1. Novel small RNAs expression libraries uncover hsa-mir30c and hsa-mir30b as key factors in anoikis resistance Miguel A. Moreno-Mateos*, Verónica Barragán*, Belén Torres and José Antonio Pintor-Toro Avda. Americo Vespucio s/n. Edif. CABIMER Parque Cientifico y Tecnolgico Cartuja 93 CP 41092 - Sevilla (SPAIN) Telephone: +34- 954467838. E-mail: miguelangel.moreno@cabimer.es *These authors contribute equally to this work. INTRODUCTION During the past ten years, microRNAs (miRNAs) and other small non-coding RNAs (ncRNAs) have been deeply studied in order to elucidate their biological functions. At present, libraries to overexpress miRNAs are generated by synthesis and cloning of individual miRNAs. In this work, a patented methodology has been developed to generate a lentiviral expression library containing the whole population of small RNAs present into the cell including matures miRNAs. Using this library, a functional screening has been completed and several miRNAs involved in anoikis (cell death by loss of attachment) resistance identified. Among these, hsa-mir30c and hsa-mir30b were selected for further analysis because their expression strongly reduced cell death. Acquisition of anoikis resistance by these miRNAs was achieved through the inhibition of apoptotic pathways by downregulation of Caspase-3. A)B) C) D) E) C TACAG TA Anoikis screening (16 days in agar) Independent clones selection Culture in attached conditions Sequencing surviving clones sRNA candidates A) B) C)C) D)D) pSSFV Overexpreesion of full lenght hsa-mir30b A) B)C) Conclusions pSSFV Overexpreesion of full lenght hsa-mir30c Small RNA isolation from MDA 231metastasic cell line 5’p OH 3’ AppddC 3’ 5’p ddC 3’ T4 RNA ligase (no ATP) 5’HO OH 3’ ddC 3’ RT-PCR BbvII U6 H1 pLENT-DUAL T4 RNA ligase + ATP BIM Act Ctr 30b 30c p53 Act Ctr 30b 30c Pro-Casp3 Act Active Casp3 Act Pro-Casp3 Active Casp3 Ctr Ctr 30b 30c CASP3 -PH +PH Ctr Ctr 30b 30c Pro-Casp3 Act Ctr 30b 30c U6 H1 AAAA AAAG pLENT-DUAL mature miRNAs ProbeYCtrmiR-19ProbeYCtrmiR-19 19 sense19 antisense 40nt 30nt 20nt ProbeYCtrmiR-21CtrmiR-21 FLProbeYCtrmiR-21CtrmiR-21 FL 21 sense21 antisense Luc 3’UTR PDCD4 Probe: Luc BS mir21(19,15) pLUC-BS Luc 3’UTR Casp3 (1570 pb) 1187-1193 Conserved 1222-1228 Poorly conserved Mut1 Mut2 A) B) Fig 1. Mature miRNAs expressed by dual convergent promoters are functionally similar to full length expressed miRNAs. A) Scheme showing the plasmid used to express mature miRNAs and other small RNAs (at the top) and the plasmid carrying the luciferase ORF followed by a binding site (BS) for miRNA 21, mir19 or mir15 (at the bottom). B) RNAse protection assay of mature hsa-mir21 and hsa- mir19 overexpressed transiently by pLENT-DUAL plasmid. Hsa-mir21 full length (FL) was expressed from a pCMV-hsa-mir21 plasmid (Ctr:control; Y: Yeast RNA). C) Luciferase assay. Transient transfections were carried out using pLENT-DUAL (hsa-mir21, mir19 or mir15), pLuc-BS (mir21, mir19 or mir15) and pCMV-Renilla (as normalization control) at 100:10:1 proportions in HEK-293T cells. Hsa-mir21 Full length (FL) was expressed from a pCMV-hsa-mir21 plasmid. All transfections were performed in triplicate, and error bars represent standard error from three independent experiments. D) Quantification of Luciferase mRNA levels by Real time PCR in the conditions described in section C. Renilla mRNA was used as normalization control. E) Western blot analysis of Luciferase protein level and quantification in the conditions described in section C. At the bottom, Luciferase assay in a similar experiment described in section C. Transfections were carried out using pLENT-DUAL-hsa-mir21 and pCMV-hsa-mir21 full length and pLUC followed by 3’UTR of pdcd4 gene carrying a binding site of mir21. Fig 2. A functional screening using a small RNAs expression library reveals hsa- mir30b and hsa-mir30c as important