Investigating the expression of some miRNAs in retinoblastoma cancer derived cell lines overexpressing TGIF2LX due to exposure to SD-208 drug

Master of Medical Biotechnology

Abstract

Scope and purpose: Retinoblastoma is one of the most common intraocular tumors in children. Although there is progress in the treatment of retinoblastoma, the failure in treatment and mortality in developed countries is remarkable. The most important reason for this failure is drug resistance and its side effects. The aim of this study is to evaluate the interaction between the anticancer drug SD-208 and the increase in TGIF2LX expression in retinoblastoma cell line Y79 cells.

Research method: In the first stage of TGIF2LX protein evaluation, the PEGFP-N1 vector containing the entire TGIF2LX gene sequence was transfected into Y79 cells and its expression was confirmed by UV microscopy and Realtime RT-PCR. Along with transfection, cells were treated with different doses of SD-208 drug (0, 1 µM, 2 µM). Then, the expression pattern of 5 miRNA including Let7g, 18a, 34a, 22, 20a in treated and untreated transfected cells was compared to non-transfected cells.

Findings: We found out that all miRNAs in the transfected cells treated with SD-208 had an increased expression p<0.05 except for miRNA20a.

Conclusion: This is the first study that showed that SD-208 increases the expression of the hemobox gene and different miRNAs that play a tumor suppressor role in retinoblastoma. Also, our results suggest that the simultaneous use of TGIF2LX and SD-208 can be a new method in retinoblastoma treatment.

Key words: retinoblastoma, TGIF2LX, miRNA, Y79 Cell line, SD-208

1.1 Retinoblastoma

Retinoblastoma is one of the most common malignant eye cancers in children, which exists in two forms: sporadic (isolated) and hereditary (familial) [1]. Approximately 4% of children's tumors are made up of retinoblastoma. This tumor is the most common primary eye malignancy. In the West, 99% of children are cured of this cancer, but more than 90% of them lose their sight, and unfortunately, in developing countries, the child's survival rate is approximately 50% [2] and [3]. 1-1) whose parents refer to Mashund[4].

Figure 1-1: The right eye of a child with leukocoria (cat's eye)

1.1.1.Epidemiology

Retinoblastoma occurs with a prevalence of approximately 1 in 15,000 and 1 in 16,600 live births in America and Northern Europe [5] and [6] and between 2005-2009, the annual prevalence Retinoblastoma in America is 4.1 per million children under the age of 15 [5] and in the whole world 5000 to 8000 children are diagnosed with retinoblastoma every year [7] The average age of diagnosis is under 2 years and almost 95% is before the age of 5. The prevalence of the disease is similar between boys and girls and black and white [5].

Approximately 1/4 of the cases Retinoblastoma is bilateral. Bilateral diseases always have a hereditary pattern. Bilateral tumors occur earlier in children, which indicates the presence of mutations in germ cells. The hereditary form of retinoblastoma requires a germline mutation that can be from either parent or from the environment (which leads to a germline mutation). On the contrary, only 15% of unilateral cases are hereditary, which are often multifocal and should be investigated for germline mutations, which usually occur in the first 2 years of life. Less than 10% of retinoblastoma patients have a positive family history.

Almost 60% of children with retinoblastoma have a non-inherited unilateral pattern. Children with non-hereditary retinoblastoma have a new mutation in a They have retinal cells that lead to tumors [8]. Genetic abnormality in the hereditary form of retinoblastoma causes the development and development of tumors such as osteogenic sarcoma, soft tissue sarcoma (especially leiomyosarcoma) and malignant melanoma. The prevalence of secondary cancer after the diagnosis of retinoblastoma in the hereditary and non-hereditary form is 51 and 5%, respectively, and more than 60% of cancers are sarcoma [9].

1.1.2. Pathogenesis[2]

Retinoblastoma is usually caused by the inactivation of both retinoblastoma alleles. With the autosomal dominant pattern of this gene in the region of chromosome 13, the long arm is located in region 14, which encodes a nuclear protein with a tumor suppressor role [10]. Clinical characteristics of hereditary and non-hereditary cases of retinoblastoma [11]. (Figure 1?2)

Figure 1?2: 2 hit model of retinoblastoma

In the hereditary model, there is a mutation in the RB1 gene in all cells, and the second hit occurs in the later stages of development, and these people are prone to bilateral and multifocal retinoblastoma. The second hit can occur or be silenced by epigenetic changes.

In the model of non-hereditary retinoblastoma, two mutations in one allele occur spontaneously in a somatic cell of the retina, which usually leads to the clinical model of a unilateral focal tumor of retinoblastoma [12]. It metastasizes to the brain 4 months after diagnosis and death occurs within a year. Most of the ways of tumor metastasis are through the optic nerve [3] to the central nervous system or spread through the choroid to the orbit [13]. It is retinoblastoma although there are other symptoms and leukocoria is not necessary for diagnosis. The most common symptoms are leukocoria (54%) and strabismus (19%), decreased vision (4%), eye infection (5%) and positive family history (5%), and other cases are heterochromic iris, vitreous hemorrhage, hyphema without trauma, glaucoma, orbital cellulitis, proptosis, eye pain, and fever [14].

1.2 Family history

The risk level among the person's generation depends on the family history of retinoblastoma or the type of tumor in the person (for example, unilateral or bilateral, one focus or two focus). The risk level is from 6% (if the proband has a unilateral and one focus disease or has a negative family history) to 50% (if the proband has a germline mutation or is suspected to have a mutation)[15].

1.3-Diagnosis

Diagnosis of retinoblastoma is done through a dilated pupil and an indirect ophthalmoscopy device under anesthesia. The findings are gray chalky retinal masses and Crispy is found. Pathology is necessary to confirm the diagnosis. Although auxiliary tests are not always necessary, they may be performed to confirm the diagnosis. Ophthalmological ultrasound or computed tomography (CT) scan shows a solid tumor with calcification characteristics. Magnetic resonance imaging [4] can also prove the presence of an intraocular mass, especially in cases where it is difficult to diagnose the disease with Kat's disease. Fundoscopy findings can distinguish retinoblastoma from Kat's disease and from exudative retinal tear disease, but it cannot show calcification, which is an important factor in diagnosing tumor size, optic nerve involvement, and the presence of intracranial damage [14].

1.4 Evaluation Before treatment

Non-genetic tests: The evaluation of children with retinoblastoma is completely unique to choose a treatment model (for example, the basic number of cells and blood biochemistry to start chemotherapy). For patients with small tumors (except in familial cases), a complete examination and examination under anesthesia, ultrasound, and MRI of the head and eyes are usually sufficient [17]. In the early stages of diagnosing the presence of metastasis cases, it is rare (testing bone marrow and spinal fluid and bone scan) and usually these cases are not tested [18]. However, if there is evidence of a tumor outside the eyeball (invasion of the optic nerve or choroidal involvement), complete evaluation of metastasis should be performed.