Preliminary Study On The Effect Of PLCE1on The Biological Behaviour And The Mutual Regulation-mechanism Of PLCE1-related MicroRNAs In Esophageal Carcinoma

Prat1The effect of PLCE1on the biological behaviour of ESCCObjective:To investigate the PLCE1expression in precancerous lesions and assesse the correlation between PLCE1expression and its clinical and pathological data. And to provide a precise quantification of the association between PLCE1protein expression and the risk of ESCC through meta-analysis. We also observe the effects of down-regulation of PLCE1expression on biological behavior of esophageal carcinoma cells and investigate the effects of PLCE1gene on the expression profile of esophageal cancer cell line by Affymetrix3’IVT microarrays.Methods:(1) Tissue microarrays (TMAs) were used for immunostaining PLCE1in112Han ethnic patients with ESCC between2009and2011from the First University Hospital, Shihezi University School of Medicine. Among the112ESCC specimens,99specimens that matched the adjacent normal esophageal tissues (NETs) were used as controls. Approximately,99precursor lesions in the adjacent mucosa were selected; among which,60were classified as low-grade intraepithelial neoplasia (LGIN) and39as high-grade intraepithelial neoplasia (HGIN). Follow-ups were conducted on75Han patients.(2) We performed a meta-analysis of literature on the correlation of PLCE1protein expression with the clinical characteristics of esophageal cancer. Eligible studies were identified from PubMed, Wanfang Data, ISI Web of Science, and the Chinese National Knowledge Infrastructure databases. Using RevMan5.2software, pooled odds ratios (ORs) with95%confidence intervals (CIs) were employed to assess the association of PLCE1expression with clinicopathological features relative to ESCC.(3) We attempte PLCE1knockdown using RNA interference. Western blot analysis was used to measurement protein expression, MTT assay was used to measure cell growth rate, colony-formation assays were used to measure cell proliferation rate, and flow cytometric analysis was used to measure the cell apoptosis of two different ESCC cell lines, namely, Eca109, and Eca9706. Invasion of Eca109cells at48h after transfection was examined by Transwell chamber invasion assay. EMT marker protein, epithelial cell marker E-cadherin, and mesenchymal marker vimentin expression were examined in Eca109and Eca9706cells transfected with si-PLCE1and controls using Western blot analysis to investigate whether PLCE1regulates EMT in esophageal cancer cells. We used the phalloidin-TRITC (F-actin) to monitor cytoskeleton dynamics in Eca109cell tresfeted with PLCE1siRNA. The changes of cell apoptosis and growth rate were observed on the Eca-109cells treated with PLCE1siRNA and/or chemotherapeutic drug, TNF-α, TRAIL, Paclitaxel and5-FU, by MTT assay and flow cytometric analysis.(4) Differences in PLCE1protein expression and clinical characteristics were compared by χ2test. Correlations between prognostic outcomes and PLCE1expression were investigated using Kaplan-Meier analysis and the Cox proportional hazards model. All statistical analyses were performed using GraphPad Prism5software. Two-way ANOVA was used for pairwise comparisons between test groups. Data points are reported as experimental averages, and error bars represent SD. Results were deemed statistically significant at the level of p<0.05. For the variables, a P value of less than0.05was considered statistically significant.Results:(1) Most esophageal tumour and precancerous lesions tissues showed strong cytoplasmic staining with PLCE1. The patients were dichotomized into two categories according to their immunoreactivity for PLCE1. PLCE1protein was upregulated in73.22%(82/112) of ESCC,72.50%(28/40) of HGIN,58.33%(35/60) of LGIN, and2.03%(2/99) of normal epithelium, displaying a gradual increased trend from normal esophageal epithelium to ESCC. The IS of PLCE1in the ESCC specimens (6.768±0.2717), HGIN (7.103±0.4312), and LGIN (6.333±0.3808) were significantly higher than those in the non-malignant specimens (1.707±0.1416)(all P<0.001).(2) Increased expression of PLCE1was correlated with advanced TNM stages (P=0.024) and lymph node metastasis (P=0.021) in patients with ESCC. Kaplan-Meier survival analysis revealed that the overall survival was significantly lower in patients with high PLCE1expression than in patients with low PLCE1expression (log-rank test, χ2=10.243, P=0.001). Multivariate survival analysis using the Cox’s proportional hazards model showed a close correlation of high PLCE1protein expression with clinical prognosis (HR=8.435,95%Cl=1.875-37.983, P=0.005).(3) Four studies examining esophageal cancer were included for the evaluation of association with PLCE1expression. PLCE1expression was associated with tumor progression (pooled OR=5.93;95%Cl=3.86-9.11).