The Regulatory Molecular Mechanism Of Mdig On Angiogenesis And Lymphangiogenesis In Non-small Cell Lung Cancer

Objective:Mineral dust-induced gene(mdig),a newly discovered lung cancer-related gene,can promote the proliferation of tumor cells.Angiogenesis and lymphangiogenesis play the key role in the development of tumors.With the rapid growth of tumor,the hypoxic microenvironment is formed inside the tumor,which further stimulates tumor angiogenesis and lymphangiogenesis.The VEGF family is one of the most important proteins regulating the above process.Therefore,this study is aimed to explore whether mdig can affect angiogenesis and lymphangiogenesis in non-small cell lung cancer by regulating the expression of VEGF family proteins.Methods:In the first part of the study,to explore the regulatory mechanisms of different oxygen environments on mdig and angiogenesis and lymphangiogenesis-related proteins.In this study,A549,H1299,and 293T cell lines were cultured in normoxia(37°C,21%O₂,5%CO₂)and hypoxia(37°C,1%O₂,5%CO₂)for 24 hours,respectively.Western blot was applied to detect the basal expression levels of mdig,EGFR,HIF-1?and VEGF-A/C/D proteins.In the second part of the study,the molecular mechanism of mdig regulating angiogenesis and lymphangiogenesis-related proteins was explored.In this study,lentivirus transfection was used to construct mdig-overexpressing A549,H1299and 293T transfected cell lines,and mdig-silenced A549 transfected cell line,and then cultured under normoxic and hypoxic conditions,respectively.After the above transfected cells were cultured and generated at least four times,the transfection effect of mdig was verified by western blot and RT-q PCR experiments.At the same time,the conditioned mediums were obtained from the A549 and H1299 transfected cell lines cultured in normoxia and hypoxia to culture human umbilical vein endothelial cells(HUVEC)and human lymphatic endothelial cells(HLEC).Western blot was used to detect the expression levels of EGFR,p-EGFR(Tyr1068),HIF-1?,and VEGF-A/C/D proteins in A549,H1299,and 293T transfected cell lines,the protein levels of VEGF receptors VEGF-R1/R2 in HUVEC and the protein levels of VEGF-R3 in HLEC cultured in conditioned medium.RT-q PCR was used to detect the m RNA levels of EGFR and HIF-1?in the above transfected cell lines cultured under normoxic and hypoxic conditions.The co-immunoprecipitation(Co-IP)experiment was used to detect the direct interaction between mdig and EGFR and HIF-1?proteins,respectively.The nuclear and cytoplasmic fractionation experiment was applied to study the effect of mdig on the expression level and intracellular distribution of HIF-1?.Finally,the tube formation assays in vitro and the xenograft tumour experiments in nude mice were used to verify the effect of mdig on tumor proliferation and the regulatory mechanisms of mdig on angiogenesis and lymphangiogenesis in non-small cell lung cancer.In the third part of the study,to explore the signalling pathways that mdig regulates angiogenesis and lymphangiogenesis.In this study,EGFR signalling pathway inhibitor was given to mdig-overexpressing transfected cell lines cultured in normoxia and hypoxia,and EGFR signalling pathway agonist was given to mdig-silenced A549 transfected cell line.Western blot was used to examine the protein levels of EGFR,p-EGFR(Tyr1068),and VEGF-A.The mdig-overexpressing A549 transfected cells treated with EGFR inhibitor were injected in nude mice,and ultimately verified the signalling pathway that mdig regulates angiogenesis in non-small cell lung cancer.At the same time,HIF-1?signalling pathway agonist was given to mdig-overexpressing transfected cell lines,and HIF-1?signalling pathway inhibitor was given to mdig-silenced A549 transfected cell line.Western blot was used to examine the protein expression of HIF-1?,VEGF-C,and VEGF-D.The mdig-overexpressing A549 transfected cells treated with HIF-1?agonist were injected in nude mice,and then explored the signalling pathways that mdig regulates lymphangiogenesis in non-small cell lung cancer.Results:Regulation of oxygen concentration on mdig and angiogenesis and lymphangiogenesis-related proteins:Western blot experiment was used to detect mdig,EGFR,HIF-1?,and VEGF-A/C in A549,H1299,and 293T cell lines cultured in normoxia and hypoxia.Under the hypoxic condition,the protein levels of EGFR,HIF-1?,and VEGF-A/C/D were significant higher than those in normoxia,while the protein level of mdig was significant decreased.The regulation of mdig on angiogenesis and lymphangiogenesis-related proteins:Lentivirus transfection was used to construct A549,H1299 and 293T transfected cell lines,and then cultured under normoxic and hypoxic conditions,respectively.Western blot and RT-q PCR experiments were used to detect the effect of mdig-overexpressing and silenced.The results showed that the levels of mdig m RNA and protein were significant increased in mdig-overexpressing A549,H1299,and 293T