respectively. After sequencing (Illumina HiSEqn 1500; 50 nt reads; Illumina Inc., San Diego, CA, USA), UMI were counted with the published Drop-seq pipeline (45,000 UMI on average per library) and cells with UMI count &lt;2.000 were removed. Data were normalized based on UMI and non-supervised clustering analyses of single-cell data were performed (SC3 and M3Drop R packages). The transcriptome profiles of all individual cells were assigned to 6 clusters with specific sets of genes. Sorting cells according to their transcriptome profiles by the CellTree R package (Bioconductor; https://bioconductor.org/packages/release/bioc/html/cellTree.html) resulted in a linear pseudo-timeline. Furthermore, this tool identified 6 groups of genes (topics). Each of them showed an over-representation of distinct Gene Ontology (GO) terms; topic 1, “translation” and “cell division”; topic 2, GO terms involved in translation, RNA splicing and cell division; topic 3, “translation”; topic 4, “ATP synthesis coupled proton transport”; topic 5, “mitochondrial translational elongation”; topic 6, “organic hydroxyl compound transport”. Moreover, increased expression of PCDH10 (protocadherin 10) was observed in the biologically pseudo-ordered more advanced blastomeres. This gene is known to be predominantly expressed in the inner cell mass (ICM) at the blastocyst stage, suggesting that these cells might become ICM. In summary, our study reveals developmental heterogeneity and hints to early lineage specification events in bovine embryos at the time of major EGA.

Association between protocadherin 8 promoter hypermethylation and the pathological status of prostate cancer.

Promoter hypermethylation of tumor suppressor genes has been confirmed to serve a pivotal role in tumorigenesis. Protocadherin 8 (PCDH8), a novel tumor suppressor gene, has been reported to be inactivated by promoter hypermethylation a number of cancer types, including bladder cancer and renal cell carcinoma. The aim of the present study was to investigate the occurrence of PCDH8 hypermethylation in prostate cancer and its potential as a novel biomarker of prostate cancer. The transcriptional levels of PCDH8 were examined by quantitative polymerase chain reaction (PCR) in 82 prostate cancer tissues as well as 30 prostate hyperplasia tissues, and verified the protein level by western blot analysis of representative samples. PCDH8 expression levels were found to be reduced to 0.30±0.10 in 70.7% (58/82) of prostate cancer tissues. To identify the possible reason for mRNA downregulation, the methylation status of the PCDH8 promoter was assessed in prostate cancer tissues and prostate hyperplasia tissues by methylation-specific PCR (MSP). A total of 47 prostate cancer patients who exhibited reduced PCDH8 expression (57.3%; 47/82) also showed promoter hypermethylation (47/58). None of the samples (0/30) in the benign prostate hyperplasia group were positive on MSP. Furthermore, the associations between the methylation status of the PCDH8 promoter and various clinicopathological features of prostate cancer were analyzed, revealing that the methylation status of PCDH8 was closely associated with tumor size, tumor shape (papillary/non-papillary), tumor stage and tumor grade (all P<0.05), while there were no correlations with the age of the patients or the number of tumors (P>0.05). Additionally, patients with hypermethylation of the PCDH8 gene promoter had a relapse rate of 36.17% and a mortality rate of 29.79%, which were significantly higher than the hypermethylation-negative patients (P<0.05), indicating a poorer prognosis. Therefore, the methylation status of the PCDH8 gene in prostate cancer may be an important marker for use in the early diagnosis and prediction of prognosis in prostate cancer.

PCDH10 gene inhibits cell proliferation and induces cell apoptosis by inhibiting the PI3K/Akt signaling pathway in hepatocellular carcinoma cells.

Protocadherin10 (PCDH10), a member of the non-clustered protocadherin (PCDH) family, functions as a tumor-suppressor gene in many cancers. Previous studies have demonstrated that the expression of PCDH10 was noticeably downregulated in the tissue and cells of hepatocellular carcinoma (HCC), when compared to those in normal liver tissue. The decreased PCDH10 expression in HCC was correlated with the aberrant methylation status of PCDH10 promoter. However, the biological functions and molecular mechanism of PCDH10 in HCC have yet to be elucidated. The aim of the present study was to identify the biological function and mechanisms of PCDH10 in HCC. Quantitative real-time polymerase chain reaction was used to detect the expression of PCDH10 in HCC cells with decreased expression of PCDH10 which were transfected with plasmid pcDNA3.1-PCDH10 or pcDNA3.1-vector using Lipofectamine 2000. The biological effects of PCDH10 in HCC cells were detected by CCK-8, colony formation and flow cytometric assays. Western blot and co-immunoprecipitation (Co-IP) assays were performed to explore the mechanism of PCDH10 in HCC cells. PCDH10 expression was downregulated in the HCC cells (HepG2, HuH7, HuH1, and SNU387) when compared to the normal liver cells (L02). Upregulation of PCDH10 inhibited cell proliferation and induced cell apoptosis in the HCC cells. More importantly, we revealed that PCDH10 inhibited the PI3K/Akt signaling pathway thus carrying out its suppressive function in HCC. This study provides insights into the tumorigenesis and progression of HCC, and puts forward the novel hypothesis that PCDH10 could be a new biomarker for HCC, or that combined with other molecular markers could increase the specificity and sensitivity of diagnostic tests for HCC. Restoration of PCDH10 could be a valuable therapeutic target for HCC.

Promoter methylation of PCDH10 by HOTAIR regulates the progression of gastrointestinal stromal tumors

HOTAIR, a long non-coding RNA (lncRNA), plays a crucial role in tumor initiation and metastasis by interacting with the PRC2 complex and the modulation of its target genes. The role of HOTAIR in gastrointestinal stromal tumors (GISTs) is remains unclear. Herein we investigate the mechanism of HOTAIR in the genesis and promotion of GISTs. The expression of HOTAIR was found to be higher in surgically resected high-risk GISTs than that in low- and intermediate-risk GISTs. Using GIST-T1 and GIST882 cells, we demonstrated that HOTAIR repressed apoptosis, was associated with cell cycle progression, and controlled the invasion and migration of GIST cells. Using a gene expression microarray and lists of HOTAIR-associated candidate genes, we suggested that protocadherin 10 (PCDH10) is a key molecule. PCDH10 expression was significantly decreased in GIST-T1 and GIST882 cells, possibly as a consequence of hypermethylation. We observed that HOTAIR induced PCDH10 methylation in a SUZ12-dependent manner. In this study, we found that the malignant character of GISTs was initiated and amplified by PCDH10 in a process regulated by HOTAIR. In summary, our findings imply that PCDH10 and HOTAIR may be useful markers of disease progression and therapeutic targets.