Research On The Expression Of WIG-1in Esophageal Cancer Tissue And Human Esophageal Squamous Carcinoma Cell Line EC109and Its Clinical Significance

BackgroundEsophageal cancer is one of the ten most common cancerous growth in today’s world.Compared with others, esophageal cancer is worse in prognostic, because in early stage itmay have distant metastasis and lymphatic spread. In recent years, genetic therapy hasbecome a hot spot in tumor treatment, which is also likely to be an effective measure in thefuture. In the esophageal cancer case, researches on the genetic level are comparatively less.Hence, it is imperative to find the molecules which play a key role in its occurring andmetastasis. WIG-1（wild-type p53-induced gene1） is a kind of zinc finger protein expressedby the induction of p53gene. After combining with double-strand RNA, it can restrain thecells from growing. Therefore, it may be a potential genetic therapy target for the treatmentof esophageal cancer.In this research, by analyzing the expression level difference of WIG-1in differentesophageal cancer tissues, its possible relationship with esophageal cancer patients’prognostic is found; by constructing human esophageal squamous carcinoma cell lineEC109of different WIG-1expression levels, possible functional mechanism of theinfluence of WIG-1on the biological behavior of esophageal cancer cell such as growthcycle, apoptosis, invasion and DNA damage repair, etc. is further studied; then possiblefunctional molecules are selected, hopefully clinical application significance can beprovided in the genetic therapy of esophageal cancer.ObjectivesMaking monoclonal antibody of WIG-1to lay a solid foundation for further researchwork. Understanding the relationship between different expression levels of WIG-1andesophageal cancer patients’ clinical pathological features and prognostic. Constructinghuman esophageal squamous carcinoma cell line EC109of high and low WIG-1expressionlevels to further study the influence of WIG-1gene on the biological behavior of EC109cell such as its growth, apoptosis, and invasion, etc. Discussing the influence of the expressionlevel of WIG-1gene on the chemotherapy drug sensitivity and tolerance of EC109cell andits influence on DNA damage repair as well as its possible molecule mechanism. Selectingpossible functional molecules based on the influence of WIG-1on gene expression profile.Methods1. Make monoclonal antibody of WIG-1by using hybridoma technology;2. Check the expression of WIG-1gene in esophageal cancer tissue and itscorresponding near and far cancer tissues by using RT-PCR and immunohistochemistrytechnology;3. Construct human esophageal squamous carcinoma cell line EC109of high and lowWIG-1expression levels by using lipofection, and appraise by using RT-PCR and WesternBlot technology;4. Draw the growth curve of EC109cell when WIG-1gene is in different expressionlevel by using MTT experiment;5. Check the change of EC109cell in cell cycle and apoptosis when WIG-1gene is indifferent expression level by using MTT experiment, colony formation assay, flowcytometry technology, and DNA fragmentation experiment;6. Examine the influence of different expression level of WIG-1gene on the invasionactivity of esophageal squamous carcinoma cell by using Transwell experiment and celldamage and scratch experiment;7. Calculate the IC50of cells on drugs, and analyze vitro drug sensitivity by usingMTT experiment;8. Test the influence of different WIG-1gene expression levels on the expression levelsof some drug resistant molecules by using Western Blot;9. Check the different DNA damage condition and repair ability caused by ultravioletradiation when WIG-1gene is in different expression level by using single cell gelelectrophoresis technology;10. Examine the influence of WIG-1gene on the gene expression profile of EC109byusing gene chip technology;11. Select possible functional molecules of WIG-1gene by using yeast twohybridization technology. Results1. Monoclonal antibody of WIG-1has been successfully made and affirmed byWestern Blot and immunohistochemistry experiments;2. The expression of WIG-1gene in esophageal cancer tumor tissue and itscorresponding near and far cancer tissues is different: the expression level of WIG-1gene inesophageal cancer tissue is obviously lower than it is in near and far cancer tissues, whilethe expression level of WIG-1gene in near cancer tissue is greatly lower than it is in farcancer tissue （p <0.05）;3. Human esophageal squamous carcinoma cell line EC109of high and low WIG-1expression levels has been successfully constructed and appraised by semi-quantitativeRT-PCR and Western Blot experiments;4. Judging from the results of MTT experiment, colony formation assay, DNAfragmentation experiment, flow cytometry technology, Transwell experiment and celldamage and scratch experiment, etc., the high expression of WIG-1gene can restrain humanesophageal squamous carcinoma cell line EC109from growing, accelerate its apoptosis,and can notably reduce its invasion and spread ability;5. The high expression of WIG-1gene can remarkably enhance the sensitivity ofEC109to chemotherapy drugs, such as5-Fu, CDDP, ADR. As for VCR, the sensitivity isnot obviously influenced. WIG-1gene may enhance the sensitivity of EC109tochemotherapy drugs and reduce its resistance to them by restraining the expression ofERCC1and P-gp.6. The high expression of WIG-1gene can reduce the damage effect of ultravioletradiation on EC109, and markedly improve its DNA repair ability;7. Gene chip examination results indicate that raising the expression level of WIG-1can influence the expression of419kinds of genes and it perhaps may also influence theexpression of301kinds of genes;8. Through the results of yeast two hybridization experiments, the possible functionalmolecules of WIG-1gene are selected as being ILF3（NM₀17620），UTP14A（NM₀06649），Sox3（NM₀05634）. ConclusionsMonoclonal antibody of WIG-1is made, hence a good foundation is laid for thisexperiment study and further deeper researches. Human esophageal squamous carcinomacell line EC109of high and low WIG-1expression levels is constructed. WIG-1gene is notonly expressed in esophageal cancer tissue cells, but also in normal tissue cells. Theexpression level of WIG-1gene in esophageal cancer tissue is obviously lower than it is innear and far cancer tissues. The high expression of WIG-1can restrain the growth of EC109cell and accelerate its apoptosis while the low expression of WIG-1can accelerate thegrowth of EC109cell and restrain its apoptosis. Raising the expression level of WIG-1canrestrain the expression of ERCC1and P-gp, then enhance the sensitivity of EC109cell tochemotherapy drugs, while the expression level is lowered, the result is exactly the opposite.The high expression of WIG-1gene can improve the ultraviolet radiation tolerance andDNA repair ability of EC109cell. Now it is known that raising or lowering the expressionlevel of WIG-1gene can influence the expression of many genes, hopefully WIG-1genecan be detected in further study. Being a control point of known anti-oncogene, its specificfunctional mechanism may offer more options in the diagnosis and treatment of esophagealcancer.