MicroRNAs as Biomarkers in Cancer: (scientist)

Author: Eunice Lozada-Delgado

Recently, due to the advancements in sequencing and transcriptomics techniques, there have been many studies trying to identify gene biomarkers that can serve as therapeutic targets for different diseases like cancer. Now, other specific areas that are being studied with this goal is by using the non-coding region of the DNA. Specifically, recently there has been great interest in studying micro-RNAs as possible biomarkers. Here we will focus on the study of microRNAs as biomarkers in cancers.

Micro-RNAs (miRNAs) are small non-coding RNAs that are known to target and regulate gene expression at the mRNA level by targeting the 3’UTR of the mature mRNA molecule (Li et al., 2012). Further, in vitro and in vivo experiments have suggested that miRNAs can be used to target and regulate the expression of genes that are known drivers of tumorigenesis. MiRNAs have been associated with different biological roles like apoptosis, cell cycle, cell division, among others that are related to cancer progression (Pink et al., 2015). Thus, ideally one can develop different personalized therapeutic strategies towards patients with specific deregulated miRNAs.

miRNA biogenesis  

http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/mirna/learning-center/mirna-introduction.html

As an example, a recent publication by Rivera-Diaz, et al. made a miRNA profile comparing differentially expressed miRNAs between WHO grades II-IV of astrocytoma patients (Rivera-Díaz et al., 2015). Here they used formalin-fixed patient samples in paraffin and compared the miRNA expression patterns between the different WHO grade patient tumor samples with both non-neoplastic surrounding tissue and control patients. They extracted the miRNA containing RNA from the different samples and conducted a microarray assay to obtain their results. They were able to obtain differentially expressed miRNA profiles distinguishing the various WHO grades. Afterwards, they validated one of the miRNAs found to be differentially expressed in astrocytoma grade IV, better known as Glioblastoma multiforme (GBM), miR-27a. When inhibiting this miRNA in CRL-1690 GBM cells an increase in apoptosis was obtained. Additionally, six potential targets for this miRNA were identified. The results obtained are of importance because GBM has a very high death rate in the United States. Moreover, the profiles obtained can now be used as biomarkers for the different stages or grades of astrocytoma tumors.

Left frontal GBM

http://www.uiowa.edu/~c064s01/nr094.htm

Another recent publication in the area is by Pink, et al. focusing on miRNA patterns of ovarian cancer cell lines resistant to cisplatin therapy (Pink et al., 2015). Here they made microarray analysis in cisplatin sensitive A2780 ovarian cancer cell line compared to the cisplatin resistant CP70 ovarian cancer cell line. Using this approach they were able to identify various miRNAs that were upregulated in the cisplatin resistant cells. Then they went and tested the role of some of these miRNAs with mimics, and of their targets with siRNAs. They identified miR-21-3p as having a role in cisplatin resistance, while it’s “sister strand” or star strand, miR-21-5p, had a contrary role by defining cisplatin sensitivity.  Afterwards, they identified a new target mRNA for the miR-21-3p, the NAV3 gene mRNA. Then to prove if this target had to do with the fact of miR-21-3p increasing cisplatin resistance they knocked down NAV3, and what resulted was an increase in cisplatin resistance. Thus suggesting that miR-21-3p increases cisplatin drug resistance through targeting of the NAV3 mRNA in ovarian cancer.  This study identifies miR-21-3p as a possible biomarker for ovarian cancer with cisplatin therapy resistance.

In summary, here we were able to demonstrate mainly through two recently published articles the interest in research to study microRNAs as biomarkers in different cancers. Here, two studies in different cancers were discussed, Glioblastoma multiforme and ovarian cancer, but the same type of study is being made in all types of cancer. This provides evidence of the emerging need to develop new types of therapies that can have personalized potential towards specific people within their type of cancer targeting their specific mutations.

Video on Profiling microRNA by pathway and disease: https://www.youtube.com/watch?=GsZpw5QEyKI

 

LI, Z.  et al. microRNA expression profiles in human colorectal cancers with brain metastases. Oncol Lett, v. 3, n. 2, p. 346-350, Feb 2012. ISSN 1792-1074. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/22740910 >.

PINK, R. C.  et al. The passenger strand, miR-21-3p, plays a role in mediating cisplatin resistance in ovarian cancer cells. Gynecol Oncol, Jan 2015. ISSN 1095-6859. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/25579119 >.

