The Effects And Mechanisms Of The Tumor Suppressor Gene ARHI On The Autophagy,Invasion And Metastasis In Gastric Cancer Line BGC823

Objective:Gastric cancer is a common malignancy,about 1/3 of the gastric cancer patients with distant metastases at diagnosis,and prognosis of such patients is poor.Distant metastasis of gastric cancer is the primary mode of treatment failure,and also the main reason of death in patients.To find and control of metastasis in gastric cancer therapy is the key of improving efficacy of treatment.Autophagy-related tumor suppressor gene ARHI was a maternally imprinted gene discovered in 1999,which was downregulated or absent in ovarian tumors,breast cancer and many other cancers,and was involved in tumor development and metastasis.ARHI functions include inhibiting the growth and reproductive development,inhibition of proliferation,migration,involved in cell cycle regulation and apoptosis,recent studies found that ARHI can also participate in autophagy inhibition of tumor metastasis.To date,systematic research on ARHI autophagy and gastric cancer invasion and metastasis has not been reported.This study aimed to investigate the effects of ARHI gene on the proliferation,invasion and autophagy,and explored the mechanisms,and to investigate the effects of ARHI gene on tumor growth and metastasis in vivo.The purpose of this study was to detect the expression and clinicopathological significance ARHI in gastric mucosa and gastric carcinoma.This study aimed to build ARHI re-expression in gastric cancer cell lines in vitro,and to investigate the effects of ARHI gene on the proliferation,proliferation,autophagy,apoptosis,and migration.This study also aimed to use tumor-bearing nude mice model to evaluate the effects of ARHI gene on gastric cancer cell xenograft growth and hematogenous metastasis,and to explore the molecular mechanism.The system is designed to fully explain the biological behavior of ARHI gene regulation mechanism of gastric cancer cells,and to objectively assess the value of ARHI as a theraputic target for gastric cancer about invasion and metastasis.Methods:1.Construction of re-expression ARHI gastric cancer cell lines.Human gastric cancer cell line such as BGC-823,SGC-7901,MKN-45,N87 immortalized cell lines and human gastric epithelial cell line GES were routinely cultured.ARHI expression levels in each gastric cancer cell lines were detected by Real-Time and Western-Blot and found the ARHI low-expressing cell line BGC823.High expression shuttle plasmid pCMV6-ARHI-AC-GFP and vector plasmid pCMV6-AC-GFP was purchased from Origene Company.BGC823 gastric cancer cells were liposome transfected.By screening and selection by G418 positive stable transfected clones obtained,and were verified by Real-Time PCR,Western-Blot and immunofluorescence method.2.Experimental observation of cytology evaluation of ARHI gene re-expression on the biological characteristics of gastric cancer cells BGC823 in vitro.The study used ARHI stably transfected BGC823 gastric cancer cell lines,analyzed re-expression of ARHI gene on the biological behavior of the gastric cancer cell line in vitro.Using MTT colorimetric assay and colony formation assay the proliferation was evaluated,using FCM the cell cycle and apoptosis was evaluated,using transwell assay and wound healing assay the migration and invasion were evaluated,using WB LC3B,p62 level was evaluated to measure the level of autophagy.Using WB evaluation related signaling pathway protein levels.Related pathway protein level was evaluated by WB.The impact of methyltransferase inhibitor 5-Aza-dC(5-Aza-2'-deoxycytidine)and histone deacetylase inhibitor trichostatin A on ARHI level was analyzed to investigate the causes of inactivation in gastric cancer.3.Effect of re-expression of ARHI gene on BGC823 gastric cancer cells in nude mice.BALB/c-nu mice were purchased from Shanghai SLAC Laboratory Animal Co.,were 4-5weeks old,male,weighing 20-22g.ARHI stably transfected BGC823 gastric cancer cell line was used.For BALB/c-nu mice subcutaneous xenograft model and hematogenous metastasis model,eperiment were divided into vector groups and ARHI group(ARHI),n=5 for each.Collecting the cells in logarithmic growth phase,cell number was counted to inoculate subcutaneously into nude mice right carotid by the number of cells per mouse5×10~6/200?L.Four weeks after subcutaneous inoculation tumor-bearing mice were sacrificed,removed the tumor to weigh.ARHI expression levels of subcutaneous tumor tissue were detected by qRT-PCR and WB.Immunohistochemical staining was used to evaluate the level of LC3B,p62,Ki67,SP1,VEGF,p-AKT proteins.Cell number was counted to inoculate into tail vein by the number of cells per mouse 5×10~6/100?L.Eight weeks after inoculation mice were sacrificed.Immunohistochemical staining was used to detect lung metastasis and liver metastasis.4.Clinicopathological significance and expression of ARHI,p62,LC3B in the gastric carcinoma and the relative gastric mucosa.A sum of 304 cases of resected gastric carcinoma(GC)specimens in surgery,and 304 cancer-free gastric mucosa specimens were collected from the First Affiliated Hospital of China Medical University.Corresponding clinical data were recorded,and follow-up.Tissue microarrayer including gastric carcinomas and gastric mucosa were constructed using Microarray,4?m consecutive sections were cut,conventional HE stained,and stored at room temperature for further immunohistochemical staining.PV9000 method and DAB color system were used to detect the expression.200 cells from two selected representative fields of each sample were counted by two independent observers.The staining intensity was classified as negative(-)or positive(+).Statistical analysis and evaluation was taken for ARHI,LC3B,p62 protein expression and clinicopathologic factors and patient prognosis.5.Statistical analysis methods.The data obtained were analyzed using SPSS 17.0 software,using single-factor analysis of variance(one-way ANOVA)method to compare,test level F=0.05.The relationship between clinicopathological factors immunohistochemistry experiments,protein expression and gastric cancer using a?2test,protein expression on survival time were using the Kaplan-Meier method analysis,using log-rank test,test level?