Genetic Variations In Microrna Genes Biology Essay

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Oral cancer or oral cavity cancer, a subtype of head and neck cancer, is any cancerous tissue growth located in the oral cavity. It may arise as a primary lesion originating in any of the oral tissues, by metastasis from a distant site of origin, or by extension from a neighboring anatomic structure, such as the nasal cavity or the maxillary sinus. Oral cancers may originate in any of the tissues of the mouth, and may be of varied histologic types: teratoma, adenocarcinoma derived from a major or minor salivary gland, lymphoma from tonsillar or other lymphoid tissue, or melanoma from the pigment producing cells of the oral mucosa. There are several types of oral cancers, but around 90% are squamous cell carcinomasHYPERLINK "#cite_note-1#cite_note-1"[2], originating in the tissues that line the mouth and lips. Oral or mouth cancer most commonly involves the tongue. It may also occur on the floor of the mouth, cheek lining, gingiva (gums), lips, or palate (roof of the mouth). Most oral cancers look very similar under the microscope and are called squamous cell carcinoma. These are malignant and tend to spread rapidly.

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Oral cancer is the sixth most common neoplasm in the World (Parkin DM et al 2000) In India, oral cancer is the leading cancer among men and within the first five cancers of women, with an incidence rate of 12% of all cancers in men and 8% of all cancers among women (Boyle et al 1989 Sankaranarayanan, et al 1990).

On the basis of a cancer registry, it is estimated that annually 75 000-80 000 new cases are reported in India (National Cancer Registry 2001). Oral Squamous cell carcinoma (OSCC) is a major health problem across the world. It is among the most common cancers seen in both men and women as can be gauged from the records of the National cancer Registry Programme (http://www.icmrnic.in/ncrp/report-pop 2001-04)

Risk factors for oral cancer

The risk factors include tobacco associated intra-oral carcinogens, which may play a synergistic role in oral tumorigenesis. From relative risk factors of alcohol and tobacco, it has been estimated, that 75% of all oral cancers are preventable. In the remaining 25% of patients who are not exposed to these substances, the cause/s of their tumors remains unknown. [Walker et al 2003] The disproportionately higher incidence of carcinoma of the head-neck in relation to other malignancies in India, may be due to use of tobacco in various forms, consumption of alcohol, low socioeconomic condition related to poor hygiene, poor diet and rampant viral infections.[ Franceschi et al 2000]

Molecular Pathogenesis of Oral Squamous Cell Carcinoma

The development of oral squamous cell carcinoma (OSCC) is a multistep process requiring the accumulation of multiple genetic alterations, influenced by a patient's genetic predisposition as well as by environmental influences, including tobacco, alcohol, chronic inflammation, and viral infection.

Tumorigenic genetic alterations consist of two major types: tumor suppressor genes, which promote tumor development when inactivated; and oncogenes, which promote tumor development when activated. Tumor suppressor genes can be inactivated through genetic events such as mutation, loss of heterozygosity, or deletion, or by epigenetic modifications such as DNA methylation or chromatin remodeling. Oncogenes can be activated through overexpression due to gene amplification, increased transcription, or changes in structure due to mutations that lead to increased transforming activity. This review focuses on the molecular mechanisms of oral carcinogenesis and the use of biologic therapy to specifically target molecules altered in OSCC. The rapid progress that has been made in our understanding of the molecular alterations contributing to the development of OSCC is leading to improvements in the early diagnosis of tumors and the refinement of biologic treatments individualized to the specific characteristics of a patient's tumor.

MicroRNAs (miRNA)

Several miRNAs have been found to have links with some types of cancer.[91]HYPERLINK "#cite_note-91#cite_note-91"[92]

A study of mice altered to produce excess c-Myc - a protein with mutated forms implicated in several cancers - shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days.[93]

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Leukemia can be caused by the insertion of a virus next to the 17-92 array of microRNAs leading to increased expression of this microRNA.[94]

Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off.[95]

By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type. miRNA profiling has already been able to determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer.[96]

Transgenic mice that over-express or lack specific miRNAs have provided insight into the role of small RNAs in various malignancies.[97]

MicroRNAs (miRNA) are a class of small noncoding RNA molecules 20 nucleotides (nt) in length. MiRNAs regulate gene expression in animals and plants through binding to the 3 untranslated region (UTR) of the mRNAs of their target genes and leading to mRNA cleavage or translation repression (3). It is estimated that f30% of human genes are regulated by miRNAs.

