Mechanism Of Carboplatin Resistance Mediated By GDNF In Human Retinoblastoma Y79 Cells

Background: Retinoblastoma(RB)is one of the most common intraocular malignancies in children under 5 years of age.It progresses rapidly and leads to blindness mainly by destroying the structure of the eyeball.43% of the world’s RB children are distributed in the Asia-pacific region,while China accounts for 13.6% of the world’s RB children.Currently,the treatment of RB is developing rapidly.Carboplatin is often used in combination with other drugs for chemotherapy in clinical practice,but the survival rate of RB children is still very low,which may be caused by the increased resistance of RB to chemotherapy drugs.Therefore,it is expected to increase the survival rate of children with RB by clarifying the mechanism of RB resistance and finding possible targets for inhibiting the resistance.Glial cell derived neurotrophic factor(GDNF)is a member of the transforming growth factor-beta(TGF-β)superfamily.It’s a secreted protein.It is reported that GDNF binding with glycosylphosphatidylinositol-inked GDNF receptor alpha(GFRα)protein to recruit Receptor Tyrosine kinase(RET)affecting target cells.GDNF is overexpressed in glia,lung cancer and pancreatic cancer,but the expression of GDNF in RB has not been reported.In recent years,micro RNA(miRNA)has been reported to be closely related to the aggressiveness and drug resistance of tumor cells.Regulation of micro RNA can promote RB drug resistance by promoting autophagy and improve chemotherapy sensitivity.Autophagy isolates organelles and proteins in autophagic vesicles and delivers cytoplasmic carriers to lysosomes for degradation,thereby ensuring tumor cells’ survival under the action of metabolic and chemotherapy drugs.However,because miRNA mimics generally have multiple binding sites,there may be a high probability of miss target.In addition,due to the instability of miRNA and the presence of a large amount of RNase degrading RNA in serum,the in vivo transport of miRNA is problematic.Finding the downstream molecular mechanism of miRNA and designing drugs combined with carboplatin and other chemotherapy drugs for the downstream targets may better treat RB.Therefore,we hope to further study the molecular mechanism of GDNF promoting RB drug resistance,so as to provide sufficient theoretical basis for the design of drug therapy targets and combined carboplatin treatment of RB to weaken its drug resistance.Methods:1.In this paper,drug-resistant cell line Y79 R was constructed from retinoblastoma cell line Y79,and RNA-seq was performed.GO and KEGG found that the differentially expression genes were significantly enriched in drug transport function.Then,GDNF and miR-211-5p with the most obvious changes were selected for further analysis.In Y79 R,the expression of GDNF was knocked down or miR-211-5p was overexpressed.IC50 of Y79 R was detected by CCK-8 assay,and apoptotic cells of Y79 R were detected by flow cytology assay.The protein expression levels of apoptosis related proteins cleaved PAPR1、Bcl-2、Bax were detected by WB.The relationship between miR-211-5p and GDNF was verified by luciferase assay and WB assay.2.In order to further explore other mechanisms of drug resistance of Y79 R caused by GDNF,we conducted further bioinformatics analysis on the sequencing results and found that differential expression genes were enriched in pathways related to autophagy.In order to determine the effect of GDNF on autophagy and apoptosis of Y79 R cells,we used CCK-8 to detect IC50 after knocking down or overexpressing GDNF in Y79 R.Apoptosis was detected by flow cytometry,m RNA expression levels of autophagy-related genes were detected by q PCR,apoptosis related proteins cleaved PAPR1,Bcl-2,Bax,autophagy related proteins LC3 I,LC3II,and P62 were detected by WB.The formation of autophagosomes was examined by electron microscopy.3.In order to prove the effect of GDNF on Y79 carboplatin resistance in vivo,we constructed a tumor-forming model in nude mice.GDNF overexpressed and silenced Y79 R cells were used to intervene in nude mouse skin tumor formation.The expression of GDNF protein in tumor tissue was