JQ1 Reduces Tongue Squamous Cell Carcinoma Malignancy By Suppressing PTK6 Through Regulating MiR-191-3p

Oral cancer is the sixth most common malignant tumor in the world,accounting for 3%of the malignant tumor mortality rate,and more than 90%of oral cancer is Oral Squamous Cell Carcinoma(OSCC).For the past 20 years,the 5-year survival rate of OSCC did not increase significantly,which still below 50%.Many scholars believe that studying the pathogenesis of OSCC and the mechanism of action of anti-tumor drugs at the molecular biology and molecular pathology level can provide new ideas for the treatment of OSCC.In recent years,BRD4 protein inhibitors have entered the field of vision as a relatively new anticancer drug.As a representative BRD4 protein inhibitor,JQ1 has the effect of inhibiting the activity of OSCC cells,but its mechanism of action is still unclear.MicroRNA,as a non-coding small RNA,regulates gene expression at the molecular level.Recent studies have shown that microRNAs have very important regulatory effects in various types of malignant tumors.Therefore,in order to explore the anti-cancer lmechanism of JQ1,we plan to perform high-throughput sequencing of mi-RNA in tongue squamous cell carcinoma cell line Ca127 before and after JQ1 treatment,and obtain mi-RNAs as well as their target genes with significant changes.Our research mainly including the following aspects.Chapter 1 JQ1 can inhibit the activity of Cal27 cellsOBJECTIVE:To test how sensitive Cal27 cells are to JQ1,and to determine the time suitable for further sequencing.METHODS:Cal27 cells were seeded in 6-well plates for 48 h,and 0 ?M,1 ?M,and 5 ?M doses of JQ1 were administered.CCK8 was used to detect the proliferation of tumor cells on 1st,2nd,3rd,and 4th day.The protein expression level of BRD4 was evaluated by Western-blot.RESULTS:JQ1 inhibited the cell viability of Cal27.When the concentration of JQ1 was 5 ?M,the cell viability on day 4 was 44.11%of the control group's(p<0.01).On day 4 with 5 ?M JQ1 treatment,BRD4 expression was significantly different from the control group(p<0.01).CONCLUSION:Cal27 cells treated with JQ1(5 ?M)for 4 days were selected as the experimental group for high-throughput sequence examination.Chapter 2 High-throughput sequence screens out miR-191-3pOBJECTIVE:To investigate which mi-RNAs have changed in the treatment;to detect the most significant changes;and to predict the target protein.METHODS:Cal27 cells were seeded in culture dishes.The cells in the experimental group were treated with 5?M of JQ1.The control group was not treated.After 4 days of culture,total RNA was extracted.The two sets of RNA were subjected to mi-RNA sequencing by high-throughput technology HiSeq 2500(Ribo).The target protein of the target mi-RNA was predicted by TargetScan,miRTarBase,miRWalk.RESULTS:Sequencing revealed significant differences in the expression of multiple genes between the two groups(p<0.01).The fold change of miR-191-3p expression was about 8 times higher than that of the control group,with the most significant change.Software analyses revealed target genes of mir-191-3p,and selected the gene PTK6 which was related to the tumor.CONCLUSION:MiR-191-3p was the most significantly changed miRNA,and the target protein PTK6 was selected for study.Chapter 3 Up-regulation of miR-191-3p causes a decrease in PTK6 expressionOBJECTIVE:To verify that miR-191-3p regulates the oncogene PTK6 in squamous cell carcinoma cells.METHODS:Cal27 cells were seeded in 6-well plates for 48 hours,and divided into four groups.The mimic,inhibitor,and negative controls of miR-191-3p were transfected with siRNA for 48 hours,and evaluated the protein and mRNA expression level of PTK6 by Western-blot and qRT-PCR.RESULTS:The results of Western-blot showed that the expression of PTK6 protein in mimic group was significantly lower than that in inhibitor group(p<0.01).qRT-PCR confirmed this result.CONCLUSION:Up-regulation of miR-191-3p in Cal27 cells causes a decrease in the expression of PTK6 protein.Chapter4 JQ1 acts on Ca127 cells to cause PTK6 down regulationOBJECTIVE:To investigate whether JQ1 causes a decrease in PTK6 expression.METHODS:Cal27 cells were divided into two groups,JQ1(5?M)group and control group.Proteins were extracted on the 4th day after treatment,and the expression of PTK6 protein was detected by Western-blot.RESULTS:The expression of PTK6 protein in the JQ1(1?M)and JQ1(5?M)groups were significantly lower than that in the control group(p<0.01,p<0.001).CONCLUSION:JQ1 can decrease the expression of PTK6 protein in Cal27 cells.After all above studies,it can be concluded that JQ1 can cause a large amount of miRNAs' expression to change,in particular,the up-regulation of miR-191-3p can cause a suppression of PTK6 protein;in addition,JQ1 can suppress PTK6 through various signaling pathways,so that inhibiting tumor growth.