Restraining The Gene Expression Of ACSS2/ACLY Enhances Sensitivity To LM-2I In Tongue Squamous Cell Carcinoma

Objective Oral squamous cell carcinoma is one of the common malignant tumors in the head and neck.It is mainly tongue squamous cell carcinoma.It has the characteristics of high recurrence,high metastasis and poor prognosis.The 5-year survival rate is no more than 50%.At present,there is still a lack of effective chemotherapeutic drugs for tongue squamous cell carcinoma.Therefore,it is urgent to find high-efficiency and low toxicity therapeutic drugs for tongue squamous cell carcinoma and explore its mechanism.LM-2I is a derivative based on spinosad a（SPA）.As an anti-tumor candidate drug,it has the advantages of high activity,good water solubility and low toxicity to mammals.Therefore,it is of great significance to further explore the effects of spa and LM-2I on tongue squamous cell carcinoma cells.Based on RNA transcriptome sequencing technology,the differential gene expression profiles of tongue squamous cell carcinoma cells treated with spa and LM-2I were screened.It was found that the key molecules catalyzing the synthesis of acetyl coenzyme A-acetyl coenzyme A synthase 2（ACSS2）and ATP citrate lyase（ACLY）were significantly up-regulated.ACSS2 and ACLY catalysed acetate and citric acid to produce acetyl coenzyme A,respectively,and jointly participated in the regulation of tumor stress metabolism.We speculate that the up-regulated expression of ACSS2 and ACLY may be the key molecules affecting the sensitivity of tongue squamous cell carcinoma cells to LM-2I.Therefore,this study intends to explore the relationship between acss2 and acly gene expression and LM-2I sensitivity,so as to provide new potential targets and strategies for the clinical treatment of tongue squamous cell carcinoma.Methods 1)the IC₅₀ values of SPA and LM-2I on tongue squamous cell carcinoma cell lines SCC15 and CAL27 were measured by MTT assay;To investigate the inhibitory effect of SPA and LM-2I on the colony proliferation of tongue squamous cell carcinoma cells.2)Transcriptome sequencing was used to screen the differential m RNA expression profiles in SPA and LM-2I treated tongue squamous cell carcinoma cell line SCC15,and q RT-PCR and Western blot were used to further verify and screen the key differentially expressed genes（ACSS2/ACLY）.3)The relationship between the protein expression of ACSS2 and ACLY and TNM stage in50 patients with tongue cancer and its adjacent tissues was analyzed by immunohistochemistry.4)MTT assay was used to analyze the effect of LM-2I combined with ACSS2 inhibitor on the proliferation of tongue squamous cell carcinoma cells.5)To construct a stable tongue squamous cell carcinoma cell line with ACSS2 overexpression and establish a tongue squamous cell carcinoma cell line with ACSS2 or ACLY gene knockout by CRISPR/Cas9 technology,and to explore the role of ACSS2/ACLY gene expression in LM-2I treatment of tongue squamous cell carcinoma.Results 1)MTT assay and plate cloning experiment showed that SPA and LM-2I inhibited the proliferation of tongue squamous cell carcinoma cells in a concentration dependent manner,and the inhibitory effect of LM-2I was significantly better than that of SPA.2)The differential expression profile analysis of scc15 cell line treated with SPA and LM-2I showed that the bioinformatics KEGG signal pathway analysis showed that the differentially expressed genes were mainly enriched in the metabolic pathway.The differentially expressed genes of SPA and LM-2I were intersected,and some differentially expressed genes were verified by q RT-PCR.At the same time,the key molecules ACSS2 and ACLY of LM-2I regulating the stress metabolism of tongue cancer were screened in combination with Western blot analysis,However,there was no significant difference in the expression of MDA-MB-231 and ACSS2/ACLY in LM-2I sensitive breast cancer cell lines.3)Immunohistochemical analysis showed that ACSS2/ACLY was highly expressed in tongue squamous cell carcinoma compared with adjacent tissues;The expression of ACLY was significantly correlated with T stage（P<0.05）,but not with N stage and TNM stage.There was no significant difference between the expression of ACSS2 and T stage,N stage and TNM stage.4)MTT analysis showed that the combination of LM-2I and ACSS2 inhibitor could enhance the sensitivity of LM-2I to tongue squamous cell carcinoma.5)The results of Western blot showed that ACSS2 overexpression and ACSS2/ACLY knockout tongue cancer cell lines were successfully constructed.Compared with the control group,ACSS2 overexpression tongue cancer cell lines and ACSS2 overexpression tongue cancer cell lines treated with LM-2I had no significant inhibitory effect on the proliferation of tongue cancer cells.ACSS2/ACLY knockout tongue cancer cell line combined with LM-2I could significantly enhance the sensitivity of tongue squamous cell carcinoma cells to LM-2I,and the IC₅₀value of tongue squamous cell carcinoma cells treated with LM-2I decreased.Conclusion:1)SPA and LM-2I significantly inhibited the proliferation of tongue squamous cell carcinoma cells in a concentration dependent manner.2)The use of ACSS2 inhibitor and ACSS2/ACLY gene knockout can enhance the sensitivity of tongue squamous cell carcinoma cells to LM-2I.ACSS2 and ACLY are key molecules regulating stress metabolism of tongue squamous cell carcinoma.3)The expression of ACSS2 and ACLY in tongue squamous cell carcinoma was significantly higher than that in adjacent tissues.The high expression of ACLY was correlated with T-stage of tongue cancer patients.