Ribonucleotide Reductase M1 Expression Research Articles

The potential of ribonucleotide reductase M2 as a novel prognostic maker and therapeutic target to inhibit medulloblastoma proliferation, migration, and invasiveness

AbstractThe crucial role of ribonucleotide reductase M2 (RRM2) enzyme in cancer occurrence and progression has been well‐established, but its specific function and significance in medulloblastoma (MB) remains largely unknown. First, we conducted a bioinformatics analysis of public genomic databases and observed highly expressed RRM2 in MB and an association of high RRM2 expression with adverse outcomes. In addition, by collecting clinical MB specimens for polymerase chain reaction (PCR), western blotting (WB), and immunohistochemistry (IHC), RRM2 was confirmed to be highly expressed in tumor tissues. Furthermore, immunohistochemical analysis linked adverse prognosis to high RRM2 expression. Moreover, knocking down RRM2 significantly inhibited MB cell proliferation, migration, and invasion in vitro. This report is the first to demonstrate the oncogenic role of RRM2 in MB, associated with adverse patient outcomes. Knocking down RRM2 contributes to weakened proliferating, migrating, and invading potentials of MB cells. RRM2 is expected to be a novel prognostic biomarker and therapeutic target for MB.

Identification and prognostic analysis of metabolic genes associated with TP53 mutation in multiple myeloma

ABSTRACT Background TP53 gene mutation is crucial in determining the prognosis of Multiple Myeloma (MM) patients. Understanding metabolic genes linked to TP53 mutation is vital for developing targeted therapies for these patients. Method We analyzed The Cancer Genome Atlas (TCGA) dataset to identify genes related to TP53 mutation and metabolism. Using univariate Cox regression and protein–protein interaction (PPI) analysis, we identified key genes. We categorized patients into high and low metabolism groups via non-negative matrix factorization (NMF) clustering, which led to the discovery of relevant differential genes. Integrating these with genes from the Gene Expression Omnibus (GEO) datasets and PPI interactions, we pinpointed crucial metabolic genes associated with TP53 mutation in MM. Additionally, we conducted prognostic analyses involving survival curves and receiver operating characteristic (ROC) charts. Results Our study reveals that the metabolic gene ribonucleotide reductase M2 (RRM2), linked to TP53 mutation, correlates positively with the International Staging System (ISS) stage in MM patients and is an independent prognostic risk factor. In the TCGA dataset, among the 767 patients, the 35 MM patients with TP53 mutation generally had poor survival outcomes. Specifically, the patients with both TP53 mutation and high RRM2 expression had a 2-year survival rate of only 38.87%, whereas those with normal TP53 function and low RRM2 expression had a 2-year survival rate of 86.31% (p < 0.001). Conclusion RRM2 significantly impacts MM prognosis and is associated with TP53 mutation, presenting itself as a potential therapeutic target and prognostic marker for MM.

RRM2‑mediated Wnt/β‑catenin signaling pathway activation in lung adenocarcinoma: A potential prognostic biomarker.

The present study aimed to investigate the role and mechanism of action of ribonucleotide reductase M2 (RRM2) in lung adenocarcinoma and its potential as a therapeutic target. Data of patients with lung adenocarcinoma from The Cancer Genome Atlas database were collected and analyzed to evaluate the potential of RRM2 as a biomarker. The expression of RRM2 was evaluated in the A549 cell line and its cisplatin-resistant A549/DDP cell line derivative by western blot and reverse transcription-quantitative PCR. The study also investigated cell proliferation and the mechanism by which RRM2 controls cellular cisplatin resistance using CCK-8 and colony-formation assays. In addition, cell migration was assessed using Transwell assays, and the cell cycle and apoptosis were examined using flow cytometry. RRM2 was highly expressed in lung adenocarcinoma and was associated with the clinical TMN stage. Functional enrichment analysis showed that RRM2 was enriched in the cell cycle. Immune cell infiltration analysis identified 12 types of immune cell that exhibited differences between patients expressing different levels of RRM2. Cellular assays revealed higher levels of RRM2 expression in A549/DDP cells than A549 cells, and its expression was induced by cisplatin. RRM2 knockdown decreased cell proliferation and migration, accelerated apoptosis and caused cell cycle arrest in the S-phase, increasing the sensitivity of A549 and A549/DDP cells to cisplatin through the Wnt/β-catenin signaling pathway. Overexpression of β-catenin reduced the effects of RRM2 knockdown on A549 cells. Lung adenocarcinoma growth may be influenced by RRM2 through the Wnt/β-catenin signaling pathway, suggesting a potential pathway for cancer progression.

