MicroRNA expression profile of MRC5-CAFs transfected with control siRNA (si-Ctrl) or Dicer1 specific siRNA (si-Dicer1) for 72 hr
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ABSTRACT: Ovarian cancer (OC) remains the leading cause of death in patients with gynecological malignancy. An improved understanding of the genomics has led to the separation of OC into histologically and molecularly defined subgroups. Based on molecular profiling in OC patients, subtype with a reactivated tumor stroma presented the worst prognosis, emphasizing the importance of tumor microenvironment especially stromal fibroblasts in fueling OC progression. Dicer1 is well recognized as the microRNA (miR) synthesis machinery, playing a crucial role in cellular maturation and development, and was generally considered to be a tumor suppressor gene that inhibited tumor initiation and metastasis. Dicer1 expression pattern and exact biological function was seldom studied in the stromal compartment. There was a recent study demonstrating that Dicer1 was involved in mouse embryonic fibroblast (MEF) development and maturation. Therefore, we are inspired to explore the expression and function of Dicer1 in stromal fibroblasts of OC patients. In this study, mRNA and microRNA gene expression profiling were conducted for MRC5-CAFs after silencing of Dicer1. By comparing the expression data of MRC5-CAFs transfected with control siRNA (si-Ctrl) or with Dicer1 specific siRNA (si-Dicer1) for 72 hr, we identified a set of differential expressed mRNA and microRNA in MRC5-CAFs after Dicer1 knockdown. This study was the first to investigate Dicer1 influence of stromal fibroblast gene expression and the underlying regulation mechanism, which held great importance in complementing our understanding of Dicer1 in OC initiation and development, and raises potent therapeutical targets in controlling OC metastasis We used microarrays to profile the microRNA expression of MRC5-CAFs before and after Dicer1 knockdown, in order to identify microRNA alteration exerted by Dicer1 in the context of stromal fibroblasts.
Project description:Ovarian cancer (OC) remains the leading cause of death in patients with gynecological malignancy. An improved understanding of the genomics has led to the separation of OC into histologically and molecularly defined subgroups. Based on molecular profiling in OC patients, subtype with a reactivated tumor stroma presented the worst prognosis, emphasizing the importance of tumor microenvironment especially stromal fibroblasts in fueling OC progression. Dicer1 is well recognized as the microRNA (miR) synthesis machinery, playing a crucial role in cellular maturation and development, and was generally considered to be a tumor suppressor gene that inhibited tumor initiation and metastasis. Dicer1 expression pattern and exact biological function was seldom studied in the stromal compartment. There was a recent study demonstrating that Dicer1 was involved in mouse embryonic fibroblast (MEF) development and maturation. Therefore, we are inspired to explore the expression and function of Dicer1 in stromal fibroblasts of OC patients. In this study, mRNA and microRNA gene expression profiling were conducted for MRC5-CAFs after silencing of Dicer1. By comparing the expression data of MRC5-CAFs transfected with control siRNA (si-Ctrl) or with Dicer1 specific siRNA (si-Dicer1) for 72 hr, we identified a set of differential expressed mRNA and microRNA in MRC5-CAFs after Dicer1 knockdown. This study was the first to investigate Dicer1 influence of stromal fibroblast gene expression and the underlying regulation mechanism, which held great importance in complementing our understanding of Dicer1 in OC initiation and development, and raises potent therapeutical targets in controlling OC metastasis We used microarrays to profile the mRNA expression of MRC5-CAFs before and after Dicer1 knockdown, in order to identify molecular alteration exerted by Dicer1 in the context of stromal fibroblasts.
Project description:Cancer-associated fibroblasts (CAFs) play significant roles in drug resistance through different ways. Antitumor therapies, including molecular targeted interventions, not only effect tumor cells but also modulate the phenotype and characteristics of CAFs, which can in turn blunt the therapeutic response. Little is known about how stromal fibroblasts respond to poly (ADP-ribose) polymerase inhibitors (PARPis) in ovarian cancer (OC) and subsequent effects on tumor cells. This is a study to evaluate how CAFs react to PARPis and their potential influence on PARPi resistance in OC. We discovered that OC stromal fibroblasts exhibited intrinsic resistance to PARPis and were further activated after the administration of PARPis. PARPi-challenged fibroblasts displayed a specific secretory profile characterized by increased secretion of CCL5, MIP-3α, MCP3, CCL11, and ENA-78. Mechanistically, increased secretion of CCL5 through activation of the NF-κB signaling pathway was required for PARPi-induced stromal fibroblast activation in an autocrine manner. Moreover, neutralizing CCL5 partly reversed PARPi-induced fibroblast activation and boosted the tumor inhibitory effect of PARPis in both BRCA1/2-mutant and BRCA1/2-wild type xenograft models. Our study revealed that PARPis could maintain and improve stromal fibroblast activation involving CCL5 autocrine upregulation. Targeting CCL5 might offer a new treatment modality in overcoming the reality of PARPi resistance in OC.
Project description:Purpose: to find the potential down-stream target gene of circFAM120A Methods: mRNA profiles of decidualized human endometrial stromal cells (hESCs) after down-regulated FAM120A and control using siRNA were generated by NGS and compared by bioinformatics analysis Results: we sequenced 26414 mRNAs in hESCs between the si-NC and si-circFAM120A groups and identified 242 differentially expressed mRNAs, between which 161 were downregulated and 81 were up-regulated in si-circFAM120A group Conclusions: Our study represents the detailed analysis of decidualized hESCs transcriptomes between si-NC and si-circFAM120A groups.
