CSUN SYMPOSIUM Final
1
DNA Small Lymphocytic Lymphoma Associated miRNA-155 Promoter Region Isolation and Characterization Department of Biology, California State University Northridge, Northridge, California Alina Adamian and Cindy S. Malone, PhD Abstract Results Discussion Background Methodology Site Directed Mutagenesis & Transcription Factors Acknowledgments MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression. miRNAs are a class of evolutionally conserved non-coding small RNAs of 18 to 24 nucleotides in length; they participate in post- transcriptional gene expression regulation through mRNA. We previously found miRNA-155HG associated with expression in indolent Small Lymphocytic Lymphomas (Figure 8), but not in aggressive Mantle Cell Lymphoma (Figure 7), suggesting its contribution to the indolent nature of this cancer. Recently, it has been found that epigenetically, BRCA1 represses expression of miR-155 (Chang et al., 2011). We hypothesize that the promoter region is found just upstream of the coding region of miRNA-155HG and will control expression of this gene. 1 CCCCTTGTGG CAGGGTCCGG GAGAAAAGAG AGAAGCGACA AAACCAAAAA TTAAAACGAC 61 CGAAGTCCCA TATGCTCCAG GAATATGTCC TGGAGATGGG AGTGGAGGGC AGGGGGAGAA 121 TGTTGTTGAG GTCAAAATTT TTGAAGTTTT AAGTCCTATA TCTTGACATC CCGAGTATAA 181 ATGCGGGTAC CAGACACAGT ACAAACGTTC TCAAAGCCCA GTTACGTATT CCAAACCAAA 241 CGCGGGCTCT TGAAGGGTGA TGAGGTAGGG ATGAAATCCA GGATCGCCTG AAGACCATTT 301 CTTCCTCTCT TAGGGACCTG CTGGTCTCGA GCTGATTCGG TCCAGGAGGA AAAACCTCCC 361 ACTTGCTCCT 1 CTCG G G CTCC CTGCAAGGAG AGAGTAGAGA CACTCCTGCC ACCCAGTTGC 421 AAGAAGTCGC CACTTCCCCC TCCAGCCGAC TGAAAGTTCG GGCGACGTCT GGGCCGTCAT 481 TTGAAGGCGT TTCCTTTTCT 2 TTAAGAACAA AGGTTGGAGC C C AAGCCTTG CGGCGCGGTG 541 CAGGAAAGTA CACGGCGTGT GTTGAGAGAA AAAAAATACA CACACGCAAT GACCCACGAG 601 AAAGGGAAAG GGGAAAACAC CAACTACCCG GGCGCTGGGC TTTTTCGACT TTTCCTTTAA 661 AAAGAAAAAA 3 GTTTTTCAAG CTGTAGGT T C C AAGAACAGG CAGGAGGGGG GAGAAGGGGG 721 GGGGGGTGCA GAAAAGGGGG CTGGTCGGTT ATGAGTCACA AGTGAGTTAT AAAAGGGTCG 781 CACGTTCGCA GGCGCGGGCT TCCTGTGCGC G Research supported by grants from: •CSUPERB Research and Development Grant •CSUN Competition for Research, Scholarship and Creative Activity Award •CSUN Biology Honors Program miRNA-155 is located on chromosome 21 and is composed of 13,024 bp. miRNA-155HG is present in normal B cells and is essential for production of high affinity antibodies. B cells infected with EBV (Ebstein-Barr Virus) overexpress MicroRNAs, including miRNA-155, and induce B-cell lymphomas in animal cells (Velkova et al., 2011). Here we have identified and initiated characterization of the putative miRNA-155 promoter region. PCR amplified cDNA of from miRNA-155 was inserted upstream of a luciferase reporter gene in the pGL3 vector. HEK293T cells were transfected with pGL3-miRNA-155 and examined for luciferase activity. Deletion constructs using site directed mutagenesis at potential transcription factor binding sites are being engineered. Analysis of changes in luciferase activity in these engineered constructs will suggest which transcription