HIF-inducible mir-191 promotes migration in breast cancer through complex regulation of TGFβ-signaling in hypoxic microenvironment.

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1 HIF-inducible mir-9 promotes migration in breast cancer through complex regulation of TGFβ-signaling in hypoxic microenvironment. Neha Nagpal, Hafiz M Ahmad, Shibu Chameettachal3, Durai Sundar, Sourabh Ghosh3, Ritu Kulshreshtha Supplementary data.- List of primers used Supplementary data.- Functional assay for confirmation of hypoxic microenvironment or HIF activity a. All the experiments have been performed by exposing the cells to.% hypoxia (.% po ) in the hypoxic chamber for 4 hrs. The hypoxic microenvironment was confirmed by exposing the cells transfected with EPO-HRE to hypoxic microenvironment. Significantly high luciferase activity was observed in the cells exposed to hypoxic microenvironment. b. Activity of the HIF-/-α overexpression constructs or HIF-/-α specific shrnas was confirmed through dual luciferase assay by cotransfecting EPO-HRE reporter plasmid in cells. Graph represents relative luciferase activity. c. cells were transfected with HIF-α overexpressing plasmid/vector control (PC) and shhif-α/sh to overexpress/ inhibit the expression of HIF-α and western blotting was done to measure the HIF-α levels. The graphical data points in a & b represent mean + S.D of at least three independent experiments. (P<.5, P<.). Error bars denote + SD. Supplementary data 3.- Confirmation of overexpression/ inhibition of specific molecules achieved by transient transfection a,b. Level of mir-9 was transiently up/ downregulated by transfecting with pre-9 (Ambion) & anti-9 oligos (Exiqon) and exposed to hypoxic microenvironment for 4 hrs. The corresponding effect on the expression of mir9 was then checked by qrt-pcr compared to that of their respective controls in MCF-7 cells. c-e. MCF-7 cells were transfected with pcdna3.- or with esirna for inhibition and exposed to hypoxic microenvironment for 4 hrs. The graph shows relative expression levels as determined by qrt-pcr (c,d) and western blotting (e) of compared to that of respective control transfected cells. f-h. MCF-7 cells were transfected with TGFβ esirna or treated with recombinant TGFβ and the efficiency of esirna mediated inhibition or overexpression was confirmed through measuring TGFβ levels through qrt-pcr (f,g) or luciferase activity of ptplux reporter construct (h). i. qrt-pcr data showing effect of ctrl or SMAD3 sirna on SMAD3 levels in cells. The graphical data points represent mean + S.D of at least three independent experiments. (P<.). Error bars denote + SD. Supplementary data 4.- Effect of mir-9 on TGFβ & levels in the presence/absence of hypoxic microenvironment a,b. Effect of mir-9 on TGFβ and at protein levels in hypoxic microenvironment. The cells with differential level of mir-9 were exposed to hypoxic microenvironment and analyzed for expression of TGFβ (a) and (b) protein by western blotting and the relative protein levels were then quantified using imagej software for band densitometery. c. Level of mir-9 was differentially modulated and cells () were exposed to hypoxic microenvironment, Immunofluorescence was done by using an antibody specific for TGFβ to compare the difference in fluorescence intensity observed. d. Cells were transfected with esictrl or esi oligos in normoxia and qrt-pcr was performed. The results show that under normoxic conditions is unable to downregulate TGFβ levels in breast cancer cell lines. e-g qrt-pcr was done to find out the effect of mir-9 on TGFβ & levels in both normoxic and hypoxic microenvironment in a panel of breast cancer cell lines (e), T47D (f) & MM3 (g). We found that the effect was minimal in the absence of hypoxic microenvironment. The graphical data points in a-g represent mean + S.D of at least three independent experiments. (P<.5, P<., ^P>.5<.). Error bars denote + SD.

