Fig 2: Functional analysis of miRNAs targeting TUSC2.(a) Lysates of 4T1 cells transfected with miRNAs were analysed by western blotting. Expression of miR-17, miR-93, miR-299-3p, miR-520a, miR-608 and miR-661 decreased TUSC2 protein levels. β-Actin on the same membranes served as a loading control. (b) The cells transfected with different miRNAs were subject to western blot analysis for expression of TIMP2 and TIMP3. Both TIMP2 and TIMP3 levels decreased in cells transfected with miRNAs as compared with the control oligo. (c) Apoptosis was analysed in cells transfected with different miRNAs. Transfection with the miRNAs decreased apoptosis; n=3, **P<0.01. (d) The migration rate was measured for the miRNA-transfected 4T1 cells. There was an increased amount of cell migration in miRNA-transfected cells compared with control cells; n=10, **P<0.01 by t-test, error bar, s.d. (e) In cell invasion assays, the cells transfected with miR-17, miR-93, miR-299-3p, miR-520a, miR-608 and miR-661 showed drastically increased in the capacity of invasion through the membrane pores compared with the control cells; n=5, **P<0.01 by t-test.

Fig 3: Expression of the TUSC2P pseudogene.(a) Analysis of miRNA targeting the 3′-UTR of TUSC2 and pseudogene TUSC2P on chromosome Y and chromosome X. (b) Expression of TUSC2P was analysed in different cancer and non-cancer cell lines by real-time PCR. The cancer cell lines had a decreased amount of TUSC2P mRNA compared with the non-cancer cell lines. The inset represents bands of reverse transcription–PCR of these cell lines; n=4, error bar, s.d. (c) TUSC2P and TUSC2 3′-UTR were cloned and inserted into pcDNA3.1 plasmid, downstream of the cytomegalovirus promoter, producing the TUSC2P and TUSC2-UTR constructs. (d) Total RNA isolated from 4T1 cells stably transfected with TUSC2P, TUSC2-UTR and control vector were analysed by real-time PCR. The TUSC2P-transfected cells had a fourfold increase compared with the endogenous TUSC2P levels in the cells. The TUSC2-UTR-transfected cells had approximately a threefold increased expression of TUSC2 compared with the control cells; n=3, **P<0.01 analysed by t-test, error bar, s.d.

Fig 4: Confirmation of TUSC2P functions.(a) HaCaT cells were transfected with an siRNA targeting Dicer1 to block the miRNA biogenesis pathway. Silencing Dicer1 decreased levels of the miRNAs tested; n=4, *P<0.05, **P<0.01 by t-test, error bar, s.d. (b) HaCaT cells were treated with siRNA targeting Dicer1 or TUSC2P followed by reverse transcription–PCR for confirmation of Dicer knockdown and western blot analysis to analyse levels of TUSC2, TIMP2 and TIMP3. Silencing Dicer increased expression of TUSC2, TIMP2 and TIMP3. HaCaT cells were transfected with luciferase constructs of TUSC2 (c), TIMP2 (d) and TIMP3 (e) combined with TUSC2P, siRNA targeting TUSC2P, and/or siRNA targeting Dicer1 followed by luciferase assays. Although silencing TUSC2P decreased luciferase activities, silencing Dicer reversed the effects; n=4, **P<0.01 analysed by t-test, error bar, s.d. (f) HaCaT cells were transfected with TUSC2P siRNA combined with or without Dicer1 siRNA. TUSC2P was silenced even in the presence of Dicer1 siRNA; n=4, **P<0.01 analysed by t-test, error bar, s.d. (g) Transfection of 4T1 cells with TUSC2P, TUSC2, TIMP2 and TIMP3 decreased cell migration; n=4, **P<0.01 analysed by t-test, error bar, s.d. (h) Transfection of HaCaT cells with siRNA targeting endogenous TUSC2P increased cell migration. Combined transfection with TUSC2, TIMP2 or TIMP3 partially, but significantly, reversed the effect of TUSC2P on cell migration; n=4, **P<0.01 analysed by t-test, error bar, s.d. (i) Transfection of 4T1 cells with TUSC2P decreased cell migration, which could be partially, but significantly, reversed by transfection with siRNAs targeting TUSC2, TIMP2 or TIMP3; n=4, *P<0.05, **P<0.01 by t-test, error bar, s.d.

Fig 5: Confirmation of TUSC2P effects by siRNA approach.(a) Lysates prepared from the siRNA-treated HaCaT cells were subject to western blot analysis. Levels of TUSC2, TIMP2 and TIMP3 decreased as a result of siRNA targeting TUSC2P. (b) Cell survival was analysed in cells transfected with siRNAs targeting TUSC2P or a control oligo. The TUSC2P siRNA-treated cells survived longer in serum-free medium than the control cells; n=5, P<0.01 by t-test, error bar, s.d. (c) In migration assay, the siRNA-treated cells migrated faster than the cells transfected with a control oligo; n=5, ** P<0.01 by t-test, error bar, s.d. (d) In invasion assay, there was an increased amount of the siRNA-treated cells that invaded through the Matrigel compared with the control; n=5, **P<0.01 by t-test, error bar, s.d. (e) Determination of cell apoptosis indicated that less siRNA-treated cells underwent apoptosis than the control cells; n=10. The luciferase constructs of TUSC2 (f), TIMP2 (g) and TIMP3 (h) were co-transfected with siRNA targeting endogenous TUSC2P in HaCaT cells. Silencing TUSC2P decreased luciferase activities; n=5, *P<0.05, **P<0.01 by t-test, error bar, s.d.