Molecular Mechanism By Which Non-Coding RNA Regulates SPRY2 Expression And Its Role In Prostate Cancer Progression

Background:Prostate cancer(PC)is a common male malignant tumor which ranking sixth incidence in malignant tumor in our country and the incidence of PC is increasing gradually in recent years.For African Americans,the 5-year survival rate for early diagnosis of prostate cancer was 100%,while it dropped to 27%when cancer cells spread far away.Although prostate specific antigen(PSA)is currently the biomarker for the diagnosis of prostate cancer,it has been proved that PSA is not associated with the mortality of prostate cancer 13 years later.The adverse consequences of PSA screening lead to overdiagnosis,overtreatment and treatment complications.Therefore,it is urgent to study new biomarkers for the diagnosis of early prostate cancer and explore new therapeutic targets for advanced prostate cancer.MicroRNAs(microRNAs/miRs)are small non-coding single RNAs with 21 to 25 nucleotides in length,which are expressed stably in human body fluid,including serum,plasma and urine.Through sequence-specific base pairing in the 3'untranslated region of the RNA,miRs play an important regulatory role,leading to degradation of the RNA or suppression of translation.Several studies have shown that prostate cancer exhibits a specific expression profile of miRs,including miR-106a,miR-223,miR-20a,miR-21,miR-141 and miR-27a.Among these microRNAs,the down-regulation of miR-27a has been found in high-grade prostate cancer,but recent studies have shown that androgen-regulated miR-27a acts as a carcinogenic gene and increases the growth of prostate cancer cells by targeting tumor suppressor and androgen receptor co-suppressor.Among other types of cancer,including pancreatic cancer,renal cell carcinoma and osteosarcoma,miR-27a is also an oncogene involved in cell proliferation,colony formation and metastasis.However,in hepatocellular carcinoma,miR-27a has been shown to be down-regulated,inhibiting metastasis by inhibiting epithelial-mesenchymal transformation.Therefore,one of the focuses of this study is to explore the role and mechanism of miR-27a in prostate development.Studies over the past decade have shown that 90%or more of the human genome transcribes produces non-coding RNAs(IncRNAs)which have longer than 200 nucleotides.LncRNAs can affect a large number of cellular processes,including normal physiological and pathological processes.In many human diseases,including malignant tumors,the expression of lncRNAs is disordered;lncRNAs may play a role as part of the posttranscriptional regulatory network.For example.lncRNAs can act as competitive endogenous RNAs(ceRNAs)through acting as miRNA response element,thus preventing the binding of miRs to their downstream target genes.According to previous studies,CASC2,PTENP1,H19,HOTAIR,MALAT1 can act as ceRNAs,which affect gene expression by adsorbing miRs,thereby regulating cell physiological activities.Among the many reported IncRNAs,CASC2 has been reported to play a tumor-suppresser role in many types of cancers,such as glioma,gastric cancer,and hepatocellular carcinoma.The role of CASC2 in cancer was first reported in human endometrial cancer by Baldinu et al.in 2004.suggesting that CASC2 may be a potential tumor suppressor gene.In non-small cell lung cancer,the expression of CASC2 was significantly down-regulated.Low expression of CASC2 could predict poor prognosis of non-small cell lung cancer patients.CASC2,as ceRNA of different miRs,including miR-18a and miR-21,regulates the expression of miRs,and then regulates cell proliferation,tumor growth and chemotherapeutic resistance of colorectal and cervical cancer.Although the down-regulation of CASC2 in cancer has been frequently reported,the role and potential mechanism of CASC2 in PC,and whether it is related to the chemotherapeutic resistance of PC to docetaxel,remain unclear.Abnormal signal transduction events mediated by receptor tyrosine kinases(RTKs),including receptors of epidermal growth factor(EGF),fibroblast growth factor(FGF)and hepatocyte growth factor(HGF),have been reported to promote canceration.In addition,in the regulation of proliferation,differentiation and survival,the induction of several important pathways,including RAS/MAP kinase and RAS/PI3K/AKT pathway,depends on RTK activated by ligand binding.Mutations affecting RTK signal transduction often lead to cell transformation,which is widely observed in many