Project description:We report the generation and characterization of DICER1-deficient hESCs. We uncover an unexpected requirement for DICER1 as well as essential pro-survival roles of members of the mir-302- 367 and mir-371- 373 clusters in hESCs. Our work provides a robust platform for interrogating microRNA function in hESC and differentiation.
Project description:We developed a genetically engineered conditional compound heterozygous Dicer1 mouse strain that fully recapitulates the bi-allelic mutations of DICER1 in DICER1 syndrome-associated cancers. Embryonic activation of bi-allelic Dicer1 mutations, driven by the anti-Müllerian hormone receptor 2 (Amhr2)-driven Cre strain (Amhr2+/cre), drove cancer development from oviduct. Small RNA sequencing was performed to compare the microRNA expression profiles between tumor and normal oviduct.
Project description:Interaction between stromal cells and the tumor greatly influences tumor initiation and progression. Cancer-associated fibroblasts (CAFs) are the major component of tumor stroma and play a key role in ovarian cancer (OC) cells adhesion and metastasis. CAFs were found abundantly in primary tumor and metastases, as well as in malignant ascites of high grade serous OC patients, underscoring that CAFs modulate OC cells phenotype and facilitate disease exacerbation along OC progression. Studies of CAFs exertion on OC mainly focused on the influence of CAFs conditioned medium (CM) influence of tumor cells phenotype, while ignoring the fact that CAFs are supposed to be in intimate contact with neoplastic cells in tumor mass, and that CAFs also form compact heterotypic spheroids with ascitic tumor cells in malignant ascites. We hypothesized that uncovering the underlying molecules that mediate tumor cells binding with CAFs could aid in developing measures destroying the pro-carcinogenic heterotypic spheroids in malignant ascites. In this study, gene expression profiling was completed for individual SKOV3, and magnetic sorted SKOV3 cells that form heterotypic spheroids with 4 cases of high grade serous OC derived CAFs. By comparing the expression data of SKOV3 cells in individual group with that in spheroid group, we identified a set of differential expressed genes. This study revealed the heterogeneity of ascitic tumor cells and raised potent therapeutical targets in destroying of the heterotypic spheroids in malignant ascites of OC. We used microarrays to profile the mRNA expression of SKOV3 tumor cells that could or not form heterotypic spheroids with CAFs, in order to identify molecules that potentially mediate the combination between tumor cells and CAFs.
Project description:Small cell lung cancer (SCLC) is a highly fatal malignancy, the complex tumor microenvironment (TME) is a critical factor affecting SCLC progression. Cancer-associated fibroblasts (CAFs) are crucial components of TME, yet their role in SCLC and the underlying mechanisms during their interaction with SCLC cells remain to be determined. In this study, a co-culture system comprising MRC5 fibroblasts and SCLC cell lines was constructed. RNA sequencing (RNA-seq) was performed on co-cultured and separately cultured MRC5 and H196 cells to identify differentially expressed genes (DEGs) and enriched signaling pathways. We obsertved that non-neuroendocrine (non-NE) SCLC-derived CAFs exhibited more abundance and DEGs than NE SCLC-derived CAFs did. Enriched glycolysis-related genes, increased glucose uptake, and upregulated glycolytic signaling proteins were found in non-NE SCLC cells when interaction with MRC5 fibroblasts, confirming CAF-mediated glycolysis promotion. Additionally, non-NE SCLC cell-educated CAFs exhibited features of antigen-presenting CAFs (apCAFs), as indicated by the expression of major histocompatibility complex (MHC) molecules. This study emphasizes the pro-tumor function of CAFs in SCLC by promoting glycolysis and impairing T cell function, providing direction for the development of novel therapeutic approaches targeting CAF in SCLC.
Project description:DICER1 plays a critical role in microRNA (miRNA) biogenesis. Recurrent somatic “hotspot” mutations at four mental binding sites within the RNase IIIb domain of DICER1, were identified in ovarian sex cord-stromal tumors and have since been described in other pediatric tumors. In this study, we identified and characterized DICER1 hotspot mutations in endometrial cancers derived from The Cancer Genome Atlas (TCGA) and our local tumor bank. DICER1 hotspot mutations are found in ~2% of endometrial tumors. Using Illumina and Sanger targeted resequencing we observed biallelic DICER1 mutations in more than 50% of cases with hotspot mutations and identified an additional recurrent mutation G1809R in 2 cases. Through small RNA deep sequencing and real-time PCR, we demonstrated mutations that add a positively charged side chain to residue 1809 have similar detrimental effects on 5p miRNA production as mutations at metal binding sites. In one case G1809R was compound heterozygous with a germline S839F mutation, which contributes to loss of DICER1 expression by promoting protein degradation. As expected, 5p miRNAs are globally reduced in tumors and cell lines with hotspot mutations. Pathway analysis of gene expression profiles indicated that genes derepressed due to loss of 5p miRNAs are strongly associated with cell cycle related pathways. Using a Dicer null cell line model, we demonstrated that DICER1 hotspot mutants abolished the inhibitory effects of wildtype DICER1 on cell proliferation upon re-expression. Furthermore, targets of let-7 family miRNAs are enriched among the upregulated genes, suggesting loss of let-7 may be impacting downstream pathways.