The Possible effect of long noncoding RNA cancer susceptibility candidate 2(CASC2)- Derived from Mesenchymal Stem Cells Exosomes- on valproic acid Induced Autism Spectrum Disorder in Rats: Targeting miR-181a/mTOR signaling Pathway

Objective To investigate the expression and the potential roles of long non-coding RNA(lncRNA)cancer susceptibility candidate 2(CASC2)and imprinted gene H19 in extrahepatic cholangiocarcinoma(ECC). Methods Four samples from patients with ECC were collected for high-throughput sequencing which was conducted to reveal the transcriptomic profiles of lncRNA CASC2 and H19.Bioinformatics tools were employed to predict the potential roles of the two genes.Another 22 ECC tissue samples and the cholangiocarcinoma cell lines(RBE,QBC939,HuH-28,and HuCCT1)with different degrees of differentiation were selected for validation.The para-carcinoma tissue and normal human intrahepatic biliary epithelial cell(HIBEC)were used as the control groups.The expression levels of lncRNA CASC2 and H19 in carcinoma tissue,para-carcinoma tissue,and cell lines were determined by real-time quantitative polymerase chain reaction(qRT-PCR).The correlation analysis was carried out for the clinical indicators of patients with the expression levels of the target genes. Results The two target genes showed significantly different expression between carcinoma tissue and para-carcinoma tissue(all P<0.05).Specifically,CASC2 had higher expression level in the carcinoma tissue than in the para-carcinoma tissue(t=1.262,P=0.025),whereas the expression of H19 showed an opposite trend(t=1.285,P=0.005).The expression levels of CASC2 in QBC939(t=8.114,P=0.015)and HuH-28(t=9.202,P=0.012)cells were significantly higher than that in the control group.The expression levels of H19 were significantly lower in RBE(t=-10.244,P<0.001),QBC939(t=-10.476,P<0.001),HuH-28(t=-19.798,P<0.001),and HuCCT1(t=-16.193,P=0.004)cells than in the control group.Bioinformatics analysis showed that CASC2 was mainly involved in the metabolic process and H19 in the development of multicellular organisms.Both CASC2 and H19 were related to catalytic activity.The expression level of lncRNA CASC2 was correlated with pathological differentiation(χ 2=6.222,P=0.022)and lymph node metastasis(χ2=5.455,P=0.020),and that of lncRNA H19 with pathological differentiation(χ2=1.174,P=0.029)and tumor size(χ2=-0.507,P=0.037). Conclusions In the case of ECC,lncRNA CASC2 and H19 have transcription disorders.lncRNA CASC2 is generally up-regulated in the carcinoma tissue,while H19 is down-regulated.Both genes have the potential to become new molecular markers for ECC.

Cancer susceptibility candidate 2 (CASC2) was found underexpressed in multiple types of human malignancies. However, the specific role of CASC2 in AML remains uncertain. The purpose of this study is to explore the expression of CASC2 in patients with AML and healthy donors and its prognostic significance in AML. Total RNA was isolated from bone marrow samples or peripheral blood samples of 87 patients with AML and 26 healthy adult donors. The expression of long noncoding RNA CASC2 was detected by quantitative real-time polymerase chain reaction. The association between CASC2 expression and other clinicopathological factors as well as its prognosis significance were analyzed. The peripheral blood mononuclear cell (PBMC) expression level of CASC2 in AML was significantly lower than that in the healthy control cohort (P = 0.0048), and in the bone marrow samples, CASC2 was significantly upregulated in patients with AML after the achievement of CR (median value: 0.041, range: 0.015-0.064) compared with that at newly diagnosis (median value: 0.017, range: 0.008-0.041) (P = 0.002). The expression of CASC2 had a significant relationship with complete remission (P = 0.019). Survival data assessed by Kaplan-Meier curves showed that patients with lower CASC2 expression had shorter overall survival and disease-free survival than patients with higher CASC2 expression. Finally, Cox proportional hazards analysis demonstrated that CASC2 was an independent prognostic indicator for both OS (P = 0.013) and DFS (P = 0.001) of AML. LncRNA CASC2 may serve as a new molecular biomarker for the early diagnosis and of AML, and may be an independent prognostic factor affecting the survival of patients with AML.

