| Objective:Hypopharyngeal carcinoma is a major malignancy in the hypopharyngeal region accounting for 1.4% to 5.0% of all head and neck malignancies and accounting for 0.5% of systemic malignancies.It is characterized by more hidden parts of the disease,patients with poor prognosis.Hypopharyngeal squamous cell carcinoma(HSCC)has the characteristics of local aggressive growth and submucosal infiltration,and is also susceptible to lymph node metastasis and distant metastasis.Hypopharyngeal squamous cell carcinoma(HSCC)has the characteristics of local aggressive growth and submucosal infiltration,and is also susceptible to lymph node metastasis and distant metastasis.There are 50% of cases,having cervical lymph node metastasis symptom at the time of treatment.Therefore,in the treatment of hypopharyngeal cancer,in order to achieve early detection and early detection of early treatment and improve patient survival,we urgently need to explore the molecular basis of hypopharyngeal cancer,find its development,invasion and metastasis of the mechanism to find Targeted therapies for hypopharyngeal cancer,these are of great significance.Micro-RNAs(miRNAs)are a class of non-protein-encoding RNAs that are widely found in eukaryotic organisms and highly conserved sequences that regulate gene expression.Research shows that microRNAs can act as oncogenes and / or tumor suppressor genes.MicroRNAs can coordinate the complex network of intra-tumor molecular proteins by simultaneously targeting several m RNAs.In the head and neck cancer system,miRNAs are differentially expressed in oral squamous cell carcinoma,tongue squamous cell carcinoma,laryngeal squamous cell carcinoma and nasopharyngeal carcinoma.However,compared to other head and neck cancers,the research on miRNAs for hypopharyngeal carcinoma is rare.If we can find the representative miRNAs and establish the molecular mechanism of hypopharyngeal cancer,we will make an important contribution to the study of mechanisms such as invasion and metastasis of hypopharyngeal carcinoma.Materials and Methods: H-E staining confirmed the tissue samples,immunohistochemical detection of hypopharyngeal carcinoma SMURF1 positive expression.Bioinformatics prediction website predicts the targeted relationship between SMURF1 and mi R-194-5p.Dual luciferase reporter gene experiments confirmed.Real-Time PCR and western blot were used to detect the expression of miR-194-5p and SMURF1 in hypopharyngeal carcinoma.Human pharyngeal squamous carcinoma cell line FaDu cell culture,observation and passage.mi R-194-5p mimics and negative control,miR-194-5p inhibitor and negative control transfected FaDu cells.The changes of cell proliferation and invasion in each transfected group were detected by CCK-8 and Transwell.Real-Time PCR and Western blot were used to detect the expression of SMURF1 mRNA and protein in each transfected group.After adding rapamycin,a specific inhibitor of mTOR signaling pathway,the changes of mTOR and p-mTOR expression were observed.The changes of proliferation and invasion ability of FaDu cells in each group were detected by CCK-8 and Transwell.siRNA interference transient silencing of SMURF1,observe the p-mTOR protein expression changes.All data were expressed as means ± standard deviation(MEAN ± STDEV).SPSS17.0 was used for statistical analysis.The independent sample t-test and paired t-test were used for comparative analysis between groups.The correlation between miR-194-5p and clinicopathological features using ?2 test,the comparison between miR-194-5p and SMURF1 using Spearman correlation analysis,* P<0.05,** P<0.01 represents a significant difference.Experimental results:1.Hypochondrogenic clinical specimens of H-E stainingH-E staining,hypopharyngeal cancer tissue cells arranged in disorder,the proportion of large nuclear staining,the nest can be seen in epithelial cells,the formation of squamous cell mass.2.Expression of miR-194-5p in hypopharyngeal carcinoma tissue specimensReal-time PCR assay confirmed that the expression of miR-194-5p was down-regulated in