RIVERA-DÍAZ, M.  et al. MicroRNA-27a distinguishes glioblastoma multiforme from diffuse and anaplastic astrocytomas and has prognostic value. Am J Cancer Res, v. 5, n. 1, p. 201-18,  2015. ISSN 2156-6976. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/25628931 >.

itstimetoshout.com  

Isolating and Identifying New Antibiotic Producing Bacteria (Non-Scientist)

Since the 1940’s a boom of antibiotics like penicillin and streptomycin were discovered, the discovery of antibiotics have revolutionized medicine.  Today because of the excessive abuse of antimicrobial drugs more resistant strains of pathogenic bacteria are emerging and threatening modern society².  It is difficult to discover or develop a new antibiotics or antimicrobial agent, most of the antibiotics were discovered by screening cultivable soil samples³. 

                About 99% of microbes have remained uncultured.  Lately uncultivable microorganisms have been reported to produce interesting compounds, the purpose of the research was to develop a method to grow uncultivable microorganisms by cultivating it in its natural environment using a multichannel device called an iChip³.  The iChip dilutes a soil sample which isolates one bacterial cell per chamber, the chamber allows the diffusion of nutrients and growth factor³.  This method presents a 50% chance of growing uncultivable bacteria (compared to a petri dish 1%)¹.  Screening for antimicrobial activity were performed by extracting isolated cells form the iChip and inoculating them on plates with Staphylococcus aureus. 

Ling and his research team detected a new antibiotic producing bacteria by using iChip methodology.  To determine the species of the bacteria the cells were sequenced by Illumina (around 800bp) and amplified on 16SrDNA using GoTaq Green Master Mix and universal primers E8F and U1510R, the sequence was submitted to RAST server to produce closest relatives.  RAST predicted relativity to: Alicycliphilus denitrificans, Leptothrix cholodnii, Methylibium petroleiphilum, and Rubrivivax gelatinosus¹.  Then DNA-DNA hybridization was performed on these genomes, the analysis revealed that the bacteria belonged to a new genus related to Aquabacteria, named Eleftheria terrae¹.  The compound produced by the new genus was purified and analyzed by NMR and mass spectrometry, the results were not reported in an available data base.  As a result they discovered a new antibiotic producing cell, the compound, named teixobactin, inhibited grampositive bacterias.

                Teixobactin is the first member of a new class of lipid II binding antibiotics, judging by the properties of teixobactin it has evolved to minimize resistance in specific microorganisms.  With the growth of resistant pathogenic bacteria, employment of new technology and genomic techniques can help isolate and identify new antibiotic producing bacterias.  Most likely more antibiotic compounds like teixobactin are found in nature and are waiting to be discovered.

References:

1.       Losee L. Ling and et al.  2015.  A new antibiotic kills pathogens without detectable resistance.  Nature.  517.

2.       POPSCI.  ICHIP: THE FUTURE OF ANTIBIOTIC DISCOVERY.  2015.  Retrived from: http://www.popsci.com/ichip-new-way-find-antibiotics-and-other-key-drugs

 

3.       WHO.  Antimicrobial Resistance. 2014.  Retrived from: http://www.who.int/mediacentre/factsheets/fs194/en/

Use of Genetic Testing for Diseases: (non-scientist)

Author: Eunice Lozada-Delgado

Recently, there has been great advances in DNA sequencing techniques where you can determine the specific sequence or parts of it of your own DNA. What DNA sequencing does is identify the specific sequence of the four different letters that make up your identity in your cells. Using this approach you can identify possible mutations or changes in specific genes that can make you prone to a disease. These gene mutations are also called biomarkers, and these have been found by different researchers. This has made emerge companies that provide this service where they do the testing for the specific mutations known in the disease that you could have inherited and are interested in testing for. Most of these tests are focused on different types of cancer, although there are tests for other inherited diseases like heart disease and Rheumatoid Arthritis^1,2. The company that began with these cancer genetic tests is Myriad genetics². They provide kits that you can order and provide your DNA either through a blood sample or an oral rinse. Then they send you your results as positive or negative for each mutation on the genes and, with the help of your physician, determine if there should be any precautions made. Also, lately the prices for these tests have dropped because more companies are providing the service. You can find these tests ranging $300- $5,000 depending on the test. Additionally, it should be noted that not all cancers emerge from inherited gene mutations, in fact most don’t. For example, only about 1 in 20 (5%) of women diagnosed with breast cancer every year carry an inherited gene mutation like BRCA1³. But, if you are part of a family with a long history of a disease, like a type of cancer, then it should most likely be because of an inherited gene mutation related to the disease.

