| Part I The expression and clinical significance of BC043614 in PDACBackgroundPancreatic ductal adenocarcinoma(PDAC)is one of the most highly malignant tumors.Its mortality rate ranks the 8th in men and 9th in women worldwide,respectively.Although advances in early detection and treatment of cancer have led to a certain reduction in the mortality of PDAC in recent years,as an extremely heterogeneous disease,PDAC remains a significant public health issue.The high mortality rate caused by PDAC is attributed to the failure on diagnose of the disease in time before the disease has been metastasized to other organs and the tolerance of cancer cells to current treatment methods.PDAC progression is caused by a variety of genetic and epigenetic changes that activate various aberrations of the cell and the appearance of various features of the tumor cell.The accumulation of aberrations promotes malignant transformation of cells and confers its tumor phenotype.Recently,the dysregulation of novel gene non-coding RNAs(lnc RNAs)as one of the causes of genetic and epigenetic disorders has attracted wide attention.In this present study wedetcting significantly differentially expressed lnc RNA in PDAC by uses GEO data combined with clinicopathologic analysis,.MethodsIn this present study,GEO data(GSE16515 and GSE32688)were analyzed,and lnc RNA-BC043614,which was significantly differentially expressed,was selected to further validate its expression in the pancreatic cancer tissues we collected.The correlation between the expression of BC043614 and clinicopathological parameters was statistically analyzed by Chi-square test and Pearson correlation.Kaplan-Meier survival(Log rank test)and Cox regression analysis were used to analyze the relationship between the expression of BC043614 and the overall survival(OS))of PDAC patients.ResultsGEO data analysis showed that 24 lnc RNAs were differentially expressed in PDAC,of in which Lnc RNA-BC043614 was the most highly expressed in PDAC tissues.RT-q PCR results showed that it was significantly higher in 42 pairs of PDAC tissues than in normal pancreatic tissue,and showed higher expression level in tissue samples at advanced stage(P = 0.0099),with tumor size> 2cm(P = 0.0176)and lymph node metastasis(P = 0.0119).The Kaplan-Meier survival curve showed that the OS of patients with high BC043614 expression was significantly lower than that of the low BC043614expression(P <0.01).The results of chi-square analysis showed that the expression of BC043614 was associated with CA19-9,tumor diameter,tumor differentiation and the presence or absence of vascular tumor thrombus(P <0.05),whereas not with gender,age and tumor location.The univariate and multivariate COX regression analysis showed that the expression of lnc RNA-BC043614(P = 0.010)was an independent factor affecting the prognosis of PDAC.ConclusionsThe lnc RNA expression in PDAC was analysedusing GEO data,and further clinical samples showed that BC043614 was highly expressed in PDAC and significantly correlated with multiple clinicopathological parameters and prognosis.AFAP1-AS1 can be used as a clinical and pathological monitoring of PDAC progression and may be a novel predictor of the prognosis of cancer patients.Part II The function of BC043614 in PDACBackgroundlnc RNAs are involved in many biological processes,such as alternative splicing,regulation of protein activity,turnover of protein localization,and epigenetic regulation(DNA methylation,chromatin remodeling,histone modification,and gene silencing)and is a widely regulatory elements.Previous studies have shown that lnc RNA-BC043614 regulates the malignant phenotype of many tumor cells,whereas whether its upregulation in PDAC is a contributing factor to the malignant development of PDAC,can promote the malignant phenotype of PDAC,or whether it is merely a concomitant phenomenon? Its specific function it is not yet clear.Clarifying the biological function of BC043614 in PDAC will help for determine its impact on PDAC and will shed light of the clinical targeted therapy.MethodsWe overexpressed or knockrd down the expression level of BC043614 in PDAC cell lines used gene clone,RNA interference,and virus deliverymethods.Then,we performed CCK-8 models and flow cytometryto observe the influence of BC043614 