Reticulocalbin-2 Research Articles

CircRNA3669 promotes goat endometrial epithelial cells proliferation via miR-26a/RCN2 to activate PI3K/AKT-mTOR and MAPK pathways

The development of receptive endometrium (RE) from pre-receptive endometrium (PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells (GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways, thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA (ceRNA) to upregulate Reticulocalbin-2 (RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol (E2) and progesterone (P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26a-RCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.

Reticulocalbin 2 promotes the prolifer ation and migration of oral squamous cell carcinoma by regulating the EGFR ERK pathway

Purpose: To investigate the expression of reticulocalbin-2 (RCN2) in human oral squamous cell carcinoma (OSCC) tissues and cell lines and its effects on the proliferation and migration of OSCC cells.Methods: Immunohistochemical (IHC) assays were performed to evaluate the expression of RCN2 in 30 histologically-confirmed OSCC patient tissues and adjacent tissues. Immunoblot assays were performed to examine the expression of RCN2 in an oral mucosal keratinocyte cell line and OSCC cell lines, while the effects of RCN2 on OSCC cell proliferation were assessed by CCK-8 and colony formation assays. The effects of RCN2 on OSCC cell motility were determined using wound closure and Transwell assays. Furthermore, the effect of RCN2 on EGFR/ERK pathway in OSCC cells was evaluated by immunoblot assay.Results: Overexpression of RCN2 occurred in OSCC tissues and cells (p < 0.01). RCN2 also increased the proliferation of OSCC cells and stimulated the motility of OSCC cells in vitro (p < 0.01). These effects occurred as a result of the regulation of EGFR/ERK signaling pathway by RCN2.Conclusion: RCN2 is a potential therapeutic target for OSCC treatment. However, further in vivo studies are required to validate these findings.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Reticulocalbin 2 as a Potential Biomarker and Therapeutic Target for Atherosclerosis.

Vascular inflammation initiated by oxidized lipoproteins drives initiation, progression, and even rupture of atherosclerotic plaques. Yet, to date, no biomarker is directly linked to oxidized lipid-induced vascular inflammation. Reticulocalbin 2 (RCN2) is a key regulator of basal and oxidized lipid-induced cytokine production in arterial wall cells. We evaluated the potential of circulating RCN2 to identify subjects with or at risk of developing atherosclerosis. Immunohistochemical analysis revealed abundant RCN2 expression in the endothelium and adventitia of normal arteries and in atherosclerotic lesions of both humans and mice. Atherosclerosis-susceptible C57BL/6 (B6) mice had higher plasma Rcn2 levels than resistant C3H mice. High-fat diet feeding raised plasma Rcn2 levels of both strains. In humans, patients with coronary artery disease (CAD) or peripheral artery disease (PAD) showed elevated serum RCN2 levels compared to healthy controls. In a cohort of 92 CAD patients, serum RCN2 exhibited a significant inverse correlation with HDL cholesterol and K+ levels and a trend toward association with white blood cell account, Na+, statin treatment, and diastolic blood pressure. HDL treatment suppressed Rcn2 expression in endothelial cells. This study suggests that circulating RCN2 is a potential non-invasive biomarker for identifying individuals with atherosclerosis and HDL protects against atherosclerosis by downregulation of RCN2 expression in endothelial cells.

Long non-coding RNA DUXAP8 elevates RCN2 expression and facilitates cell malignant behaviors and angiogenesis in cervical cancer via sponging miR-1297

BackgroundCervical cancer (CC) endangers women’s health in the world range. Accumulating studies have revealed the crucial regulatory role of long non-coding RNAs (lncRNAs) in multiple malignancies, including CC. Our study aimed to explore the role of lncRNA double homeobox A pseudogene 8 (DUXAP8) in cervical carcinogenesis.MethodsGene expressions in CC were assessed by RT-qPCR. Function experiments and tube formation assays were performed to evaluate the role of DUXAP8 in CC cells. Subcellular fractionation and FISH assays were conducted to determine the subcellular location of DUXAP8. Luciferase reporter, RNA pull down and RIP assays were conducted to investigate the mechanism of DUXAP8.ResultsDUXAP8 was notably upregulated in CC cells. Downregulation of DUXAP8 repressed cell malignant behaviors and angiogenesis in CC. Mechanically, DUXAP8 boosted the expression of reticulocalbin-2 (RCN2) through relieving the binding of miR-1297 to RCN2 3’-UTR. Moreover, miR-1297 inhibition and RCN2 overexpression could counteract the inhibitory effects of DUXAP8 knockdown on the malignant phenotypes of CC cells. Besides, enhanced RCN2 expression restored the tumor growth in vivo that was inhibited by DUXAP8 repression.ConclusionsDUXAP8 promotes malignant behaviors in CC cells via regulating miR-1297/RCN2 axis.Graphical

