The dual roles of miRNA as tumour suppressors or oncogenes create a great mystery about its nature and function in cancer. These phenomena of miRNAs were also applicable for prostate cancer [352-355]. Still, the large portion of miRNAs function in hormone-refractory prostate cancer is unknown. MiRNA expression profiling of prostate cancer patient and cell lines now has been documented [356-358]. Previous microarray data analysis reported by Mattie et al. [356] revealed miR-148a remain silenced in the advanced prostatic tumour (Gleason score 8) and prostatic lymph node metastasis than the normal adjacent tissues. Another group Porkka et al. [357] showed the expressional difference between prostate carcinoma samples and benign prostatic hyperplasia (BPH) where it was acknowledged that miR-148a remain downregulated in hormone-refractory carcinomas compared with BPH. In this chapter, it was shown that the miR-148a
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expression was downregulated in PC3 and DU-145 cell lines than normal immortalized keratinocyte cell line HaCaT by RT- PCR. In order to identify the down-regulated miRNA that acts like tumour suppressor and can be a viable therapeutic target for hormone- refractory prostate cancer here, miR-148a has been studied. Previous studied already identified the downregulation of miR-148a is not due to a defect in p53 in PC3 cells [348]. In primary breast cancer tissue samples and metastasized lymph node derived cell line shows aberrant hypermethylation of the CpG island in the promoter causes the silencing of miR-148a [4, 359]. But the exact mode of silencing of miR-148a in prostate cancer is still not studied. Ectopic expression of miR-148a in head and neck cancer SIHN- 011B cells inhibits cell motility and proliferation. It also reduced the tumour size and inhibits metastasis in xenograft models [4]. In this study, it is demonstrated that ectopic expression of miR-148a induces apoptosis in part by downregulating DNMT1. This results suggested that dysregulation of miR-148a can initiate the metastatic potential of PC3. DNA methyltransferase plays a significant part in the aberrant gene expression related to tumour initiation and progression [360]. Previously it was reported miR-148a directly targets DNMT3b in MCF-7 and Hela cell line. In PC3 cells downregulation of DNMT3b inhibits cell migration and cell growth which suggest the gene responsible for the inhibition of migration and growth may be silenced by DNMT3b [361].
This study proves that ectopic expression of miR-148a leads to initiation of apoptosis in part by targeting DNMT1 the maintenance DNA methyltransferase. Variation of DNMT1 expression level modifies the global transcriptomes because downregulation of DNMT1 de-repress the silenced genes. The overall effect of the global change is cell context dependent. The previous study on bladder cancer shows DNMT1 expression was increased in stage-dependent manner [362]. Additionally, it also reported that it inhibit cell proliferation and induce apoptosis [363]. Dhawan et al. demonstrated in canine model targeting DNMT1 can be used a viable therapeutic model for future discoveries [364]. Here it was shown from the patient databases in prostate cancer DNMT1 expression was also dependent on the stages of cancer. The metastatic stages show a higher level of DNMT1 expression. The de-regulation of DNMT1 plays a dynamic role in prostate cancer progression, but the cause remains unknown. In this chapter, it was also shown that the survival of the patient depends on the DNMT1 expression. Repression of miR-148a plays an imperative protagonist in prostate cancer progression. Previously it was reported miR-148a increase the chemosensitivity of PC3 cells [348]. This study reveals targeting DNMT1 by miR-148a increased cellular apoptosis in cells. Previously in
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colorectal cancer, it was reported miR-148a repress the expression of BCL2 [365]. This study reveals in prostate cancer cell line PC3 miR-148a inhibits BCL2 and induces apoptosis. It also increases the expression of BAX and cleaved PARP. Here it was also shown that the increasing level of miR-148a in prostate cancer helps in survival and delay cancer recurrence. miR-148a inhibits cell proliferation and inhibits cell growth was determined by cellular assays. It causes cell death by anoikis is also evidenced by this study. From this, it can be concluded that miR-148a inhibit metastasis by causing the death of cells by anoikis. This report suggests miR-148a target DNMT1 in a region which is conserved, and it was proved by luciferase assays. In cell cycle analysis the results indicate it affect the cellular apoptosis and reduce the cells in G0 phase. The increasing
rate of apoptosis was also observed when PC3 cells were transfected with DNMT1 siRNA. So the role of DNMT1 in prostate cancer can be defined as an oncogene. Then the cause of silencing of miR-148a was revealed by inhibiting and overexpressing DNMT1 by siRNA and vector. Targeting DNMT1 induces miR-148a expression whereas overexpression downregulated its expression. From this results it can be concluded miR- 148a remain downregulated in PC3 and DU-145 by repression of miR-148a gene by DNMT1. These results demonstrate miR-148a has a potential as a novel therapeutic agent in the treatment of hormone-refractory prostate cancer. Not only targeting DNMT1 its role can be extended as an apoptosis inducer. MiRNA has the ability to target multiple mRNA targets simultaneously, and it gives it a cutting edge advantage to the fight with cancer. In conclusion here in this chapter the role of miR-148a has been elucidated for a novel therapeutic for prostate cancer.
Chapter 5
Objective 3
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5. To decipher the role of miR-193a in regulation of histone modifier MLL1 and a