A Tool to Change Human Antigen R Protein Expression: May Have Implications in both Cancer & Diabetes >

 

Copyright © 2016 by Dr. Kerry Masterson  ·  All Rights reserved  ·  E-Mail: km@humanantigenr.com
      Modulating HuR protein expression may improve clinical-outcomes in cancer1 and moderate inflammatory Diabetes.2  Tools which change HuR expression may stream-line the study of HuR activities. For a picture of HuR activities click here. This page shows that a relatively inexpensive, commercially available, water-soluable, and cell permeable without DMSO oglionucleotide attenuates the regulation of HuR protein expression. The HPS reduces PC3 prostate cancer cell line growth rates.

     MicroRNAs regulate HuR mRNA translation rates, HuR protein abundance.
MicroRNAs (miR) are short endogenous 18 to 25 nucleotide long non-coding RNAs that regulate the protein expression of nearly 60% of all human protein coding genes.3 MicroRNAs regulate mRNA translation when incorporated into multi-protein RNA-induced silencing complexes (RISC). RISC use microRNA 3p or  5p arms to target the decay of mRNAs, a process known as RNA interference (RNAi).4
   
      The targeting and decay of a mRNA by a RISC is primarily determined by WC base-pairing between nucleotides 2-8 (extended seed sequence) in the microRNAs 5’ end to complementary nucleotides (NTs) within a targeted mRNAs 3’ untranslated region (3’UTR). Additional WC pairing outside the extended seed sequence may improve or even enable the targeting of an mRNA by a RISC. An Adenosine at mRNA target 1 nucleotide (t1) in the mRNA 3’UTR has been shown to interact with the RISC machinery to compound mRNA decay by RNAi.
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     Figure 1 Illustrates binding patterns between microRNA-9-5p and four targeted mRNA sequences. Lower: the least repressive 6-mer seed sequence. Upper: the most repressive or optimal M8-A1 8 mer extended seed sequence. This diagram is modeled after diagram presented in “Determinants of targeting by endogenous and exogenous microRNA and siRNA.”
6 Nielsen et al, RNA 2007 13: 1894-1910.

     According to Nielsen et al, the predicted target-rank of a mRNA, which is the probability of mRNA being decayed by microRNA sequence within a RISC, is based primarily upon the type of mRNA target-site. Said target sites are ranked from most to least repressive: M8-A1 8mer > (M8 7mer > or = A1 7 mer) > seed match 6 mer. Other target-rank factors include the base at position t9, destabilizing AU content flanking the target-site, site conservation, AU content of the 50 nucleotides which precede the target-site, and the number and variety of target-sites for a specific microRNA sequence within a mRNA.6 MRNAs with lower target-ranks have a higher probability of repression by a specific microRNA sequence; whereas, mRNAs with higher target-ranks have a lower probability of repression by the same microRNA sequence.

    When a RISC binds to a mRNA it does not always result in mRNA translation repression, decay.  However, w
hen a RISC is bound to an mRNA, or bound to an mRNA-decoy which effectively mimics a mRNA target site, that RISC is temporarily or permanently prevented from decaying other mRNA targets. Thus; sufficiently abundant mRNAs or mRNA target-site decoys with similar target-ranks or binding affinities may intercede to “protect” mRNAs from RISC (RNAi) mediated mRNA decay. 7 Protected mRNAs are not decayed and protein expression is increased or disconnected from microRNA regulation.

     
RISCs target mRNA transcripts for Argonaute 2 enzyme cleavage or deadenylation, both of which repress mRNA translation rates.8 A common misperception is the extent of protein repression that microRNAs exert over an mRNAs. In Lim et al transfection experiments of excess short interfering microRNAs into HeLa cells showed that the average decline in all targeted protein expression was 2%.9  Lim et al also showed that multiple microRNAs acting in concert on a single mRNA target produced and average decline in protein expression of 5% to 10%.  Thus; blocking concerted microRNA repression of a mRNA, with a target-site decoy modeled after a specific target-site within that mRNA, may compound the reduction in RNAi and change protein expression beyond 5% to10%.
     
     HuR expression been shown to be repressed by several microRNAs including miR-125a, miR-133, and miR-9.10  MiR-9 when blocked systemically with an anti-microRNA was shown to increase the protein expression of the Dicer enzyme and HuR protein in Hodgkin lymphoma tumors outgrowths.
11  MiR-133 was shown to represses HuR protein expression in HeLa cells.12 MiR-125a and not miR-125b reduced MCF7 Breast cancer cell growth by targeting HuR mRNA 3’UTR between NTs 671-692.13  Selectivity for HuR repression by miR-125a over miR-125b is an interesting nuance. According to work done by Dr. Hartley’s group at the University of New Mexico, the miR-125a HuR mRNA target-site is enabled by NTs out-side of the extended seed sequence, Figure 2 A and B. My research into bioinformatics on that site indicated that two strongly-repressive extended seed sequences for miR-133 and miR-9 are also present within NTs 671-692.  The strongest microRNA regulators of HuR mRNA according to TargetSCAN are listed as miR-125, miR-133 and miR-9.


