cancer tissues compared with the adjacent normal tissues.There was no significant correlation between miR-194-5p expression and age and gender.But correlated with the stage of primary tumor(T)and the presence and extent of regional lymph node metastasis(N).3.Effect of miR-194-5p on Proliferation and Invasion of Fa Du CellsAfter miR-194-5p expression was up-regulated,the proliferation ability of FaDu cells was decreased and the number of invasive cells was decreased.After miR-194-5p expression was down-regulated,the proliferation ability of FaDu cells increased and the number of invasive cells increased,indicating that miR-194-5p negative Regulates proliferation and invasion of FaDu cells.4.Expression of SMURF1 in hypopharyngeal carcinoma tissue specimensImmunohistochemical staining of the specimens found that the positive rate of SMURF1 in cancerous tissues was higher than that in adjacent tissues.Real-time PCR and Western Blot also confirmed that SMURF1 mRNA and protein levels were up-regulated in cancerous tissues and down-regulated in normal tissues.Statistical analysis was negatively correlated with the expression of miR-194-5p.5.Dual luciferase reporter gene detection of miR-194-5p has a targeted relationship with SMURF1SMURF1-wtUTR and hsa-miR-194-5p mimic co-transfected FaDu cells,compared with the control group,significantly decreased fluorescence activity,SMURF1 and miR-194-5p targeted binding.6.Change of SMURF1 expression after overexpression or low expression of miR-194-5pThe expression of SMURF1 was inhibited in miR-194-5 pmimic group and upregulated in miR-194-5 pinhbitor group.7.The expression of mTOR and p-m TOR protein in each transfection group after adding rapamycinmiR-194-5p mimic + rapamycin significantly reduced the expression of p-mTOR,indicating that overexpression of miR-194-5p down-regulated p-mTOR,and inhibitor rapamycin has synergistic effect.miR-194-5p inhbitor + rapamycin group p-mTOR content increased significantly,although not up to miR-194-5p inhbitor group level,but compared with rapamycin alone group increased significantly,indicating that The low expression of miR-194-5p sensitively down-regulates the level of p-mTOR.The addition of a specific inhibitor,rapamycin,partially attenuates some of the effects.The reverse side proves that the mTOR pathway is regulated by miR-194-5p Regulation.8.Effect of miR-194-5p on the Proliferation and Invasion of FaDu Cells in Hypopharyngeal Carcinoma Induced by mTOR Signal PathwayOver expression of miR-194-5p and inhibition of mTOR signaling pathway have a synergistic effect,inhibition of FaDu cell proliferation and invasion than mimic alone transfected alone or more inhibitors.The proliferation ability of FaDu cells in miR-194-5p inhbitor + rapamycin group was significantly higher than that of rapamycin alone group,but it did not reach the level of miR-194-5pinhbitor,indicating that rapamycin could inhibit mTOR pathway Is not enough to completely antagonize the down-regulation of mTOR signaling pathway induced by miR-194-5p downregulation on the proliferation and invasion of FaDu cells.9.FaDu cells transfected SMURF1 siRNA,p-mTOR expression changesSilencing SMURF1,the expression of p-mTOR was significantly reduced,demonstrating that the down-regulation of SMURF1 resulted in the decrease of p-mTOR protein expression and the inhibition of mTOR signaling pathway.Conclusion:1.Hypopharyngeal carcinoma tissues and cells in low expression of mi R-194-5p,its target protein SMURF1 high expression.2.miR-194-5p targeted binding to SMURF1,when miR-194-5p overexpression,SMURF1 downregulation,cancer cell proliferation and invasion were inhibited.3.miR-194-5p targets SMURF1 to regulate mTOR signaling pathway.When miR-194-5p is downregulated,SMURF1 is upregulated and mTOR signaling is activated.On the contrary,miR-194-5p is highly expressed and SMURF1 is downregulated and mTOR signaling is inhibited.This study is to explore hypopharyngeal cancer-specific tumor markers,looking for reliable targeted therapy to provide new ideas. |
PDF Full Text Request