Decreased Expression Of TCF12 Contributes To Progression And Predicts Biochemical-recurrence In Patients With Prostate Cancer

Background and purpose:Prostate cancer(PCa),one of the most common noncutaneous cancer among males,represents the second leading cause of cancer-related deaths worldwide,particularly in the Western countries[1].Although there have been great advances in early diagnosis and prevention,the incidence and mortality of PCa are increasing constantly.The patients hospitalized were frequently diagnosed as advanced diseases characterized by metastasis during which cancer cells may be moved from the point of origin within the prostate gland to multiple distant organ sites throughout the body[2].Various clinicopathological characteristics,such as serum levels of prostate-specific antigen(PSA),tumor-node-metastasis(TNM)stage,and Gleason score,have been extensively used to divide PCa patients into different risk groups[3].However,these parameters all have limitations and can't be used as a sole criterion[4].Therefore,it is necessary to identify more accurate biomarkers for PCa diagnosis and prognosis.Recently,studies on the biological role of microRNA(miRNAs)in various tumors become more and more popular.miRNAs represent the best characterized class of small(19-25 nt in length)non-coding RNA transcripts.Mature miRNAs regulate a variety of gene expression by directly degrading mRNA or suppressing post-transcriptional protein translation by pairing with complementary nucleotide sequences in the 3'-UTR of specific target genes.The dysregulation of miRNAs have been observed in many types of human malignancies.miRNAs are involved in the progression and tumorigenesis of PCa via directly targeting coding genes.Moreover,miRNAs control more than 50%of the coding genes.Recently,many studies have proven that miRNAs also play an important role in prostate cancer,such as miR-30d,miR-373 and miR-520c.Therefore,getting deeper insight into the molecular functions of miRNAs is an indispensable part.miR-30d has been proven to be up-regulated in multiple tumors and acted as an tumor suppressor,including melanoma,lung cancer,hepatocellular carcinoma and malignant peripheral nerve sheath tumour.According to these studies,miR-30d promoted the progression of tumors and controlled the cellular cycle,cellular apoptosis,cellular migration,cellular invasion and angiogenesis by targeting its downstream genes.However,because the biological functions of miRNAs have highly tissue and cell specificity,miR-30d was found to inhibit tumor cells proliferation,migration and invasion by controlling TGF-?1.Previous studies and our previous experiments have demonstrated that miR-30d is upregulated in prostate cancer and plays a key role in cancer progression.Therefore,we used gene expression profiling technique to analyze the gene expression profiles of LNCaP and DU145 cell lines overexpressing miR-30d and NC control,and obtained a number of differentially down-regulated genes of miR-30d regulation.Using software prediction network system Expression of down-regulated genes was screened for transcription ?related genes,and TCF12 was identified as a candidate gene.Transcription factor 12(TCF12,also known as HEB or HTF4),as a member of helix-loop-helix(HLH)protein family,has the direct DNA(E-box)binding ability[5].TCF12 protein is found to be extensively expressed in many tissues and can form homodimers or heterodimers[6].Functionally,TCF12 is responsible for cell development and differentiation[7].It has been reported that TCF12 can regulate the differentiation of lymphocytes or the development of neural or mesenchymal tissues[8];The mutations in this gene or the translocation fusion of its fragment with other molecules may result in craniosynostosis or mesenchymal malignancies[9].Growing evidence shows that TCF12 may function as either an oncogene or a tumor suppressor in various human cancers.For example,He et al.[10]found that TCF12 expression was elevated in gallbladder cancer tissues and correlated with poorer overall survival in patients;Tang et al.[11]indicated that TCF12 were obviously elevated in cancer-associated fibroblasts,leading to extracellular matrix remodeling and triggering the invasion and metastasis of breast cancer cells both in vitro and in vivo;Lee et al.[12]determined that TCF12 may act as a transcriptional repressor of E-cadherin and its overexpression may be closely correlated with the occurrence of colorectal cancer metastasis;In contrast,Chen et al.[13]identified TCF12 as a direct target of miR-211 in oral squamous cell carcinoma,and confirmed that it might function to suppress the oncogenicity of this malignancy.However,there are no reports on the involvement of TCF12 in PCa.As a member of helix-loop-helix protein family,TCF12 functions as either an oncogene or a tumor suppressor in various human cancers[10-13].However,there are no reports on its involvement in PCa.To investigate clinical relevance of TCF12 in PCa and to evaluate its roles in malignant phenotypes of this cancer,we here examined the expression patterns of TCF12 protein in PCa and benign prostate tissue specimens by immunohistochemistry.Then,associations of TCF12 expression with various clinicopathological characteristics and patients' prognosis of PCa were evaluated.Moreover,its involvements in cancer cell proliferation,migration,invasion and tumor growth were respectively determined by in vitro and in vivo experiments.Materials:1.For the immunohistochemistry analysis,a tissue microarray(TMA)of 50 primary PCa tissues were purchased from Xi'an Ailina Biotechnology Co,Ltd(Xi'an,People's Republic of China;catalog number:PR807a).Patients received chemotherapy or radiotherapy before surgery were excluded from the study.For the evaluation of the clinical relevance of TCF12,a publicly available dataset named the Taylor dataset including 150 primary PCa tissues and 29 adjacent non-cancerous prostate tissues with mRNA microarray expression data was collected[14].2.Animal:8 BALB/c nude mice(4?6-week-old males)were purchased from Zhongshan Medical Animal Center P.R.China.3.Cell culture:Normal human prostate epithelial cells(PREC)were purchased from Lonza Company and were cultured in PrEGM Bullet kit(Lonza,USA)with antibiotics.Human PCa cell lines(LNCaP and DU145)were purchased from the American Type Culture Collection(Manassas,VA,USA).Methods:1.Construction