MiR-320a Targets And Regulates MRP2 To Inhibit The Biological Function And Mechanism Of Tongue Cancer Cells

Objective:1.MiRNA microarray technology was used to screen serum miRNA in patients with tongue squamous cell carcinoma and normal controls.2.Bioinformatics technology was used to analyze differentially expressed miRNA and predict its target genes.Methods :1.The expression level of miR-320 a in the serum of 48 cases of tongue squamous cell carcinoma and 50 healthy volunteers was detected by tissue microarray.The data of microarray were confirmed by qRT-PCR.2.The relevant bioinformatics technology is used to conduct a detailed discussion on the differentially expressed miRNAs 320 a and identify the target genes therein to perform prediction work.Results:1.A total of 21 miRNAs abnormalities were detected by microarray,and miR-320 a expression was significantly decreased,P < 0.05.In order to confirm the discovery of microarray,4 miRNAs with significant abnormal expression in OTSCC samples and healthy people,including miR-320 a,miR-129,and miR-106 a and miR-31,were all tested by qRT-PCR,and the expression trend was consistent with the discovery of microarray,P < 0.05.2.In situ hybridization and immunohistochemistry showed that the expression level of miR-320 a in tongue cancer tissues was significantly lower than that of miR-320 a in normal tongue tissues,P<0.01,while the expression level of MRP2 in tongue cancer tissues was significantly higher than that in normal tongue tissues P<0.01.Conclusion:The expression level of miR-320 a in the serum of patients with tongue cancer was significantly lower than that in normal human serum,and the expression level in the tissue of tongue cancer was also lower than that in normal tissues.However,the level of expression of MRP2 is increased.It is suggested that the abnormal reduction of miR-320 a expression and the increase of MRP2 can be used as reference indicators for the diagnosis of squamous cell carcinoma of the tongue.Objective:1.To observe the effect of exogenous miR-320 a on proliferation,migration and invasion of SCC cell line.2.To verify whether MRP2 is a natural target gene of miR-320 a in SCC cell lines,and explore the molecular mechanism of mi R-320 a reducing MRP2 expression.3.To further observe whether MRP2 can effectively reverse the inhibitory effect of mi R-320 a on the proliferation of tongue squamous cell carcinoma cells.Methods:1、The content of mi R-320 a in different cell lines and normal keratinocytes was detected by q RT-PCR using human SCC cell line as the research object.2、The effects of mi R-320 amimics on the proliferation,migration and invasion of SCC cells were observed by CCK8,clone formation,Edu,Transwell in vitro migration and invasion experiments,and cell scratch test by transfection of mi R-320 a simulated double stranded oligonucleotide(mimics).3、QRT-PCR and Western blot were used to detect the effect of transfected mi R-320 a on the expression of related MRP2 m RNA and protein in the abovementioned cell lines,and to explore the molecular mechanism of mi R-320 a to reduce the expression of MRP2;and the luciferase test was used to verify that MRP2 was a natural target gene for mi R-320 a.4、The eukaryotic expression plasmids of MRP2 were constructed to mediate the overexpression of the corresponding protein in the SCC cell line.Edu test showed that the expression of MRP2 could effectively reverse the inhibitory effect of mi R-320 a on the proliferation of squamous cell carcinoma of the tongue.Results:1、QRT-PCR showed that the level of mi R-320 a in the normal oral keratinocyte line was significantly higher than that of the tongue squamous cell carcinoma cell line,and the difference between them has a certain statistical value,P<0.001.2 、 CCK8 test results showed that the proliferation of SCC cells was significantly inhibited in group mi R-320 a,and the 450 nm absorption value was significantly lower than that in NC group,P<0.01.After two weeks in vitro culture,there were cell clones in both group NC and mi R-320 a group in SCC cell line.Compared with group NC,the clones formed in group mi R-320 a were smaller and dyed lighter,and the cloning formation efficiency of mi R-320 a group(CFE)was significantly lower than that in NC group,and the difference between them has a certain statistical value,P< 0.01.3、In vitro scratch migration test showed that the clearance rate of SCC in mi R-320 a group(relative to 0h%)was significantly higher than that in group NC,P<0.001.The results of Transwell in vitro migration showed that the number of cells passing through the cellulose membrane holes in the SCC mi R-320 a group was significantly less than that in the corresponding NC group,and the difference between them has a certain statistical value,P < 0.001.The results of the invasion test in vitro showed that the number of cells passing through the matrix glue(Matrigel)in the SCCmi R-320 a group was less than t that of the NC group,and the difference between them has a certain statistical value,P < 0.001.4、QRT-PCR test showed that the MRP2 content of SCC cell line mi R-320 a group was significantly lower than that of Scr group,the difference between them has a certain statistical value,P<0.05.The expression level of MRP2 protein in the SCC cell line mi R-320 a group was significantly lower than that of the NC group.The statistical analysis of the gray value of the Gelpro measurement showed that the difference was significant,P<0.05.5 、 The luciferase test of mi R-320 a target MRP23 ’UTR showed that the luciferase activity of mi R-320a-WT group group,and the difference between them has a certain statistical value,P<0.001,but there was no statistical difference between the Mock-WT group and NC-WT group,P>0.05;at the same time,differences in three mutant plasmids transfected group(mi R-320a-MT,Mock-MT and NC-MT)were not statistically significant,P>0.05.6、The CCK-8 test showed that the 450 nm absorbency of the three cell line 320a+MRP2 group(co transfected mi R-320 a mimics and MRP2 eukaryotic expression plasmid)had no significant difference between the observation time point and the NC group,P>0.05,and the 48 h was significantly higher than the mi R-320 a group after the transfection,and the difference was statistically significant,P<0.001.In the Edu experiment,the rate of Edu positive cells in the SCC cell line NC group was significantly higher than that in the mi R-320 a group,and the difference between them has a certain statistical value,P<0.001.The positive rate of Edu in the 320a+MRP2 group was higher than that in the mi R-320 a group,and the difference between them has a certain statistical value,P<0.01~0.001.Conclusion:1.The level of mi R-320 a in tongue squamous cell carcinoma cell line was significantly lower than that in oral keratinocyte line.2.Exogenous mi R-320 can effectively inhibit the ability of proliferation,migration and invasion.3.Exogenous miR-320 a can effectively induce the degradation of MRP2 mRNA and reduce the expression of MRP2 protein.4.MRP2 is a natural target gene of mi R-320 a.Mi R-320 a can reduce the expression level of MRP2 by binding to the binding site of m RNA 3 ’UTR region.5.Overexpression of MRP2 can effectively reverse the inhibitory effect of miR-320 a on the proliferation of tongue squamous cell carcinoma cells,and MRP2 can inhibit the inhibitory effect of mi R-320 a on the proliferation of squamous cell carcinoma of the tongue to a certain extent.