The Role Of β-arrestin2 In Regulating Inflammation In A549 Induced By Toll-like Receptor 9 Agonist

The lung cancer is one of the most common malignant tumor in the world, and it has become the leading cause of cancer deaths. In fact,85% of all lung cancer patients are non-small cell lung cancer(NSCLC) with an overall 5-year survival not more than 15%.β-arrestin2 is visual arrestins, whose role in diseases has been studied extensively. In recent years, researchers discovered that β-arrestins may play an important role in tumourgenesis. Once GPCR activated, β-arrestins can activate downstream signaling pathways, for example, MAPK signaling pathway.In addition, a large amount of studies show that MAPK signaling pathway involves in apoptosis signaling pathways and inflammatory signaling pathways. Inflammation is a basic protective measure in the process of immunity in an organic.More and more evidences show that inflammation is an important initiator,and it can induce other numerous diseases.In the process of tumorigenesis in lots of tumours, inflammation can promote tumorigenesis through inducing angiogenesis and cell proliferation and cell metastasis.Increasing evidences indicate that immune system palys an important role in regulating tumorigenesis. And some constituents of innate immunity and adaptive immunity both involved in this process.Some constituents can suppress the cell growth of tumours,and then they cause tumours cell death through immunosurveillance.We have known that bacterias and bacterias extract can bind unmethylated CG sequence,and then obtain antitumor activity,in addition to,they also can influence the immune system. Besides,some researches showed that TLR-9 may paly an important role in the process of tumorigenesis.And other researches indicated that TLR-9 is highly expressed in the tissue of patients who had got non-small cell lung cancer.However,other studies demonstated that β-arrestin2 is lowly expressed in the serum of non-small cell lung cancer patients.This indicate that both TLR-9 and β-arrestin2 play a role in the process of tumorigenesis of non-small cell lung cancer.However, what the relationship between them is still unknown.In this paper,we would begin the experiment from this pointcut.This paper we would study whether the protein β-arrestin2 takes part in the inflammation mediated by TLR-9,and how it works in this process.Results1.TLR-9 agonist ODN2216 can promote the growth of A549In this paper,we first detected the effect of TLR-9 agonist ODN2216 on the livability of A549.After we treated A549 with ODN2216,we counted the livability of the cell with SRB.We found that ODN2216 can promote the growth of A549 at 10ug/ml.2. Overexpression β-arrestin2 can inhibit the growth of A549Because we wanted to study the function of β-arrestin2,we tested whether β-arrestin2 affect the cell line of A549. After overexpression β-arrestin2 in A549,we detected the livability of the cell with SRB as well.We found that β-arrestin2 can inhibit the growth of A549.3. Overexpression β-arrestin2 will inhibit the mRNA lever of inflammatory cytokineFrom the results we got,we know that β-arrestin2 can contribute to the growth of A549.There was large amount of researches show that inflammation can contribute to the growth of tumors.So we detected the mRNA of inflammatory cytokine with RT-PCR after we overexpressed β-arrestin2 in A549.Our results showed that β-arrestin2 can inhibit the mRNA lever of IL-6 and IL-8.4.The relationship between β-arrestin2 and MAPK signal pathwayMAPK signal pathway is also seen as participating in inflammation.It has been reported that the phosphorylation of p38MAPK and the lever of TNFa and IL-6 in trauma patient was increased.Other researches show that when activated p38MAPK,the lever of inflammatory cytokine could also be increased.In this paper,the lever of inflammatory cytokine was decreased when we overexpressed β-arrestin2 in A549.Then we suppose that whether MAPK signal pathway was involved in this process. So we detected the phosphorylation lever of p38 and JNK,and we found that the phosphorylation lever of p38 and JNK was both decreased.5. β-arrestin2 cannot inhibit the increased level of inflammatory cytokine induced by TLR-9 agonist ODN2216It has been reported that the agonist of TLR-9 can induce the cell to release inflammatory cytokine.Therefore, we first overexpressed β-arrestin2,then treated the cell with ODN2216,at last we detected the mRNA lever of IL-6 and IL-8.However,we found that β-arrestin2 cannot inhibit the increased level of inflammatory cytokine induced by TLR-9 agonist ODN2216.ConclusionFrom the results,we know that the agonist of TLR-9 ODN2216 can promote the growth of A549.Overexpressed β-arrestin2 in A549 can decrease the level of IL-6 and IL-8. Overexpressed β-arrestin2 in A549 can decrease the phosphorylation lever of p38 and JNK.But when we used the siRNA of β-arrestin2 in A549,we did not see an increase of the phosphorylation lever of p38 and JNK.Overexpressed β-arrestin2 in A549,and then treated the cell with ODN2216,we found that β-arrestin2 cannot inhibit the increased level of inflammatory cytokine induced by TLR-9 agonist ODN2216.