Study On The Anti-cancer Mechanism Of Pristimerin In Non-small Cell Lung Cancer

BackgroundCancer is a malignant disease caused by abnormal cell proliferation.Being different from primary tumor,cancer has the ability to spread from primary focus to distant tissues or even the whole body,which is very harmful.According to statistics,there were 18.1 million cancer patients around the world in 2018,including 9.6 million death caused by cancer.In China,cancer has become the first cause of death related to various diseases,among which lung cancer is particularly harmful.Non-small cell lung cancer（NSCLC）and small cell lung cancer（SCLC）are the most common types of lung cancer.NSCLC accounts for 80-85%of all lung cancer.Clinically,only a small number of NSCLC patients（about 20-25%）were diagnosed in the early stage of cancer（stage Ⅰ or Ⅱ）,and more than 60%of lung cancer patients were diagnosed as locally advanced or metastasized（stage Ⅲ or Ⅳ）.Natural compounds and their derivatives are important sources of novel drugs.As a triterpenoid compound,the strong antitumor activity of pristimerin has been widely reported.Pristimerin is involved in several key biological processes of cancer cells,including mitochondrial apoptosis,autophagy and angiogenesis.In molecular mechanism,it can regulate nuclear factor kappa B（NF-κB）,Akt（protein kinase B,PKB）,mitogen activated protein kinase（MAPK）and other pathways.Pristimerin shows strong antitumor activity against oral,colorectal,breast cancer and other cancer cells,however,there are few reports on lung cancer cells.In addition,the existing researches are mainly about the regulation of pristimerin on protein level and mRNA level,whether pristimerin exerts anti-cancer activity by regulating lncRNA remains unclear.Therefore,the aim of this study is to explore the antitumor activity and the related molecular mechanism of pristimerin against non-small cell lung cancer H1299 cells.Besides,lncRNA chip was used to find out the key lncRNAs related to the antitumor activity of pristimerin,which provide a scientific basis for the role of pristimerin in treatment of lung cancer.MethodsMTT assay was used to detect the inhibitory effect of pristimerin against H1299cells and determine the IC₅₀ value;plate cloning assay was used to investigate the proliferation ability of cells;annexin V-FITC/PI double staining assay was used to detect apoptosis;scratch test was used to investigate the effect of the drug on cell migration ability;transwell test was applied to detect the invasion ability of cells;the expression of vimentin,F-actin,integrinβ1,MMP2 and Snail was detected after the action of drugs by western blotting;the mRNA expression of integrinβ1,Snail and MMP2 was measured by RT-qPCR;lncRNA microarray was used to analyze the differentially expressed lncRNA and mRNA under the treatment of concentration gradient or time gradient of pristimeirn;bioinformatics analysis was applied to explore the ceRNA regulatory network.Results1.Pristimerin has a strong inhibitory effect on the proliferation of H1299 cells with IC₅₀ of 2.2±0.34μM.Moreover,the inhibiting action on proliferation was increased with the increase of concentration of pristimerin.2.Plate cloning assay showed that the ability of cloning forming of H1299 cells was inhibited by pristimerin in a time-dependent manner,which was identified by the decrease of the number and size of the colony cells.3.Annexin V-FITC/PI double staining assay showed that pristimerin induced the apoptosis of H1299 cells in a dose-dependent manner.4.The protein level of vimentin,F-actin,integrinβ1,MMP2 and Snail which is in EMT pathway can be regulated by pristimerin in a time-dependent manner,suggesting that the mechanism of anti-migration and invasion effect of pristimerin is associated with the inhibition of EMT pathway.5.RT-qPCR showed that the mRNA expression of integrinβ1,Snail and MMP2 could be down regulated by pristimerin,which indicated that the inhibition of pristimerin on integrinβ1,Snail and MMP2 was related to their mRNA expression.6.Using lncRNA microarray,we obtained the differentially expressed lncRNA and mRNA in H1299 cells after treatment of the time or concentration gradient of pristimerin.7.After the treatment of pristimerin,we found 27 lncRNAs up-regulated in a time-dependent manner,and 100 lncRNAs up-regulated in a dose-dependent manner.Moreover,7 lncRNAs were up-regulated in a time and dose-dependent manner.8.By predicting the mechanism for ceRNA,we finally screened out lncRNA RP11-83B20.1,which can indirectly target multiple mRNA related to migration and invasion through biding miR and thus exert an anti-cancer activity.ConclusionsIn this study,it was found that pristimerin has significant antitumor effect on NCI-H1299 cells,which showed its ability to inhibit the proliferation,induce apoptosis,inhibit migration and invasion of H1299 cells in a time or concentration dependent manner.In the mechanism,the expression of vimentin,F-actin,integrinβ1,MMP2 and Snail which is in EMT pathway can be down-regulated by pritimerin in a time-dependent manner,and the mRNA expression levels of integrinβ1,Snail and MMP2can be down-regulated at the same concentration of pristimerin.Using the lncRNA microarray,we obtained the differentially expressed lncrna and mRNA under the action of time or concentration gradient of pristimerin.Finally,Rp11-83B20.1 is identified,which can indirectly target multiple mRNA related to migration and invasion through miR through the prediction of ceRNA mechanism.