factors may regulate miRNA-155. • PCR of the promoter region of miRNA-155 was performed and PCR products were inserted into pJET1.2 (Figure 2). The miRNA-155 promoter was extracted from pJET1.2 using BglII and inserted into the luciferase reporter vector, pGL3. Orientation of inserts was confirmed by restriction digest using BglII and SmaI (Figure 4). DNA sequence of miRNA-155 promoter region was confirmed by sequencing (Laragen). • The pGL3-miRNA-155 construct was transfected into HEK293T cells using Effectene (Promega) transient transfection when cells were 80% confluent (Figure MicroRNA-155, an oncomiR, is epigenetically repressed by BRCA1’s association with HDAC2 due to histone deacetylation. BRCA1 binds to HDAC2 aiding in deacetylation modifications to DNA (Figure 1). In the absence of BRCA1, miRNA-155 is overexpressed, resulting in acceleration of tumor growth, whereas a knockdown of miRNA-155 reduces its effect on tumor growth (Velkova and Monteiro, 2011). Characterization of the promoter region of miRNA-155 will permit us to understand the mechanism under which miRNA-155 contributes to tumor growth. Sp1 Elf-1 Nde1 Kpn 1 Xho1 Sma 1 1 Xma1 2 Sac1 3 BamH1 OVEREXPRESSION of miRNA-155 Figure 2: CloneJet (pJET) Cloning Vector Utilizing Escherichia coli, miRNA-155-Promoter construct was inserted into CloneJet. Figure 3: pGL3 Cloning Vector with Luciferase Reporter 1 2 3 4 5 6 7 8 9 10 11 1KB ladder 10,000 bp Figure 4: Construct confirmation gel. From left: Lane 1: 1 kb ladder. Lanes 2-11: alternating samples of cut and uncut vector using restriction digest BglII. miRNA-155 with 811 bp confirmed. Figure 5: 293T HEK cells at 80% confluency used for transfection of mirRNA-155-PGL3 construct Figure 6: Luciferase activity measured in relative light units (RLU). Mantle Cell Lymphoma vs. Small Lymphocytic Lymphoma 1000 bp Figure 1 Figure 8 Figure 7 Figure 6 Figure 5 Figure 4 Figure 3 Figure 2 HDAC2
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Transcript of CSUN SYMPOSIUM Final
DNADNA
Small Lymphocytic Lymphoma Associated miRNA-155 Promoter Region Isolation and Characterization
Department of Biology, California State University Northridge, Northridge, CaliforniaAlina Adamian and Cindy S. Malone, PhD
Abstract Results Discussion
Background
Methodology
Site Directed Mutagenesis & Transcription Factors
Acknowledgments
MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression. miRNAs are a class of evolutionally conserved non-coding small RNAs of 18 to 24 nucleotides in length; they participate in post-transcriptional gene expression regulation through mRNA. We previously found miRNA-155HG associated with expression in indolent Small Lymphocytic Lymphomas (Figure 8), but not in aggressive Mantle Cell Lymphoma (Figure 7), suggesting its contribution to the indolent nature of this cancer. Recently, it has been found that epigenetically, BRCA1 represses expression of miR-155 (Chang et al., 2011).
We hypothesize that the promoter region is found just upstream of the coding region of miRNA-155HG and will control expression of this gene.
