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Summer Research Fellowship Programme of India's Science Academies

Evaluation of MicroRNAs expression in oral squamous cell carcinoma

Sourav Mukherjee

National Institute of Technology Durgapur, Durgapur 713209

Dr. Mausumi Bharadwaj

Scientist 'G' (Director Grade) & Head, Molecular Biology Group, NICPR, Noida 201301

Abstract

​Cancer of the oral cavity is one of the major cancers in India, both in men and women. Oral cancer combines cancers of the lips, tongue, cheeks, the carpet of the mouth, hard and soft palate, sinuses, and pharynx (throat). Squamous cell carcinoma is the predominant (~95%) histological kind and therefore the expression ‘oral cancer’ points to be practiced mutually with oral squamous cell carcinoma (OSCC). The extensive habit of smoking or grinding tobacco and alcohol drinking, aside from inadequate oral hygiene, poor diet and Human Papilloma Virus (HPV) infections may reveal this disproportionately greater number of OSCC in India. Therefore, this investigation focused on the association of biomarkers for advanced diagnosis of OSCC. Pointers of excellent or poor prognosis is undoubtedly crucial. MicroRNAs (miRNAs) are small (19-to-25 nucleotide) single stranded non-coding RNAs, which attach to complementary sequences of the 3’ untranslated region (UTR) of destination messenger RNAs resulting inhibition of their translation by the RISC, an RNA induced silencing complex. Considering >30 % of the human genes is foretold to be controlled by miRNAs, certain small RNAs oversee every cellular, physiological and developmental process. Therefore, miRNA deregulation is well-known in every human cancer including OSCC and miRNA stamps have been significant at any levels directly from diagnosis to the perception of medication acknowledgment. The preponderance of the miRNA expression profiling investigations executed in OSCC continuously now describe either oral cancer cell line models or tissue samples of the head, neck carcinoma totally. Therefore, the aim of the current work is to identify miRNAs regulated differently in OSCC cancerous tissue as compared to healthy adjacent controls.

Keywords: RISC(RNA Induced Silencing Complex), HPV(Human Papilloma Virus), UTR(Untranslated Region), ncRNAs(non-coding RNAs)