Aberrant expression of miRNAs contributes to the etiology of many common human diseases including cancer (3). Numerous recent studies have shown that alteration of miRNAs plays a critical role in cancer development (3, 4) by regulating the expressions of proto-oncogenes or tumor suppressor genes (3-5).

MiRNA genes are first transcribed by RNA polymerase into primary miRNAs (pri-miRNAs) with several hundred nucleotides. Processing of primary miRNAs (pri-miRNA) by the nuclear RNase DROSHA within the microprocessor complex also including DGCR8 produces the 70- to 100-nt pre-miRNAs. The pre-miRNAs are then exported into the cytoplasm by the Exportin-5/Ran-GTP complex (6) and cleaved by DICER as part of the RNA-induced silencing complex's loading complex including TARBP2 and AGO2 (7). This complex also includes GEMIN3 and GEMIN4 and contributes to both miRNA processing and target gene silencing (8, 9).

The importance of miRNA as apotential cancer indicator is underscored by their involvement in regulation basic cellular process such as cell proliferation , differentiation , apoptosis. miRNA are small non coding RNAs that mediate gene expression at post transcriptional level by degrading or repressing target miRNA

Cancer & MicroRNAs (miRNA)

Cancer is a genetic and epigenetic disease that requires inactivation of tumour-suppressor genes and activation of proto-oncogenes. Mutated DNA sequences are transcribed to mRNA, which is finally translated into functionally aberrant proteins. However, RNA is not a 'passive intermediate product' between DNA and proteins.16 The expression of genes is also dependent on RNA-based mechanisms, including nonsense-mediated decay, alternative splicing, RNA editing and miRNA. Functional regulation of these RNAbased mechanisms could constitute one or more of the steps involved in cancer development.

Several miRNAs have been found to have links with some types of cancer.[91]HYPERLINK "#cite_note-91#cite_note-91"[92]

A study of mice altered to produce excess c-Myc - a protein with mutated forms implicated in several cancers - shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days.[93] Leukemia can be caused by the insertion of a virus next to the 17-92 array of microRNAs leading to increased expression of this microRNA.[94]

Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off.[95]

By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type. miRNA profiling has already been able to determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer.[96]

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Transgenic mice that over-express or lack specific miRNAs have provided insight into the role of small RNAs in various malignancies.[97]

Genetic polymorphism:

A difference in DNA sequence among individuals, groups, or populations. Sources include SNPs, sequence repeats, insertions, deletions and recombination. (E.g. a genetic polymorphism might give rise to blue eyes versus brown eyes, or straight hair versus curly hair). Genetic polymorphisms may be the result of chance processes, or may have been induced by external agents (such as viruses or radiation). If a difference in DNA sequence among individuals has been shown to be associated with disease, it will usually be called a genetic mutation. Changes in DNA sequence which have been confirmed to be caused by external agents are also generally called "mutations" rather than "polymorphisms."[Source: PHRMA Genomics Lexicon]

Single Nucleotide Polymorphism

A Single Nucleotide Polymorphism is a source variance in a genome. A SNP ("snip") is a single base mutation in DNA. SNPs are the most simple form and most common source of genetic polymorphism in the human genome (90% of all human DNA polymorphisms) (Kaleigh Smith et al., 2002).

Epigenetics

Chromatin alterations have been associated with all stages of tumour formation and progression. The best characterized are epigenetically mediated transcriptional-silencing events that are associated with increases in DNA methylation - particularly at promoter regions of genes that regulate important cell functions. Recent evidence indicates that epigenetic changes might 'addict' cancer cells to altered signal-transduction pathways during the early stages of tumour development (Stephen et al 2006). Although miRNA has effects on epigenetic machinery, its control is also affected by epigenetic mechanisms. DNA methylation enzymes (DNA methyltransferases) are predicted to be potential targets of miRNA. on the other hand, DNA methylation inhibitors up-regulate the expression of some miRNAs. Therefore, epigenetic drugs may exert their anti-tumour effects by activating tumoursuppressor genes silenced epigenetically, and also by turning on tumour-suppressor miRNAs that down-regulate oncogenic mRNA.

