The Effect Of GPR40 Receptor On The Anti Lung Cancer Of Docosahexaenoic Acid

G protein coupled receptor 40(GPR40),also known as free fatty acid receptor 1(FFAR1),is a member of the rhodopsin family.While GPR40 is activated by different medium and long-chain free fatty acids,it mediates the transmission of extracellular signals to intracellular,and plays diverse biological functions.As a new target of type Ⅱ diabetes,GPR40 can participate in insulin secretion,nervous tissue development,tumor development and other biological functions.Further study on GPR40 function is of great significance for tumor treatment and drug development.Docosahexaenoic acid(DHA)is a kind of omega-3 unsaturated fatty acid,which is a kind of essential fatty acid in the human body.After ingestion,DHA can enter the cell through passive diffusion,and take the form of glycerophospholipid as the component of biofilm.It can be hydrolyzed into a variety of metabolites by biological enzymes,and can be involved in many important physiological activities such as neuroprotection,inflammatory response,blood pressure regulation and tissue regeneration.However,the molecular mechanism of anti-tumor effect of DHA is not clearly understood,and there is no literature report on how many doses of DHA can play an anti-tumor role or play a synergistic anti-tumor role in vivo.DHA can induce apoptosis and cell cycle arrest in cancer cells.Clinical application of DHA can greatly improve the prognosis and survival rate of tumor patients.In addition,DHA and a variety of anti-tumor chemotherapy drugs have the synergistic anti-tumor effect,which can enhance the therapeutic effect of drugs.This study investigated whether GPR40 receptor mediates the antitumor effect of DHA and whether GPR40 mediates the synergistic antitumor effect of DHAIn this study,we first constructed the plasmid vector pHBLV-CMV-GPR40-3FLAG-EF1-ZsGreen-T2A-PURO,then packaged lentivirus through three plasmid expression system,and finally transfected cells to obtain H460 cell line with high GPR40 expression,named H460-GPR40H.The vector LV-U6-Stuffer-Scaffold-EFS-hCas9-T2A-Purowas constructed by biological company,and the GPR40 knockout H460 cell line was then obtained and named H460-GPR40L.The effect of GPR40 receptor on the antitumor effect of DHA alone or in combination with paclitaxel was observed by using GPR40 overexpression or gene knockdown cell lines or the use of GPR40 receptor inhibitor GW 1100In this study,SRB test,scratch test and Transwell test were used in determining whether DHA can inhibit the proliferation,migration and invasion of lung cancer cell H460 and A549.The inhibition rate of DHA(120 μmol/1)on the proliferation of H460 cells was 56.5%,while when combined with GW1100,an inhibitor of GPR40 receptor,the inhibition rate was 37.6%,The inhibition rate of DHA(120 μmol/1)on GPR40 overexpression H460 cells was 66.5%,and that on GPR40 knockdown H460 cells was 36.8%.The results showing that DHA could inhibit cell proliferation through GPR40 receptor.In the scratch test,the migration rate of H460 cells treated with DHA(150 μmol/l)was 9.1%,while when combined with GW1100 the migration rate was 22.3%.The migration rate of DHA(150 μmol/1)on GPR40 overexpression H460 cells was 4.6%,and that on GPR40 knockdown H460 cells was 22.7%Indicating that DHA could inhibit cell migration through GPR40 receptor.In the Transwell experiment,the average number of invasive cells of H460 cells with high GPR40 expression was 34.0±2.6 and that of H460 cells with low GPR40 expression was 71.3±6.8 after DHA(120 μmol/1)treatment,indicating that DHA can inhibit cell invasion through GPR40 receptor.The apoptosis rate of H460 cells induced by DHA(180 μmol/1)was 47.1%,while the inhibition rate was 35.7%when it was combined with GW 1100,showing that the apoptosis of H460 cells was induced by DHA through GPR40 receptor.In cell cycle experiment,after treated with DHA(300μmol/l),the ratio of G0/G1 phase of H460 cells increased from 57.6%to 86.3%,which indicating that DHA could induce G0/G1 phase arrest of H460 cellsWestern blot assay was used to detect the phosphorylation levels of key kinases PI3K,Akt,ERK,JNK and p38 in PI3K/Akt or MAPKs signaling pathway in H460 cells.The results showed that DHA significantly inhibited the PI3K/Akt signaling pathway,but had no significant effect on MAPKs signaling pathway.DHA-induced inhibition of PI3K/Akt signaling pathway was mediated by GPR40In the experiment of combination of DHA and PTX,the growth inhibition rate of PTX(1 ng/ml)on H460 cells was 28.3%,while the inhibition rate of PTX combined with DHA(30 μmol/l)was 61.9%.The inhibition rate of PTX combined with DHA and GW1100 on H640 cells was 46.1%,indicating that DHA can enhance PTX cell proliferation through GPR40.In the migration experiment,the migration rate of H460 cells treated with PTX+DHA(30 μmol/L)was significantly lower than that of PTX alone(31.7%),and the migration rate of PTX+DHA+GW1100 group(26.1%)was significantly higher than that of PTX+DHA group.The migration rate of GPR40 overexpression cells(7.1%)was significantly lower than that of GPR40 knockdown cells(24.4%).It is suggesting that DHA can inhibit H460 cell migration by enhancing PTX through GPR40.In the invasion experiment,the average number of invasive cells in PTX+DHA(30 μmol/L)was 33 ± 6.8,while that in PTX+DHA+GW1100 group was 58 ± 7.5.Under PTX+DHA treatment,the average number of invasive cells with high GPR40 expression was 15.9 ± 4.4,and the number of GPR40 knockdown cells was 56.7±9.8.It is suggesting that DHA can enhance PTX and inhibit H460 cell invasion through GPR40.The apoptosis rate of H460 cells induced by PTX(1 ng/ml)was 38.7%,while the inhibition rate of H460 cells combined with DHA(60 μmol/l)was 64.8%.When PTX combined with DHA and GW1100,the inhibition rate of proliferation of H640 cells was 54.2%,indicating that DHA can enhance PTX induced apoptosis through GPR40In conclusion,we found that DHA exerted its anti lung cancer activity through receptor GPR40.