Abbreviations

OSCC - Oral Squamous Cell Carcinoma

HPV - Human Papilloma Virus

miRNA - MicroRNA

UTR - Untranslated Region

IARC - International Agency for Research on Cancer

TAS - Total-Analysis Systems

OCT - Optical Coherence Tomography

HNSCC - Head and Neck Squamous Cell Carcinoma

HSV - Herpes simplex virus

RISC - RNA-Induced Silencing Complex

CAFs - Carcinoma-Associated Fibroblasts

MiR - MicroRNAs

OLP - Oral Leukoplakia

FIH - Factor-Inhibiting Hypoxia-Inducible Factor

HIF - Hypoxia-Inducible Factor

EMT - Epithelial-Mesenchymal Transition

hTERT - human Telomerase Reverse Transcriptase

NOKs - Normal Oral Keratinocytes

IRAK1 - IL-1 Receptor-Associated Kinase 1

TRAF6 - TNF Receptor-Associated Factor 6

WWOX - WW domain-containing Oxidoreductase

MCPH1 - Microcephalin 1

INTRODUCTION

Background

Cancer is a combination of diseases concerning unusual cell growth which can penetrate or expand to different portions of the body. It explains the disorder that occurs while cellular alterations produce the independent growth and breakdown of cells. A few kinds of carcinoma influence speedy cell growth, while the rest cause cells to develop and distribute at a long-delayed scale. In 2015, approximately 90.5 million people had carcinoma [1].The chance of malignancy progresses significantly with time, also several carcinomas happen more regularly in advanced nations [2].More than 100 kinds of carcinomas harm people, oral cancer is one of them. Oral carcinoma is a dangerous and rising quandary in various portions of the world. Carcinoma of the oral cavity is one of principal cancer in India both in men and women. Oral cancer connects carcinomas of the lips, tongue, cheeks, the carpet of the mouth, hard and soft palate, sinuses, and pharynx (throat). Squamous cell carcinoma is the predominant (~95%) histological variety and accordingly the appearance ‘oral cancer’ objects to be handled respectively with oral squamous cell carcinoma (OSCC). The widespread addiction to smoking or grinding tobacco and alcohol drinking, apart from inadequate oral hygiene, puny diet and Human Papilloma Virus (HPV) infections may exhibit the disproportionately more prominent figure of OSCC in India. During 2018, It is considered that there will be 51,540 distinct circumstances of and 10,030 mortality attributable to oral cavity carcinoma and pharynx in the United States (U.S.) [3].Though the performance of tobacco and alcohol as significant hazard representatives for oral carcinoma are adequately confirmed, the probable performance of oral hygiene practices and diet are frequently assumed as self-governing peril determinants. MicroRNAs (miRNA) are short (19 to 25 nucleotides), non-protein-coding RNA-molecule which regulates up to 70% of the human genome by controlling essential processes like cell proliferation, differentiation or apoptosis [4] [5] [6].MicroRNAs connect to complementary sequences of the 3’ untranslated region (UTR) of target messenger RNAs occurring restraint of their translation by the RISC which is an RNA induced silencing complex [7]. The principal role of miRNAs is the post-transcriptional silencing of mRNAs by complex base-paring synergies that appear in translational constraint or mRNA splitting [8]. Numerous investigations have witnessed unique miRNA-expression profiles in individual tumours that recognize for increased specific discernment of various disorders and tissues associated including messenger RNA profiling [9] [10].Circulating miRNAs have been expressed to explicate varied levels in the body fluids of OSCC victims analyzed to normal people [11] [12].Circulating miRNAs are not assimilated by RNase and also are enduring at raised pH, following boiling, and subsequent various freeze-thaw cycles [13] [14].Considering growing testimony implies that miRNAs have an effective performance in cell function, including various miRNAs are straight associated in oncogenesis, functioning as tumor suppressors (e.g. miR-16) or also oncogenes (e.g. miR-17-92 cluster) [15] [16].Also, the pattern of circulating miRNAs has been recommended as a marker for various kinds of carcinoma with large accomplishment and repeatability [17] [18], also for the investigation of OSCC examples [19].

Objectives of the Research

 1) To extract the RNA from oral pre-cancer/cancer and control tissue biopsy samples by miRNAs Mini Kit.

2) Preparation of complementary DNA by using cDNA synthesis kit.

 3) Evaluation of miRNAs expression in oral pre-cancer/cancer and normal control tissue biopsy samples by using Real Time PCR (qRT-PCR).

 4) Statistical analysis.

LITERATURE REVIEW

Cancer

Cancers are a huge accumulation of disorders that affect unusual cell extension with the potential to overrun or reach to different portions of the body. The chance of carcinoma rises significantly with age; also various carcinomas happen more regularly in advanced nations [2]

 

Types of Cancer

Cancers are specified for the region in which they occur and also the kind of cell they are composed of.

 

1. Carcinoma is cancer that begins in the skin or even in the tissues that bound different organs.

2.  Sarcoma is a cancer of tissues that are connected like bones, blood vessels, muscles and cartilage.

3. Leukaemia is a bone marrow cancer, which generates blood cells in the body.

4. Lymphoma and myeloma are immune system cancers.

 

Epidemiology of Cancer

Global Scenario:

The latest global cancer data insinuates that global cancer weight has increased to 18.1 million events and resulting in 9.6 million cancer mortality [20]. The International Agency for Research on Cancer (IARC) determines that one-in-five men and one-in-six women globally will evolve cancer during their continuance, and also will result in one-in-eight man and one-in-eleven women deaths [20]. Several determinants resemble to be encouraging this progress, especially a growing and ripening global community and an increment in susceptibility to cancer prospect circumstances connected to cultural and commercial improvement [20].Asia considers for approximately half of the fresh cancer incidents and higher than half of the cancer mortality. Approximated imply that Asia and Africa have a giant dimension of cancer mortality (7.3% and 57.3% sequentially) correlated with their frequency (5.8% and 48.4% respectively) [20]. IARC recommends the trend mentioned above is possible due to the immense incidence of cancer varieties connected with a more inferior prognosis and also an insufficient entrance to suitable diagnosis and medication [20].

GLOBOCAN 2018 Global burden maps.jpg
    : Global cancer incidence [20]
    GLOBOCAN 2018 5 most commonly diagnosed cancer types.jpg
      : Percentages of new cancer cases and cancer deaths worldwide in 2018. [20]

      Indian Scenario:

      The recent global cancer data mentions that the Indian cancer burden has expanded to 1.2 million fresh issues and ending in 0.8 million cancer deaths [20]. In 2018, breast cancer is the most regular cancer in India and also causing maximum deaths while oral cancer stands second most death causing cancer in India [20].

