LKB1 Regulates Fatty Acid Synthesis To Inhibit Migration And Invasion Of Cervical Cancer

Background Cervical cancer is one of the most common cancers among women,with about500,000 new cases and 280,000 deaths each year worldwide.In underdeveloped areas,cervical cancer ranks second in morbidity and mortality among female reproductive system tumors.Local invasion and distant metastasis of cervical cancer are the main reasons leading to poor efficacy and poor prognosis of surgical resection,radiotherapy and chemotherapy.Meanwhile,the popularity of HPV vaccine in China is not comprehensive enough,so finding new therapeutic targets to optimize the therapeutic effect of cervical cancer has become an urgent problem to be solved.As a recognized tumor suppressant gene,the mutation rate of Lkb1 in cervical cancer is as high as 20%,and the inactivation of LKB1 is significantly associated with the poor prognosis of cervical cancer.At present,most studies on LKB1 in the process of tumorigenesis and development focus on the role of LKB1 as an antitumor agent through the activation of its substrate,Adenosine Monophosphate-activated protein kinase?AMPK?.Metabolic changes of tumor include metabolic abnormalities of glucose,lipids and glutamine,among which lipid is the main storage form of cell energy and an important component of biofilm.The effect of metabolic changes on tumor progression has been widely studied in recent years.Studies have shown that the increase of fatty acid synthesis is an important metabolic phenotype of tumor cells,in which the important regulatory factor Sterol Regulatory Element binding Protein-1?SREBP1?can regulate the de novo synthesis of endogenous fatty acids,and its expression increase can be detected in pancreatic cancer,prostate cancer,hepatocellular carcinoma and other malignant tumors.However,it has not been reported whether LKB1 can affect tumor progression through regulating fatty acid synthesis in cervical cancer,and what role does SREBP1 play in it.Objectives This study aims to observe the relationship between LKB1 and the development and prognosis of cervical cancer,and to explore whether LKB1 can regulate fatty acid synthesis through SREBP1 to affect the migration and invasion of cervical cancer cells,in order to provide theoretical basis for searching new targets for the treatment of cervical cancer.Methods1.Relationship between LKB1 expression and cervical cancer?1?Immunohistochemical staining was performed on 21 cases of cervical tissues?including 5 cases of cervicitis,6 cases of cervical squamous intraepithelial lesion,and 10 cases of cervical cancer?obtained by clinical cervical conization,to observe the LKB1 in different cervical lesions Expression.?2?Used the bioinformatics website http://www.cbioportal.org to search the expression of LKB1 in cervical cancer patients in the TCGA database to explore the correlation between LKB1 expression and Overall Survival?OS?and disease-free Survival?DFS?of cervical cancer patients.?3?Cervical cancer cell line He La and the cell He La-LKB1 with He La-GFP-LKB1 plasmid stable expressed were used for cytological experiments.The effect of LKB1 expression on He La cell migration ability was observed through scratch experiment;the effect of LKB1 expression on He La cell invasion ability was observed through Transwell invasion experiment.2.The regulatory effect of LKB1 on fatty acid synthesis of cervical cancer cells?1?The effect of LKB1 expression on lipid accumulation in He La cells was detected by oil red O staining and Nile red staining;?2?To detect the activation of LKB1 expression on its downstream target protein AMPK and the effect on SREBP1 expression by Western Blot experiment;to detect the correlation between LKB1 and SREBP1 expression in cervical cancer patients by immunohistochemical staining;3.SREBP1 inhibitor inhibits cervical cancer cell migration and invasion Fatostatin,a specific inhibitor of SREBP1,was used to inhibit the expression of the active form of SREBP1 to explore the inhibitory effect of fatostatin on cervical cancer cell migration and invasion.?1?Through the CCK-8 cell proliferation-toxicity test,detect and calculate the inhibitory rate of fatostatin on He La cell proliferation ability,and the IC₅0 value;?2?Western Blot and RT-q PCR experiments were used to detect the effect of fatostatin on the expression level of SREBP1 protein and m RNA in He La cells,as well as the regulation of m RNA expression of target protein related to fatty acid synthesis downstream of SREBP1;?3?The effect of fatostatin on lipid accumulation in cervical cancer cells was detected by oil red O staining