Figure 4: LZTS1 is a direct target of miR-135b.

(a) Co-transduction of CL1-0 cells with a control vector (Ctrl vector) or a miR-135b-expressing plasmid with firefly luciferase fused with 3′-UTR sequences of putative miR-135b target genes. Luciferase activity was measured, and the relative ratio of the activity in the miR-135b groups to that in the control vector group is presented as the mean±s.d., *P<0.05 by Student’s t-test. (b) CL1-0 cells were co-transfected with Ctrl vector- or miR-135b-expressing plasmids and firefly luciferase fused with wild-type (wt) LZTS1 3′-UTR or seed sequence-mutated (mut) 3′-UTR (1mut, seed 1 mutated; 2mut, both seeds mutated). Mean±s.d. is shown, *P<0.05 by Student’s t-test. (c) CL1-0 cells were co-transfected with miR-135b, wild-type LZTS1 3′-UTR along with Scramble or miR-135b-specific antagomir (Antago-135b). Luciferase activity was measured and is presented as described in (a). Mean±s.d. is shown, *P<0.05; **P<0.005 by Student’s t-test. (d) Western blot analysis of endogenous LZTS1 expression in CL1-0 cells after transduction with different multiplicities of infections of control (Neo) or miR-135b-expressing lentiviral vectors. (e) CL1-5-F4 and UACC-257 cells were administered with 100 nM of negative control (NC) or antago-135b for 48 h. Total cell lysates were harvested for western blot analysis. (f) The cells were transduced with different amounts of LZTS1-expressing lentiviral vector, and western blot analysis of LZTS1 expression along with analyses of invasion and migration ability were conducted as described above. Mean±s.d. is shown, *P<0.05; **P<0.005 by Student’s t-test. (g) UACC-257 cells were transfected with control (NC) or LZTS1-specific siRNAs (si-LZTS1 no. 2 and no. 3) for 48 h and subjected to invasion and migration assays as described above. Mean±s.d. is shown, *P<0.05; **P<0.005 by Student’s t-test. (h) CL1-0 cells stably expressing control vector, miR-135b or co-expressing miR-135b and LZTS1 were analysed in transwell invasion assays. Mean±s.d. is shown, *P<0.05 by Student’s t-test. (i) The statistical incidence of lung nodule generation in the mice after injecting CL1-0 cells with different vector-transduced cells. The animals were killed 8 weeks after injection(n=6). Mean±s.e.m. is shown, *P<0.05 by Student’s t-test. (j) The number of colonies of soft agar assay by CL1-0 cell derivatives with Neo-control, miR-135b or miR-135b+ LZTS1 (n=3). Mean±s.d. is shown, *P<0.05 by Student’s t-test.