Effects Of PLCE1 On The Autophagy Of Esophageal Cancer Cell Lines And Primarily Study The Molecular Mechanism

Objective: To Explore the effect of PLCE1 on the autophagy level of esophageal squamous carcinoma; to explore the impact on apoptosis when downregulated the level of autophagy which induced by silencing PLCE1 expression in esophageal squamous carcinoma cancer; Preliminary study the molecular mechanisms of PLCE1 regulated autophagy level in esophageal squamous carcinoma.Methods:(1) Use PLCE1 siRNA transfected the esophageal squamous cancer cell lines, Eca-109, TE-1, through MDC staining, AO staining and cellular immune fluorescence detection method, to detected the influence of after downregulated the PLCE1 expression on the level of autophagy in cancer cells;(2) Then use autophagy specific inhibitor 3-MA or Beclin-1 si RNA, inhibit autophagy induced by silence PLCE1 expression, use the MDC staining detected the autophagy level in the esophageal squamous cancer cells, by MTT method and flow cytometry instrument methods to detected the apoptosis and proliferation level of tumor cell, using Western Blot method to detect autophagy related molecules expression level of Beclin-1 and LC3, and apoptosis related molecular the protein expression level of cleaved-PARP, Bax, Bcl-2, cleaved-caspase-3;(3) By Western Blot assay, detected the expression level of MCM7 after knockdown of PLCE1, then according to the previous findings, combining the analysis of target gene prediction software, such as Target Scan, mi Randa, DIANAm T, mi RDB, mi RWalk, using the luciferase report test targeting relationship between mi R-106b-5p and autophagy related gene Beclin-1;(4) In ECA109 transfected both mi R-106b-5p mimics and PLCE1 si RNA, through the MDC and AO staining, to detected the autophagy in cancer cells. By Western Blot method to detect the expression level of autophagy-related gene, Beclin-1 and LC3.Results:(1) To analyze the machinery involved in the autophagic process, AO and MDC staining were employed to detect the AVO formation. AO-label and MDC-label cells were visualized after 10 n M and 50 n M PLCE1 si RNA treatment respectively at particularly 48 h time point. Eca-109 cells were transfected with LC3 antibodys. Si-PLCE1-treated cells showed a punctate pattern of LC3 fluorescence, representing recruitment of LC3-II to autophagosomes and the formation of autophagic vacuoles, whereas the vehicle-treated control cells exhibited diffuse LC3-associated green fluorescence;(2) Pretreatment with 3-MA remarkably attenuated the formation of acidic autophagic vacuoles in the presence of PLCE1 of si RNA. Pretreatment of cells with 3-MA or Beclin-1 si RNA increased the number of viable si-PLCE1-treated cells, as assayed by MTT. Pretreatment with 3-MA or Beclin-1 si RNA obviously increased LC3 II, Beclin-1, decreased cleaved-caspase-3 and cleaved-PARP expression, and increased Bax to Bcl-2 ratio.(3) Western blot results showed that the expression level of MCM7 was downregulated after treated with PLCE1 si RNA. To elucidate the cancerogenic effects of mi R-106b-5p in ESCC, we further exploited mi R-106b-5p’s targets through through several target predictors to search for assumed human protein-coding gene targets of mi R-106b-5p. It showed a complementary match between mi R-106b-5p seed sequence and the 3’UTR of Beclin-1. We performed luciferase reporter assay with a vector containing the putative Beclin-1 3’-UTR target site downstream of the luciferase reporter gene and co-transfected Beclin-1 vectors together with mi R-106b-5p mimics or its negative control into Eca-109 cells. The results showed that, Beclin-1 was a downstream target of mi R-106b-5p through targeting at 3’UTR. The Western Blot showed that mi R-106b-5p mimics could downregulated the expression of Beclin-1.(4) As we had proved knockdown of PLCE1 could trigger the recruitment of autophagosomes and the formation of autophagic vacuoles, the results detected by AO and MDC staining indicated that mi R-106b-5p-Mi restrain the formation of autophagy vesicles in Eca109 cells, however, co-transfected with PLCE1 si RNA and mi R-106b-5p mimics could partially improve this phenomenon in cells. The results of Western Blot showed that, the esophageal cancer cells transfected with mi R-106b-5p mimics showed a lower expression level of Beclin-1 than control group, the cells co-transfected with mi RNA mimics and PLCE1 si RNA showed a lower expression level of Beclin-1 than the group only treated by si RNA.Conclusion:(1) The overexpression of PLCE1 could promote the proliferation of esophageal cancer through inhibit the level of autophagy and apoptosis in cancer cells;(2) the molecular mechanism of PLCE1 modulating autophagy could be through upregulating mi R-106b-5p to inhibit the expression of Beclin-1;(3) our study indicated that upregulated the level of autophagy through targeting block PLCE1 gene in esophageal cancer, could be a new strategy to cure the esophageal cancer.