Project description:To analyse gene expression differences between HeLa tranfected cells with mutant COL1 (COL1G610C) compared to their relative scramble controls
Project description:Prostate cancer (PCa) is the second most frequently diagnosed cancer and the fifth leading cause of death from cancer in men worldwide. Increased understanding of the prostate cancer metastasis mechanisms will help identify more efficient intervention strategies to prevent or treat this deadly disease in the future. To identify the candidate proteins that contribute to metastasis of PCa, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis was performed to explore differentially expressed proteins between two homologous human prostate cancer cell lines including highly-metastatic PC-3M-1E8 cell line and poorly-metastatic PC-3M-2B4 cell line. Here, a total of 58 proteins were identified to be significantly differentially expressed between PC-3M-1E8 and PC-3M-2B4 cells, which were further verified using real-time quantitative PCR and western blot analysis. The bioinformatic analysis suggested that the differentially expressed proteins, like MMP1 and FHL1, may contribute to the higher metastatic ability of PC-3M-1E8 cells than PC-3M-2B4 cells. In addition, functional analyses proved MMP1's positive effect on the higher metastatic ability of PC-3M-1E8 cells than PC-3M-2B4 cells. These findings provided a unique resource to specifically reveal the complex molecular regulatory mechanisms underlying the progression of prostate cancer from poorly-metastatic to highly-metastatic stage.
Project description:Single cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing polyA tails of mRNAs, which excludes many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single cell level. In this study, we have developed a measurement protocol for nc886 as a model case, as an initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5’-reading in scRNA-seq. We then produced sequencing libraries for standard 5’ gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines which express high, low, and zero levels of nc886, respectively. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows us for a combined analysis of ncRNAs and gene expression phenotype.
Project description:Prostate cancer is the most common cancer of men in the Western world, and novel approaches for prostate cancer risk reduction are needed. Plant-derived phenolic compounds attenuate prostate cancer growth in preclinical models by several mechanisms, which is in line with epidemiological findings suggesting that consumption of plant-based diets is associated with low risk of prostate cancer. The objective of this study was to assess the effects of a novel lignan-stilbenoid mixture in PC-3M-luc2 human prostate cancer cells in vitro and in orthotopic xenografts. Lignan and stilbenoid -rich extract was obtained from Scots pine (Pinus sylvestris) knots. Pine knot extract as well as stilbenoids (methyl pinosylvin and pinosylvin), and lignans (matairesinol and nortrachelogenin) present in pine knot extract showed antiproliferative and proapoptotic efficacy at ≥ 40 μM concentration in vitro. Furthermore, pine knot extract derived stilbenoids enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis already at ≥ 10 μM concentrations. In orthotopic PC-3M-luc2 xenograft bearing immunocompromized mice, three-week peroral exposure to pine knot extract (52 mg of lignans and stilbenoids per kg of body weight) was well tolerated and showed anti-tumorigenic efficacy, demonstrated by multivariate analysis combining essential markers of tumor growth (i.e. tumor volume, vascularization, and cell proliferation). Methyl pinosylvin, pinosylvin, matairesinol, nortrachelogenin, as well as resveratrol, a metabolite of pinosylvin, were detected in serum at total concentration of 7-73 μM, confirming the bioavailability of pine knot extract derived lignans and stilbenoids. In summary, our data indicates that pine knot extract is a novel and cost-effective source of resveratrol, methyl pinosylvin and other bioactive lignans and stilbenoids. Pine knot extract shows anticarcinogenic efficacy in preclinical prostate cancer model, and our in vitro data suggests that compounds derived from the extract may have potential as novel chemosensitizers to TRAIL. These findings promote further research on health-related applications of wood biochemicals.