The long noncoding RNA cancer susceptibility candidate 9 (CASC9) has been revealed to be an oncogenic gene in several types of cancer, and high CASC9 expression is related to tumorigenesis and cancer progression. However, the role of CASC9 in bladder cancer (BC), particularly during epithelial-mesenchymal transition (EMT), has not been characterized. RT-qPCR, EdU, CCK-8, wound scratch, Transwell and flow cytometric assays were performed to detect CASC9 expression, miR-758-3p expression and their functions in BC. RNA FISH was used to detect CASC9 subcellular localization. Luciferase reporter assay, RT-qPCR assay and western blotting were used to explore the relationship of CASC9, miR-758-3p and TGF-β2. In the present study, it was revealed that CASC9 regulated EMT in BC. CASC9 expression was significantly upregulated in BC cell lines and specimens compared to that in adjacent normal bladder tissues. Upregulated CASC9 was associated with increased invasion ability and poor prognosis of BC. CASC9 knockdown inhibited BC cell proliferation, migration and invasion. Furthermore, a bioinformatics study and luciferase reporter assays revealed that CASC9 functioned as a ceRNA for miR-758-3p. CASC9 inhibited microRNA (miR)-758-3p activity and resulted in the de-suppression of its target transforming growth factor (TGF)-β2. TGF-β signaling driven by TGF-β2 was crucial for CASC9 to promote EMT in BC. Collectively, these results indicated that CASC9 sponged miR-758-3p to regulate the expression of TGF-β2, which activated the TGF-β signaling pathway and promoted proliferation and EMT in BC.

Diabetic nephropathy (DN) is one of the primary complications of diabetes. Long non-coding RNA cancer susceptibility candidate 2 (CASC2) has been established to function in DN, while its role in high glucose (HG)-induced human mesangial cells (HMCs) remains limited. The expression level of CASC2 and miR-133b was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay. Extracellular matrix (ECM) accumulation was monitored through the expression levels of collagen IV (Col IV) and fibronectin (FN) using qRT-PCR and western blot analyses. Oxidative stress was observed through the expression of NADPH oxidase2 (NOX2) and the activity of malondialdehyde (MDA) and superoxide dismutase (SOD) using western blot or corresponding detection kit. The expression of forkhead box P1 (FOXP1) at mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. The relationship between miR-133b and CASC2 or FOXP1 was predicted by online bioinformatics tools and verified by dual-luciferase reporter assay or RNA pull-down. The expression of CASC2 was reduced in serum from DN patients and HG-induced HMCs. CASC2 upregulation inhibited HG-induced HMCs proliferation, ECM accumulation and oxidative stress. MiR-133b was a target of CASC2 with a high level in serum from DN patients and HG-induced HMCs, and its enrichment reversed the effects of CASC2 upregulation. Besides, FOXP1 was a target of miR-133b with a low level in HG-induced HMCs, and its knockdown abolished the impacts of CASC2 upregulation. CASC2 upregulation suppressed HG-induced proliferation, ECM accumulation and oxidative stress of HMCs through miR-133b /FOXP1 regulatory axis, suggesting that CASC2 was a novel biomarker for DN treatment.

