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Initial Study on the Regulatory Mechanism of miR-205in the Adrenocortical Carcinoma

Author: XuWenQing
Tutor: HuWeiLie
School: Southern Medical University,
Course: Department of Urology
Keywords: MicroRNA Adrenocortical cancinoma Apoptosis Cell cycleinvasiion and metastasis
CLC: R737.11
Type: Master's thesis
Year: 2013
Downloads: 1
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Abstract


Backgroud:MicroRNAs (miRNA) are small non-coding RNAs (18-25nucleotides) that negatively regulate the expression of taget genes.Their biogenesis in animals is a two-step process.The primary transcript,called primary miRNA(pre-miRNA), is transcribed by RNA pol III, and several hundred nucleotides(nts) long. In the first step, which takes place in the nucleus, pri-miRNA is processed by a multiprotein complex containing an enzyme called Drosha to give rise to approximately70-nt long,stem-loop,precursor miRNA(pre-miRNA). Exportin5(Exp-5) utilizes Ran-GTP and exports pre-miRNA to cytoplasm,where the second step takes place. The pre-miRNA is cleaved by an enzyme called Dicer, to produce a21-nt long mature duplex miRNA, one strand of which associates with the RNA-induced silencing complex(RISC),which the other one is degraded by cellular nucleases. The miRNA-RISC complex then bind to its comlementary3’untranslated region(3’UTR) in a specific target mRNA,leading to translational repression or cleavage of these mRNAs.Thus,miRNAs modulate protein expression either by promoting RNA degradation,or inhibiting mRNA translating.Studies had found about1000species of miRNA in the human body, and control about one-third of human gene expression process. One miRNA can control a variety of mRNAs, wheres as various miRNA can be controled by the same mRNA. MiRNA3’UTR region can contain more than one role of miRNA loci and associated with the degree of inhibition of translation, miRNA loci.A complex and precise network is formed by the miRNAs that regulates genes expression and psysiogical functions.Diease can happen when the miRNA expression levels loss balance.Genes stable expression ensures the procedures of cell division, differentiation, and apoptosis process orderly.Abnormal expression of human genes lead to extraordinarily cell proliferation and apoptosis,than result in tumorigenesis.Current research found that mirnas are related to lung cancer, breast cancer, pancreatic cancer, leukemia, etc.Abnormal expresion of miRNA could fould in these tumor cells, but their working mechanism in toumors is not completely clear.The relationship between mircoRNA and tumors mainly based on the following findings:1.MiRNA genes mainly locate in tumor related genomes areas and instability genome areas。2.The miRNA expression levels in normal tissue cells and tumor cells are not the same, the miRNA expressions in tumor cells are specificity dsyregultion,indicates the miRNA may create conditions for tumorigenesis.Studies have found that some of the miRNA can directly control the proliferation, differentiation and apoptosis of tumor cells.Numerous studies show that micrornas are association with tumorigenesis, progression, and metastasis and prognosis.MicroRNAs exists in a variety of tinterstitial fluid, and it’squality is stable.Chen used Solexa sequencing technology to sequence for RNAs in healthy individuals,and found there are a variety of small RNAs in serum such as microRNA rRNA and mRNA,but the main are microRNAs.They checked results by using quantitative PCR for some randomly selected several microRNAs,and found those microRNAs also expressed in serum.Then some studies found that microRNAs exist in most of human body fluid include urine, cerebrospinal fluid, saliva, semen,hydrothorax and peritoneal dropsy. MicroRNAs were thougt as an instability state the serum because there are RNA enzymes in serum. Unexpected, the study pointed out that microRNAs can in serum resistant to degradation of RNA enzyme in serum. Serum levels of microiRNAs can be placed for a long time.The quantity of microRNA did not obviously change after store at room temperature for1week.The quality of miRNA did not be seen significant change under conditions of strong acid or strong alkaline conditions or pressure at the boil.Micrornas can still be detected in the malin fixed, paraffin embedded sections for years. Micrornas plays an important role in the study of tumor pathogenesis and molecular diagnosis and prognosis ability in view these characteristics of microRNAs.Adrenocortical carcinoma is a rare and highly aggressive and strong invasive malignancy,accounting for an estimated0.02%of all cancers.They usually had metastases to distant sites at the time of diagnosis.They are high in tumor recurrence.5-year survival of adrenocortical carcinoma is less than30%.It is difficult to preoperative diagnosis of adrenocortical carcinoma to identify adrenocortical form adrenal cortical benign tumors under the microscope.lt is particularly important to search for better biomarkers in the diagnosis of adrenal cortical carcinoma, treatment and prognosis.A lot of research on biomarkers of adrenocortical carcinoma had been researched in recent years.They are defective in clinical application because of the low specificity and sensitivity.As other malignancies,the development and progression of adrenocortical carcinoma involves in many factors, such as activating of oncogene, invalidating of cancer suppressor genes and abnormal expressing of apoptosis genes.Gene mutation and chromosome abnormality paly an important role in the process.The epigenetic changes such as abnormal expression of oncogenes and tumor-suppressor genes caused by microRNAs also plays an important role in the process.The researchers found that a variety of micrornas were dsyregulation in adrenocortical carcinoma and play an important role in tumorigenesis, development and prognosis of the cancers.MiRNA expression profiles were determined in a series of adrenocortical carcinoma(ACC),adenoma(ACA) and normal cortices using microarray by Ozata.A subset of miRNAs showed distinct expression patterns in ACC compared with adrenal cortices and adenomas.Among others, miR-483-3p,miR-483-5-p,miR-210,and miR-21were found over expressed,while miR-195,miR-497and miR-1974were under expressed in ACC.Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195or miR-497reduced cell proliferation in human NCI-H295R ACC cells.In addition, downregulation of miR-483-3p,but not miR-483-5p,and increased expression of miR-195or miR-497led to significant induction of cell death. Patterson found a total of23differentially expressed miRNAs between ACCs and ACAs,among they miR-100,miR-125b and miR-195were down-regulated while miR-483-5p was up-regulated in a study of adrenocortical carcinoma and adenoma. Soon found that ACC patients with up-regulated miR-483-5p and down-regulated miR-195identified a subset with poorer disease specific survival.Cherradia found that the quantitation of miR-139-5p confirmed its marked over-expression in recurrent adrenocortical carcinoma tumors.Interestingly,miR-139-5p level in no-recurrent adrenocortical carcinoma tumors was not significantly different from its level in the normal adrenal cortex,suggesting that this miRNA maybe used as prognostic biomarker of recurrent ACC. More and more microRNAs in adrenocortical carcinoma have been confirmed to play an important role in oncogenesis with continuously study.Identifing expression and investigating regulation mechanism the expression, and investigating regulation mechanism of these miRNAs can promote people to clarify the pathogenesis of adrenocortical carcinoma at an early date and provide important basis for explore potential treatment targets and new treatment.We found that the expression level of miR-205in adrenocortical carcinoma was lower in than that in normal adrenal tissue in our preliminary study,suggesting the expression of miR-205is negatively correlated with the tumorigenesis and development of adrenocortical carcinoma. MiR-205could inhibit the growth of adrenocortical carcinoma.Objective:To find potential target genes of miR-205through the miRNA related websites. Up-regulation the expression of miR-205in human adrenocortical carcinoma cell line SW-13,analysis the expression of target genes by real-time quantitative PCR,test the change of protein which involved in cell cycle, apoptosis, invasion.Explore mechanism of miR-205in adrenocortical carcinoma.Method:1.To find potential target genes of miR-205through the miRNA related websites such as PicTar (http://pictar.mdc-berlin),Scan (http://www.targetscan.org) and miRSVR (http:/www.microrna.org/microrna/home.)2.Acquire miR-205up-regulation cells by miR-205eukaryotic expression vector transfection into human adrenocortical carcinoma cell line SW-13.Set in the control group and blank group at the same time:control group cells were transfected blank plasmid,blank group SW-13cell were transfected no reagent3.Extracted total RNA and then reverse transcriptase to cDNA, analysis target genes of miR-205expression by real-time quantitative PCR.Extraction of proteins of cell line and analysis its protein expression by western blot at the same time.4.Analysis the expression of the proteins involved in cell cycle, apoptosis and invasion by western blot after transfected miR-205.These proteins included Bax、 Caspase-3、p27、cyclingD1、P16、caspase-9and E-cadherin.Outcome:1.We found PCAF、E2F1、VEGF-A and Bcl-2are the target genes of miR-205through the miRNA related websites such as PicTar,Scan and miRSVR.2.Western blot results showed that miR-205the group PCAF, E2F1, VEGF-A, the Bcl-2protein expression levels were lower in the miR-205group than the blank group and the control group (P values<0.05), and the expression levels were no difference between control group and blank group (P>0.05);3.Real-time quantitative PCR results showed that PCAF,E2F1, VEGF-A, Bcl-2mRNA expression were lower in the miR-205group than the blank group and the control group, and the difference had statistical significance (P<0.05).These genes mRNA expression no statistical difference between.control group and blank group.4.Western blot results showed that the protein expression levels of Bax, Caspase3, Caspase-9, E-cadherin protein in miR-205group were higher than blank group and the control group (all P values<0.05), the expression level of cyclinDl was lower in miR-205group whereas the protein expression level of P27was no difference among the three groups.Conclusion:1.MiR-205can inhibit the expression of Bcl-2, down-regulated mitochondrial apoptotic pathways downstream protein Bax, caspase-3,9, promote cell apoptosis.MiR-205can inhibit tumor growth in adrenocortical carcinoma.2.MiR-205can inhibit the expression of E2F1, induce G1arrest, then inhibit the growth of cell, suppressed tumor occurrence and development.3.MiR-205can mediate the expression of VEGF-A from mRNA and protein levels. Increased the expression of miR-205can down-regulation VEGF-A,result in increasing expression of intercellular adhesion factor E-cadherin expression, accordingly increased cell adhesion effect. miR-205can inhibit tumor invasion and metastasis by inhibit the expression of VEGF-A and raise the expression E-cadherin expression.4.MiR-205in SW-13cell can inhibit the expression of PCAF in mRNA and protein levels, then inhibit cancer cell proliferation,tumor occurrence and development.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor
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