The Expression Of ARHI In Colon Cancer Cell Line And Its Biological Behavior

BackgroundColon cancer is one of the most common cancers The incidence of a disease in China and abroad are rising year by year.Among various kinds of metastatic tumor,colon cancer incidence and mortality in the male and female in the third.It is not easy to find out the clinical symptom and signs,colon cancer has commonly transferred and research suggested that about a half of patients have lung or other organ metastases,and a fifth of patients already have liver cancer when diagnosed colorectal cancer.The rate of colon cancer case fatality is very high,the 5-year survival rate is about 92% of early signs,and the advanced colon cancer is only 16%.So the treatment of colon cancer is very difficulty and prognosis is poor.Although there have been major advances in the diagnosis and treatment of colon cancer,one significant malignancy in the gastrointestinal tract in recent years,Tumorigenesis results from various kinds of tumor suppressors and dysregulation of oncogenes that influence cellular proliferation,differentiation,cycle,apoptosis and senescence.The anti-oncogene ARHI can take part in the relating pathway to regular cell cycle and apoptosis.The biological activities of ARHI is by influencing the pathways downstream related protein expression.It can inhibit the cycle regulatory proteins such as cyclin D1,to influence the cell cycle and inhibiting growth.at the same time can affect the classic apoptosis related proteins,such as the Bcl-2 and Bax whom have been known as apoptosis regulatory proteins,and then promotes cancer cell apoptosis.Here we show that up-regulated of ARHI expression,through recombinant plasmid,influences various biological processes in two kinds of colon cancer cell lines to discuss its behavior and function in signal pathway in colon cancer.It is possible for ARHI to be a alternative target of gene therapy or provide a new treatment method for clinical treatment of colon cancer.ObjectiveThe purpose of our study was to find out the effects of ARHI on cellular apoptosis,cycle,proliferation,invasion and migration.We built colon cancer cells Sw480 and Ca Co2 deletion of ARHI expression through recombinant plasmid.at the same time,We study the two cell lines in vitro and its modulations of apoptosis and cell cycle by PI3K/AKT pathway.Through these studies we can have a new insight into the possible diagnostic and therapeutic applications of ARHI in colon cancer.MethodsIn this study,we commissioned Shanghai Jokai gene Biotechnology Co.Ltd.to provide the plasmid vectors: pc DNA3.1-flag and pc DNA3.1-flag-ARHI.The cells were divided into two groups: ARHI(transfected with pc DNA3.1-flag-ARHI for overexpression of the ARHI gene),vector(transfection with pc DNA3.1-flag empty viral vectors).Expression of ARHI target genes in normal colonic epithelium tissue and four colon cancer cell lines: Ca Co2,Sw480,HCT116 and HT29 was measured using Semi-quantitative RT-PCR(RT-PCR).Using Western blot technique for validation of higher expression of target gene ARHI and testing related proteins expression.The monoclonal formation and CCK8 assays were used to determine the cell proliferation.Using flow cytometry to research the effect of cell apoptosis and the cell cycle.Cellular motility through invasiveness and migration experiments was further determined.All statistical analyses were performed with SPSS 19.0 software.Results1,The expression of ARHI was upregulated in the normal colonic epithelium tissue compared with colon cell lines,and Ca Co2 and SW480 cell lines were used in this study.2,The two cell lines were transfected with the pc DNA3.1-flag-ARHI constructs or with an empty vector pc DNA3.1-flag plasmid as a control.3,The CCK-8 assay showed inhibition of cell growth in the two cell lines after ARHI re-expressed.Flow cytometry results suggest that the cell cycle and apoptosis were also affected by ARHI.Moreover,over-expression of ARHI induced apoptosis.Transwell migration and invasion suggest that the migration and invasion ability of pc DNA3.1-flag-ARHI groups were weaker than pc DNA3.1-flag vector groups.4,With pc DNA3.1-flag-ARHI-transfected,GSK-3β was expressed at high levels and reduced downstream Cyclin D1,leading to cell cycle arrest.Moreover,with pc DNA3.1-flag – transfected,p53 and Bax were expressed at low levels and the expression of p-AKT and Bcl-2 were induced,leading to cell apoptosis arrest,while AKT in both pc DNA3.1-flag-ARHI-transfected group and pc DNA3.1-flag-transfected group had no significant difference.ConclusionIn colonic epithelium tissue group,ARHI was high expressed,but it low or not expressed in colon c ancer cell lines groups.This indirect illustrates the ARHI takes a part in the development of colon cancer,and it may play a protect role in colon cancer.We up-regulation the expression of ARHI by recombinant plasmid in Ca Co2 and SW480.Our results demonstrate that ARHI can induces cells apoptosis and cell cycle arrest in the G0-G1 phase.At the same time,ARHI inhibits Ca Co2 and Sw480 cells clonogenicity,proliferation,invasiveness and migration These effects may be mediated by negative modulation of the PI3K/AKT signaling pathway,Promote downstream GSK3βprotein expression,which promote the inhibit abilitiy of GSK3βto cyclin D1.Thus can inhibit cell cycle.On the other hand,ARHI can inhibit PI3K/AKT pathway and inhibit p-AKT,up-regulation the tumor-suppressor p53 nd apoptosis-related protein Bax,but down-regulation Bcl-2 protein.But for ARHI specific work sites also need to explore in the further research.