The Role Of Wnt Signaling Pathway Inhibitor Chibby In The Occurrence Of Laryngeal Carcinoma

Objective: More studies have shown that there was close relationship between Wnt/β-cateninsignaling pathway and the occurrence and development of tumour. Chibby a new antagonistfactor in wnt/β-catenin signaling pathway block Wnt signaling through inhibiting the activationof β-catenin, so it maybe a potential tumour suppressor. In recent years, studies have shown thatthere is a certain relationship between Chibby and the occurrence of some tumors. But there isnot study about Chibby and Laryngeal carcinoma. This study will systemly described therelationship between Chibby and Laryngeal carcinoma in the level of expression and function. Itwill establish basis for further clarifying the pathogenesis of Wnt/β-catenin signaling pathway inLaryngeal carcinoma and finding a new target for its gene therapy.Methods:1. Detect the expression differences of Chibby mRNA and protein between cancertissues and corresponding adjacent normal laryngeal mucosa through real-time PCR andimmunohistochemistry (SP method), respectively. Statistical analysis the expression differencesof Chibby mRNA and protein in different age, gender, clinical stage and tumor differentiationusing statistical software SPSS17.0.2. Coding sequence of the mRNA of human Chibby wassearched in GeneBank. Primers for obtain Chibby fragment was designed using primer designsoftware primer premier5. The recombine plasmids plv-cs2.0-cby was conceived through dualenzyme digestion by XbaⅠ/MIuⅠ. Viral supernatant were obtained after lentiviral packagingplasmids through liposomes (TurboFectTM) method. Laryngeal carcinoma cell lines Hep-2wereinfected with the same titers of viral supernatant. Stable cell lines plv-cs2.0-Cby sereeningthrough puro were established. The expressive efficiency of Chibby in Stable cell lines wasdetected through Real time PCR and Western blot, respectively.3. Detect cells proliferation,cells clones forming ability; cells cycle distributionand cells apoptosis using MTT assay; platecolony forming assay; flow cytometry and Tunel assay, respectively.Results:1. Compareing with normal laryngeal mucosa, the expression efficiency of ChibbymRNA and protein in cancer tissues was reduced56.67%(17/30) and80.00%(24/30),respectively. The difference was statistically significant (p＜0.05). There was no significantrelationship between the expression differences of Chibby mRNA and protein in tumor tissue and the different gender, age, clinical stage and tumor differentiation of laryngeal cancer patients(p＞0.05).2. Gene421bp size of fragment was obtained through PCR amplification.Fragment the size of8750bp and414bp were obtained from recomninant vector afterdouble-digested by XbaⅠ/MIuⅠ. Fragment the size of6886bp,1384bp and894bp wereobtained from recomninant vector after double-digested by XbaⅠ/kpnⅠ. The size of thesefragment were consistent with the theory. The result of sequencing was consistent with thepublished in GeneBank.The Chibby mRNA was overexpressed276.92times by RT PCR(P＜0.01). There was only exogenous Chibby protein but not endogenous Chibby protein bands (p＜0.01).3. Compared with the control group and plv-cs2.0group, cells proliferations werereduced in the group of plv-cs2.0-Cby. The more the time, the more obvious differences (p＜0.05). The number of colony-forming cells and cloned area in the group of plv-cs2.0-Cby weresignificantly lower than ones in control groups and plv-cs2.0group (p＜0.01). Flow cytometryanalysis showed that plv-cs2.0-Cby cells remained in G1phase, but decreased in S phase (p＜0.05). Tunel staining was found that, compared with control group, the rate of apoptosis in thegroup of plv-cs2.0-Cby was significantly increased(p＜0.01). But the cells proliferation,colony-forming ability, cell cycle distribution and apoptosis ratio between control group andplv-cs2.0group were no difference (P＞0.05).Conclusions:1. Compared with normal laryngeal mucosa, the expression level of ChibbymRNA and protein were lower in tumor tissue. There was no significant relationship betweenthe expression differences of Chibby mRNA and protein and the patient’s gender, age, clinicalstage and tumor differentiation.2. The plv-cs2.0-Cby lentiviral eukaryotic overexpressedvectors was successfully constructrd. The Chibby was effectively over-expression in Hep-2afterinfected with lentiviral plv-cs2.0-Cby. The foundation was laid for the functional expression.3. After the over-expression of Chibby, inhibited the proliferation and colony-forming ability ofHep-2, remained Hep-2in G1phase and promoted apoptosis of Hep-2. Therefore, theoverexpression of Chibby was expected to become an effective means for the targeted genetherapy of laryngeal carcinoma.