=0.05.Results:1.A stable ARHI re-expression gastric cancer cell line ARHI-BGC823 was successfully obtained.ARHI mRNA expression relative levels in gastric cancer cell lines MKN-45,SGC-7901,NCI-N87,BGC-823 in normal gastric cell lines were2.88,0.97,0.89,0.17 times GES-1,and we selected BGC823 gastric cancer cell lines to transfect for the further experement.After selection of G418-resistant clones and screening for fluorescence positive clones,we ultimately successful obtained ARHI stable re-expression gastric cancer cell clone,named ARHI-BGC823.Using Real-Time PCR,Western-Blot and immunofluorescence method ARHI-BGC823 gastric cancer cells were proved to express ARHI mRNA and protein at a high level.2.Re-expression of ARHI significantly inhibited malignant biological behavior in gastric cancer cell line BGC823 in vitro.Re-expression of ARHI gene induced autophagy and apoptosis in gastric cancer cell line BGC823,inhibited the proliferation,caused the cell cycle arrest in G0/G1 phase,and inhibited the migration and invasion,possibly by inhibition of PI3K-AKT-mTOR signaling pathway and MMP2,MMP9 expression.Low expression of ARHI gene in gastric cancer cells may caused by promoter methylation or histone deacetylation.3.Re-expression of ARHI gene inhibited the growth of subcutaneous xenograft and hematogenous liver and lung metastasis of gastric cancer cells.Subcutaneous xenograft experiment showed that,re-expression of ARHI gene in gastric cancer cells inhibited the growth of subcutaneous xenograft,P<0.05,reduced autophagy level of subcutaneous tumor,inhibited the proliferation activity,reduced angiogenesis,and inhibited the level of p-AKT signaling pathway.Hematogenous metastasis study showed that,re-expression of ARHI gene inhibited hematogenous liver and lung metastasis of gastric cancer cells,P<0.05.4.ARHI,p62 protein expression in gastric cancer was positively correlated with lymph node metastasis and prognosis.The positivity rates of ARHI and LC3B expression were significantly lower in gastric carcinoma(GC)than in normal gastric,and the positivity rate of p62 was significantly higher in gastric carcinoma(GC)than in normal gastric.ARHI expression in gastric carcinoma was positively correlated with LC3B expression,but not correlated with p62 protein expression;expression of LC3B protein and p62 protein were not relevant.ARHI positive rate in N staging N1-N3 patients was less than that in N0patients,and ARHI positive rate in TNM stage?and?patients was less than patients with stage?and?.The positive rate of p62 in Borrmann type?and?was higher than that in patients with type?and type?,and the positive rate of p62 in N stage N1-N3 patients is higher than that in N0 patients.LC3B positive rate in T stage T3 and T4patients was less than that in T1 and T2 patients.ARHI negative expression or p62negative expression implied a good prognosis.Conclusion:1 A stable ARHI re-expression gastric cancer cell line ARHI-BGC823 was successfully obtained.Re-expression of ARHI gene induced autophagy and apoptosis in gastric cancer cell line BGC823,inhibited the proliferation,caused the cell cycle arrest in G0/G1 phase,and inhibited the migration and invasion.2.Subcutaneous xenograft experiments showed that Re-expression of ARHI inhibited the growth of subcutaneous xenograft.Hematogenous metastasis study showed that,re-expression of ARHI gene inhibited hematogenous liver and lung metastasis of gastric cancer cells.3.Low expression of ARHI gene in gastric cancer cells may caused by promoter methylation or histone deacetylation.Re-expression of ARHI inhibits proliferation,migration,invasion in gastric cancer celsl and tumor growth and metastasis of hematogenous in vivo,possibly by inducing G0/G1 arrest,apoptosis,inhibition of gastric cancer cell proliferation,and by inhibition of PI3K-AKT-mTOR signaling pathway and MIIP,MMP2,MMP9 expression to inhibit invasion;through inhibition of angiogenesis to inhibit the growth and metastasis of gastric cancer cells in vivo.4.The positivity rates of ARHI and LC3B expression were significantly lower in gastric carcinoma(GC)than in normal gastric,and the positivity rate of p62 was significantly higher in gastric carcinoma(GC)than in normal gastric.ARHI expression in gastric carcinoma was positively correlated with LC3B expression,but not correlated with p62protein expression;protein expression of LC3B and p62 protein expression were not relevant.ARHI expression in gastric carcinoma was correlated with lymph node metastasis and TNM staging,p62 expression was correlated with lymph node metastasis,LC3B expression was correlated with the invasion depth of gastric cancer negatively.The negative expression of ARHI or p62 negative expression implied a good prognosis.ARHI and p62 were independent prognosis factor in gastric cancer.