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Summer Research Fellowship Programme of India's Science Academies

MicroRNAs in colorectal cancer: Small big players in cell cycle

Anubhuti Kashyap

Tripura University (A Central University), Suryamaninagar, Tripura (W) 799022

Professor M Radhakrishna Pillai

Director, Rajiv Gandhi Centre for Biotechnology (RGCB), Thycaud Post, Poojappura, Thiruvananthapuram 695014

Abstract

Colorectal cancer (CRC) stands as the 3rd most prominent cancer (globally) leading to high mortality all over the world. In India it is the 4th most common cancer in males and 3rd most common cancer in females. This study focuses on the role of microRNAs in the regulation of genes with oncogenic potential involved in various stages of cancer progression. microRNAs have been shown to participate in the post transcriptional regulation of cell cycle proliferation and progression. These are small noncoding RNAs (about 20-30 nucleotides) specifically binding to the 3’ untranslated region of mRNAs thereby regulating translation of that mRNA. The microRNA binds to the gene encoding proteins which are responsible for expression of cell cycle components such as cyclins, cyclin dependent kinases, etc. one such gene is the Forkhead box M1 (FOXM1) located at 12p13.3. The protein encoded by this gene has been reported as a transcriptional activator involved in cell proliferation. The protein encoded by FOXM1 could be phosphorylated in M phase and regulates the expression of several cell cycle genes for example cyclin B1, cyclin D1. Studies have shown that the FOXM1 gene is ubiquitously expressed in embryonic tissues whereas in adult stages this gene is downregulated in resting or terminally differentiated cells except for some actively dividing cells such as thymus, testis, colon and intestine. The overexpression of FOXM1 has been however reported in a broad range of cancer cell lines and cancer types examined. Genetic reporter systems like Dual Luciferase Assay have been widely used for the study of eukaryotic gene expression including study of mRNA processing. The objective of this study has been designed so as to observe how microRNAs plays a role in the expression of FOXM1 gene in CRC cell line (HT-29). The Dual Luciferase Assay is the methodology employed to identify the specific microRNA and its role in regulating FOXM1

Keywords: FOXM1, Dual Luciferase Assay

Abbreviations

The following table enlists the abbreviations for all short forms in the report that will follow:

Abbreviations
CRC Colorectal Cancer
FOXM1 Forkhead Box M1
miRNA Micro RNA
miR-149 Micro RNA- 149
ATCC American Type Culture Collection
DMEM Dulbecco’s Modified Eagle’s Medium
FBS Fetal Bovine Serum
DMSO Dimethyl Sulfoxide
PBS Phosphate Buffered Saline
3’UTR 3’ Untranslated Region
RISC RNA induced silencing complex
nL nano Litres 
µLmicro Litres 

INTRODUCTION

Background/Rationale

According to World Health Organization, cancer causes 13% of deaths, CRC being the third most common form of cancer in the world. CRC is the third most common cause of female cancer followed by lung and breast cancers. However, it stands as the third most common form of cancer in females and fourth most common form of cancer in males. Generally, most CRCs are considered sporadic hence there is lesser risk that the cancer is passed from one generation to the next. The inherited colorectal cancers are less common. Although the common forms of treatment for cancer is chemotherapy, radiation and surgery there are always incidences where the patient is confronted with recurrence of the cancer and hence it is important for more target specific research.

Symptoms of CRC:

The symptoms of CRC may include irregular bowels, diarrhoea, constipation, blood in stool, abdominal pain, fatigue or tiredness, unexplained weight loss, any appearance of lump in the abdomen or back. https://fightcolorectalcancer.org/prevent/symptoms/

Age and CRC: Age has been a huge risk factor for colorectal cancers. Majority of colorectal cancers are diagnosed in the people older than 50 which makes it even more difficult for the treatment courses as the older people face unique challenges with regard to treatment procedures. Meanwhile studies have also shown that the incidence rate of CRC has increased by about 2% in adults who are younger than 50 attributing to the changing lifestyle and food habits. https://www.cancer.net/cancer-types/colorectal-cancer/risk-factors-and-prevention

Gender and CRC: The appearance of CRC in older women population is higher than those in men which suggests that CRC may be a major threat to aging women. Scientists have suggested that patients with proximal colon cancer are more often in females than males . Hormonal factors may attribute to the increased incidence of CRC in females. https://www.cancer.net/cancer-types/colorectal-cancer/risk-factors-and-prevention

Heredity and CRC: Studies suggest that most CRCs are sporadic and they appear only after birth. However, there are also minimal chances that they are hereditary and may pass from parents to their children. If a person has a family history of CRC, then there is a higher risk on incidence of CRC. The risk may increase if other close relatives have also developed CRC. https://www.cancer.net/cancer-types/colorectal-cancer/risk-factors-and-prevention

Lifestyle choices and CRC: People who are physically inactive and overweight, and are having habits like smoking, consumption of alcohol may have a higher risk of developing CRC. https://www.medicalnewstoday.com/articles/155598.php

Statement of the Problems

Problems related to CRC and its treatment:

Cancer as we all know is a fatal disease and so far, no absolute treatment has been found. Some patients diagnosed with cancer go through various forms of treatment like radiation, chemotherapy, surgery. But the same patient may return after years complaining with the recurrence of cancer. This happens because some cancer cells are chemo resistant and they survive the drug treatments that the patient undergoes. These surviving cells have been termed as cancer stem cells which remain dormant for some time and reappear at a later stage. These resistant cells are not even detected by CT-scans. https://www.medicalnewstoday.com/articles/155598.php·

Colorectal cancer already been established as the 3rd most common cancer American Cancer Society, is one of the major interests among researchers today. Although many potential treatment procedures like colostomy, radiation, chemotherapy have been found, the consequences or more specifically the side effects of these treatment procedures are harsh. Radiation and chemotherapy to cure cancers has huge side effects which include tiredness, skin reactions, hair loss, infertility. These treatment procedures can therefore hugely affect the lifestyle of the patients although treated of cancer. These treatment procedures hence can hugely affect the lifestyle of the patients although treated of cancer. https://www.cancer.net/cancer-types/colorectal-cancer/risk-factors-and-prevention·

Recent studies suggest that CRC incidence has increased in age groups below 50-60 years. This can be attributed to the lifestyle choices. But again, there are cases where a person was leading a healthy life and still is diagnosed with cancer. Hence more work should be done at the molecular level to make cancer diagnosis and treatment more effective.

