Supplementary Figures

Transcription

1 Supplementary Figures Supplementary Figure 1 Experimental schema for the identification of circular RNAs in six normal tissues and seven cancerous tissues.

2 Supplementary Fiure 2 Comparison of human circrnas identified from different studies. We compared the circular RNAs identified in this study (Zheng et al., 27,226 circrnas) to previously published databases obtained from circbase ( 92,061 human circrnas from 4 studies including 87,351 in Salzman et al., PLoS Genet, 2013 and 9,276 in others which comparised 7,771 in Jeck et al., RNA, 2013; 1951 in Memczak et al., Nature, 2013; 103 in Zhang et al., Mol Cell, 2013) and a most recent study (Rybak-Wolf et al., Molecular Cell, 2015; 65,731 human circrnas identified mainly from human brain tissues). Notably, there are totally 148,701 unique human circrna candidates from all the studies, indicating that circrnas may contain one of the largest RNA families in human transcription.

3 Supplementary Figure 3 Validation of 36 commonly expressed circrna candidates using RT-PCR and real-time PCR with divergent primers in HEK-293T cells. (A) RT-PCR products with divergent primers showing a single, distinct product of the expected size from HEK293T cdna. (B) Real-time PCR showing resistance of circrnas to RNase R digestion. Actin and GADPH mrnas were served as negative controls.

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5 Supplementary Figure 4 Validation of 20 novel circrna candidates by qrt-pcr with RNase R treatment and RT-PCR with Sanger sequencing. (A) qrt-pcr showing resistance of circrnas to RNase R digestion. Actin mrnas were served as negative control. (B) RT-PCR products with divergent primers showing a single, distinct product of the expected size. (C) Sanger sequencing showing the back-spliced events of candidate circrnas. circmat1a was not shown due to low RT-PCR product.

6 Supplementary Figure 5 The highly reverse complement of the inverted repeated Alu elements flanking circhipk3 loci. (A) The genomic loci of circhipk3, HIPK3 gene and repeating elements. (B) BLAST alignment showing the highly reverse complement of the inverted repeated Alu elements.

7 Supplementary Figure 6 circhipk3 was significantly upregulated in liver cancer compared with matched normal tissues. Real-time PCR was performed to determine the expression of circhipk3 in HCC and adjacent non-cancerous liver tissue samples (NT) (n=16), the relative expression level of 18s rrna as an internal control.

8 Supplementary Figure 7 The micrographs of Edu assay of HCT-116 and HeLa cells in figure 5G. DNA synthesis assessed using an EdU (5-ethynyl-2 -deoxyuridine) assay in HCT-116 (A) and HeLa (B) cells transfected with sirnas for 48 h. Cells were fluorescently stained with EdU (red). Nuclei were stained with DAPI (blue). Micrographs represent at least three experiments. Scale bar=200 μm.

9 Supplementary Figure 8 The proliferation of HEK293T cells after deletion the large upstream intron by four sets of paired grnas. The measurement of cell growth rate was performed using CCK8 kit in the third day.

10 Supplementary Figure 9 qrt-pcr analysis of the expression level of circhipk3 in HEK-293T cells after transfection with mirna mimics. Data are the means ± SEM of three experiments.

11 Supplementary Figure 10 Luciferase reporter assay for the luciferase activity of LUC-cHIPK3 in HEK-293T cells co-transfected with mirna mimics, circhipk3 overexpression plasmid or mutant circhipk3 overexpression plasmid. Data are the means ± SEM of three experiments.

12 Supplementary Tables Supplementary Table 1 Summary of RNA-seq datasets from 6 normal tissues and 7 cancerous tissues sample total_reads mapped_reads circ_num Brain_rep Colon_rep Heart_rep Liver_rep Lung_rep Stomach_rep Brain_rep Colon_rep Heart_rep Liver_rep Lung_rep Stomach_rep BLCA BLCA_NT BRCA BRCA_NT CRC CRC_NT HCC HCC_NT GC GC_NT KCA KCA_NT PRAD PRAD_NT

