The Expression Of ZNF703in Gastrointestinal Cancer And Its Role In Promoting Tumor Progession

Background and ObjectiveGastrointestinal cancer (GIC) is the most common malignant tumors ofdigestive system. In the past20years, with the improvement of living conditions, the change of dietary structure and the deterioration of environment, the morbidity and mortality of gastrointestinal cancer in our country has been increasing straightly.Colorectal cancer, including colon cancer and colorectal cancer is a common malignancy, and its incidence big differences in different regions of the world, North America, Oceania, the highest in Europe centered lower in Asia and Africa, southern China, especially the southeast coast of significantly higher in the north. According to reports, in2008, the global total of1.2million new cases, and there are60.9million people died due to the disease. About9%of cancer-related deaths in colorectal cancer. The incidence of colorectal cancer is also not optimistic, the incidence of colorectal cancer showed a clear upward trend. In2010,the incidence of colorectal cancer accounts for our fourth common cancer after lung cancer, stomach cancer and liver cancer. In some developed regions of colorectal cancer incidence rate ranks first two incidence of malignant tumors, a serious threat to human life and health.Recent research indicates that multiple genes were involved in the development process of colorectal cancer.Inl988,Vogelstein polygenic firstly proposed multi-gene, multi-stage, multi-step process of colorectal cancer incidence patterns. Therefore, there is an growing number of genes have been confirmed to participate in the occurrence and development of colorectal cancer.Gastric carcinoma (GC), one of the most common malignan tumors in the world, is the third cause of cancer mortality in China, incidence of which in our country has been increasing significantly. Also, it’s the world’s most common cause of cancer-related deaths, five-year survival rate of which is less than30%.The number of new global gastric is more than100million per year,China accounted for42%.Deaths of global gastric is about800,000,accounting for35%of our country, which is one of the countries with the highest incidence of gastric cancer and mortality.morbidity and mortality rates of our courtry are more than twice the world average.Many factors that influence the prognosis of gastric cancer, among which invasion and metastasis are a complex process of many pathological changes and is an important cause of death in patients with gastric cancer.One of the main causes of death in patients caused by cancer is the distant metastasis and complications accordingly in the course of tumor growth, which is regulated by many genes.That is currently a hot area of research. In-depth study of the mechanism of invasion and metastasis of gastrointestinal cancer, will contribute to the development of molecules for invasion and metastasis and gene targeted therapy has important clinical significance.Zinc finger protein expression in eukaryotes are widely involved in cell differentiation, proliferation and apoptosis of a variety of important life processes. In recent years, about the role of zinc finger proteins in tumors caused widespread concern, and many studies have confirmed the occurrence of zinc finger protein family evolution by regulating the transcription and expression of related genes mediated tumors.Zinc finger protein703(ZNF703) who is a member of NET/N1Z family of zinc finger transcription factors is identified as a novel oncogene in Luminal B breast cancer, It contributes to aspects of developmental growth and patterning across evolutionarily diverse species.Current studies have shown that ZNF703in human breast cancer and endometrial cancer showed overexpression, which is closely related to the occurrence of abnormal expression of tumor invasion and metastasis. It suggests that ZNF703is associated with the the occurrence and development of human tumors. Although more research suggests that the key factor for human ZNF703tumor development However, so far, the role of ZNF703in gastrointestinal cancer remains unclear, and the expression of ZNF703in both gastrointestinal cancer and related research at home and abroad have not yet been reported.Meanwhile Helicobacter pylori infection is also a significant contributory factor