| Objective:This study intends to investigate the role of heat shock proteins 90(HSP90α)and hypoxia inducible factor-1(HIF-1α)in Ba P regulated molecular chaperone autophagy(CMA)by RNA interference technology and subcutaneous tumorigenesis in nude mice.Methods:1.To construct and screen the silencing cell line A549.The stably transfected A549 cell line was identified by the methods of q PCR and Western blot.After successful identification,the sh Control group(transfected the negative control plasmid)and sh HSP90ɑgroup(transfected the silencing HSP90ɑplasmid)were collected and inoculated into the forearm of nude mice to observe tumor formation.When the tumor volume to be transplanted reached 100 mm~3,nude mice in the two tumor-forming groups were randomly divided into two groups,with 10 mice in each group,and a total of four groups(sh Control group,sh Control+Ba P group、sh HSP90ɑgroup、and sh HSP90ɑ+Ba P group).The experimental group was intragastrically treated with Ba P-corn oil solution 1.80 mg/kg/d,while the Control group was intragastrically treated with corn oil solution.The length and diameter of the tumor were measured and weighed every 10 days,and draw the growth curve of the transplanted tumor and the weight change curve of the nude mice.Sixty days later,the neoplastic mice were put to death(drug administration and food were stopped the day before execution),and the size and weight of the transplanted neoplasm in each group were measured.HE staining was used to observe the structural and morphological changes of the xenograft tumor,and the effects of Ba P on the xenograft tumor in nude mice were observed with the small animal in vivo imaging system.The effects of Ba P on the expression of CMA-related gene and protein HSP90ɑ,HSC70 and Lamp-2A were detected by the methods of q PCR,Western blot and immunohistochemistry;2.The methods of Enzyme linked immunosorbent assay(ELISA)and Western blot were used to detect the effects of different concentrations of Ba P(0.1μmol/L,1μmol/L and 10μmol/L)on the concentration of EPO and HIF-1ɑin A549 cells.3.Western blot assay was utilized to detect the effects of different concentrations of Ba P(0.1μmol/L,1μmol/L and 10μmol/L)on HIF-1ɑprotein expression in A549 cells and statistical analysis was conducted;4.The methods of q PCR,Western blot and immunofluorescence were used to investigate the effects of Ba P on the expression of CMA-related genes and proteins after HIF-1ɑinhibition.Results:1.We successfully constructed a stable cell line that could get down HSP90ɑm RNA and protein expression,and the differences were statistically significant(P<0.05)compared with the sh HSP90ɑgroup;2.We had successfully built up a tumor transplanted into 40 nude mice 12 days after being inoculated with the corresponding group A549 cells,including two deaths in the sh Control group,three deaths in the sh Control+Ba P group and two deaths in the sh HSP90ɑ.They were also able to get down the volume and weight of the xenograft tumor from the sh Control+Ba P group,and the differences were statistically significant compared with the sh Control group(P<0.01),however,the sh HSP90ɑ+Ba P group were also able to get down the volume and weight,which was significantly lower than that of the sh Control+Ba P group(P<0.01);3.q PCR results showed that the m RNA expression levels of HSP90、HSC70 and Lamp-2A of sh Control+Ba P were significantly higher than that of the sh Control group(P<0.01、P<0.01、P<0.01),while the m RNA expression levels of HSP90、HSC70 and Lamp-2A were significantly lower than that of the sh Control+Ba P group(P<0.01,P<0.01,P<0.05);4.Western blot revealed that the levels of HSP90ɑ,HSC70 and Lamp-2A in the sh Control+Ba P group were significantly higher than those in the sh Control group(P<0.01,P<0.01,P<0.05),however,the protein expression levels of HSP90ɑ,HSC70and Lamp-2A of sh HSP90ɑ+Ba P were significantly decreased compared with that of the sh Control+Ba P group(P<0.05,P<0.01,P<0.05);5.The results of HE staining showed that different degrees of necrosis were observed in the tumors in each group.Among them,the area of necrosis in the sh Control group was the largest with large patches of necrosis,while the necrosis area in the sh Control+Ba P group was smaller and the tumor tissues of each group have large atypia,more intercellular substance,deep staining of nuclei,more nucleo-cytoplasmic ratio,disordered cell arrangement,and conform to the pathological characteristics of cancer cells.There is no significant difference in the tissue structure of sh HSP90ɑgroup and sh HSP90ɑ+Ba P group;6.The results of in vivo animal imaging showed that the total bioluminescence photon number of transplanted tumors in the sh Control+Ba P group was significantly higher than that of the sh Control group(P<0.01),while the total number of bioluminescence photons in the sh HSP90ɑ+Ba P group was significantly lower than that in the sh Control+Ba P group,which was statistically significant(P<0.01);7.The results of the enzyme-linked immunosorbent assay(Elisa)showed that when the concentration of Ba P was 10μmol/L,compared with the control group,the concentrations of EPO and HIF-1ɑwere significantly higher,and the difference was statistically significant(P<0.05);8.Western blot results showed that with the increase of Ba P concentration,the expression of HIF-1ɑprotein increased significantly in a dose-dependent manner.When the concentration of Ba P was 10μmol/L,compared with the Control group,the expression of HIF-1ɑprotein was statistically significant(P<0.05);9.The q PCR results showed that the m RNA expression levels of HSP90ɑ,HSC70 and Lamp-2a were significantly increased in the Ba P group compared with the Control group(P<0.01,P<0.05,P<0.01),however,the m RNA expression levels of HSP90ɑ,HSC70,and Lamp-2A were significantly decreased compared with the Ba P group after the HIF-1ɑinhibitor was added(P<0.05,P<0.01,P<0.05);10.The results of immunofluorescence showed that the fluorescence intensity of HSP90αin Ba P group was significantly higher than that in Control group,and it was significantly lower than that in Ba P group when HIF-1αinhibitor was added;11.Western blot results showed that the protein expression levels of HSP90ɑ,HSC70 and Lamp-2A were significantly increased in the Ba P group compared with the Control group(P<0.05,P<0.01,P<0.05),however,the protein expression levels of HSP90ɑ,HSC70,and Lamp-2A were significantly lower than those of the Ba P group when the HIF-1ɑinhibitor was added(P<0.05,P<0.05,P<0.05).Conclusions:1.Ba P can promote the growth of human lung cancer A549 cells xenografts in nude mice,and the growth of xenografts in nude mice is inhibited after silencing HSP90ɑ;2.Ba P can promote the expression of CMA related genes at the overall animal level;3.Ba P can promote the occurrence of CMA by promoting the expression of HSP90αand HIF-1α.HSP90αand HIF-1αplay an important role in Ba P regulated CMA. |
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