High Ribonucleotide Reductase M2 Expression as an Independent Prognostic Biomarker and Correlated with Immune Infiltrates in Live Hepatocellular Carcinoma

Hepatocellular carcinoma (LIHC) is a prevalent cancer worldwide. High expression of Ribonucleotide reductase M2 (RRM2), a gene expressed in various tumors including LIHC, has been associated with poor prognosis, yet its correlation with immune cell infiltration in LIHC remains unclear. We aimed to develop a prognosis model for patient prognosis analysis and immunotherapy using RRM2 expression data and corresponding clinical data collected from The Cancer Genome Atlas (TCGA) database. Patients were divided into high- and low-risk groups based on their median risk score. Our analysis revealed that high expression of RRM2 was associated with T-stage, pathological stage, histologic grade, AFP levels, and age. Further analysis showed that cell cycle checkpoints, B cell receptor, cell cycle, MHC class II antigen, cancer pathway, and TP53 were differentially enriched in the RRM2 high expression phenotype. Additionally, RRM2 correlated with the majority of immune cell infiltration levels and was positively correlated with Th2 cells and negatively correlated with neutrophil cells, DC, CD8 T cells, and NK cells. The levels of these immune cells were significantly different between the RRM2 high and low expression groups. Our study suggests that overexpression of RRM2 is associated with poor prognosis in LIHC and may represent a valuable prognostic biomarker for this cancer.

Morphological predictors of the efficacy of mitotane therapy in adrenocortical cancer

Adrenocortical cancer (ACC) is an orphan malignant tumor of the adrenal cortex with a predominantly poor prognosis and an aggressive clinical course. Nowadays, mitotane is a non-alternative drug in the treatment of ACC. The search for prognostic parameters that determine the sensitivity of ACC to ongoing treatment is currently an urgent task. Expression levels of the large subunit of ribonucleotide reductase M1 (RRM1), cytochrome P450 2W1 (CYP2W1), and sterol- O-acyltransferase-1 (SOAT1) are considered as potential predictors of response to mitotane therapy. To assess the immunohistochemical expression of RRM1, CYP2W1 and SOAT1 in ACC as markers of clinical outcomes and response to the therapy with mitotane. The study included 62 patients older than 17 years of age with a diagnosis of ACC confirmed histologically and immunohistochemically. Mitotane therapy was initiated in 29 patients in the postoperative period, 33 patients were under dynamic observation without concomitant drug treatment. Antibodies to RRM1, CYP2W1, SOAT1 were used diluted in accordance with recommendations of firms-manufacturers for immunohistochemical detection. In the group of patients with low and moderate RRM1, CYP2W1 and SOAT1 immunoreactivity in the tumor and no antitumor therapy, a better DFS was noted (p=0.037, p=0.020 and p=0.001, respectively) compared to the group of patients receiving mitotane therapy at this level of marker expression. With high immunoreactivity of the markers, no statistically significant differences in DFS were found. Consistent with the findings in our study, low expression of RRM1, CYP2W1 and SOAT1 was associated with worse DFS with antitumor therapy. The results of the work indicate the need to assess the levels of immunoreactivity of these markers in patients with ACC before starting treatment with mitotane in order to predict the efficiency of therapy.

RRM2 regulates osteogenesis of mouse embryo fibroblasts via the Wnt/β‑catenin signaling pathway.

