Exosomes Derived From HBV-Associated Liver Cancer Induce Chemoresistance By Activating Chaperone-Mediated Autophagy Pathway

Objectives Primary liver cancer,one of the most common cancer in our country,has been poorly prognosis for its serious chemoresistance.Hepatitis B virus?HBV?infection in PLC patients is negatively associated with therapy efficiency to Oxaliplatin?Oxa?treatment.Exosomes play a key role in cells communication and tumor progression.In our research,we identified the mechanisms that exosomes derived from HBV associated liver cancer could modulate tumor sensitivity to Oxaliplatin?Oxa?through upregulating chaperone-mediated autophagy?CMA?pathway,which can provide a new strategy for effective treatment of liver cancer patients.Methods 1 Data has been collected of PLC patients who undergone Oxaliplatin?Transarterial chemoembolization,TACE:130mg/m²?therapy.Child-Pugh grade A-B and BCLC system B stage in PLC patients are for our research.PLC patients were divided into two groups according to HBV infection?HBsAg and HBV DNA were positive?,HBV-positive liver cancer group and HBV-negative liver cancer group?HCV is negative?.And patients indicator such as sex and age were further statistically analyzed.In two PLC patients groups,tumor volume reduction was evaluated by statistical analysis to compare patients'responses to Oxaliplatin therapy.2 HepG2 cells were cultured in vitro,HBV patients serum and health volunteer serum were collected from North China University of Science and Technology affiliated hospital.Cells were separately infected with HBV serum?HBV DNA>1.0×10⁹copies/ml?and health individual serum?HBV?HCV negative?for 48 h.Removed HBV supernatant and washed it for 8-10 times with phosphate buffer saline?PBS?,the last washing supernatant was preserved for HBV DNA identification to testify HBV residue.After removing the HBV serum,cells were cultured with DMEM and the culture supernatant was collected at 0 h?12 h?24 h?48 h?72 h and 96 h for further HBV DNA identification by real-time fluorescent quantitative PCR analysis;After serum infection,exosomes in culture supernatant were isolated using Exosomes Isolation Kit and identified by Western Blot analysis and transmission electron microscope.3 Treated with HBV-associated liver cancer exosomes for 12 h and further cultured with Oxaliplatin for 24 h,HepG2 cells apoptosis was tested by flow cytometry analysis and apoptotic-related proteins such as Cleaved caspase-3 and Bcl-2 were identified by Western Blot analysis.In addition,the levels of Lamp2a expression,which is a key criteria for chaperone-mediated autophagy?CMA?activation,was identified by Western Blot analysis;4 Lamp2a gene in HepG2 cells was silenced by shRNA interfere technology;Treated with exosomes and Oxa,sh-Lamp2a HepG2 cells apoptosis was determined through flow cytometry analysis.5 Student's t test or one-way ANOVA was complemented for statistical analysis through SPSS17.0 software.Results 1 Data was collected from PLC patients,Child-Pugh classification at A-B grade and BCLC at B stage PLC patients were chosen to be researched and their information such as sex and age has no statistically difference in two PLC patients groups;Tumor volume reduction in HBV-related PLC patients was?36.81±15.27?%,which was lower than non-HBV-associated PLC patients group?61.45±14.35?%after three months treatment of Oxaliplatin?TACE:130mg/m²?,P<0.05.2 Real-time fluorescent quantitative PCR analysis showed that HBV DNA contents in supernatant was reached1.5×10⁴copies/ml at 96 h and exosomes in the supernatant were isolated using Exosomes Isolation Kit.The exosomes markers CD63 and CD9 were identified by Western Blot analysis.In addition,transmission electron microscope discovered that there were a lot of exosomes which were round vesicles.Nanosight tracing analysis observed that the diameter of exosomes was approximately 150 nm and,evaluated that the concentration of HBV-associated liver cancer exosomes was?5.25±0.63?×10¹⁰particle/ml and?4.95±0.87?×10¹⁰particle/ml in non-HBV-associated liver cancer exosomes group,there was no statistically significance.3 Treated with HBV-associated liver cancer exosomes and Oxa,pro-apoptosis protein Cleaved caspase-3 was decreased while anti-apoptosis protein Bcl-2 expression was increased which was showed by Western Blot analysis,at the same time,flow cytometry analysis indicated that,cells apoptosis was?45.21±7.69?%in HBV-associated liver cancer exosomes group,which was obviously lower than the negative group?54.21±7.69?%.Moreover,Western Blot indicated that the levels of Lamp2a expression,which is a key molecule in CMA activation,was significantly upregulated when cells treated with HBV-associated liver cancer exosomes;4 Lamp2a gene was knocked down in HepG2 cells which were given exosomes and Oxa,and it showed that sh-Lamp2a HepG2 cells apoptosis was?71.34±12.98?%which was increasingly upregulated compared with negative group?56.16±11.62?%.Conclusions 1 HBV-associated liver cancer exosomes could downregulate cell chemoresistance to Oxaliplatin;2 Exosomes derived from HBV-associated liver cancer could decrease cells chemosensitivity to Oxaliplatin therapy through activating CMA pathway.Figure7;Table1;Reference 41...