Many studies have shown that circular RNAs (circRNAs) play a key regulatory role in the whole biological process of tumors. The purpose of this study was to explore the biological function and molecular mechanism of circ_0001666 in non-small cell lung cancer (NSCLC), so as to provide new targets for the diagnosis and treatment of NSCLC. Gene expression profiles were downloaded from Gene Expression Omnibus (GEO, GSE101586) and the differential genes were obtained by using GEO2R analysis. The quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression level of circ_0001666 in NSCLC cells. Cell counting kit-8 (CCK-8) and Annexin V-FITC apoptosis detection kit were respectively used to assess the cell proliferation and apoptosis, where circ_0001666 was knockdown in NSCLC cells. The targeted relationship among mircoRNA 330-5p (miR-330-5p), circ_0001666, and high mobility group A2 protein (HMGA2) was verified by bioinformatics prediction, dual-luciferase reporter gene, RNA immunoprecipitation (RIP) and RNA pull down assay. The results showed that the expression of circ_0001666 in NSCLC cells was significantly up-regulated than that in normal lung epithelial cells. Circ_0001666 knockdown reduced the cell viability and promoted the apoptosis of NSCLC cells, which could be reversed by miR-330-5p inhibitors. MiR-330-5p is the downstream target of circ_0001666 and can be adsorbed by circ_0001666. HMGA2 is a target gene of miR-330-5p, which can be indirectly regulated by circ_0001666. The results suggest that circ_0001666 promotes the proliferation and inhibits apoptosis of NSCLC cells via miR-330-5p/HMGA2 axis.
Apoptosis/genetics* ; Carcinoma, Non-Small-Cell Lung/genetics* ; Cell Proliferation/genetics* ; HMGA2 Protein ; Humans ; Lung Neoplasms/genetics* ; MicroRNAs/genetics* ; RNA, Circular

OBJECTIVE: To investigate the regulatory effects of RBM47 on HMGA2 and the function of RBM47 in human chronic myeloid leukemia cell K562. METHODS: K562 cells were transduction by the overexpressed and knockdown RBM47 lentiviral vector. CCK-8 assay was used to detect the effect of RBM47 on the proliferation of K562 cells. Flow cytometry assay was used to detect the effect of RBM47 on the cell cycle progression of K562 cells. RNA immunoprecipitation assay was used to detect the association between RBM47 and HMGA2 mRNA. RT-qPCR was used to detect the effects of RBM47 on the stability of HMGA2 mRNA. Western blot was used to evaluate the effect of RBM47 on HMGA2 protein expression. RESULTS: The overexpressed RBM47 could inhibit the proliferation and cell cycle progression of K562 cells. However, the inhibitation of RBM47 could improve the proliferation and cell cycle progression of K562 cells. RBM47 combined with HMGA2 mRNA could promote the degradation of HMGA2 mRNA. Thus, the overexpressed RBM47 could decrease the expression of HMGA2 protein in K562 cells. CONCLUSION RNA binding protein RBM47 can inhibit the proliferation of K562 cells by regulating HMGA2 expression.
Apoptosis ; Cell Proliferation ; HMGA2 Protein/genetics* ; Humans ; K562 Cells ; RNA, Messenger/genetics* ; RNA-Binding Proteins/genetics*

