The novel circSLC6A6/miR-1265/C2CD4A axis promotes colorectal cancer growth by suppressing p53 signaling pathway

Background Colorectal cancer (CRC) is one of the most frequent malignancy and a leading cause of cancer-related deaths. Therefore, further researches are required to identify novel and more effective diagnoses and to identify molecular targets in treatment of CRC. Methods C2CD4A expression in CRC tissues and cell lines was detected by qRT-PCR and western blot. The biological functions of C2CD4A were performed both in vitro and in vivo. Western blot, cDNA array, IP-MS, Co-immunoprecipitation assay, and Ubiquitination assay were used to analyze the interaction between C2CD4A and p53. Bioinformatics analysis, FISH, RNA sequencing, luciferase reporter assay, RNA immunoprecipitation, RNA pull-down and rescue experiments, were deployed to detect upstream regulation mechanism of C2CD4A. Results C2CD4A was elevated in CRC tissues compared with adjacent normal colorectal tissues. C2CD4A knockdown significantly promoted cell apoptosis and with inhibited proliferation in vitro, and tumorigenicity in vivo, whereas C2CD4A overexpression led to opposite effects. Moreover, circSLC6A6 was upregulated and shown to positively regulate C2CD4A expression via sponging miR-1265. Fundamentally, C2CD4A inhibited p53 signaling pathway through interacting with p53 and increasing its ubiquitination and degradation. Conclusion Our results identified that circSLC6A6/miR-1265/C2CD4A axis, which was involved in CRC via the p53 signaling pathway, may serve as a therapeutic target for CRC.

Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.

The Novel CircSLC6A6/miR-1265/C2CD4A Axis Promotes Colorectal Cancer Growth by Suppressing P53 Signaling Pathway

Background: Colorectal cancer (CRC) ranks as the third most frequently diagnosed cancer and is a leading cause of cancer-related deaths. Therefore, further researches were required to identify novel and more effective diagnoses and to identify molecular targets in treatment of CRC.Methods: CRC fresh frozen tissues and cell lines were used to detect C2CD4A expression by qRT-PCR and western blotting. The biological functions of C2CD4A were performed in vitro and in vivo. Western blotting, cDNA array, IP-MS, Co-IP, and Ubiquitination assay were used to analyze the interaction between C2CD4A and p53. Bioinformatics analysis, FISH, RNA sequencing, luciferase reporter assay, RNA immunoprecipitation, RNA pull-down and rescue experiments, were deployed to detect upstream regulation mechanism of C2CD4A.Results: C2CD4A was aberrantly upregulated in CRC tissues compared with adjacent normal colorectal tissues. C2CD4A knockdown significantly promoted cell apoptosis and with inhibited proliferation in vitro, and tumorigenicity in vivo, whereas C2CD4A overexpression had displayed an opposite effect. Moreover, circSLC6A6 was upregulated and positively associated with C2CD4A expression in CRC tissues. C2CD4A was positively regulated by circSLC6A6 via sponging miR-1265. Fundamentally, C2CD4A inhibited P53 signaling pathway through interacting with P53 and increasing its ubiquitination and degradation.Conclusion: Our results identified that circSLC6A6/miR-1265/C2CD4A axis, which was involved in CRC via the P53 signaling pathway, could be as a therapeutic target for CRC.

Upregulation of SNTB1 Correlates with Poor Prognosis and Promotes Cell Growth in Colorectal Cancer