Objectives of the Research

The principal objective of the research is to look for epigenetic abnormalities for incidence of CRC.

The research focuses on the role of miR-149 which is already established as a potent inhibitor of FOXM1 transcription factor.

The research looks forward to establish actions of novel microRNAs which might affect the FOXM1 transcription factor by taking miR-149 as a positive control.

The experiments focus on optimizing the HT-29 cell line for double transfection with FOXM1 containing plasmids and miRNA containing plasmids for the study of above-mentioned objectives.

LITERATURE REVIEW

Information

Colorectal cancer (CRC) is the development of cancer in the colon or rectum which are the regions/organs towards the distal end of gastrointestinal tract. CRC has been shown to originate from the epithelial cells which line the colorectal region. Mostly CRC incidences have been reported from the age groups above 50. Although studies have also suggested that lifestyle choices like increased consumption of red meat, fat, alcohol, smoking are increasing the risks of CRC incidences in populations under the age of 50.

Most CRC cases show progression through a series of morphological changes. The first signs include appearance of excess cells at the surface of the large intestine. As these polyps occurs with time (which maybe hyperplastic or dysplastic) , these are seen as outgrowing masses from the surface of the intestine. The tumor is called an adenoma if the polyp is dysplastic i.e., the disordered cellular organization in the affected tissue. ​Frank SA, et al, 2007​.

Studies suggest that epigenetic alterations i.e., alterations that are not involved in the DNA sequence are more frequent in colon cancer than genetic alterations. These alterations include changes in the activity and expression of genes and they may persist through the lifetime of the cell and may also last for several generations.

The progression of chronic inflammation into CRC includes many epigenetic changes which include DNA methylation, the dynamics of chromatin modification, histone modification, RNA mediated regulation of gene expression. These may lead to activation of carcinogenic signaling pathways and ultimately develop cancer metastasis. The action of miRNAs in the regulation and expression of cell cycle associated genes have also been documented. ​Yang ZH, et al​ . According to the miRBase database (v.21), 1881 miRNAs have been identified in humans and these miRNAs are responsible for the regulation and expression of more than half of all the protein coding genes. miRNAs play major role in the regulation of cell division, differentiation, programmed cell death but any abnormality or changes in the expression of these miRNAs may lead to fatal diseases like cancer. miRNAs can decrease the expression levels of their target mRNAs but doesn’t necessarily extinguish their expression totally. ​Michelle M.J.Mens. MicroRNAs do not extinguish the expression of their target mRNAs but reduce their expression levels Bartel, 2009 .

microRNAs are small, 20-30 nucleotide long non-coding RNAs and they are responsible for the suppression of gene expression by either causing degradation of their target mRNA or by inhibiting the translation of mRNA into their respective proteins. The primary miRNA is produced by the RNA polymerase II and this primary miRNA is then cut by Drosha to form a pre-miRNA hairpin which is then cleaved by DICER-I to form the mature miRNA. This single stranded miRNA is integrated into RISC and then targeted to its mRNA 3’-UTR to regulate its expression. Cell cycle entry and progression through the cell cycle may be regulated by the oncogenic miRNAs and cell cycle arrest may be induced by tumor suppressor miRNAs which are capable of downregulating various components required for the proper functioning of cell cycle Yang ZH, et al . Oncogenic miRNAs may regulate cell cycle entry and progression whereas tumor suppressor miRNAs may induce cell cycle arrest by downregulating various components required for the cell cycle Bueno MJ .

The eukaryotic cell cycle has mainly four stages, G1 phase, S phase, G2 phase and M phase. Many proteins required for DNA synthesis and replication are synthesized in the G1 phase which has been studied as the most unpredictable stage and this stage is responsible to determine the fate of the cell, followed by the S phase which is the DNA synthesis phase. The cell prepares itself for division in the G2 phase and enters into the M phase which gives rise to two daughter cells. The daughter cells now formed may either enter the G1 stage for further division or they may enter the quiescent stage a.k.a G0 phase. The G1 phase has been reported to be the most variable phase and the duration of the G1 phase determines the fate of the cell Michelle M.J.Mens . When a cell enters the G1 phase, higher expression of a protein cyclin D occurs in response to stimuli for mitosis. The cyclin D binds to CDK4/6 (cyclin dependent kinases) and these cyclin CDK complexes are responsible for the control of the different phases of above-mentioned cell cycle, for example phosphorylation of the Retinoblastoma family proteins (Rb). RB family targets the E2F genes and the Rb tumor suppressor proteins acts a negative regulator. Studies have shown that the pRb can inactivate E2F transcription factors and thus inhibit cell from entering into the S phase from G1. Likewise, cyclin E-CDK2 complex, cyclinB-CDK1 complex are responsible for regulation of cell cycle entry into S phase and G2 to M respectively. It is well established that one of the main reasons for instability in cell cycle and division is the loss of p53 because the cells lacking p53 or an abnormal copy of p53 have an abnormally functioning G1-S checkpoint. Michelle M.J.Mens. Now expression of these transcriptional factors involved in cell cycle like E2F, p53 have been shown to be regulated by the miRNAs. Cancer may occur due to the abnormal expression of these important transcriptional factors. Bueno MJ. The abnormal expression of these cell cycle regulatory proteins plays a critical role in cancer initiation and progression Michelle M.J.Mens. It has been established that the overexpression of cyclin D has an important role to play in the pathogenesis of human cancers Musgrove et al, 2011 .

