The Function And Mechanism Reaserch Of MicroRNA-1225-5p In Laryngeal Squamous Carcinoma

Purpose:This study is aim to investigate the function and mechanism of MicroRNA-1225-5p(miR-1225)in laryngeal cancer(LC).Method:20 cases of laryngeal cancer were collected from our hospital,qPCR was used to detect the expression of miR-1225-5p in these clinical samples.Also,the expression of miR-1225-5p in cellular level was tested in Hep-2 cell by using realtime PCR.The overexpression and inhibition of miR-1225-5p expression was accessed by miR-1225-mimic and miR-1225-inhibitor,and MTT technique was used to test the influence of miR-1225-5p on cell multiplication,colony number in different groups was detected by clonalexpansion experiment.Cell viability was evaluated by BrdUimmunofluorescence.The function of miR-1225-5p in Hep-2 cell cycle and progression was tested by flow cytometry.The influence of miR-1225-5p on Hep-2 apoptosis was detected by Hoechst staining and AnnexinV-FITC/PI.Furthermore,qPCR and western blot were used to evaluate the expression of Bcl-2/Bax mRNA and protein.Western blot was used to detect the change of Caspase-3protein expression.The target gene of miR-1225-5p was found through bioinformatics,and CDC14B gene was confirmed to be potential target of miR-1225-5p in this research.The interreaction of miR-1225-5p with 3’UTR in CDC14B was detected by Luciferase Reporter Gene detection.Realtime PCR and western blot were used to test the expression level of CDC14B in Hep-2 cells and U20S cells.Realtime PCR and western blot were also used to detect the expression level of CDC14BmRNA and protein in the condition of overexpression and inhibition of miR-1225-5p.Co-transfection of miR-1225 inhibitor with CDC14B interference vector and co-transfection ofmiR-1225-mimic with CDC14B overexpressionvector was conducted,both of the co-transfection were conducted with Hep-2 cells.At last,western blot were used to test the expression level of CDC14B in different groups,MTT technique was used to test the change of cell multiplication,flow cytometry was used to detect cell cycle and cell apoptosis.Results:We found that in clinical laryngeal cancer,the miR-1225-5p expression level in cancer tissues was lower than adjacent tissue.In laryngeal cancer cells,miR-1225-5p expression level is significance lower than that in U20S cells.The overexpression of miR-1225-5p can lead to the inhibition of Hep-2cell proliferation,and Hep-2 cell proliferationcan be promoted by inhibiting the expression of miR-1225.The percentage of cells in G0/G1 phase can be increased by miR-1225-5p,which can decrease the percentage of cells in G2/M phase.The results of Hoechst staining and AnnexinV-FITC/PI showed that the number of dead Hep-2 cells was significant increased,when miR-1225-5p was overexpressed.When miR-1225-5p was overexpression,the expression of Bcl-2 was reduced,and the expression of Bax was improved.On the contrary,when miR-1225-5p was inhibited,the expression of Bcl-2 was improved,and the expression of Bax was reduced.At the same time,the caspase 3 activity was increased when miR-1225-5p was overexpression,and the activity of caspase 3 was decreased when miR-1225-5p was inhibited.Bioinformatics and luciferase reporter gene analysis suggested that miR-1225-5p and CDC14B 3 ’UTR could bind together and regulate the expression level of CDC14B in Hep-2 cells of laryngeal carcinoma.Thus,CDC14B might be a potential target for miR-1225-5p to regulate the malignant phenotype of laryngeal carcinoma cells.After overexpression and inhibition of miR-1225-5p,Western blot and qPCR experiments confirmed that overexpression of miR-1225-5p could inhibit the expression level of CDC14B,while interference of miR-1225-5p could up-regulate the expression level of CDC14B.Co-transfection of CDC14B interference vector with miR-1225 inhibitor,and co-transfection of CDC14B overexpression vector with miR-1225 mimic in hep-2 cells confirmed that the expression of CDC14B regulated by miR-1225-5p in hep-2 cells could reverse proliferation,cell cycle changes and apoptosis of laryngeal cancer cells casued by miR-1225-5p.Conclusion:miR-1225-5p in laryngeal cancer samplewas locatedat a lowexpression levels.Through enhancing the expression of miR-1225-5p,the proliferation and survival of laryngeal cancer cell could be reduced,and led to cell divisionstagnate at the phase of G1/S.By contraries,laryngeal cancer cellsurvival can be improved by reducing the expression of miR-1225-5p.Thus,miR-1225-5p could lead to cell divisionstagnate at the phase of Gl/S and increase the apoptosis rate of cells.miR-1225-5p could target at 3’ UTR of CDC14B,inhibit the CDC14B expression.The inhibition influence of miR-1225-5p on laryngeal cancer cell can be counteractedby restoring the expression of CDC14B gene.Our study provides direct evidence of miR-1225-5p’s function on the development of laryngeal cancer.We also investigate the mechanism of miR-1225-5p in the development of laryngeal cancer preliminarily.