Background: Colorectal cancer (CRC) is one of the most highly malignant tumors and has a complicated pathogenesis. A preliminary study identified syntrophin beta 1 (SNTB1) as a potential oncogene in CRC. However, the clinical significance, biological function, and underlying mechanisms of SNTB1 in CRC are unknown. Thus, the present study aimed to investigate the function of SNTB1 in CRC.Methods: The expression profile of SNTB1 in CRC samples was evaluated by database analysis, cDNA array, tissue microarray, Quantitative real-time PCR (qPCR), and immunohistochemistry. SNTB1 expression in human CRC cells was silenced using short hairpin RNAs and its mRNA and protein levels were assessed by qPCR and western blotting, respectively. Cell proliferation, colony formation, cell cycle and apoptosis were determined by the cell counting, colony formation, and flow cytometry assays, respectively. A xenograft nude mouse model of CRC was established for validating the roles of SNTB1 in vivo. Immunohistochemistry was used to score the expression of SNTB1 in tissue samples. The isobaric tags for relative and absolute quantification (iTRAQ) was used to analyze the differentially expressed proteins after knockdown of SNTB1 in CRC cells.Results: SNTB1 expression was increased in CRC tissue compared with adjacent noncancerous tissues and the increased expression was associated with shorter overall survival of CRC patients. Silencing of SNTB1 suppressed cell viability and survival, induced cell cycle arrest and apoptosis in vitro, and inhibited the growth of CRC cells in vivo. Further elucidation of the regulation of STNB on CRC growth by iTRAQ analysis identified 210 up-regulated and 55 down-regulated proteins in CRC cells after SNTB knockdown. A PPI network analysis identified protein kinase N2 (PKN2) as a hub protein and was up-regulated in CRC cells after SNTB1 knockdown. Western-blot analysis further confirmed that SNTB1 knockdown significantly up-regulated PKN2 protein expression in CRC cells and decreased the phosphorylation of both ERK1/2 and AKT. Conclusion: These findings indicate that SNTB1 is overexpressed in CRC. Elevated SNTB1 levels are correlated with shorter patient survival. Importantly, SNTB1 promoted tumor growth and progression of CRC, possibly by reducing the expression of PKN2 and activating the ERK and AKT signaling pathway. Our study highlights the potential of SNTB1 as a new prognostic predictor and therapeutic target for CRC.

High Expression of PPFIA1 Is Associated With Tumor Progression and a Poor Prognosis in Esophageal Squamous Cell Carcinoma

Background: PTPRF interacting protein alpha 1 (PPFIA1) is reportedly related to the occurrence and progression of several types of malignancies. However, its role in esophageal squamous cell carcinoma (ESCC) remains unknown. We aimed to investigate the expression and clinical value of PPFIA1 in ESCC.Methods: The Oncomine, Gene Expression Profiling Enrichment Analysis (GEPIA), and Gene Expression Omnibus (GEO) databases were utilized to explore PPFIA1 mRNA expression in esophageal cancer. The associations of PPFIA1 expression with clinicopathological variables and prognosis were evaluated in the GSE53625 dataset and verified in quantitative real-time polymerase chain reaction (qRT-PCR)-based cDNA array and immunohistochemistry (IHC)-based tissue microarray (TMA) datasets. The interactions between PPFIA1 and other genes based on the protein-protein interaction (PPI) network was analyzed via the STRING website.Results: PPFIA1 expression was obviously upregulated in ESCC tissues versus adjacent normal tissues according to online database analyses (all P<0.05). High PPFIA1 expression was significantly associated with several clinicopathological features, including tumor size, histological grade, tumor invasion depth, lymph node metastasis, and tumor-node-metastasis (TNM) stage. High PPFIA1 expression was related to worse outcomes and was identified as an independent prognostic indicator of overall survival (OS) in ESCC patients GSE53625 dataset, P=0.004; cDNA array dataset, P<0.001; TMA dataset, P=0.039). PPI analysis demonstrated that PPFIA1 was highly correlated with multiple genes, including UNC13B, RAB3A, PTPRD, and SYT1.Conclusion: PPFIA1 may be associated with ESCC progression and could be used as a biomarker for prognostic evaluation in ESCC patients.