Lipopolysaccharide induces vascular endothelial cell pyroptosis via the SP1/RCN2/ROS signaling pathway

Endothelial cell pyroptosis is a novel cause of endothelial dysfunction in sepsis. Reticulocalbin-2 (RCN2) is involved in regulating vascular inflammation and plays an important role in the cardiovascular system. However, the role of RCN2 in inflammation-induced endothelial cell pyroptosis remains to be explored. Here, we found that RCN2 was upregulated after lipopolysaccharide (LPS) treatment in a concentration‐ and time-dependent manner. RCN2 knockdown resulted in a significant decrease in pyroptosis, reduced LDH and IL-1β release and ROS production and inhibited the expression of pyroptosis‐related proteins (NLRP3, cleaved caspase-1, and cleaved GSDMD) (all p < 0.05). N‐acetyl‐L‐cysteine (NAC) counteracted the effects of RCN2 on pyroptosis (all p < 0.01). The silencing of RCN2 antagonized the inhibitory effect of LPS on the phosphorylation of eNOS (p < 0.05). We predicted and confirmed that specificity protein-1(SP1) could directly bind to the RCN2 promoter and regulate RCN2. RCN2 overexpression rescued the inhibitory effect of SP1 inhibitor on HUVEC pyroptosis induced by LPS (all p < 0.05). These findings suggested that the activation of the SP1/RCN2/ROS signaling pathway could promote LPS-induced endothelial cell pyroptosis.

Osteogenic differentiation and calcification of human aortic smooth muscle cells is induced by the RCN2/STAT3/miR-155-5p feedback loop

BackgroundVascular calcification (VC) is associated with the high morbidity and mortality of cardiovascular diseases in dialysis patients and is a process in which vascular smooth muscle cells (VSMCs) actively differentiate into osteoblast-like cells. Reticulocalbin-2 (RCN2) is involved in the process of osteogenic differentiation under diabetic conditions, but its regulatory role under hyperphosphatemic conditions and the related mechanisms remain unclear. In this study, the importance of the interactions among RCN2, STAT3 and miR-155-5p during the osteogenic differentiation and calcification of human aortic VSMCs (HASMCs) were investigated. MethodsRCN2 was measured in femoropopliteal artery plaque specimens from 6 peripheral arterial disease (PAD) patients with chronic kidney disease (CKD) and 6 PAD patients without CKD. RCN2 protein and mRNA expression were assessed in the high phosphate-induced aortic rings culture ex vivo model. In vitro calcification assays and molecular mechanism studies were performed in HASMCs. ResultsImmunohistochemical staining results revealed increased RCN2 expression in the calcified plaques of femoral arteries of patients with CKD and in a high phosphate-induced aortic culture ex vivo model. RCN2 promoted HASMCs osteogenic differentiation and calcification by inducing STAT3 phosphorylation. Furthermore, inhibition of STAT3 activation by cryptotanshinone (CT) promoted miR-155-5p expression in HASMCs. In turn, miR-155-5p inhibited RCN2 mRNA expression, while RCN2 overexpression partially offset the miR-155-5p-mediated inhibition of HASMC calcification, acting as a positive feedback loop. ConclusionThese results demonstrate that RCN2 is a crucial regulator of VC under hyperphosphatemic conditions. RCN2/STAT3/miR-155-5p feedback loop is important in VC and targeting each member of this feedback loop could potentially reverse high phosphate-induced VC.

