1. What is siRNA and why are they utilizing it in this study? siRNA are a form of molecular RNA,

Mutations in this gene are responsible for an increased incidence of melanoma in a very small amount of families. The incidence of melanoma of CDK4 is similar to the families that have mutations in the gene CDKN2A. The gene CDK4 is a proto-oncogene that makes a protein that helps control cell division. However, when the CDK4 gene is mutated, it products an abnormal CDK4 protein that is too active. This abnormal protein makes cell divide uncontrollably fast, which could lead to tumor formation. Clinically, CDK4 and CDKN2A are mostly similar.

Hu, Jing, Hui Jing, and Hening Lin. “Sirtuin Inhibitors as Anticancer Agents.” Future medicinal chemistry 6.8 (2014): 945–966. PMC. Web. 9 Apr. 2017.

CRISPR can be used to treat cancer and other conditions. On October 28, the first clinical trial using CRISPR to treat cancer began in China. In this trial, CRISPR was used to disable the gene PD-1, which cancers use to prevent an immune system response (Cyranoski, 2016). A previous gene-editing method, TALEN, has been used to successfully treat leukemia. A one-year-old girl with leukemia had her immune cells replaced with TALEN-edited cells, which allowed her to survive until a matched T cell donor was found for her (Reardon, 2015). CRISPR is a simpler process and more efficient than TALEN and is predicted to lead to a biomedical race to adopt this new technology between the United States and China (Cyranoski, 2016). Other conditions, such as AIDS, can be treated as well. CRISPR can be used to disable a gene called CCR5, which would prevent someone from developing AIDS regardless of if they are HIV positive. Alternatively, the HIV virus itself could be destroyed using CRISPR (Maxmen,

1a. To select sRNA, mRNA, and lncRNA unique to Cn but not present in mammalian hosts.

At present, chemotherapy is the main systemic treatment option for TNBC patients. This study suggests a potential role for CDK7 in modulating the sensitivity of TNBC cells to the chemotherapeutic agent doxorubicin. This may provide additional therapeutic strategies for the fraction of TNBC patients with poor inherent response to doxorubicin, as well as those displaying residual disease following good initial response to treatment. A moderate level of increased sensitivity was also observed following CDK7 knockdown with carboplatin treatment, but not with docetaxel treatment, suggesting that the apparent protective effect mediated by CDK7 may be limited to genotoxic agents. Previous studies demonstrated that CDK7 played a positive role in DNA

Three to four replicates of the same patient mutations will run on consecutive weeks. On day one, one nearly confluent 60 mm dish of HEK cells is plated on a 96- well plate; the total volume per each well is 100ul. On day two, 5ul of 20X antagonist solution is added to each well. Then the patient DNA that will be used for that week’s experiments are prepped into 0.02 g/l DNA mixes. Those respective DNA mixes are then incorporated into another mixture 3ul of Fugene, 5 ul DMEM, and 5ul of the patient DNA mix (0.02 g/l). The cells are then transfected with this new DNA mixture of fuguene, DMEM, and DNA. On day three, the surface expression and total expression of protein in the cells is assessed. To asses for total protein expression, media is removed from half of the cells transfected with a specific DNA mix. Those cells are then washed with a HBSS/HEPES wash buffer, then triturated in water. The plate in then incubated for 30 minutes. After 30 minutes, the media on the surface cells is removed, the surfaces cells are washed with a HBSS/HEPES wash buffer. Finally, 100 mM of nitrocefin solution is added to the surface cells and 200mM of nitrocefin solution is added to the total cells. The 96-well plate is then put into a microplate reader to measure nitrocefin

MicroRNAs are short non-coding RNAs play main role in human diseases; it deregulates the RNA and represses the translation process by controlling gene expression. Some deregulated miRNAs express deferentially such as mir-125b, mir-145, mir-155, and mir-21 leads for partition between cancer and normal tissues. miRNAs expression with particular cancer biopathologic features in breast, such as tumor stage, estrogen and progesterone receptor expression, vascular invasion, or proliferation index. Exiqon microarrays analysis revealed that the most of the miRNAs down regulated in hereditary breast cancer by comparing the normal and cancer tissues, and by using mechanism of tumor progression and also pathway enrichment analysis showed

Part B: The longevity gene, sirtuin 6 (SIRT6) has been studied as a factor of cellular aging. This gene can be the difference between life and death. The example was given about mice; studies conclude that mice who lack this gene age quickly and those who have abundance will live longer than those who do not. SIRT6 is responsible for repairing DNA strands when they are broken. Gorbunova and Seluanov conclude that the source of activation for this gene is protein. In response to oxidative stress, the protein JNK activates the gene and the DNA strand can then be repaired. This is backed up because when JNK is inhibited and unable to activate or signal information the gene is inactive and the strand remains broken. By adding phosphate groups to proteins, amino acid residues that are on the SIRT6 gene are modified which attracts the enzyme Polymerase to the broken area to repair the site. Further studies can inform how to get this occurance to happen in absence of stress, which could save lives.