detected by immunohistochemistry and the apoptosis level of tumor tissue was detected by TUNEL.Results:1.After the successful construction of carboplatin resistant cell model Y79 R,it was found that the expression level of GDNF was most significantly increased in drug-transport-related genes,and the expression level of miR-211-5p was most significantly decreased in genes that might interact with GDNF.Dual luciferase assay showed that miR-211-5p could directly bind to GDNF 3’-UTR,and WB assay showed that miR-211-5p could inhibit the expression of GDNF protein.In a word,GDNF knockdown and miR-211-5p overexpression decreased THE IC50 of Y79 R cells and increased the apoptosis of Y79 R cells.2.Bioinformatics analysis suggested that carboplatin resistance caused by GDNF in Y79 R cells might be related to autophagy.After GDNF overexpressed in Y79 R cells,IC50 was increased in Y79 R cells by CCK-8 assay.Flow cytometry showed that apoptosis of Y79 R cells decreased.WB results showed that apoptosis related protein decreased,autophagy related protein LC3 I increased to LC3 II and p62 decreased.The increase of autophagosome was also observed by electron microscopy.After adding 3-MA,an early autophagy inhibitor,to Y79 R cells overexpressing GDNF,the IC50 of Y79 R cells decreased and the apoptosis of Y79 R cells increased.The transformation of autophagy related protein LC3 I to LC3 II decreased,the expression of P62 increased,and the number of autophagosomes decreased.The results suggest that GNDF may influence RB drug resistance by inducing early autophagy.3.CCK-8 assays showed that overexpression of miR-211-5p could reduce IC50 of Y79 R cells,increase apoptosis of Y79 R cells,increase expression level of apoptosis-related protein cleaved PAPR1,decrease transformation of autophagy related protein LC3 I to LC3 II,and increase p62 expression.However,after overexpressed GDNF in Y79 R cells overexpressing miR-211-5p,IC50 of Y79 R cells increased,apoptosis of Y79 R cells decreased,apoptosis related protein cleaved PAPR1 expression level decreased,LC3 I transformation increased to LC3 II,and p62 expression decreased.The results showed that miR-211-5p promoted the autophagy and drug resistance of Y79 R cells through GDNF,and inhibited cell apoptosis.4.QPCR and WB results showed that the transformation of GFRA1,RET,P-SRC,P-AMPK and autophagy related protein LC3 I to LC3 II was increased in overexpressed GDNF of Y79 R cells,while p62 was decreased.CCk-8 assays showed that overexpression of GDNF increased IC50 in Y79 R cells,while knockdown of GFRA1 inhibited IC50 in Y79 R cells.Flow cytometry results showed that overexpression of GDNF reduced apoptosis of Y79 R cells,while knockdown of GFRA1 increased apoptosis.The results indicate that GDNF induces the activation of the downstream autophagy pathway by forming a complex with its receptor GFRA1 on the surface of the cell membrane,which then reduces cell apoptosis and ultimately leads to carboplatin resistance.5.Compared with normal Y79 cells,the growth rate and size of subcutaneous tumor in nude mice were significantly increased by Y79 R intervention,and the apoptosis of tissue cells was decreased.The growth rate and size of subcutaneous tumor in nude mice were significantly decreased by GDNF-silenced Y79 R cells,and the apoptosis of tissue cells was increased.The growth rate and size of subcutaneous tumor in nude mice were significantly increased by GDNF-overexpressed Y79 R cells,and the apoptosis of tissue cells was decreased.The autophagy inhibitor chloroquine can significantly inhibit the growth rate and size of subcutaneous tumor in nude mice under the intervention of Y79 R cells overexpressing GDNF,and promote the apoptosis of tissue cells.The results in vivo were consistent with those in cells.Conclusion:1.Downregulation of miR-211-5p leads to upregulation of GDNF expression,leading to carboplatin resistance of Y79 R and reducing apoptosis of Y79 R cells.2.GDNF binds to receptor GFRA1 to form GDNF/GFRA1 complex,which activates SRC and SRC-AMPK signaling pathway in the form of complex,and then activates early autophagy,inhibits apoptosis,and promotes carboplatin resistance of Y79 R.