| ObjectiveTo detect the expression of Yes-Associated Protein (YAP) in Laryngeal Squamous Cell Carcinoma (LSCC) patients' cancer tissues and corresponding adjacent tissues, and to identify the influence of low YAP expression on laryngeal cancer cell line HEp-2, the changes caused in cell cycle, migration and sensitivity to cisplatin chemotherapy drug. We analyzed the relationship between different YAP expression levels and different clinical characteristics in different LSCC patients. Clinical characteristics include gender, age, TNM classification, clinical stage, pathologic grade, lymph node metastasis, recurrence and tumor sizes. Then we explored the different expression of YAP in different LSCC patients'tissues and the molecular mechanism. We are trying to find clues of diagnosis for laryngeal cancer in the early stage, and scientific basis of the mechanism of development, progress invasion and metastasis.MethodsWe used Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology to screen different kinds of proteins expression in LSCC patients'cancer tissues and corresponding adjacent tissues, then used RT-PCR and Western Blot to testify the results of the iTRAQ. According to Western Blot results, we analyzed the results with statistical software SPSS 16.0. We compared the differences between YAP expression quantity and gender with two independent sample t-tests; the differences between YAP expression quantity and age with chi-square test; the differences between YAP expression quantity and different TNM classification, clinical stage, pathologic grade with One-Way ANOVA, the differences between YAP expression quantity and lymph node metastasis, recurrence with two independent sample t-tests; the correlation between YAP expression quantity and tumor sizes. We were regarded the difference was significant if P value was less than 0.05.We used shRNA technology to decrease the expression of YAP protein in laryngeal cancer cell line HEp-2, and then we detected the change of cell cycle with FACS, the ability of migration with Scratch test, the sensitivity to cisplatin chemotherapy drug with WST-1.Results1. The difference between YAP expression quantity and gender was significant (t=2.039, P=0.048).2. The expressions of YAP in 37 cases of cancer tissues were higher than corresponding adjacent tissues in LSCC patients, in which there were 16 patients younger than 63 years old. The expressions of YAP in 7 cases of corresponding adjacent tissues were higher than cancer tissues, in which there was six patient who were older than 63. The difference was not significant (χ2=2.082, P=0.149).3. The differences between the expression of YAP in cancer tissue and corresponding adjacent tissue in the same patient was significant (t=5.239, P=0.000).4. The YAP expressions were different in different TNM classification. The YAP expression differences between T1 and T2, T2 and T3 patients were significant (P=0.033,0.045). The difference between T3 and T4 was not significant (P=0.706).5. The YAP expressions were different in different clinic stages. The YAP expression difference betweenⅠandⅡpatients was significant (P=0.045). The differences betweenⅡandⅢ,ⅢandⅣpatients were not significant (P=0.220, 0.930).6. The differences among different pathologic grades were not significant (P=0.457,0.710).7. The differences between LSCC patients with lymph node metastasis and without was not significant (t=0.427, P=0.673).8. The differences between LSCC patients with recurrence and without was not significant (t=1.178, P=0.860).9. There was not a linear relevance between YAP expression quantity and tumor sizes (r=-0.012 P=0.468).10. We detected the expression of YAP in laryngeal carcer cell line HEp-2 with Immunofluorescence, YAP expresses in both nucleus and cytoplasm. 11. With the low expression of YAP laryngeal carcer cell line HEp-2, the ratio of the G2/M phase increased from(11.232±0.231)% to (13.810±0.934)%, the difference was significant, t=5.545, P=0.005; the ratio of the S phase decreased from (20.261±0.675)% to (18.688±0.939)%, the difference was significant, t=6.484, P=0.003; the ratio of the G0/G1 phase decreased from (68.206±1.000)% to (67.502±1.488)%, the difference was not significant, t=0.876, P=0.430. The G2/M was cycle arrest after YAP was knockdown.12. The ability of migration of laryngeal carcer cell line HEp-2 decreased after YAP was knockdown, the difference was significant (t=5.378, P=0.001).13. After YAP was knockdown, laryngeal cancer cell line HEp-2 showed high sensitivity to cisplatin chemotherapy drug. The difference was significant (t=11.395, P=0.000).ConclusionThe protein YAP played different roles in men or women patients with LSCC. It participated in the occurrence and the development of LSCC, and may affect the LSCC in an early stage by controlling the G2/M and S phase in cell cycle. YAP is one of the factors can promote cancer migration in LSCC. YAP protein can decrease the sensitivity to cisplatin chemotherapy drug in laryngeal cancer cell line.But we need more correlational researches to identify whether YAP protein could be a marker on estimating the pathological grade, metastasis, recurrence and the tumor size, whether YAP protein can be a prognostic indicator and therapeutic target. |
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