      356-india-fact-sheets-1.jpg
        : Scenario of cancer in India [20]

        Oral Cancer

        Oral cancer involves cancers of the lips, tongue, cheeks, the carpet of the mouth, hard and soft palate, sinuses, and pharynx (throat), which can be dangerous if not diagnosed and negotiated quickly.

         

        Epidemiology of Oral Cancer

        Global Scenario:

        The latest global cancer data proposes that In 2018, oral cancer happened worldwide in nearly 354,864 people, and ended in approximately 177,384 deaths [21].

        Indian Scenario:

        The current global cancer data imply that oral cancer strain has grown to 119992 fresh cases and resulting in 72616 cancer deaths in India [20]. The comprehensive obsession with smoking or grinding tobacco and alcohol drinking, apart from incompetent oral hygiene, diminutive diet and Human Papilloma Virus (HPV) infections may manifest the disproportionately more obvious structure of OSCC in India.

         

        Stages of Oral Cancer

        The subsequent 4 stages are adapted to explain lip and oral cavity cancer:-

        Stage I- The cancer is smaller than 2 centimeters in dimension and has not reached to lymph nodes in the region.

         

        Stage II- The cancer is larger than 2 centimeters in dimension, however smaller than 4 centimeters, and has not reached to lymph nodes in the region.

         

        Stage III- There may be two possibilities: The cancer is an unspecified dimension that simply has reached hardly one lymph node on the corresponding neck surface as cancer. The lymph node that comprises cancer strokes no longer than 3 centimetres. The cancer is larger than 4 centimetres in dimension.

         

        Stage IV- Several possibilities are there: cancer has reached tissues nearby the lip and oral cavity. Cancer has reached different components of the body. The lymph nodes in the region may or may not carry cancer. The cancer is an unspecified dimension and has reached to larger than one lymph node on the corresponding neck surface as cancer, to lymph nodes on either one or both surfaces of the neck, or to an unspecified lymph node that holds larger than 6 centimetres.

        The TNM staging method:- In this TNM system T represents the tumor, N represents the lymph nodes, and M represents faraway metastasis.

         

        · TX- Initial tumor cannot be imposed.

        · T0- No indication of the initial tumor.

        · Tis- Carcinoma in situ

        · T1- Tumor 2 cm or smaller in the most prominent size.

        · T2- Tumor larger than 2 cm still not larger than 4 cm in size.

        · T3- Tumor larger than 4 cm in size. (Lip) Tumor penetrates nearby structures.

        · T4- Oral cavity Tumor penetrates contiguous structures.

         

        N-stages:

        · NX- Local lymph nodes cannot be exacted.

        · N0-No local lymph node metastasis.

        · N1- Metastasis in a particular ipsilateral lymph node, 3 cm or less in size.

        · N2- Metastasis in a particular ipsilateral lymph node, larger than 3 cm though not larger than 6 cm in size and also in various ipsilateral, bilateral or contra lateral lymph nodes, not larger than 6 cm in size.

        · N2a- Metastasis in a particular ipsilateral lymph node larger than 3 cm however not larger than 6 cm in dimension.

        · N2b- Metastasis in various ipsilateral lymph nodes, not larger than 6 cm in dimension.

        · N2c - Metastasis in bilateral or contra lateral lymph nodes and not larger than 6 cm in dimension.

        · N3- Metastasis in a lymph node larger than 6 cm in size.

        M-stages:

        · MX- Appearance of faraway metastasis cannot be imposed.

        · M0- No faraway metastasis.

        · M1- Faraway metastasis.

         

        The grades of Mouth and Oropharyngeal Cancer

        The descriptions of the G classifications refer to every head and neck localities without a thyroid.

        · G – Histopathological Grading

        · GX – Grade of differentiation cannot be imposed.

        · G1 – Fully differentiated.

        · G2 – Slightly differentiated.

        · G3 – Unwell differentiated.

        · G4 – Undifferentiated.

         

        Screening methods of Oral Cancer

        There are several advanced diagnostic advancements in the investigation of oral cancer.

        a) Lab on a Chip- Microfluidics technology is additionally associated with lab-on-a-chip or micro-total-analysis systems (TAS) which is the evolution, miniaturization, integration, and computerization of scientific laboratory methods within a particular device or “chip”.The disclosure of oral pre-cancer and cancer cells inside the chip will catch the benefit of membrane-associated cell proteins that are uniquely displayed on cancer cells.The evaluated portrait is matched with archived gene transcription portraits to ascertain the cancer kind as well as stage [22].