on tumor cell proliferation and apoptosis.We also performed Transwell cell Matrigel invasion assay to observe the influence of BC043614 on tumor metastasis.ResultsThe relative expression levels of BC043614 in six PDAC cell lines and normal pancreatic ductal epithelial cells were detected,and the relative expression level of BC043614 in PDAC cell lines was significantly higher than that in normal human pancreatic ductal epithelial cells(P <0.05).In the CCK-8 experiment in PDAC cell lines,we found overexpression of BC043614 can significantly enhance the proliferation of tumor cells(P <0.05),knockdown of BC043614 significantly inhibited the proliferation of tumor cells(P <0.05).The results of flow cytometry showed that BC043614 overexpression could significantly reduce the apoptosis rate of tumor cells(P <0.05),and knockdown of BC043614 promoted the apoptosis of tumor cells(P <0.05).Transwell experiments results suggested that BC043614 overexpression could significantly promote the invasion and migration of PDAC cells(P <0.05),and the migration and invasion of BC043614 knockdown cells were significantly inhibited(P <0.05).ConclusionsIn vitro experiments show that BC043614 can promote the proliferation,migration and metastasis of PDAC cells,inhibit the apoptosis of PDAC cells and promote the malignant progression of PDAC.Up-regulation of BC043614 in PDAC is a contributing factor to the malignant behavior of PDAC,The specific downstream molecular mechanism by which BC043614 functions in PDAC need to be elucidated.Part III Screening and identification of BC043614downstreammolecular mechanismBackgroundA lnc RNA may play a variety of roles in tumor progression.BC043614 is involved in tumor development through multiple pathways and mechanisms.It is known that BC043614 can plays rolesby regulating Wnt / β-catenin,Rho A / Rac2 and PTEN / The p-AKT signaling pathway,as well as affecting gene transcription through cis-and trans-actions.However,the mechanism of BC043614 in PDAC has not yet been elucidated.MethodsThe Targetscan 7.0,DIANA and micro RNA.org databases were used to detect mi RNAsthat could bind to BC043614,andthe number of mi RNAs then were further reduced by the data ofdownregulatedmi RNAsin PDACs in the TCGA database.RT-q PCR was used to detect mi R-133 expression in PDAC tissues and cells,The correlation between BC043614 and the expression of mi R-133 a and IGF1 R in PDAC was analyzed by Spearman analysis.The luciferase assay was used to detect the binding of mi R-133 a with BC043614.The repression experiment was used to detect whether AFN1-AF1-AS1 in PDAC is involved in the regulation of IGF1 R expression by mi R-133 a.ResultsThe binding sites of BC043614 and mi R-133 a were found by a series of bioinformatic analysis.The expression of mi R-133 a in 42 pairs of pancreatic cancer tissues was significantly lower than that in normal pancreatic tissues(P <0.01).The relative expression level of mi R-133 a in PDAC cell lines was significantly lower than that in normal cells.The results of RT-PCR showed that overexpression of BC043614 could significantly reduce the expression of mi R-133 a,knockdown of BC043614 could promote the expression of mi R-133 a.The expression level of BC043614 and mi R-133 a was negatively correlated.Luciferase results show that mi R-133 a and BC043614 was complementarily binding.Further,we found that the promotion of IGF1 R by mi R-133 a inhibitor was knocked down by BC043614,and the inhibition of IGF1 R by mi R-133 a mimics was relieved by overexpression of BC043614.IGF1 R was highly expressed in PDAC tumor tissues.Knockdown of mi R-133 aabolished the effects of BC043614 on the proliferation,migration and invasion,while rescure the effects of BC043614 on the apotosis in the BC043614-knockdown PDAC cells.Confirmed that lnc RNA-BC043614 in PDAC regulates target gene IGF1 R expression through mi R-133 a and plays a role of promoting cancer in PDAC.ConclusionsIn PDAC,BC043614 can regulate the expression of IGF1 R,a target gene of mi R-133 a,through its complementary binding with mi R-133 a,and play a role in promoting tumorigenesis in PDAC.The BC043614-mi R-133a-IGF1R-related signaling pathway may be an important research directionfor targeted therapy. |
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