The Effect And Mechanism Of MiR-345on The Progression Of Prostate Cancer Androgen-independent State

【Background】Prostate cancer (PCa) is one of the most commonly diagnosed malignancies inold men in western countries. The American Cancer Society2012report estimates848,170new cancer cases and301,820deaths cases in male, of which the incidence ofPCa account for the top, about29%, and the mortality of PCa account for the second,about9%. In recent years, an upward trend in prostate cancer incidences has also beenobserved in our country, possibly due to the generally improved healthcare and theaging society. The disease seriously impacts on the quality of life and life expectancyof men over the age of50in our country. It is estimated that by2020, the incidence ofprostate cancer in our country will reach40/100000new cases with a total of350000new cancer cases, which is similar to the incidence of liver cancer. Prostate cancerposes a major public health problem in the world. Prostate cancer is diagnosed aslocal or advanced clinically, and treatments range from surveillance to radical localtreatment or androgen-deprivation treatment. The current standard therapy is stillusing androgen-deprivation treatment in combination with radiation or traditionalchemotherapy for non-surgical advanced prostate cancer. Initially, patients respondfavourably to this therapy, and in this stage called androgen-dependent prostate cancer(ADPC) the valid rate is up to70-80%. But most tumours relapse within2years to anincreased invasion, proliferation and malignancy, androgen-independent state (AIPC).In this stage almost any therapy shows poor effect, the cancer cells spread into otherorgans rapidly, and lead to death eventually. Thus, understanding the changes andmechanisms of prostate cancer cells on growth, proliferation and invasion under theemasculation hormone environment will show a new strategy for the clinicaldiagnosis and treatment of prostate cancer.MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs of~22nucleotides that discovered recently in a variety of eukaryotes and viruses. MiRNAsare involved in a range of normal physiological processes, notably proliferation,differentiation and apoptosis by negatively regulating gene expression at theposttranscriptional level, primarily through base pairing to the3' untranslated region(UTR) of target mRNAs. MiRNAs play an important role in the activities of life andregulate tumor development, progression, transformation through the similar oncogenes, tumor suppressor genes or otherwise. Through bioinformatics prediction,up to30%of all genes coding proteins are negatively regulated by miRNAs in humancells, especially those in the signal transduction pathway. Recent research showedmiRNAs became differential expression in prostate cancer, and play important role inthe development and progression of tumor. That provides the new approach forunderstanding expression and function of gene. Prostate cancer-related miRNAs arehighly likely to be important biomarkers of diagnosis, treatment and prognosis ofprostate cancer.【Objective】Gene screening was performed through miRNA chip on the basis of ADPC andAIPC specimen. The miRNA differential expression database was constructed andverified through prostate cancer cell lines. The differentially expressed miR-345wasselected for further investigation. It was studied that the effects and mechanisms ofmiR-345on regulation of cell biological function and malignant progression ofprostate cancer. That would provide theoretical basis for miR-345as an aggressivebiological marker and therapeutic target of prostate cancer.【Methods】1. MiRNAs screening were performed on the basis of ADPC and AIPC specimen,and the differential expression profile of miRNAs were constructed. LNCaP-AIcell line was constructed in emasculation environmemt which wasandrogen-independent prostate cancer cell line and was applied to confirm thedifferentially expressed miRNA through qRT-PCR compared with LNCaP cellline. And differentially expressed miR-345was selected for further investigation.qRT-PCR were performed to evaluate the expression of miR-345inandrogen-dependent prostate cancer cell line (LNCaP) andandrogen-independent prostate cancer cell lines (LNCaP-AI, PC3, and C42).That became a basis of follow-up study.2. The functions of miR-345were examined on the ADPC and AIPC cell lines. Thesynthetic miR-345mimics or anti-miR-345mimics were transfected into the celllines to examine the effects of miR-345on the development, proliferation,migration and invasion.3. The mechanisms of miR-345were identified on prostate cancer cell. The effects of miR-345on cell cycle and expression of NSE, PSA, AR were examined onADPC and AIPC cell lines through overexpression or knockdown of miR-345.The miRNA target genes were seeked through bioinformatics and miRNAdatabase, and verified by RT-PCR and western blot.4. As serum tumor marker, miR-345was detected in patients with advancedprostate cancer.