         

        b) Vital Tissue Staining Toludine Blue Staining & Lugol’s Iodine- Oral cancer in situ and initial invasive oral cancer reveals an affinity for toluidine blue dye. Lugol’ iodine and toluidine blue have been applied mutually in the exposure of newly cancers [22].

         

        c) Brush Cytology- Brush cytology (Oral CDX), originated in 1999 and has enhanced successful dental exercise now. Oral CDx is beneficial in the estimation of dysplastic variations in several speculated injuries, particularly in oral cancer [22].

         

        d) Optical Coherence Tomography- Optical coherence tomography (OCT) has been confirmed to be a beneficial method for oral disease investigation. Based on the scanning pictures of a swept-source OCT system, several useful analysis pointers for oral cancer and pre-cancer have been recognized [22].

         

        e) Biomarkers: The common auspicious of the molecular markers consequently incomparably convenient and appraised in OSCC growth comprises of the TSG p53 protein expression, chromosomal polysomy, and variations in chromosomes 3p or 9p [22].

         

        f) Chemiluminescence Vizilite: It requires the performance of a hand-held, single-use, disposable chemiluminescent light pole that releases light at 430, 540 and 580 nm wavelengths. The advantage of the light stick is designed to enhance the optical contrast between natural mucosa and oral white injuries [22].

         

        g) Velscope System: Practicing the tissue auto fluorescence idea for investigation of dysplastic injuries in the oral cavity couples on the variations in the arrangement and metabolism of the epithelium and the sub epithelial stroma during communicating with light [22].

         

        h) Saliva as a Diagnostic Tool: Saliva from victims has been practiced in a unique alternative to identify molecular biomarkers for oral cancer recognition [22].

         

        2.8 Signs and Symptoms of Oral Cancer:

        Potential signs and symptoms of the oral cavity and oropharyngeal cancers comprise of the followings-

        1. A sore in the mouth which doesn't recover.

        2. Pain in the mouth.

        3. Difficulty chewing or swallowing.

        4. A lump or thickening in the cheek, neck.

        5. A white or red scrap on the gums, tongue, tonsil, or covering of the mouth.

        6. Loosening of the teeth or discomfort neighboring the teeth or jaw.

        7. Struggle to move the jaw or tongue.

        8. Swelling of the jaw which makes dentures to suit inadequately.

        9. A sore throat or a perception that something is detected in the throat which doesn’t removable.

        10. Insensitivity of the tongue or other regions of the mouth.

        11. Voice differences etc.

         

        Risk Factors for Oral Cancer

        Various risk factors for oral cancer have been listed below-

        Chemical Factors:-

        1. Tobacco: Plentiful proofs are recommending that tobacco in different modes, like smoking, chewing and in betel quid, have carcinogenic effect in oral cavity [23]. The most popular mode of tobacco usage is smoking.

         

        2. Alcohol: Various investigations have proposed alcohol to be a significant risk factor for oral cancer. Although there is a definite order of contention whether alcohol separately may have carcinogenic effect [23].

         

         

        Biological Factors:-

         Viruses: Oncogenic viruses’ performance is an significant field of investigation in human cancer.

        1. Human papillomavirus (HPV): In almost 23.5% of oral cancer cases HPV has been recognized [24]. HPV-16 is the most regularly identified HPV in head and neck squamous cell carcinoma (HNSCC), accompanied by HPV-18, HPV-31, and HPV-33 [23].

        2. Herpes simplex virus (HSV): HSV-1 or "oral herpes" is generally connected with sores nearby the mouth and lip. It has been proposed to be a risk factor in oral cancer [25].

        3. Syphilis: The information on the causal relationship within syphilis and oral cancer is ineffective [23].

        4. Candida: Candida has been recommended to perform a task at the beginning of oral cancer [23].

         

        Dental Hygiene and Related Factors:-

        Between oral hygiene and the prevalence of oral cancer, there is an inverse relationship. In the growth of oral cancer, puny oral hygiene and continued annoyance from edged teeth have been observed for their attainable function [23].

        Nutritional Factors- Dietary lacks are additionally recommended to perform a task in the growth of oral cancer [23].

