INVESTIGATING HUMAN LONG INTERSPERSED ELEMENT 1 (LINE 1) ACTIVITY IN HEALTH AND DISEASES

Abstract

LINE 1 (L1), is the only active autonomous retrotransposon that propagate via RNA intermediate, comprising 20% of the human genome. The ongoing activity of L1 turned to be the major instrumental force that shapes the genomic architecture but whether the effect is detrimental or beneficial is still an enigma. Although most of the L1s are “molecular fossils” (due to truncation in 5’UTR), around 80-100 human specific L1 (L1Hs) are competent to retrotranspose. Normal somatic cells have evolved their own defensive mechanism to prevent L1 mobility. It is being postulated that L1 retro-transposition event have to occur in the germ-line or during early development in-order to ensure their evolutionary success; but with what extent this process impact somatic cells is yet to reveal. Strikingly, L1 shows high activity in brain encouraging the intriguing hypothesis of L1 mediated retrotransposition might generate neuronal genomic diversity. In recent years, expression of L1 encoded proteins is indicated as a hallmark feature for most of the cancers. Malignancy and ageing are commonly associated with accumulation of deleterious mutation leading to altered function within the cell. Here in, I tried to decipher somatic L1 retrotransposon activity in normal human brain and in oral squamous cell carcinoma (OSCC) samples. An active full-length L1 is ~6 kb in length encoding two proteins designated as ORF1p (40 kDa) and ORF2p (150 kDa) that are prime essential to facilitate its transposition. My first aim was to investigate the activity of L1 in different anatomical regions of normal human brain by looking the expression of L1ORF1p with an in-house human L1ORF1p (hL1ORF1p) antibody. I have characterized the hL1ORF1p antibody and my data showed that our in-house L1ORF1p antibody is very sensitive and specific towards detecting L1ORF1p and will be very useful to study retrotransposon biology in human brain and other tissues. Next, I employed immune histological staining (IHC) to check the expression of L1-ORF1p, in different anatomical regions of normal human brain. My data elucidated significant amount of L1ORF1p expression in different anatomical regions of postmortem normal human brain; old age brain showed more expression compared to the young age group. Further investigation was conducted to detect full length L1 RNA and L1ORF2p expression in the postmortem brain samples. Cumulatively my data showed that higher L1ORF1p Abstract ii expression in different anatomical regions of aged brain compared to younger one. Overall, my data indicates that L1 might have some role with aging and age related neurodegenerative diseases. In Indian population OSCC is prevalent and having panacea is challenging due to its high mortality rate and late diagnosis. Studies regarding L1 in OSCC are scanty; hence I have investigated the expression of L1 proteins (ORF1p and ORF2p) in small cohort of operated OSCC samples obtained from Surgical Oncology Department, AIIMS, Rishikesh, India. My data showed around 50% samples expressed L1 proteins (L1ORF1p and L1ORF2p). Further investigation was conducted to find out the methylation status of L1 promoter in paired normal cancer tissues. Overall my study revealed that L1 retrotransposon is active in this particular cancer in stage independent manner and thus L1 proteins (L1ORF1p and L1ORF2p) can be used as potential biomarker. The outline of Ph.D. thesis is divided into four chapters. A brief description of each chapter is mentioned below: Chapter 1: Introduction and literature review: In this chapter, rationale behind the study will be described. The three objectives will be elaborated and the link among objectives will be presented in details. A comprehensive and up-to-date literature review related to each objective will be narrated. The chapter will also include to the point description regarding retrotransposable elements (with special preference to LINE1 retrotransposon) and their propagation mechanism. Detailed description of the following will also be included in this chapter: Activity of LINE1 in neuronal genome and its capability to shape the neuronal genome architecture, regions of brain supporting L1 activity and its possible role in age related neurological disorder, upregulation of L1 activity in different cancer that induce genomic instability, status of oral squamous cell carcinoma in India, and activation of L1 in oral squamous cell carcinoma. Chapter 2: Materials and Methods: This section consists the detailed description about the recipes for reagent, solution and experimental protocols performed to answer all the objectives. The protocol which will be described in details are animal cell culture and transfection, over expression of proteins in bacterial system, immunization protocol for antibody generation, tissue processing, SDS- Abstract iii PAGE gel electrophoresis, Western blotting, Northern blotting, retrotransposoition assay, immunofluroscence, immunoprecipitation, immunohistochemistry and semi qRT-PCR. Chapter 3: Results: The chapter includes objective wise major finding of this study: The main objectives are 1. Characterization of in house generated human L1ORF1p antibody to detect L1 encoded ORF1p in different cancer cell lines 2. Detection of L1ORF1p in different anatomical regions of human brain 3. Investigating the expression of L1 proteins (ORF1p and ORF2p) in Oral squamous cell carcinoma 1. Characterization of inhouse generated human L1ORF1p antibody to detect L1 encoded ORF1p in different cancer cell lines i. Characterisation of αhL1ORF1 antibody by western blotting: Here western blotting was performed to characterize the hL1ORF1p antibody by looking its expression in different human and murine cancer cell line (MCF7, Hela, Du145, NIH3T3, and HEK293T). The dilution to be used to perform Western blotting was calibrated and data showed that the inhouse hORF1p antibody detects ORF1p as