In the present study, we studied the expression status of CRNN in clinical ESCC specimens and ESCC cell lines by quantitative and semiquantitative kinase inhibitor Ivacaftor RT-PCR respectively. Both in vitro and in vivo functional assays were used to investigate the tumor suppressive effect of CRNN in ESCC cell lines. The results demonstrated that CRNN had strong tumor suppressive function. In addition, the tumor suppressive mechanism of CRNN and its clinical significance in ESCC were also addressed. Materials and Methods Cell lines and primary ESCC specimens Chinese ESCC cell lines HKESC1, EC18 and EC109 were kindly provided by Professor Srivastava (Department of Pathology, The University of Hong Kong) [11]. Six Japanese ESCC cell lines (KYSE30, KYSE140, KYSE180, KYSE410, KYSE510, KYSE520) were obtained from DSMZ, the German Resource Center for Biological Material [12].

Primary ESCC tumors and their adjacent non-tumorous tissues were collected immediately after surgical resection at Linzhou Cancer Hospital (Henan, China). All patients did not receive preoperative treatment. Samples used in this study were approved by the Committees for Ethical Review of Research involving Human Subjects at Zhengzhou University (Henan, China). Written informed consents for the original human work that produced the tissue samples were obtained. The study was also approved by the Institutional Review Board at Cancer Center, Sun Yat-sen University. Quantitative and semiquantitative RT-PCR Total RNA was extracted by Trizol (Invitrogen, Carlsbad, CA) and 2��g of total RNA was used to synthesize cDNA with the Advantage RT-for-PCR Kit (Clontech, Mountain View, CA), following the standard protocols provided by the manufacturer.

Semiquantitative RT-PCR was performed by using AmpliTaq (Applied Biosystems, Foster City, CA). The GAPDH or 18s were used as internal controls. For qRT-PCR, cDNA were amplified using a SYBR Green PCR Kit (Roche, Basel, Switzerland). The sequences of primers were listed in Table 1. Amplification protocol consisted of incubations at 95��C for 15sec, 60 ��C for 1min for 40 cycles. Quantification was done using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). All gene expression values were normalized using the internal control and calculated using the comparative CT method (����CT method) [13]. Downregulation was determined if relative quantification (RQ) value of Dacomitinib non-tumor tissue was more than 2-fold change than RQ of corresponding tumor tissue. Table 1 Primers�� sequences. Tissue microarray (TMA) and immunohistochemistry (IHC) A TMA composed of 300 ESCC tumor specimens were collected from Linzhou Cancer Hospital (Henan, China).