Animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. BALB/c male nude mice aged of 8–12 weeks were used for this study. 5637 or H-bc cells were embedded in BD Matrigel Matrix (Becton, Dickinson, Franklin Lakes, NJ, USA)51 (link) and subcutaneously injected into the four sites at the back of mice as following: 1.7 × 107 cells/site for 5637, 0.7 × 107 cells/site for H-bc, 4 sites/mouse, 6 mice for 5637, 6 mice for H-bc, respectively. From the fourth day after cell injection, all 5637-generated tumors were intratumorally injected with 2nM miR-193a-3p agomiR or miR-193a-5p agomiR, while H-bc-generated tumors were injected with 4 nM miR-193a-3p/-5p/Mock antagomiR/PBS in a similar manner. From the sixth day after cell injection, three mice from 5637 and three from H-bc intraperitoneally received Pa (45 ug/ mouse) once in 2 days. The remaining six mice (three from 5637 and three from H-bc) received PBS as a mock treatment control. Mice were humanely killed on day 25, and the tumors were weighed and photographed. The tumor weight was described as the mean±S.D.
Expression levels of SRSF2, TP73 and Ki67 proteins were measured using immunochemical analysis on 5-mm slices of formalin-fixed paraffin-embedded tumor xenografts in nude mice. To avoid inter-treatment bias, the tissue slides from all the six groups were made on a single slide and subjected to the same immuno-staining simultaneously. Antigens were retrieved by pretreating dewaxed sections in a microwave oven at 750 Watts for 5 min in a citrate buffer (pH 6) processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). The enzymatic activities were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. Following counter staining with Mayer hematoxylin (Invitrogen), slides were mounted in an aqueous mounting medium (glycergel, Dako).Pictures were taken using the LEICA DM 4000B microscope (Wetzlar, Germany), while the relative level of each protein was calculated using the LEICA software (Wetzlar, Germany), and the percentage of the mock over the chemotherapeutic treated tumors was calculated and plotted.
Expression levels of SRSF2, TP73 and Ki67 proteins were measured using immunochemical analysis on 5-mm slices of formalin-fixed paraffin-embedded tumor xenografts in nude mice. To avoid inter-treatment bias, the tissue slides from all the six groups were made on a single slide and subjected to the same immuno-staining simultaneously. Antigens were retrieved by pretreating dewaxed sections in a microwave oven at 750 Watts for 5 min in a citrate buffer (pH 6) processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). The enzymatic activities were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. Following counter staining with Mayer hematoxylin (Invitrogen), slides were mounted in an aqueous mounting medium (glycergel, Dako).Pictures were taken using the LEICA DM 4000B microscope (Wetzlar, Germany), while the relative level of each protein was calculated using the LEICA software (Wetzlar, Germany), and the percentage of the mock over the chemotherapeutic treated tumors was calculated and plotted.