single bands with MW ~40 kDa in human cancer cell lines (HEK293T and MCF7). ii. Application of the generated antibody for immunofluroscence (IF): Here the IF technique was employed to see endogenous and exogenous expression of ORF1p in MCF7 and HeLa respectively. The Hela cells didn't not show any endogenous expression. In MCF7 cells ORF1p was detected in cytoplasmic fraction. iii. Validation of the antibody for Immunoprecipitation (IP): Next, I purified the antibody using affinity capture mechanism by protein A agarose and the purified antibody detected L1ORF1p in HEK 293T cell line at a concentration of 1:5000 dilution indicating that the antibody is more efficient than the commercially available antibodies against L1ORF1p. Thus my data depicts that the antibody can be used for immunoprecipitation of over expressed ORF1 protein in bacterial expression system. Abstract iv 2. Detection of L1 ORF1p in different anatomical regions of human brain i. Characterisation of generated antibody for Immunohistochemistry (IHC): The generated hL1ORF1p antibody was characterised and employed for immunohistochemistry. I took commercialized L1ORF1p antibody from Merk Millipore (Cat no: MABC1152) as a positive control. Primary anti-His and primary non-immune sera was used as negative control. The data revealed that inhouse L1ORF1p antibody can significantly detect L1 ORF1p at a concentration of 1:500 in brain as compared to the commercial antibody. ii. Detection of L1 encoded ORF1p in different human tissue: Next my focus was to elucidate the expression of L1ORF1p in different anatomical region of human brain. My data indicated that expression of L1ORF1p increases significantly in older brain as compared to young brain. Non brain somatic tissue (kidney, heart, liver and lungs) shows no reactivity to L1ORF1p. The expression of ORF1p was specifically in the neurons and the protein tends to localise in the neuronal nucleus with age. Comprehensively the data indicate expression of ORF1p increase with age and the expression is more in frontal cortex, hippocampus and basal ganglia than other region of the brain. iii. Detection of L1 transcription in human brain sample: To dissect the L1 transcription level we next employed Northern blotting to detect the amount of L1RNA transcript in old and young aged brain samples. The data depicted increased expression of L1RNA in old aged (80 years) samples as compared to young age (15years). Scarcity of fresh normal brain tissue was a great limitation for the study. Hence on the same samples we tried to check expression level of both ORF1 and ORF2 by semi quantitative PCR. Together the data exhibited enhanced expression of L1 ORF1 and ORF2 in the old age sample suggesting lower transcription of L1 in younger age. Further to validate the data we performed western blotting to detect the L1ORF1p in old aged brain sample and as expected we got distinct band at 42 kDa for L1ORF1p. Abstract v 3. Investigating the expression of L1 proteins (ORF1p and ORF2p) in Oral squamous cell carcinoma i. Sample collection, Block preparation and histology: The operated OSCC samples were collected from surgical oncology department of AIIMS, Rishikesh following institutional ethical clearance and proper consent from the patient’s family. We focused mainly on samples having previous habit of tobacco addictions. Upon collection of the samples fresh tissue were further stored in RNA later solution in -200C and formalin fixed tissues were used to make paraffin blocks. In this section detailed study indicating altered histology during cancer progression is also elaborated. ii. Validation of αhL1ORF1 and αhL1ORF2 by immunohistochemistry in collected OSCC samples. In this section the antibody concentration for L1ORF1p and L1ORF2p was optimised for performing IHC in cancer tissues. The positive and negative controls were also established to screen OSCC samples. iii. Detection of L1 encoded ORF1p and ORF2p in OSCC samples: On a small cohort of 27 samples, the expression of both the L1 proteins (ORF1p and ORF2p) was investigated. Data revealed more than 50 % of the samples were positive for both the proteins. We took Ki67 and CK19 as cellular proliferation marker and squamous cell carcinoma marker respectively. We divided the pattern of expression in three different group i.e high, moderate and low. The OSCC samples showing high expression of L1 proteins was further used for methylation studies to check activation of L1 5’UTR. Our data indicate that expression of L1 proteins are elevated in OSCC that might be used as a marker protein. iv. Patho-clinical significance of ORF1p and ORF2p This section we will describing the stage wise expression of both the L1 proteins. For a deeper insight we tried to correlate the expression of L1 with the TNM and grade of squamous cell carcinoma. Samples showing higher expressions are well differentiated carcinoma but distinct stage dependent variation was not found. The percentage of expression throughout all the samples screened was quite significant but in a stage independent manner. Expression in early stage OSCC indicates that both the L1 proteins could be exploited as a potential biomarker. Abstract vi Chapter 4: Discussion This chapter includes the discussion part of the thesis which concludes the inferences obtained from the results. Further conclusion and future prospective of the work has been discussed. Overview of the drawn conclusion is as follows: 1. The in house generated novel αhL1ORF1p antibody can be used for western blotting, immunofluorescence, and immunohistochemistry. 2. L1 ORF1p is expressed in a significant amount in different anatomical region of the brain. 3. Frontal cortex among the other entire brain exhibited robust expression. 4. Expression of L1ORF1p and L1 RNA increases as age progress in human. 5. L1 encoded ORF1p and ORF2p are overexpressed in oral squamous cell carcinoma in a stage independent manner. Both the proteins ORF1p and ORF2p can be exploited as OSCC biomarker.