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

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

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

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

High mobility group A2 protein (HMGA2), an architectural factor, is highly expressed in various cancer types including lung cancers. It is a candidate target for cancer therapy. RNAi is an effective gene silencing method with low cost and less time-consuming. It is possible to exploit this technology in therapy. Here, 5 siRNAs targeting Hmga2 gene (HMGA2 siRNA1-5) were designed and synthesized. MTT assay, colony formation assay, transwell assay and flow cytometry were used to evaluate the effects of these siRNAs on lung cancer cell lines (NCI-H446 and A549). Results from cell proliferation, clone formation, migration and apoptosis showed that HMGA2 siRNA1, 3, 5 could affect these aspects for both lung cancer cell lines. Among the five siRNAs, HMGA2 siRNA5 showed the greatest inhibition effects. The inhibition effects of HMGA2 siRNA5 are sequence specific and are not due to the induction of interferon response. Taken together, siRNAs targeting Hmga2 gene are potential candidates for lung cancer gene therapy.
Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colony-Forming Units Assay ; Gene Silencing ; Genetic Therapy ; HMGA2 Protein ; genetics ; metabolism ; Humans ; Interferons ; metabolism ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Point Mutation ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo detect the expression of high mobility group protein A (HMGA) in male mouse testicular cell lines TM4, GC-1spg and GC-2spd(ts), and to pave the theoretical ground for further investigation of the action mechanism of the HMGA gene in male mouse spermatogenesis.

METHODSWe detected the expressions of HMGA1 and HMGA2 by RT-PCR and Western blot in the male mouse testicular cell lines TM4, GC-1spg and GC-2spd(ts).

RESULTSHMGA1 and HMGA2 were expressed in the male mouse testicular cell lines TM4, GC-1spg and GC-2spd(ts) at both mRNA and protein levels. Western blot and RT-PCR methods showed similar results.

CONCLUSIONThe expression of HMGA may be involved in the cell division and proliferation of TM4, GC-1spg and GC-2spd(ts) and play an important role in spermatogenesis of male mice.

Animals ; Cell Division ; Cell Line ; Cell Proliferation ; Gene Expression ; HMGA1a Protein ; genetics ; HMGA2 Protein ; genetics ; Male ; Mice ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatogenesis ; Testis ; cytology ; metabolism

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