Some twenty years ago, the miRNA regulating cell cycle was first studied which showed that miRNAs lin-4 and let-7 were able to induce cell cycle arrest in the nematode C.elegans.​​ ​Reinhart BJ, 2000​ ; Lee RC, et al., 1993

Tumor initiation, invasion, metastasis and chemoresistance can be attributed to the Cancer Stem Cells (CSCs) which are essentially a class of cancer cells having the properties of stem cells. However, the miRNA regulation of transcription are seen to vary among the cancer stem cells from that of the normal stem cells. Michelle M.J.Mens. In several types of cancer cells like lung cancer, colon cancer, liver cancer, various miRNAs like miR34 were studied as downregulated ​Engkvist ME, 2017​; ​Okada N, 2014​; ​Bommer GT, 2007​; ​He L, 2007​​Tazawa H, 2007​; ​Sun F, et al, 2008​. In colon cancer cells, miR-34 has been shown to suppress cell cycle progression by regulation of transcriptional factors like CDK 6, cyclin D1 and E2F. ​Tazawa H, 2007​​; Sun F, et al, 2008​​; Bao B, 2012​. Influence in many cancer cell line death have also been reported by the action of miRNAs which may also suggest that the downregulation of these abnormally expressed transcriptional factors can trigger programmed cell death in the tumor cells. Therefore, miRNAs have been potent in arresting cell cycle as well as induce apoptosis.​​Choi et al, 20122007201220092012​​.

The Forkhead Box M1 (FOXM1) is a protein coding gene and the protein encoded by this gene is a transcriptional activator involved in cell proliferation. It has been shown in previous works to regulate the expression of various cell cycle proteins like cyclins. This gene is located in the short arm of chromosome 12 (12p13.3). Usually the terminally differentiated cells and G0 stage cells have a low expression of this gene but studies have shown its overexpression in the cancer or tumor cells. In the foetal tissues during the development of the embryo, FOXM1 is highly expresses however, in adult tissues its expression is greatly diminished apart from few actively dividing cells like the cells of thymus, testis, colon and intestines.

miR-149 which has already been established as downregulated in CRC tissues has also been shown to significantly inhibit the cancer progression. The miR-149 targets the FOXM1 and can thus inhibit the growth, migration and invasion of the CRC cells. Studies refer to an inverse relationship between the levels of FOXM1 expression and miR-149. Luciferase Assay studies performed by various scientists prove that miR-149 can bind to the 3’UTR of FOXM1 and thus inhibit its expression in the CRC cells. Chemoresistance of CRC cells against 5Fluorouracil has also been shown to be reversed by direct target of miR-149 on FOXM1.​Xu K, et al​. The FOXM1 oncogenic transcription factor was established as a direct target for miR-149 in non-small cell lung cancer also where Cyclin D1 served as downstream target for FOXM1 transcription factor​ Zhao L, et al​.

Summary

Since the relationship between cell cycle, microRNAs and genes with oncogenic potential like FOXM1 has already been established, more work on the same needs to be carried out for proper cancer diagnostics and treatment. In our experiments we are focusing on the action of microRNAs which might be affecting the upregulation or downregulation of transcriptional factor FOXM1 by keeping the action of miR-149 as a positive control by carrying out Dual Luciferase Assay methodology. The HT-29 colorectal cancer cell line is being used for the experiments to validate the possible action of other novel microRNAs on FOXM1 expression.

MATERIALS AND METHODOLOGIES

Concepts

Morphology of cancer cells

Cancer cells have irregular size and irregularly shaped nucleus, with prominent nucleoli. Cytoplasm is very little, they usually have a small cytoplasmic amount, with vacuoles. There occurs accumulation of rRNA and mRNA in the cytoplasm which makes the cytoplasm basophilic with poorly developed golgi apparatus, high variability in mitochondrial size. Reports suggest that glycogen is present in high amounts and it is a characteristic of malignancy. Malignant cells have got unusual microvilli, pseudopods and vesicles. https://www.ncbi.nlm.nih.gov/books/NBK9553/

Cell culture

Cell culture is a major technique which includes removal of cells from an animal or plant and their successive growth into an artificial environment favourable to their survival or proliferation. The temperature, substrate, appropriate growth medium are artificially applied for their optimal growth. The pH and osmorality is kept at a check.

Cell line

A cell line is a cell culture expanded from a single cell and hence consists cells of the exact same genes. It is a permanently established cell culture that will indefinitely proliferate if provided with appropriate fresh medium and space for their growth in the artificial conditions.

It allows us to examine the cells for any alterations in their structure, biology, and genetic makeup under the provided environment for their optimal growth.

 HT-29 cell line

This is a human colorectal adenocarcinoma cell line. They are adherent and morphologically of the epithelial type when in culture. HT-29 cells form a tight monolayer. HT-29 cells overexpress the p53 tumor antigen, but have a mutation in the p53 gene at position 273 resulting in a histidine replacing an arginine. Though these cells can proliferate in cell culture lacking growth factors with a doubling time of around 4 days, it can be reduced to one day with added fetal bovine serum. https://atcc.org/Products/All/HTB-38.aspx .

PASSAGE

A passage number is the number of times a cell culture has been subcultured. Passaging or subculturing of cells is a common procedure where cells from a given culture are divided or “split” into new cultures and fed with fresh media to facilitate further expansion

Cell culture medium

An important step in cell culture is the selection of an appropriate growth medium for the optimal growth of the cells of our interest. It is a liquid or gel prepared to support the growth of microorganisms, cells, or small plants. Cell culture media generally constitutes compounds that can regulate the cell cycle. A typical culture medium is composed of amino acids, vitamins, inorganic salts, glucose and serum as a source of growth factors, hormones and attachment factors. In addition to nutrients, the medium also helps to maintain pH and osmolarity. Examples of cell culture mediums include DMEM, RPMI-1640, etc. The DMEM media was used to culture HT-29 cell line.

