Effects of small interfering RNA inhibit Class Ⅰ phosphoinositide 3-kinase on human gastric cancer cells

AIM:To investigate the effects of small interfering RNA(siRNA)-mediated inhibition of Class Ⅰ phosphoinositide 3-kinase(Class Ⅰ PI3K) signal transduction on the proliferation,apoptosis,and autophagy of gastric cancer SGC7901 and MGC803 cells.METHODS:We constructed the recombinant replication adenovirus PI3K(I)-RNA interference(RNAi)-green fluorescent protein(GFP) and control adenovirus NCRNAi-GFP,and infected it into human gastric cancer cells.MTT assay was used to determine the growth rate of the gastric cancer cells.Activation of autophagy was monitored with monodansylcadaverine(MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment.Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3(LC3).Mitochondrial membrane potential was measured using the fluorescent probe JC-1.The expression of autophagy was monitored with MDC,LC3 staining,and transmission electron microscopy.Western blotting was used to detect p53,Beclin-1,Bcl-2,and LC3 protein expression in the culture supernatant.RESULTS:The viability of gastric cancer cells was inhibited after siRNA targeting to the Class Ⅰ PI3K blocked Class Ⅰ PI3K signal pathway.MTT assays revealed that,after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP,the rate of inhibition reached 27.48% ± 2.71% at 24 h,41.92% ± 2.02% at 48 h,and 50.85% ± 0.91% at 72 h.After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAiGFP,the rate of inhibition reached 24.39% ± 0.93% at 24 h,47.00% ± 0.87% at 48 h,and 70.30% ± 0.86% at 72 h(P < 0.05 compared to control group).It was determined that when 50 MOI,the transfection efficiency was 95% ± 2.4%.Adenovirus PI3K(I)RNAi-GFP(50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells,and the results described here prove that RNAi of Class Ⅰ PI3K induced apoptosis in SGC7901 cells.The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity,indicating increased formation of autophagosomes.The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low.After incubating with adenovirus PI3K(I)-RNAi-GFP(50 MOI),Beclin-1,LC3,and p53 protein expression was significantly increased from 24 to 72 h.We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP(50 MOI).A number of isolated membranes,possibly derived from ribosomefree endoplasmic reticulum,were seen.These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles.We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)RNAi-GFP(50 MOI) treatment.Control cells showed a round shape and contained normal-looking organelles,nucleus,and chromatin,while adenovirus PI3K(I)-RNAiGFP(50 MOI)-treated cells exhibited the typical signs of autophagy.CONCLUSION:After the Class Ⅰ PI3K signaling pathway has been blocked by siRNA,the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.

Distinct Rab7-related Endosomal-Autophagic-Lysosomal Dysregulation Observed in Cortex and Hippocampus in APPswe/PSEN1dE9 Mouse Model of Alzheimer＇s Disease

Background：Amyloid-β deposition and accumulation of autophagic vacuoles are pathologic features of Alzheimer＇s disease （AD）.Dysregulation of the endosomal-autophagic-lysosomal （EAL） pathway,which impairs amyloid precursor protein processing,is one of the earliest changes in AD.However,the precise role of EAL pathway in neurodegeneration remains unclear.This study aimed to investigate the role of EAL pathway in AD and further study the mechanism of EAL dysfunction.Methods：We used 3-,7-,and 12-month-old APPswe/PSEN1dE9 （APP/PS1） mice to model different stages of AD with age-and gender-matched wild-type littermates as controls （4-7 mice per group） and detected the changes of EAL markers,endosomal organizers Rab5 and Rab7,autophagosome marker LC3B,and lysosomal proteins Lamp 1/2 in cortex and hippocampus by immunohistochemistry and Western blotting analysis.To further explore the mechanism of EAL dysregulation in AD,components of the class Ⅲ phosphatidylinositol 3-kinase （PI3KC3） complex,activators ofRab7 （Beclin1 and UVRAG）,and the negative regulator of Rab7 （Rubicon） were also measured in this two brain regions.Results：In 7-month-old APP/PS1 brain that amyloid beta initiated to accumulate intracellularly,EAL pathway,and related PI3KC3 members,UVRAG and Beclin1 were upregulated both in cortex and hippocampus （all P 〈 0.05）.By the age of 12 months old,when abundant amyloid plaques formed,EAL markers,UVRAG,and Beclin 1 were also upregulated in the cortex （all P 〈 0.05）.However,Rab7 was decreased significantly （P =0.0447）,accompanied by a reduction of its activating PI3KC complex component Beclin1 （P =0.0215） and enhancement of its inhibiting component Rubicon （P =0.0055） in the hippocampus.Conclusions：Our study implies that EAL pathway,represented as Rab7 and its PI3KC3 regulators＇ expressions,showed temporal and spatial variation in brains at different stages of AD.It provides new insights into the role of EAL pathway in pathogenesis and indicates potential therapeutic targets in neurodegenerative diseases.