| Background and Objective: Cervical cancer is a malignant tumor threatening global women’s health. Autophagy is not only involved in the malignant tumor occurrence, development, erosion and metastasis.on each stage, but also relates to chemoradiation-resistance,and enhancement of chemoradiation cytotoxicity The newly found gene ER membrane protein complex subunit 6(EMC6) is an important gene that has regulatory effect on autophagy. EMC6 can interact with the known tumor suppressor gene Beclin1 and tumor metastasis related gene Rab5 a to participate in the metastasis and differentiation of cervical cancer together. Therefore, the present study aimed to investigate the expression and correlation of autophagy gene EMC6, Rab5 a and Beclin1 in cervical cancer tissues, and the in vivo and in vitro differential effects of gene expression plasmid and specific RNA interference plasmid on cell growth and autophagy of cervical cancer as well as their interaction. In addition, the present study also aimed to learn about the role of autophagy gene in the development and progression of cervical cancer, explore the feasibility of autophagy induction in the treatment and prevention of cervical cancer, and discuss the potential autophagy targeted cervical cancer treatment strategy, so as to provide a new target for gene therapy of cervical cancer.Methods: 1) The expression levels of EMC6, Beclin1 and Rab5 a in cervical cancer tissues, cervical intraepithelial neoplasia(CIN) tissues and normal cervical tissues were determined using SP method; their correlation with clinical pathological factors of cervical cancer such as clinical stage, pathological grade and lymph node metastasis was analyzed. 2) Correlation of EMC6, Beclin1 and Rab5 a expression levels in cervical cancer tissues, CIN tissues and normal cervical tissues was analyzed. 3) EMC6 gene eukaryotic expression plasmid p Lenti-CMV-EMC6-PGK-T2A-Puro, specific interference EMC6 plasmid p Lenti-u6-shrna-CMV-T2A-Puro and blank plasmid p Lenti-u6-ccdb-CMV-T2A-Puro were constructed. He La cells were transfected with three plamids, and cell strains with stable expression were screened. Changes of EMC6 gene expression in He La cells of three groups after plasmid transfection were detected using Real time-PCR. 4) GFP-LC3 in cell strains of treatment group 、 blank plasmid group after transfection and He La cell line were observed using laser scanning confocal microscope. Then, He La cell strains of three groups were treated with bafilomycin A and 3MA, followed by a second observation on GFP-LC3 in cell strains using laser scanning confocal microscope. 5) The expression levels of Rab5 a and Beclin1 in EMC6 gene stably over-expressed He La cells and EMC6 gene stably non-expressed He La cells were determined using Real time-PCR and Western blot, respectively. 6) He La, He La-EMC6 and He La-sh RNA expression cell strains were inoculated into the subcutaneous tissues of nude mice to form tumor and establish the female nude mouse allograft cervical cancer model. The tumor growth rate and tumor size of three groups were observed and compared. Tumors were removed, and the expression levels of EMC6 protein, Rab5 a protein and Beclin1 protein in tumor tissues of three groups were determined using immunohistochemistry. 7) Statistical methods: Rank-sum test was used for ranked data. χ2 test was used for emumeration data. Rank-sum test was used for paired comparison of multiple groups and statistical analysis of ranked data. Kruskal-wallis test, a nonparametric test, was used for paired comparison of multiple groups of data. Nonparametric Spearman rank correlation method was used for correlation analysis. SPSS22.0 statistical software was used. α=0.05 was significant level(bilateral).Results: 1) In cervical squamous cell carcinoma tissues, negative EMC6 protein expression accounted for 36.0%(36/100), weakly positive expression accounted for 18.0%(18/100) and strongly positive expression accounted for 46.0%(46/100); in CIN tissues, negative EMC6 protein expression accounted for 6.3%(5/80), weakly positive expression accounted for 6.3%(5/80) and strongly positive expression accounted for 87.5%(70/80); in normal cervical epithelial tissues, negative EMC6 protein expression accounted for 18.8%(15/80), weakly positive expression accounted for 25.0%(20/80) and strongly positive expression accounted for 56.2%(45/80). EMC6 protein expression was the lowest in cervical cancer tissues and the highest in CIN tissues, and there was significant difference(P<0.05). 2) In cervical cancer tissues, negative Beclin1 protein expression accounted for 38.0%(38/100), weakly positive expression accounted for 50.0%(50/100) and strongly positive expression accounted for 12%(12/100); in CIN tissues, negative EMC6 protein expression accounted for 6.25%(5/80), weakly positive expression accounted for 62.50%(50/80) and strongly positive expression accounted for 31.25%(25/80); in normal cervical tissues, negative expression accounted for 18.75%(15/80), weakly positive expression accounted for 31.25%(25/80) and strongly positive expression accounted for 50.0%(40/80). The proportion of negative Beclin1 protein expression in cervical cancer group was higher than that in CIN group(P<0.05), the proportion of positive Beclin1 protein expression in CIN tissues was higher than that in normal cervical tissues, and there was significant difference(P<0.05). 3) In cervical cancer tissues, negative Rab5 a protein expression accounted for 25%(25/100), weakly positive expression accounted for 37.0%(37/100) and strongly positive expression accounted for 38.0%(38/100); in CIN tissues, negative expression accounted for 31.25%(25/80), weakly positive expression accounted for 25.0%(20/80) and strongly positive expression accounted for 43.75%(35/80); in normal cervical tissues, negative expression accounted for 75.00%(60/80), weakly positive expression accounted for 17.50%(14/80) and strongly positive expression accounted for 7.5%(6/80). The proportion of postive Rab5 a protein expression in cervical cancer group and in CIN group was higher than normal cervical tissues,but there was no significant difference between in CIN tissues and normal cervical tissues(P>0.05). 