2 Supplementary data 5.- is a direct target of mir-9. a. Diagram showing sequence and position of mir-9 binding sites (wild type: B & B, mutant type Mut B) in 3 UTR. b,c. 3 UTR luciferase activity of lucifease constructs (individually cloned: B, B, Both together: B&) in response to differential mir-9 levels. It was found that mir-9 overexpression led to decrease in luciferase activity with B and B& constructs (b) while lesser effect was observed with B (b) or mutated B sites (c). Therefore, mir-9 mediated downregulation of is mediated mainly through binding to the B site present in the 3 UTR. The graphical data points in b & c represent mean + S.D of at least three independent experiments. (P<.5, P<., ^P>.5<.). Error bars denote + SD. Supplementary data 6.- Diagram showing putative mir-9 and binding sites in TGFβ 3 UTR Diagram showing in-silico analysis data of the 3 UTR region of TGFβ for the putative consensus sequences and mir-9 binding sites using RNAhybrid, ARE score and catrapid softwares. The 3 UTR region containing the mir-9 and binding sites of TGFβ-3 UTR was cloned and checked for luciferase activity. Primer details are given in Supplementary data. Supplementary data 7.- Validation of hypoxic microenvironment and transfection efficiency in the tumor spheroids a. To recapitulate the endogenous hypoxic microenvironment 3D tumor spheroids were manifested and the level of VEGFA was sought as a marker of hypoxic microenvironment. b. The 3D tumor spheroids of were transfected with anti-mir9/n and the effectiveness of transfection was confirmed by qrt-pcr. The graph shows mir-9 levels in antimir-9/n treated spheroids on day and 7. The readings were normalized using U6.. The graphical data points in a and b represent mean + S.D of at least three independent experiments. (P<.5, P<.). Error bars denote + SD.

3 Supplementary Data - List of Primers Primer Detail Primer Sequence Annealing Temperature mir-9 RT primer GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCA GCTG 4⁰C RNU6B RT primer GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAA AATATGGAAC 4⁰C mir-9 forward primer CGCGCAACGGAATCCCA 6⁰C RNU6B forward primer GCCCCTGCGCAAGGATGAC 6⁰C Stem-loop reverse universal GTGCAGGGTCCGAGGT 6⁰C Primers designed to detect transcript levels of various shortlisted target genes Primer Detail Forward Primer Reverse Primer Annealing Temperature PTGS TCAGTAGGTGCATTGGAATCAAGC TAGCAGTCCTGAGCTGAGGTTTACC 6⁰C BMP4 AGCATGTCAGGATTAGCCGATCG AGTCATTCCAGCCCACATCGC 6⁰C SMAD3 GAGATGATGGGCTAAACAGGCAAC CACTGTCCTTGGGAGGAGAGACC 6⁰C TGFβ AGTCATACCACCTTTCCGATTGCC AAAGCATGGTGCAAACATCTCTCTG 6⁰C TGFβ TCCTGGCGATACCTCAGCAAC TGAACCCGTTGATGTCCACTTG 6⁰C JUN GTGTGCACGAGTGGGAAGG GATCGAATGTTAGGTCCATGGAG 58⁰C FOS AATGACCCTGAGCCCAAGCC AGCTCTGTGGCCATGGCC 58⁰C CTGF AGCGTGCTCACTGACCTGCC TCACTTGCCACAAGCTGTCCAG 6⁰C VEGFA GCTACTGCCATCCAATCGAGAC CTATGTGCTGGCCTTGGTGAG 58⁰C ACCAATGTGAAAGTGATCC TATGAATTCTTATTTGTGGGACTTGT TG 55⁰C