of miR-30d,TCF12 inhibitory expression and overexpression plasmid(Danvers,MA,USA)and transfection into DU145 and LNCaP cell lines using TransDux virus transfection reagent(model:LV850A-1,SBI,USA)2.The gene expression profiles of LNCaP and DU145 cell lines overexpressing miR-30d and NC were analyzed by gene expression profiling technique.A number of differentially expressed genes were down-regulated by miR-30d.Screening of genes related to transcription in the expression down-regulation gene using software predictors of the network system.3.The expression of miR-30d and TCF12 in DU145 and LNCaP cell lines were detected by Western blotting respectively.The expression of miR-30d LNCaP and DU145 Expression of miR-30d and TCF12 in cell lines.4.The statistical analysis of the expression of TCF12 in the clinical miRNA microarray database of Taylor clinical prostate cancer was correlated with the clinical characteristics and prognosis of patients with prostate cancer,such as PSA level,Gleason score,biochemical recurrence and so on.Through the survival curve analysis and Cox regression analysis,the relationship between the expression of TCF12 and the overall survival rate,biochemical recurrence survival rate and other indicators on the overall survival of prostate cancer and no biochemical recurrence of survival,whether it can be used as a prognostic factor.5.The effect of TCF12 gene up-regulation or down-regulation on the proliferation,migration and invasion of prostate cancer cells was analyzed by cell function assay,including cell proliferation assay,apoptosis test,scratch test and invasion experiment.Nude mice were subcutaneously inoculated with DU 145 cell line to form a subcutaneous transplanted tumor model of nude mice to analyze the effect of TCF12 on tumorigenesis of prostate cancer in vivo.Statistical analysis:The statistical analysis of data is handled by SPSS 19.0 statistical software package.Continuous variable in the form of a show.Count data were analyzed using Pearson chi-square test.Quantitative analysis of QRT-PCR,immunohistochemistry,Western blot and cell function experiments was performed using two independent samples of t-test.The LSD method is used for comparison between two or more groups.Repeated measurement of variance analysis was performed at different time points between the two sets of data.The relationship between the level of TCF12 gene expression and the clinical characteristics of prostate cancer patients in the Taylor clinical prostate cancer tissue database was statistically analyzed by two independent sample t tests and One-way ANOVA test.Clinical study of prostate cancer patients by Kaplan-Meier method and log-rank test.COX proportional hazards regression model single factor and multivariate analysis to assess whether TCF12 can be used as an independent prognostic indicator of prostate cancer prognosis.P<0.05 for the difference was statistically significant.Result:1.In the gene expression profile analysis of overexpressing miR-30d,we obtained 25 down-regulated genes.In addition,we performed Western blot analysis.The results showed that there was a negative correlation between TCF12 and miR-30d mRNA and protein expression levels compared with the blank control group.2.Immunohistochemical staining of tissue microarray showed that the low expression of TCF12 in prostate cancer tissue was significantly correlated with high Gleason score(P=0.009).Because prostate cancer tissue specimens(TMA data)do not include more clinical and pathological features,such as preoperative PSA levels,overall survival,biochemical recurrence,metastasis and pathological staging,we use the prostate cancer clinical chip data set(Taylor common database)The correlation between TCF12 expression and clinical features and the prognosis of patients with prostate cancer were analyzed.The results showed that the down-regulated expression of TCF12 was negatively correlated with Gleason score,postoperative metastasis and biochemical recurrence(P=0.025,0.001 And 0.019),while there was no statistically significant correlation between TCF12 expression level and age,preoperative PSA levels,and whether death and pathological staging were present.3.The Kaplan-Meier and Log rank methods were used to evaluate the value of TCF12 expression in the prognosis of prostate cancer.The results showed that low expression of TCF12 was associated with poor prognosis in patients with prostate cancer,and the overall survival rate of patients with high expression and low expression of TCF12 in prostate cancer(P=0.0037),and the low recurrence rate of TCF12 in patients with high expression of TCF12 was lower than that in TCF12 low expression group(P=0.004),indicating that low expression of TCF12 may be a risk factor for biochemical recurrence.The single factor and multivariate analysis showed that the TCF12 gene expression level[P=0.046],Gleason score[P<0.001]and pathological stage P=0.012]were independent predictors of the risk of biochemical recurrence after radical prostatectomy.4.The expression of TCF12 protein was detected by Western blotting.The results showed that the level of TCF12 overexpression was significantly higher than that of NC group(P<0.05),while TCF12 was down-regulated(P<0.05),indicating that the transfection effect was good.In vitro cell function experiments showed that the proliferation,migration and invasion of DU 145 and LNCaP cell lines with TCF12 overexpression were significantly attenuated(P<0.05).In contrast,the proliferation,migration and invasion of DU 145 and LNCaP cell lines were significantly enhanced by TCF12(P<0.05).5.The overexpression of TCF12 could significantly inhibit the tumor size and growth rate of transplanted tumor formed by DU 145(P<0.05).It was confirmed that TCF12 inhibited the proliferation and invasion of tumor cells.Conclusion:1.TCF12 is an important tumor suppressor gene in prostate cancer and inhibits progression of prostate cancer and predicts the risk of biochemical recurrence and metastasis after radical prostatectomy as an independent predictor.2.The low expression of TCF12 in prostate cancer specimens was significantly correlated with the high Gleason score,and it was expected to detect the expression of TCF12 in prostate cancer tissues and to help the traditional clinicopathological parameters to distinguish between low-grade and highly malignant prostate cancer.Treatment programs.