(4) Proliferation of Eca109and Eca9706cells decreased in si-PLCE1transfected cells compared with the respective controls. Colony-formation assays showed a similar pattern to that MTT. The number of colonies decreased following the downregulation of PLCE1in the two different ESCC cell lines. Flow cytometric analysis results showed that the early apoptosis rate increased by8.3-fold and4.1-fold in Eca-109-si-PLCE1and Eca9706-si-PLCE1-cells, respectively. Western blot analysis showed that the apoptosis-related protein Bax and cleaved-PARP increased.(5) The tumor cell migration and invasion assay indicated that transfection of si-PLCE1could reduce the invasion and migration power of the ESCC cell line. The epithelial marker E-cadherin was significantly upregulated, whereas the mesenchymal marker vimentin was significantly reduced in cells with knockdown of PLCE1compared with the control siRNA groups. Furthermore, the formation of lamellipodia and filopodia was inhibited by the knockdown of PLCE1compared with control cells using phalloidin-TRITC (F-actin) to monitor cytoskeleton dynamics in Eca109cell.(6) We found that TNF-a induced a dose-dependent decrease in cell viability and increased cell apoptosis rate in Eca109cell transfected with the si-PLCE1compared with the si-control Eca109cells. TRAIL induced strong apoptosis in Eca109cells transfected with si-PLCE1than that in si-control. Eca109cells were more sensitive to Paclitaxel-induced cell death when PLCE1expression was knocked down in the esophageal cancer cells. Similarly, suppression of PLCE1protein in Eca109made the cells more sensitive to5-FU-induced cell death and apoptosis.(7)223different expression genes whose fold-change was larger than2were screened.168were up-regulated and55were down-regulated in PLCE1-siRNA and control-PLCE1siRNA cells. These genes were involved in cell proliferation, differentiation, apoptosis, signal transduction and protease activity, invasion and metastasis of tumor, and so on.Conclusion:(1) PLCE1is a powerful poor prognostic marker and potential therapeutic target in ESCC.(2) Downregulation of PLCE1suppresses ESCC cell growth, and induces apoptosis. PLCE1induces ESCC cell migration and invasion and regulates cytoskeleton dynamics and contributes to the resistance of ESCC cell to TNFa, TRAIL, Paclitaxel and fluorouracil (5-FU)-induced apoptosis. Prat2Preliminary study on the mutual regulation-mechanism of PLCE1-related miRNAs in ESCCObjective:(1) Overexpression of the PLCE1protein has been found in esophageal cancer tissues. However, the mechanisms by which PLCE1becomes over-expressed in ESCC remain unclear. MicroRNAs (miRNAs) can regulate target gene expression through translational control. In the present study, we investigated whether microRNA (miR-145) regulates PLCE1expression in ESCC.(2) To study the microRNAs that could regulated by PLCE1in ESCC and research PLCE1upregulated miR-106b-5p and its downstream genes on the effect of esophageal cancer cells biological characteristics.Methods:(1) Luciferase reporter assay and ESCC cells transfected with miR-145mimics or inhibitors were used to determine whether miR-145directly targets PLCE1. To investigate the effect of miR-145on ESCC biological behavior, cell proliferation and invasion were analyzed using ESCC cells transfected with miR-145mimics or inhibitor or transfected with specific siRNA to deplete PLCE1(siRNA-PLCE1). We also evaluated the expression of miR-145and PLCE1in ESCC tissues and adjacent non-tumor esophagaela tissues by qRT-PCR and Immunohistochemistry, respectively, and analyze the correlation between them.(2)To investigate the consequences of downregulation of PLCE1on miRNA expression levels, we used Agilent miRNA microarrays to determine miRNA expression levels in ESCC cells transfected with siRNA-PLCE1compared with cells transfected with si-NC.(3) To investigate the effect of PLCE1or miR-106b-5p on ESCC biological behavior, we attempted PLCE1knockdown using siRNA-PLCE1or transfected with miR-106b-5p mimics or inhibitor and studied in the MTT assay for the measurement of cell growth rate, flow cytometry for the measurement of cell apoptosis rate, clonogenic assays for the measurement of cell proliferation rate, and transwell without metrigel for the measurement of cell metastasis rate of ESCC cell lines.(4) Luciferase reporter assay and ESCC cells transfected with miR-106b-5p mimics was used to determine whether miR-106b-5p directly targets RBL1or RBL2.(5) We also evaluated the expression of PLCE1, miR-106b-5p and RBL2in ESCC tissues and non-tumor esophagaela tissues by qRT-PCR and immunohistochemistry, respectively, and analyze the correlation between them.Results:(1) Online miRNA target prediction databases (TargetScan, miRanda, miRDB) were used to determine the miRNAs that can target PLCE1. The databases predicted miR-145as potential miRNA that targets PLCE1. The dual-luciferase reporter assays showed that miR-145significantly attenuated the luciferase activity of reporter vector with the wt-3’UTR of PLCE1, whereas this effect was abrogated when the3’UTR-binding site was mutated. Western blot analysis revealed that PLCE1protein expression decreased in the in Eca109cell transfected with miR-145-mimic. On the other hand, miR-145mRNA expression level decreased with miR-145-inhibitor in TE-1cells; Western blotting analysis revealed that inhibition of miR-145in TE-1cells enhanced PLCE1protein expression.