The Effects Of Inhibition Of IL1B On The Function Of Tongue Cancer Cell Lines

Due to the unique histological features of the tongue,which is rich in lymphatic circulation and has highly muscular structure,making tongue cancer which is the most common type of oral cancer more prone to local invasion and distant metastasis than other types of oral cancer,which is also an important cause of poor prognosis of tongue cancer.Despite the medical technology has been greatly improved in the past20 years,the survival rate of tongue cancer has only increased by 5%,in the meantime,the 5-year survival rate of tongue cancer remains at around 60%^[1].Therefore,it is of long-term and profound significance to study the molecular mechanisms involved in the growth and metastasis of tongue cancer and to develop new treatments based on these molecular mechanisms.IL-1βwhich is an important inflammatory mediator,has been conformed to participate in many cellular activities,such as proliferation,differentiation and apoptosis^[2].In recent years,a number of studies have emphasized the importance of IL-1βon tumors,however,rare study has been taken to investigate its role in tongue cancer.Therefore,our study aims to make an exploration of its gene IL1B in tongue cancer and its effect on the function of tongue cancer cells.ObjectiveThe purpose of this study is to make an inquiry of IL1B expression in tongue cancer and figure out whether IL1B has an effect on the biological function of tongue cancer cell lines.MethodPart I:The RNA-seq measure of tongue cancer tissue and its adjacent normal tissues in 5 patients with tongue cancer were taken to observe whether IL1B was differentially expressed in tongue cancer tissues.In order to further clarify the reliability of the sequencing results,we collected the tongue cancer tissues and adjacent tissues of 58 patients for qRT-PCR to detect the expression of IL1B mRNA.In addition,we also analyzed the expression of IL1B in head neck squamous cell carcinoma,oral squamous cell carcinoma and tongue cancer relative to normal tissues through TCGA and Oncomine databases.Part II:After designing IL1B siRNA,we transfected it into CAL27 and SCC9tongue cancer cell lines,using qRT-PCR and Western blot to verify the knockdown of IL1B at mRNA and protein level after transfection of siRNA respectively.ELISA was taken to detect IL-1βprotein secreted by CAL27 into the culture supernatant after transfection of siRNA.Thereby two effective siRNAs were selected.Then we studied whether IL1B had an effect on the proliferation ability of two tongue cancer cell lines after knocking it down by conducting CCK8 proliferation assay and plate cloning assay.The effect of inhibition of this gene on the migration and invasion ability of CAL27 and SCC9 tongue cancer cell lines was studied by invasion chamber experiments.ResultPart I:5 cases of tongue cancer tissue and its adjacent normal tissues were detected by RNA-seq,showing that compared to adjacent normal tissues,IL1B is highly expressed in tongue cancer tissues.Follow-up detection of IL1B in 58 cases of tongue cancer and its adjacent normal tissues also showed that IL1B is highly expressed in tongue cancer tissues.TCGA database analysis showed that IL1B is abnormally highly expressed in head neck squamous cell carcinoma,and high expression of IL1B may indicate a shorter overall survival of patients;Oncomine database analysis also showed IL1B is highly expressed in in oral cancer and tongue cancer tissues.Part II:After transfection with IL1B siRNA into CAL27 and SCC9 tongue cancer cells,qRT-PCR and Western blot results showed that siRNA-489 and siRNA-853 could knock down IL1B at mRNA and protein levels,and ELISA results showed that the concentration of IL-1βsecreted by CAL27 cells into the culture supernatant was lowered.CCK8 proliferation assay and plate cloning assay were performed using two valid siRNAs screened,showing that IL1B knockdown could reduce the proliferation ability of two tongue cancer cells.Invasive chamber experiments were performed after IL1B siRNA transfection,showing that knocking IL1B down could reduce the migration and invasion ability of two tongue cancer cells.ConclusionTaking all experimental results above together,we can come to a conclusion that IL1B is overexpressed in tongue cancer tissues,and IL1B knockdown can significantly reduce the ability of proliferation,migration and invasion of tongue cancer cells,indicating that IL1B is an oncogene in tongue cancer,which may play an important role in the growth and metastasis of tongue cancer,while the specific mechanism needs further research.