Is of cancer [24,25].Suppression of miR-27a and induced expression of the miR-27a-regulated gene ZBTB10 mediated inhibition of tumor growth in breast cancer [18] in vitro and in vivo. These studies have demonstrated the important role for miRA27a and its target gene ZBTB10 in regulating tumor growth, metastasis and chemotherapy resistance, which suggests that miR27a might be a clinically useful marker for selecting high-risk cancer patients with distant metastasis.locked nucleic acid-modified, 59digoxigenin (DIG)-labeled oligonucleotide probe complementary to miR-27a or a scrambled control probe was added to 100 ml of the hybridization solution and hybridized at a temperature of 51uC overnight. The sections were rinsed twice in 26standard saline citrate, followed by three washes of 20 minutes at 50uC in 50 formamide/ 26standard saline citrate. Then, the samples were washed five times in PBS/0.1 Tween-20 and blocked in blocking solution (2 sheep serum, 2 mg/ml bovine serum albumin in phosphate buffered saline with Tween-20) at room temperature for 1 hour. An anti-DIG antibody (1:1000; Abcam, Cambridge, MA, USA) was applied, and the sections were incubated at 4uC overnight. After washing in staining solution, the sections were incubated with the NBT/BCIP developing solution for 2 hours at 37uC 
and counterLecirelin biological activity stained with nuclear fast red.Immunohistochemistry (IHC)IHC was performed using standard techniques. Briefly, 4-um paraffin-embedded specimens were dewaxed in xylene and rehydrated in graded alcohols. Endogenous peroxidase was blocked using 3 hydrogen peroxide. Antigen retrieval was accomplished in citrate buffer (pH 6.0) using a microwave. Polyclonal rabbit anti-human ZBTB10 antibody (1:50, Santa Cruz, CA, USA) was added and the samples were incubated at 4uC overnight. The sections were then treated with a secondary antibody, followed by further incubation with HSS-HRP, DAB chromogen staining and counterstaining with hematoxylin. Negative controls were obtained by replacing the primary antibody by an isotope IgG.Methods EthicsThe use of tissues for this study has been approved by the Ethics Committee of Sun Yat-Sen Memorial Hospital, Sun-Yat-Sen University. At the time of initial diagnosis, all patients had provided consent in the sense that their tumor samples could be used for investigational purposes. Written informed consents were received from all participants involved in the study.Scoring of ISH and IHCThe expression of miR-27a and ZBTB10 in 102 paraffinembedded breast invasive cancer specimens was examined and scored separately by two independent investigators Anlotinib blinded to the histopathological features and patient data for the samples. In each section, 561000 tumor cells were counted randomly, and the scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The proportion of positively stained tumor cells was graded as follows: 0, no positive tumor cells; 1, ,10 positive tumor cells; 2, 10 to 50 positive tumor cells; and 3, .50 positive tumor cells. The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light blue or yellow), 2 (moderate staining, blue or yellow), and 3 (strong staining, dark blue or yellow). For tumors that showed heterogeneous staining, the predominant pattern was taken into account for scoring. The staining index (SI) was calculated as follows: staining index = proportion of positively stained tumor.Is of cancer [24,25].Suppression of miR-27a and induced expression of the miR-27a-regulated gene ZBTB10 mediated inhibition of tumor growth in breast cancer [18] in vitro and in vivo. These studies have demonstrated the important role for miRA27a and its target gene ZBTB10 in regulating tumor growth, metastasis and chemotherapy resistance, which suggests that miR27a might be a clinically useful marker for selecting high-risk cancer patients with distant metastasis.locked nucleic acid-modified, 59digoxigenin (DIG)-labeled oligonucleotide probe complementary to miR-27a or a scrambled control probe was added to 100 ml of the hybridization solution and hybridized at 
a temperature of 51uC overnight. The sections were rinsed twice in 26standard saline citrate, followed by three washes of 20 minutes at 50uC in 50 formamide/ 26standard saline citrate. Then, the samples were washed five times in PBS/0.1 Tween-20 and blocked in blocking solution (2 sheep serum, 2 mg/ml bovine serum albumin in phosphate buffered saline with Tween-20) at room temperature for 1 hour. An anti-DIG antibody (1:1000; Abcam, Cambridge, MA, USA) was applied, and the sections were incubated at 4uC overnight. After washing in staining solution, the sections were incubated with the NBT/BCIP developing solution for 2 hours at 37uC and counterstained with nuclear fast red.Immunohistochemistry (IHC)IHC was performed using standard techniques. Briefly, 4-um paraffin-embedded specimens were dewaxed in xylene and rehydrated in graded alcohols. Endogenous peroxidase was blocked using 3 hydrogen peroxide. Antigen retrieval was accomplished in citrate buffer (pH 6.0) using a microwave. Polyclonal rabbit anti-human ZBTB10 antibody (1:50, Santa Cruz, CA, USA) was added and the samples were incubated at 4uC overnight. The sections were then treated with a secondary antibody, followed by further incubation with HSS-HRP, DAB chromogen staining and counterstaining with hematoxylin. Negative controls were obtained by replacing the primary antibody by an isotope IgG.Methods EthicsThe use of tissues for this study has been approved by the Ethics Committee of Sun Yat-Sen Memorial Hospital, Sun-Yat-Sen University. At the time of initial diagnosis, all patients had provided consent in the sense that their tumor samples could be used for investigational purposes. Written informed consents were received from all participants involved in the study.Scoring of ISH and IHCThe expression of miR-27a and ZBTB10 in 102 paraffinembedded breast invasive cancer specimens was examined and scored separately by two independent investigators blinded to the histopathological features and patient data for the samples. In each section, 561000 tumor cells were counted randomly, and the scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The proportion of positively stained tumor cells was graded as follows: 0, no positive tumor cells; 1, ,10 positive tumor cells; 2, 10 to 50 positive tumor cells; and 3, .50 positive tumor cells. The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light blue or yellow), 2 (moderate staining, blue or yellow), and 3 (strong staining, dark blue or yellow). For tumors that showed heterogeneous staining, the predominant pattern was taken into account for scoring. The staining index (SI) was calculated as follows: staining index = proportion of positively stained tumor.
