| Background Esophageal cancer(EC) is the eighth most common cancer and the sixth most common cause of cancer deaths worldwide. The total new cases of EC in China each year account for nearly half of all new cases across the world. Esophageal squamous cell carcinoma(ESCC) is a major histological subtype in China and accounts for more than 90% of EC. Although significant advances in multi-modality treatment have been made in last decades, the prognosis of ESCC remains poor. The five year survival time of ESCC after radical esophagectomy is still less than 30%. The main cause leading to worse prognosis of ESCC lies in that the majority of patients with ESCC are at advanced stage at presentation and 50% patients have distant metastasis. TAGLN2, a SM-22α homologous protein, is one of members of calponin family. Through interaction with actin, TAGLN2 is involved in cell stress fibers and maintains the stability of actin. Numerous studies reported overexpression of TAGLN2 in a variety of malignancies, such as lung, colorectum, pancreas, cervical, head and neck, etc., while low expression of TAGLN2 occurred in breast cancer, prostate cancer and Barrett’s esophageal adenocarcinoma. Hippo signaling pathway is commonly regarded as a tumor inhibition pathway and inactivation of this pathway due to a variety of reasons, results in the activity of YAP/TAZ, the target molecules of Hippo signal pathway, increased, which promotes tumor cell proliferation, migration and invasion. This study determined the correlation between expression levels of TAGLN2, YAP and TAZ and clinical parameters of ESCC, shedding lights on the roles of TAGLN2 in esophageal carcinogenesis.Aims To assess TAGLN2 expressions in normal esophageal epithelium(NEE), low grade intraepithelial neoplasia(LGIN), high grade intraepithelial neoplasia( HGIN) and different TNM stages of ESCC, and to determine the correlation between TAGLN2 expression with clinicopathological parameters in esophageal squamous cell carcinoma, overall survival after esophagectomy, which helps elucidate the roles of TAGLN2 in the formation and progression of ESCC. In addition, correlation analysis of the expression of TAGLN2 and YAP/ TAZ in ESCC was evaluated as well.Materials and Methods 1.1 Cell lines and tissue samples of ESCC ESCC cell lines EC9706, EC1, NE3, TE1 and immortalized esophageal epithelial cell line NEC,NE6 were maintained at Henan key laboratory of cellular and molecular immunology(The University of Henan). One hundred ESCC tissue samples of TNM I-IV stage with adjacent non‐tumorous tissue(25 cases of each stage) were obtained from the pathology section of Cancer Hospital of Linzhou from 2010 to the end of 2012. One hundred sixty-two cases with ESCC were available for postoperative survival analysis with 5-year follow-up data, which included 86 cases on an ESCC tissue microarray from Shanghai Outdo Biotech and 76 cases from the Huaihe Hospital of Henan University from 2007 to 2012. None patients received preoperative radio- or chemo-therapy. 1.2Methods 1.2.1 Real-time Polymerase Chain Reaction(RT-PCR) The mRNAs were extracted from 25 pairs of ESCC and corresponding adjacent tissue samples followed by mRNA concentration measurement. Reverse transcription was performed using Promega A3800 Im Prom II Reverse Transcription Kit. The reaction system was 20 ul, including Mg Cl2, 5*buffer, primer, RNA enzyme inhibitor, reverse transcriptase. RT-PCR reaction system was 20 ul, including 2 x Mixture Ultra SYBR(ROX with), primers, DEPC water and c DNA. Differential expressions of TAGLN2 mRNA in ESCC and adjacent nontumor tissue samples were detected by quantitative PCR. 1.2.2 Western blot Proteins from tissue and cell samples are extracted using 2D lysis buffer. The tissue samples included precancerous lesions comprising 5 NEEs, 5 LGINs and 5 HGINs, and 25 pairs of ESCCs from each TNM stage. Cell lines included NEC, NE6, EC1, TE1, NE3 and EC9706. Protein concentration was measured by Bradford kit. Electrophoresis of SDS-PAGE was used to separate proteins followed by electroblotting to PVDF membrane. PVDF membrane was incubated with 5% skimmed milk for 1 h at room temperature, primary antibodies at 4℃ overnight and corresponding secondary antibody for 1 h. ECL was used to detect protein bands and semi-quantitative analysis was performed using Quantity One software. 1.2.3 Immunohistochemical staining and semi-quantitative analysis Immunohistochemistry(IHC) was performed to detect TAGLN2 protein expressions in 162 cases of ESCC with 5-year follow-up data tissues, and 110 cases of adjacent precancerous lesions. According to the staining intensity and positive cells, semi-quantitative analysis was performed by multiplication of intensity and percentage of cell positivity. 