| Background and objectiveProstate cancer(PCa), an emerging threat to the health of aging men, accounts for the second site for global cancer incidence and six site for cancer mortality[1]. The incidence of PCa in China has been rising rapidly in recent years. In economically developed and healthcare-rich cities like Beijing, Shanghai and Guangzhou et al., incidence of prostate cancer has located in the top ten ranking tumors. Further data from the municipal center for disease control shows that prostate cancer has been the leading site in morbidity of male urinary malignancy since 2002. Epidemiological data also shows that the incidence of prostate cancer in China has risen from 1.71/100,000 male population in 1993 to7.9/100,000 male population in 2005, with 13% of annual increase[2]. According to the above analysis, the total number of prostate cancer in 2020 is expected to be more than40/100,000 male population, which is equal to the Europe and America and may raise serious health crisis.Different from advanced western countries, most patients of PCa diagnosed in China have been in the late phage of tumor processing with distant metastasis[3]. Although cancer metastasis has been the research focus for many years, there is no exact molecular mechanism to illustrate prostate cancer metastasis. Besides, previous studies focusing on gene transcription and protein translation failed to fully elucidate the mechanism. Thus, we try to open a new angle on this classical issue to clarify the molecular mechanisms of prostate cancer metastasis.Genomics studies on the mammalian genome transcription have shown that only 2%translated into protein and more than 98% of the transcription are non-coding RNA(non-coding RNA, nc RNA)[4]. According to the molecular size, nc RNA can be divided into small non-coding RNA(small nc RNA) and Long non coding RNA(Lnc RNA). Lnc RNA was classically defined as gene transcripts greater than 200 nucleotides without coding protein, which is considered to be "noise" transcription of the genome and byproduct of RNA polymerase II without biological effect. However, mounting researches suggested that Lnc RNA involved in the genomic imprinting, chromatin modification, promoting transcription, regulating post-transcription and protein function et al. Although, the role of Lnc RNA has not yet been fully understood.Multiple tumors were found to be characterized by Lnc RNA disorders and its contribution to tumor progress has been the research focus recently[5]. Moreover, thediscovery of Lnc RNA provides a new perspective to explore the mechanism of metastasis.Several researches have reported the role of Lnc RNA role in tumor metastasis.Lnc RNA-ATB promoted the epithelial-mesenchymal transition of tumor and stabilized IL-11 m RNA to inhibit tumor localization, which facilitates the liver cancer cell metastasis[6]. HOTAIR combined with PRC2 and LSD1 / REST simultaneously and mediated the binding specificity to locus of the genomes, which inhibited expression of the related metastasis suppressor, promoting tumor metastasis [7]. These results show that Lnc RNAs may exert more crucial roles in tumor metastasis compared with tumor associated protein.Therefore, this study mainly on prostate cancer processing related long-chain non-coding RNA, aims to further explore the role of Lnc RNAs in prostate cancer metastasis and may offer a new perspective or targets for prostate cancer improvements.We collected 66 tissue samples from patients with prostate and performed deep analysis of transcriptome study on carcinoma tissue and tissue adjacent to carcinoma respectively.After differently expressed Lnc RNAs were explored, the tumors were further classified according to differentiation grade, T stage and metastasis and bioinformatics analysis were performed. Finally, we successfully identified 17 prostate cancer processing related Lnc RNAs. Besides, ENST00000503625(Lnc RNA625) inhibited prostate cancer cell metastasis significantly in vitro.Part I: Screening and identification of prostate cancer processing related long non-coding RNA.Objective:To find out the Lnc RNAs which play key role in prostate cancer progression.Methods:(1) 66 pairs of carcinoma tissue and tissue adjacent to carcinoma were collected in Changhai hospital.(2) Deep sequence analysis of transcriptome.(3) Explore the differently expressed Lnc RNAs between carcinoma and tissue adjacent carcinoma.(4)Screening of prostate cancer progressing related Lnc RNAs based on tumor process according to the clinical data.(5)Identify the association between the selected Lnc RNA and clinical features of prostate cancer.(6) Assess the coding potential of Lnc RNA and subcellular localization.(7) The full length sequence of Lnc RNA625 was verified by RACE experiment.Results:(1) After patient agreement and the hospital ethics committee approval, we collected 66 pairs of prostate carcinoma specimens.(2) Properly kept tissues were send and the extraction of total RNA qualified for computer test.(3) 439 differently expressedLnc RNAs were select, of which 188 increased, 251 decreased.