Study On The Development Mechanism Of MiR-196b Targeting PCDH-17 To Regulate Human Laryngeal Squamous Cell Carcinoma

Part ?:Differential expression of miR-196 b and PCDH-17 in human laryngeal squamous cell carcinoma and correlation analysis with patient prognosisObjective:The differential expressions of miR-196 b and PCDH-17 in human laryngeal squamous cell carcinoma and adjacent normal tissues were investigated to determine their oncogenic or tumor suppressive properties,and the relationship between miR-196 b and PCDH-17 and the prognosis of patients was analyzed.Materials and Mehotds:A total of 79 cases of human laryngeal squamous cell carcinoma diagnosed by pathology and operated in our hospital from June 2010 to June 2013 were selected.The stage of lymph node metastasis(TNM)was in accordance with the AJCC/UICC 8th edition of the head and neck tumor stage standard jointly developed by the International Union for Cancer Control(UICC)and the American Joint Commission on Cancer(AJCC).The general sociological data of 79 patients were counted.Characteristics of clinicopathological data,Immunohistochemical staining was used to analyze the positive expression of PCDH-17 protein in the cancer tissues and adjacent tissues.Expression of miR-196 b and PCDH-17 m RNA in human laryngeal squamous cell carcinoma and adjacent tissues were detected by RT-PCR.The correlation between miR-196 b and PCDH-17 m RNA expression was determined.79 patients were followed up for 5 years,and survival rate was calculated.Correlation analysis was conducted between miR-196 b and PCDH-17 m RNA expression and prognosis of the patients.Results:Analysis of general clinical data:the average age of the patients was 60.58±14.25 years.Male patients accounted for the majority;Patients with laryngeal squamous cell carcinoma were associated with a higher proportion of smoking and alcohol abuse,with the lowest proportion of grade I in histological classification,while there was little difference in the proportion of grade II and III.T1 stage is the lowest,and T2~T4 stage has little difference.The TNM stage shows the same trend,among which the stage I has the lowest proportion and the stage II~IV has a small difference.m RNA expression levels of miR-196 b and PCDH-17 in laryngeal squamous cell carcinoma and adjacent tissues were detected by RT-PCR:the expression level of miR-196 b in laryngeal squamous cell carcinoma was significantly higher than that in normal adjacent tissues,showing obvious cancer-promoting characteristics.The expression of PCDH-17 was significantly lower than that of the adjacent normal tissues,showing the characteristics of cancer inhibition.Compared with the two groups,the difference was statistically significant(P<0.05).Immunohistochemical staining was used to detect the positive expression rate of PCDH-17 protein in laryngeal squamous cell carcinoma and adjacent tissues:The positive expression rate of PCDH-17 protein in carcinoma tissues was significantly lower than that in adjacent normal tissuesthe difference was statistically significant(P<0.05).The expression of miR-196 b and PCDH-17 was correlated with the clinicopathological characteristics of laryngeal squamous cell carcinoma patients:there was little difference in the expression of miR-196 b and PCDH-17 in different age groups,sex ratio,smoking history and alcoholism history,all P values were greater than 0.05,and the difference was not statistically significant.With the increase of histological grade,T stage and N stage,and TNM stage,the expression of miR-196 showed an increasing trend,and the expression of PCDH-17 showed a decreasing trend,all P values were less than 0.05,and the difference was statistically significant.Pearson analysis of the expression of miR-196 b and PCDH-17 m RNA:With the increase of Mir-196 b expression in the cancer tissues,the expression of PCDH-17 showed a downward trend.The correlation coefficient R value was 0.586,P was less than 0.001,and the difference was statistically significant.The expression of miR-196 b and PCDH-17 was correlated with the survival rate of patients with laryngeal squamous cell carcinoma:In the 5-year follow-up of 79 patients,it was found that 42 patients were still alive,with a survival rate of 53.16%.The median m RNA expression of miR-196 was 4.922 as the truncation value,and the number of patients in the miR-196 high expression group was lower than that in the miR-196 low expression group at different time periods.Statistical analysis showed thatthe difference was statistically significant(P<0.05).The median Expression of PCDH-17 m RNA was 0.228 as a truncation value,and the number of patients in the high expression group of PCDH-17 was higher than that in the low expression group of PCDH-17 at different time periods.Statistical analysis showed thatthe difference was statistically significant(P<0.05).Conclusions:In the pathological development of laryngeal squamous cell carcinoma,Mir-196 b showed the cancer-promoting characteristics,while PCDH-17 showed the anticancer characteristics.The expression of the two showed a negative correlation,and was also correlated with the clinical histological grade and TNM stage of laryngeal squamous cell carcinoma,which provided the basis for the following study.Part ?:Study on the development mechanism of miR-196 b targeting PCDH-17 to regulate human laryngeal squamous cell carcinomaObjective:To fully investigate the targeted regulation of miR-196 b on PCDH-17 in the pathological process of laryngeal squamous cell carcinoma and their regulatory role in the biological activity of laryngeal squamous cell carcinoma.Materilas and Methods:The cell lines of LARYNgeal squamous cell carcinoma Hep-2 were selected as the research object.Primary culture and third-generation monobayers were selected as the research object.The cells were transfected with Mir-196 b inhibitor,miR-NC,miR-196 b mimic and PCDH-17 