DMEM (Dulbecco’s Modified Eagle’s Medium)

ATCC DMEM has 4500 mg/L of glucose and a reduced sodium bicarbonate concentration (1500mg/mL) for use with 5% CO2. https://www.atcc.org/products/all/30-2002.aspx

Cells in culture produce but require only small amounts of it for growth and survival but this affects the pH of medium. With increase in atmospheric, pH of medium decreases.to avoid this problem, sodium bicarbonate is used as buffer in this media. The media turns more acidic with more production of as the cells undergo metabolism. The medium contains an indicator called phenol red which turns yellow as the pH decreases or as the medium turns acidic. In culture media, dissolved is in equilibrium with bicarbonate ions and many cell culture media take advantage of this/bicarbonate reaction to buffer the pH of the media. Dissolves freely into the culture media and reacts with water to form carbonic acid.

DMEM is a basal medium i.e., it contains no proteins or growth promoting agents and hence requires protein to be supplemented for it to become a “complete medium”. It is mostly provided with 10% FBS.

Methods

Cryopreservation of HT-29 Cell line

The cells from previous culture was checked for contamination from bacteria, cryopreservation, mycoplasma and viruses before cryopreservation.

A freeze medium was prepared consisting of FBS: DMSO in the ratio 9:1, i.e., 90%FBS and 10% DMSO v/v. Undiluted DMSO should not be added to a cell suspension as dissolution of DMSO in aqueous solutions gives off heat (exothermic).

The residual media from the cell culture plates were discarded and the cells were treated with trypsin so as to detach them from the culture vessel surface. After adding trypsin, the cells were kept to incubate for about 2-5 minutes.

Little media was added to the trypsinized cells and given a gentle mix. Now this content was taken into an Microfuge tube with the help of a pipette and centrifuged for 3-5 minutes at 2000rpm. The supernatant was discarded and the cell pellet was mixed gently with the prepared freeze medium.

The vials were labelled appropriately with the name of cell line and date. The suspension thus prepared was put into the vials and sealed.

Place the vials into a controlled rate freeze chamber and place the chamber in a -80⁰C mechanical freezer unit until the cryovials achieve a temperature below -80⁰C.

The vials were then stored at -80⁰C until next revival or transfer to liquid nitrogen.

 Revival of HT-29 cryopreserved cells

The cryovials containing the cells are taken out from the liquid nitrogen and carefully placed in lukewarm water taken in a beaker until the suspension was in a semi-thawed condition.

After thawing the vials are decontaminated by spraying with 70% ethanol. Strict aseptic conditions were maintained in a laminar flow tissue culture hood for all further manipulations.

The top of the vial was unscrewed and the contents were transferred to a sterile centrifuge tube containing complete growth medium. The cryoprotectant agent was removed by gentle centrifugation.

The supernatant was discarded taking care not to disturb the soft pellet, and the cells were resuspended in 1ml or 2ml of complete growth medium. Pipette gently to loosen the pellet and break apart clumps. The cell suspension was transferred into the medium in the culture vessel and mixed thoroughly.

The cultures were examined after 24 hours and maintained as per requirement.

  Culture/Passaging of HT-29 Cell line

The cells were checked under a phase contrast microscope before beginning the work. The confluency and morphology of the cells were observed. The medium color and clarity should be checked for possibility of contamination.

The medium from flask was discarded and given a short rinse with 1X PBS. Decant it.

For a T-25 flask, dispense 200-250µL of trypsin EDTA solution in the flask. Spread it over the cell layer by gently tilting the flask.

Incubate at 37⁰C for 2-3 minutes. The cell layer can be seen coming off the bottom surface.

Added 1-2ml of complete medium to the flask and resolve the cell clumps by gently pipetting in and out.

Transfer these cells to a sterile 1.5ml Microfuge tube. Centrifuge the cells at 2000rpm for 3 minutes.

Meanwhile, dispense 10ml of fresh complete medium to a fresh sterile T-75 cell culture flask.

After centrifugation, decant the supernatant and resuspend the cell pellet in 1-2 ml of fresh complete medium by gently pipetting in and out.

Labelled the flask for next passage.

Kept it in 37⁰C incubator. Check the least once a day for growth, contamination and color of medium.

Added sodium hydroxide pellets to the liquid discard and discarded properly after dissolving the pellets.

Cloning procedure for FOXM1 3'UTR containing plasmids

psiCHECK-2 vector:

Description-the psiCHECK-2 is a 6273bp vector designed for optimizing RNA interference assays and monitors changes in expression of target gene which is incorporated with a reporter gene. Reporter genes present in the psiCHECK-2 vector are Renilla luciferase and Firefly Luciferase and decrease in activity of these luciferases can be corelated to RNA interference. The vector has an Ampicillin resistance gene which acts as a selectable marker to select the required colonies of bacteria from the Ampicillin containing medium. It has specific restriction sites for Notl and Xhol restriction endonucleases. (ix)

psicheck2 final.png
    psiCHECK-2 vector  Promega

    Protocol:

    The size of FOXM1 3’UTR is 954 bps. Taking additional 100 bps upstream and downstream of the target sequence, a stretch of 1154 bps was used to design the primers. These primers spanned a stretch of ~1 kb. Platinum Taq polymerase (high fidelity, Invitrogen) at an annealing temperature of 54⁰C according to the manufacturer’s protocol. Hence the resulting amplicon size is 1kb.

    This initial amplicon was cut by double restriction digestion at 37⁰C. The psiCHECK2 vector was also cut using the same two restriction endonucleases as the initial amplicon. The restriction enzymes used were Xhol and Notl. The psiCHECK2 vector has an Ampicillin resistance gene.

    These restriction digestion products were then kept for ligation with T4 ligase (Invitrogen) overnight at 4⁰C following the manufacturer’s protocol.