Gene regulation by microRNAs:

The role of small RNAs as key regulators of mRNA turnover and translation has been well established. Recent advances indicate that the small RNAs termed microRNAs play important roles in animal development and physiology. Cellular activities such as proliferation, morphogenesis, apoptosis and differentiation are regulated by microRNAs. The expression of various genes are regulated by microRNAs, and several microRNAs act in reciprocal negative feedback loops with protein factors to control cell fate decisions that are triggered by signal transduction activity. These observations implicate small RNAs as important mediators of gene regulation in response to cell-cell signaling. The mechanism by which microRNAs silence gene expression is post-transcriptional, possibly influencing the stability, compartmentalization and translation of mRNAs Carthew RW et al 2006)

NEED FOR STUDY/RESEARCH

Close to 36,000 Americans will be diagnosed with oral or pharyngeal cancer this year. It will cause over 8,000 deaths, killing roughly 1 person per hour, 24 hours per day. Of those 36,000 newly diagnosed individuals, only slightly more than half will be alive in 5 years. This is a number which has not significantly improved in decades. The death rate for oral cancer is higher than that of cancers which we hear about routinely such as cervical cancer, Hodgkin's lymphoma, laryngeal cancer, cancer of the testes, and endocrine system cancers such as thyroid, or skin cancer (malignant melanoma). If you expand the definition of oral cancers to include cancer of the larynx, for which the risk factors are the same, the numbers of diagnosed cases grow to approximately 50,000 individuals, and 13,500 deaths per year in the US alone. Worldwide the problem is much greater, with over 640,000 new cases being found each year.

The death rate associated with this cancer is particularly high not because it is hard to discover or diagnose, but due to the cancer being routinely discovered late in its development. Often it is only discovered when the cancer has metastasized to another location, most likely the lymph nodes of the neck. Prognosis at this stage of discovery is significantly worse than when it is caught in a localized intra oral area.

Besides the metastasis, at these later stages, the primary tumor has had time to invade deep into local structures. Oral cancer is particularly dangerous because in its early stages it may not be noticed by the patient, as it can frequently prosper without producing pain or symptoms they might readily recognize, and because it has a high risk of producing second, primary tumors. This means that patients who survive a first encounter with the disease, have up to a 20 times higher risk of developing a second cancer. This heightened risk factor can last for 5 to 10 years after the first occurrence. There are several types of oral cancers, but around 90% are squamous cell carcinomas. It is estimated that approximately $3.2 billion is spent in the United States each year on treatment of ORAL cancers.

 

Understanding the causative factors of cancer will contribute to prevention of the disease. The emergence of miRNA knowledge and its potential interactive action with such alterations therefore creates a new understanding of cell transformation. These alterations could represent new targets for cancer diagnosis and treatment in future.

AIMS AND OBJECTIVES OF STUDY

To asses the role of biological/ biochemical parameters related to oral squamous cell carcinoma (OSCC)

Identification and classification of miRNA genes related to OSCC

To study the association between miRNA polymorphism and risk in oral squamous cell carcinoma

To examine the significance of following miRNA

Mir 137

Mir 21

Mir 200a

Mir 205

And related miRNA genes in case control studies

To measure the relationship between DICER & miRNA in oral squamous cell carcinoma.

OutCome of the Study

Over Expression of miRNa may reduce protein products of tumour suppressor genes. On other hand use of tumour suppressor miRNA expression may cause elevated levels of oncogenic proteins. One or both of these alterations could represent new targets for cancer diagnosis and treatment in future.

The emergence of miRNA knowledge and its potential interactive action with such alterations therefore creates a new understanding of cell transformation.

Research methodology for the proposed work

Materials & Methods

Sample collection and Analysis:

Oral cancers diagnosed based on clinical examination as well as pathological examination by oncologists and pathologists will be studied.