Figure 2 Details binding between between microRNAs miR-125, miR-133 and miR-9 and the HuR 3'UTR  mRNA target site between NTs 671-692 or the HuR Protective Sequence (HPS).  A shows binding between miR-125a-5p  to the A1 7 mer extended seed sequence plus 8 additional  binding NTs.  B shows binding between miR-125b-5p to the A1 7 mer extended seed sequence plus 5 additionail  binding NTs. The miR-125 binding site it is not conserved; however, the addition of 8 NTs in miR-125a is thought enable decay. The addition of 5 NTs in miR-125b is insufficient to enable decay.  C, D, and E shows strongly repressive and broadly coserved amoung vertibrates M8-A1 8 mer targeting between specific miR-133 and miR-9 microRNA arms. F shows the HuR mRNA sequence which used to construct the ssDNA HuR Protective Sequence (HPS). Note: The above diagram is a composite of diagrams within the dissertation, please use it or redraw it and credit the dissertation. Figure 2 A and B are redrawn.13         

                                       
Diagram in which the HPS Blocks MicroRNAs from Targeting HuR mRNA.


























































     
Figure 3 Boxes 1 & 4: MicroRNAs are processed by Dicer (RNASEH) into ~22 NT double-stranded microRNA duplexes. One strand is within the RNA Induced Silencing Complex to guide (capture) mRNAs in the cytoplasm. The other strand is ejected.  Box 2:  The guide strand binds to HuR mRNA and the Argonaute 2 enzyme cleaves both the guide strand and HuR mRNA. Cleaved HuR mRNA is not processed in the ribosome and HuR protein expression is repressed (regulated). Box 3: The remaining HuR mRNA is translated into a regulated amount of HuR protein.  Box 5: The HPS binds to the microRNA guide strand forming a temporary RNA/ DNA hybrid.  The Argonaute 2 enzyme cleaves the guide strand not DNA in a hybrid.  HuR mRNA is protected. The HPS is recycled.  Box 6: HuR mRNA transits through the cytoplasm with less regulation by microRNA hsa-miR-9, hsa-miR-125a, or hsa-miR-133ab; thus, HuR expression is deregulated. NTs 671-692 in HuR mRNA 3’ UTR in ssDNA is the HPS.
 
                  A ssDNA oglionucleotide changes HuR protein expression
                                       5’-ATGGACCAAAGAGTTTCAGGGA-3’                                                                                                                 
            
Human Antigen R mRNA protective sequence (HPS) MW 6832
      The HPS decoys miR-125a, miR-133, and miR-9 away from attacking a conserved microRNA degradation prone          "sweet-spot"  in HuR mRNAs  3' UTR at NTs 671-692. The HPS also reduces attacks by miR-125a, miR-133, and miR-9 at     other sites within HuR mRNA. The HPS partially disconnects HuR protein expression from microRNA regulation. At higher      HPS concentrations HuR protein expression is decreased in PC3 Human Prostate cancer cells, at lower HPS concentrations HuR protein expression is increased in PC3 cancer cells. Please see the Dissertation  for more details.

HuR: A Cancer  and Diabetes Target          HuR mRNA Protective Sequence (HPS)           HPS Experiment Results          Contact
Human Antigen R mRNA Protective Sequence Site
      All experimental information within the dissertation is given freely for you to use and to publish; reference the dissertation if you publish on the HPS.  Changing HuR protein expression with the HPS may be effective in combating both Cancer and Diabetes. The HPS in combination with Paclitaxel, Tamoxifen,  Doxorubicin, or Gemcitabine  may reduce cancer drug dosage  by over-comming HuR linked cancer drug resistance. The HPS may improve overall clinical efficacy by inhibiting cancer cell migration ( cancer metastatics.)  If you have access to the NCI-60 consider submitting the HPS for drug screening.

 
In Diabetes,  Decreased AUF1 activity is a result of the diabetes drug metformin. HuR is a AUF1 competator; therefore, changing HuR protien expression may compete with AUF1 and function as a metformin alternative in diabetes. The HPS was shown to decreases phospho-AKT Serine 473 in PC3 cancer cells. Decreasing AKT phosphorylation or othe HUR regulated pathway changes may effect phospho-AMPK THR 172.  Changing AMPK activity in-turn shound increase sugar metabolism at the cellular level.  HuR controls the metabolism of cancer cells and reduces sugar metabolism in cancer cells; thus, it stands to reason the HuR protein expression is also behind decreased sugar metabolism in normal cells and in Type II diabetes. Diabetes is an inflamatory condition and changing HuR protein expression is how the Human body responds to stress at the cellular level.