Osteoporosis is a widespread bone metabolic disease characterized by reduced bone mass and bone microstructure deterioration. Ribonucleotide reductase M2 (RRM2) is a key enzyme in DNA synthesis and repair. The present study investigated the effect of RRM2 on osteogenesis of mouse embryo fibroblasts (MEFs) and its molecular mechanism. Bioinformatics analysis revealed that RRM2 expression was increased during osteogenesis of MEFs triggered by bone morphogenetic protein 9. Subsequently, MEFs were used as a mesenchymal stem cell model and osteogenic inducing medium was used to induce osteogenic differentiation. RRM2 protein expression was measured by western blotting during osteogenic differentiation induction of MEFs. RRM2 levels in MEFs were upregulated and downregulated by RRM2-overexpressing recombinant adenovirus and small interfering RNA-RRM2, respectively. Bone formation markers (RUNX family transcription factor 2, osterix, distal-less homeobox 5, collagen type I α1 chain, osteopontin and osteocalcin) were detected by reverse transcription-quantitative (RT-q) PCR and alkaline phosphatase (ALP) and Alizarin Red S staining were examined. The protein expression levels of β-catenin and the ratio of phosphorylated (p-)GSK-3β to GSK-3β were detected by western blotting and the RNA expression of downstream related target genes (β-catenin, axis inhibition protein 2 (AXIN2), transcription factor 7 like 2, lymphoid enhancer binding factor 1, c-MYC and Cyclin D1) in the Wnt/β-catenin signaling pathway was measured by RT-qPCR. RRM2 protein expression increased as the osteogenic differentiation induction period was extended. RRM2 overexpression increased osteogenic marker RNA expression, ALP activity, bone mineralization, the protein expression levels of β-catenin, the ratio of p-GSK-3β to GSK-3β and the RNA expression of downstream related target genes in the Wnt/β-catenin signaling pathway, whereas RRM2 knockdown had the opposite effect. The findings of the present study revealed that RRM2 overexpression enhanced osteogenic differentiation, while RRM2 knockdown reduced osteogenic differentiation. RRM2 may regulate osteogenic differentiation of MEFs via the canonical Wnt/β-catenin signaling pathway, providing a possible therapeutic target for osteoporosis.

RRM2 Improves Cardiomyocyte Proliferation after Myocardial Ischemia Reperfusion Injury through the Hippo-YAP Pathway.

Objective Ribonucleotide reductase M2 (RRM2) as an enzyme that catalyzes the deoxyreduction of nucleosides to deoxyribonucleoside triphosphate (dNTP) has been extensively studied, and it plays a crucial role in regulating cell proliferation. However, its role in ischemia-reperfusion injury (I/RI) is still unclear. Methods SD rats were used as the research object to detect the expression of RRM2 in the myocardium by constructing an I/RI model. At the same time, primary SD neonatal rat cardiomyocytes were extracted, and hypoxia/reoxygenation (H/R) treatment simulated the I/RI model. Using transfection technology to overexpress RRM2 in cardiomyocytes, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of RRM2, Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and immunofluorescence staining was used to detect Ki67 and EdU-positive cells. Western blot (WB) technology was used to detect YAP and its phosphorylation expression. Results qRT-PCR results indicated that the expression of RRM2 was inhibited in the model group, and cardiomyocytes overexpressing RRM2 can obviously promote the proliferation of primary cardiomyocytes and improve the damage of cardiac structure and function caused by I/R. At the same time, RRM2 can promote the increase of YAP protein expression and the increase of Cyclin D1 mRNA expression. Conclusion RRM2 expression was downregulated in myocardial tissue with I/R. After overexpression of RRM2, cardiomyocyte proliferation was upregulated and the Hippo-YAP signaling pathway was activated.

The High Expression of RRM2 Can Predict the Malignant Transformation of Endometriosis.

A large number of epidemiological studies have revealed that women with endometriosis (EMS) have a higher risk of developing endometriosis-associated ovarian cancer (EAOC). At present, there are few studies on predicting the malignant transformation of ovarian endometriosis (OE). The purpose of this study is to identify and verify the molecules that may be able to predict the malignant transformation of OE. The gene expression profiles of ovarian cancer and OE were downloaded from Gene Expression Omnibus (GEO), and a common hub gene ribonucleotide reductase M2 (RRM2) was identified. A total of 44 patients with EAOC and 44 with OE were enrolled in this study. Immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to detect the expression of RRM2, while the relationship between RRM2 and Ki-67 was analyzed by IHC co-localization. Bioinformatics analysis showed that the expression of RRM2 was low in EMS and high in ovarian cancer. RRM2 was obviously positively expressed in eutopic endometrium (EU), ectopic endometrium (EC), and cancer tissues of EAOC patients. The IHC signal and mRNA levels of RRM2 were higher in the EC of EAOC patients compared with OE patients (P<0.01). In addition, there was a correlation between the expression of RRM2 and Ki-67 in EC of EAOC patients (P<0.01). The upregulated expression of RRM2 in the EC of OE patients may indicate malignant transformation. High expression of RRM2 promotes abnormal proliferation of histiocytes. RRM2 can be used as a potential marker of malignant transformation of OE.

