| Objective: To investigate the expression of Beclin1in ESCC and normalesophageal mucosa tissues, and explore their correlation with theclinicopathological parameters. The possible function of Beclin1on theproliferation, autophagy, apoptosis and the drug sensitivity were furtherinvestigated in ESCC TE1cells under over-expression condition. Our resultsmay provide theoretical foundation for the etiology of ESCC.Methods:1Immunohistochemical staining and Real time-PCR were used to detect theprotein and mRNA expressions of Beclin1in57specimens ESCC and normalesophageal mucosa tissues.2Gene over-expression, Real time-PCR and Western blot were used toinvestigate the influence of exogenous Beclin1on ESCC TE1cells.3MTT assay was used to evaluate the effect of Beclin1over-expression on theproliferation and growth of the transfected cells.4Cell cycle and apoptosis were measured by flow cytometry (FCM).5Autophagy phenomenon was observed under fluorescence microscope.6pCMV6-Entry-Beclin1and3-MA were used to ESCC TE1cells，detectedthe growth inhibition rate, autophagy and apoptosis of cells in eachgroup(control group, DDP group, Beclin1+DDP group and3-MA+DDPgroup).Results：1The Immunohistochemical staining results showed that the expression ofBeclin1protein in ESCC were29.82%(17/57), which was significantly lowerthan those in normal esophageal mucosa tissues (57/57)(P＜0.000); Beclin1expression was significantly correlated with differentiation (χ~2=6.158,P=0.046) and lymph node metastasis (χ~2=5.664,P=0.017); but not significantly correlated with sex, age, tumor size or invasion depth of the ESCC. Realtime-PCR analyses showed that the expression of Beclin1mRNA in ESCCwere0.168±0.038, which was significantly lower than those in normalesophageal mucosa tissues0.280±0.052(P＜0.000).2The expression of DDK at protein level was detected after transfected48hwith pCMV6-Entry-Beclin1and pCMV6-Entry in TE1cells by Western Blotmethod. In comparison with control group, DDK protein could be detectedafter transfection, suggesting that this expression vector could be used in thenext experiments.3To further investigate the biological effects of Beclin1in ESCC, we usedinstant transfection to enhance the Beclin1expression in TE1cells to observethe role of Beclin1on mRNA, protein, cell proliferation, cell cycle distribution,and the changes of autophagy vesicles.3.1The expressions of Beclin1mRNA and protein in TE1cells aftertransfected with pCMV6-Entry-Beclin1were significantly higher than thoseafter transfected with blank plasmid (TE1-PE group) and non-transfectiongroup (TE1group).3.2MTT assay showed that the growth of TE1cells withpCMV6-Entry-Beclin1transfection was significantly inhibited compared withthe TE1-PE group and the TE1group. The inhibition rates inpCMV6-Entry-Beclin1transfected cells were10.30%,19.29%,21.94%and21.82%at24h,48h,72h,96h and120h, respectively. The results revealed thatpCMV6-Entry-Beclin1transfection could inhibit the proliferation of TE1cells3.3The results of FCM showed that after over-expression of Beclin1in TE1cells, the proportion of cells at G0/G1phase increased significantly, theproportion of cells at S stage decreased significantly, cell proliferation wasinhibited(P＜0.01); the apoptosis rate was5.91%, which was significantlyhigher than those in TE1-PE group and TE1group (P＜0.01).The resultsindicated that pCMV6-Entry-Beclin1can lead cells to be arrested at G0/G1phase.3.4pCMV6-Entry-Beclin1transfection of TE1cells produced a significantly higher MDC fluorescent intensity than TE1-PE group and TE1group. Theresults revealed that after pCMV6-Entry-Beclin1transfection could enhancethe levels of autophagy in TE1cells.4pCMV6-Entry-Beclin1and3-MA were used to ESCC TE1cells，detectedthe cell proliferation, the expression of Beclin1protein, the change ofautophagy vesicles, cell cycle distribution and apoptosis of cells in eachgroup(control group, DDP group, Beclin1+DDP group and3-MA+DDPgroup).4.1MTT assay showed that cells proliferation inhibition rate of control group,DDP group, Beclin1+DDP group and3-MA+DDP group were1.06%,31.59%,18.93%, and51.44%, respectively. DDP group can effectively inhibit theproliferation of TE1cells. Compared to DDP treatment alone, the proliferationinhibition rate of TE1cells were decreased by Beclin1+DDP treatment andincreased by3-MA+DDP treatment(P＜0.01).4.2Western blot showed that the expression of Beclin1protein in controlgroup, DDP group, Beclin1+DDP group and3-MA+DDP group were0.319±0.029,1.175±0.060,1.537±0.076,0.910±0.047, respectively.Differences were significant after compare with between groups by One-WayANOVA and SNK test(P＜0.01).4.3The number of MDC-labeled autophagic vacuoles from high to low wasBeclin1+DDP group, DDP group,3-MA+DDP group and control group.Beclin1+DDP group produced significantly higher MDC-labeled autophagicvacuoles than control group, DDP group and3-MA group. DDP groupproduced significantly higher MDC-labeled autophagic vacuoles than controlgroup and3-MA group. While, there was no significant differences between3-MA group and control group.4.4The results of FCM showed that the proportion of cells at G0/G1phase ineach group was44.820±0.437,56.250±0.624,27.553±0.085,66.480±0.638,respectively；at S phase in each group was34.850±0.235,24.417±0.607,51.800±0.721,19.167±0.666, respectively。The apoptosis rate in each groupwas1.237±0.015,4.813±0.025,1.320±0.010,6.117±0.035, respectively。 Differences were significant after compare with between groups by One-WayANOVA and SNK test(P＜0.01).Conclusions:1Beclin1expression is down-regulated in ESCC, which may relate totumorigenesis and development of ESCC.2The expression of Beclin1was correlated with lymph node metastasis,suggested that the expression of Beclin1was associated with the invasion andmetastasis of ESCC.3Beclin1over-expression can inhibit the proliferation and growth of TE1cells in vivo, suggesting its potential role in gene therapy of ESCC.4DDP could induce TE1cells autophagy and apoptosis; the increase ofreactive autophagy activity in the course is related to the formation of cisplatinresistance, which indicates that monitoring and regulating the autophagylevels of tumor cells may provide a new idea for reversing DDP resistance inESCC.5This study demonstrated that the down-regulated autophagy couldincrease chemotherapeutic sensitivity of DDP to ESCC. |
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