malignant tumors.These mutations may affect the components of RTK or downstream pathways,such as MAP kinase and PI3K/AKT,leading to overproliferation,survival,invasion and metastasis of transformed cells.Therefore,targeting the RTK signaling pathways remains a challenge in cancer prevention.Some small molecule inhibitors and antibodies have been exploring to target RTK and its downstream pathways.Sprouty2(SPRY2)is the main antagonistic regulator of the RTK signal.If it is inhibited,it may accelerate the proliferation and angiogenesis of malignant tumors.In many cancers,the expression of SPRY2 has been reported to be down-regulated,including renal cancer,colon cancer,pancreatic cancer,and PC.In the cancer reported above,through direct targeting,miRs can inhibit the expression of SPRY2,suggesting that CASC2 may participate in the resistance of PC to docetaxel by adsorbing miRs.This project explores the molecular mechanism by which non-coding RNA regulats SPRY2 and its role in the progression of prostate cancer from two aspects.On the one hand,our results show that the expression of miR-27a in tumor tissues and serum of patients with PCR is excessive.Overexpression of miR-27a is associated with low survival rate and increased proliferation of cancer cells.In addition,we also found that SPRY2 is the direct target of miR-27a,and the induced expression of SPRY2 can save the proliferation of cancer cells mediated by microRNA-27a in PC cells.On the other hand,we first examined the expression of CASC2 and SPRY2 in PC tissues and cell lines,and evaluated the effects of CASC2 and SPRY2 on the proliferation and apoptosis of PC cells under docetaxel,and the related factors of the ERK signal downstream.Bioinformatics analysis revealed that microRNA-183 had potential binding sites of CASC2 and SPRY2,respectively.The interaction of miR-183 with CASC2 and SPRY2 was verified.Finally,we studied the role of CASC2/miR-183/SPRY2 in the resistance of PC to docetaxel.Methods:1.Collect the tissue samples of human prostate cancer and the patient's medical records.Conventionally cultured prostate cancer cell lines,including LNPC,PC-3,RWPE-1,C4-2,DU145,etc.2.The expression of microRNA-27a,CASC2,microRNA-183 and SPRY2 in tissues and cells was detected by qRT-PCR,and the correlation between the expression of these molecules and clinicopathological characteristics was evaluated by Pearson correlation analysis.The expression of SPRY2 and ERK signaling pathway-related proteins was detected by Western blotting.3.The inhibitors and mimics of microRNA-27a,microRNA-183 were used to interfere with or over-express the two microRNAs.The siRNAs of CASC2.SPRY2 and pcDNA3.1 were used to interfere with or over-express the two RNAs respectively The two RNAs were transfected with liposome 2000.4.Luciferase assay was used to verify the target genes of microRNA-27a and microRNA-183.5.CCK-8,MTT and flow cytometry were used to detect the proliferation and apoptosis of prostate cancer cells.The results:Results of part 1:1.The expression of microRNA-27a is high in prostate cancer and serum samples.The serum level of microRNA-27a may be used as a diagnostic marker for early PC and may be related to patient survival.2.MiR-27a directly inhibits the expression of SPRY 2,and their expression level is negatively correlated with that in PC tissue.3.MiR-27a promotes the proliferation of PC cells,and overexpression of SPRY2 can reverse this function.4.MiR-27a/SPRY2 regulates PC cell cycle.Results of part 2:1.Expressions of CASC2 and SPRY2 in prostate tissues and cells and their correlation analysis.2.Overexpression of CASC2 and SPRY2 enhanced the sensitivity of PC cells to paclitaxel.3.CASC2 and SPRY2 can inhibit ERK signaling pathway.4.CASC2 adsorbs and reduces the expression of microRNA-183.SPRY2 is the downstream target gene of microRNA-183 and can be negatively regulated by microRNA-183.5.Interference with SPRY2 expression can reverse the sensitivity of PC cells to paclitaxel mediated by inhibitors of microRNA-183.Conclusions:1.Serum microRNA-27a may be a new non-invasive biomarker for the diagnosis and prognosis of PC patients,and microRNA-27a/SPRY2 may be a therapeutic target for PC.2.CASC2 combines with SPRY2 to compete with microRNA-183 to rescue the expression of SPRY2 in PC cells,thereby enhancing the sensitivity of PC cells to docetaxel through ERK signaling pathway downstream of SPRY2.CASC2 and SPRY2 may be new adjuvants for docetaxel-based PC chemotherapy.