Abstract 1240: RRM2 and mutant p53 as therapeutic targets in TNBC

Abstract Triple negative breast cancer (TNBC) is a highly aggressive type of cancer associated with poor prognosis and characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2). TNBC accounts for 10 - 15% of all new cases of breast cancer. TNBC is heterogenous in nature (70% basal-like) and currently doesn't have effective targeted therapy. As such, dose-dense chemotherapeutic agents used in combination, such as doxorubicin (DOXO) with cyclophosphamide, is the regimen of choice for TNBC. However, DOXO has limited efficacy as a therapeutic agent owing to acquired drug resistance and its cardiotoxic effects. Ribonucleotide reductase (RR) is the rate limiting enzyme required for the conversion of ribonucleotide to deoxyribonucleotide in DNA synthesis during cell proliferation. Didox (3,4 dihydroxybenzohydroxamic acid) is a unique RR inhibitor with iron chelating and free radical scavenging properties. We have previously identified that ribonucleotide reductase M2 (RRM2) is upregulated in TNBC cells and is a key contributor to acquired drug resistance. We found that Didox not only inhibits RRM2, but it also reduces mutant p53 protein levels and inhibits the activation of the NF-κB pathway. Through this inhibition, we found that Didox works synergistically with DOXO to inhibit TNBC tumor growth while also minimizing DOXO-induced cardiac damage in mice. In our present study, we have observed that RRM2 expression is upregulated in TNBC patients from three independent data sets and that high RRM2 levels are associated with lower recurrence free survival (RFS) and overall survival (OS). Interestingly, high levels of RRM2 were only predictive of lower RFS and OS in patients with wild-type p53 but not mutant p53. Additionally, we have found that Didox downregulates mutant p53 protein levels in TNBC cell lines but does not alter total p53 in cells with functioning p53. Our data confirms that Didox induced downregulation of mutant p53 is not a transcriptional effect. Finally, we have identified that RRM2 protein levels increase in TNBC with progression of stage as measured in patient tissue microarray analysis. Our data presents a novel and promising approach for the treatment of TNBC that involves inhibition of RRM2, NF-kB activation, and mutant p53 that merits further clinical investigation in human models. Citation Format: Nahid Sultana, Elizabeth A. Wilson, Howard L. Elford, Jesika S. Faridi. RRM2 and mutant p53 as therapeutic targets in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1240.

Overexpression of RRM2 is related to poor prognosis in oral squamous cell carcinoma.

Ribonucleotide reductase M2 (RRM2) is a rate-limiting enzyme involved in DNA repair and synthesis. This study aimed to investigate the expression level, clinicopathological significance, and prognostic value of RRM2 in oral squamous cell carcinoma (OSCC). Human OSCC tissue microarrays were used to detect the expression of RRM2, cancer stem cell (CSC) markers CD44 and aldehyde dehydrogenase 1 (ALDH1), and the epithelial-mesenchymal transition (EMT) marker Slug. The correlation of RRM2 expression with clinicopathological parameters was evaluated. The effects of RRM2 on cell proliferation, migration, and apoptosis were investigated. Compared with normal and dysplastic tissues, the expression of RRM2 in human primary OSCC was significantly increased, and its overexpression was correlated with advanced pathological grade. The overall survival rate of patients with high RRM2 expression was lower than that of patients with low RRM2 expression. The overexpression of RRM2 was significantly associated with OSCC recurrence, and its overexpression was correlated with the CSC markers CD44 and ALDH1 and the EMT marker Slug. The expression of RRM2 promotes the proliferation and migration of human OSCC cells and inhibits apoptosis. Ribonucleotide reductase M2 may be a novel target in the diagnosis, prognosis, and therapy of OSCC.