Reticulocalbin 2 enhances osteogenic differentiation of human vascular smooth muscle cells in diabetic conditions

AimDiabetes accelerates pro-atherogenic and pro-osteogenic phenotypes of vascular smooth muscle cells (VSMCs), an important process for vascular calcification. Reticulocalbin 2 (RCN2) is a candidate gene for atherosclerosis and involved in vascular remodeling in hypertension. However, the role of RCN2 in VSMCs calcification under diabetic conditions is unclear. Materials and methodsExpression of RCN2 and Runt-related transcription factor 2 (Runx2) in femoropopliteal arterial plaques was compared between type 2 diabetes mellitus (DM) and non-DM patients using immunohistochemical staining (IHCS). Human aortic VSMCs (HAVSMCs) were analyzed under RCN2 gene knockdown and overexpression conditions. Alizarin red staining and intracellular calcium deposition quantification were used to observe calcification induced in vitro under normal glucose or high glucose combined with β-glycerol phosphoric acid conditions. The cells were investigated for gene modulation of osteogenic differentiation markers using Western blotting. Key findingsThe expression of RCN2 and Runx2 in femoropopliteal artery plaques was significantly higher in DM than in non-DM patients. In addition, a significant positive correlation was observed between RCN2 and Runx2 levels. RCN2 was highly expressed when HAVSMCs were treated with high glucose and the expression levels correlated with the calcification characteristics. RCN2 upregulated osteogenic transformation markers Runx2 and Osterix in HAVSMCs and downregulated contractile phenotype markers α-SMA and SM22α. SignificanceThe results from this study indicate RCN2 is a major factor in mediating the calcification process of HAVSMCs in diabetic conditions. Thus, RCN2 may serve as a future therapeutic target for vascular calcification in diabetes.

Downregulation of microRNA‑183‑5p inhibits the proliferation and invasion of colorectal cancer cells by inactivating the reticulocalbin‑2/Wnt/β‑catenin signaling pathway.

microRNAs (miRNAs) are frequently aberrantly expressed in colorectal cancer (CRC) and are considered to serve a critical role in the onset and development of CRC by binding to its target transcription factor. The aim of the present study was to examine the role of miRNA (miR)‑183‑5p in the proliferation, invasion and migration of CRC cells, in addition to its underlying mechanism. Reverse transcription‑quantitative polymerase chain reaction analysis was used to detect the expression level of miR‑183‑5p. MTT and Transwell assays were performed to examine proliferation and invasion in SW620 cells. Western blot analysis was performed to determine the protein expression of reticulocalbin‑2 (RCN2), matrix metalloproteinase‑2, β‑catenin, cyclin D1 and c‑Myc. miR‑183‑5p expression was significantly upregulated in the CRC tissues compared with adjacent normal tissues. In addition, the inhibition of miR‑183‑5p suppressed proliferation, invasion and migration in SW620 cells. miR‑183‑5p downregulation or overexpression regulated the CRC cell cycle, invasion and migration by modulating RCN2 expression. Furthermore, the Wnt/β‑catenin pathway was observed to be involved in the inhibitory effect of miR‑183‑5p downregulation in CRC cell proliferation, invasion and migration. These results provided evidence that the downregulation of miR‑183‑5p inhibits CRC proliferation and invasion by regulating the RCN2/Wnt/β‑catenin pathway. miR‑183‑5p and RCN2 may serve an important role in the molecular etiology of CRC and have potential applications in CRC treatment.

Reticulocalbin-2 enhances hepatocellular carcinoma proliferation via modulating the EGFR-ERK pathway.

This corrects the article DOI: 10.1038/onc.2017.230.

Reticulocalbin-2 enhances hepatocellular carcinoma proliferation via modulating the EGFR-ERK pathway.

