Suppression Of Survivin Expression By Short Hairpin RNA Induces Apoptosis In Human Laryngeal Carcinoma Cells

Objective and background:As an antiapoptosis gene survivin is a member of the IAP(inhibitor of apoptosis proteins)family.It is the smallest one of the eight IAP members and has a promising future in basic and clinical area.So survivin has became the most attentive gene in the study of tumor.The gene plays important roles in inhibiting apoptosis, enhancing cell proliferation and regulating cell division.It is the stronggest antiapoptosis gene.There are several explanations about the mechanism of its antiapoptosis action.One theory indicates that the mechanism of antiapoptosis is mediated by caspases which survivin can bind with to resist apoptosis.The other theory shows that survivin suppresses apoptosis by co-regulation with NF-κB,repression of apoptosis enzymes and interaction with the receptor of serine/thereonine kinase.Most of the studies have shown that survivin plays an important role in mitochysis.It regulates the microtubule organizing centre and the formation of the mitotic spindle.At the mitosis telophase,survivin binds with the microbule which constructs the cyto-centrum and distributes into two daughter cell,and then degradates rapidly.This process demonstrates that survivin plays an important role in cell division.The mitotic division cannot run smoothly if survivin gene is suppressed.The cell cycle is regulated by survivin through its binding with mitotic spindle.Surivin is highly expressed in G2/M and it is a regulating gene of the G2/M checkpoint of cell cycle.The overexpression of survivin can help the tumor cell escape from the checkpoint of the cell cycle and proliferate abnormally.So we can see survivin anticipates the generation and the development of the carcinoma.Survivin is expressed during embryonal development but either undetectable or expressed at a very low level in terminally differentiated adult tissues.Interestingly,it becomes over-expressed in transformed cell lines and almost all types of human malignancies.So survivin may be a promising target protein in cancer gene research and therapy.RNA interference surppresses the expression of target gene using 21-23nt double small RNAs.Several scientists have applyed the survivin specific shRNA or siRNA into many kinds of tumors and obtained a significant suppression effect.Much research shows that the shRNA vector can get a higher effect of RNAi.But the application of survivin specific shRNA in laryngeal carcinoma has not been reported.In the present study,we detected the expression of survivin in 60 cases of laryngeal squamous cell carcinoma(LSCC)and 10 cases of normal laryngeal mucosa using immunohistochemistry and constructed three plasmid vectors encoding shRNA against survivin to determine whether plasmid-mediated RNAi targeting survivin would suppress the proliferation of the tumour cell and enhance the apoptosis of Hep-2 cells.Methods:1.Immunohistochemistry was used to detected the expression of survivin in 60 cases of laryngeal squamous cell carcinoma(LSCC)and 10 cases of normal laryngeal mucosa.2.Three sequences were selected as the positive-sense strand of siRNA. Nine oligonucleotides were added between the positive-sense strand and the antisense strand to construct a hairpin structure,and then cloned on the plasmid vector.So three plasmid vectors targeted survivin were construced and named survivin-shRNA-1,survivin-shRNA-2,survivin-shRNA-3,respectively.The sequences of the vectors were confirmed to be correct.3.The cells were transfected with RPMI-1640 media(untreated cells), nonsense shRNA and survivin shRNAs,respectively.48 hours after transfection we collected the cells and performed a series of experiments. Western blot and RT-PCR analysis were performed to detect the down-regulation of survivin on the mRNA and the protein level.Growth curve was used for determination of the cells proliferation.PI single staining was applied for detecting the constituent ratio of the cell cycle.The apoptosis condition of the cells was analyzed by flow cytometry with annexin V-FITC/PI double staining and PI single staining.Results1.The expression of survivin cannot be detected in the 10 cases of normal laryngeal mucosa compared with the 60 cases of LSCC among which 41 cases were positive,the positive rate was 68.33%.In the male the positive rate was 71.43%(40/56);in the female the positive rate was 25%(l/4).Survivin was expressed in 22 of 32 cases over 60 in age and in 19 of 28 cases under 60(including 60);The positive rate of supraglottic carcinoma was 72.22%(13/18)compared with the glottic carcinoma (66.67%).The survivin expression positive rates in gradeⅠ,ⅡandⅢtumors were 71.43%(20/28),63.16%(12/19),69.23%(9/13),repectively and in clinical stageⅠ-ⅡandⅢ-Ⅳtumors being 54.84%(17/31)and 82.76%(24/29).The positive rate of expression was higher in lymphatic metastasis group(93.33%)than in non-lymphatic metastasis group(60%). Analyzed by statistical methods,the significant difference of survivin expression was observed between the LSCC and the normal laryngeal mucosa.The expression of survivin was significantly correlated to clinical stage and lymphatic metastasis.On the contrary there was no obvious correlation between the survivin expression with the sex,the age, the differentiation and the tumor arised site.2.Three recombinant plasmid vectors were confirmed to be exactly correct and inserted into expected site by DNA sequence analysis.3.After transfection,all the three recombinant survivin-shRNAs can depressed the mRNA level of survivin in Hep-2 cells significantly compared with the negative control group and the untreated group.The inhibition rates were 65.77±0.82%,56.66±0.70%,63.26±1.0%, respectively.And also the survivin shRNAs depressed the protein level of survivin in Hep-2 cells significantly compared with the negative control group and the untreated group.The inhibition rates were 61.50±0.79%, 52.91±1.43%,59.36±0.53%,respectively.The cell proliferation was reduced obviously at 72 hours after transfection.The arrest of G2/M was observed in the three groups which was transfected with surviyin shRNAs. The apoptosis rate was detected using PI single staining which displays apoptosis of late stage and annexin V-FITC/PI double staining which exposes apoptosis of early stage.The results showed that the apoptosis rates of survivin shRNAs were significantly higher than the two control groups.Statiscal analysis showed that the three survivin shRNAs were effective and the survivin shRNA-2 had a weaker effect than the other two shRNAs.Conclusion:Survivin shRNA is capable of suppressing survivin expression.The shRNA targeted survivin can significantly inhibit cellular proliferation due to cell cycle arrest and apoptosis.This study suggested that survivin specific shRNA maybe a useful and promising approach in the treatment of human laryngeal carcinoma and other malignant tumors with highly survivin expression.