This research would not have been successful without the kind help and extensive support of many mentors, especially my main advisor Assoc. miRNA146a polymorphisms were not significantly associated with ALL susceptibility and clinicopathological variables in Thai childhood.
LIST OF TABLES
- Background
- Objectives of research
- Research hypothesis
- Scope of the research
- Benefits of the research
Therefore, the genotyping of miRNA 146a (rs2910164) was determined in this study and the gene frequency was compared between childhood ALL and healthy children. The association of miRNA146a (rs2910164) G>C polymorphism with childhood ALL was investigated in this study.
LITERATURE REVIEWS
Hematopoiesis
Leukemia
Some forms of acute leukemia respond well to treatment and many patients can be cured. The four main types of leukemia are acute lymphocytic leukemia (ALL), which is most common in children, acute myeloblastic leukemia (AML), which is most common in adults, and chronic lymphocytic leukemia (CLL), which is most common in older adults (more than 60 years). years) and chronic myelocytic leukemia (CML), which is most common in adults.
Acute lymphoblastic leukemia (ALL)
The terms myeloid and lymphoid refer to the types of cell lines with which leukemia first began. Both adults and children can develop leukemia, but certain types are more common in different age groups.
Epidemiology of ALL
Genome-wide association studies suggest that genetic polymorphisms of some susceptibility genes are associated with childhood ALL development (Treviño, et al., 2009). Long-term event-free survival (EFS) rates for childhood B-ALL approach 90%, although infants are associated with poorer prognosis and lower EFS rates (Inaba, et al., 2013).
Signs and symptoms of ALL
A platelet deficiency can cause easy bruising and bleeding (Figure 2.5), frequent or severe nosebleeds, or bleeding gums. Occasionally, deposits of leukemic cells can occur in the bone itself, which can cause local pain.
Risk factors
These types of factors are important in many adult cancers, but are unlikely to play a role in most childhood cancers. There is evidence that viral infections may play a role in the development of ALL in some children.
Diagnosis of ALL
The number of blast cells in the bone marrow examination helps diagnose ALL. Tendency to be eccentric, round, oval, or sometimes complex, especially in the form with a monocytic component.
The standard treatments of ALL
Radiation therapy to the brain and spinal cord is sometimes used as CNS sparing therapy to treat high-risk children. A stem cell transplant is a method of giving very high doses of chemotherapy and sometimes radiation therapy to kill the leukemia cells, and then.
MicroRNAs (MiRNAs)
MiRNA processing
MiRNA gene polymorphism
SNPs can occur in miRNA biogenesis pathway genes include pri-miRNA, pre-miRNA or mat-miRNA (Deel, et al., 2010). Recently, the most common pre-miRNA SNPs that have been studied include SNPs of miRNA146a, miRNA196, miRNA149, and miRNA499 (Srivastava and Srivastava, 2012).
MiRNA146a
Target molecules Cellular system Biological consequences TRAF6, IRAK1 (adaptor . molecules downstream of Toll-like receptors and cytokines). IL-8, RANTES Lung epithelial alveolar cells Innate immune response CCL (MCP2) HIV-infected microglial cells Innate immune response.
Laboratory investigation of SNPs
TaqMan probes are oligonucleotides constructed with a fluorescent reporter dye attached to the 5' end of the probe and a quencher at the 3'. The short pieces of DNA called primers are designed to bind to the beginning and end of the DNA target. Nucleotides are added to the PCR reaction mixture, the nucleotides consist of the four bases adenine (A), thymine (T), cytosine (C) and guanine (G).
Billions of copies of the original DNA can be generated within hours after multiple cycles of replication in a computer-controlled thermocycler that provides alternating cycles of heating and cooling. The annealing temperature is related to the melting temperature (Tm) of the primers and must be determined for each pair of primers used in the PCR. The third step, extension of the DNA chain by adding nucleotides from the primers using DNA polymerase as a catalyst in the presence of Mg2+ ions.