to overexpress or partially silence the cells in laryngeal squamous cell carcinoma HEP-2,and the groups were set as follows:Control group(no treatment of cancer cells),miR-196 b inhibitors(partial silencing treatment of miR-196 b inhibitor in cancer cells),Mir-NC(empty plasmid of miR-196 b transfected in cancer cells),PCDH-17(pc DH-17 was transfected in cancer cells to make them overexpressed in cells),miR-196 b Mimics + PCDH-17(miR-196 b and PCDH-17 were transfected in cancer cells to make them overexpressed in cells),and mir-196 b mimics + PCDH-17 were treated in cancer cells.After the cells were cultured for 48 hours,relevant project verification analysis was carried out:dual-luciferase gene was used to detect the targeted regulation effect of miR-196 b on PCDH-17;The m RNA expression of PCDH-17 in each group was detected by RT-PCR to determine the targeted regulation of miR-196 b on PCDH-17.CCK-8 was used to detect the proliferation trend of cells in each group.Annexin V assay was used to detect apoptosis in each group.The cell migration performance of each group was detected by scratch test.The invasion property of each group was measured by Transwell method.Western-blot assay was used to analyze the expression levels of cell proliferation,apoptosis,migration and invasion of each group.Results:Target analysis of miR-196 b on PCDH-17:Data analysis using Target Scan software found that a PCDH-17 binding site was predicted in the 3 'non-coding region of miR-196b'S m RNA.Luciferase wild-type(WT-PGL3-miR-196b-3' UTR)and mutant reporter gene vector(MUTation-PGL3-miR-196b-3 'UTR)of Mir-196 b were constructed respectively,and the binding ability of miR-196 b to PCDH-17 was identified through co-transfection experiment.Different combinations of wild-type and mutated PGL3-miR-196b-UTR expression vectors were transfected into HEP-2 cells using co-transfection technology.The results showed that the activity of the wild-type expression vector reporter gene was significantly decreased compared with the control group,and the difference was statistically significant(P<0.05).In the data analysis of miR-196 b,there was a small difference in mir-196 b expression in the control group,the blank plasmid group and the PCDH-17 overexpression group,with statistically significant differences.Data in the partially silenced miR-196 b group were significantly decreased,while data in the overexpressed miR-196 b group were significantly increased(P<0.05),with statistically significant differences.From the data analysis of PCDH-17,it can be seen that the expression difference of PCDH-17 in the control group,the blank plasmid group and the co-expression group was small,and the difference was not statistically significant compared with the data of the control group.However,after miR-196 b was inhibited and PCDH-17 was overexpressed,the expression concentration of PCDH-17 was significantly increased,the difference was statistically significant(P < 0.05).CCK 8 testing each cell proliferation activity:with the extension of time,the trend of each cell proliferation were significantly increased,when the miR-196 b is restrained and PCDH-17 expression,cell proliferation trend low increase rate lower,the control group,group,and miR-196 b blank plasmid and PCDH-17 cell proliferation trend expression group was obviously promoted,P<0.05,the difference is statistically significant;Annexin V assay was used to detect apoptosis in each group:After miR-196 b was inhibited and PCDH-17 was overexpressed,apoptosis trend was significantly enhanced,the apoptosis rate was(13.20±1.17)%and(13.37±1.51)%,respectively,the difference was statistically significant(P < 0.05).The apoptosis trend of the cells in the control group,the blank plasmid group and the mir-196 a and PCDH-17 overexpression group showed little difference,which were(5.61±0.75)%,(5.49±1.01)% and(5.37±1.51)%,respectively.The difference between the two groups was not statistically significant compared with the control group.Scratch test and Transwell test:with the extension of time,cells in each group showed certain invasion and migration ability.When miR-196 b was inhibited and PCDH-17 was overexpressed,the invasion and migration ability of cells was significantly reduced,P>0.05,and the difference was not statistically significant.However,the invasion and migration ability of the cells in the control group,the blank plasmid group and the overexpression group of miR-196 a and PCDH-17 were significantly increased(P<0.05),and the difference was statistically significant.Western-blot analysis:After miR-196 b was inhibited and PCDH-17 was overexpressed,the expression levels of Survivin,Bcl-2,MMP-2 and MMP-9 proteins were decreased,the expression levels of Bax protein were increased,the proliferation,migration and invasion trends of cells were significantly decreased,and the apoptotic ability was significantly increased,P<0.05,and the differences were statistically significant.The expression levels of Survivin,Bcl-2,MMP-2 and MMP-9 protein in the control group,the blank plasmid group and the miR-196 a and PCDH-17 overexpressing groups were increased,while the expression levels of Bax protein were decreased,the proliferation activity,migration and invasion ability were significantly increased,and the apoptotic activity of the cells was decreased,P>0.05,but the difference was not statistically significant.Conclusions:In the pathological development of laryngeal squamous cell carcinoma,Mir-196 b negatively targets PCDH-17 to regulate the physiological behavior of laryngeal squamous cell carcinoma cells.Overexpression of miR-196 b can significantly inhibit the expression of PCDH-17,promote the proliferation activity,migration and invasion ability of laryngeal squamous cell carcinoma cells,and reduce the apoptosis.After miR-196 b times inhibition,the opposite trend was observed,which provided certain clinical inspirations.