| ObjectiveOur research group has identified high expression of long non-coding RNA CUDR(CUDR)in pancreatic ductal adenocarcinoma(PDA)by high-throughput chip screening and bioinformatics analysis.CUDR expression was significantly correlated with differentiation,lymph node metastasis and regional invasion of PDA.Overexpression of CUDR promoted the proliferation,migration,invasion and metastasis of PDA,and induced resistance to chemotherapy drugs.Based on our previous work,in this study,we further explore the effect and mechanism of CUDR overexpression or knockdown on PDA autophagy,and the relationship between autophagy and the proliferation,migration and invasion of CUDR overexpression or knockdown PDA cells.Methods1.In the established CUDR overexpression(oe CUDR)and its control(oe SHAM),CUDR knockdown(sh CUDR)and its control(sh SHAM)cell lines,western blot(WB)was used to study the effect of CUDR expression level on autophagy.The main indicator for evaluating PDA cell autophagy is the conversion of microtubule-associated protein 1 light chain 3(MAP1LC3B,LC3B).MTT,scratch healing experiment,transwell and other methods were used to study the effects of autophagy inhibitors on the proliferation,migration,invasion of CUDR overexpressed or knockdown cell lines.2.Real-time quantative polymerase chain reaction(RT-q PCR)and WB were used to study CUDR overexpression or knockdown of autophagy-related genes and their proteins(ATG),and the expression level or functional status of other key proteins of autophagy pathway such as ULK1,Beclin-1,m TORC1.Bioinformatics methods were used to screen micro RNAs(mi RNAs),which are competitive endogenous RNA(ce RNA)to CUDR,and their corresponding autophagy regulatory proteins.3.The relationship between CUDR expression and ATG expression was verified by RT-q PCR in 35 pairs of PDA tissue samples,and its clinical pathological significance was analyzed.Results1.WB results showed that LC3B-II in CUDR overexpressed PANC-1 wassignificantly higher than that in the control group,and LC3B-II in CUDR knockdown CFPAC-1 was significantly lower than that in the control group.MTT results showed that the proliferation of CUDR highly expressed cell lines oe CUDR and CUDR normally expressed sh SHAM cell lines was inhibited by the autophagy inhibitor chloroquine(CQ)to a greater extent than the control group.The results of scratch test and transwell invasion experiment showed that CQ can significantly inhibit the migration and invasion of CUDR highly expressed cell line oe CUDR and CUDR normally expressed sh SHAM cell line.2.The expression of ATG7 m RNA and protein in oe CUDR was significantly increased compared to oe SHAM,and the expression of ATG7 m RNA and protein in sh CUDR was significantly reduced compared to sh SHAM.Overexpression or knockdown of CUDR did not cause changes in m TORC1 expression and phosphorylation.Ten mi RNAs with ce RNA relationship to CUDR and ATG7 m RNA were preliminarily screened by bioinformatics methods.3.CUDR and ATG7 are highly expressed in most PDA tissues,and their expressions are positively correlated.Patients with high ATG7 expression were not significantly different from those with low or normal expression in terms of gender,age,tumor differentiation,and nerve invasion,but had significant differences in tumor size,lymph node recidivism,and p TNM staging.Conclusion1.Overexpression of CUDR enhances autophagy of pancreatic cancer cells,and knockdown of CUDR inhibits autophagy of pancreatic cancer cells.Overexpression of CUDR promotes the proliferation,migration and invasion of pancreatic cancer cells and is attributable to activation of autophagy.2.CUDR may affect autophagy by affecting ATG7 protein expression.3.In tissues,the expression of ATG7 m RNA is positively correlated with the expression of CUDR.The high expression of ATG7 is associated with larger tumors,more lymph node metastases,and later pathological stages. |