during gastric carcinogenesis.However, in1994, it was classified as a class I carcinogen by WHO (World Health Organization). Since1983, the Australian scholar Marshall and Warren for the first time successfully isolated and cultured Helicobacter pylori (Helicobacter pylori, Hp) from the human gastric mucosa. Since then, Hp has become the object of many experts and scholars at home and abroad. Hp can lead to gastric cancer.It has not been reported whether Hp is associated with the cancer gene ZNF703.Therefore, we researched the expression and distribution of ZNF703in gastrointestinal cancer, analysed the relationship between ZNF703mRNA and colorectal cancer (CRC) clinicopathologic significance, and explored the effects of RNA interference (RNAi) ZNF703on the bioactivity of CRC cell line LoVo. It provides a theoretical basis for clinical diagnosis and treatment and judgment of prognosis. For further investigation, we also detected the association between the infection of helieobaeter Pylori and the expression of ZNF703protein in the present study to discuss if Hp infection is related with ZNF703expression during the development and progression of gastric cancer(GC).METHODS(1) Expression of ZNF703mRNA(ZNF703protein) in human colorectal cancerThirty fresh frozen CRC and corresponding normal colorectal mucosa tissue samples(more than10cm away from the edge of the CRC) were taken from patients with CRC.The expression of ZNF703was examined in fresh, paired CRC tissues by reverse transcriptase polymerase chain reaction (RT-PCR; n=22) and Western blot analysis (n=26).(2)The relationship between ZNF703expression and clinicopathologic parametersThe expression of ZNF703was examined on paraffin embedded specimens (including112CRC specimens,58matched normal specimens and23paired metastatic lymph node samples) by immunohistochemistry(IHC).To investigate the relationship between ZNF703expression and clinicopathologic parameters(age, gender, tumor size, tumor location, tumor differentiation, depth of wall invasion, lymph node metastasis and AJCC stage) and prognosis of CRC patients.(3)Effect of ZNF703down-expression on the biological behaviors of human CRCTo analysize the ZNF703expression in eight CRC cell lines by Western blot,and select the CRC cell lines which have the overexpression of ZNF703.We designed and synthesized specific small interfering RNA of ZNF703(siRNA-ZNF703) and transacted it into LoVo cells. Western blot was performed to determine the effects of siRNA-ZNF703on ZNF703expression. In vitro, using RNA interference, we investigated the effects of ZNF703depletion on tumor proliferation and migration.(4)60cases of GC and the corresponding normal gastric tissues just around the tumor were collected.Then, all the samples were detected by immunohistochemistry (IHC)、 Western blot and reverse transcription-polymerase chain reaction (RT-PCR) to investigate the expression of ZNF703. Moreover, to analysize the ZNF703expression in CRC cell lines and normal.(5) H.pylori bacterial lysates were incubated with the nomal gastric mucosa cells(GES-1)for24h at37℃. Protein was extracted from infected cells. We attempted to analysed the association between the infection of helieobaeter Pylori and the expression of ZNF703protein, using cell line models and Western blot.(6)Statistical Methods:The analysis of date is performed by SPSS16.0. As P<0.05is considered statically significant.The chi-square test and speanrman correlation analysis are used as statistical methods.RESULTS(1) RT-PCR demonstrated that the expression of ZNF703mRNA in colorectal cancer tissues was significantly higher than in adjacent normal tissues. Western blot showed ZNF703protein showed high positive expression rate of65.38%(17/26), normal mucosa adjacent low or no expression, the positive rate34.62%(9/26) in colorectal cancer, the difference was statistically significant (p<0.05). Immunohistochemistry found that ZNF703expression mainly in the nucleus (78/112,69.64%), partially expressed in the cytoplasm or the cell membrane. ZNF703expression in colorectal carcinoma was77.68%(87/112), while the positive expression in normal mucosa adjacent cancer was9.4%(3/32), the difference was statistically significant (p<0.05). ZNF703protein expression in23cases of metastatic lymph node tissue was91.3%(22/23).