The Mechanism Of Necroptosis Of Lung Cancer Cells Induced By MiR-101 Inhibitor And FA-GNR Mediated PTT

Background:Lung cancer is the most lethal cancer among men,and the second most common cancer leading to women’s death（after breast cancer）.The incidence of lung cancer in China accounts for about 1/3 of the world’s total incidence.Since the pathogenesis of lung cancer is not clear,there is a lack of effective treatment in clinic.Therefore,fooding more sensitive molecular markers can provide a new theoretical and experimental basis for the early diagnosis and treatment of lung cancer.Objectives:This study intends to construct a nano gene vector with tumor targeting,drug loading ability and Photothermal therapy（PTT）with gold nanorods as the core GNR@Si O₂-SA-PEI-FA（FA-GNR）,detect the toxicity,transfection efficiency and photothermal effect of the nano gene vector,clarify the relationship between mi R-101and RIPK1 and the effect of FA-GNR mediated PTT on necroptosis,and explore the role and mechanism of mi R-101 on necroptosis under FA-GNR mediated PTT.Methods:1.A novel composite nano material FA-GNR was prepared using a layer-by-layer synthesis method,and the characterization of FA-GNR was detected by UV absorption spectrum,zeta potential meter,nuclear magnetic resonance hydrogen spectrum and transmission electron microscope.2.The photothermal conversion ability of FA-GNR is detected by near infrared laser emitter and infrared camera.3.Gel migration test was used to detect the loading capacity of FA-GNR to mi RNA.4.The cytotoxicity and killing ability of FA-GNR on A549 cells were detected by CCK-8 method.5.The transfection efficiency of Cy3-mi R-101 in A549 cells was detected by fluorescence microscope and flow cytometry.6.The tumor targeting of FA-GNR was detected by small animal in vivo imager and frozen section.7.The relationship between mi R-101 and RIPK1 was determined by double luciferase,q-PCR and Western blot.8.q-PCR and Western-blot were used to detect the effect of FA-GNR transfection of mi R-101 mimics,mi R-101 inhibitor and their negative controls on the expression level of RIPK1 in A549 cells.9.In A549 cells,the effects of transfected mi R-101 mimics and mi R-101inhibitor on their proliferation,invasion,migration and colony formation were detected by CCK-8 method,Transwell,scratch test and clone formation test.10.RIPK3-KO A549 cells were constructed by CRISPR/Cas9 gene editing technology,and the target gene was successfully knocked out by q-PCR,Western blot and DNA sequencing.WT A549 cells and RIPK3-KO A549 cells were treated with PTT,the cell viability was detected by CCK-8 method,and the expression of mi R-101 was detected by q-PCR method.11.FA-GNR/mi R-101 mimics and FA-GNR/mi R-101 inhibitor were transfected into WT A549 and RIPK3-KO A549 cells respectively,and then treated with PTT.The cell viability was detected by CCK-8 method,and the expression levels of RIPK1,RIP3,MLKL and p-MLKL were detected by Western-blot.Results:1.Compared with GNR,the UV absorption spectrum of FA-GNR has a significant red shift;Zeta potential increased significantly;In the NMR spectrum,the nano material was successfully attached with folate molecules;TEM results showed that the size of the nanoparticles was the same,the silicon layer was evenly wrapped on the GNR surface,and has good dispersion.2.FA-GNR has good photothermal conversion ability,which is related to illumination time,material concentration and illumination intensity.3.mi RNA could be completely loaded by FA-GNR when the mass ratio of FA-GNR to mi RNA reached 6:1 in the Gel migration test.4.The results of CCK-8 showed that the concentration of FA-GNR was 105μg/m L,the cell viability was about 90%,indicating that the cytotoxicity of the material was low.5.FA-GNR has excellent photothermal conversion ability and its concentration is 60μg/m L and treated with PTT,the killing efficiency of cancer cells can reach about 80%.6.The transfection efficiency of FA-GNR on mi R-101 was very high,which could reach more than 95%of transfection rate.7.The small animal in vivo imager and frozen section showed that Cy3-mi R-101had obvious accumulation of red fluorescence in tumor tissue after FA-GNR mediated transfection,indicating that FA-GNR had good tumor targeting.8.Double luciferase assay showed that RIPK1 was the target gene of mi R-101;The expression level of RIPK1 in cells transfected with mi R-101 mimics was higher,while the expression level of RIPK1 in cells transfected with mi R-101 inhibitor was lower.9.mi R-101 was transfected into A549 cells by FA-GNR.The expression of RIPK1 in mi R-101 inhibitor group was significantly lower than that in negative control group and mi R-101 mimics group.10.Overexpression of mi R-101 inhibited the proliferation,invasion,migration and colony formation of A549 cells,while mi R-101 inhibitor had the opposite effect.11.RIPK3-KO A549 cell line was successfully established by CRISPR/Cas9method.CCK-8 showed that WT A549 had higher cell death rate after PTT treatment than RIPK3-KO A549;The results of q-PCR showed that the expression level of mi R-101 in WT A549 cells decreased significantly after PTT treatment.12.When RIPK3 in A549 cells was knocked out,the necroptosis pathway induced by PTT（mediated by FA-GNR）combined with mi R-101 inhibitor was inhibited.Conclusion:1.FA-GNR has excellent biocompatibility and photothermal effect,extremely low toxicity and easy clearance.It has high drug loading capacity and transfection ability,and can efficiently deliver the loaded drugs to the tumor in vivo.2.The expression of RIPK1 in mi R-101 inhibitor group was significantly lower than that in negative control group and mi R-101 mimics group.mi R-101 mimics can inhibit the proliferation,invasion,migration and colony formation of A549 cells,while mi R-101 inhibitor can enhance the proliferation,invasion,migration and colony formation of A549 cells.3.FA-GNR mediated PTT effect can reduce the expression level of mi R-101 in A549 cells and start the process of necroptosis.4.The PTT effect of mi R-101 inhibitor combined with FA-GNR significantly increased the necroptosis of A549 cells.