MATERIALS AND METHODS
- Instrument and equipment
- Chemical reagent
- Study population
- DNA isolation
- DNA extraction in healthy control group
- DNA extraction in patients group
- Genotyping of the miR 146a (G>C) polymorphism
- Polymerase chain reaction-restriction fragment length polymorphisms (PCR- RFLP) for miR 146a (G>C) polymorphism
- Polyacrylamide gel electrophoresis
- Quality control procedures
- Statistical analysis
Genomic DNA was isolated from 3 ml of peripheral blood by proteinase K digestion and salting out method. Red blood cells were lysed with 1X Rbc lysis buffer for 10 minutes and incubated at 4◦C for 10 minutes, then centrifuged at 4000 rpm for 10 minutes at 4◦C. The white blood cell pellet was incubated in 1X nuclear lysis buffer 3 ml, 25 µl 20 mg/ml proteinase K and 200 µl 10% sodium dodecyl sulfate (SDS), incubated at 37◦C overnight or incubated at 57◦C for 4 hours.
Remove DNA pellet to microtube 1.5 ml and DNA pellet was washed with 1 ml of cold 75% ethanol, centrifuged 13,000 rpm for 10 minutes at 4◦C, the washing step was repeated three times. Genomic DNA in patient was isolated from 3 ml of peripheral blood by phenol chloroform method. To remove traces of phenol, add 4 ml of chloroform-isoamyl alcohol and mix briefly, then centrifuge at 4000 rpm at 4◦C for 10 minutes.
RESULTS
Genotyping of the miR146a (G>C) polymorphism
PCR products were found to be 147 bp in length by 2% agarose gel electrophoresis, stained with 0.5 µg/ml ethidium bromide, and then visualized using gel documentation.
Polymerase chain reaction-restriction fragment length polymorphisms (PCR- RFLP) for miR 146a (G>C) polymorphism
Polymerase chain reaction-restriction fragment length polymorphisms (PCR- RFLP) for miR 146a (G>C) polymorphism in ALL patients
Lane 2, ALL 13 is a PCR product in ALL patients and the genotype is G/G Lane 3, ALL 15 is a PCR product in ALL patients and the genotype is C/G Lane 4, ALL 18 is a PCR product in ALL patients and the genotype is C/C Lane 5, ALL 19 is PCR product in ALL patients and genotype is C/G Lane 6, ALL 20 is PCR product in ALL patients and genotype is C/C Lane 7, ALL 21 is PCR product in ALL patients and genotype is G /G Lane 8, ALL 22 is PCR product in ALL patients and genotype is C/C Lane 9, ALL 23 is PCR product in ALL patients and genotype is C/G Lane10,ALL 24 is PCR product in ALL patients and genotype is C /G. Lane 2, ALL 36 is a PCR product in ALL patients and the genotype is C/G Lane 3, ALL 38 is a PCR product in ALL patients and the genotype is C/C Lane 4, ALL 40 is a PCR product in ALL patients and the genotype is C/G Lane 5, ALL 41 is PCR product in ALL patients and genotype is C/C Lane 6, ALL 43 is PCR product in ALL patients and genotype is C/C Lane 7, ALL 44 is PCR product in ALL patients and genotype is C /G Lane 8, ALL 46 is PCR product in ALL patients and genotype is C/G Lane 9, ALL 47 is PCR product in ALL patients and genotype is C/G Lane10,ALL 48 is PCR product in ALL patients and genotype is C /G. Lane 2, ALL 50 is a PCR product in ALL patients and the genotype is C/C Lane 3, ALL 53 is a PCR product in ALL patients and the genotype is C/C Lane 4, ALL 56 is a PCR product in ALL patients and the genotype is C/C Lane 5, ALL 57 is PCR product in ALL patients and genotype is C/G Lane 6, ALL 58 is PCR product in ALL patients and genotype is C/G Lane 7, ALL 62 is PCR product in ALL patients and genotype is C /C Lane 8, ALL 63 is PCR product in ALL patients and genotype is C/C Lane 9, ALL 64 is PCR product in ALL patients and genotype is C/C Lane 10, ALL 65 is PCR product in ALL patients and genotype is C /C.