    The ligated product thus formed was processed for bacterial transformation in DH5-α strain of E. coli.

    After transformation, the bacterial cells were allowed to grow overnight on agar plate containing Ampicillin at 37⁰C. The colonies which had undergone recombination are ampicillin resistant and hence these survived in the ampicillin containing medium. The recombinant colonies were thus selected and inoculated in Luria Bertani broth (LB) and grown overnight at 37⁰C, 200 RPM. These cultures were used for plasmid isolation.

    For the confirmation of clones for FOXM1 3’UTR, PCR was performed using the same primer pair that was used for initial amplification and double restriction digestion using the restriction enzymes used for cutting the vector and the initial amplicon, viz. Xhol and NotI.

    The PCR products and restriction digestion products were run on a 1.8% agarose gel in Tris-Acetate EDTA buffer and the following results were obtained as shown in the figures below.

    FOXM1 3UTR , RE original.png
      confirmation of FOXM1 3’UTR clones by (A) PCR for the same primer pairs used for initial amplification (B) double restriction digestion using Xhol and Notl; the enzymes used for cutting the vector and the initial amplicon

      Cloning procedure for MicroRNA containing plasmids

      Taking additional 100 bps upstream and downstream of the target sequence, a target stretch was used to design the primers. Platinum Taq polymerase (high fidelity, Invitrogen) at an annealing temperature of 54⁰C according to the manufacturer’s protocol.

      This initial amplicon was cut by double restriction digestion at 37⁰C. The pRIP vector was also cut using the same two restriction endonucleases as the initial amplicon. The restriction enzymes used were Xhol and Notl. The pRIP vector has a Kanamycin resistance gene.

      These restriction digestion products were then kept for ligation with T4 ligase (Invitrogen) overnight at 4⁰C following the manufacturer’s protocol.

      The ligated product thus formed was processed for bacterial transformation in DH5-α strain of E. coli.

      After transformation, the bacterial cells were allowed to grow overnight on agar plate containing Kanamycin at 37⁰C. The colonies which had undergone recombination are Kanamycin resistant and hence these survived in the Kanamycin containing medium. The recombinant colonies were thus selected and inoculated in Luria Bertani broth (LB) and grown overnight at 37⁰C, 200 RPM. These cultures were used for plasmid isolation.

      For the confirmation of clones for microRNAs, PCR was performed using the same primer pair that was used for initial amplification and double restriction digestion using the restriction enzymes used for cutting the vector and the initial amplicon, viz. Xhol and NotI.

      The PCR products and restriction digestion products were run on a 1.8% agarose gel in Tris-Acetate EDTA buffer.

       Hemocytometer and cell counting

      The hemocytometer was invented by Louis-Charles Malassez. It is basically a counting-chamber device and it was made so as to obtain main grids inside the chamber for the ease of counting. A rectangular concavity is inscribed to create a chamber. The chamber is divided into small squares. By determining a required area of the grid, the number of cells are counted which are present in a specific volume of the fluid. The concentration of cells in the fluid can hence be calculated.

      Principle:

      The gridded area of the device has nine 1X1 mm (1 mm2) squares. These are subdivided into the following three types- 0.25X0.25 mm (0.0625 mm2), 0.25X0.20 mm ( 0.05 mm2) and 0.20X0.20mm (0.04 mm2) , the central square is further subdivided into 0.05X0.05mm (0.0025 mm2) squares.

      The coverslip is held 0.1mm above the grid by the lifted edges of the hemocytometer to provide each square a defined volume. The coverslip used in the hemocytometer device is thicker so that they are heavy enough to overcome the surface tension of a drop of liquid.

      Dimensions of gridded area of hemocytometer
      Dimensions Area (in mm2) Volume at 0.1mm depth
      1X1 mm 1 100nL
      0.25X0.25mm 0.0625 6.25nL
      0.25X0.20mm 0.05 5nL
      0.20X0.20mm 0.04 4nL
      0.05X0.05mm 0.0025 0.25nL

      Dilution factor:

      The solution of the sample is most often too concentrated which can lead to overlapping of the cells and hence the counting inferred would not be correct. Hence, such concentrated solutions are diluted with an appropriate solution. This dilution needs to be factored into the calculations also and hence the final answer obtained by the calculations are multiplied by the dilution factor.

      Dilution factor = volume of the diluted sample (after dilution)/volume of the original mixture in the sample (before dilution)

      Procedure:

      The polished surface of the hemocytometer and the coverslip were carefully cleaned using 70% ethanol.

      The cover slip was carefully placed on the counting surface of the hemocytometer prior to putting the cell suspension on it.

      10µL of the diluted cell suspension was introduced into the V shaped well using a micropipette. The area under the coverslip automatically fills with the suspension due to capillary action.

      The counting surface was then placed under the microscope and the counting grid was focused to count the cells.

      The average cell count from each of the corner squares was taken. Multiplied this value by the dilution factor.

      This value obtained would let us know how much of the cell suspension needs to be added in each well of the 24 well plate to obtain 2 x 105 cells per well for further downstream processing

      Double transfection in suspension

      Transfection is described as a process of introducing naked or purified nucleic acids into eukaryotic cells Temporary pores are created on the membrane of the cells where the nucleic acid of our interest has to be inserted. Transfection can be carried out via various processes which include electroporation, lipofection (physical treatments) while other methods include chemical or biological particles, viruses which can be used as carriers. Transfection at the US National Library of Medicine Medical Subject Headings(MeSH).

