obtained before chemotherapy or radiotherapy. Twenty patients www.selleckchem.com/products/chir-99021-ct99021-hcl.html were receiving radical surgery, and 13 and 21 patients were receiving post operative radiother apy and concomitant chemoradiotherapy, respectively. For each tissue, total RNA was e tracted and subjected to miR 196a and miR 196b analyses as described previ ously. To define the relative levels of miR 196 in the clinical samples, the e pression level of each tumor sam ple was normalized to an internal control and compared with that of normal tissue from the same pa tient. The cutoff points were determined after calculat ing the receiver operating characteristic curve for best fit of sensitivity and specificity. E pression levels greater than 15 fold for miR 196a and 7 fold for miR 196b in tumor tissues compared to the e pression in normal tissue were defined as high.

Level. The Pearson chi square test was used to e amine the association of miR 196 e pression with clinicopathologic features, in cluding TNM stage. Survival curves were calculated by the Kaplan Meier method with a log rank test. All P values were two sided, and the significance level was set at P 0. 05. Results Both miR 196a and miR 196b promote cell migration and invasion without affecting cell growth To determine the carcinogenic functions of miR 196a and miR 196b, in vitro loss of function e periments using antagomir oligonucleotides and gain of function e periments using miRNA plasmid transfections were performed. The results indicated that the antagomirs against miR 196a and miR 196b substantially inhibited their e pression by 79 80 and 62 73%, respectively, in OECM1 and SAS cells after 1 day.

Plasmid transfection upregulated miR 196a and miR 196b levels by 2. 8 5. 7 and 4. 6 7. 1 fold, respectively, in OECM1 and SAS cells after 1 day. The potential ef fect of miR 196a or miR 196b on cell growth was e am ined in OECM1 and SAS cells. As shown in Additional file 2 Figure S1, silencing of miR 196a or miR 196b had no effect on cell proliferation. Similarly, over e pression of miR 196a or miR 196b has no significant effect on cell colony growth. These results suggested that miR 196 has minimal effect on growth regulation. The potential effect of miR 196 on and chemo radio sensitivity was also e amined using a clonogenic survival assay. Silencing of miR 196a or miR 196b had no effect on cell survival in response to cisplatin treatment.

However, miR 196a and miR 196b had differential GSK-3 effects on radiosensitivity. Whereas miR 196b depletion had no effect, both cell lines were significantly more sensitive to radiation after miR 196a silencing. This result suggests that miR 196a, but not miR 196b, pro tects cells against radiation damage. Cell migration and invasion were ne t analyzed using in vitro wound healing and Matrigel invasion assays. As shown in Figure 1B, miR 196 silencing resulted in slower migration toward the gap area in both OECM1 and SAS cells, with Volasertib CAS reductions of 28 50% and 41 62%, respect ively, for miR 196a and mi