Yan Zou

The purpose of the present study was to investigate the expression profile of leucine‑rich repeat‑containing protein 8A (LRRC8A) in osteosarcoma and normal cortical bone, as well as its association with sex, age and tumor malignancy. Immunohistochemical staining of osteosarcoma tissue microarrays (TMAs) was performed to determine the protein expression of LRRC8A and compare them among different subgroups. The expression of LRRC8A in the nuclei and cytoplasm of U2OS tumor cells and MC3T3‑E1 osteoblast‑like cells was determined using reverse transcription‑quantitative PCR. Of all samples of the TMA for patients with osteosarcoma that were tested, 94% featured high cytoplasmic expression of LRRC8A, while in all normal bone tissue control groups, the gene was mainly expressed in the nucleus. In MC3T3‑E1 osteoblasts, the expression of LRRC8A at the RNA level was mainly in the cytoplasm. The difference in expression of LRRC8A between microarrays and osteoblasts was statistically significant. In U2OS osteosarcoma cells, LRRC8A mRNA was concentrated in the nuclei and cytoplasm. In osteosarcoma, the expression level of LRRC8A was not significantly associated with sex or age. In conclusion, LRRC8A was highly expressed in the cytoplasm of osteosarcoma cells and the degree of expression may be associated with the degree of tumor malignancy.

Vascular complications are the primary reason for disability and mortality associated with diabetes mellitus (DM), and numerous microRNAs (miRNAs/miRs) are involved in the process, such as miR‑122, miR‑24 and miR‑423. It has been reported that miR‑328 regulates DM and cardiovascular disease; however, the role and mechanism of action underlying miR‑328 in HUVECs is not completely understood. The present study aimed to investigate the role and mechanism of action underlying the effects of miR‑328 on the functions of HUVECs. To simulate hyperglycemia combined with ischemia‑induced tissue starvation, HUVECs were cultured in endothelial cell medium with 25 mmol/l D‑glucose and 2% FBS for 24 h [high glucose (HG) + 2% FBS group]. HUVEC miR‑328 expression levels were detected by reverse transcription‑quantitative PCR. Cell migration, cytotoxicity and tube‑like structure formation were analyzed using wound healing, Cell Counting Kit‑8 and tube formation assays, respectively. Following transfection with miR‑328 inhibitor, miR‑328 expression was downregulated in HUVECs. Protein expression levels were determined by western blotting. Compared with the control group, the migration and tube‑like structure formation of HUVECs were decreased, and cell cytotoxicity was increased in the HG + 2% FBS group. The protein expression levels of vascular endothelial growth factor were also decreased, and the expression levels of miRNA‑328 in the HG + 2% FBS group were increased compared with the control group. However, miRNA‑328 downregulation reversed the aforementioned effects. Further experiments indicated that the AKT signaling pathway was inhibited in the HG + 2% FBS group; however, miR‑328 downregulation activated the AKT/mTOR signaling pathway, which was blocked by the AKT signaling pathway inhibitor, perifosine. Gene prediction and western blotting demonstrated that miR‑328 displayed a regulatory role via Pim‑1 proto‑oncogene, serine/threonine kinase (PIM1). In conclusion, miR‑328 expression was upregulated and angiogenesis was inhibited when HUVECs were subjected to high glucose and low serum conditions. miR‑328 downregulation enhanced angiogenesis by increasing PIM1 expression and activating the AKT/mTOR signaling pathway in HUVECs under high glucose and low serum conditions.