13 Supplementary Table 2 List of circular RNA validated by divergent primers name circ_loci strand genomic spliced length length circatp5c1 chr10: circankrd12 chr18: circvapb chr20: circatxn1 chr6: circvmp1 chr17: circfoxp1 chr3: circash1l chr1: circnrd1 chr1: circfam120a chr9: circctbp1 chr4: circtraf5 chr1: circslc39a8 chr4: circznf277 chr7: circkiaa0564 chr13: circcntrob chr17: circspg21 chr15: circptpn12 chr7: circmpp6 chr7: circklhdc10 chr7: circrasa3 chr13: circubap2 chr9: circcep135 chr4: circxpo1 chr2: circezh2 chr7: circhipk3 chr11: circspecc1 chr17: circmet chr7: circplce1 chr10: circpvt1 chr8: circdhrs3 chr1: circsmarca5 chr4: circslc8a1 chr2: circzmynd8 chr20: circfoxk2 chr17: circcamsap1 chr9: circrtn4 chr2: circalpk2 chr18: circcamsap2 chr1:

14 circfhod3 chr18: circmyom1 chr18: circmlip chr6: circpalld chr4: circpopdc2 chr3: circsorbs2 chr4: circc3 chr19: circitih2 chr10: circmat1a chr10: circpah chr12: circc10orf90 chr10: circhydin chr16: circslc4a10 chr2: circehbp1 chr2: circmyh11 chr16: circlrp5 chr11: circtpm1 chr15: circcdh5 chr16: Supplementary Table 3 Primers and RNA sequences used in this study List of oligonucleotide sequences 5'--> 3' primers for Real-time PCR and RT-PCR HIPK3-L-qF TGGAGACTGGGGGAAGATGA HIPK3-L-qR CACACTAACTGGCTGAGGGG Actin-qF TTGTTACAGGAAGTCCCTTGCC Actin-qR ATGCTATCACCTCCCCTGTGTG GAPDH-qF GTCAAGGCTGAGAACGGGAA GAPDH-qR AAATGAGCCCCAGCCTTCTC 18S-qF TTAATTCCGATAACGAACGAGA 18S-qR CGCTGAGCCAGTCAGTGTAG IL6R-qF TCACTGTGTCATCCACGACG IL6R-qR CTGGATTCTGTCCAAGGCGT Dlx2-qF TCAACAACGTCCCTTACTCCG Dlx2-qR AAGGTCCTCCTTCTCAGGCTC circatp5c1-f GCTGTCATCACAAAAGAGTT circatp5c1-r TCCATATATTCGAGCTGGT circankrd12-f AGAGGAGTTGGAGTTGCTA circankrd12-r GTTTTGTGAACCCAGATTT circvapb-f ACCAATAGTGTCTAAGTCTCTG circvapb-r CACATCTAAGTTTTGAATCC

15 circatxn1-f GATTGAAGACAGCCATAGCC circatxn1-r CTGATAAACGGAAAGTCACATT circvmp1-f ACACCTTTCTGCTTTATCTG circvmp1-r CCATTATGATGTTCCTTGTT circfoxp1-f CCTCTGTTACCTGAAGCAA circfoxp1-r GAGATAAGGCAAGATGGGA circash1l-f TTTCTTTAATTCTCTTGGACCC circash1l-r ACCCTCATCACCAGCCTTG circnrd1-f CAGTTCTTCATCCACCCAC circnrd1-r GCATCAAATCCATTCTCATC circfam120a-f TGGTCCAGAACATCCACTA circfam120a-r ATAATCAGAGTCATACGCAAC circctbp1-f AGGGCACACGAGTCCAGAGC circctbp1-r TGCGTCGGGGTCAAAGTCT circtraf5-f ACACAGTGCCAATCTGCCC circtraf5-r TTCCAACCGCTCCACAAAC circslc39a8-f TTTTGATAATGTCAGTGTGG circslc39a8-r GGTCTTGTTTTGTGCTTGG circznf277-f CTGATGTCAAGTTGGTTGC circznf277-r CCTTCTAAAGTGGTGGTGC circkiaa0564-f GAACTTTGCCGATACCTTA circkiaa0564-r GAGCTTCCTGAATCTTGAA circcntrob-f CCAGACCTCACTTCATCCACAG circcntrob-r AGGCATAGTCCAGACCCTCCT circspg21-f GACACCTCTATCTTCAACCA circspg21-r TTAAGGGGAACTGTACCTC circptpn12-f GAATCTCGTAGGCTGTATC circptpn12-r TTCCCATCTCAAATTCTCG circmpp6-f CAAGGTGTAGGCCGAAGAA circmpp6-r GGCAGCTCCGTAAGGTTTT circklhdc10-f CAGACCTGCACAAGTTAGA circklhdc10-r CCAAATGGGATGCCCGTAC circrasa3-f CAGAAGGAGGACTTGCAGAA circrasa3-r CGGGTAAGAGGGAAGGTTT circubap2-f GAGTTTGGGCCAGTTTACC circubap2-r GAGCAGATGAGGCAGTGAA circcep135-f CCACGAGAAGGATACAGAA circcep135-r CATAAGCATGATGGGCATT circxpo1-f CCAAGGAACCAGTGCGAAG circxpo1-r GAAATCAAGCAGCTGACGA circezh2-f TGGAAACAGCGAAGGATAC circezh2-r TCGTCTGAACCTCTTGAGC circhipk3-f TATGTTGGTGGATCCTGTTCGGCA