repressors of anoikis. A) Scheme of the small RNA cloning system to develop expression libraries based on pLENT-DUAL. B) Immortalized and sensitive to anoikis RPE1 cells were infected with a lentiviral library containing a small RNAs collection from MDA231 cell line. RPE1 infected cells were cultured for 16 days in agar dishes at low confluence avoiding any contact. Surviving clones were transferred to adhesive culture dishes to allow expand anoikis resistant cells. DNA from these clones was isolated and sequenced. C) Table showing small RNA sequences obtained from pLENT-DUAL of anoikis resistant clones. D) RPE1 cells were infected with lentivirus containing mature miRNA candidates or empty vector (control). Three days after infection, cells were deprived of serum for 24 hours and then were cultured in polyhema (PH) plates and metyl cellulose (2%) serum free medium for another 24 hours. Apoptosis was determined by FACS Fig 3. Full-length hsa-mir30b and hsa-mir30c downregulate anoikis and Caspase-3 expression. A) Levels of mature miRNAs expressed from a lentiviral plasmid carrying full length hsa-mir30b and full length hsa-mir30c-1 (empty vector as control). On the right, percentage of apoptosis in the infected cells. B) Western blot analysis of putative targets proteins of hsa-mir30b and hsa-mir30c (by TargetScan analysis) and related to anoikis. Active Caspase-3 was analysed in cells treated as described in section 2D. On the right, both protein and mRNA levels (measured by densitometry real time PCR respectively) of pro-Caspase-3 from three independent experiments. Actin was used as normalization control. C) Caspase-3 complementation. RPE1 cells were coinfected with two lentiviruses expressing miRNAs and cDNA of Caspase-3. Western blot analysis of Pro-Caspase-3, active Caspase-3 and Actin is shown. At the bottom, apoptosis determined by FACS of Caspase-3 infected cells growing in normal conditions or as described in section 2D. Fig 5. Hsa-mir30b and hsa-mir30c reduce cell death induced by TRAIL in MCF10A Cells. Cells were infected with lentivirus containing full-length hsa-mir30b and hsa- mir30c. Four days after infection, cells were cultured in presence of Trail (500 ng/ml) for seven hours. Apoptosis was determined by FACS. At the bottom, western blot analysis of Pro-Caspase-3 in these infected cells. Actin was used as control. Fig 4. Hsa-mir30b and hsa-mir30c downregulate Casp3 through its 3’ UTR. A) The 3’UTR of Caspase-3 mRNA was cloned downstream of Luciferase open reading frame in the pLUC-BS plasmid. The 3’UTR of Caspase-3 contains two putative miRNA30b/30c regulatory elements (MRE); one of them is highly conserved and the other one poorly conserved (by TargetScan analysis). B) HEK-293T cells were transiently contransfected with plasmids overexpressing miRNAs (hsa-mir30b, hsa- mir30c or control), pLUC-3’UTR casp3 (3’UTR WT) and pCMV-Renilla at 100:10:1 proportions. The same experiment was carried out using two mutant versions of pLUC-3’UTR casp3, where six nucleotides were changed both in the conserved (Mut 1) and poorly conserved (Mut 2) sites. *This work is supported by grants from the Spanish MEC and the DGUI of the Junta de Andalucía. - An expression library of the complete population of the cellular small RNAs has been performed. - Mature miRNAs expressed by this system and full length expressed miRNAs prove a similar functionality - Using this library, hsa-mir30b/c have been identified as important negative modulators of anoikis. - Caspase-3 is downregulated by hsa-mir30b/c through translational repression. Hsa-mir30b targets 3’UTR conserved MRE and hsa-mir30c targets both conserved and non-conserved MRE. - Complementation experiments suggest that hsa-mir30b/c decrease anoikis mainly targeting Caspase-3. - Other forms of Casp3-dependent cell death are also downregulated by hsa-mir30b/c.