(4) Based on the clinical data of patients, 17 prostate cancer progression associated Lnc RNA were successfully identified. Combining with auxiliary public database analysis, ENST00000503625 were identified to be further studied.(6) ENST00000503625 shows significant association with the clinical features of prostate cancer.(7) Coding Potential Calculator calculate(CPC)algorithm demonstrated that Lnc RNA625 have not protein coding ability.nuclear/cytoplasmic separation assay, FISH suggested that Lnc RNA625 mainly located in the cytoplasm.(8) Full-length sequence of Lnc RNA625 was identified via RACE and it was confirmed that Lnc RNA625 was actually the transcript EPHA5-AS1.Conclusion: A new Lnc RNA is identified in our experiments via large clinical samples collected, deep analysis of transcriptome, multiple bioinformatic analysis and a series of experiments validated. The results show that Lnc RNA625 closely associated with clinical characteristics and prognosis of prostate cancer. Online analysis of Lnc RNA coding ability confirmed its inability of protein-coding. And Further experiment verified the cytoplasmic localization of Lnc RNA625. Sequence identificated by RACE find out that Lnc RNA625 is actually EPHA5-AS1.Part II: Lnc RNA625 inhibited the invasion and migration of prostate cancer cellsObjective:To evaluate the role of Lnc RNA625 play in malignant behavior of the tumor cells.Methods:(1) Screening for the effective small interference RNA of Lnc RNA625.(2)Selecting cell lines with high expression of Lnc RNA625 for subsequent experiment.(3)Flow cytometry to detect the cell cycle, Ed U mixing experiment and CCK 8 experiments to detect cell proliferation; After Annexin V/PI double dye, flow cytometry to detect cell apoptosis; Transwell and scratch test to assess cell migration and invasion.Results:(1) 2 Small interference RNA were successfully selected with interference efficiency more than 70%.(2) Cell line LNCa P and 22Rv1 showed high expression of Lnc RNA625 and selected for subsequent study.(3) si- Lnc RNA625 significantly promoted cell migration and invasion, however, didn’t show significant effects on cell proliferation and apoptosis.Conclusion: Lnc RNA625 located in cytoplasm and can be effective knockdown by si RNA. Expression of Lnc RNA625 was closely associated with the malignant degree of cell lines. Altered expression of Lnc RNA625 enhanced the ability of cell migration and invasion, but did not affect cell proliferation and apoptosis.Part III: Lnc RNA625 altered the expression of tumor suppressor protein EPHA5Objective: To illustrates the molecular mechanisms how Lnc RNA625 inhibited the invasion and migration of prostate cancer cells.Methods:(1) Exploring the possible target gene of Lnc RNA625 through the sequence analysis.(2) Confirming the expression of target gene and Lnc RNA625 and the regulatory relationship.(3) The Rescue experiment validates that Lnc RNA625 works by regulating target genes.(4) Explore the action mechanism of Lnc RNA625 regulating target gene.Results:(1) We predict that EPHA5 was the target gene of Lnc RNA625 by sequence alignment.(2) It was demonstrated that expression of EPHA5 was regulated by Lnc RNA625 validated by changed EPHA5 protein expression after altered Lnc RNA625.(3) Lnc RNA625 works through EPHA5 validated by rescue experiment, in which ov-EPHA5 reversed the phenotype of si-Lnc RNA625.(4) Further research showed that Lnc RNA625 stabilized EPHA5 m RNA, which was demonstrated by half-life experiments.Conclusion: Lnc RNA625 is actually the antisense strand of EPHA5 m RNA, which is a tumor suppressor protein in prostate cancer. Here, we demonstrated EPHA5 was the target gene of Lnc RNA625, which improved the stabilization of EPHA5 m RNA through its 5 ’ sequences complemented with EPHA5 m RNA, thus affecting EPHA5 protein levels.ConclusionsThis study based on the large sample collection and high throughput RNA sequencing analysis and focused on prostate cancer processing related Lnc RNA. After analysis of the differently expressed Lnc RNA based on further classification of different tumor grade,stage, we finally found Lnc RNA ENST00000503625(Lnc RNA625), which decreased in the progress of prostate cancer. Besides, public data analysis shows that Lnc RNA625 is an independent predictor of biochemical recurrence of prostate cancer. Tumor phenotype experimental study shows that Lnc RNA625 plays an important role in prostate cancer cell migration and invasion, but does not affect cell proliferation and apoptosis. Further study suggested that Lnc RNA625 regulated tumor-suppressor protein EPHA5 in prostate cancer and EPHA5 carried crucial role in the effect of Lnc RNA625. Finally we found that the sequence of Lnc RNA625 is complementary to EPHA5 m RNA, which delay EPHA5 m RNA degradation in the cytoplasm. This study offers a new bio-marker for prostate cancer recurrence and metastasis and may optimize the anti-tumor strategy for prostate cancer. |
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