The Role Of Hsa-mir-143-3p And Its Target Gene MAGE-A9 On Biological Behaviour For Laryngeal Squamous Cell Carcinoma

Expression and prognostic value of MAGE-A9 in laryngeal squamous cell carcinomaMelanoma-associated antigen(MAGE) family genes have been reported to play important roles in the development of human cancers. However, the relationship between expression of MAGE-A9 and clinicopathological characteristics in human laryngeal carcinoma is not well defined. This study aimed to examine the expression level of MAGE-A9, and to evaluate the clinical significance of its expression in human laryngeal squamous cell carcinoma(LSCC).Methods: Quantitative real-time reverse transcription-PCR(q PCR) and immunohistochemistry(IHC) were performed to characterize the expression of MAGE-A9 in LSCC tissues and tumor-adjacent normal tissues. Kaplan-Meier survival and Cox regression analyses were carried out to evaluate the prognosis of LSCC.Results: The expression of MAGE-A9 in LSCC was significantly higher than that in tumor-adjacent normal tissues. The cytoplasm expression of MAGE-A9 was detected in 70 of 123(56.9%) LSCC tissue. The expression level of MAGE-A9 in LSCC was related to histopathological grade(P=0.024). Kaplan-Meier survival and Cox regression analysis revealed that MAGE-A9 expression level and lymph node metastasis were independent prognostic factors of LSCC, respectively(P=0.005; P=0.001).Conclusions: The study suggests that MAGE-A9 expression is a prognostic biomarker for LSCC patients. High expression level of MAGE-A9 indicates unfavorable survival outcomes in LSCC patients.The role of hsa-mir-143-3p and its target gene MAGE-A9 on biological behaviour for laryngeal squamous cell carcinoma ObjectiveTo investigate the expression and function of hsa-mir-143-3p in laryngeal squamous cell carcinoma. We also aimed to the interaction between hsa-mir-143-3p and its predicted target gene MAGE-A9, which could be a key role in the genesis of laryngeal squamous cell carcinoma.Materials and Methods: The expression of MAGE-A9 in laryngeal TU212 and TU686 cell lines were detected by Western blot and q PCR. we selected cell lines in TU686. After transfected with hsa-mir-31-5p, hsa-mir-124-3p, hsa-mir-138-5p, hsa-mir-143-3p or cel-mir-67, TU686 cell line, with a higher expression of MAGE-A9, was collected for MAGE-A9 expression detection by Western Blot and q PCR. The expression of MAGEA9 was inhibited in all groups, with most significantly inhibited by Hsa-mir-143-3p. Bioinformatics and molecular biology techniques were introduced to predict and construct carrying hsa-mi R-143-3p mimics the MAGE-A9 3’UTR specifically binding reporter vector. Dual-luciferase reporter gene experiments was used to clarify the relationship of hsa-mir-143-3p and MAGEA9 gene. MTT assay, cell invasion and cell scratch test were introduce to detect cell proliferation, apoptosis and invasion and metastasis of tumor cell transfected by hsa-mir-143-3p. A laryngeal squamous cell carcinoma xenograft model in nude mice was founded to observe the the tumor′s weight and volume. Fluorescence quantitative RT-PCR, Western blot and immunohistochemistry were used to detect m RNA and protein of MAGE-A9. Tumor tissues apoptosis was detected with TUNEL staining. The laryngeal cancer sample were collected in 15 different patient. The expression of hsa-mir-143-3p and MAGE-A9 in near the tumor and cancer tissues were tested by fluorescence quantitative q RT-PCR and immunohistochemistry, in order to verify the negative feedback relationship between hsa-mir-143-3p and the MAGE-A9Results: 1.To detected MAGE-A9 expression after transfection by Western Blot and q PCR. Hsa-mir-143-3p inhibition of gene expression MAGEA9.is the best. The difference was statistically significant(P < 0.05).2. Gene sequencing results showed that the p GL3-wt3’UTR and hsa-mi R-143-3p mimics co-transfection significantly reduced luciferase activity(P <0.001), in addition, the mutant plasmid p GL3-mu3’UTR with p GL3-control cotransfection not change luciferase activity, no significant effect(p> 0.05).3. MTT results suggest that the ability to inhibit cell proliferation was significantly higher than other group after transfection of hsa-mir-143-3p. The difference was statistically significant(p <0.05). In vitro cell invasion assay show, hsa-mir-143 cells was significantly lower than the control group and cel-mir-67, the difference was statistically significant(P <0.05), while the control group and the cel-mir-67 group of no significant difference between the number of cells, suggesting that the expression of hsa-mir-143 could significantly reduce invasion of laryngeal TU686 cells. Cell scratch test indicated that hsa-mir-143 cells slow healing scratches, no healing. Shows the construction of hsa-mir-143 effectively inhibited cell migration, the difference was statistically significant(P <0.05).4, hsa-mir-143-3p-TU686 and TU686 group nude mice, there are differences cel-mir-67-TU686 tumor weight(P <0.05).Tumor volume of hsa-mir-143-3p-TU686 group,TU686 group, cel-mir-67-TU686 group were significant differences after 18 days there(P <0.05) hsa-mir-143-3p-TU686 group tumor mass expression of MAGE-A9 decreased(P <0.05). hsa-mir-143-3p-TU686 tumor tissue apoptosis is significant.5.The expression of hsa-mir-143-3p in human laryngeal carcinoma than is lower than in adjacent tissues. The expression of hsa-mir-143-3p target gene MAGE-A9 in human laryngeal carcinoma is higher than adjacent tissues.Conclusion:. 1.Hsa-mir-143-3p play a tumor suppressor role in laryngeal carcinoma, MAGE-A9 is one of its target genes. 2. Hsa-mir-143-3p inhibites the expression of MAGE-A9 protein,thus inhibites proliferation and invasion capacity of the cells.3. A method with stabilize and effective reduction of hsa-mir-143-3p expression for the biological treatment of laryngeal squamous cell carcinoma needs further study.