The United Analysis Of The Influence Of Pazopanib Upon MRNA And MiRNA Transcriptomes As Well As Its Involvement In Metabolism

Background and purpose:Lung cancer is one of the most threatening malignant tumors,the mortality of which ranges high both in China and in the world.During the progression of various kinds of tumors,there are always some hallmarks in common,including sustaining proliferative sigmals,evading growth suppressors and resisting cell death,activating invasion and metastasis,and inducing angiogenesis.So far some treatments have been established targeting tumor angiogenesis,like the monoclonal antibody bevacizumab and tyrosine kinases inhibitor pazopanib.Whereas the pre-clinical studies had confirmed the inhibition efficacies of pazopanib on cancer cell proliferation,the clinical trials did not show any advantages of pazopanib in terms of overall survival.Therefore,more investigation upon the mechanisms of inhibition as well as resistence in angiogenesis remedies like pazopanib are desperately required.Method:(1)In this study,4 non small cell lung cancer cell lines: A549,H460,L9981 and YTMLC-90 were choiced as experimental subjects.(2)By MTT assays the appropriate dosages range and time range were judged.By flowcytometry,wound healing assay,and transwell assay the cell cycle phase ratios,and capabilities of migration and invasion were tested between the pazopanib treatment groups and control groups.(3)After distracting the total RNA of pazopanib treatment groups and control groups,the m RNA microarrays and miRNA microarrays were used to screen the differently transcripted m RNAs and miRNAs.(4)The results of microarrays were affirmed by realtime PCR and western blot.The PCK2 gene was cloned into the p EGFP-N1 plasmid,and the efficacy of hybrid plasmid was tested by western blot.(5)Si RNAs targeting PCK2 were tested by realtime PCR.(6)By upregulating by fusion expression plasmid and downregulating by siRNAs,the flowcytometry was employed to detect the relationship between PCK2 and cell cycle.(7)By upregulating by fusion expression plasmid and downregulating by siRNAs,the glucose and lactate dynamics in the medium were monitored to confirm the effect of PCK2 on glyconeogenesis.(8)The MTT assay was used to detect the influence of PCK2 upon inhibiton of lung cancer cell proliferation.Results:(1)In MTT assay the IC50 of pazopanib on A549,H460,L9981 and YTMLC-90 at48 hours were 15.913±2.57μM,36.161±4.53μM,9.81±1.34μM and11.448±3.28μM,respectively;at 96 hours were 11.693±0.79μM,13.472±1.3μM,5.395±1.38μM,7.001±3.33μM,respectively.(2)The flowcytometry showed pazopanib in range of 0-10μM could arrest A549,H460 and L9981 at G0/G1 phase.(3)The wound healing assay revealed that pazopanib could inhibit migration of A549 and L9981,instead of that of H460 and YTMLC-90.(4)In transwell assay pazopanib triggered the invasion potential of all 4 cell lines.(5)Microarray showed that pazopanib enhanced the transcription of genes PCK2,DDIT3 and CHAC1,and decreased the transcription of has-miR-1233-5p.(6)Realtime PCR confirmed that pazopanib did upreagulate PCK2,DDIT3 and CHAC1,and downregulate has-miR-1233-5p.(7)By western blot the fusion expression plasmid did enhance the expression of PCK2.(8)By realtime PCR si PCK2-806 、 si PCK2-1363 showed comparatively better knockdown efficacy upon PCK2.(9)By flowcytometry,PCK2 did have a positive correlation with the ratio of S phase.(10)Upregulation of PCK2 leveled up the growth curve of A549,and opsitely downregulation of PCK2 leveled down the growth curve of A549.(11)Upregulation of PCK2 decreased the glyconeogenesis peak and increased the production of lactate;oppositely pazopanib increased the glyconeogenesis peak and delayed the production of lactate.(12)Upregulation of PCK2 augmented the resistence of A549 to pazopanib,and the IC25 increased 2.56 times to 12.11μM.Conclusion:(1)Pazopanib could directly inhibit the proliferation of lung cancer cell,and arrest some of them into G1/S phase as well as hamper their migration capalibity;but it may also improve their invasion potential.(2)Pazopanib does upregulate the expression of PCK2.(3)The activated PCK2 helps to support the proliferation of cancer cell,improve glyconeogenesis level as well as increase the portion of s phase cell,and thus leads to resistence to pazopanib treatment.