BACKGROUND:The main component of cartilage, type Ⅱ col agen gene expression in chondrocyte is positively correlated with SOX9 concentration in a dose-dependent manner.
OBJECTIVE:To observe the variation of SOX9 and type Ⅱ col agen mRNA content at different periods in the differentiation process (osteogenic, chondrogenic, adipogenic induction) of mesenchymal stem cel s, and to explore the correlation of SOX9 expression and type Ⅱ col agen.
METHODS:Bone marrow mesenchymal stem cel s were isolated from 4-week-old Kunming mice, and cultured in vitro to passage 3. The cel phenotype was identified with flow cytometry. Cel s were divided into three groups and subjected to three kinds of induction conditions favorable for adipogenic, chondrogenic and osteogenic differentiation, and each group was observed at three time points. In addition, the non-induced cel s were used as a control group. The total RNA of cel s was extracted at 3, 7, 14 days after induction, and SOX9 and type Ⅱ col agen mRNA was quantified with reverse transcription-polymerase chain reaction. The induced cel s were stained by immunofluorescence to observe the differentiation and perform statistical analysis.
RESULTS AND CONCLUSION:Passage 3 bone marrow mesenchymal stem cel s grew wel , and cel phenotype was confirmed as stem cel s by flow cytometry. The staining results showed that, the cel s differentiated into chondrocytes, adipocytes and osteoblasts. The SOX9 mRNA levels in the induced cel s were the highest in chondrogenic differentiation group, then in osteogenic differentiation group, and the lowest in adipogenic differentiation group. Type Ⅱ col agen mRNA levels in the induced cel s were the highest in chondrogenic differentiation group, then in adipogenic differentiation group, and the lowest in osteogenic differentiation group. SOX9 expression in chondrogenic differentiation group increased at 3 and 7 days, and then decreased at 14 days. While type Ⅱ col agen expression increased at 3, 7, 14 days. SOX9 mRNA levels increased as the osteogenic differentiation, while type Ⅱ col agen expression gradual y decreased. There was no significant difference in the SOX9 mRNA expression between adipogenic differentiation group and control group (P>0.05), while type Ⅱ col agen expression was not regularly changed. Experimental findings suggest that, critical effect of SOX9 in chondrogenic differentiation is better than that in osteogenic and adipogenic differentiation. SOX9 is associated with type Ⅱcol agen, which may alter along with the SOX9 in the early chondrogenic differentiation;SOX9 may play a fine-tuning role in the process of chondrogenic and osteogenic differentiation.

[Abstract] Objective: To explore the role of adhesion molecule with Ig like domain 2 (AMIGO2) in the proliferation of nasopharyn‐ geal carcinoma (NPC) cells and its mechanisms. Methods: A total of 10 NPC tissue samples and 10 normal nasopharyngeal epithelial tissue samples collected at Fujian Cancer Hospital during September 2017 and November 2017 were used for this study; in addition, NPC cell lines (CNE-1, CNE-2, SUNE-1, 6-10B, C666-1) and human immobilized nasopharyngeal epithelial cell line NP69 were also collected. The relative expression of AMIGO2 mRNAin above mentioned tissues and cell lines was detected by qPCR. Lentivirus vectors were constructed to interfere AMIGO2 mRNA expression, and qPCR was used to verify its interference efficiency. CCK-8 method, Clonal formation and Flow cytometry were performed to evaluate the effect of AMIGO2 interference on proliferation, clone formation and apoptosis of NPC cells. The influence of AMIGO2 interference on PI3K/AKT/mTOR signaling pathway and proliferation related molecular markers in NPC cells was assessed by Western blotting. Results: The results of qPCR showed that AMIGO2 was highly expressed in NPC tissues, CNE-2, and SUNE-1 cells (all P<0.01). The interference efficiency of AMIGO2 in CNE-2 and SUNE-1 cells could reach over 50%. The interfering of AMIGO2 expression significantly inhibited the proliferation and clone formation of CNE-2 and SUNE-1 cells (all P<0.01), promoted cell apoptosis (all P<0.01), reduced the phosphorylated protein expression levels of PI3K, AKT and mTOR in SUNE-1 cells (all P<0.01), as well as down-regulated the protein expressions of survivin and PCNA (all P<0.01). Conclusion: AMIGO2 may promote the proliferation as well as inhibit apoptosis of NPC cells by activating the PI3K/AKT/mTOR signaling pathway, suggesting that AMIGO2 may be a potential target for NPC therapy.