16 circhipk3-r circspecc1-f circspecc1-r circmet-f circmet-r circplce1-f circplce1-r circpvt1-f circpvt1-r circdhrs3-f circdhrs3-r circsmarca5-f circsmarca5-r circslc8a1-f circslc8a1-r circzmynd8-f circzmynd8-r circfoxk2-f circfoxk2-r circcamsap1-f circcamsap1-r circrtn4-f circrtn4-r circalpk2-f circalpk2-r circcamsap2-f circcamsap2-r circfhod3-f circfhod3-r circmyom1-f circmyom1-r circmlip-f circmlip-r circpalld -F circpalld -R circpopdc2 -F circpopdc2 -R circsorbs2 -F circsorbs2 -R circc3 -F circc3 -R circitih2 -F circitih2 -R TGGTGGGTAGACCAAGACTTGTGA ACTAAAGGCAAACGGTGAA GGAAGAAGTTGGACTGGAC TATTGTTTATACTCCCACT TACTACTCTAAGAACCTGG ACAGTGGAGATAAGGCAAG AGACTTTTACTCGGGATTG TTCAGCACTCTGGACGGACTT TATGGCATGGGCAGGGTAG CCAGCACCGAGATGTTCCA TCATCACTGTCCATTAGGCTCTT ATGGATACAGAGTCAAGTG ATCTTCATCAGTGATCTCA CAGAGGTGGAGGGGAGGAT CCATTGAAAAGGTGGGTGA GCTCCTATCACGACGAAAA TCTCTTGCATGGCACTGTT TAATGGCTGACAACTCACA GCTTCTCTCTCTTCTCGCT AGTGCCTCGAAAGAACTTC TCCTGCTCATACTGGTCAA AGTAATTCTGCTCTTGGTC GTCTTCTTAATGTCTCTCC GTCTCCAAAGAAGGCAACA AGCCGAGATTTGATAGACAGC CCCGTCCTCAAGTAGTAAA AAGCCATCAACATAAGACATAGA TTATCACTCCTCAAGACCCTCA CTTCCTCCTCCTCCTCCCT TGGAAGCAACCAGATAAGA GGGCCAGGCACAATACCCT GGAGGAAGTCTATGAACCC GTCAGCCAACTGAGTATGG AAGAAGTAGCAGAAGGGAGC TTCAGTAGGAAGGCCAGAG GAGGTGAAGGGAGAACTAACA CCGAGAAGAGGCAGGAGAT TGTGATGACGGCTGGTTTG TGGGTCTTTCCAGGCTTCT GGAAGTCCCTGAAGGTCGTG TTTCCACCTGCTCGTTTCG CAAGGATCTCAGGTGCTAGAGT GAGGTCAGCGGAGTCACAA