Sample collection

Saliva & mouthwash from patients and control: water rinsed thoroughly will be collected in sterile test tubes and centrifused. The cells will be collected

DNA Isolation:

DNA will be isolated from all the tissue by a rapid enzymatic method by salting out the cellular proteins by dehydration and precipitation with saturated sodium chloride solution

Polymerase Chain Reaction:

PCR will be performed on the isolated DNA samples using primers specific for the genes chosen for analysis. DNA will then be amplified with specific primers using DNA as the template.

Restriction Fragment Length Polymorphism

Restriction endonucleases cut DNA molecule at a limited number of specific nucleotide sequences generating fragments of different length. These fragments separate on electrophoresis into distinct bands depending on the size of the fragment, which can be determine using appropriate marker.

Single Strand conformation polymorphism

In denatured condition, each single strand of a double strand DNA molecule can undergo intrastrand hybridization by formation of hydrogen bonds with in the complementary bases. This leads to the formation of specific configuration other than the linear one. Change of even one nucleotide can effect the configuration of the strand. This change of configuration detecting change or polymorphs of nucleotides can be detected by the change in the movement of the denatured strands along an appropriate concentration of polyacrylamide gel in an electric field. This process is called as Single Strand Confirmatory Polymorphism type of electrophoresis.

RNA extraction

Total RNA will be extracted from the liver tumors using TRIZOL, after pulverizing the tumours in a chilled stainless steel mortar. Total RNA will be briefly exposed to RNAase-free DNAase I.

Real Time PCR

The expression of the miRNA precursors will be determined using real-time quantitative PCR.

Procedure for real time PCR: TaqMan real time PCR is one of the two types of quantitative PCR methods . unlike the other real time PCR ,the CYBR Green method, which uses florescent dye that can bind to any double stranded DNA, TaqMan uses a flourogenic probe which is a single stranded oligonucleotide of 20- 26 nucleotidesand is designed to bind only the DNA sequence between the two PCR primers. Therefore only specific PCR product can generate florescent signal in TaqMan PCR. To do TaqMan PCR , besides reagents required forregular PCR, additional things required area realtime PCR machine,two PCR primers with a preffered product size of 50-150 bp, a probe with a fluorescent reporter or fluorophore such as 6- carboxyfluorescein(FAM) and Tetrachlorofluorescin(TET) and quencher such as tetramethylrhodamine(TAMRA) covalently attached to its 5' and 3' ends respectively.

MicroRNA Northern Blotting

Northern analysis is a widely used method for miRNA analyses because it is generally a readily available technology

Period of Study 3 years

Scope of the Study

The goal of this initiative is to encourage applications utilizing gene silencing by miRNA that are focused on the treatment and prevention of oral and craniofacial diseases and disorders.  Therapeutic applications of miRNA are very broad and range from acquired diseases such as viral infections, to genetic disorders, particularly where there is a gain-of-function mutation.  Most research areas would benefit from the application of miRNA strategies, including bone disorders, oral cancer, chronic inflammatory conditions, viral infections, autoimmune disorders, and craniofacial birth defects.  The accessibility of the oral cavity to miRNA delivery makes it an attractive target for gene silencing strategies.   Examples of research areas that would be appropriate for this RFA include, but are not limited to, the following:

Silencing inflammatory mediators, matrix metalloproteinases, and osteoclastic factors that cause bone erosion in periodontal diseases

Silencing immunological and pro-apoptotic factors leading to the development of Sjogren's syndrome

Identification and validation of drug targets for head and neck cancers

Dissection of key molecules and pathways involved in head and neck carcinogenesis

Validation of target genes involved in pain signaling

Treatment of neuropathic pain and other pain conditions that are refractory to current therapeutics

Manipulation of molecular pathways to enhance tooth repair or regeneration

The proposed initiative is intended to encourage and facilitate translational studies that take advantage of the efficiency and cost- savings of gene silencing.  Gene silencing by miRNA takes advantage of an endogenous defense mechanism for protecting cells from invading viruses and transposable genetic elements.    Since the description of the phenomenon mi RNAi has emerged as a powerful strategy for silencing genes and has become a widely used tool due to its great simplicity and high efficiency.