Cancer susceptibility candidate 2 (CASC2) and long noncoding RNA (lncRNA) have been identified asa tumor suppressor in colorectal, lung, renal, and stomach cancer as well as in patient gliomas, but the function of CASC2 in papillary thyroid carcinoma (PTC) is not yet clear. The present study aimed to explore the effects of CASC2 in PTC. The CASC2 expression was measured in PTC samples and normal tissues by using quantitative real-time polymerase chain reaction. The lentiviral vectors were used to establish CASC2 overexpression models in PTC cell lines to determine the effects of CASC2 on cell proliferation, apoptosis, migration, and invasion. A tumor xenograft animal model was used to examine the functions of overexpression CASC2. CASC2 expression was significantly decreased in PTC tumor tissues than adjacent normal tissues. CASC2 downregulation in PTC tissues significantly correlated with the tumor size, the presence of multifocal lesions, and the advanced pathological stage. CASC2 overexpression suppressed the cell proliferation and promoted apoptosis in PTC cell lines and CASC2 overexpression resulted in the inactivation of protein kinase B (PKB/AKT) and extracellular signal-regulated kinases (ERK1/2). The regulatory effects of CASC2 on PTC cell biological behavior were further enhanced by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or AKT1/2/3 inhibitor MK-2206 2HCl. CASC2 overexpression suppressed tumor growth in PTC cells in xenograft mouse models. Our results indicated that CASC2 significantly suppressed tumorigenesis in PTC and CASC2 may serve as a novel prognostic marker or therapeutic target.

CASC15 promotes epithelial to mesenchymal transition and facilitates malignancy of hepatocellular carcinoma cells by increasing TWIST1 gene expression via miR-33a-5p sponging

BackgroundDiabetic nephropathy (DN) is a common complication of diabetes. Long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is reported to exert a protective role in DN by a previous study. The working mechanism underlying the protective role of CASC2 in DN progression was further explored in this study.MethodsThe expression of CASC2 and microRNA-135a-5p (miR-135a-5p) was determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation ability was assessed by Cell Counting Kit-8 (CCK8) assay and 5-ethynyl-29-deoxyuridine (EDU) assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the production of inflammatory cytokines in the supernatant. Western blot assay was performed to analyze protein expression. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the target relationship between miR-135a-5p and CASC2 or tissue inhibitors of metalloproteinase 3 (TIMP3).ResultsHigh glucose (HG) treatment reduced the expression of CASC2 in human glomerular mesangial cells (HMCs) in a time-dependent manner. CASC2 overexpression suppressed HG-induced proliferation, inflammation and fibrosis in HMCs. miR-135a-5p was validated as a target of CASC2, and CASC2 restrained HG-induced influences in HMCs partly by down-regulating miR-135a-5p. miR-135a-5p bound to the 3ʹ untranslated region (3ʹUTR) of TIMP3, and CASC2 positively regulated TIMP3 expression by sponging miR-135a-5p in HMCs. miR-135a-5p silencing inhibited HG-induced effects in HMCs partly by up-regulating its target TIMP3. CASC2 overexpression suppressed HG-induced activation of Jun N-terminal Kinase (JNK) signaling partly through mediating miR-135a-5p/TIMP3 signaling.ConclusionsIn conclusion, CASC2 alleviated proliferation, inflammation and fibrosis in DN cell model by sponging miR-135a-5p to induce TIMP3 expression.

Abnormal expression of long non-coding RNA cancer susceptibility candidate 9 (CASC9) has been found to play vital roles in many human tumors. However, the role and the regulatory mechanism of CASC9 have not yet been demonstrated in retinoblastoma (RB). Hence, we performed this study to explore the function and mechanism of CASC9 in RB. CASC9 expression was firstly detected in human RB tissues and cells. The influence of CASC9 on the malignant phenotypes of RB cells, including cell proliferation, invasion, epithelial–mesenchymal transition (EMT) and apoptosis, was analyzed by overexpressing or silencing CASC9. The association between CASC9, miR-145-5p and E2F transcription factor 3 (E2F3) was determined by dual-luciferase reporter assay and RNA immunoprecipitation. We found that CASC9 expression was elevated in RB tissues and cells. Overexpression of CASC9 significantly facilitated the proliferation, invasion and EMT of RB cells. On the contrary, knockdown of CASC9 inhibited the proliferation, invasion and EMT, while enhanced the apoptosis of RB cells. CASC9 acted as a competing endogenous RNA (ceRNA) for miR-145-5p to regulate E2F3. Additionally, miR-145-5p inhibitor and E2F3 overexpression both partly reversed the malignant phenotypes of RB cells affected by CASC9 knockdown. However, miR-145-5p overexpression further strengthened these features induced by CASC9 downregulation. These findings suggested that CASC9 contributed to RB development by regulating E2F3 via sponging miR-145-5p. CASC9 might be a possible therapeutic target for RB.