Acquired tamoxifen resistance is surmounted by GW8510 through ribonucleotide reductase M2 downregulation-mediated autophagy induction

Tamoxifen resistance is a major roadblock in the treatment of patients with breast cancer. Ribonucleotide reductase M2 (RRM2) was found to be involved in acquired resistance of breast cancer cells (BCCs) to tamoxifen. Here, we used GW8510, which has been identified as a potential RRM2 inhibitor, to evaluate the effect of RRM2 inhibition on reversing resistance of BCCs to tamoxifen and investigate its mechanisms. We showed that RRM2 overexpression played a key role in the development of acquired tamoxifen resistance in BCCs through downregulation of autophagy level. Combination treatment with tamoxifen and GW8510 significantly inhibited survival of the tamoxifen-resistant BCCs through induction of autophagic cell death compared to either of the two drugs. Furthermore, combination of tamoxifen and GW8510 resulted in marked growth inhibition of tamoxifen-resistant BBC xenograft tumor in vivo compared to tamoxifen or GW8510 alone. In conclusion, tamoxifen in combination with GW8510 can overcome acquired tamoxifen resistance in BCCs and may be a rational therapeutic approach against breast cancer with high RRM2 expression.

Expression of Ribonucleotide Reductase M2 in Patients with Multiple Myeloma and Its Mechanism Inhibiting Tumor Cell Proliferation

To investigate the expression of ribonucleotide reductase M2 (RRM2) in patients with multiple myeloma and its mechanism of inhibiting human multiple myeloma cell proliferation. Thirty-Six patients with multiple myeloma in our hospital from July 2016 to September 2018 were selected as MM group, at the same time simple iron deficiency anemia patients were taken as the control group. RT-qPCR and Western blot techniques were used to determine the mRNA and protein expression of RRM2 in bone marrow mononuclear cells, respectively. siRNAs oligos targeting RRM2 was transfected into RPMI8226 cells to establish the RRM2 silence model. CCK-8 and flow cytometry were used to analyze the cell proliferation and the cell cycle, and Western blot was used to detect the expression of cell cycle related proteins. The expression level of RRM2 mRNA and the expression level of RRM2 protein in multiple myeloma group were significantly higher than those in control group (P＜0.05). Further analysis indicated that the level of RRM2 expression closely correlated with ISS staging, bone destruction and extramedullary infiltration (P＜0.05). Transfection with siRNAs targeting RRM2 could significantly down regulate the RRM2 expression. And the RRM2 down regulation inhibited the cell proliferation and arrested the cell cycle at S stage (P＜0.05). Further study indicated that RRM2 silencing influenced cell cycle-related proteins expression. RRM2 overexpressies in bone marrow mononuclear cells of MM patients, moreover significantly relates with the degree of malignancy. The silencing RRM2 in multiple myeloma cells can inhibit cell proliferation through arresting cell cycle at S phase. RRM2 may be a marker to predict the prognosis of patients with multiple myeloma and novel target for new drug development.

Knockdown of lncRNA KCNQ1OT1 inhibits glioma progression by regulating miR-338-3p/RRM2

AbstractThe dysregulated lncRNA play essential roles in glioma development. This study aimed to investigate the role and mechanism of lncRNA potassium voltage-gated channel subfamily Q member 1 opposite strand/ antisense transcript 1 (KCNQ1OT1) in glioma progression. Tumor tissues and adjacent normal samples were collected from 30 glioma patients. The expression levels of lncRNA KCNQ1OT1, microRNA (miR)-338-3p and ribonucleotide reductase M2 (RRM2) were detected by quantitative real-time polymerase chain reaction or western blot analyses. Levels of cell viability, apoptosis, cell migration and invasion in glioma cell lines were determined using cell counting kit-8, flow cytometry with annexin V-FITC and trans-well assays, respectively. The role of KCNQ1OT1 in glioma development in vivo was investigated using a xenograft model. The target association between miR-338-3p and KCNQ1OT1 or RRM2 was validated by luciferase reporter assay. The results found that expression of KCNQ1OT1 was enhanced in glioma tissues and cells, and KCNQ1OT1 knockdown inhibited cell viability, migration and invasion, and xenograft tumor growth, but promoted apoptosis. miR-338-3p was targeted via KCNQ1OT1 and could reverse the effect of KCNQ1OT1 on glioma progression. RRM2 was targeted via miR-338-3p and attenuated the suppressive effect of miR-338-3p on glioma cell viability, migration and invasion. Besides, KCNQ1OT1 overexpression increased RRM2 expression, and this event was weakened via miR-338-3p up-regulation. In conclusion, the present finding suggest that silencing of KCNQ1OT1 can suppress the development and progression of glioma by up-regulating miR-338-3p and down-regulating RRM2.