1 CCCCTTGTGG CAGGGTCCGG GAGAAAAGAG AGAAGCGACA AAACCAAAAA TTAAAACGAC
61 CGAAGTCCCA TATGCTCCAG GAATATGTCC TGGAGATGGG AGTGGAGGGC AGGGGGAGAA
121 TGTTGTTGAG GTCAAAATTT TTGAAGTTTT AAGTCCTATA TCTTGACATC CCGAGTATAA
181 ATGCGGGTAC CAGACACAGT ACAAACGTTC TCAAAGCCCA GTTACGTATT CCAAACCAAA
241 CGCGGGCTCT TGAAGGGTGA TGAGGTAGGG ATGAAATCCA GGATCGCCTG AAGACCATTT
301 CTTCCTCTCT TAGGGACCTG CTGGTCTCGA GCTGATTCGG TCCAGGAGGA AAAACCTCCC
361 ACTTGCTCCT1CTCGGGCTCC CTGCAAGGAG AGAGTAGAGA CACTCCTGCC ACCCAGTTGC
421 AAGAAGTCGC CACTTCCCCC TCCAGCCGAC TGAAAGTTCG GGCGACGTCT GGGCCGTCAT
481 TTGAAGGCGT TTCCTTTTCT2TTAAGAACAA AGGTTGGAGC CCAAGCCTTG CGGCGCGGTG
541 CAGGAAAGTA CACGGCGTGT GTTGAGAGAA AAAAAATACA CACACGCAAT GACCCACGAG
601 AAAGGGAAAG GGGAAAACAC CAACTACCCG GGCGCTGGGC TTTTTCGACT TTTCCTTTAA
661 AAAGAAAAAA3GTTTTTCAAG CTGTAGGTTC CAAGAACAGG CAGGAGGGGG GAGAAGGGGG
721 GGGGGGTGCA GAAAAGGGGG CTGGTCGGTT ATGAGTCACA AGTGAGTTAT AAAAGGGTCG 781 CACGTTCGCA GGCGCGGGCT TCCTGTGCGC G
Research supported by grants from: •CSUPERB Research and Development Grant•CSUN Competition for Research, Scholarship and Creative Activity Award•CSUN Biology Honors Program
miRNA-155 is located on chromosome 21 and is composed of 13,024 bp. miRNA-155HG is present in normal B cells and is essential for production of high affinity antibodies. B cells infected with EBV (Ebstein-Barr Virus) overexpress MicroRNAs, including miRNA-155, and induce B-cell lymphomas in animal cells (Velkova et al., 2011). Here we have identified and initiated characterization of the putative miRNA-155 promoter region. PCR amplified cDNA of from miRNA-155 was inserted upstream of a luciferase reporter gene in the pGL3 vector. HEK293T cells were transfected with pGL3-miRNA-155 and examined for luciferase activity. Deletion constructs using site directed mutagenesis at potential transcription factor binding sites are being engineered. Analysis of changes in luciferase activity in these engineered constructs will suggest which transcription factors may regulate miRNA-155.
• PCR of the promoter region of miRNA-155 was performed and PCR products were inserted into pJET1.2 (Figure 2). The miRNA-155 promoter was extracted from pJET1.2 using BglII and inserted into the luciferase reporter vector, pGL3. Orientation of inserts was confirmed by restriction digest using BglII and SmaI (Figure 4). DNA sequence of miRNA-155 promoter region was confirmed by sequencing (Laragen).
• The pGL3-miRNA-155 construct was transfected into HEK293T cells using Effectene (Promega) transient transfection when cells were 80% confluent (Figure 6). Analysis of promoter activity in cells transfected with pGL3-miRNA-155 was compared to controls, pGL3 Basic and pGL3-SV40
(Figure 6).
MicroRNA-155, an oncomiR, is epigenetically repressed by BRCA1’s association with HDAC2 due to histone deacetylation. BRCA1 binds to HDAC2 aiding in deacetylation modifications to DNA (Figure 1). In the absence of BRCA1, miRNA-155 is overexpressed, resulting in acceleration of tumor growth, whereas a knockdown of miRNA-155 reduces its effect on tumor growth (Velkova and Monteiro, 2011). Characterization of the promoter region of miRNA-155 will permit us to understand the mechanism under which miRNA-155 contributes to tumor growth.
Sp1 Elf-1 Nde1 Kpn1 Xho1 Sma1 1Xma1 2Sac1 3BamH1
OVEREXPRESSION of miRNA-155
Figure 2: CloneJet (pJET) Cloning VectorUtilizing Escherichia coli, miRNA-155-Promoter construct was inserted into CloneJet.
Figure 3: pGL3 Cloning Vector with Luciferase Reporter
1 2 3 4 5 6 7 8 9 10 11
1KB ladder
10,000 bp
Figure 4: Construct confirmation gel. From left: Lane 1: 1 kb ladder. Lanes 2-11: alternating samples of cut and uncut vector using restriction digest BglII. miRNA-155 with 811 bp confirmed.
Figure 5: 293T HEK cells at 80% confluency used for transfection of mirRNA-155-PGL3 construct
Figure 6: Luciferase activity measured in relative light units (RLU).
Mantle Cell Lymphoma vs. Small Lymphocytic Lymphoma
1000 bpFigure 1
Figure 8Figure 7
Figure 6Figure 5
Figure 4
Figure 3Figure 2
HDAC2