1.2.4 Construction of stable cell lines NE6 Cells and 9706 cells were transfected with lentivirus carring TAGLN2 gene,to build the cell line NE6-TAGLN2 and NE6-T2 control cells, 9706-sh-TAGLN2 and NE6-T2 control cells. 1.2.5 Cell nuclear and cytoplasmic extraction We extracted the nuclear and cytoplasmic protein according to NE-PER nuclear and cytoplasmic extraction reagents kit protocol. Proteins were detected by Weatern blot. 1.3 Statistical analyses Software SPSS 17.0 was used to for statistical analyses. T-test was used for comparison between two groups. Wilcoxon test or Mann-Whitney test was used for statistical analysis of semi-quantitative results for RT-PCR, Western blot and IHC. Gamma test was used to evaluate the correlation between TAGLN2 and lesions. Chi-square test was used to evaluate the significance of difference between clinicopathological parameters and TAGLN2. Survival curve was estimated by Kaplan-Meier method and Log-rank was used to assess difference between two survival curves. Cox regression model was used to assess the significant prognostic factors. All results was considered to be a significant difference if P < 0.05.2 Results 2.1 The levels of TAGLN2 mRNA in ESCC were significantly higher than those in the corresponding adjacent nontumor tissues(P < 0.05) and positively correlated with the development of ESCC(P < 0.05). 2.2 From NEE to LGIN, and then to HGIN, TAGLN2 expressions gradually increased. Furthermore, TAGLNs expressions increased stepswise with progression of clinical TNM ESCC stage of. The expressions of TAGLN2 protein in esophageal epithelial cells NE6 and NEC were lower than those in ESCC cell lines. 2.3 IHC staining results showed that TAGLN2 was highly expressed in ESCC, mainly localized in the cell membrane and cytoplasm compared with adjacent nontumor cells and stromal. X2 test showed that TAGLN2 expression gradually increased from NEE to LGIN to HGIN(P < 0.05) but not significantly different from HGIN to ESCC. Mann-Whitney test showed that the levels of TAGLN2 were positively correlated with progression of ESCC precancerous lesions and progression of ESCC. The expressions of TAGLN2 were positively correlated with lymph node metastasis and TNM stage, respectively(P < 0.05) but there were no significant correlations between TAGLN2 and sex, TAGLN2 and age, TAGLN2 and depth of invasion, TAGLN2 and differentiation(P > 0.05). 2.4 Correlations between TAGLN2 and clinicopathological data, TAGLN2 and prognosis TAGLN2 expression was negatively correlated with the postoperative 5-year overall survival(P < 0.05). At early clinical stage of TNMⅠ-Ⅱ, there were negative correlations between TAGLN2 expression, lymphnode metastasis with 5-year overall survival but no correlation at late TNM stage(III and IV). 2.5 Univariate and multivariate analysis of prognostic factors of ESCC Univariate Cox regression analysis showed that gender, depth of tumor invasion, lymph node metastasis, TNM stage and TAGLN2 were significant prognostic factors for ESCC(P < 0.05), and age and grade were not important prognostic factors(P > 0.05). Cox multivariate regression analysis showed that TAGLN2 was an independent risk factor for ESCC(P < 0.05). Sex, depth of invasion, lymph node metastasis and clinical TNM stage were not considered to be independent risk factors for ESCC prognosis(P > 0.05). 2.6 Correlations between TAGLN2 and YAP /TAZ protein The expressions of YAP and TAZ protein in ESCC were higher than those in adjacent nontumor tissues with statistical significance(P < 0.05). There were positive correlations between expressions of TAGLN2 and TAZ, TAGLN2 and YAP evidenced by co-upregulation in 75% and 62.5% ESCC respectively. In 9706-sh-TAGLN2 and NE6-TAGLN2 cell nuclears, the YAP/TAZ protein were significantly decreased compared with that in the nucleus of the control cells.3 Conclusions 3.1 Aberrant TAGLN2 expression occurred in precancerous lesions of ESCC and increased stepwise with progression of precancer lesions, indicating that TAGLN2 may be a biomarker for diagnosis, prevention and therapeutic targets of ESCC. 3.2 TAGLN2 was correlated with clinical stage of ESCC and was one of independent prognostic factors for postoperative overall survival of ESCC. 3.3 The expression level of TAGLN2 was positively correlated with clinical stage of ESCC and negatively correlated with postoperative overall five year survival of ESCC. TAGLN2 was an independent prognostic factor for postoperative overall survival of ESCC. We hypothesized that TAGLN2 might be one of potential risk molecules after esophagectomy. 3.4 The positive correlations between TAGLN2 and YAP, TAGLN2 and TAZ, suggests that there may exists a crosstalk between TAGLN2 and Hippo signaling pathway, and TAGLN2 promotes YAP/TAZ to enter the nucleus. It indicated that TAGLN2 may promote progression of ESCC through increasing activity of Hippo pathway targets, YAP and TAZ. |
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