Rheumatoid arthritis (RA) is a perennial inflammatory condition. Preliminary research indicated that long non-coding (lnc)RNA cancer susceptibility candidate 2 (CASC2) was downregulated in the serum of RA patients. Our study was designed to reveal the roles of lncRNA CASC2 in RA and the latent mechanisms underlying its role. Bioinformatics method (Starbase) and dual-luciferase reporter assay revealed that microRNA (miR)-18a-5p directly interacted with lncRNA CASC2. Furthermore, lncRNA CASC2 and miR-18a-5p expression in the serum samples of RA patients and healthy controls were measured via reverse transcription-quantitative PCR. Compared with the healthy subjects, lncRNA CASC2 was downregulated, whereas miR-18a-5p was upregulated in patients with RA. Overexpression of lncRNA CASC2 decreased the viability of human fibroblast-like synoviocytes (HFLSs) and induced apoptosis, as revealed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry analyses. Furthermore, the Western blotting assay suggested that Bax was upregulated and Bcl-2 was downregulated in lncRNA CASC2 up-regulated HFLSs. Downregulation of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, matrix metalloproteinase (MMP)1, and MMP3 levels by lncRNA CASC2 up-regulation was determined using enzyme-linked immunosorbent assays (ELISAs). However, HFLSs co-transfected with miR-18a-5p mimic exhibited opposite effects compared with the case for the overexpression of lncRNA CASC2. The aforementioned methods were used to verify that a binding site exists between B-cell translocation gene 3 (BTG3) and miR-18a-5p. The effects of miR-18a-5p inhibitor on HFLSs were reversed by BTG3 silencing. Overall, lncRNA CASC2 alleviated RA by adjusting the miR-18a-5p/BTG3 signaling axis and could serve as a novel therapeutic option for RA.

BackgroundHigh glucose (HG) induced podocytes injury plays an important role in diabetes nephropathy (DN) development. Long noncoding RNA cancer susceptibility candidate 2 (CASC2) was found to be decreased in serum of DN patients. We aimed to explore the function and possible mechanism of CASC2 in HG induced podocytes injury.MethodsUnder normal glucose (NG), HG and mannitol stimulated podocyte conditions, the levels of CASC2, microRNA-9-5p (miR-9-5p) and peroxisome proliferator-activated receptor gamma (PPARγ) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Podocyte injury was evaluated by measuring cell viability and apoptosis of CIHP-1 cells were checked by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Western blot was used to detect all protein levels. Dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to confirm the relationship between CASC2 and miR-9-5p.ResultsHG stimulation inhibited the expression levels of CASC2 and PPARγ, but promoted the expression of miR-9-5p. HG could restrain cell viability, autophagy and facilitate apoptosis in CIHP-1 cells, while CASC2 overexpression could reverse HG-induced podocytes injury. Furthermore, CASC2 could be used as a ceRNA to adsorb miR-9-5p, and miR-9-5p mimic overturned the effects of CASC2 on cell viability, autophagy and apoptosis in HG-stimulated podocytes. Additionally, PPARγ was a target gene of miR-9-5p, and CASC2 could weaken the HG-induced podocytes injury by up-regulating PPARγ.ConclusionCASC2 increased cell viability, autophagy and inhibited cell apoptosis by regulating miR-9-5p/PPARγ axis, thus reducing the HG-induced podocytes injury.

