Investigation Of The Effect Of LncRNA UCA1/miR-485-5p/FZD7 Axis On Proliferation,Invasion And Migration Of Pdac Cells

Pancreatic cancer(PC),an aggressive digestive system malignancy,characterized with highly aggressive biological behaviors and dismal prognosis.Pancreatic ductal adenocarcinoma(PDAC),originating from glandular epithelium,occupies about 90% of PC with a high ability of infiltration and migration.Surgical resection is the preferred method for the treatment of PC.However,because of its difficulty in early diagnosis and low surgical resection rate,the five-year survival rate after surgery is less than 5%,and even many patients have lost the chance of surgery when they are diagnosed with PC.Under such circumstances,the diagnosis and treatment have been greatly limited.Therefore,it is of great significance to uncover the potential molecular mechanism of the initiation and progression of PDAC and to seek sensitive diagnostic biomarkers,which may greatly facilitate clinical diagnosis and treatment.And because of an independent clinical and pathological characteristics of different pathological types,this study focused on the PDAC.In our previous study,the gene expression profiles of PDAC(173 cases)and normal tissues(27 cases)were detected by in situ hybridization(ISH),and differentially expressed genes were obtained.Twelve PDAC microarray gene chips(PDAC=362,control=202,Affymetrix,Inc.)were obtained by searching GEO(Gene Expression Omnibus)database,and R software was used for differential analysis.One hundred and forty-six cases of PDAC sequencing data were extracted from public database for prognostic analysis.The results showed that lnc RNA UCA1 was differentially expressed in PDAC and correlated with prognosis.Lnc RNA UCA1 is one of the tumor related lnc RNAs,which has been reported to dysregulated in various human cancers,such as bladder cancer,non-small cell lung cancer,liver cancer,and so on,and associated with malignant biological behaviors.Based on literatures,lnc RNA UCA1 has been up-regulated in PC tissues and cell lines,and it may function as an oncogene via targeting miR-135 a or miR-96.Besides miR-135 a and miR-96,whether there are other target miRNAs for UCA1? Neither,to date,the molecular mechanism of UCA1 in PDAC is still unclear.According to the principle of base complementary pairing,online prediction software was used to predict the target gene of UCA1,and 2 potential binding sites for UCA1 and miR-485-5p were found.Verified by public database sequencing data,UCA1 expression was negatively correlated to micro RNA-485'(the precursor of miR-485-5p).Micro RNA-485-5p(miR-485-5p)was shown to be down-regulated in all published tumor-related literature,such as bladder cancer,liver cancer and melanoma.And it was associated with proliferation,invasion,or metastasis in each tumor.In the literatures,many target genes of miR-485-5p were verified by the double fluorescein enzyme.Among them,frizzled class receptor 7(FZD7)was highly expressed in other tumors,related to the biological function of proliferation,invasion or metastasis,and no reported in pancreatic cancer.The binding site of FZD7 and miR-485-5p had also been obtained through the above online prediction software.FZD7 was up-regulated in hepatocellular carcinoma,gastric cancer,breast cancer,oral squamous carcinoma and so on.It promoted the invasion and migration of tumor cells via Wnt signaling pathway.Meanwhile,PDAC sequencing data showed that FZD7 was a co-expression gene of lnc RNA UCA1.From these,the expression of UCA1,miR-485-5p and FZD7 conforms to the basic apparent conditions of the pathway,and the biological function of them are all related to the proliferation,invasion and metastasis of tumors.Given above,we speculated that UCA1 might suppress miR-485-5p,therefore regulating target gene FZD7 and being involved in Wnt signaling pathways,which may influence the initiation and progression of PDAC,especially the proliferation,invasion and metastasis of PDAC.Thus we came up with a hypothesis: the regulatory axis of lnc RNA UCA1/miR-485-5p/FZDF may affect the aggressive progression