         

        MicroRNAs

        MicroRNAs is short (19 to 25 nucleotides), single-stranded, evolutionary conserved, non-coding RNA molecules that attach objective mRNA to inhibit protein making [26].It controls up to 70% of the human genome by regulating crucial methods like cell proliferation, differentiation or apoptosis. Above 1900 miRNAs may be encoded in humans [27].The miRNAs biogenesis starts with the pri-miRNAs transcription by RNA polymerase II. Later, Drosha cleaves Pri-miRNAs in the nucleus, originating pre-miRNAs [28] [29]. Pre-miRNAs are consequently shipped within the cytoplasm and treated by Dicer to produce adult miRNAs ( around 21bp), which are consolidated within the RNA-induced silencing complex (RISC) [7] [28].MiRNAs combine to corresponding sequences of the 3’ untranslated region (UTR) of quarry messenger RNAs happening inhibition of their translation by the RISC [7].

         

        Role of MicroRNAs in oral cancer

        In fresh studies, miRNAs have been considered as essential determinants for tumorogenesis [30]. Proof implies that miRNAs work as either oncogenes or tumor suppressors [31].The enduring presence of miRNAs in natural body fluids addresses them excellent biomarkers for investigation or diagnosis of oral cancer. Certain Investigations discovered that hsa-miR-31-3p, hsa-miR-424-5p, hsa-miR-196b-5p, hsa-miR-877-5p, hsa-miR-7-5p, hsa-miR-135b-5p, hsa-miR-31-5p, hsa-miR-142-3p, hsa-miR-187-3p, hsa-miR-19a-3p, hsa-miR-708-3p,hsa-miR-223-3p, hsa-miR-32-5p, hsa-miR-18a-5p, hsa-miR-301a-3p, hsa-let-7a-3p, hsa-miR-21-5p, hsa-miR-455-5p, hsa-miR-92b-3p, hsa-miR-21-3p, hsa-miR-142-5p, hsa-miR-944, hsa-miR-20a-5p are revealing up-regulated expression in oral cancer whereas hsa-miR-204-5p, hsa-miR-375, hsa-miR-4497, hsa-miR-1291, hsa-miR-4492, hsa-miR-3196, hsa-miR-6087, hsa-miR-4508, hsa-miR-4485-3p, hsa-miR-3195, hsa-miR-3687, hsa-miR-3648, hsa-miR-6510-3p, hsa-miR-4516, hsa-miR-7704, hsa-miR-3656, hsa-let-7c-5p, hsa-miR-4532, hsa-miR-4488, hsa-miR-99a-3p, hsa-miR-125b-5p, hsa-miR-139-5p, hsa-miR-3651, hsa-miR-125b-2-3p, hsa-miR-99a-5p are explicating down-regulated expression in case of oral cancer [32].An adequately investigated miRNA, miR-21 is over expressed and to control various biological roles in OSCC [33].By targeting PTEN, miR-21 is recognized for controlling cell growth and proliferation, furthermore, its over-expression is correlated with the activation of that Pi3k/Akt pathway and also active cell increase [34].An expanding collection of testimony implies that miR-21 might be essential in the regulation of carcinoma-associated fibroblasts (CAFs) initiation and their action [35]. Also, the more crucial stromal appearance of miR-21 was correlated with inferior diagnosis in OSCC [35].MiR-31 and its rider strand miRNA (miR-31∗) have an oncogenic function in OSCC tumorogenesis, are upregulated in oral leukoplakia (OLP) and OSCC [36].Both in the case of in vitro and in vivo, Liu et al. described that the ectopic appearance of miR-31 inhibited its destination factor-inhibiting hypoxia-inducible factor (FIH) character to initiate hypoxia-inducible factor (HIF) supporting normoxic circumstances. Furthermore, in OSCC cells, miR-31-FIH-HIF-VEGF governing cascade was observed to influence various biological methods like cell proliferation, migration, and epithelial-mesenchymal transition (EMT) [37].miR-31 was determined to cooperate with human telomerase reverse transcriptase (hTERT) to canonize normal oral keratinocytes(NOKs), symbolizing that it might provide something to early-stage oral carcinogenesis [36]. On the other hand, miR-31∗ controlled apoptosis, cell proliferation, migration, and invasion in OSCC cells likewise [38].Both in the in vitro and in vivo mouse xenograft prototype, miR-146a has been confirmed to be over expressed in OSCC and to heighten OSCC tumorogenesis [39]. The oncogenic purposes of miR-146a were observed to be correlated with the simultaneous down regulation of IL-1 receptor-associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6), and NUMB [39].the miR-134 appearance was upregulated in HNSCC tissue parts and cells (HSC-3, OECM-1, and SAS cell-lines)correlated to the identical healthy controls. Practical investigation exhibited that miR-134 appearance intensified the oncogenicity of HNSCC cells in vitro, tumor growth including metastasis of HNSCC cells in vivo by targeting WW domain-containing oxidoreductase (WWOX) [40].In a different research,miR155 was observed to be over expressed in OSCC cells and tissues correlated to the normal controls [41]. In OSCC, the Oncogenic impacts of miR-155 were recommended to be because of the down regulation of a tumor suppressor CDC73 [42]. Furthermore, miR-27a was determined to down regulate the appearance of and also to hinder tumor suppressor role of microcephalin 1 (MCPH1) in OSCC cells [43].