17 circmat1a -F circmat1a -R circpah -F circpah -R circc10orf90 -F circc10orf90 -R circhydin -F circhydin -R circslc4a10 -F circslc4a10 -R circehbp1 -F circehbp1 -R circmyh11 -F circmyh11 -R circlrp5 -F circlrp5 -R circtpm1 -F circtpm1 -R circcdh5-f circcdh5-r GCTGCGGCCTGACTCTAAG TGTGCTTGATGGTGTCCCT TGCCATGAGCTGTTGGGACA ACAGGTCGGAGGCGGAAAC CATTCCTGGCTGGAGTTAC TTGGTGAAGTCAGATTTTGC CCAGGATTCTCAACCTATGC CTTTCCCAATATCCAGCAA CCCAGGAGAAGAGGAAGAT GGCTGGAGCAGACTGAGGA GTAGGAGAATTGGCAGCAC GACTAGCATTTGGCTTTCG GCGAGGTGAACGCACTCAA GCTGGGACTCCTCCTCTGC CCCTTCCCGCACGAGTATGT GGGCGGCTTGGTGATTTCT AAGGTCCTTTCCGACAAGC CTGGGCACGATCCAACTCT GACATAACACCACGAAACG TTGTCATTCTCATCCAAAAC Oligos for plasmid construction chipk3-nest-f GCCTCAATAAACTCACATC chipk3-nest-r ATAGCATTACCCATAGCAT chipk3-alu-hind-f GACAAGCTTACATTGAGAAGAATGGGAACA chipk3-alu-hind-r GCAGAATTCCACGGGAGCCAAAGAAGTA chipk3-del-alu-hind-f GACAAGCTTTCTTGGGCTTTAGGTTTAA chipk3-del-alu-hind-r GGAGATTTCTATCCTGCTT Oligos for cloning of grnas grna-hipk3-1intron-1f CCCCGTACTTATGTGGCCTACTTA grna-hipk3-1intron-1r AAACTAAGTAGGCCACATAAGTAC grna-hipk3-1intron-2f CCCCGCTTGGTATGTAGTTAAGAG grna-hipk3-1intron-2r AAACCTCTTAACTACATACCAAGC 5 - alu- grna1-f CCCCGGCTTTAGGTTTAAGAAATA 5 - alu- grna1-r AAACTATTTCTTAAACCTAAAGCC 5 - alu- grna2-f CCCCGCACCGTTTCTAGGGCATAT 5 - alu- grna2-r AAACATATGCCCTAGAAACGGTGC 3 - alu- grna1-f CCCCTAAGATTCAGATATACAGCC 3 - alu- grna1-r AAACGGCTGTATATCTGAATCTTA 3 - alu- grna2-f CCCCTCTATCCACATATATGCTAT 3 - alu- grna2-r AAACATAGCATATATGTGGATAGA

