Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: A functional screening using a novel small RNAs expression library reveals hsa-miR-30b and hsa-miR-30c as repressors of anoikis. ( A ) Immortalized RPE-1 cells were infected with a lentiviral expression library containing a small RNAs collection from the MDA-MB-231 cell line. RPE-1-infected cells were cultured for 19 d in agar dishes at low confluence avoiding any contact and surviving clones transferred to adhesive culture dishes to allow the growth of anoikis-resistant cells. DNA from these clones was isolated and sequenced. ( B ) Table showing the small RNA sequences overexpressed in anoikis-resistant clones. ( C ) Flow cytometry analysis of the levels of apoptosis in RPE-1 cells (SubG1/SubG1 control + polyHEMA) infected with lentivirus containing candidate mature miRNAs or empty vector (control). Three days after infection, cells were deprived of serum for 24 h and then cultured in polyhema (PH) plates plus methylcellulose (2%) in serum-free medium for another 24 h. ( D ) RNase protection assay of miR-30b and miR-30c transiently overexpressed in HEK-293 cells from pLENT-DUAL plasmid. Control (Ctr) cells expressed the empty vector pLENT-DUAL; Y corresponds to yeast RNA; M, Decade RNA markers (Ambion). ( E ) Levels of apoptosis in RPE-1 cells (SubG1/SubG1 control) infected with lentivirus carrying full-length miR-30b, miR-30c-1 (FL), miR-30b/c mature miRNAs (Lent-30b/c), and the corresponding empty vectors as controls in a similar assay as described in C . Results are shown as the averages ± standard errors of the means from at least three independent experiments and were analyzed by one-way ANOVA, followed by Bonferroni post-test for significance versus control cells. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Functional Assay, Expressing, Infection, Cell Culture, Clone Assay, Isolation, Flow Cytometry, Plasmid Preparation, Rnase Protection Assay

Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: Hsa-miR-30b and hsa-miR-30c down-regulate CASP3 expression through its 3′ UTR-binding. ( A ) Western blot analysis of p53, CASP3, and BCL2-like 11 (BIM), three putative target proteins of miR-30b/c related to anoikis. RPE-1 cells were infected with lentivirus expressing full-length miR-30b, miR-30c-1 (miR-30c), or empty vector as a control. Active CASP3 was analyzed in cells cultured under anoikis conditions as described in C. Actin (ACTB) was used as the loading control. ( B ) Densitometric analysis of pro-CASP3 and active CASP3 levels. ACTB protein was used as the normalization control. Results are shown as the averages ± standard errors of the means from at least three independent experiments and were analyzed by one-way ANOVA, followed by Bonferroni post-test (* P < 0.05; ** P < 0.01). ( C ) Schematic representation of the caspase 3 3′ UTR containing two putative binding sites for miR-30b/c. The 3′ UTR of CASP3 mRNA was cloned downstream from the open reading frame of luciferase (pLuc-BS). Both broadly (1187–1193) and poorly (1222–1228) conserved putative miRNA regulatory elements of the CASP3 3′ UTR were mutated (Mut1 and Mut2, respectively) and cloned along with the wild-type 3′ UTR (WT). HEK-293T cells were transiently cotransfected with plasmids overexpressing full-length miRNAs (miR-30b, miR-30c-1, or empty vector as control), pLuc-3′ UTR CASP3 (WT, Mut1, or Mut2) and pCMV- Renilla at 100:10:1 proportions, respectively. Firefly and Renilla activities were measured 40 h after transfection. The luciferase activity normalized to Renilla is shown ( lower panel). Transfections were performed in triplicate, and results are shown as the averages ± standard errors of the means from at least three independent experiments and were analyzed by one-way and two-way ANOVA, followed by Bonferroni post-test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (ns) not significant.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Expressing, Binding Assay, Western Blot, Infection, Plasmid Preparation, Cell Culture, Clone Assay, Luciferase, Transfection, Activity Assay

Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: Caspase 3 is the main target of miR-30b/c in the anoikis-resistance context. ( A ) RPE-1 cells were co-infected with both lentiviruses expressing full-length miR-30b/c and the ORF of caspase 3. Western blot analysis of pro-CASP3, active CASP3, and ACTB is shown for RPE-1 cells growing either in complete medium under adherent conditions (−PH) or in anoikis-inducing conditions (+PH). Flow cytometry analysis of the levels of apoptosis (SubG1/SubG1 control) in CASP3-infected cells growing under anoikis-inducing conditions ( lower panel). ( B ) RPE-1 cells were infected with either lentivirus expressing a shRNA to silence the expression of caspase 3 or a scramble shRNA (control). Experiments were carried out with RPE-1 infected cells previously selected in puromycin for 12 d. The diagram to the left shows CASP3 mRNA levels measured by real time PCR after puromycin selection. HPRT mRNA was used as the normalization control. On the right , levels of apoptotic cells in anoikis-inducing conditions are shown. Results are shown as the averages ± standard errors of the means from three independent experiments. The data were subjected to two-tailed Student's t -test. (**) P < 0.01, (***) P < 0.001. ( C ) Number of colonies after plating 2 × 10 4 cells from B per well in six-well plates with a bottom layer of 0.5% agar and a top layer of 0.35% agarose. Colonies were photographed and counted after 4 wk. Results are shown as the averages ± standard errors of the means from three independent experiments. The data were subjected to two-tailed Student's t -test. (**) P < 0.01.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Infection, Expressing, Western Blot, Flow Cytometry, shRNA, Real-time Polymerase Chain Reaction, Selection, Two Tailed Test

Journal: RNA

Article Title: Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance

doi: 10.1261/rna.039461.113

Figure Lengend Snippet: Hsa-miR-30a and -30d do not confer anoikis resistance in RPE-1 cells. ( A ) Western blot analysis of pro-CASP3 in RPE-1 cells infected with lentivirus expressing full-length miR-30a, miR-30d, or their corresponding empty vectors as control. Actin (ACTB) was used as the loading control. On the right , densitometric analysis of pro-CASP3 is shown. ACTB protein was used as a normalization control. ( B ) Flow cytometry analysis of the levels of apoptosis (SubG1/SubG1 control) in infected cells growing under anoikis-inducing conditions. ( C ) Endogenous levels of mature miR-30a/d ( left panel) and miR-30b/c ( right panel) expressed in RPE-1 and MDA-MB-231 (clone 4175) cells. miR-16 and miR-324 were used as normalization controls for miR-30b/c and miR-30a/d, respectively. Similar results were observed when human U6 snRNA primer and U6-snRNA ( RNU6B ) were used as controls. Results are shown as the averages ± standard errors of the means from three independent experiments. The data were subjected to two-tailed Student's t -test. (*) P < 0.05.

Article Snippet: Immortalized human retinal pigment epithelial cell line hTERT RPE-1 (Clontech) was maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 units/mL penicillin, 50 μ/mL streptomycin).

Techniques: Western Blot, Infection, Expressing, Flow Cytometry, Two Tailed Test