of PDAC.Part 1.The expression and clinical significance of UCA1,miR-485-5p and FZD7 in clinical PDAC tissues and its bioinformatics verification Materials and Methods1.The expression of UCA1,miR-485-5p and FZD7 was detected by using RT-q PCR in 50 PDAC formalin fixed paraffin embedded(FFPE)tissues and 40 FFPE non-cancerous pancreas tissues(NPT).Pearson was used to evaluate the correlations among UCA1,miR-485-5p and FZD7 level;T test,ROC curve and Kaplan-Meier curve were applied to investigate their significance in clinicopathological parameter,diagnosis and prognosis,respectively.2.All PDAC gene microarrays and miRNA arrays were screened from public databases: Gene Expression Omnibus(GEO),Array Express and Oncomine;Quantitative analysis was performed to verify the expression general trend of UCA1,miR-485-5p and FZD7 in all corresponding arrays.Online public databases were employed to analyze the relationship between genes and clinicopathological parameters and potential prognostic value.Results1.UCA1,miR-485-5p and FZD7 expressed differently in clinical PDAC and control tissues,and had significance for clinical factors,diagnosis and prognosis.UCA1 m RNA level was up-regulated in 50 PDAC and 40 controls(P=0.003).UCA1 was associated with tumor size(P=0.003)and had certain diagnostic value(AUC=0.697,P=0.001)and prognostic value(Log Rank P=0.040).Mi R-485-5p m RNA level was down-regulated(P=0.018).Mi R-485-5p was associated with tumor size(P=0.032),TNM stage(P=0.004),lymphatic metastasis(P=0.013),and had certain prognostic value(Log Rank P=0.022).FZD7 m RNA level was up-regulated(P=0.012).FZD7 was associated with TNM stage(P=0.004),lymphatic metastasis(P=0.013)and grade(P=0.001).The expression of miR-485-5p and UCA1 and FZD7 m RNA were negatively correlated(R=-0.534,P<0.001;R=-0.447,P=0.001),and UCA1 was positively correlated with FZD7 m RNA expression(R=0.031,P=0.027).2.By bioinformatics analysis,UCA1,miR-485-5p and FZD7 were dysregulated expression in PDAC and control tissues,and related to clinicopathological parameters and prognosis.A total of 14 PDAC gene microarrays and 3 miRNA arrays were included.Among the gene microarrays,12 came from GEO,2 came from Array Express.All miRNA arrays came from the GEO.Among the 14 PDAC gene microarrays,8 contained UCA1 expression data.The forest plots indicated that UCA1 was highly expressed in PDAC tissues compared with controls(SMD=0.49,95%CI=0.27,0.71,P<0.001).Surv Express database showed that UCA1 was prognosis risk factors for PDAC.All 3 miRNA microarrays contained miR-485-5p expression data.Forest plot indicated that miR-485-5p was lower expressed in the PDAC(SMD=-0.74,95%CI=-1.30,-0.17,P=0.011).Onco MIR database suggested that miR-485-5p expression was related to gender,tumor grade,TNM stage,tumor size and distant metastasis of PDAC patients.And 12 gene chips contained FZD7 expression data,and the forest plots indicated that FZD7 was highly expressed in PDAC tissues(SMD=1.22,95%CI=0.60,1.84,P<0.001).Surv Express suggested FZD7 also was prognosis risk factors for PDAC.Conclusions1.In the clinical PDAC tissues,UCA1 and FZD7 m RNA expression was up-regulated.The expression of miR-485-5p m RNA was down-regulated.UCA1,miR-485-5p and FZD7 level were associated with PDAC progression(proliferation,invasion or metastasis).2.Bioinformatics showed that UCA1 and FZD7 m RNA were up-regulated in PDAC tissues with being prognostic factors.The expression of miR-485-5p m RNA was down-regulated and was related to the clinicopathological parameters of pancreatic cancer progression.Part 2.The effect of lnc RNA UCA1 on the proliferation,invasion and migration of PDAC cellsMaterials and Methods1.Cell lines screening: the expression of UCA1 m RNA was detected in 5 pancreatic cancer cell lines(As PC-1?Bxpc-3?Capan-1?Panc-1 and SW1990).Two cell lines with higher UCA1 level were screened for follow-up experiments.2.Lentivirus transfection and in vitro experiments: transfected UCA1-RNAi Lentivirus into Bxpc-3 and Panc-1 to knockdown UCA1 expression.Plate clone formation assay,CCK8 assay,cell adhesion array,invasion and migration