18 Primers to amplify genomic deletions 1intron-deletion -F ATGTCCTAACTGGTTCTGG 1intron-deletion-R GCTATTCTGAGTATTGCGT 5 -del-alu-nest-f TGTGGTAAGAATGTTGGCTAT 5 -del-alu-nest-r AAGGCACTTGACTGAGTTTGA 5 -del-alu-clone-f ATTGAGAAGAATGGGAACA 5 -del-alu-clone-r GCTATTCTGAGTATTGCGTTAA 3 -del-alu-clone-f GTTTGAGTAGCCCTTATCT 3 -del-alu-clone-r GGTAATCATTAGTTAGCCTCT Actin-Genomic-F TGACTTAGTTGCGTTACACCCTT Actin-Genomic-R CACCTTCACCGTTCCAGTTTT FISH probes FISH-dig- F FISH-dig-T7-R GCTTTCAGCACCGTAACCA TAATACGACTCACTATAGGGAGACTTGCGCTTCAATCCACAT northern probes chipk3-dig-f chipk3-dig-t7-r CGGCAGCCTTACAGGGTTA TAATACGACTCACTATAGGGAGACAACTGCTTGGCTCTACTT primers for site-directed mutation mir-124-mut1-f AGTCAAGTCGGATTTGTAGTGTGAAGAAACTCAAAGTA mir-124-mut1-r ACTACAAATCCGACTTGACTGAGTTTGATAAACATATG mir-124-mut2-f AGGGTAGTCGAATTCAGACAAAGATACCATTTAATAGA mir-124-mut2-r TGTCTGAATTCGACTACCCTTAGTGGGAGGATGAGAAT mir-152-mut-1-f TTGTCGATCTTATGTCCATACTTCCTGCAATGTTGCAAACC mir-152-mut-1-r GTATGGACATAAGATCGACAATCTGCATTGCGCTGCTATGA mir-152-mut2-f GGCCACTGGTGAGAAAAAATTGAAAAGTCTTGGTTTAATT mir-152-mut2-r ATTTTTTCTCACCAGTGGCCACTTGTTGAAGAATGGGCCG mir-193a-mut1-f CCACTAAGGGTTCACCTTTTCAGACAAAGATACCATTTAAT mir-193a-mut1-r GAAAAGGTGAACCCTTAGTGGGAGGATGAGAATTTCCAAAG mir-193a-mut2-f TGGGTCGCGGTGTCATGTATCAAAGACTGTTTGTTCAACA mir-193a-mut2-r ATACATGACACCGCGACCCAAAGTCTATTACTTTAACCCT mir-29a/b-mut-f CACTGCAGCACGTACAAAGGTCATAGCAGCTCAGGCACAGC mir-29a/b-mut-r ACCTTTGTACGTGCTGCAGTGTTTTTCAAAACAACAGCACT mir-338-mut1-f AGTTAAATCGACGAAAAGAGGGACAAATGAAATTGTAGCA A mir-338-mut1-r CCTCTTTTCGTCGATTTAACTACCTGGCCAAACGTGCCTCG mir-338-mut2-f TTTGAGATCGACGAACAAAACTTGTATGACTTTCTGAAAC mir-338-mut2-r TTTTGTTCGTCGATCTCAAAGACTAAACAAGTATGGTTAC mir-338-mut3-f AATATTATCAACGTGGATCCTGTTCGGCAGCCTTACAGGG mir-338-mut3-r GGATCCACGTTGATAATATTCTCTGGCTTGAGATCAGCAT mir-379-mut1-f GTCTTGGTGATGCCACCATATGTTTATCAAACTCAGTCAAG

19 mir-379-mut1-r mir-584-mut-1-f mir-584-mut-1-r mir-584-mut2-f mir-584-mut2-r mir-584- mut3-f mir-584- mut3-r mir-584-mut4-f mir-584-mut4-r mir-584-mut5-f mir-584-mut5-r mir-654- mut1-f mir-654- mut1-r mir-654-mut2- F mir-654-mut2- R ATATGGTGGCATCACCAAGACTTGTGAGGCCATAC TCTACCCACGTATTGTTTATCAAACTCAGTCAAGTGCCTTT GATAAACAATACGTGGGTAGACCAAGACTTGTGAGGCCAT GGAAAGAATGATTCCACGGACCTATGTGAATGGTAGAAACT GTCCGTGGAATCATTCTTTCCTGGAATACACAACTGCTTGG TTGGATAATGTATGCAGCGCAATGCAGATTGTCGATGAAT GCGCTGCATACATTATCCAACTCCTCACTCTTGCGCTTCA CACCGTAACGTATCTTGTTTAGTCTTTGAGATGCTGGAACA TAAACAAGATACGTTACGGTGCTGAAAGCATTCATAAGCTC GATCTCAACGGTGAGAATATTATGTTGGTGGATCCTGTTCG AATATTCTCACCGTTGAGATCAGCATGAATTAAACCAAGAC TCACGTGCACTGTAGTCAGATTGGGGCGTGGCGAAACAGAT ATCTGACTACAGTGCACGTGAGCTTGCTGTGCCTGAGCTGC TTCATTGCACTGTAGTGCTGTTGTTTTGAAAAACACTGCAG ACAGCACTACAGTGCAATGAAAAGTTGTGTCCTCGAGGTCT sirnas si-mhipk3 si-circhipk3 si-both-hipk3 GCUGAUUGAUGCAGAUUUA CUACAGGUAUGGCCUCACA GAAGUGAGCAUAUUAGCAA