        Schematic-illustration-demonstrating-biogenesis-and-function-of-miRNA-miRNA-genes-are.png
          : Schematic diagram describing the biogenesis and function of miRNA. [44]

          METHODOLOGY

          Patients and specimen

          In this study, 3 histo-pathologically confirmed oral tissue biopsy samples (pre-cancer, cancer & control) were collected from collaborating Hospitals of NICPR-ICMR, Noida (Delhi, NCR). Fresh tissue biopsies were collected in RNA later and stored at -80°C deep freezer for further processing.

           

          MiRNA extraction from oral tissue biopsies

          MiRNA was extracted from 3 samples (pre-cancer, cancer and control) by using miRNAs Mini Kit (Qiagen, USA). The tissue biopsy approximately 5gm was homogenized in a petri plate kept on the ice by adding 200 ul of Qiazol (lysis buffer provided in the Qiagen Kit). The minced tissue was incubated for 5 min at room temperature. Then 140 μl of chloroform was added and mixed properly till a milky appearance was observed. The tubes were centrifuged at 12000 rpm at 40ºC for 15 minutes. After centrifugation two layers of aqueous phase and organic layer were formed. The upper aqueous phase was transferred to a new tube. The following steps were performed to purify the miRNA-enriched fraction.

          1. Discard the miRNeasy Mini spinColumn.

          2. Add 450 μl of 100% ethanol (0.65 volumes) to the flow-through

          3. and mix thoroughly by vortexing. Do not centrifuge.

          4. Close the lid and centrifuge at 8000 x g (≥10,000 rpm) at room temperature (15–25°C). Discard the flow through.

          5. Pipette 700 μl of the sample into an miRNeasy MinElute spin column placed in a 2 ml collection tube. Close the lid gently and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) at room temperature (15–25°C).

          6. Add 700 μl Buffer RWT to the miRNeasy MinElute spin column. Close the lid gently and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the column.

          7. Pipette 500 μl Buffer RPE into the miRNeasy MinElute spin column. Close the lid gently and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through. After centrifugation, remove the RNeasy MinElute spin column from the collection tube carefully so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.

          8. Place the miRNeasy MinElute spin column into a new 2 ml collection tube. Open the lid and centrifuge for 5 min at 8000 x g (10,000 rpm). Place the miRNeasy MinElute spin column into a 1.5 ml collection tube and pipet 14 μl RNase-free water onto the spin column membrane.

          9. Close the lid gently and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the miRNA-enriched fraction. The miRNA enriched fraction of RNA separated is stored at -700C for further use.

           

          Quantification of miRNA

          Spectrophotometric quantification of RNA:

          To ensure significance, A260 readings should be greater than 0.15. An absorbance of 1 unit at 260 nm corresponds to 44µg of miRNA per ml. This relation is valid only for measurements at a neutral pH. Therefore, if it is necessary to dilute the miRNA sample, this should be done in a buffer with neutral pH the ratio between the absorbance values at 260 and 280 nm gives an estimate of miRNA purity. This can be accomplished by washing cuvettes with 0.1 M NaOH, 1 mM EDTA, followed by washing with RNase free water. Use the buffer in which the miRNA is diluted to zero the spectrophotometer.

