Expression And Significance Of CHD1L In Esophageal Squamous Cell Carcinoma And Expression Of CHD1L In Esophageal Carcinoma Cell Lines

Background and ObjectiveEsophageal cancer (EC) is one of the six most commom malignant tumors, which is ranked as the fourth frequent cause of cancer-related death in China and the sixth in the world. Esophageal carcinoma is characterized by its significant regional differences, mainly concentrated on the Taihang mountain area, and the incidence of EC from high to low in concentric circles to outside distribution. Linzhou and the adjacent district Huixian of Henan province are the highest incidence region in China, where the major histological subtype of EC is esophageal squamous cell carcinoma (ESCC). Because of the high incidence of ESCC and its poor prognosis, the worldwide especially Chinese scholars extensively pay attention to nosetiology of ESCC. The same with other sorts of tumors, the development of ESCC is also a genetic multi-step process. In the process of cell cancerization, most of the adjustment factors in the cell cycle have an important function, but the most significant molecular basis is the activation of oncogenes and the inactivation of tumor suppressor genes (TSGs). Normally, pre-oncogene pays an important role in the process of cells signal transduction and proliferation, however, when pre-oncogene activated as oncogene, the high expression or expression of abnormal function protein can lead to tumorigenesis. Presently, the genes associated with ESCC contain:ras, C-myc, EGFR, c-Jun, MDM2, MTA1, hFRI, c-fos, CyclinD, C-erbB2, int-2et al.Using Chromosome Microdissection and Comparative Genomic Hybridization (CGH), Xin-yuan Guan, isolates a novel oncogene CHD1L (Chromodomain helicase/ATPase DNA binding protein1-like gene) within the1q21amplicon, who is the professor of Departments of Clinical Oncology of the University of Hong Kong.CHD1L is also named as ALC1(Amplified in liver cancer1, GENE BANK: AF537213) and the full-length mRNA (2980bp) encodes an89kDa protein. In HCC, CHD1L was found to up-expression and was further confirmed as the new candidate hepatoma gene by investigating the function and mechanism of cells trasnsfected the above gene. However, there were not literatures about CHD1L connected with ESCC.The study of CHD1L in hepatocellular carcinoma (HCC) demonstrates through downregulation of p53and its downstream gene p21, upregulation of Cdk2and CyclinE,it promotes DNA synthesis and G1/S transition and inhibits apoptosis in response to cellular stress. Reverse transcription-polymerase PCR (RT-PCR) and real time PCR (q-PCR) were used to detect the expression of CHD1L in primary ESCC and corresponding nontumorous tissues in RNA level, immunohistochemistry (IHC) to detect its expression in protein level, in order to investigate its clinical meaning in ESCC. Through Reverse Transcription-polymerase PCR (RT-PCR) and real time PCR (q-PCR) detected the expression of CHD1L in7ESCC cell lines,we selected not only relatived-downexpression ESCC cell lines KYSE30and KYSE510, but up-regulated ESCC cell lines KYSE180and HKESC. Medicine G418and Puromycin separately selected KYSE30, KYSE510, KYSE180and HKESC1, to provide the foundation of selecting stable clones and researching function and molecular mechanism.Materials and Methods1. Fifty EC samples by surgical removal and its responding normal esophageal mucous membrane (nontumorous tissues be apart from tumor beyond5cm) were obtained from the People’s Hospital of LinZhou city from July2010to October2010,289paraffin_embedded tissues from the same Hospital is from February2002to December2008,which were all evaluated as esophageal squamous cell carcinoma through pathologist detection. There was not one of these cases treated by radiotherapy, chemotherapy and immunotherapy.2. Reverse transcription-polymerase PCR (RT-PCR) and real time PCR(q-PCR) were used to mesure the expression of CHDIL in50primary ESCC and their paired normal esophageal mucous membrane in RNA level.3. Immunohistochemistry (IHC) was used to detect CHDIL expression in245primary ESCC tissues and their paired normal esophageal mucous membrane in protein level.4. The experimental data was analyzed by SPSS16.0statistical package, through Student’s test and Chi-square