| OBJECTIVES: By combining the small sample for high throughput genemicroarray screening with the large sample of clinical tissues for verification, weaimed to find some gastric cancer related genes. Evaluate the clinical significance andprognostic value of candidate gene THY-1, which is over expression in gastric cancertissues. And in order to provide theoretical basis for the development of newtherapeutic targets for the treatment of gastric cancer, we also investigated its effecton gastric cancer cell biological function in vitro together with its ability oftumorigenicity in nude mice.METHODS:(1) The differential profiling of mRNA expression in5pairs of gastriccancer and adjacent tissues was studied by Arraystar Human LncRNA Microarray. Bycombining with4similar platforms in GEO DataSets and further verifying the clinicalsignificance and prognostic value of those candidate genes using qRT-PCR, wefinally aimed those genes that were over expression in gastric cancer tissues forfurther investigation.(2) A tissue microarray was established basic on a total of423human gastric cancer FFPE specimens that were obtained from patients admitted inour hospital between2005and2008. The protein expression of THY-1was detectedby immunohistochemical staining. Then the relationship between THY-1expressionand clinicalpathological data and prognosis was analyzed.(3) CCK-8assay, colonyformation assay and cell cycle experiments were used to investigate the effect ofTHY-1on cell growth and proliferation in vitro. Transwell and wound healing assaywas also performed to examine migration and invasion of GC cells. The cell apoptosisexperiment was analyzed by using FITC-Annexin V/PI double staining assay. To identify the role of THY-1in tumorigenic ability of gastric cancer cells in vivo, wealso established the gastric cancer xenografts by injecting HGC-27cellsubcutaneously into nude mice.RESULT:(1) By combining the result of gene chip together with the microarraydata obtained from GEO DataSets, we screened out12candidate genes which wereover expression in gastric cancer tissues. Further verification through qRT-PCR in alarge sample of clinical tissues resulted in5particular genes up-regulation in gastriccancer tissues (P<0.05). They were CEACAM6, HEYL, MSLN, PLXDC1and THY1.Furthermore, the over expression of THY-1was positively correlated with degree oftumor differentiation (P=0.032), Lauren classification (P=0.035), cancer embolus(P=0.013) and neural invasion (P=0.019). In addition, high expression level of THY-1was also related to poor prognosis (Log-Rank-test, P=0.017).(2) Immuno-histochemistry anaysls in423gastric cancer cases showed that THY-1wasoverexpression in40.5%(126/311) tumor tissues, which was significantly higher thanthose in adjacent tissues (P<0.001). Moreover, the expression of THY-1was closelyrelated with tumor volumn (P=0.013), Lauren classification (P<0.001), degree oftumor differentiation (P=0.003), cancer embolus (P=0.039), lymph node metastasis(P=0.036), and TNM stage (P=0.047). The Cox’s proportional-hazard model formultivariate analysis revealed that THY-1expression, together with Laurenclassification, cancer embolus, T stage, N stage, and M stage, were independentlyunfavorable prognostic factors for OS.(3) CCK-8assay in vitro indicated that theproliferation of HGC-27and MGC-803after THY-1interference was obviouslyinhibited. Until Day5, the proliferation rate had already reduced30%as comparedwith sh-nc group (P<0.01). In contrast, exogenous THY-1significantly promote thegrowth of AGS cells. Until Day4, the proliferation rate had already risen25.7%ascompared with lenti-nc group (P=0.003). Colony formation assay showed that thecolony formation rate of HGC-27cells was obviously inhibited in sh-2and sh-3groups (4.17±0.67%and3.30±0.59%) as compared with sh-nc group (7.50±0.80%)(P=0.005and0.002). In AGS cells, the colony formation rate of lenti-THY-1group (8.53±1.14%) was remarkably higher than that in lenti-nc group (4.00±0.90%)(P=0.006). Cell cycle analysis revealed that exogenous THY-1could reduce theG0/G1arrest and induce AGS cells to enter into S phase (P=0.008). KnockdownTHY-1may causeing the G0/G1arrest in HGC-27cells and inhibited cellproliferation (P<0.001). Transwell assay verified that knocking down THY-1couldreduce the migration and invasion ability of MGC-803cells, and exogenous THY-1could induce this ability of AGS cells in contrarily. By using wound healing assay, wealso found out that knockdown of THY-1remarkably decreased migration of HGC-27cells. Over24hours of incubation without serum, the space of the wound in sh-2andsh-3were84.7±7.0and100.6±7.6, which was significantly lower than63.9±10.4,the space of sh-nc group (P=0.016and0.001). In the Annexin V/PI double stainingassay, the apoptotic rate of lenti-THY-1AGS cells was7.89±1.08%after48hoursstarvation, while in the lenti-nc group this rate was incressed to11.90±0.45%(P=0.004). Meanwhile, the apoptotic rate of HGC-27cells in THY-1interferencegroup was37.88±5.47%, which is significantly higher than22.84±1.50%in thesh-nc group (P=0.01). Nude mice subcutaneous tumor experiments showed that tumormass increased rapidly12days after injection. Untill Day21, the mean volume oftumors was up to1018.33±521.48mm3and the weight was up to81.47±41.72mg.On the other hand, tumor volume curve of the interference group was relatively flat.Untill Day21, the mean volume of tumors was only213.72±111.94mm3and theweight was only17.10±9.00mg (P<0.01).CONCLUSIONS:(1) CEACAM6, HEYL, MSLN, PLXDC1and THY1wereuprelated in gastric cancer tumor tissues. Furthermore, the over expression of THY-1was positively correlated with degree of tumor differentiation, Lauren classification,cancer embolus and neural invasion.(2) THY-1protein was overexpression in gastrictumor tissues, which was significantly higher than those in adjacent tissues. Theexpression of THY-1was also closely related with tumor volumn, Laurenclassification, degree of tumor differentiation, cancer embolus, lymph node metastasis,and TNM stage. THY-1can be treated as molecular markers to assess the prognosis ofgastric cancer patients.(3) THY-1could increase the proliferation of gastric cancer cell, promote gastric cancer cell migration, invasion and reduce the rate of apoptosisin no serum environment. In nude mice subcutaneous tumor experiments, we alsofound THY-1could promote the formation and growth of subcutaneous tumor mass. |
PDF Full Text Request