A recent example that has been made public of a celebrity that tested for the BRCA1 and 2 genes, mutations known to provide high susceptibility to woman to acquire breast and/or ovarian cancer, was Angelina Jolie. She tested for these two genes because her own mother died of ovarian cancer and she wanted to know if she was prone too to be able to take the precautions needed to live for her children. She actually turned out to have a mutation in one of these genes, the BRCA1, which in her case gave her an 87% probability of developing breast cancer and 50% of developing ovarian cancer. This is why she took drastic measures to lower her probability of acquiring breast cancer by having a preventive double mastectomy. She has even made it public herself to serve as an example for women that don’t know that these options are available, even if they still are too expensive for most people. She even wrote an article for The New York Times telling her story for the benefit of others⁴.

In summary, we now know the options of genetic testing for the detection of gene mutations related to specific diseases like cancers. The Angelina Jolie case was used as an example even though this doesn’t mean everyone is the same. Not all should end up doing the surgery as she did, since everybody is different and there are other options like intensive surveillance using mammograms or MRI scans. The purpose of this entry was to provide a brief and general sense of what is currently happening in this emerging area of genetic testing. I hope it has been of good information to the readers. You can comment below with any questions. 

References:

11.      MedlinePlus. Genetic Testing. http://www.nlm.nih.gov/medlineplus/genetictesting.html

22.     Myriad Genetics. https://www.myriad.com

33.   Scowcroft, Henry. Angelina Jolie, inherited breast cancer and the BRCA1 gene. (2013). Cancer Research UK Science Blog. <http://scienceblog.cancerresearchuk.org/2013/05/14/angelina-jolie-inherited-breast-cancer-and-the-brca1-gene/>

44. Jolie, Angelina. My Medical Choice. (2013). The New York Times. <http://www.nytimes.com/2013/05/14/opinion/my-medical-choice.html?_r=2&> 

Plasmodium falciparum Resistance to Sulfadoxine in Cambodia (Scientist)

Plasmodium falciparum is the agent that causes malaria in humans, the pathogen is well distributed in developing countries and has a high mortality rate.  In Africa alone there are hundreds of strains of falciparum malaria making it impossible to sequence the genome.  Malaria is a medical, social and economic burden in endemic population, over the years more drug resistant malaria have been reported¹. 

Chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) are the least expensive and widely available antimalarial drugs.  The synergetic effects between SP and antifolate is used to treat CQ resistant or uncomplicated falciparum malaria.  Sulfadoxine works by inhibiting the enzyme dihydropteroate synthase (dhps) of the folate biosynthesis pathway².  Mutations in Plasmodium falciparum crt, dhfr and dhps genes have lead to resistance in CQ and SP antimalarial drugs². 

In Sumiti Vinayak investigation, they study the origins of dhps alleles resistant in Cambodia.  Blood was collected from five sites in Cambodia, DNA was extracted using QIAmp Mini kit, and a total of 234 P. falciparum were isolated and amplified.  The dhps genotypes were analyzed for eight neutral microsatellite loci on chromosome 2, ten microsatellite loci of the dhps gene were found.  Microsatellites were used to determine genetic differentiation and origin of the dhps alleles.  In Cambodia, ancestral wild type dhps alleles SAKAA were present in 11% of the isolated DNA, wild type dhps is resistant to sulfadoxine.  Mutants of dhps were as followed: single mutant were at 10%, double mutant at 7.5%.  and triple mutants at 51%.  It is believed that from the ancestral wild type the single mutant evolved followed by the double and then the triple mutant. 

Sulfadoxine resistant falciparum malaria has evolved because of sulfadoxine-pyrimethamine pressure.  Countries that use sulfadoxine-pyrimethamine as antimalarial drug will experience an increase in malaria resistance to SP.  It is also likely that cross resistance to related drugs like cotrimoxazole (trimethoprim plus sulfamethoxazole), may have a role in selection of this allele³.  With more strains of Plasmodium falciparum resistance antimalarial drug, the task to control and treat malaria is becoming impossible, there for new methods or treatment must be created to treat or cure malaria.

            Genetic techniques are a useful tool to detect mutations in pathogenic organisms that can lead to resistance in drug treatment.  Although no new treatment was proposed for Sulfadoxine resistant falciparum malaria, genetic sequencing can provide information on developing specific drug treatments for these resistant strains.

Healthy Bacteria Promotes a Healthy Colon (Non-Scientist)

For maximum colon health butyrate producing bacteria is needed to maintain colon balance.  Previous studies already demonstrated that gut microbiota are essential for maintaining health, still the microbiota is not the same throw out the digestive system.  Phylogenetic identification and butyrate synthesis pathway analysis on the colon microbiota can detect butyrate producing bacteria which has a beneficial or synergetic relationship with colonocytes (colon cells).  If butyrate producing bacteria in the colon were at an imbalance by inhibiting butyrate synthesis pathway then ulcerative and type II diabetes would arise³.  Butyrate producing bacteria are key in maintaining homeostatis and epithelial integrity. 

