Preliminary Research On Effects Of MiR-192 Inhibitors On Proliferation And Apoptosis Of Human Osteosarcoma Cell Line SOSP-9607

Background miRNA are a class of 18 to 25 nucleotides,non-coding small RNAs that regulate gene expression post-transcriptionally by binding to their cognate target mRNAs. Hundreds of miRNA genes have been found in diverse animals and plants. miRNA regulate a variety of biological processes, including tumor, growth and development, stem cell differentiation and so on. Study on the efforts of miRNA to cancer become the hot spot of resent research and thus, miRNA maybe become markers for cancer diagnostics and promising targets for therapeutics.Osteosarcoma is the most common primary malignant bone tumor that is usually contracted by children and young and tumor metastasis is the most important reason for the failure of cancer treatment. About half of the cases who have this disease have been in metastasis period when they begin to recognize they have this disease and metastasis is the fist reason for treatment failure. Our laboratory established two pair cell lines with different metastasis capability by monoclone on the basic of osteosarcoma SOSP-9607. E10 and F5 had high metastatic capability but H9 and F4 had low metastasis capability. We detected the different expression of miRNA by microarray between the two pair cell lines. We found that miR-192 expression significantly increased in high metastatic cell lines E10 and F5 but significantly decreased in low metastatic cell lines H9 and F4. So we choose miR-192 as experiment aim in order to study and research on the effects of miR-192 on proliferation and apoptosis of human osteosarcoma cell line SOSP-9607.Objective To describe the effects of miR-192 on proliferation and apoptosis of human osteosarcoma cell line SOSP-9607 and establish element experience for further research.Methods (1) miR-192 inhibitors were used to inhibit the function of miR-192 . RT-PCR was used to detect the expression of miR-192 in two pair cell lines with different metastasis capability. RT-PCR was used to detect the expression of miR-192 in SOSP-9607 which was transfected by miR-192 inhibitors and SOSP-9607 which was not transfected by miR-192 inhibitors. Cultured SOSP-9607 cells were divided into two groups: control group and experimental group. The control group included miR-192 inhibitor-negative control group, liposome control group and the control cells. (2)MTT assay was used to examine the inhibition percentage of miR-192 inhibitors to the proliferation of SOSP-9607 cells. It contained three groups with different doses.(3)The cell cycle were measured by flow cytometry.The cell apoptosis were measured by flow cytometry and TUNEL. Cultured SOSP-9607 cells were also divided into two groups: control group and experimental group.Result (1) Real-time quantitative PCR showed: The expression of miR-192 was about 10 times in E10 and F5 comparing to the expression of miR-192 in H9 and F4. The expression of miR-192 in SOSP-9607 cells which was transfected by miR-192 inhibitors significantly decreased than the expression in SOSP-9607 cells without transfection (about 1/160). (2) miR-192 inhibitors inhibited SOSP-9607 cell proliferation in a dose-dependent manner (P<0.01), while the control group had no obvious effect on cell proliferation(P>0.05). With the increment of concent ration of inhibitors from 50 nmol/L to 200 nmol/L , the inhibition percentage increased significantly (P<0.01).(3) The apoptosis ratio of experimental group[(13.6±2.1)%] was obviously higher than the apoptosis ratio of control group[(7.1±0.5)%] (P<0.01). miR-192 inhibitors increased the cell ratio of G1 phase, simultaneously decreased significantly the cell ratios of G2 phase and S phase (P<0.01). Apoptosis cells in experimental groups (about 10 in every scope) were significantly more than those in control groups (about 1to2 in the same scope) (P<0.01).Conclusion (1) The expression of miR-192 was high in E10 and F5 but low in H9 and F4. The activity of miR-192 was significantly decreased by miR-192 inhibitors. (2) miR-192 inhibitors significantly inhibited SOSP-9607 cell proliferation.(3) miR-192 inhibitors significantly enhanced the apoptosis ratio of SOSP-9607 and caused the cell cycle retention of SOSP-9607. miR-192 inhibitors induced the alteration of proliferation and cell cycle of SOSP-9607 but the reason needed deep research.