           

          Quantification by gel electrophoresis:

          The integrity of isolated miRNA was examined by electrophoresis on an ethidium bromide stained 1.5% agarose gel in MOPS buffer. The purity of RNA was determined by the presence of at least two bands corresponding to 28S and 18S RNA in the ratio of 2:1.

          cDNA preparation

          The isolated miRNA was used for cDNA preparation by using cDNA synthesis kit purchased from system bioscience. cDNA was prepared from range of 10pg-10µg/µl miRNA sample. The initial step of cDNA preparation is Poly (A) tail addition. In which, Total RNA taken of concentration ~ 800 ng, 5X PolyA buffer, 25mM MnCl2, Poly A Polymerase are added and incubated for 30 min. at 370C.Then annealing of anchor dT adapter (0.5ul) is added. Heat the mixture for 5 min at 600C. Let cool to room temp for 2 min. Add 5X RT buffer, dNTPs mix,0.1M DTT, nuclease-free H2O, Reverse Transcriptase enzyme to prepare total reaction mixture to be 25 ul. Incubate for 60 min at 420C. Heat for 10 min at 950C.Allow it to cool and store at -200C. The cDNA prepared was checked on 1% agarose gel electrophoresis stained with ethidium bromide in 1X TE buffer.

          Real Time PCR (qRT-PCR)

          The prepared cDNA was further used for miRNA expression profiling using miRNome microRNA Profilers kit (System Biosciences) which is designed for amplification of whole genomic miRNA using miRNA primers. Small endogenous RNAs Human U6 snRNA, served as positive controls and, qRT-PCR mix without template was used as negative control. The Syber green master mix was used as a detection dye. The PCR conditions were pre incubation at 500C for 2 min, 950C for 10 min, 2 step amplification at 950C for 15 seconds and 600C for 1 min and 72 for 15 seconds with 50 cycles and the melting conditions were, 950C for 15 sec, 650C for 1 min and 970C for continuous and cooling at 370C for 30 seconds. Amplification of all miRNAs in samples was carried out according to the manufacture’s instruction and was analyzed on the 96 well plates Real Time Light Cycler (Roche). Differentially expressed miRNAs identified by comparing their expression with normal Vs. pre-cancer & cancer to calculate the delta Ct. Each experiment was performed in duplicate and average Ct value was used for the analysis.

           

          Data Analysis

          The miRNA data was analyzed by using the software provided by the System biosciences (Sanger miRBase version 15). In which Ct values of cases and controls were compared to reach normalized miRNA expression level. The relative quantification was determined by taking housekeeping gene as reference genes.

          RESULTS AND DISCUSSION

          miRNAs isolation and quantification from oral precancer, cancer and control tissue biopsies

          Total RNA extracted from oral cancer tissue biopsies by column purification, (miReasykit,Qiagen) were checked for their quality by electrophoresis on 1.5 % agarose gel. The integrity of RNAs was found to be intact shown in fig 5.

          Picture1.jpg
            : miRNA isolated from oral pre-cancer/cancer and control tissue biopsy samples and checked on 1.5% agarose gel (A), cDNA was checked on 1% agarose gel stained with EtBr (B).LanePC1:Oral pre-cancer sample, Lane OC1:Oral cancer sample, Lane N:Normal control

            miRNAs gene expression by using Real Time- PCR

            We profiled 47 microRNAs that are annotated till date by various human microRNAs databases by quantitative real time polymerase chain reactions (qRT-PCR). We have checked the expression of 47 miRNAs including housekeeping gene (GAPDH) from oral pre-cancer, cancer and control samples. Amplification curve of amplified miRNAs in oral cancer and control samples are shown in Fig 6. The up and down regulated miRNAs are shown in Fig 7 & Table 1.

            Picture2.png
              : Representation of the amplified 47 miRNAs in oral cancer tissue sample
              Picture3.png
                : Common differentially expressed miRNAs in oral pre-cancer/cancer and control tissue samples
                : List of common expressed miRNAs in Oral pre-cancer and cancer samples
                miRNAs in NormalPre-cancerCancer
                Up regulationDown regulationUp regulationDown regulation
                let-7d       miR-7      miR-9      miR-10a   miR-10b  miR-15a  miR-18a  miR-22   miR-25    miR-29b  miR-30c  miR-32   miR-33a  miR-125a-5p miR-138 miR-143  miR-149 miR-181a miR-181b miR-181d miR-186 miR-187  miR-188-5p miR-191 miR-193b miR-196a miR-197 miR-210  miR-224 miR-375  miR-382 miR-411  miR-425 miR-432  miR-455-5p miR-502-3p miR-520g miR-520h miR-532-5p miR-548a-3p miR-548a-5p miR-548d-3pmir-let-7d miR-9       miR-10b    miR-15a    miR-18a    miR-22      miR-25      miR-29b    miR-30c    miR-32      miR-125a-5p miR-138     miR-143     miR-149     miR-181a    miR-181b    miR-181d    miR-186      miR-187      miR-188-5p miR-191      miR-193b     miR-196a     miR-197      miR-210      miR-224      miR-375      miR-382      miR-425      miR-432      miR-502-3p miR-520g    miR-548a-5p miR-548d-3p  miR-7        miR-10a     miR-33a     miR-411     miR-455-5p miR-520h    miR-532-5p miR-548a-3plet-7d        miR-7        miR-10b     miR-15a      miR-18a     miR-22       miR-25       miR-29b     miR-30c     miR-32      miR-33a     miR-125a-5p miR-143     miR-181d   miR-186     miR-187     miR-188-5p miR-191     miR-193b   miR-196a miR-197 miR-210  miR-224 miR-375  miR-382  miR-425  miR-455-5p miR-502-3p miR-520g miR-548a-5p miR-9           miR-10a        miR-138        miR-149        miR-181a       miR-181b       miR-411         miR-432         miR-520h       miR-532-5p    miR-548a-3p   miR-548d-3p   

