| Objective: As one of the most common malignant tumors,gastric cancer(GC)shares high morbidity and mortality.In spite of the significant improvements in gastric cancer’s diagnosis and treatment during recent years,most patients with GC were found to be at advanced stage when dignosed.These patients thus failed the chance for radical resection,and even if they received some treatments the rate of relapse and metastasis remained high.Patients with gastric cancer in advanced level had low five-year survival rates as only 30%-50%,probably because GC is a disease with high heterogeneous.Activation of epithelial to mesenchymal transition(EMT)is found to be one of the key mechanisms in invasion and metastasis ofgastric cancer.Autophagy has been reported by researchers to be highly related to cancer cells’ s EMT and invasion,and regulated by some molecules.Our research group previously established expression profiles of lncRNAs related to gastric cancer by Human LncRNA Microarray,and found that lncRNA AK022914 was highly expressed in gastric cancer,but its function role and its molecular mechanism in gastric cancer remains unknown.Methods:1.We investigated the expression patterns of lncRNAs in GC tumor samples(T)and their paired adjacent non-tumor tissues(N)in five GC patients with GC family history;and selected high expressed lncRNA AK022914 for further research.2.Fluorescence real-time quantitative PCR was used to detect the mRNA level of AK022914,autophagy related molecules like LC3 B and Atg7 in 120 pairs of gastric tumor tissues and paired adjacent normal tissues,then analysis was conducted to find the relationships between AK022914 expression and patients’ s age,gender,location,degree of differentiation,size,depth,lymph node metastasis,distant metastasis,helicobacter pylori infection,five year survival and molecules related to autophagy like LC3 B and Atg7.3.Quantitative PCR assay was used to detect the expression of AK022914 in gastric cell line GES1 and some GC cell lines,we selected cell lines AGS and BGC823 as experiments subjects.4.Lentivirus vector with over-expressed AK022914 and AK022914 small interference RNA were constructed respectively,by liposome transfections in AGC and BGC823 cell lines to decrease or increase AK022914 expression,weobserved the effects on cancer cells proliferation,clone formation,migration,invasion and EMT related molecules like E-cadherin and N-cadhein.Nude mouse models were used to observe effects of over expression of AK022914 on tumor formation,and protein levels of E-cadherin and N-cadherin in cancer cells.5.In order to get well knowledge of AK022914’s function in inducing EMT and promoting invasion,we carried out experiments on levels of clinical specimen,cells and nude mouse models to investigate potential molecular mechanisms of AK022914 in inducing EMT and promoting invasion.Results:1.LncRNA chip detection revealed that AK022914 is highly expressed in gastric cancer tissues.2.Using fluorescence real-time quantitative PCR to detect mRNA level of AK022914,then based on the level of expression the samples were divided into two groups and each group had 60 paired samples.It was found that AK022914 has significantly correlation with tumor invasion(p = 0.000),lymph node metastasis(p=0.001),distant metastasis(p=0.001),TNM stage(p=0.000)and 5 year survival(p=0.000),but no correlation with patients’ gender(p=0.853),age(p=0.350),tumor diameter(p=0.10),tumor location(p=0.924),tumor differentiation(p=0.077).Further studies showed that AK022914 is positively related with the expression of autophagy molecules LC3 B and Atg7.3.Detection of cell lines showed that,compared with normal gastric GES-1 cell line,AK022914 is up-regulated in varying levels in different gastric cancer cell lines.We used cell lines of AGS and BGC823 forfollowing research.4.Molecular knockout test was performed on AGS and BGC823 cells,CCK8 assay,clone formation test,scratch repair assay,and Transwell method was conducted to detect the proliferation,migration and invasion of cancer cells.Results showed that,compared with the blank control group and the empty control(Emepty Vector,EV)group,the ability of cell proliferation,the number of clones,ability of cell migration,and the ability of invasion in cells with overexpressed AK022914 were significantly increased,and the differences were of statistically significance.In addition,the level of E-cadherin protein in AK022914 over expressed cells was decreased while N-cadherin protein increased;and in siRNA group these situation were in the opposite.Compared with the blank control group and the EV group,the ability of cell proliferation,the number of clones,ability of cell migration,and the ability of invasion in cells with AK022914 siRNA were decreased significantly,and the E-cadherin protein increased with N-cadherin protein decreased.Further in vivo studies in vivo showed that the tumor growth rate,volume and quality in AK022914 over-expression group were significantly higher than those of control group.Compared with the control group the E-cadherin protein in the AK022914 overexpression group were decreased,while N-cadherinincreased.These results suggested that the overexpression of AK022914 may promote EMT in gastric cancer cells.5.In studies of clinical specimens,we found that the EMT related molecule E-cadherin was negatively correlated with autophagy related genes LC3 B and Atg7(r =-0.888,r =-0.788,respectively)in gastric carcinoma,and N-cadherin was positively correlated with the expression of LC3 B and Atg7(r = 0.964,r = 0.946,respectively).In studies of mouse model,we found that,compared with EV group,the expression of LC3 B and Atg7 protein in AK022914 over-expressed group were highly increased.In vitrostudies,it was showed that,when compared with the EV group,the autophagy related gene LC3 fluorescent plaque and its numbers in the AK022914 over-expressed group were increased,and the expression of autophagy gene Atg7 was also in high level;while LC3 fluorescent plaque and its numbers in the siRNA group were decreased,and the autophagy related gene Atg7 expression was decreased.According to the researches results which were based on clinical,nude mouse model and cytological levels,we may get suggestion that over expression of AK022914 may be closely related to EMT and autophagy in gastric cancer cells.6.We conducted co-transfection experiment with AK022914 plasmid and Atg7 siRNA in the AGS cell line,and then obtained cells with high expressed AK022914 and low level of autophagic gene Atg7.Western blot detection found that after co-transfected with AK022914+siAtg7,the level of E-cadherin was higher in co-transfection cells than cells with simple high expression AK022914,but the N-cadherin level was decreased and the EMT level was reduced too.On the other hand,when compared with the blank control,however,the EMT level was still in a higher level.All of these suggest that AK022914 may promote EMT through autophagy in gastric cancer cells.Conclusions:1.LncRNA AK022914 is highly expressed in gastric cancer,may act as negative factor of patients prognosis;2.Induction of EMT may be one of the important mechanisms by which AK022914 can play an important role in promoting cancer;3.AK022914 may regulate gastric cancer cell’s EMT through autophagy pathway. |