      Lipofectamine is a common transfection reagent used to increase the transfection efficiency of RNA or plasmid DNA into in vitro cell cultures by lipofection. The lipid-based transfection reagent and nucleic acids forms compact complexes which would protect the nucleic acids and these complexes are then incorporated to the cells via endocytosis Dalby B, et al, June 20042003. Lipofection is a lipid mediated DNA-transfection process using liposomes as vectors. Liposomes and the target cell membrane merge with each other since both their membranes are made of phospholipid bilayer. The positively charged lipid mixture forms aggregates with the negatively charged nucleic acids. Lipofectamine used here in the experiments is a transfection reagent which increases the transfection efficiency of RNA or plasmid DNA into in vitro cell cultures by lipofection. Lipofectamine consists of the cationic mixture which encapsulates the negatively charged nucleic acids and allows them to overcome the electrostatic repulsion of the cell membrane. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/lipofection​ .

      Opti MEM is an improved Minimal Essential Medium (MEM) that allows for a reduction of FBS supplementation by at least 50% with no change to growth rate or morphology. The Opti-MEM is supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements and growth factors. The Opti-MEM uses a sodium bicarbonate buffer system (2.4 g/L) and hence requires a 5-10% CO2 environment to maintain physiological pH. https://www.biocompare.com/20024-MEM-w-Lglutamine-Powder/68084-GIBCO-OptiMEM-I-ReducedSerum-Medium-powder/.​

      Protocol:

      The cells cultured in T-75 flask reached about 70% confluency.

      Discarded the media from the culture, gave a PBS wash and then added trypsin. The suspension thus formed was centrifuged at 2000rpm for 3 minutes.

      The pellet obtained was resuspended in fresh media and then took 10µL of it into hemocytometer (after dilution of suspension 20 times) for cell counting.

      Required number of cells were suspended in fresh media so as to obtain 6 x 105 cells per tube. 9 such tubes were prepared, such that each tube had 1.2mL of cell suspension.

      In another 9 Microfuge tubes, 200µL opti-MEM and 10µL lipofectamine each was taken and mixed gently.

      9 Microfuge tubes were taken and each was filled with 200µL opti-MEM, 2µL PLUS enhancer and required amount of plasmid (0.5µg per well). Mixed them gently.

      The opti-MEM, PLUS and plasmid mixture was added dropwise to the opti-MEM and lipofectamine containing tubes.

      The 300µL of lipofectamine + plasmid + opti-MEM mixture was mixed with the 1.2mL cell suspension to obtain 1.5mL transfection mixture per Microfuge.

      Of this 500µL was seeded onto each well (in triplicates) in a 24 well plate and kept for incubation for 24 hours.

      Gave a media change at 24 hours. At 72 hours, the plate was taken for observation under confocal microscopy.

      Transfection in suspension for standardization

      The cells in the culture were let to reach about 70% confluency.

      Discarded the media from the culture, gave a PBS wash and then added trypsin. The suspension thus formed was centrifuged at 2000rpm for 3 minutes.

      The pellet obtained was resuspended in fresh media and then took 10µL of it into the hemocytometer (after dilution) for cell counting.

      Required number of cells were suspended in fresh media so as to obtain 1 x 105 cells per well (3 wells) and 1.5 x 105 cells per well (3 wells) of a 24 well plate.

      Two mixtures were prepared as follows-

      1.     Mixture A:

      100µL opti MEM + 5µL lipofectamine for 2 wells

      2.     Mixture B:

      100µL opti MEM + 1µL PLUS enhancer + 0.8µL plasmid for FOXM1 3’UTR + pRIP (0.5, 0.75, 1µg/well) for 2 wells.

      ·       Mixture ‘B’ was added to the tube containing mixture ‘A’ dropwise. Let this resulting mixture incubate for 5 minutes.

      ·       100µL of above mixture was put with 400µL cell suspension per well w.r.t the following arrangement in a 24 well plate.

      showing the arrangement of wells of 24-well plate for standardization protocol
      pRIP/cell count 1 x 105 cells 1.5 x 105 cells
      0.5µg    
      0.75 µg    
      1.0 µg    

      Figure: showing the arrangement of wells of 24-well plate for standardization protocol

      Incubated at 37⁰C and gave a media change at 24 hours. Kept for further incubation up to 72 hours before observation under confocal microscopy.

      Dual luciferase assay

      Concept: this Assay can be used to study the expression of eukaryotic genes. There occurs expression of two individual reporter enzymes in the same system and hence its name. There is an “experimental” reporter which is simultaneously measured with a “regulated” reporter to study their regulated expressions. The changes in their expression can be studied by comparingchanges in expression of thecoupled promoter. Bruce A. Sherf . The firefly luciferase reporter is measured first by addition of Luciferase Assay Reagent II (LARII) to generate a stable luminescence. After measuring the firefly luminescence, this reaction is eliminated and Renilla luciferase reaction is subsequently initiated by adding Stop & Glo Reagent to that same tube. The Stop & Glo reagent also produces a stable signal from the Renilla luciferase which would slowly decay over the course of measurement.

      Procedure:

      The required amount of LARII is added into appropriate number of luminometer tubes for the number of DLR Assays required.

      Carefully the Cell lysate is transferred into the luminometer tube containing the LARII, and mixed by pipetting. The tube is placed on luminometer to start the reading.

      The Firefly luciferase activity is measured and recorded.

      Using a reagent injector, added Stop & Glo Reagent. And vortex for a short time to mix. The sample is placed again in the luminometer and reading is begun.

      The Renilla luciferase activity thus measured and recorded.

      RESULTS AND DISCUSSION

      Observation for Double Transfection

      Transfection of the HT-29 cells was done according to the above-mentioned protocol. The experiment includes a double transfection in which two plasmids were given for transfection into the HT-29 cells such that one plasmid had the 3’-UTR region and the other had microRNAs of our interest. It was expected that both the plasmids should transfect the cells such that both 3’-UTR and the miRNA are incorporated into the same cells simultaneously and their interaction could be studied. But after the transfection experiment, when the cells were taken for observation under confocal microscopy, it was seen that double transfection had rarely occurred.

      Instead some cells showed the presence of 3’-UTR (green fluorescence) while some other cells showed the presence of miRNA (red fluorescence), but rarely, very few cells showed the presence of both 3’-UTR and miRNA transfected into the same cells indicating that the double transfection experiment had failed.