(2) ZNF703overexpression was significantly associated with the depth of the infiltration (P=0.028, P<0.05) and lymphonodes metastasis of colorectal cancer (P=0.006, P<0.05). Immunohistochemical results with clinical and pathological data found that the expression levels of ZNF703with colorectal cancer patient’s age (p=0.524), gender (p=0.152), tumor size (p=0.063), site (p=0.108), differentiation (p=0.742) and AJCC staging (p=0.406) had no significant correlation, the differences were not statistically significant (P>0.05). ZNF703breakthrough serosa serosa and did not break the expression in colorectal cancer rates were52.9%(36/68) and31.8%(14/6), the difference was statistically significant (P<0.05). ZNF703and lymph node metastasis in colorectal cancer without lymph node metastasis positive expression rate was55.2%(32/58) and29.6%(16/54), the difference was statistically significant (P<0.01). Survival analysis showed that, ZNF703overexpression is a poor prognostic factor in patients with colorectal cancer. The deeper of invasion, as well as lymph node metastasis,ZNF703has the lower expression that suggesting ZNF703may be associated with the prognosis in colorectal cancer.(3) Western blot demonstrated that ZNF703was expressed in human colon cancer cell lines (LS174T, SW480, HT29, SW620, DLD1, SW1116, LoVo, Caco-2cells),which,ZNF703expression in LoVo cells was highest in CaCo cells expression to a minimum.Western blot assay revealed that ZNF703was expressed in LoVo cell membrane,cytoplasm and nucleus.(4) RT-PCR and Western-blot showed that specific siRNA-ZNF703significantly inhibited ZNF703expression, with inhibition by siRNA-ZNF703being highest. MTT assay revealed that, LoVo cells after transfection of siRNA-ZNF70348hours before the start appear proliferation inhibition, transfected with the extension of time, the cell growth inhibition rate increasingly high, with statistical significance (P<0.01) and negative interference group and the control group; scratch experiments showed, LoVo cells after24h and scratches48h, si-ZNF703treatment group was significantly slower speeds healing of scratches on negative interference group and the control group (P<0.01). The Transwell assay revealed that LoVo cells after transfection of siRNA-ZNF703migration was significantly reduced, siRNA-ZNF703group to reach the lower chamber of the cells with the control group and blank control group were significantly different (P<0.01). Downregulation of ZNF703by specific siRNA could obviously inhibit CRC cells proliferation and migration significantly.(5) ZNF703showed high positive expression in gastric cancer (positive rate of85%,51/60), no or low expression (positive rate of12%,3/25) in normal gastric mucosa by immunohistochemistry, two with a statistically significant difference between (P=0.021). Western-blot showed that ZNF703protein was expressed in gastric cancer tissues, but not in normal gastric mucosa low expression. RT-PCR analysis found that gastric cancer has ZNF703mRNA expression, but not in normal gastric mucosa low or no expression of ZNF703mRNA expression.(6)ZNF703mRNA(protein) was no expressed in normal gastric cell line(GES-1), while there was a significant increase in the expression of ZNF703mRNA(protein) in gastric cancer cell lines (AGS, SGC7901, MGC823, BGC802) by RT-PCR and western blot analysis.(P=0.029).(7)Western blot results showed that H. pylori reference strain NCTC11637(CagA+) may increase normal gastric cell line (GES-1) expression of ZNF703protein, but the expression of a mutant strain of H. pylori SS1on GES-1cells ZNF703protein no effect (P=0.037).Conclusions(1)ZNF703expression is higher in colorectal cancer. ZNF703overexpression was significantly associated with lymphonodes metastasis, the depth of the infiltration. Overexpression of ZNF703might be associated with GC development and progress.(2)Downregulation of ZNF703by specific small interfering RNA could obviously inhibit the proliferation and migration of colorectal cancer, suggesting that over-expression of ZNF703is closely related to the progress of colorectal cancer and ZNF703gene is important to promote the invasion and metastasis of colorectal cancer,which may become a potential target for therapy.(3) ZNF703expression in gastric cancer; Hp wild strains can upregulate the expression of ZNF703in normol gastric mucosacells,suggesting that ZNF703may be involved in the pathogenesis of Helicobacter pylori carcinogenic.