【Results】1. Tissue microarrays and qRT-PCR were performed to evaluate differentiallyexpressed miRNAs. The studies revealed that AIPC specimen showeddifferentially expressed miRNAs compared with ADPC specimen, and fivemiRNAs (miR-345, miR-221, miR-222, miR-663, and miR-106a) wereup-regulated significantly, while two miRNAs (miR-29c and miR-148a) weredown-regulated significantly.2. The expression level of miR-345was the lowest in normal prostate epithelialcells (RWPE-1), and the highest in androgen-independent prostate cancer cells(LNCaP-AI). And miR-345level was higher in androgen-independent prostatecancer cells (LNCaP-AI, PC3, C42) than that in androgen-dependent prostatecancer cells (LNCaP).3. Cell growth was assessed by using the CCK-8cell proliferation assay. Theresults showed transfection of LNCaP cells with miR-345significantly enhancedthe ability of cell growth, and transfection of LNCaP-AI cells with anti-miR-345significantly reduced the growth capacity.4. The wound migration assay showed the scratches distance of LNCaP cellstransfected with miR-345were significantly shorter compared with controlgroup, and distance of LNCaP-AI cells transfected with anti-miR-345were notsignificantly changed.5. Transwell invasion assay showed the number of cells transfected with miR-345through the Matrigel was not significantly changed compared with control group,so does the cell transfected with anti-miR-345.6. The flow cytometric analysis revealed that LNCaP cells transfected withmiR-345in S-phase were significantly increased compared with control group,and LNCaP-AI cells tranfected with anti-miR-345in S-phase were significantlydecreased. 7. In emasculation environment for three days, LNCaP transfected with miR-345showed obvious neuroendocrine manifestation. The NSE expression level wassignicantlly higher than control group by RT-PCR.8. RT-PCR showed treatment with miR-345had no significantly effect on the ARexpression level in LNCaP, but significantly reduced the DHT-induced PSAexpression compared with control cells; and treatment with anti-miR-345had nosignificantly effect on the AR expression level in LNCaP-AI, but significantlyincreased the DHT-induced PSA expression compared with control cells.9. Screened through bioinformatics and miRNA database and verified by RT-PCRand western blot, that was found miR-345regulates CDC25B expression level.10. The levels of miR-345were analyzed in the serum samples from ADPC andAIPC patients by qRT-PCR. Serum miR-345levels were found to correlate toserum PSA levels in patients with ADPC and AIPC, and elevated significantly inAIPC patients compared with ADPC patients.【Conclusions】1. Tissue microarrays and qRT-PCR revealed that AIPC specimen showeddifferentially expressed miRNAs compared with ADPC specimen, and fivemiRNAs (miR-345, miR-221, miR-222, miR-663, and miR-106a) wereup-regulated significantly, while two miRNAs (miR-29c and miR-148a) weredown-regulated significantly.2. The expression level of miR-345in LNCaP-AI was significantly higher thanthat in LNCaP, PC3, C42and RWPE-1cell lines. It suggests that miR-345couldplay important role in androgen-independent progression of prostate cancer.3. CCK-8and wound migration assay showed that miR-345significantly promotesprostate cancer cell proliferation and migration. And flow cytometric analysisrevealed that miR-345significantly increased prostate cancer cells in S-phase.To a certain extent, it proved the miR-345promotes spread of advanced prostatecancer.4. RT-PCR showed that miR-345promotes neuroendocrine transformation ofprostate cancer cell detected by NSE expression and miR-345has nosignificantly effect on the AR expression level, but it significantly reduced theDHT-induced PSA expression. It suggested that miR-345would play a role inandrogen-independent progression of prostate cancer. 5. Screened through bioinformatics and miRNA database and verified by RT-PCRand western blot, that was found miR-345regulates CDC25B expression level.6. The levels of miR-345were analyzed in the serum samples from ADPC andAIPC patients by qRT-PCR. And serum miR-345levels were elevatedsignificantly in AIPC patients. MiR-345could be offered as a new specificserum tumor marker, and an important target of future studies on prostate canceradvanced stage.