      It was also observed that transfection efficiency of the plasmids containing 3’UTR was very high which suggests that concentration of plasmids used to transfect the 3’-UTR into the seeded cells was correct.

      On the other hand, it was observed that efficiency of miRNA containing plasmids into the seeded cells was very low, hence these parameters require to be standardized.

      Therefore, these cells can’t be processed further for the Dual Luciferase Assay. To nullify the above observed problems for future experiments, it seems necessary to first standardize the transfection protocol with respect to-

      1.     Plasmid concentration

      2.     Number of cells seeded

      ht-29 untransfected.jpg
        HT-29 cell line before double transfection
        double transfection_1.jpg
          HT-29 cell line after double transfection

          Observation for Standardization

          From the standardization experiments it was observed that pRIP concentration of 1.0µg against a cell count of 1lakh shows successful double transfection.

          When observed under confocal microscopy, it is seen that both green and red fluorescence are present in the same cell. The green fluorescence corresponds to FOXM1 containing plasmids and the red fluorescence corresponds to miRNA containing plasmid.

          This indicates that 1µg concentration of pRIP for transfection of miRNA into the 1 x 105 cells is optimal for co-transfection with FOXM1 containing plasmids.

          Hence, these standardized values can be successfully used for double transfection in the HT-29 cell line for further downstream processing with the Dual luciferase Assay to see the action of microRNA in the upregulation or downregulation of the FOXM1 transcription factor.

          green.jpg
            HT-29 cell transfected with FOXM1 3'-UTR containing plasmid shows green fluorescence under confocal microscopy
            red.jpg
              The same HT-29 cell transfected with microRNA containing plasmid , showing red fluorescence under confocal microscopy

              Discussion

              Transfection protocols may vary based in the type of cell to be transfected, transfection method and transfection reagent used. The most common causes for transfection failures may be reduced transfection efficiency and reduced cell viability. For optimal transfection results, routine subculturing procedures and passaging of cultures are performed once or twice a week such that they become nearly confluent before the next passage. The optimal cell density for transfection varies for different cell types as stated above and it needs to be determined for every new cell line to be transfected. Maintaining proper seeding protocol, the cells were allowed to reach about 70-90% confluency at the time of transfection to ensure proper cell viability after transfection. Although in general serum in culture medium enhances transfection with DNA but some serum proteins may interfere with DNA-lipid complex formation during lipofection i.e., when transfection is performed by cationic lipid-mediated transfection. Also, RNA transfection into cells are preferably performed using reduced serum media to avoid possible contamination with RNAs. This justifies the use of Opti-MEM which is a reduced serum media instead of DMEM which is a complete media.

              Transfection was done at suspension rather than adherent cells because trypsinization of the cells makes them more vulnerable and these cells can take up foreign nucleic acids better than the adherent ones. Too high cell density as against concentration of plasmids used to transfect them can also result in poor nucleic acid uptake or decreased expression if the transfected gene. To ensure this, standardization protocol was performed as described in the methods.

              During the first transfection protocol followed, the efficiency of transfected cells was very low. Rarely few cells had undergone double transfection. To ensure proper double transfection though, standardization protocol was up taken which gave the exact number of cells to be used for successful double transfection as against the required concentration of plasmid containing miRNAs.

              On successful double transfection, such that the plasmid containing FOXM1 and plasmid containing miRNA are incorporated in the same cells, these can be harvested and further proceed with the Dual Luciferase Assay to study the regulation of the FOXm1 gene by the miRNAs.

              CONCLUSION

              Upon successful double transfection of the HT-29 cell line, the Dual Luciferase Reporter Assay would allow to infer the proper conclusion if the novel miRNAs of our interest have a regulatory function on FOXM1 transcription factor. miR-149 has already been established as a potent downregulator for FOXM1 in cancer cell lines. Using the DLR Assay protocol the novel miRNA expressions after transfection into the HT-29 cells can be determined by using miR-149 as a positive control.

              ACKNOWLEDGEMENTS

              First and foremost, I would like to express my sincere gratitude to my guide Professor M. Radhakrishna Pillai for giving me the opportunity to gain experience under his supervision.

              I would like to extend by sincere gratitude towards Dr. S Asha Nair for her continuous support, motivation and immense knowledge whose guidance helped me in all the time of the training period.

              My sincere thanks also go to Ketakee Mahajan for helping me in each step throughout the training period and without whose help I could not have imagined finishing my project in such ease.

              I would also take the opportunity to thank the committee members of Indian National Science Academy for offering me the summer internship opportunity at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram.

              Last but not the least I would like to extend my heartfelt thanks to my parents for supporting me spiritually throughout my life.

              REFERENCES

              1.     https://fightcolorectalcancer.org/prevent/symptoms/

              2.     https://www.cancer.net/cancer-types/colorectal-cancer/risk-factors-and-prevention

              3.     https://www.medicalnewstoday.com/articles/155598.php

              4.     https://www.cancer.net/cancer-types/colorectal-cancer/types-treatment

              5.     https://www.ncbi.nlm.nih.gov/books/NBK9553/

              6.     https://atcc.org/Products/All/HTB-38.aspx

              7.     https://atcc.org/Products/All/HTB-38.aspx#characteristics

              8.     https://www.atcc.org/products/all/30-2002.aspx

              9.       https://www.promega.in/products/quantitation-and-analysis/rna-interference/psicheck-1-and-psicheck-2-vectors/?catNum=C8011

              10.  Transfection at the US National Library of Medicine Medical Subject Headings(MeSH)

              11.  Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, Ciccarone VC (June 2004). "Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications". Methods33 (2): 95–103. doi:10.1016/j.ymeth.2003.11.023PMID 15121163

              12.  https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/lipofection

              13.  https://www.biocompare.com/20024-MEM-w-Lglutamine-Powder/68084-GIBCO-OptiMEM-I-ReducedSerum-Medium-powder/

              CITATIONS

              1. Frank SA. (2007) Dynamics of Cancer: Incidence, Inheritance and Evolution. Princeton (NJ): Princeton University Press

              2. Yang ZH, Dang YQ, Ji G. Role of epigenetics in transformation of inflammation into colorectal cancer

              3Michelle M.J.Mens and Mohsen Ghanbari. Cell cycle regulation of stem cells by microRNAs.