Integrative microRNA and gene expression analysis identifies new epigenetically regulated microRNAs mediating taxane resistance in ovarian cancer

Taxane is a family of front-line chemotherapeutic agents against ovarian cancer (OC). The therapeutic efficacy is frequently counteracted by the development of chemoresistance, leading to high rates of relapse in OC patients. The role(s) of microRNAs (miRNAs) in cancer chemoresistance had been supported by many evidences Epigenetic regulation by miRNAs has been reported to influence cancer development and response to therapeutics, however, their role in OC resistance to paclitaxel (PTX) is unclear. Here, we conducted miRNA profiling in the responsive and PTX-resistant OC cell lines before and after treatment with epigenetic modulators. We reveal 157 miRNAs to be downregulated in the PTX-resistant cells compared to parental controls. The expression of five miRNAs (miRNA-7-5p, -204-3p, -501-5p, -3652 and -4286) was restored after epigenetic modulation, which was further confirmed by qPCR. In silico analysis of the signaling pathways targeted by the selected miRNAs identified the PI3K-AKT pathway as the primary target. Subsequent cDNA array analysis confirmed multiple PI3K-AKT pathway members such as AKT2, PIK3R3, CDKN1A, CCND2 and FGF2 to be upregulated in PTX-resistant cells. STRING analysis showed the deregulated genes in PTX-resistant cells to be primarily involved in cell cycle progression and survival. Thus, high throughput miRNA and cDNA profiling coupled with pathway analysis and data mining provide evidence for epigenetically regulated miRNAs-induced modulation of signaling pathways in PTX resistant OC cells. It paves the way to more in-depth mechanistic studies and new therapeutic strategies to combat chemoresistance.

Transcriptome profile and association study revealed STAT3 gene as a potential quality marker of bovine gametes

SummaryThe present study was aimed to investigate differences in molecular signatures in oocytes derived from Holstein-Friesian heifers with different genetic merit for fertility, euthanized during day 0 or day 12 of the estrous cycle. Moreover, association between single nucleotide polymorphisms (SNPs) of ODC1 and STAT3 genes and bull fertility traits was investigated. The gene expression patterns were analyzed using cDNA array and validated with quantitative real-time polymerase chain reaction (PCR). The result revealed that several genes have shown not only to be regulated by fertility merit but also by the day of oocyte recovery during the estrous cycle. The STAT3 gene was found to be upregulated in oocytes recovered from animals with high fertility merit at both day 0 and day 12. Some other genes like PTTG1, ODC1 and TUBA1C were downregulated at day 0 and upregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In contrast, the transcript abundance of TPM3 was upregulated at day 0 and downregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In addition, ODC1 and STAT3 were found to be associated (P < 0.05) with sperm quality traits as well as flow cytometry parameters. Therefore, the expression of several candidate genes including ODC1 and STAT3 was related to the genetic merit of the cow. In addition polymorphisms in these two genes were found to be associated with bull semen quality.

Differential gene expression in porcine oviduct during the oestrous cycle

The oviduct undergoes changes under the influence of steroid hormones during the oestrous cycle. However, the molecular mechanisms underlying oviductal regulation are not fully understood. The aim of the present study was to identify the gene expression profile of the porcine oviduct in different stages of the cycle using microarray technology. A systematic study was performed on animals at four different stage: prepubertal gilts, and sows in the preovulatory, postovulatory and luteal phase of the oestrous cycle. The porcine oviduct expressed a total of 4929 genes. Moreover, significant differences in the expression of several genes were detected as the oestrous cycle progressed. Analysis of the differentially expressed genes indicated that a total of 86, 89 and 15 genes were upregulated in prepubertal gilts, preovulatory and luteal sows respectively compared with levels observed in postovulatory sows. Moreover, 80, 51 and 64 genes were downregulated in prepubertal, preovulatory and luteal animals respectively compared with the postovulatory sows. The concentrations of 10 selected transcripts were quantified by real-time reverse transcription–polymerase chain reaction to validate the cDNA array hybridisation data. Conversely, for some genes, localisation of corresponding protein expression in the oviduct was analysed by immunohistochemistry (i.e. cholecystokinin, glutathione peroxidase 2, mucin 1, phosphatidylethanolamine binding protein 4 and tachykinin 3) and mass spectrometry analysis of oviductal fluid allowed identification of peptides from all five proteins. The results of the present study demonstrate that gene expression in the porcine oviduct is clearly regulated during the oestrous cycle, with some oviductal proteins that could be related to several reproductive processes described here for the first time.