To investigate the effect of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells on apoptosis induced by chemotherapeutics. 
 Methods: A total of 30 osteosarcoma tissues of sensitive and resistant to chemotherapeutics were divided into a chemotherapy-sensitive group and a chemotherapy-resistant group. The mRNA expression levels of miR-30a and high mobility group protein A2 (HMGA2) in the chemotherapy-sensitive group and the chemotherapy-resistant group, and the mRNA expression levels of miR-30a in osteosarcoma U2-OS cells treated by cisplatin, doxorubicin and methotrexate at different concentrations were detected by real-time PCR. The expression levels of autophagy related protein Beclin 1, microtubule associated protein 1 light chain 3B (LC3B) and autophagy factor P62 were detected by Western blotting. The osteosarcoma U2-OS cells were transfected with miR-30a mimics and miR-30a inhibitors to construct a miR-30a high expression group, a miR-30a low expression group and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after treatment of cisplatin and doxorubicin in these 3 groups were detected by Western blotting; the level of autophagy was detected by monodansylcada (MDC) staining; the level of ROS was detected by dihydroethidium (DHE); the level of cell surviving rate was detected by cell counting kit-8 (CCK-8); the level of apoptosis was detected by annexin APC/PI double staining; the level of mitochondria oxidative damage was detected by mitochondrial membrane potential assay kit with JC-1 (JC-1 method). The interaction between miR-30a and HMGA2 was detected by dual luciferase reporter assay. The osteosarcoma U2-OS cells were transfected with HMGA2 mimics and HMGA2-shRNA to construct a high HMGA2 group, a low HMGA2 group, and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after the treatment of cisplatin were detected by Western blotting.
 Results: The level of miR-30a in the chemotherapy-resistant tissues was significantly lower than that in the chemotherapy-sensitive tissues (P<0.05), and the expression of HMGA2 was opposite comparing to that of miR-30a (P<0.05). After the treatment by low concentration (5 μmol/L) of chemotherapeutics, the level of miR-30a was down-regulated in osteosarcoma U2-OS cells, accompanied with up-regulation of Beclin 1 and LC3B (P<0.01) and down-regulation of P62 (P<0.01). Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly decreased (P<0.05), and the expression level of P62 was significantly increased (P<0.05) in the miR-30a high expression group, which was opposite in the miR-30a low expression group. In the miR-30a high expression group treated by chemotherapeutics, the level of autophagy and the cell survival rate were lower than those in group with low expression of miR-30a, while the levels of ROS, the mitochondrial oxidative damage and the apoptosis were higher than those in group with low expression of miR-30a (all P<0.05). The targeting interaction between HMGA2 and miR-30a were verified by dual luciferase reporter assay. Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly increased (P<0.05), and the expression level of P62 was significantly decreased (P<0.05) in the HMGA2 high expression group, which was opposite in the HMGA2 low expression group.
 Conclusion: Suppression of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells is likely to enhance the therapeutic effect of chemotherapeutics.
Apoptosis ; Apoptosis Regulatory Proteins ; Autophagy ; Beclin-1 ; Bone Neoplasms ; Cell Line, Tumor ; HMGA2 Protein ; metabolism ; Humans ; MicroRNAs ; genetics ; Osteosarcoma

Animals ; Apoptosis ; Biomarkers, Tumor ; metabolism ; Cell Cycle ; Cell Proliferation ; Epithelial-Mesenchymal Transition ; HMGA2 Protein ; genetics ; metabolism ; physiology ; Humans ; Lymphatic Metastasis ; Neoplasm Invasiveness ; Neoplasms ; blood supply ; metabolism ; pathology ; Neovascularization, Pathologic

BACKGROUNDThe management of Barcelona Clinic Liver Cancer (BCLC) stage B hepatocellular carcinoma (HCC) is controversial due to the early recurrence after curative hepatectomy, and many variables were related to the prognosis. The purpose of this study was to predict the tumor recurrence in early postoperative period of the patients with BCLC stage B HCC.

METHODSFrom January 2004 to January 2012, 104 patients with BCLC stage B HCC underwent hepatectomy. Clinicopathological factors and follow-up data were statistically analyzed to establish a predicting scoring system.

RESULTSThe overall survival rates for one, three, and five years were 69.2%, 52.7%, and 42.3%, and the disease-free survival rates for one, three, and five years were 52.9%, 47.3%, and 37.5%, respectively. The multiple factors analysis showed that the micro-vessel invasion, lymph nodes metastasis, multiple lesions, and the high expression of HMGB1 were independent factors (P < 0.05). A scoring system was established to predict the early recurrence within one year after the surgery for BCLC stage B HCC, according to the analysis results with a specificity of 85.1% and a sensitivity of 80.3%.

CONCLUSIONVariant clinicopathological factors were associated with early postoperative recurrence for BCLC stage B HCC and recurrence early after hepatectomy was more likely in patients with a higher score of the scoring system.

Carcinoma, Hepatocellular ; metabolism ; pathology ; surgery ; Disease-Free Survival ; Female ; HMGA1a Protein ; metabolism ; Hepatectomy ; Humans ; Liver Neoplasms ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Treatment Outcome

BACKGROUNDOncofetal protein high-mobility-group AT-hook protein 2 (HMGA2) is reactivated in serous ovarian cancer (SOC) and its overexpression correlates with poor prognosis. To explore the mechanism, we investigated whether HMGA2 could avoid microRNA regulation due to gene truncation or 3' UTR shortening by alternative polyadenylation.

