MiR-192 Inhibits Cell Growth And Invasiveness By Targeting TCF7 In Human Osteosarcoma

Background Osteosarcoma(OS) is the most common primary bone tumor, mainly affects children and adolescents. OS is characterized by high propenssity to metastasize and thus leads to many patients die of lung metastasis. Although great efforts have been made in the treatment of OS, the 5-year survival rate remains approximately 60–70%. Nowdays, human have foused on the research of post-genome. Therefore, it’s particularly important to investigate the molecular mechanism of osteosarcoma from the gene levels and look for the new methods for the diagnosis and prevention of osteosarcoma. Micro RNAs(mi RNAs) are a class of conserved and endogenous small non-coding RNAs, which regulates gene expression at post and post-transcriptional levels. mi RNAs are involved in many biological processes, including cell proliferation, angiogenesis, cell metabolism and so on. The abnormal expressions of mi RN As are closely related to the tumor occurrence and progress. The new research reported: the downregulation of mi R-192 has been found in a wide variety of cancer, such as lung cancer, colorectal cancer, liver cancer, breast cancer, bladder cancer and osteosarcoma. However, the influence of mi R-192 on the biological behaviours of osteosarcoma has not yet been reported. Therefore, discussing the effect of mi R-192 on proliferation, apoptosis, migration and invasion of osteosarcoma and revealing the related mechanism will provide new thoughts and strategies for the clinical prevention and treatment of osteosarcoma.Purpose This study used human osteosarcoma specimens as the objective to investigate the expression levels of mi R-192 and TCF7 in OS tissues and the matched adjacent tissues. Meanwhile, we used human osteosarcoma cell lines U-2OS and MG63 as the objective to investigate the effect of mi R-192 on cell proliferation, apoptosis, migration and invasion when cells were transfected with mi R-192. Then we explored the targets of mi R-192 and its regulatory mechanism to provide the experimental basis and new targets for the functions of the micrornas in the prevention of osteosarcoma.Methods 1. We obtained the osteosarcoma specimens(n=20) from the First Affiliated Hospital of Zhengzhou University(from June 2013 to March 2015), and preservated at-80°C. We used RT-q PCR assay to quantify the expression levels of mi R-192 and TCF7 m RNA in 20 paired osteosarcoma samples and the matched adjacent tissues and analyzed the relationship. Besides, we used the Western blot assay to determine the expression level of TCF7 protein. 2. We used the RT-q PCR assay to quantify the expression levels of mi R-192 and TCF7 m RNA in osteoblast cell h FO B1.19 and human osteosarcoma cell lines U-2OS and MG63. 3. We used the liposomes to transfect synthetic mi R-192 agomir or mi R-192 negative control into human osteosarcoma cell lines U-2OS and MG63. The experimental groups for each group as follow:(1) mi R-192 group: cells transfected with mi R-192 agomir.(2) NC group: cells transfected with mi R-192 negative control.(3) Blank group: cells transfected with only liposomes. 4. CCK-8 growth experiment and colony formation assay were used to evaluate the growth effect of mi R-192 on osteosarcoma cells. 5. Flow cytometry and Caspase activity assay were used to evaluate the apoptosis effect of mi R-192 on osteosarcoma cells. 6. Transwell assay was used to evaluate the invasion effect of mi R-192 on osteosarcoma cells. 7. Wound-healing assay was used to quantify the the migration effect of mi R-192 on osteosarcoma cells. 8. We used Pic Tar、mi Rwalk、Target Scan and mi Randa informatics softwares to predicte the targets of mi R-192. Wild type and mutant type recombinant vectors that contained TCF7 3’-UTR region were constructed and then co-transfected them with mi R-192 agomir or mi R-192 negative control into U-2OS and MG63. We used Luciferase reporter assay and Western blot assay to confirm the targets of mi R-192. 9. We used western blott assay to investigate the expressions of Cyclin D1,apoptosis inhibitory protein Survivin and ce ll invasion and migration related protein Snail in osteosarcoma cells that transfected with mi R-192 agomir.Results 1. Compared to the adjacent tissues, the relative expression level of mi R-192 in OS tissue was significantly lower(P<0.01), and the expression level of TCF7 m RNA was significantly higher(P<0.01). The expression levels of mi R-192 and TCF7 m RNA were negatively correlated.Besides, the TCF7 protein expression in osteosarcoma tissues was increased compared with matched adjacent tissues. 2. The expression of mi R-192 was lower in U-2OS and MG63 cell lines than osteoblast h FOB1.19 cell(P<0.01). And the TCF7 m RNA expression levels were significantly higher in U-2OS and MG63 cells than osteoblast h FOB1.19 cell(P<0.01). 3. After the cells transfected mi R-192 agomir, the mi R-192 expression level of mi R-192 group was significantly higher than NC group and Blank group and the difference was statistically significant(P<0.01). This showed that the mi R-192 expression level of cells increased after the osteosarcoma cells transfected with mi R-192 agomir,. 4. CCK-8 growth experiment and colony formation assay showed that,compared to the NC and Blank group, the O D450 values and the colony numbers of the osteosarcoma cells in mi R-192 group were decreased, and the differences were significant(P<0.05), while the OD450 values and the number of colony formation of the osteosarcoma cells between the NC group and Blank group were no significant difference(P >0.05). 5. Flow cytometry and Caspase activity assay showed that, compared to the NC and Blank group, the apoptotic capacity and caspase-3 activity of the osteosarcoma cells in mi R-192 group were both enhanced, and the differences were statistically significant(P<0.01), but the apoptotic capacity and caspase-3 activity of the osteosarcoma cells between the NC group and Blank group were no significant difference(P >0.05). 6. Transwell assay showed that, compared with the NC and Blank group, the number of cells penetrating the membrane of mi R-192 group was decreased, and the difference was significant(P<0.05), but the number of cells penetrating the membrane between the NC and Blank group was no significant difference(P >0.05). 7. Wound-healing assay showed that, compared with the NC and Blank group, the capacity for wound healing of the osteosarcoma cells was weaker in the mi R-192 group, but there was no difference in the NC and Blank group. 8. Western blot assay and luciferase reporter assay showed that TCF7 act as the target of mi R-192. 9. Western blot assay showed that, compared to the NC and Blank groups, the expression levels of C yclin D1,apoptosis inhibitory protein Survivin and cell invasion and migration protein Snail of mi R-192 group were reduced, while the NC group and the Blank group comparison, the expressions of C yclin D1,Survivin and Snail have no difference.Conclusions 1. In osteosarcoma tissues and cells, mi R-192 shows high expression, while TCF7 is low expression, and we also identify an inverse correlation between the levels of mi R-192 and TCF7. 2. mi R-192 negatively regulates the expression of TCF7, and then down-regulates the expression of C yclin D1, apoptosis inhibitory protein Survivin and cell invasion and migration protein Snail, thus playing the biological function. 3. Up-regulation of mi R-192 can effectively inhibit the growth, invasion and migration and promote apoptosis of osteosarcoma cells.