              4. Bartel (2009)

              5. Bueno MJ, Malumbres M. MicroRNAs and the cell cycle.

              6. Musgrove et al., (2011)

              7. Lee RC, Feinbaum RL, Ambros V (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. ;75(5):843–854.)

              8. Reinhart BJ, et al., (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature.;403(6772):901–906)

              9.Engkvist ME, et al., (2017). Analysis of the miR-34 family functions in breast cancer reveals annotation error of miR-34b. Science Reporter.;7(1):9655.)

              10. Okada N, et al. (2014). A positive feedback between p53 and miR-34 miRNAs mediates tumor suppression. Genes & Development.;28(5):438–450.), 

              11.Bommer GT, et al., (2007). p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Current Biology.;17(15):1298–1307.),

              12. He L, et al., (2007). A microRNA component of the p53 tumour suppressor network. Nature.;447(7148):1130–1134.)

              13.Tazawa H, et al., (2007). Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proceedings of the National Academy of Sciences of the United States of America.;104(39):15472–15477.)

              14. Sun F, et al., (2008). Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Letters.;582(10):1564–1568

              15. Bao B, et al., (2012). Targeting CSC-related miRNAs for cancer therapy by natural agents. Current Drug Targets.;13(14):1858–1868.),

              16. Choi et al., (2012); Goga et al., (2007); Horuichi et al., (2012); Molenaar et al., (2009); Sawai et al., (2012)

              17. Xu K, Liu X, Mao X, Xue L, Wang R, Chen L, Chu X. MicroRNA-149 suppresses Colorectal cancer cell migration and invasion by directly targeting forkhead box transcription factor FOXM1.

              18. Zhao L, Liu L, Dong Z, Xiong J. miR-149 suppresses human non-small cell lung cancer growth and metastasis by inhibiting the FOXM1/cyclin D1/MMP2 axis.

              19). Bruce A. Sherf, Shauna L. Navarro, Rita R. Hannah and Keith V. Wood. Dual-Luciferase reporter Assay: An Advanced Co-Reporter Technology Integrating Firefly and Renilla Luciferase Assays

              References

              • https://fightcolorectalcancer.org/prevent/symptoms/

              • https://www.cancer.net/cancer-types/colorectal-cancer/risk-factors-and-prevention

              • https://www.medicalnewstoday.com/articles/155598.php

              • American Cancer Society

              • Frank SA. (2007) Dynamics of Cancer: Incidence, Inheritance and Evolution. Princeton (NJ): Princeton University Press

              • Yang ZH, Dang YQ, Ji G. Role of epigenetics in transformation of inflammation into colorectal cancer

              • Michelle M.J.Mens and Mohsen Ghanbari. Cell cycle regulation of stem cells by microRNAs.

              • Bartel (2009)

              • Bueno MJ, Malumbres M. MicroRNAs and the cell cycle.

              • Musgrove et al., (2011)

              • Reinhart BJ, et al., (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature.;403(6772):901–906)

              • Lee RC, Feinbaum RL, Ambros V (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. ;75(5):843–854.)

              • Engkvist ME, et al., (2017). Analysis of the miR-34 family functions in breast cancer reveals annotation error of miR-34b. Science Reporter.;7(1):9655.) 

              • Okada N, et al. (2014). A positive feedback between p53 and miR-34 miRNAs mediates tumor suppression. Genes & Development.;28(5):438–450.),  

              • Bommer GT, et al., (2007). p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Current Biology.;17(15):1298–1307.),

              • He L, et al., (2007). A microRNA component of the p53 tumour suppressor network. Nature.;447(7148):1130–1134.)

              • Tazawa H, et al., (2007). Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proceedings of the National Academy of Sciences of the United States of America.;104(39):15472–15477.)

              • Sun F, et al., (2008). Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Letters.;582(10):1564–1568

              • Bao B, et al., (2012). Targeting CSC-related miRNAs for cancer therapy by natural agents. Current Drug Targets.;13(14):1858–1868.)

              • Choi et al., (2012); Goga et al., (2007); Horuichi et al., (2012); Molenaar et al., (2009); Sawai et al., (2012)

              •  Xu K, Liu X, Mao X, Xue L, Wang R, Chen L, Chu X. MicroRNA-149 suppresses Colorectal cancer cell migration and invasion by directly targeting forkhead box transcription factor FOXM1

              • Zhao L, Liu L, Dong Z, Xiong J. miR-149 suppresses human non-small cell lung cancer growth and metastasis by inhibiting the FOXM1/cyclin D1/MMP2 axis

              • https://www.ncbi.nlm.nih.gov/books/NBK9553/

              • https://atcc.org/Products/All/HTB-38.aspx

              • https://www.atcc.org/products/all/30-2002.aspx

              • Promega

              • Transfection at the US National Library of Medicine Medical Subject Headings(MeSH)

              • Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, Ciccarone VC (June 2004). "Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications". Methods33 (2): 95–103. doi:10.1016/j.ymeth.2003.11.023. PMID 15121163

              • https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/lipofection​

              • https://www.biocompare.com/20024-MEM-w-Lglutamine-Powder/68084-GIBCO-OptiMEM-I-ReducedSerum-Medium-powder/

              • Bruce A. Sherf, Shauna L. Navarro, Rita R. Hannah and Keith V. Wood. Dual-Luciferase reporter Assay: An Advanced Co-Reporter Technology Integrating Firefly and Renilla Luciferase Assays

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