4 Primers designed to amplify HIF binding sites in mir-9 promoter for CHIP assay AGGCGCGCTTCGATGACG AGGCAGGCGAGGGCATGG 6⁰C H GCTCCCTGTGCCATGTTGTCC TCACCATGTTGGCCAGGCTG 6⁰C H AGCCTGGCCAACATGGTGAAAC TGCCTCCTTCAGTGTGATGCC 6⁰C H3 ACTAACTGCACGGTGACTCCTGC ATCAGGACTGCAGCTTGGCTG 6⁰C H4 CATCTGACTCTGGCTCCCAAGG AGGGTTCACCATGTTGGCCAG 6⁰C H5 ACGACAAATCCACGCAGCCTC TGATAAACGGAAACCGCGTGC 6⁰C H6 TGTTCTGTGGCCCAGGTGAGC AGCTGCTTTTGGGATTCCGTTG 6⁰C Primers designed to clone the 3 UTR luciferase reporter constructs TGFβ ATTGAGCTCTGCTTTGGCTTT CTGGTTCTATG ATTAAGCTTGAGGAGTCTGGTCTT GTAGGTAGC 57⁰C Mut-TGFβ CAGATATAACAAGAGCCACG TGCTTTCTCCCCTTGGTTGTT TGGGATCAGCTACTTGC GCAAGTAGCTGATCCCAAACAAC CAAGGGGAGAAAGCACGTGGCTC TTGTTATATCTG 57⁰C B ATAACTAGTGGCAATTGGCG TGTAATGATG ATTAAGCTTCACATGGTCATGGTC AAAGAGG 57⁰C B ATAACTAGTCACCTCTTTGAC CATGACCATGTG ATTAAGCTTTGGGAATCGGTTAAG AGAGCCTC 57⁰C B & ATAACTAGTGGCAATTGGCG TGTAATGATG ATTAAGCTTTGGGAATCGGTTAAG AGAGCCTC 6⁰C Mut- B TCCACCAGAAGAGAAGCCTT TTGGCTTGGTTTGGGGCCACC TCTTTGAC ATAACGCGTAGCCTGGCCAA CATGGTGAAAC GTCAAAGAGGTGGCCCCAAACCA AGCCAAAAGGCTTCTCTTCTGGTG GA ATACTCGAGAGAGTGCTTGGTTCC AAGGTATGTG 66⁰C ATACTCGAGGCATGAGGTAT GGCAGAGG ATAACGCGTACCACTGCCCTTATC TTGCCTG 6⁰C H-HRE H3-HRE 6⁰C

5 Supplementary Data 7 b c 6 6 Relative Luciferase Activity Relative Luciferase Activity a EPO-HRE EPO-HRE HIFα HIFα shgfp shhifα shhifα

6 b Pre9 5 RNU6B c..8.6 N.4 Anti9. RNU6B MIR esi.4 esi. f..8.6 esitgfβ..5 TGFβ TGFβ i..8.6 Relative Fold Change Relative ptplux reporter luciferase activity h TGFβ.5.5 esi.4 g e MIR d a Supplementary Data sismad3. esi esitgfβ TGFβ si SMAD3

7 Supplementary Data 4 Relative band density (arbitrary unit).5 Pre9 N Anti9.5 Relative fluorescence intensity of TGFβ b.5 β-actin 3.5 esi.5 esi.5 f Relative mrna expression level 3.5 T47D T47D Norm Pre9 Norm.5 Hypo Pre9 Norm TGFβ N Norm.5 Pre9 Norm Hypo Pre9 Hypo.5 TGFβ MM3.5 Norm.5 Pre9 Norm Hypo.5 Pre9 Hypo TGFβ Pre9 N β-actin Anti9.5 g Pre9 3.5 MM3 3.5 Relative mrna expression level e TGFβ Relative mrna expression level Relative TGFβ expression level d c Relative band density (arbitrary unit) a Anti9

8 Supplementary Data 5 b.5 a B (47-433) mir-9.5 Pre9 N ^ Anti9.5 B (47-477) B B B& mir-9 Mut-B (47-433) c.5 mir-9 Relative luciferase activity 3 UTR Relative luciferase activity.5 Pre9 Anti9.5 B Wild type N B Mutant type

9 Supplementary Data 6 TGFβ

10 Supplementary Data 7 a Relative VEGFA levels in the 3D tumor spheroids Day3 Day7.5.5 b T47D. Relative mir-9 expression level Day.8.6 N Anti9.4. day day7 3D tumor spheroids