The use of microRNA for the treatment of prostate cancer: in vitro and in vivo studies

Author(s)

Alexandre Iscaife

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2016-06-10

Examining board members

Katia Ramos Moreira Leite; Valdemar Ortiz; José Pontes Junior; Eduardo Moraes Rego Reis; Miguel Srougi

• Advisor: Katia Ramos Moreira Leite

Abstract

Introduction: Prostate cancer (PCa) is the most common neoplasia of man in Western countries and the second cause of death by cancer in men in the US, Europe and Brazil. The localized cancer has high cancer-specific survival when treated properly, however metastatic disease still presents low effective treatments with 28% of global survival. microRNAs (miRNAs) are a group of small RNA molecules containing from 19 to 25 nucleotides of noncoding protein with fundamental action in the regulation of gene expression. They are involved in key processes in normal and neoplastic cells as cell cycle, proliferation, apoptosis, energy metabolism, invasion and metastasis. Objectives: To carry out studies in vitro and in vivo using miRNA in a novel model of metastatic prostate cancer in our country in order to evaluate its potential as a therapeutic agent of this neoplasia. Methods: In the in vitro studies, three cell lines were used (PC3, DU145 and LNCaP). These cell lines were transfected with miRNAs 100, 145 and 373 and their antiMiRs using lipofectamine. We analyzed the gene expression of mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 by real-time polymerase chain reaction (qRT-PCR). We also performed studies of apoptosis, cell cycle and ploidy using flow cytometer. Changes in the invasion potential were evaluated by the technique of matrigel. The pre-clinical model in vivo was developed by intracardiac injection of PC-3MLuc-C6 cell line in NUDE mice with 9 weeks. Tumor growth was evaluated with an in vivo image system (IVIS). After the full establishment of metastases on day 21, the animals were treated with three injections into the tail vein containing the miRNA plus atelocollagen. The animals were sacrificed on day 48 for tissues analysis. Results: MiR-100 increases apoptosis in LNCaP and reduces apoptosis in DU145. The anti-miR-100 increased apoptosis in 14% in PC3. In cell line DU145, miR-100 inhibited proliferation. In the analysis of gene expression, the miR-100 inhibits SMARCA5 in DU145 and PC3 and mTOR in LNCaP, anti-miR-100 stimulates mTOR and SMARCA5 in LNCaP. The miR-145 promoted an increased in apoptosis by 24% in DU145. In PC3 cell line miR-145 acts by inhibiting the proliferation, with an absolute difference of 18% compared to control. MiR-145 inhibits KRAS and CMYC in the three cell lines and anti-miR-145 stimulates CMYC in DU145 and KRAS in the three cell lines. The miR-373 reduced apoptosis by 29% in DU145 and reduces proliferation with an absolute difference of 13% relative to control. MiR-373 stimulates MMP9 in DU145 and LNCaP cells and inhibits CD44 in PC3. The anti -miR-373 inhibits MMP9 in DU145 and LNCaP. In the in vivo studies of metastatic PCa, miR-100 shows a tendency to decrease tumor growth (p=0.23) and miR-145 reduces tumor growth on day 34 (p=0.02). After those days, the tumor grows back aggressively. Animals treated with anti-miR-373 showed no changes relative to controls. Conclusion: The miR-100 is a context-dependent miRNA, with tumor suppressor role in aggressive tumor cell lines. The miR-373 acts in vitro as oncomiR and miR-145 acts as a tumor suppressor in vitro and in an animal model with consistent therapeutic response and can be used in the therapeutic arsenal against this neoplasia. Future studies should evaluate the use of miRNAs alone or adjuvant in the treatment of metastatic prostate cancer (AU)

• FAPESP's process: 13/07350-7 - The microRNA use for the treatment of prostate cancer: studies in vitro and in vivo
• Grantee: Alexandre Iscaife
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)