Up-regulation Of MiR-101-3p Enhances Sensitivity To Cisplatin Via Regulation Of ATG4D And Autophagy In NSCLC

Part Ⅰ Expression of miR-101-3p in tissues and cells of NSCLC and its effect on sensitivity to cisplatin ObjectiveBy analyzing the related microarray of non-small cell lung cancer in GEO database,the miRNA expression microarray dataset GES135918 was selected to screen the differential miRNA between NSCLC patients and healthy people.The effect of differential miRNA on cisplatin treated NSCLC cells was studied.MethodsThe Geoquery package was used to download the miRNA expression microarray dataset GSE135918,and the annotation information of GPL18058 platform was extracted.Differentially expressed miRNAs was screened by the limma package.We analyzed the survival curve of the differentially expressed miRNAs in NSCLC by the Starbase 3.0 online database,and to determine the meaningful miRNAs for subsequent research.The clinical information of NSCLC patients,postoperative tumor tissues and normal tissues were collected.The differential expression of miR-101-3p in different tissues was detected by qRT-PCR.The expression of miR-101-3p was detected in MCR5 cell line and A549,hcc827,PC-9 and NCI-H1299 cells.miR-101-3p mimic and miR-101-3p inhibitor were transfected into NSCLC tumor cells A549 and NCI-H1299 by gene transfection technology.We used CCK-8test,EDU test and PI staining kit to detect the effects of miR-101-3p on proliferation and apoptosis of cisplatin treated NSCLC cells.ResultsA total of 34 differential miRNAs were screened by GSE135918,including 24down-regulated miRNAs and 10 up-regulated miRNAs.The results of Starbase 3.0online database indicated that only miR-101-3p had clinical significance.The survival time of Patients with different expression levels of miR-101-3p had statistically different survival time.miR-101-3p was selected as the follow-up study object.q PCR detection of NSCLC tissue and cell results showed that compared with normal lung tissue and normal lung epithelial cells,the expression level of miR-101-3p was significantly lower in NSCLC.CCK-8 test and edu test showed that up regulating the expression of miR-101-3p could increase the sensitivity of tumor cells to cisplatin and inhibit the proliferation of NSCLC cells.Up regulation of miR-101-3p expression can increase the sensitivity of tumor cells to cisplatin and promote the apoptosis of NSCLC cells.ConclusionmiR-101-3p is a differential miRNA between normal control and NSCLC patients.It is low expressed in tumor tissues and cells,and lower expressed in cisplatin resistant patients.Up regulation of miR-101-3p can increase the sensitivity of NSCLC cells to cisplatin.Part Ⅱ miR-101-3p increases the sensitivity of NSCLC cells to cisplatin by targeting ATG4 D to regulate autophagyObjective In this part,we studied the targeting effect of miR-101-3p and ATG4 D,and confirmed that miR-101-3p increased the sensitivity of NSCLC cells to cisplatin by targeting ATG4 D to regulate autophagy.Methods miR-101-3p mimic and miR-101-3p inhibitor were transfected into NSCLC tumor cells A549 and NCI-H1299 by gene transfection.Double luciferase reporter gene experiment was used to verify the targeting relationship between miR-101-3p and ATG4D;qRT-PCR experiment was used to verify the effect of different expression levels of miR-101-3p on ATG4 D expression;CCK-8 experiment,Western blot experiment,and transmission electron microscope experiment were used to verify that miR-101-3p regulates autophagy and ATG4 D to increase the sensitivity of cisplatin to NSCLC cells.Results This part of the experiment proved that miR-101-3p directly targeted autophagy related gene ATG4 D.Bioinformatics predicted that miR-101-3p was complementary to the 3’-UTR region of ATG4 D gene.The results of dual luciferase assay showed that miR-101-3p inhibited the activity of wild type ATG4 D,but had no effect on the activity of mutant ATG4 D.miR-101-3p directly targeted ATG4 D.qRT-PCR showed that up-regulation of miR-101-3p inhibited the expression of ATG4 D,down-regulation of mir-101-3p increased the expression of ATG4 D,and miR-101-3p regulated the expression of ATG4 D.Western blot showed that up-regulation of miR-101-3p could increase p62 protein expression,while down-regulation of ATG4 D and LC3 protein expression;transmission electron microscopy showed that miR-101-3p could inhibit the production of autophagosomes,and silencing ATG4 D reversed the effect of miR-101-3p on cisplatin sensitivity.Conclusion In this part,we demonstrated that miR-101-3p enhanced the sensitivity of cisplatin to NSCLC cells by targeting ATG4 D to inhibit autophagy.Part Ⅲ miR-101-3p targeting ATG4 D inhibits autophagy and increases the sensitivity of tumor to cisplatin in NSCLC nude miceObjectiveTo study miR-101-3p targeting ATG4 D to inhibit autophagy and increase the sensitivity of xenotransplanted tumor of NSCLC to cisplatin in nude mice.MethodsA xenotransplanted tumor model of NSCLC was established in nude mice using the A549 cells.The tumor volume was measured and the tumor growth curve was drawn.The nude mice were imaged and photographed before death.The subcutaneous tumor was taken and weighed.The expression of miR-101-3p and ATG4 D in tumor tissue was detected by qRT-PCR,apoptosis was detected by TUNEL kit,the expression of ATG4 D was detected by immunohistochemistry,and the expression of autophagy related protein was detected by Western blot.ResultsmiR-101-3p can enhance the ability of cisplatin to inhibit the growth volume and weight of xenotransplanted tumor tissue,and promote the apoptosis of NSCLC xenotransplanted tumor tissue.miR-101-3p can inhibit the protein expression of ATG4 D and autophagy related genes in xenotransplanted tumor tissue.ConclusionThis part of the study demonstrated that miR-101-3p enhanced the sensitivity of cisplatin to xenotransplanted tumor of NSCLC by targeting ATG4 D to inhibit autophagy in nude mice.