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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
The RNA methyltransferase NSUN6 suppresses pancreatic cancer development by regulating cell proliferation
Ruimeng Yang1, Xing Liang2, Hui Wang3
1The Core Laboratory in Medical Centre of Clinical Research, Department of Endocrinology, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
Background:
Pancreatic cancer (PC) is one of the most lethal solid malignancies in the world due to its excessive cell proliferation and aggressive metastatic features. Emerging evidences revealed the importance of posttranscriptional modifications of RNAs in PC progression. However, knowledge about the 5-methylcytosine (m5C) RNA modification in PC is still extremely limited. In this study, we attempted to explore the expression changes and clinical significances of 12 known m5C-related genes among PC patients.
Methods:
A total of 362 normal and 382 tumor specimens from PC patients were examined for candidate m5C-related gene and protein expression by using quantitative PCR (qPCR) and immunohistochemistry (IHC). The proliferation rate of PC cells was detected by MTS assay. Xenograft mouse models were used to assess the role of NSUN6 in PC tumor formation.
Findings:
Through analyzing the four Gene Expression Omnibus (GEO) databases, six m5C-related genes shown significant and consistent alterations were selected for further examination in our 3 independent PC cohorts. Finally, we identified the reduction of NSUN6 as a common feature of all PC sample sets examined. NSUN6 expression correlated with clinicopathologic parameters including T stage, and Ki67+ cell rate. Further assessing the transcriptional profiles of 50 PC tissues, we found biological processes associated with cell proliferation like cell cycle and G2M checkpoint were enriched in NSUN6 lower expression group. Helped by in vitro PC cell lines and in vivo xenograft mouse models, we confirmed the role of NSUN6 in regulating cell proliferation and PC tumor growth. Last but also importantly, we also show the good performance of NSUN6 in evaluating tumor recurrence and survival among PC patients.
Interpretation:
Our data suggested that NSUN6 is an important factor involved in regulating cell proliferation of PC, and highlights the potential of novel m5C-based clinical modalities as a therapeutic approach in PC patients.
Funding:
This study was supported by the National Natural Science Foundation of China (Grant Nos. 81803014, 81802424, and 81802911).
Insights
Reduced expression of NSUN6 is a key feature in pancreatic cancer (PC), impacting cell proliferation and tumor growth. This finding highlights NSUN6 as a potential biomarker for PC recurrence and survival.
Area of Science:
- Oncology
- Molecular Biology
- Epigenetics
Background:
- Pancreatic cancer (PC) is a highly lethal malignancy characterized by rapid cell proliferation and metastasis.
- Posttranscriptional RNA modifications, particularly 5-methylcytosine (m5C), are increasingly recognized for their role in PC progression.
- The specific involvement and clinical significance of m5C-related genes in PC remain largely unexplored.
Purpose of the Study:
- To investigate the expression patterns of 12 known m5C-related genes in pancreatic cancer.
- To determine the clinical significance of these genes, focusing on their association with clinicopathological parameters and patient outcomes.
- To explore the functional role of identified m5C-related genes in PC cell proliferation and tumor development.
Main Methods:
- Analysis of gene and protein expression of m5C-related genes in PC tissues using quantitative PCR (qPCR) and immunohistochemistry (IHC).
- Utilized Gene Expression Omnibus (GEO) databases for initial screening of candidate genes.
- Employed MTS assays in vitro and xenograft mouse models in vivo to assess the functional role of NSUN6 in PC cell proliferation and tumor formation.
Main Results:
- Six m5C-related genes showed significant alterations in PC, with reduced NSUN6 expression identified as a consistent feature across multiple cohorts.
- NSUN6 expression levels correlated with advanced T stage and higher Ki67+ cell rates in PC tissues.
- Lower NSUN6 expression was associated with enriched cell proliferation pathways (cell cycle, G2M checkpoint) and demonstrated potential as a predictive biomarker for PC recurrence and survival.
Conclusions:
- NSUN6 plays a crucial role in regulating cell proliferation and tumor growth in pancreatic cancer.
- The study identifies NSUN6 as a potential diagnostic and prognostic biomarker for PC.
- These findings suggest novel m5C-based therapeutic strategies for pancreatic cancer treatment.
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