The SNHG16/miR-30a axis promotes breast cancer cell proliferation and invasion by regulating RRM2.

Long noncoding RNAs (lncRNAs) have been suggested to play vital roles in tumor initiation and progression. Recent studies have reported that the lncRNA small nucleolar RNA host gene 16 (SNHG16) is highly expressed in breast cancer tissue. In the present study, we demonstrated that SNHG16 is an oncogene involved in cell proliferation and invasion in breast cancer. First, we examined the functional role of SNHG16 in breast cancer cells by knocking down SNHG16 expression via siRNA. We found that SNHG16 inhibition reduced the proliferation and invasion of breast cancer cells in vitro. Then, based on bioinformatic prediction and functional assay validation, we demonstrated SNHG16 interaction with miR-30a and its role in breast cancer cells. Finally, we examined the functional role of RRM2 in breast cancer cells by knocking down RRM2 expression via siRNA. Our results indicated that the SNHG16/miR-30a axis regulated the expression of ribonucleotide reductase M2 (RRM2) in breast cancer cells. These results provide novel insight into breast cancer tumorigenesis and suggest that SNHG16 could serve as a therapeutic target in breast cancer.

Silencing RRM2 inhibits multiple myeloma bytargeting the Wnt/β‑catenin signaling pathway.

Ribonucleotide reductase M2 (RRM2) is one of the two subunits that comprise ribonucleotide reductase (RR), the enzyme that catalyzes the conversion of ribonucleotide 5′-diphosphates into 2′-deoxyribonucleotides, which are required for DNA synthesis. RRM2 is a stress response factor important for the development of several tumors. However, its role in multiple myeloma (MM) remains to be fully elucidated. The present study aimed to investigate the role of RRM2 in MM. The expression of RRM2 in patients with MM was analyzed using the Oncomine database. The results demonstrated that RRM2 expression was higher in MM compared with healthy subjects. Reverse transcription-quantitative polymerase chain reaction and western blot results revealed that RRM2 expression was decreased following transfection with a small interfering RNA targeting RRM2 into NCI-H929 cells. RR activity and Cell Counting Kit-8 assays demonstrated that RRM2 silencing reduced RR activity and inhibited cell proliferation. Annexin V-propidium iodide staining indicated that the percentage of apoptotic NCI-H929 cells was increased following RRM2 silencing compared with that in the control group. Increased phosphorylation of H2AX indicated that RRM2 silencing may activate the DNA-damage response pathway in NCI-H929 cells. Western blot analysis revealed that protein levels of the apoptosis-associated factor Bcl-2 were reduced, whereas Bax, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1 were upregulated following RRM2 silencing compared with the control group. In addition, the results demonstrated that RRM2 silencing may inhibit target gene expression in the Wnt/β-catenin signaling pathway by increasing the phosphorylation of glucose synthase kinase 3β. These findings indicated that RRM2 may be involved in the proliferation and apoptosis of MM cells via the Wnt/β-catenin signaling pathway, suggesting that RRM2 may represent a novel therapeutic target for MM.

Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

Hepatitis C virus (HCV) establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HCV life cycle is closely associated with host factors that promote or restrict viral replication, the characterization of which could help to identify potential therapeutic targets. To this end, here we performed a genome-wide microarray analysis and identified ribonucleotide reductase M2 (RRM2) as a cellular factor essential for HCV replication. We found that RRM2 is up-regulated in response to HCV infection in quiescent hepatocytes from humanized chimeric mouse livers. To elucidate the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes from chimeric mice and hepatoma cells infected with the HCV strain JFH1. Both models exhibited increased RRM2 mRNA and protein expression levels. Moreover, siRNA-mediated silencing of RRM2 suppressed HCV replication and infection. Of note, RRM2 and RNA polymerase nonstructural protein 5B (NS5B) partially co-localized in cells and co-immunoprecipitated, suggesting that they might interact. RRM2 knockdown reduced NS5B expression, which depended on the protein degradation pathway, as NS5B RNA levels did not decrease and NS5B protein stability correlated with RRM2 protein levels. We also found that RRM2 silencing decreased levels of hPLIC1 (human homolog 1 of protein linking integrin-associated protein and cytoskeleton), a ubiquitin-like protein that interacts with NS5B and promotes its degradation. This finding suggests that there is a dynamic interplay between RRM2 and the NS5B-hPLIC1 complex that has an important function in HCV replication. Together, these results identify a role of host RRM2 in viral RNA replication.

SiRNA-Mediated RRM2 Gene Silencing Combined with Cisplatin in the Treatment of Epithelial Ovarian Cancer In Vivo: An Experimental Study of Nude Mice.

Introduction: We aimed to explore small interfering (si)RNA silencing of ribonucleotide reductase M2 (RRM2) gene combined with cisplatin for the treatment of human ovarian cancer in nude mice models of subcutaneous transplantation of tumor cells.Methods: After conventional cultivation of human ovarian cancer cell line SKOV3 in vitro, SKOV3 cells were injected into the right back of nude mice by subcutaneous injection to establish the subcutaneous tumor models. Twenty-four tumor-burdened rats were randomly divided into four groups (n=6): siRNA group, siRNA in combination with cisplatin group, cisplatin group, and control group. Intraperitoneal injection of cisplatin and subcutaneous injection of siRNA were performed weekly. Tumor volume was measured, and tumor growth inhibition rate was calculated. RRM2 expression at the mRNA and protein levels was detected by reverse transcription-polymerase chain reaction and immunohistochemistry.Results: In the siRNA group, the tumor volume and tumor growth inhibition rate were 249.60±20.46 mm³ and 36.39%, respectively. The tumor growth inhibition rate and tumor volume were significantly different between the siRNA and control groups (p<0.05). In the cisplatin group, the tumor volume and tumor growth inhibition rate were 249.86±12.46 mm³ and 41.10%, respectively. The tumor growth inhibition rate and tumor volume were significantly different between the cisplatin and control groups (p<0.05). In the siRNA + cisplatin group, the tumor volume reduced to 180.84±16.25 mm³ and the tumor growth inhibition rate was increased to 64.33%, which were significantly different compared with the control group (p<0.01). Significant downregulation of RRM2 mRNA and protein expression in the tumor tissues was detected by reverse transcription polymerase chain reaction and immunohistochemistry assay (p<0.05).Discussion: siRNA alone or combined with cisplatin can effectively inhibit the growth of human ovarian cancer in nude mice models of subcutaneous transplantation of tumor cells. RRM2 gene silencing may be a potential treatment regimen for ovarian cancer in future.

DSCAM-AS1 promotes tumor growth of breast cancer by reducing miR-204-5p and up-regulating RRM2.