4) In cervical squamous cell carcinoma, expression of EMC6 protein and Beclin1 had no correlation with age,clinical stage, tumor size, gross type, tumor pathological type, tumor pathological grade and lymph node metastasis(p>0.05). In cervical squamous cell carcinoma, Rab5 a protein expression had no correlation with clinical stage and tumor size(P>0.05), the expression in poorly differentiated group was higher than that in moderately differentiated group and well differentiated group(p<0.05), and the expression in lymph node metastasis group was higher than that in non-lymph node metastasis group(p<0.05). 5) Immunohistochemistry showed that the EMC6 protein expression level in cervical cancer tissues, CIN tissues and normal cervical tissues had no correlation with Beclin1 protein and Rab5 a protein expression level(p>0.05). 6) RT-PCR showed that the expression of EMC6 m RNA in He La cells was significantly increased after p Lenti-CMV-EMC6-PGK-T2A-Puro plasmid transfection(p<0.05), the expression of EMC6 m RNA in He La cells was significantly decreased after p Lenti-u6-shrna-CMV-T2A-Puro plasmid transfection(p<0.05),while the expression of EMC6 m RNA in cells had no significant change after p Lenti-u6-ccdb- CMV-T2A-Puro(blank plasmid) transfection(p>0.05). 7) Laser scanning confocal microscope observed that the intracellular GDP-LC3 in the treatment group was significantly increased after transfection compared with that in the blank plasmid group and in He La cell line; Bafilomycin A1 can make the aggregation of GFP-LC3 in the three groups, and the LC3 spots in the treatment group are the most abundant.3-MA can reduce the LC3 spots in the treatment group 、blank plasmid group and He La cell line; in the cell strains of treatment group 8) PCR and Western blot showed that the expression levels of Rab5 am RNA, Rab5 a protein, Beclin1 m RNA and Beclin1 protein were increased in EMC6 over-expressed He La cell strains; the expression levels of Rab5 am RNA and Rab5 a protein, Beclin1 m RNA and Beclin1 protein were decreased in EMC6 specifically interfered or suppressed He La cell strains. 9) Compared with the nude mice in He La group(not transfected with plasmid), the nude mice in Hela-sh RNA group(EMC6 interfered) had significantly enhanced tumorigenicity, rapid growth and significantly increased tumor size(P<0.05). The nude mice in He La-EMC6 group(EMC6 over-expressed) had significantly reduced tumorigenicity, slow growth and significantly decreased tumor size(P<0.05). The average tumor weight of the nude mice in Hela-sh RNA group was 1.03±0.12 g, which was increased by 28.75% compared with that in He La group(P<0.05); the average tumor weight in He La-EMC6 group was 0.39±0.14 g, which was decreased by 51.25% compared with that in He La group(P<0.05). 10) Immunohistochemistry showed that the expression levels of Rab5 a protein and Beclin1 protein in the tumor were higher in the He La-EMC6 group than in the Hela-sh RNA group and He La group; the expression levels of Rab5 a protein and Beclin1 protein in the tumor were higher in the Hela-sh RNA group than in the He La group.Conclusions: 1) Autophagy gene EMC6 is expressed in normal cervical tissues, CIN tissues and cervical cancer tissues. In the continuous pathological process of “normal cervix-CIN-cervical cancer”, the expression level of EMC6 in CIN tissues is increased, which suggests that autophagy activity is enhanced in precancerous lesion stage; however, the expression level of EMC6 is decreased when CIN develops to cervical cancer, which suggests that suppression of autophagy activity promotes the malignant transformation of cervix. In addition, negative or down-regulated EMC6 expression occurs in the early stage of cervical cancer, and thus the expression of EMC6 has no correlation with the size, stage, differentiation degree and lymph node metastasis of cervical cancer tissues, which provides research directions for EMC6 gene targeted cervical cancer prevention. 2) In cervical cancer tissues, CIN tissues and normal cervical tissues, the EMC6 protein expression level has no direct correlation with the Beclin1 and Rab5 a protein expression level. 3)Up-regulation or down-regulation of EMC6 in cervix cancer cells can result in corresponding up-regulation or down-regulation of intracellular Rab5 a and Beclin1. It is speculated that EMC6 protein may act at the upstream of Rab5 a protein and have anti-tumor effect. 4) In the present study, female nude mouse transplanted tumor model was used to investigate the effect of EMC6 on the tumorigenicity and growth of cancer cells. According to the result analysis, EMC6 protein can significantly inhibit the growth of He La cells in nude mice and reduce the tumorigenicity. Immunohistochemistry for the tumors of nude mice showed that Rab5 a and Beclin1 were up-regulated in smaller tumors over-expressing EMC6, while Rab5 a and Beclin1 were down-regulated in larger tumors with down-regulated expression of EMC6. 5) According to the experimental results above, it is speculated that if autophagy gene EMC6 is selected as the target of gene therapy, it can not only inhibit the action of Rab5 a protein in cancer metastasis and invasion, but also promote the autophagic anti-tumor effect of Beclin1. The present study provides new research directions for the targeted therapy of cervix cancer and even another tumors. |
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