test, the test level a being0.05. Kaplan-Meier was used to analyze the relationship between the expression of CHD1L and survival of the ESCC patients.5. Using Reverse Transcription-polymerase PCR (RT-PCR) and real time PCR (q-PCR) to detect the expression of CHDIL in7ESCC cell lines, we selected not the two most low-expression but up-regulated ESCC cell lines.6. KYSE30and KYSE510which were low-expression of CHD1L were cultured in the media supplemented with G418and KYSE180and HKESC1which were up-regulation of CHD1L were cultured in the media supplemented with puromycin to get the appropriate concentration.Result1. Of these50samples of esophageal squamous cell carcinoma, up-regulation of CHD1L mRNA was detected in31cases of ESCC compared to their paired nontumorous tissues, and the frequency of over-expression is62.0%(31/50). Down-regulation of CHD1L mRNA was absent in5cases of ESCC compared to their paired nontumorous tissues, and the frequency of low-expression is10%(5/50). The results of PCR demonstrated that there was a significant difference in expression of CHD1L between esophageal squamous cell carcinoma and their paired normal esophageal mucous membrane in mRNA level (P<0.05).2. The result of Immunohistochemistry(IHC) showed in245primary esophageal squamous cell carcinoma102cases were found expression of CHD1L protein(41.6%) and143cases were not found expression of CHD1L protein(58.4%). In their paired nontumorous tissues52cases up-rugulation of CHD1L protein level (21.2%) and193cases expressed normally (78.8%). In protein level, the expression ratio of CHD1L in esophageal squamous cell carcinoma is41.6%(102/245),which was significantly higher than the ratio21.2%(52/245)of their paired normal esophageal mucous membrane,and there was a significant difference between them(P<0.05).3. In245primary esophageal squamous cell carcinoma tissues, the over-expression ratio of CHD1L protein in Tis group was25.0%(1/4), the over-expression ratio of CHD1L protein in T1group was7.1%(1/14), the over-expression ratio of CHD1L protein in T2group was26.3%%(5/19), the over-expression ratio of CHD1L protein in T3group was25.0%(13/52), the over-expression ratio of CHD1L protein in T4group was52.6%(82/156). Of invasion level, the expression of CHD1L protein had statistic difference (P<0.05).In the TNM standard stages, the over-expression ratio of CHD1L protein in Ⅲ～Ⅳstage group was60.0%(48/80), which was obviously over the over-expression ratio of CHD1L protein in0～ⅡB stage group32.7%(54/165), and the two groups had significant difference in statistics (P<0.05). The over-expression ratio of CHD1L protein in the group with lymphatic metastasis is52.4%(55/105),which was over the group of no lymphatic metastasis33.6%(47/140),and the two groups had significant difference in statistics (P<0.05).4. In245cases of esophageal squamous cell carcinoma, the abnormal expression of CHD1L protein had no significant difference in statistics with age, gender and cell differentiation (P>0.05).5. The5-year overall survival of CHD1L over-expression in ESCC cases (28.8%)was significantly lower than that in ESCC cases with normal CHD1L expression(36.7%,p<0.05).6. The expression of CHD1L mRNA in ESCC cell lines KYSE30, KYSE410, KYSE510was relatively low, whereas CHD1L mRNA was up-expression significantly in ESCC cell lines EC18, KYSE180, HKESC1, KYSE140.7. The drug concentration of G418in ESCC cell line KYSE30was500ug/ml, and the drug concentration in ESCC cell line KYSE510was800ug/ml. The drug concentration of puromycin in ESCC cell line KYSE180was0.3ug/ml, and the drug concentration in ESCC cell line HKESC1was0.1ug/ml.Conclusion1. CHD1L in esophageal squamous cell carcinoma shows up-regulation,and the over-expression is associated with ESCC invision, clinicopathological staging, lymph node metastasis and5-year overall survival. It is suggested that CHD1L may correlate with the progression and prognosis of ESCC.2. Through detecting the expression of CHD1L in7ESCC cell lines, it is selected the appropriate drug concentration of G418in ESCC cell lines KYSE30and KYSE510, and the appropriate drug concentration of puromycin in ESCC cell lines KYSE180and HKESC1, for providing the foundation about selecting stable clones and the researching function and molecular mechanism.