Michael Qian

I/M 1/11 AP

Status Quo H19 and HuR Review Paper

Introduction

The purpose of the intestinal epithelium is to control the flux of water, ion, and

macromolecules between the luminal contents, or content within the hollow space within the

intestine, and the underlying tissue and immune system. The intestinal immune system itself is a

part of the dynamic mucosal layer that separates intestinal lumen from underlying submucosal

layer while facilitating water, nutrient, and ion transport. The intestinal epithelial barrier is

constantly regulating its structure to maintain homeostasis of the water, ion, and macromolecule

diffusion and permeability during disease and disease-free periods.

The barriers function is primarily determined by the epithelial monolayer, which acts as

a blockade for transmucosal and water flux. This monolayer is sealed with tight junction (TJ)

proteins and adherens junction (AJ) proteins. TJ proteins are made of transmembrane and

cytosolic proteins accompanied by cytoskeletal and regulatory proteins, and function as

paracellular protein connection. AJ proteins are composed mainly of cadherins and other binding

proteins, and supply adhesion for maintenance of cell-cell interaction. TJ and AJ protein

production and presence are often used as dependent variables when measuring for intestinal

epithelial barrier integrity.

The intestinal epithelial barrier also has several main pathways that facilitate flux in and

out of the intestine. There is the pore pathway, a size and charge selective route with high

capacity, with permeability determined by subset of claudins expressed. There also exists the

leak pathway, with more limited selectivity with low capacity and permeability determined by

Xo1, occludin, and myosin light chain kinase (MLCK). There are unrestricted pathways, which

are high capacity and nonselective due to lack of TJ proteins and can lead to large proteins and
 Michael Qian
I/M 1/11 AP

whole bacteria crossing the pathway. This pathway only occurs at sites of epithelial damage, and

is the main area of transport for unintentional influx of solutes and water 9.

This paper will review the regulation regarding H19 and HuR (ELAV1) within the

intestinal epithelial barrier. H19 is a long-noncoding RNA, or lnc RNA. It can both act as

precursors for smaller non-coding RNA transcripts, or micro-RNAs (miRNA). HuR is a RNA

binding protein (RBP) that influence editing, splicing, and other RNA translation factors.

H19 Background

The H19 described assumes the form of long non-coding RNA. The focused study will be

on the regulation regarding H19 within the intestinal epithelial barrier; however, it is important to

have background knowledge of this gene outside of the studied region. Within muscle tissue,

overexpression of H19 causes hypertrophy and hyperplasia 2. It is highly expressed in satellite

cells, or muscle stem cells, that repair damaged tissue. In mice with overexpressed H19, the

study shows an increase of 8-10% in mass. H19 deletion causes depletion of satellite cells and

also controls proliferations of myoblasts. This genes role in muscle regeneration is clearly linked

to cell proliferation, suggesting a similar role in the intestinal epithelial barrier.

Regulation of Intestinal H19 by Other Factors

H19 can be regulated by competing pseudogene RNAs, which are a subclass of lnc RNA.

Certain pseudogenes, or an imperfect copy of a normal gene, maintain 5 and 3 UTR from

parental genes that allow them to compete for miRNA. HMGA1P7 acts as a competitive

endogenous miRNA decoy and upregulates lnc H19 expression. H19 can also be regulated by

competition with other noncoding RNAs as well 3.These findings suggest potential undiscovered

regulatory pseudogenes independent of the described HMGA1P7 gene.

Intestinal H19 Regulation on Other Factors

 Michael Qian
I/M 1/11 AP

The long non-coding RNA H19 has a negative regulatory effect on the overall integrity of

the intestinal epithelial barrier. This is demonstrated through its ability to destabilize TJ, ZO-1

and AJ, e-cadherin protein production 14. Translating fraction rates for ZO-1 and E-cad mRNAs

decreased dramatically in cells with overexpressed H19. Other potential targets of regulation

revealed unremarkable results. H19 overexpression results in intestinal barrier dysfunction,

shown by the significantly higher transepithelial electrical resistance (TEER) measurement of the

control cells versus the H19 overexpression. Therefore, although H19 is necessary for cell

proliferation in muscle cells, it plays a negative regulatory role specifically regarding TJ and AJ

proteins within the intestinal epithelial barrier.