assays were performed to verify the effect of down-regulation of UCA1 m RNA level on proliferation,invasion and migration of pancreatic cancer cells.Results3.Cell lines screening: UCA1 m RNA level was higher in Bxpc-3 and Panc-1(P<0.05),and the two cell lines were selected for follow-up experiments.4.In vitro experiments: the cloning efficiency,ability of cell proliferation,invasion and migration were significantly suppressed in PDAC cells with UCA1 being knocked-down as compared to controls(P<0.05).Conclusions1.Bxpc-3 and Panc-1 were selected for in vitro experiments.2.Down-regulation the expression of UCA1 could inhibit the proliferation,invasion and migration of PDAC cells.Part 3.The effect of miR-485-5p on the proliferation,invasion and migration of PDAC cellsMaterials and Methods1.Lentivirus transfection: Constructed miR-485-5p over-expressed(OE)lentivirus,transfected them into Bxpc-3 and Panc-1 to obtain the stable high miR-485-5p-expressed cell lines.2.In vitro experiments: transfected miR-485-5p over-expressed(OE)Lentivirus into Bxpc-3 and Panc-1 to up UCA1 level.CCK8 assay,invasion and migration assays were performed to verify the effect of up-regulation of miR-485-5p m RNA level on proliferation,invasion and migration of pancreatic cancer cells.ResultsIn vitro experiments: the ability of cell proliferation,invasion and migration were significantly suppressed in PDAC cells with miR-485-5p being over-expressed as compared to controls(P<0.05).ConclusionsUp-regulation the expression of miR-485-5p could inhibit the proliferation,invasion and migration of PDAC cells.Part 4.The verification of the regulation relationship of lnc RNA UCA1/mir-485-5p/FZD7 and the discussion on the influence of PDAC cell biology functionMaterials and Methods1.RT-q PCR was applied to assess miR-485-5p m RNA level after knock-down UCA1;2.RT-q PCR was applied to assess UCA1 and FZD7 m RNA level after up-regulated miR-485-5p;Western blot(WB)was to detect the FZD7 protein level.3.Dual luciferase report gene experiment was to verify the relationship of sequence complementation between UCA1 and miR-485-5p.4.Retrieved the regulatory relationship between miR-485-5p and FZD7 in the literatures.Results1.After down-regulated UCA1 m RNA,the expression of miR-485-5p m RNA was up-regulated.2.After up-regulated miR-485-5p m RNA,the expression of UCA1 and FZD7 m RNA were down-regulated(P<0.05).But no significant change was found in FZD7 protein expression.3.Dual luciferase reporter assay demonstrated that miR-485-5p may specifically bind to UCA1-3' UTR.4.In the literatures,FZD7 was the direct target gene of miR-485-5p which confirmed by dual luciferase reporter assay.FZD7 is an important receptor for classical Wnt pathway.Conclusions1.The expression of miR-485-5p m RNA could negatively affected by UCA1.2.The expression of UCA1 and FZD7 m RNA could negatively affected by miR-485-5p.But no effect on FZD7 protein expression.3.UCA1 has a direct binding site with miR-485-5p.4.FZD7 is the direct target gene of miR-485-5p,which is involved in tumor development through the classical Wnt pathway.Conclusions for Full Text1.In PDAC,UCA1 and FZD7 expression were up-regulated,and the expression of miR-485-5p was down-regulated,which was related to the clinical pathological parameters of PDAC development(proliferation,invasion and migration).UCA1 and miR-485-5p have certain prognostic significance.2.LncRNA UCA1 maybe an oncogene in PDAC,and interference with UCA1 expression could inhibit the proliferation,invasion and migration of PDAC cells.3.Mir-485-5p maybe a tumor suppressor gene in PDAC,and overexpression of miR-485-5p could inhibit the proliferation,invasion and migration of PDAC cells.4.There may be mutual regulatory relations between UCA1 and miR-485-5p.FZD7 is a direct target gene of miR-485-5p.UCA1/miR-485-5p/FZD7 axis may exist and influence PDAC development by participating in the classical Wnt pathway.Sum up,UCA1 may inhibit the expression of miR-485-5p,affect the expression level of FZD7,and indirectly participate in the Wnt pathway to regulate the proliferation and metastasis of PDAC cells.