and Nile red staining;?4?Observed the effect of fatostatin on the migration and invasion of cervical cancer cells through scratch test and Transwell invasion test.Results1.Relationship between LKB1 expression and cervical cancer?1?Immunohistochemical staining results showed that compared with patients with cervicitis,the expression level of LKB1 in cervical tissue of patients with cervical cancer was significantly reduced?P <0.05?;there was no significant difference among the expression of LKB1 in cervical tissue of patients with cervical squamous intraepithelial lesion and patients with cervicitis;?2?The search results of the bioinformatics website showed that when LKB1 was mutated,the overall survival time of cervical cancer patients was significantly shortened?P = 0.0173?,and the disease-free survival time was also significantly shortened?P <0.001?.?3?The scratch test results showed that,compared with He La cells,the cell migration rate was significantly reduced with overexpression of LKB1?P <0.001?;the results of the Transwell invasion test showed that the number of He La cells with LKB1 overexpressed passed through the basement membrane was significantly decreased after 48 hours?P <0.001?.2.The regulatory effect of LKB1 on fatty acid synthesis of cervical cancer cells?1?The results of Oil Red O staining showed that the relative content of red lipid droplets accumulated in the cytoplasm of He La-LKB1 cells decreased compared with He La cells?P <0.01?;the results of Nile red staining showed that the intensity of red fluorescence in He La with LKB1 overexpressed is significantly reduced?p <0.001?.?2?Western Blot results show that overexpression of LKB1 in cervical cancer cells can phosphorylate and activate AMPK,and p AMPK expression is up-regulated,while SREBP1 expression level is significantly reduced?P <0.001?;Immunohistochemical staining results show that in cervical cancer patients when LKB1 is highly expressed in tissues,SREBP1 is lowly expressed;on the contrary,when LKB1 is lowly expressed,SREBP1 is highly expressed.3.SREBP1 inhibitor inhibits cervical cancer cell migration and invasion?1?The result of CCK-8 cell proliferation-toxicity experiment showed that with the increase of the concentration of fatostatin,the inhibition rate of He La cell viability gradually increased,and the IC₅0 value of the effect of fatostatin on He La cells was 12.56?mol;?2?Western Blot results showed that the expression of SREBP1 protein was significantly reduced?P<0.001?after 48 hour-treatment of fatostatin on He La cells;meanwhile,RT-q PCR experimental results showed that after 48 hour-treatment of fatostatin on He La cells SREBP1 and its downstream fatty acid synthesis-related enzymes FASN,ACLY m RNA expression levels were significantly reduced?P<0.001?;when using fatostatin to treat LKB1 overexpressing He La-LKB1 cells,the decrease in expression level of SREBP1 protein,m RNA levels,and FASN,ACLY m RNA were more significant?P <0.001?;?3?The results of oil red O staining showed that the relative content of red lipid droplets accumulated in the cytoplasm of He La cells was significantly reduced?P<0.001?after 48 hours of fatostatin treatment;the results of Nile red staining showed that under the same treatment conditions the red fluorescence intensity of He La cells was significantly reduced?P <0.001?;similarly,the relative content of red lipid droplets in the cytoplasm of He La-LKB1 cells overexpressing LKB1 under the condition of fatostatin was further reduced,and the red fluorescence intensity of cells was further reduced at the same time?P <0.001?;?4?The results of the scratch experiment showed that compared with the DMSO solvent control group,the migration rate of He La cells was significantly reduced?P<0.001?after 48 hours of treatment with fatostatin;the results of the Transwell invasion experiment showed that after 48 hours of treatment with fatostatin,The number of He La cells passing through the basement membrane was significantly reduced?P <0.001?;48 hours after treatment of LKB1 overexpressing He La-LKB1 cells with fatostatin,the cell migration rate and the number of cells passing through the basement membrane were further reduced?P <0.001?.Conclusions1.Lack of LKB1 expression in cervical cancer is associated with the development and poor prognosis of cervical cancer.2.LKB1 can activate AMPK through phosphorylation to inhibit the expression of SREBP1 and inhibit the ability of fatty acid synthesis in cervical cancer cells.3.Fatostatin can cooperate with LKB1 overexpression inhibit migration and invasion of cervical cancer cells by regulating fatty acid synthesis.