In addition, lncRNA H19 also acts as a competitive RNA regarding water channels within

the intestinal epithelial barrier 10. AQP3 codes for a water channel protein in the cellular

membrane and also plays a role in glycerol regulation, making it relevant to intestinal barrier

permeability and function. AQP3 is regulated by miR-874 inversely depending on miR-874

concentration. miR-874 is regulated by H19 RNA competition, similar to the function of

pseudogenes and RNA transcript mentioned in the regulation of H19. H19 knockdown resulted

in upregulation of miR-874 and subsequent downregulation of AQP3 protein. H19

overexpression resulted in downregulation of miR-874 and subsequent upregulation of AQP3

protein. These results suggest that H19 plays a positive regulatory role regarding AQP3 protein

production for water channel function within the intestinal epithelial barrier.

HuR Background

The HuR described is a RNA binding-protein (RBP) with functions described within the

intestinal epithelium. It is frequently overexpressed in cancers, such as color and ovarian

cancer1,5.
 Michael Qian
I/M 1/11 AP

Within colon cancer, HuR post-transcriptionally regulates genes through its interaction

with 3'UTR AU-rich elements (AREs). HuR has been shown to increase cell division by

enhancing stability of mRNAs that regulate proliferation and growth. In addition, small molecule

MS-444 that inhibits HuR expression minimizes growth of colorectal cancer cells in vitro and in

vivo.

Within ovarian cancer, There is an observed two-six fold increased HuR expression in

ovarian cancer cells compared to ovarian primary cells. The suppression of HuR resulted reduced

tumor cell population by 32%, clearly linking HuRs role in cell proliferation. This suppression

was caused by RNAi-mediated silencing, which significantly decreased cell proliferation and

anchorage-independent growth, and impaired migration and invasion. These studies clearly link

HuRs role in cellular proliferation within certain cancers.

Regulation of Intestinal HuR by Other Factors

The factors involved in regulating HuR expression are mostly undiscovered. However,

miR-29B exhibits a clear regulatory role regarding HuR 6. The miR-29B destabilizes 3 UTR of

HuR mRNA, repressing translation. Deletion of miR-29B binding site on LRP6 and RBP HuR

prevents translation inhibition, and the overexpression of miR-29B has an inverse reaction. This

study suggests other RNAs regulating HuR pre-translation and pre-transcription through a

competitive RNA mechanism.

Intestinal HuR Regulation on Other Factors

HuR plays a net positive regulatory role in regard to intestinal epithelial barrier integrity.

RBP HuR has an essential role in the regeneration of small intestinal mucosa regeneration rates

through Wnt signaling pathway7. This is supported by its calculated relationship with

regenerative abilities. HuR deletion causes mucosal atrophy and lowers the regenerative potential
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I/M 1/11 AP

of crypt progenitors, suggesting its importance in cell proliferation, and maintains mRNA Wnt

stability and translation. HuR directly regulates LRP6, as silencing LRP6 leads to reduced LRP6

protein by 85% and decreasing LRP6 mRNA by 40%. HuR silencing inhibits IEC proliferation

in vitro, once again proving its key role in cellular proliferation.

In addition, HuR competes with CUGBP1 to regulate occludin mRNA 13. Occludin is a TJ

protein. CUGBP1 downregulates occludin mRNA whereas HuR upregulates the occludin. HuR

regulates the production of occludin mRNA in conjunction with CUGBP1 through a competition

mechanism. HuR overexpression inhibited CUGBP1 because of HuRs greater concentration and

promoted TJ occludin translation and barrier integrity. Likewise, CUGBP1 overexpression

inhibited HuR due to CUGBP1s greater concentration and decreased TJ occludin translation.

Suppressed HuR leads to lower binding affinity and more dysfunctional TJ barrier function.

Conclusion

H19 plays a net negative role in regulating the intestinal epithelial barrier, and HuR plays

a net positive role. These effects were recorded by measuring the transcriptional and translational

changes of genes related to tight junction proteins. Although the regulation by the genes H19 and

HuR are well-researched, there is still much to be learned about the regulation of H19 and HuR

within the intestine. Specifically, a double-knockout of H19 and HuR and undiscovered potential

factors regulating these gene products could provide a more advanced understanding of this

topic.
 Michael Qian
I/M 1/11 AP