                CONCLUSION

                In India, OSCC is a significant life-threatening carcinoma [45]. Not with standing multimodal treatment, the 5-year durability rates of OSCC sufferers have not traversed 50 % for the previous four decades [46]. Numerous investigations have correlated miRNA expression as well as clinical parameters in OSCC with recurrence, metastasis, and durability in the last few years. A meta-analysis to appraise the prospect of miRNAs as biomarkers to identify OSCC was performed by Lin et al and there they picked eight profiling investigations and obtained inconsistent and conflicting outcomes [47].Separate fresh meta-analyses of OSCC and miRNAs have been handled by Zeljic et al and Troiano et al, which revealed few similarities and few variations in outcomes [48] [49].The scholars settle that feasible inclination in meta-analysis is the root of the samples. Assembling pieces of evidence imply that miRNAs are beneficial as diagnostic and prognostic markers for cancer, as well as contribute to the competency for targeted treatments [50].Real Time-quantitative PCR and microarray have been extensively practiced by several investigators to recognize miRNAs that are differentially exposed in oral cavity cancers [51].Therefore, appraising worldwide miRNA appearance and examining their relationship with tumor diagnostics is an encouraging essential to enhance diagnosis, advances to treatment and to diminish the weight of OSCC in India.

                Cervigne, Nilva K., et al revealed over-expression of miR-520g in their investigation on growing dysplasias and OSCCs [52]. Another study explained the up-regulation of miR-let-7d in Head and neck cancer cell lines [53]. A separate investigation also described the down-regulation of miR-149 in their research on Tongue squamous cell carcinoma samples [53]. Schneider, Augusto, et al explicated that hsa-miR-21-5p was one of the greatest expressed miRNAs, also remained up-regulated in cancerous correlated to normal tissue [32]. According to Chan, John K., et al, hsa-miR-21 overexpression was described to be inversely associated with drug sensitivity in chemotherapy and progression-free endurance [54]. In our research, we have observed that miR-let-7d, miR-30c, miR-181d, miR-188-5p, miR-425, miR-520g, miR-159 are up regulated and miR-138a, miR-149, miR-548a-3b are down regulating in both cancer and pre-cancer samples while comparing with the normal control. MiR-let-7d, mir-25, miR-30c and miR-15a are highly expressed in cancer samples with the comparison of controls an it may also help in the progression of the disease.

                ACKNOWLEDGEMENTS

                Firstly I would prefer to reveal my gratitude to Indian Academy of Sciences (IAS-INSA-NASI) for allowing me to participate on this project. I owe my sincere gratitude to Dr. Mausumi Bharadwaj, Scientist 'G'(Director Grade) & Head, Molecular Biology Group, National Institute of Cancer Prevention and Research, Noida-201301, for viewing me as a suitable applicant for SRFP-2019 and affording me continuous encouragement, support and supervision.

                I am thankful to Dr. Surabhi Chaudhuri, Professor, Department of Biotechnology, NIT Durgapur for giving me letter of recommendation for this IAS summer fellowship program.

                I cordially thank Mrs.Upma Sharma, who took acute interest on my project, and supervised me till its fulfilment.

                Finally, I would like to thank Dr. Vineeta, Mr. Rajeshwar Patle, Ms. Heena Gautam, Mr. Mohammad Mousin, and Mr. Mabood Khan for their timely support and valuable suggestions.

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