Lane 2, ALL 98 is PCR product in ALL patients and genotype is C/C Lane 3, ALL 99 is PCR product in ALL patients and genotype is C/G Lane 4, ALL 100 is PCR product in ALL patients and genotype is C/C Lane 5, ALL 101 is PCR product in ALL patients and genotype is C/C Lane 6, ALL 102 is PCR product in ALL patients and genotype is C/C Lane 7, ALL 103 is PCR product in ALL patients and genotype is C /C Lane 8, ALL 104 is PCR product in ALL patients and genotype is C/G Lane 9, ALL 105 is PCR product in ALL patients and genotype is C/G Lane10, ALL 106 is PCR -product in ALL patients and genotype is C /C. Lane 2, ALL 107 is PCR product in ALL patients and genotype is C/G Lane 3, ALL 108 is PCR product in ALL patients and genotype is C/C Lane 4, ALL 109 is PCR product in ALL patients and genotype is C/G lane 5, ALL 110 is PCR product in ALL patients and genotype is C/G lane 6, ALL 111 is PCR product in ALL patients and genotype is C/C lane 7, ALL 112 is PCR product in ALL patients and genotype is G /G Lane 8, ALL 113 is PCR product in ALL patients and genotype is C/G Lane 9, ALL 114 is PCR product in ALL patients and genotype is C/C Lane10, ALL 115 is PCR -product in ALL patients and genotype is G /G. Lane 2, ALL 117 is PCR product in ALL patients and genotype is C/C Lane 3, ALL 118 is PCR product in ALL patients and genotype is C/G Lane 4, ALL 119 is PCR product in ALL patients and genotype is C/C Lane 5, ALL 120 is PCR product in ALL patients and genotype is C/G Lane 6, ALL 121 is PCR product in ALL patients and genotype is C/G Lane 7, ALL 122 is PCR product in ALL patients and genotype is C /C Lane 8, ALL 123 is PCR product in ALL patients and genotype is C/G Lane 9, ALL 124 is PCR product in ALL patients and genotype is C/G Lane10, ALL 125 is PCR -product in ALL patients and genotype is C /G.
Polymerase chain reaction-restriction fragment length polymorphisms (PCR- RFLP) for miR 146a (G>C) polymorphism in controls
The allele frequencies for miR146a genotype in childhood ALL and controls
The allele frequencies for miR146a genotype in childhood ALL and controls with clinico-pathological variables
DISCUSSIONS AND CONCLUSIONS
Jazdaewski, et al., 2008, first reported that the miRNA146a (rs2910164) G>C was associated with increased susceptibility to papillary thyroid carcinoma. Yin-Hung Chu et al. study the impact of miRNA gene polymorphisms on the sensitivity of environmental factors leading to carcinogenesis in oral cancer. Saki et al., 2014 used Jakrat T cells as a model for in vitro investigation of T-ALL to investigate the role of miR146a on gene expression involved in T cell differentiation.
It is possible that the mature miR146a cannot be produced due to the polymorphic form of miR146a. Therefore, RUNX1 cannot be upregulated by miR146a in all cases of T-cell ALL that is a genotype of miR146a G>C. As shown that in this study, mature miR146a did not occur, because all cases of T-cell ALL have genotype of miR146a G>C.
A common polymorphism in pre-microRNA-146a is associated with lung cancer risk in a Korean population.” Gene. Acute Lymphocytic Leukemia in Children and Adolescents: Prognostic Factors and Analysis of Survival.” Rev Bras Hematol hemoter. Smith, M., Arthur, D., Camirra, B., “Uniform Approach to Risk Classification and Treatment Allocation for Children with Acute Lymphocytic Leukemia.” J Clin Oncol.
A functional genetic variant in microRNA-196a2 is associated with increased susceptibility to lung cancer in Chinese.”. Management of adult and pediatric acute lymphoblastic leukemia in Asia: resource-stratified guidelines from the Asian Oncology Summit. Lancet Oncol. Polymorphism of the pre-miRNA146a is associated with the risk of cervical cancer in a Chinese population.
APPENDICES
APPENDIX A
REAGENT PREPARATION
Prepare a stock solution of 10x Tris –borate-EDTA (TBE) buffer
Prepare 10% ammonium persulfate (APS)
Prepare 10% Acrylamide gel
APPENDIX B SUMMARY DATA
BIOGRAPHY