Oral squamous cell cancer (OSCC) is a primary malignant tumor of the head and neck. Long non-coding RNA cancer susceptibility candidate 2 (CASC2) is related to the chemoresistance of diverse tumors. At present, the resistance of OSCC to first-line chemotherapy drug cisplatin (DDP) is still a giant problem. Herein, we investigated the role and mechanism of CASC2 in OSCC resistance to DDP. Expression levels of CASC2, miR-31-5p, and KANK1 in OSCC tissues and cells were determined by qRT-PCR. The half-maximal inhibitory concentration (IC50) value of DDP-resistant OSCC cells and apoptosis of DDP-resistant OSCC cells were determined via CCK-8 or flow cytometry assays. The relationship between CASC2 or KANK1 and miR-31-5p was verified with a dual-luciferase reporter or RIP assays. The role of CASC2 in vivo was confirmed by xenograft experiments. We observed that CASC2A and KANK1 were downregulated while miR-31-5p was upregulated in DDP-resistant OSCC tissues and cells (p<0.05). CASC2 overexpression enhanced cell DDP sensitivity and accelerated cell apoptosis in DDP-resistant OSCC cells in vivo and in vitro (p<0.05). Notably, KANK1 acted as a target for miR-31-5p. Also, CASC2 modulated KANK1 expression via sponging miR-31-5p in DDP-resistant OSCC cells (p<0.05). Both CASC2 and KANK1 introduction-mediated impacts on the DDP sensitivity and apoptosis of DDP-resistant OSCC cells were restored by miR-31-5p elevation (p<0.05). To conclude, CASC2 boosted the DDP sensitivity and apoptosis of DDP-resistant OSCC cells by upregulating KANK1 via sponging miR-31-5p, and CASC2 might be a potential target for DDP-resistant OSCC treatment.

BackgroundRecently, it has been reported that long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2), a novel tumor suppressor, participates in regulating the carcinogenesis and suppresses tumor progression by sponging microRNAs (miRNAs). However, the expression and function of CASC2 in hepatocellular carcinoma (HCC) remain unclear.MethodsThe expression of CASC2 and miR-367 in HCC specimens and cell lines were detected by real-time PCR. Western blotting and immunohistochemistry were carried out for detection of epithelial-to-mesenchymal transition (EMT) markers in HCC. Transwell assays were used to determine migration and invasion of HCC cells. A mouse model for lung metastasis was established to evaluated HCC metastasis in vivo. The correlation among CASC2, miR-367 and F-box and WD repeat domain containing 7 (FBXW7) were disclosed by a dual-luciferase reporter assay, RIP assay and biotin pull-down assay.ResultsHere, CASC2 expression was significantly downregulated in HCC tissues, especially in aggressive and recurrent cases. In accordance, CASC2 underexpression was observed in HCC cell lines compared to LO2. In vitro and in vivo experiments revealed that CASC2 inhibited migration and invasion of HCC cells. Additionally, CASC2 repressed EMT process of HCC cells. Further studies demonstrated that CASC2 could function as a competing endogenous RNA (ceRNA) by sponging miR-367 in HCC cells. Functionally, gain- and loss-of-function studies showed that miR-367 promoted migration, invasion and EMT progression of HCC cells. Moreover, further investigations disclosed that FBXW7 was a downstream target of miR-367 and CASC2 prohibited EMT progression and subsequently exerted its anti-metastatic effects via CASC2/miR-367/FBXW7 axis in HCC cells. Clinically, CASC2 underexpression and miR-367 overexpression were closely correlated with the metastasis-associated clinicopathologic features. Notably, CASC2 low-expressing and miR-367 high-expressing HCC patients showed the poorest clinical outcome.ConclusionsOverall, we conclude that the CASC2/miR-367/FBXW7 axis may be a ponderable and promising therapeutic target for HCC.

ObjectiveSevere pneumonia occurs commonly in children and is the main cause of clinical infant mortality. This study tested the expression pattern of long noncoding RNA cancer susceptibility candidate 2 (CASC2) in the serum of children with severe pneumonia and explored its clinical values.MethodsSerum levels of CASC2 were detected in 145 children with severe pneumonia. All cases were divided into two groups based on their respiratory failure (RF) condition. Receiver operating characteristic (ROC) and Kaplan–Meier (K‐M) curves were plotted for the diagnostic and prognostic ability evaluation. Multivariate cox regression analysis was done for the examination of independent influence factors.ResultsThe serum levels of CASC2 significantly decreased in children with severe pneumonia in contrast with healthy individuals and reached the lowest value in those with RF. Serum CASC2 can distinguish severe pneumonia and predicted the development of RF. Based on the 28‐day survival data, cases with low CASC2 levels had a poor survival rate. CASC2 (hazard ratio [HR] = 0.068, 95% confidence interval [CI] = 0.016–0.292, P < 0.001) and age (HR = 2.806, 95% CI = 1.240–6.394, P < 0.001) were independent influence factor for the poor prognosis of children with severe pneumonia.ConclusionDownregulation of serum CASC2 was related to the occurrence of RF in children with severe pneumonia and may be a predictor of the poor prognosis. This study will provide a potential biomarker for severe pneumonia treatment in clinic.