transfected cell lines compared to those in the LV-con group,while the levels of mdig m RNA and protein were significant decreased in the mdig-silenced A549 transfected cell line compared to the LV-mdig-RNAi-con group.Western blot was used to detect EGFR,p-EGFR(Tyr1068),HIF-1?,and VEGF-A/C/D in the above transfected cell lines,and detected VEGF-R1/R2/R3 proteins in HUVEC and HLEC cultured in conditioned medium.The results showed that the protein levels of EGFR,p-EGFR(Tyr1068),VEGF-A,and VEGF-R1/R2 in the mdig-overexpressing group were significant increased compared to those in the LV-con group,while those of HIF-1?,VEGF-C/D and VEGF-R3 were significant reduced.The results were reversed in mdig-silenced A549 transfected cell line.In addition,in order to explore the molecular mechanism of mdig on EGFR and HIF-1?,RT-q PCR experiment was used to detect the regulation of mdig on EGFR and HIF-1?m RNA.Under normoxic or hypoxic conditions,there was no significant difference between mdig m RNA and EGFR and HIF-1?m RNA,respectively.Subsequently,the Co-IP experiment was used to detect the direct interaction between mdig and EGFR and HIF-1?at the protein levels.The results showed that there was no direct effect between mdig and the latter two proteins.In order to explore the mechanism which mdig inhibits HIF-1?protein,the nuclear and cytoplasmic fractionation experiment was applied to detect the effect of mdig on the expression and intracellular distribution of HIF-1?.The results show that mdig inhibits HIF-1?translocating from cytoplasm to nucleus.Results in the tube formation assays indicated that the density of angiogenesis was significantly increased in HUVEC cultured using the conditioned media of cells from the LV-mdig group compared with that in HUVEC cultured using the conditioned media of cells from the LV-con group.However,the density of lymphangiogenesis was notably decreased in HLEC cultured using conditioned media of cells in the LV-mdig group compared with that in HLEC cultured using the conditioned media of cells in the LV-con group.Finally,the tumor volumes formed by the mdig-overexpressing A549 transfected cell lines was significant larger than those of the LV-con group after three weeks of implantation in vivo experiment,but none of the cells in the mdig-silenced group were tumorigenic.According to the immunohistochemistry experiments,it was found that the density of angiogenesis in the mdig-overexpressing group was significant increased compared to that in the LV-con group,and the expression levels of mdig and VEGF-A were also enhanced compared with the vector group,while the density of lymphangiogenesis and the expression levels of VEGF-C/D were significant increased.The signalling pathways of angiogenesis and lymphangiogenesis regulated by mdig:mdig-overexpressing transfected cell lines cultured under normoxic and hypoxic conditions were treated with EGFR inhibitor,and western blot was used to detect the protein levels of EGFR,p-EGFR(Tyr1068),and VEGF-A.The results showed that the protein expression levels of EGFR,p-EGFR(Tyr1068),and VEGF-A in the mdig-overexpressing group given EGFR inhibitor were significant reduced,whether in normoxia or in hypoxia,compared with those in the mdig over-expressing group without intervention.The above protein levels were significant promoted in the mdig-silenced group with EGFR agonist compared with the mdig-silenced group without intervention.The xenograft tumour experiments indicated that the tumour volumes and angiogenesis density were significantly decreased by mdig over-expressing cells treated with EGFR inhibitor.Similarly,the mdig-overexpressing transfected cell lines were treated with HIF-1?agonist,the protein levels of HIF-1?,VEGF-C,and VEGF-D were detected by western blot.The results showed that the expression levels of HIF-1?and VEGF-C/D proteins in the mdig over-expressing group with HIF-1?agonist were significant increased compared to those in the mdig-overexpressing group without intervention,while those in mdig-silenced A549 transfected cell line with HIF-1?inhibitor were significant reduced.The xenograft tumour experiments indicated that the lymphangiogenesis density was significantly increased by mdig over-expressing cells treated with HIF-1?agonist.Conclusion:1.mdig is an oxygen-sensitive protein whose expression level is related to oxygen concentration.Hypoxia inhibits the expression of mdig.2.mdig promotes tumor proliferation and can induce tumor angiogenesis.In both normoxia and hypoxia,mdig exists the upstream of EGFR and HIF-1?,and promotes EGFR but inhibits the expression and intranuclear distribution of HIF-1?protein.mdig regulates the protein levels of EGFR and HIF-1?during translation or post-translation process.3.mdig promotes tumor angiogenesis by activating the EGFR/p-EGFR(Tyr1068)/VEGF-A/VEGF-R1/R2 signalling pathway,and may be inhibiting tumor lymphangiogenesis by blocking the HIF-1?/VEGF-C/D/VEGF-R3 signaling pathway.