METHODSReal-time reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the abundance of different regions of HMGA2 mRNA in 46 SOC samples. Rapid amplification of cDNA 3' ends (3' RACE) and Southern blotting were used to confirm the shortening of 3' untranslated region (UTR). 5' RACE and Southern blotting were used to prove the mRNA decay.

RESULTSNo significant difference in the ratio of the stable coding region to the fragile region was observed between SOC and control normal fallopian tubes, indicating that the HMGA2 gene is not truncated in SOC. Varying degrees of 3' UTR shortening in SOC samples were observed by comparing the abundance of the proximal region and distal region of the HMGA2 3' UTR. The ratio of the proximal to the distal region of the 3' UTR correlated significantly with expression of the HMGA2 coding region in SOC (r = 0.579, P < 0.01). Moreover, although the abundance of the HMGA2 coding region varied, all samples, including the very low expressed samples, exhibit relatively high levels of the proximal 3' UTR region, suggesting a dynamic decay of HMGA2 mRNA from the 5' end. The shortening of 3' UTR and the decay from the 5' end were confirmed by 3' RACE, 5' RACE and subsequent Southern blotting.

CONCLUSIONHeterogeneous 3' UTR lengths render HMGA2 susceptible to different levels of negative regulation by microRNAs, which represents an important mechanism of HMGA2 reactivation in SOC.

3' Untranslated Regions ; genetics ; Cystadenocarcinoma, Serous ; genetics ; metabolism ; Female ; HMGA2 Protein ; genetics ; metabolism ; Humans ; Ovarian Neoplasms ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Hematopoietic stem cells (HSCs) comprise a rare population of cells that can regenerate and maintain lifelong blood cell production. This functionality is achieved through their ability to undergo many divisions without activating a poised, but latent, capacity for differentiation into multiple blood cell types. Throughout life, HSCs undergo sequential changes in several key properties. These affect mechanisms that regulate the self-renewal, turnover and differentiation of HSCs as well as the properties of the committed progenitors and terminally differentiated cells derived from them. Recent findings point to the Lin28b-let-7 pathway as a master regulator of many of these changes with important implications for the clinical use of HSCs for marrow rescue and gene therapy, as well as furthering our understanding of the different pathogenesis of childhood and adult-onset leukemia.
Animals ; Cell Differentiation ; *Cell Lineage ; Embryonic Stem Cells/cytology/*metabolism ; HMGA2 Protein/genetics/metabolism ; Hematopoietic Stem Cells/cytology/*metabolism ; Humans ; Leukemia/etiology/metabolism/surgery ; MicroRNAs/genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism

OBJECTIVETo explore the effects of lentivirus-mediated RNA interference silencing HMGA2 on proliferation and expressions of cyclin B2 and cyclin A2 in HL-60 cell line.

METHODSThe protein and mRNA expressions of HMGA2 in HL-60 cells transduced by recombinant lentivirus producing HMGA2 gene short hairpin (shRNA) were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis; The effects of the lentivirus on cell proliferation inhibiting rate, the ability of cell proliferation and cell cycle were analyzed by soft agar colony formation assay and FCM, respectively; The protein and mRNA expressions of cyclin B2 and cyclin A2 were also examined by Western-blot and RT-PCR.

RESULTSRecombinant lentivirus producing HMGA2 shRNA was successfully constructed, which was identified by PCR and sequencing; Stable HMGA2-deficient HL-60 cell line was established by puromycin, its mRNA and protein expression inhibition rates were (80.66 ± 7.98)% and (76.35 ± 12.72)%, respectively. Silencing of endogenous HMGA2 resulted in efficient inhibition of the cellular proliferative activity, low and flat of the cell growth curve and the lack of typical character of exponential growth. FCM revealed significant more cell cycle G(2)/M arrest \[(30.00 ± 5.78)%\] in HL-60 cell line transfected specific shRNA than control group \[(13.90 ± 4.07)%\] (P < 0.05). The cyclin B2 mRNA and protein expression inhibition rates in stable HMGA2-deficient HL-60 cell line were (67.55 ± 7.69)% and (51.77 ± 4.81)%, respectively, while the expression of cyclin A2 had no significant change compared with control group.

CONCLUSIONRNAi silencing of HMGA2 down-regulated cyclinB2, significantly inhibited the proliferation of HL-60 cells and induced the accumulation of HL-60 cells in the G(2)/M phase. Thus, HMGA2 may be an important target for anti-leukemia therapy.