Chemoresistance is the leading cause of recurrence in non-small cell lung cancer (NSCLC). The long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) inhibits the tumorigenesis of various cancers. However, the regulatory function of CASC2 on the chemoresistance of NSCLC remains unclear. The levels of CASC2 and miR-18a in cisplatin (DDP)-resistant NSCLC tissues and cell lines were evaluated by quantitative Polymerase Chain Reaction (qPCR). The role of low CASC2 levels on overall survival in patients with NSCLC was tested using the log-rank test. The Chi-squared test was used to assess the relation between CASC2 expression and clinicopathological features of NSCLC patients. Cell Counting Kit-8 (CCK-8) assays tested the cell proliferation of cisplatin-resistant NSCLC cells (H226/DDP and A549/DDP). The underlying regulatory mechanism between CASC2 and miR-18a or miR-18a and interferon regulatory factor 2 (IRF-2) was predicted by bioinformatics and verified by a Dual-Luciferase reporter assay, RNA transfection, qPCR, and Western blotting. Chromatin immunoprecipitation (ChIP) assay was done to exam the relation between E74 like factor 1 (ELF1) and CASC2 gene. Mice xenografts were applied to exam the function of CASC2 on chemosensitivity of cisplatin of NSCLC cells in vivo. Low CASC2 expression is more likely to present in patients with advanced TNM stage (IV), cisplatin-resistance, and poor overall survival. The expression of CASC2 sharply decreased in cisplatin-resistant NSCLC tissues and cell lines (H226/DDP and A549/DDP). CASC2 overexpression strongly inhibited proliferation, migration, and invasion of cisplatin-resistant NSCLC cells (H226/DDP and A549/DDP) in vitro and inhibited tumor growth in vivo. Besides, CASC2 repressed miR-18a function by binding to the complementary sites of miR-18a as competing endogenous RNAs (ceRNAs). MiR-18a released by the declining expression of CASC2 reduced the protein concentration of IRF-2 in NSCLC cells. Furthermore, the transcription factor ELF1 was found to be promotor of CASC2 and increased its levels in cisplatin-resistant NSCLC cells. IRF-2 expression mediated by the ELF1/CASC2/miR-18a axis is markedly associated with the proliferation, migration, and invasion of cisplatin-resistant NSCLC, resulting in inferior survival. These findings suggest that this regulatory axis may serve as a novel therapeutic target in NSCLC.

Long noncoding RNA (lncRNA) has been identified to serve a critical role in the development of various types of cancer. Cancer susceptibility candidate 2 (CASC2) is a cancer‑associated lncRNA. However, whether CASC2 regulates osteosarcoma progression remains unclear. Reverse transcription‑quantitative polymerase chain reaction, western blot, invasion and migration assays were used to evaluate the role of CASC2 in osteosarcoma. The present study reported that CASC2 may inhibit osteosarcoma development. Osteosarcoma tissues demonstrated reduced CASC2 expression compared with normal adjacent tissues. In addition, CASC2 transduction may decrease proliferation, migration and invasion of osteosarcoma cell lines whereas knockdown of CASC2 displayed opposing effects. Patients with low CASC2 levels were predicted to have a poor survival. Invivo implantation studies using pcDNA‑CASC2 or short interfering‑CASC2 exhibited decreased or increased tumor weight, respectively. These results suggested that CASC2 may serve as a potential tumor suppressor lncRNA in osteosarcoma and may provide potential insight into targeted intervention.