Hepatocellular carcinoma (HCC) is a major health threat worldwide. Although the involvement of reticulocalbin-2 (RCN2) in cell differentiation has been reported, its function in oncogenesis is poorly understood. Here, we showed that RCN2 was upregulated in tumors compared with adjacent non-tumorous tissues in HCC patients and RCN2 expression clinically correlated with tumor size, disease recurrence and survival rate. Both knockdown and knockout of RCN2 significantly inhibited HCC cell proliferation by inducing G1/S transition arrest and downregulating cyclin D1 expression, while the proliferative ability was restored in knockout HCC cells with exogenously expressed RCN2. Mechanistically, we demonstrated that RCN2 interacted with the epidermal growth factor receptor (EGFR). Knockout of RCN2 in HCC cells not only inhibited activation of the EGFR-ERK pathway by blocking EGF-mediated EGFR dimerization and internalization but also suppressed cell proliferation and EGFR phosphorylation under long exposure to EGF. We further showed that knockout of RCN2 inhibited EGFR phosphorylation, Ki-67 expression and tumor growth in nude mice. Moreover, we demonstrated that RCN2 knockout sensitized HCC cells to tyrosine kinase inhibitors, including erlotinib, lapatinib and sunitinib. Taken together, our results indicate that RCN2 plays a pivotal role in HCC cell proliferation and tumor growth presumably through regulating activation of the EGFR-ERK pathway. Our work also suggests that RCN2 is a potential therapeutic target of HCC.

Phosphatidylinositol glycan anchor biosynthesis, class X containing complex promotes cancer cell proliferation through suppression of EHD2 and ZIC1, putative tumor suppressors.

We identified phosphatidylinositol glycan anchor biosynthesis, class X (PIGX), which plays a critical role in the biosynthetic pathway of glycosylphosphatidylinositol (GPI)-anchor motif, to be upregulated highly and frequently in breast cancer cells. Knockdown of PIGX as well as reticulocalbin 1 (RCN1) and reticulocalbin 2 (RCN2), which we found to interact with PIGX and was indicated to regulate calcium-dependent activities, significantly suppressed the growth of breast cancer cells. We also identified PIGX to be a core protein in an RCN1/PIGX/RCN2 complex. Microarray analysis revealed that the expression of two putative tumor suppressor genes, Zic family member 1 (ZIC1) and EH-domain containing 2 (EHD2), were upregulated commonly in cells in which PIGX, RCN1, or RCN2 was knocked down, suggesting that this RCN1/PIGX/RCN2 complex could negatively regulate the expression of these two genes and thereby contribute to human breast carcinogenesis. Our results imply that PIGX may be a good candidate molecule for development of novel anticancer drugs for breast cancer.

Stem cell markers in gastrointestinal cancer

21095 Background: Cancer cells with stem cells (CSC) properties have been identified in different tumors. It is suggested that CSC are responsible for the continuous growth of tumors, metastasis and drug-resistance. Markers for stem cells have been described. Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. ABCG2 transporter (MDR1) gene expression has been described as surrogate for the side-population phenotype. PTTG1 has also been recently identified as a component of the molecular signature of human (hu) ES cell-lines. Methods: Using Digital Northern we have demonstrated a significant tag counts for PTTG1 and reticulocalbin 2 (RCN2) in 11 huES cell-lines of the CGAP. The objective of our work has been to assess gene expression of these SC markers in a panel of new gastrointestinal cancer (GC) cells lines (CL) developed in our laboratory. Quantitative assessment was obtained by real-time PCR relative to normal bone marrow (BM), colonic mucosa and established cell-lines. GCCLs have been developed from ascitic fluid obtained of pancreatic carcinoma (MBQ-OJC1) and colon cancer (JJPF-OJC4, LCM-OJC5 and JAC-OJC6). GCCLs had been fully characterized by cytomorphology, epithelial and tumor markers (keratins, EGFR, EpCAM, p53), karyotype and tumor spheroids cultures. Results: Expression for ABCG2, Nanog, Oct4, PTTG1 and RCN2 were clearly detected in all the GCCL. Relative levels for each mRNA shown wide variety. For example, ABCG2 mRNA was highly expressed (2–26 fold) in colon cancer CL relative to BM. RCN2 was overexpressed (more than 2 x 102 fold) in 3 GCCL. Conclusions: Our results show that expressions of different “stemness” genes are maintained in cultured cancer cells. These data suggest that CSC are present in metastatic sites and can be maintained in continuous culture. We hypothesized that PTTG1 and RCN2 could be tested as a new cancer stem cells markers. No significant financial relationships to disclose.