                In Marius Vital and teams study, bacteria with genes coding for butyrate synthesis pathway within the Integrated Microbial Genome (IMG) database and the sequences were analyzed with HMM model¹.  Then the genes were used to construct phylogenetic trees to compare butyrate producing bacteria genes using program MEGA¹.  The majority of the potential butyrate producing bacteria, butyrate could be produced by acetyl-CoA and lysine pathway¹.  The main producers of butyrate producing bacteria are the phylums Firmicutes, Fusobacteria, Spirochaetaceae, and Bacteroidetes¹.  Phylogenetic analysis demonstrated neighboring and joining trees indicting coevolution and that not all members of the same family exhibited the same pathway¹.  In the study specific pathway genes were detected, it does not imply its functionality in the butyrate synthesis pathway, for that more testing would be needed at a biochemical level. 

In the end, genetic sequencing and analysis was employed to study gene expresions of butyrate producing pathways, the main pathway that produce butyrate are acetyl-CoA, so to maintain the acetyl-CoA producing bacteria a steady diet of plant-derived polysaccharide such as starch and xylan is needed in order to maintain a healthy colon.  So to avoid ulcerative colitis, type II diabetes, and colon inflammation it’s highly recommended to eat your vegetables.

References:

1.       Marius Vital, Adina Chuang Howe and James M. Tiedjea.  2014.  Revealing the Bacterial Butyrate Synthesis Pathways by Analyzing (Meta)genomic Data.  America Association of Microbiology.  5(2).

2.       Marius Vital and et al.  2013.  A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community.  Microbiome Journal.   

3.       Ulcerative Colitis.  2015.  Retrieved from: http://www.medicinenet.com/ulcerative_colitis/page2.htm

Characterizing HIV Mutations While Lowering NGS Bias (Scientific)

It’s estimated that 38 million human beings are infected with HIV worldwide, using next generation sequencing (NGS) technology to understand the virus mutation and resistance with determination to design successful therapies to treat HIV¹.  Today sequencing is more cost efficient and faster than it was almost 10-15 years ago, now companies have developed diverse next generation sequencers that vary on the read length and error rates.  Still sequencing the HIV virus presents its challenges; library containing large quantity of reference DNA, when large reference sequences aren’t available PCR is utilized which affect analysis of diversity in the sample³.  To sequence large quantities of HIV virus isolated from clinical samples using Illumina NGS, then the amplified nucleic acid is aligned to a poorly defined reference genome in order to characterize entire viral populations².  One of the difficulty of working with HIV it’s the high rate of viral production and error prone mechanism of viral reverse transcriptase that leads to a heterogeneous viral population making it somewhat impossible to sequence globally the HIV virus. 

In Stephanie M. Willerth and teams study, to develop Illumina libraries without relying on PCR and primers a large quantity of viral dsDNA collected from clinical in vitro CD4+T cells was isolated into RNA using QIAamp viral RNA Mini Kit³.  Then the RNA is converted into an extensive ssDNA, and the ssDNA was converted to double strand using NuGEN WT-Ovation³.  The cDNA was processed into an Illumina library using the Illumina Genomic DNA Sample Prep kit³.  To validate the procedure the prepared Illumina library was compared to a homogenous control, HIV stain NLA-3, using MAQ program³.  After computational analysis for insertion and deletions by MAQ the sequence was scanned against Stanford University HIV Drug Resistance Database which identified drug resistant mutations in the clinical samples³. 

The study successfully proves that the HIV genome can be sequenced efficiently and rapidly by eliminating primers there, for lowering the bias of characterizing HIV population while using next generation sequencing.  This method could be applied to study larger HIV populations for mutations and viral resistance to drugs, also the method can assist in providing genomes of organisms that are hard to cultivate.

References:

1.       CDC. HIV.  Retrieved from: http://www.cdc.gov/hiv/library/factsheets/index.html

2.       Daniel Aird, Michael G Ross, Wei-Sheng Chen, Maxwell Danielsson, Timothy Fennell, Carsten Russ, David B Jaffe, Chad Nusbaum and Andreas Gnirke.  2011.  Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.  Genome Biology. 

3.       Stephanie M. Willerth, He ´lder A. M. Pedro, Lior Pachter, Laurent M. Humeau, Adam P. Arkin and David V. Schaffe.  2010.  Development of a Low Bias Method for Characterizing Viral Populations Using Next Generation Sequencing Technology.  PLoSONE.  5(10)