The Clinical Significance And The Mechanism Of Drug Resistance Of Long Non-coding RNA HOTTIP In Gastric Cancer

Part 1 Long noncoding RNA HOTTIP as potential novel diagnostic and prognostic biomarker test for gastric cancerBackground:Long noncoding RNA HOTTIP plays important roles in the generation and progression of human cancers.Exosomes participate in cellular communication by transmitting moleculars between cells and are regarded as suitable candidates for non-invasive diagnosis.However,the existence of HOTTIP in the circulating exosomes and the potential roles of exosomal HOTTIP in gastric cancer(GC)was poorly understood.This study aims at investigating the clinical roles of exosomal HOTTIP in GC.Methods:1.Exosomal HOTTIP was detected in the culture medium of GC cells using RT-qPCR.2.Evaluation the stability of exosomal HOTTIP in serum after treated samples with prolonged exposure to room temperature or mutiple freeze-thaw cycles.The serum exosomal HOTTIP from 246 subjects(126 GC patients and 120 healthy people)were detected by RT-qPCR.3.Receiver operating characteristic curve(ROC)was built to estimate the diagnostic value of serum biomarkers.4.Kaplan-meier curve was drawn to analyze the correlation between the expression levels of serum biomarkers and the 5-year survival rate of GC patients.5.Cox proportional hazards models were performed to identify independent prognostic factors for GC.Results:1.Exosomal HOTTIP could be detected in the culture medium of GC cells and the levels were increased with the incubation time extended.2.The expression levels of serum exosomal HOTTIP remained stable when samples were treated with exposuring to room temperature for 12h or repeating freeze-thaw cycles for 5 times.Its expression levels were typically higher in GC than in normal control(P<0.001)and were significantly correlated with invasion depth and TNM stage of tumor(P<0.05).2.The area under the ROC curve(AUC)was 0.827,which was higher than CEA,CA 19-9 and CA72-4(AUC=0.653,0.685 and 0.639,respectively)(P<0.001).At the optimal cut-off value(1.720),the sensitivity and specificity of exosomal HOTTIP for GC were 69.8%and 85.0%,respectively(P<0.001).3.The Kaplan-Meier analysis showed a correlation between the increased exosomal HOTTIP levels and poor overall survival(OS)(P<0.001).However,there was no significant relationship between these traditional tumor markers and OS(all P>0.05).4.Univariate and multivariate COX analysis revealed that exosomal HOTTIP overexpression was an independent prognostic factor in GC patients(P=0.027).Conclusion:The expression levels of exosomal HOTTIP were typically higher in GC and it might be a novel biomarker for GC in diagnosis and prognosis.Part 2 The clinical and mechanism study of long noncoding RNA HOTTIP in chemotherapy resistance of gastric cancerBackground:Radical gastrectomy and perioperative chemotherapy based on platinum and fluorouracil have become the recommended first-line treatment for advanced gastric cancer by NCCN.However,the tumor recurrence because of chemotherapy resistance is one of the most important factors leading to the failure of GC chemotherapy.The roles of HOTTIP in tumor resistance have been reported,but its role in cisplatin resistance in GC remains unknown.Our study aims at investigating the important roles of HOTTIP in GC chemotherapy resistance.Methods:1.Expression levels of HOTTIP in 106 cases of GC tissues and matched noncancerous tissue and GC cells were detected by RT-qPCR.2.The relation between HOTTIP and tumor recurrence was analysed by ROC and Kaplan-Meier analysis.3.The effect of HOTTIP expression levels on cisplatin resistance in GC was detected by CCK8 proliferation assay,apoptosis assay and autophagy assay.4.The interaction among HOTTIP,miR-216a and Bcl-2 were predicted by StarBase v2.0 and confirmed by double luciferase reporter system and western blot.Results:1.The expression levels of HOTTIP in GC tissues was higher than in the matched non-tumor tissues(P<0.001),and its expression levels in recurrent patients was higher than that in the non-recurrent(P<0.001).The expression levels of HOTTIP was positively correlated with tumor invasion depth,lymph node metastasis and TNM stage in GC patients(P<0.01).ROC curve analysis showed that the AUC for diagnosing the recurrence of GC was 0.728,and at the optimal cut-off value(2.52),the sensitivity and specificity were 70.7%and 70.8%,respectively(P<0.001).The Kaplan-Meier survival analysis showed that high levels of HOTTIP in GC tissues were negatively correlated with disease-free survival and 5-year overall survival(P<0.001).2.HOTTIP was more highly expressed in SGC7901/DDP than in SGC7901(P<0.001).3.HOTTIP promoted cell chemoresistance via inhibiting autophagy.4.HOTTIP could sponge miR-216a to promote the expression of Bcl-2,then decrease the release of Beclinl from Bcl-2/Beclinl complex,and finally inhibit autophagy.Conclusion:HOTTIP was associated with the recurrence of GC patients who received chemotherapy and was negatively associated with the 5-year overall survival.HOTTIP could affecte the cisplatin resistance of GC cells by regulating miR-216a/Bcl-2/Beclinl pathway.