Breast cancer (BC) is a common malignancy worldwide. More than 3 700 000 women die of BC every year. DSCAM-AS1 was overexpressed several kinds of cancer and miR-204-5p was lowly expressed, which indicated that miR-204-5p had anti-tumor activity and DSCAM-AS1 had pro-tumor activity. We intended to analyze DSCAM-AS1, miR-204-5p, and ribonucleotide reductase M2 (RRM2). Microarray analysis and quantitative Real Time fluorescence Polymerase Chain Reaction (qRT-PCR) were employed to determine DSCAM-AS1 and miR-204-5p expression. Luciferase reporter assay was applied to examine the target relationship between DSCAM-AS1, miR-204-5p, and RRM2. Cell Counting Kit-8 (CCK-8 assay), transwell assay, and flow cytometry were used to detect cell proliferation, invasion, and apoptosis. The expression of DSCAM-AS1, miR-204-5p, and RRM2 were confirmed by Western blot. We also conducted in vivo assay to verify the effect of DSCAM-AS1. DSCAM-AS1 was up-regulated, while miR-204-5p was down-regulated in BC tissues and cells. DSCAM-AS1 directly targeted miR-204-5p. DSCAM-AS1 promoted the proliferation and invasion of BC cells by reducing miR-204-5p and inhibiting miR-204-5p expression. DSCAM-AS1 expression was related to the expression of RRM2, and miR-204-5p could reverse the function of DSCAM-AS1. RRM2 was up-regulated in BC cells, and miR-204-5p inhibited RRM2 expression by targeting RRM2. Overexpression of RRM2 stimulated proliferation and cell invasion and impeded apoptosis. In vivo experiments showed that knockdown of DSCAM-AS1 decreased the tumorigenesis of BC cells, increased the expression of miR-204-5p. DSCAM-AS1 promoted proliferation and impaired apoptosis of BC cells by reducing miR-204-5p and enhancing RRM2 expression. DSCAM-AS1/miR-204-5p/RRM2 may serve as novel therapeutic targets for BC.

The promoted delivery of RRM2 siRNA to vascular smooth muscle cells through liposome-polycation-DNA complex conjugated with cell penetrating peptides

Peripheral vascular disease (PVD) is a prevalent vascular disease that affect a large number of patients. The establishment of optimal treatments to mitigate the intimal hyperplasia (IH)-induced restenosis would help relieve the health burden of the PVD. Ribonucleotide reductase M2 (RRM2) is critical to cellular migration and proliferation. We have previously demonstrated that suppression of RRM2 expression could substantially inhibit hepatocellular carcinoma cell proliferation and migration. We hereby developed RRM2 small interfering RNA (siRNA)-loaded cell penetrating peptides-conjugated liposome-polycation-DNA complex (LPD) (RRM2-CLPD), aiming to inhibit the migration and proliferation of vascular smooth muscle cells (VSMCs) crucial for IH. RRM2-CLPD is of a small size (∼150 nm) and high siRNA encapsulation efficiency (∼90%). Further, we demonstrated that RRM2-CLPD could significantly inhibited RRM2 gene and protein expression by ∼80%. Notably, RRM2-CLPD was able to effectively bind to VSMCs, resulting in significant cellular proliferation and migration inhibition. Taken together, RRM2-CLPD represent a very promising treatment for IH.

MiR-211 regulates the expression of RRM2 in tumoral metastasis and recurrence in colorectal cancer patients with a k-ras gene mutation.

Colorectal cancer (CRC) ranks as the third-leading cause of cancer-associated mortalities in Taiwan. The expression of ribonucleotide reductase M2 (RRM2) and p53R2 is associated with tumoral malignancy and progression in several types of cancer. The aim of the present study was to determine the association of p53R2/RRM2 with the upstream expression of microRNA (miR)-211 and the association of expression levels of p53, APC and k-ras with clinical outcomes in patients with CRC. The study consisted of 192 tumor tissue samples obtained from patients with CRC. Immunohistochemistry and direct sequencing of DNA were performed to analyze p53R2/RRM2 protein expression and p53/APC/k-ras gene mutations in these samples. The expression level of miR-211 was detected by reverse transcription-quantitative polymerase chain reaction. The results showed that the expression of p53R2 was lower and that of RRM2 was higher in patients with lymph node metastasis, distant metastasis, and late-stage CRC compared with patients without lymph node metastasis, distant metastasis and early-stage CRC. A high expression of RRM2 in patients had a negative effect on overall survival (OS) and disease-free survival (DFS) in CRC. Positive expression of RRM2 was detected in tumor tissues, and expression associated with the presence of k-ras gene mutation. Furthermore, it was detected that the upstream miR-211 expression was negatively associated with RRM2 expression in tumor tissues of patients with CRC. miR-211 expression was associated with survival and tumoral recurrence in patients with k-ras mutations. The present authors suggest that the downregulation of miR-211 and overexpression of RRM2 in tumor tissues of patients with CRC could be used to predict metastases and disease prognosis, particularly in patients with k-ras gene mutations.