Cell Proliferation ; Cyclin A2 ; genetics ; Cyclin B2 ; genetics ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HL-60 Cells ; HMGA2 Protein ; genetics ; Humans ; Lentivirus ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo elucidate the role of let-7d in regulating the biological behavior of ovarian cancer cells and their expressions of HMGA2 and ras proteins.

METHODSThe pre-let-7d sequence was synthesized and inserted into pcDNA6.2GW/EmGFPmiR and transfected into ovarian cancer IGROV1 cells to cause pre-let-7d overexpression. Real-time quantitative RT-PCR was employed to examine the expression levels of let-7d miRNA and HMGA2 mRNA, and Western blotting was performed to detect the expressions of HMGA2 and ras protein in the transfected cells. The effect of pcDNA6.2GW-let-7d transfection on IGROV1 cell proliferation was determined using MTT assay and the cell apoptosis rate was measured using flow cytometry.

RESULTSThe eukaryotic expression vector containing the target gene let-7d was successfully constructed and transfected into IGROV1 cells. The transfected cells showed a marked reduction of HMGA2 expression but a less obvious down-regulation of ras expression. Transfection with pcDNA6.2GW-let-7d to suppress the expression of HMGA2 caused alterations of the phenotype of IGROV1 cells shown by a reduced proliferative activity and increased cell apoptosis.

CONCLUSIONLet-7d plays an important role in altering the malignant cell phenotype of ovarian cancer IGROV1 cells by regulating the expression of HMGA2.

Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; HMGA2 Protein ; genetics ; metabolism ; Humans ; MicroRNAs ; genetics ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; ras Proteins ; genetics ; metabolism

BACKGROUNDThe high mobility group A1 (HMGA1) proteins are architectural transcription factors found to be overexpressed in lung adenocarcinoma. Lentivirus-mediated RNA interference (RNAi) technology is a powerful tool for silencing endogenous or exogenous genes in human cancer cells. Our preliminary study shows that gemcitabine inhibits growth of the human lung cancer cell line SPCA-1 and induces apoptosis, and this effect might link with down-regulation of HMGA1 expression. This study aimed to investigate the chemosensitivity change of the lung adenocarcinoma cells SPCA-1 after HMGA1 inhibition by lentivirus-mediated RNAi.

METHODSWe studied a highly malignant lung adenocarcinoma cell line (SPCA-1 cells). Lentiviral short-hairpin RNA (shHMGA1) expression vectors targeting HMGA1 were used for generation of lentiviral particles. After being transfected into the lung adenocarcinoma cell line SPCA-1, the expression of HMGA1 was determined by retrotranscriptase polymerase chain reaction (RT-PCR) and Western blotting. The effect of gemcitabine on proliferation of positive and negative cells was observed by methyl thiazolyl tetrazolium (MTT) assay and clonogenic survival assay. Apoptosis was observed by flow cytometery. Chemosensitivity to gemcitabine was determined by IC50 analysis. Caspase activity was quantitated by a caspase colorimetric protease assay kit.

RESULTSHMGA1-siRNA silenced its target mRNA specifically and effectively in SPCA-1 cells. The apoptotic rates of the scramble control group were (7.43 ± 0.21)%, (11.00 ± 0.20)%, and (14.93 ± 0.31)%, and the apoptotic rates in the silenced group were (9.53 ± 0.42)%, (16.67 ± 0.45)%, and (25.40 ± 0.79)% under exposure to 0.05, 0.5 and 5.0 µg/ml of gemcitabine (P < 0.05). The IC(50) of the silenced group was (0.309 ± 0.003) µg/ml which was significantly lower than in the scramble control group, (0.653 ± 0.003) µg/ml (P < 0.05). It reduced cancer cell proliferation and increased apoptotic cell death after being treated with gemcitabine compared with the scramble control group. HMGA1 silencing resulted in reduction in the phosphorylation of Akt, and promoted the activation of caspases 3, 8 and 9 upon exposure to gemcitabine.

CONCLUSIONSLentivirus-mediated RNA interference of HMGA1 enhanced chemosensitivity to gemcitabine in lung adenocarcinoma cells. The mechanism may be associated with the PI-3K/Akt signal pathway. HMGA1 may represent a novel therapeutic target in lung cancer.

Antimetabolites, Antineoplastic ; pharmacology ; Blotting, Western ; Calcium-Transporting ATPases ; genetics ; metabolism ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Flow Cytometry ; Genetic Vectors ; genetics ; HMGA Proteins ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; RNA Interference ; physiology ; Reverse Transcriptase Polymerase Chain Reaction