A large amount of data highlighted the different molecular mechanisms the 90 primarily tumor suppressive function of macroH2A1, which was reviewed 91 elsewhere (1, 18). Briefly, current data support a tumour-suppressive role for 92 macroH2A1.1, whereas the function of macroH2A1.2 is dependent on the specific 93 cancer context (18). MacroH2A1 expression often is downregulated as disease 94 progresses or when the tumor is de-differentiated; its artificial over-expression 95 reduces the metastatic potential of melanoma and hepatocellular carcinoma (HCC) 96 (13, 20, 21), whereas siRNA-mediated depletion of macroH2A1 was shown to 97 increase the aggressiveness of HCC, teratoma and breast cancer cells (15, 20, 22). 98 This could be due to the

The reduction can occur through genetic modification or treatment with a reagent such as short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. Gene knockdown or RNA interference (RNAi) is a natural process which cells use to turn down, or silence the activity of specific genes. It is a biological process where RNA molecules inhibit gene expression, which is caused by the destruction of specific mRNA molecules. RNAi destroys the molecular messengers that carry information, which is coded in genes to the cell’s protein factories. The molecular messengers that are being destroyed are called messenger RNAs (mRNAs) which are destroyed by the RNAi molecules as they bind to the mRNA and deactivate the mRNA, so stop it from producing polypeptide, these mRNAs are used to carry out a critical function, without this a gene is essentially inactive. The double-stranded RNA molecules that trigger RNAi get cut into small fragments by the enzyme called ‘Dicer’. These small fragments serve as guides which lead the cells RNAi machinery to mRNAs that match the genetic sequence of the fragments. After this, the machinery then slices these cellular mRNAs, which effectively destroy their messages and shut off the corresponding gene. RNAi exists in many living things, including single-celled organisms, plants and

Human sirtuins are often characterized as nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases (Michan 2007). Sirtuins remove acetyl groups (O=C-CH3) from the lysine residues of histone proteins. The primary ramification of this reaction is a tighter binding between DNA and histones. When DNA binds tightly to the histones, less nucleotides are

We have therefore further characterized the functional roles of IL-6 in the metastasis of breast cancer and demonstrated that IL-6 is a promising molecular target for the development of molecularly-targeted therapeutic strategies against IL-6. In this study, to characterize the extent to which IL-6 is capable of inducing a metastatic phenotype, we established IL-6 over-expression cell lines MCF-7IL-6 2# and MCF-7IL-6 14# and IL-6 knockdown cell lines MDA-MB-231/IL-6-/+. We employed the breast cancer model in conjunction with gene expression profiling with RNA-seq data as an efficient tool for high-throughput analysis and to screen for differentially expressed genes. Our aim was to identify the gene candidates essential for IL-6-promoted tumor metastasis and invasion by comparing IL-6 over-expression cell lines MCF-7IL-6 2# and MCF-7IL-6 14# with MCF-7, IL-6 knockdown cell lines MDA-MB-231/IL-6-/+ with MDA-MB-231. We found that IL-6 significantly promoted breast cancer metastasis in vivo and in vitro. Furthermore, we also suggested a novel role for the CDKN1A and EPAS1 as a crucial factor in the IL-6-induced migration and invasion of breast

miRNAs can be encoded by their own genes or can sometimes be found in the introns of the genes they regulate thus undergoing the same transcriptional regulation. After transcription they are folded, processed and exported from the nucleus as a precursor dsRNA. In the cytoplasm they bind the Dicer endonuclease that cuts the RNA into short segments of approximately 21 nucleotides, which then bind the Argonaut protein where one strand is selected and remains bound to the protein and the other one is degraded. The guide strand together with the Argonaut and other proteins form the RNA Induced Silencing Complex (RISC). Through base complementarity between the guide strand and the mRNA, the complex can either inhibit translation or catalyze cleavage of the mRNA after which it is degraded. Unlike siRNAs that have perfect complementarity with the mRNA, the miRNAs have a partial complementarity with a

A common form of gene therapy is to take a genetically engineered virus, or a viral vector, like the Herpes simplex virus (HSV) or Adenovirus. These viruses purposes are to infect cancer cells while leaving the healthy cells in the body alone (Cross). Viruses are modified to rid the virus of the toxicity, but maintain the virus’s high gene transfer capability (El-Aneed). The genes are modified by deleting parts or all of the coding regions in the viral genome (Thomas). An RNA guided editing tool called CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats) is capable of editing multiple genes simultaneously (Xiao-Jie). CRISPR-Cas9 is a cost effective tool in gene therapy and while it is relatively new, with just being introduced for mammalian use in 2013, it is already proving to be very useful. This type of gene therapy is where problems arise with a patient’s low immune system and high risk of infections, the biological system can create an infection in the patient.

To realise clinical application of iPSC therapy, several new methods to produce iPSCs were developed using non-integrating approach such as recombinant proteins, Sendai virus, mRNA, and episomes1. The new lines of iPSCs eschew the risk of tumorigenicity