(2) We performed a colony formation analysis to validate whether miR-145regulates ESCC cell growth by transfecting miR-145or miR-145-in into Eca109or TE-1cells. Our results showed that miR-145-mimic transfected cells displayed fewer and smaller colonies than the control transfectants. Correspondingly, after transfection with miR-145-inhibitor, TE-1cells displayed more and larger colonies than the control transfectants. Analysis of apoptosis exhibited that the numbers of apoptotic cells were significantly increased in Eca109cells transfected with miR-145, but were conspicuously decreased in TE-1cells transfected with miR-145-in. Western blot analysis revealed that the protein expression of Bax and cleaved-PARP increased in Eca109with miR-145, but decreased in TE-1with miR-145-in. We observed that enhancing miR-145expression in Eca109cells by introducing the miR-145mimic could effectively suppress Eca109cell migration, whereas the motility of TE-1cells conspicuously increased by transfecting with miR-145-inhibitor. We also tested whether miR-145is involved in the EMT to influence esophageal cancer metastasis. MiR-145mimic resulted in highly increased E-cadherin expression and reduced Vimentin expression in Eca109cells, whereas miR-145inhibitor caused opposite alterations in TE-1cells. F-actin was used to monitor the cytoskeleton dynamics in Eca109cells. Eca109cell lines transfected with the miR-145mimic inhibited the formation of lamelipodia and filopodia compared with those transfected with miR-145inhibitor.We also examined whether the knockdown of PLCE1by siRNA could potentiate the antitumor effects of miR-145. We found that co-transfection of si-PLCE1and miR-145-mimic in Eca109cells efficiently decreased PLCE1expression with enhanced expression of miR-145and partially increased the antitumor effects induced by miR-145.(3)We confirmed that miR-145expression was highly expressed in noncancerous tissues. The expressions of miR-145negatively correlated with N classification and clinical stage. We performed real-time PCR analysis for16paired ESCC from Han ethnic, of which PLCE1was significantly overexpressed in ESCC based from the immunohistochemistry analysis. As expected, the data showed that thirteen out of sixteen (81.25%) esophageal cancer tissues expressed lower level of miR-145compared with the matched non-tumor tissues, indicating that the majority of ESCC tissues followed the expected miR-145-PLCE1regulation pattern where tumors showed lower level of miR-145and higher level of PLCE1when compared with the matched non-tumors tissues. Moreover, pearson correlation showed that a significant inversely correlation existed between miR-145and PLCE1protein expression in esophageal carcinoma tissues from Chinese Han ethnic (R=-0.472,**P=0.008).(4)Using microRNA array detected the miRNAs that possible regulated by PLCE1in ESCC cell lines, we found that miR-106b-5p and miR-93can be significantly upregulated by PLCE1.Preliminary confirmed that between PLCE1and miR-106b-5p or miR-93there may be positive relationship in esophageal cancer cells. Real-time quantitative PCR showed that the miR-106b-5p and miR-93expression significantly downregulated after the PLCE1silenced.(5)MiR-106b-5p can significantly promote the esophageal cancer cell proliferation and migration, and suppress esophageal cancer cell apoptosis. We also found that miR-106b-5p overexpression can be partially reversed esophageal cancer cell proliferation and migration ability weaken which caused by siRNA mediated PLCE1gene silencing,and miR-106b-5p overexpression can block si-PLCE1-induced apoptosis.(6)Compared with normal esophageal tissue, miR-106b-5p expression in cancerous tissue significantly increased (P=0.0211), RBL2expression in cancerous tissue significantly decreased (P<0.0001). In esophageal cancer tissue samples, there is a positive correlation relationship between PLCE1protein expression and miR-106b-5p expression (R=0.4814, P=0.0431), there is a negative correlation relationship between PLCE1protein expression and RBL2protein expression(R=-0.2934, P=0.0015), there is a negative correlation relationship between miR-106b-5p-5p expression and RBL2protein expression (R=-0.6143, P=0.0014).Conclusion:(1) These results demonstrated that miR-145is a direct regulator of PLCE1and may function as a tumor suppressor by regulating the abnormal activity of PLCE1in human ESCC patients.(2) PLCE1upregulates miR-106b-5p-5p expression and result in the esophageal cancer cell proliferation and migration promoted, apoptosis inhibited. Focus on PLCE1/miR-106b-5p-5p/RBL2signaling pathway treatment strategies are expected to benefit the patients with esophageal cancer.