The Mechanism Of MTSS1 Targeting HK2 Regulating Aerobic Glycolysis Affecting Breast Cancer Metastasis

BackgroundBreast cancer is a malignant tumor that seriously threatens women’s health and ranks first in the incidence of women’s cancer.Complications caused by invasion and migration are one of the main causes of death in breast cancer patients.The tumor metastasis suppressor gene MTSS1 can regulate the invasion and migration of a variety of tumor cells,but its mechanism is still unclear.Studies have shown that aerobic glycolysis of tumor cells can mediate tumor metastasis,but whether aerobic glycolysis is involved in the regulation of tumor metastasis by MTSS1 is unclear.Our previous studies have found that high expression of MTSS1 can significantly reduce aerobic glycolysis in breast cancer cells,and at the same time can cause a decrease in the activity of the key rate-limiting enzyme of aerobic glycolysis,HK2,suggesting that MTSS1 may regulate tumor aerobic glycolysis through HK2 kinase.Affect the metastasis of breast cancer.Clarify the mechanism of MTSS1 regulating cancer metastasis and provide new potential targets for the treatment of breast cancer.ObjectiveClarify the effect of MTSS1 on aerobic glycolysis of breast cancer,clarify the effect of aerobic glycolysis on MTSS1’s regulation of breast cancer cell migration and invasion,and clarify the role of HK2 in MTSS1’s regulation of aerobic glycolysis and breast cancer cell migration and invasion.To explore the interaction mechanism between MTSS1 and HK2,and provide a new direction for clinical treatment of breast cancer.Methods1.Western Blotting was used to detect the expression of MTSS1 protein in breast cancer cell lines.Screened breast cancer cell lines with low MTSS1 expression.2.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of MTSS1 mRNA in breast cancer cell lines.3.Use adenovirus transgene technology to enhance the expression of MTSS1 in breast cancer cells MDA-MB-231 and SKBR3,use RNA interference technology to reduce the expression level of MTSS1 in breast cancer cells MCF7,and use Western blotting and qRT-PCR to verify protein and mRNA levels.4.The PCR array was used to detect the effect of overexpression of MTSS1 gene on the expression of genes related to carbohydrate metabolism in breast cancer cells MDAMB-231.5.Glucose detection and lactate detection technology to detect the effect of overexpression of MTSS1 gene on the glucose uptake and lactate production of breast cancer cells MDA-MB-231 and SKBR3.6.Transwell migration test and scratch test were used to detect the effect of inhibiting aerobic glycolysis on the migration of breast cancer cells MDA-MB-231,SKBR3 and MCF7 regulated by MTSS1.Transwell invasion test was used to detect the change of cell invasion ability.7.Kinase detection technology detects the effect of overexpression of MTSS1 gene on breast cancer cells MDA-MB-231 and SKBR3 glycolysis-related kinases.8.Western Blotting and qRT-PCR were used to detect the expression of HK2 protein and mRNA in breast cancer cell lines,and the HK2 low-expressing breast cancer cell lines were screened out.9.Gene silencing technology was used to knock down the expression level of HK2 in breast cancer cells MDA-MB-231 and SKBR3 cells,and the protein and mRNA levels were verified by Western blotting and qRT-PCR.10.Glucose detection and lactate detection technology to detect the effect of knocking down HK2 gene on the glucose uptake and lactate production of breast cancer cells MDAMB-231 and SKBR3.11.Transwell migration test and scratch test were used to detect the effect of inhibiting HK2 kinase activity on the migration of breast cancer cells MDA-MB-231,SKBR3 and MCF7 regulated by MTSS1.Transwell invasion test was used to detect the change of cell invasion ability.12.The tail vein injection technique was used to establish a breast cancer metastasis model,and glycolysis inhibitors and HK2 inhibitors were given to observe the role of glycolysis and HK2 in the regulation of breast cancer liver and lung metastasis by MTSS1.13.The HE staining technique was used to stain and analyze the liver and lungs in animal experiments.14.Use immunoprecipitation technique to detect the binding of MTSS1 and HK2 protein.15.The mitochondrial isolation technique was used to detect the expression of HK2 in the cytoplasm and mitochondria of breast cancer cells after high expression of MTSS1.Results1.The expression of MTSS1 in different breast cancer cell lines: Western Blotting and qRT-PCR results showed that MTSS1 was significantly differentially expressed in 5breast cancer cell lines(P<0.05),and the higher MTSS1 expression was in MCF7 cells.The relatively low amounts are MDA-MB-231 and SKBR3 cells.Therefore,these 3 cell lines were selected for follow-up related experiments.2.Construct high-expressing cell lines MDA-MB-231-NC,MDA-MB-231-MTSS1;SKBR3-NC,SKBR3-MTSS1,and use qRT-PCR and Western Blotting methods to further verify the expression of the constructed cell line MTSS1.The results showed that the effect of overexpression of MTSS1 was obvious,and the difference was statistically significant(P<0.05).3.PCR array test results show that high expression of MTSS1 can significantly increase the expression of tricarboxylic acid cycle-related genes in breast cancer cells MDA-MB-231,and reduce the expression of glycolysis-related genes.4.The test results of the glucose uptake and lactate production test kit show that high expression of MTSS1 can significantly reduce the glucose uptake and lactate production of breast cancer cells.5.The results of the scratch healing experiment,Transwell migration experiment and invasion experiment show that inhibiting glycolysis can significantly reduce the inhibitory effect of migration and invasion caused by high expression of MTSS1.The difference was statistically significant(P<0.05).6.The results of the kinase detection kit showed that the activity of hexokinase(HK)decreased significantly after high expression of MTSS1,and the activity of other kinases(PFK,PK,LDH)did not change significantly.The difference was statistically significant(P<0.05).7.The expression of HK2 in different breast cancer cell lines: Western Blotting and q RTPCR results show that HK2 has significant differential expression in 5 breast cancer cell lines(P<0.05),and the higher expression of HK2 is MDA-MB-231 and SKBR3 cells.Therefore,these two cell lines were selected for follow-up related experiments.8.Construct the HK2 low expression cell lines MDA-MB-231-NC,MDA-MB-231-sh HK2;SKBR3-NC,SKBR3-sh HK2,and use qRT-PCR and Western Blotting methods to further verify the expression of the constructed cell line HK2.The results showed that the effect of interference with HK2 was obvious,and the difference was statistically significant(P<0.05).9.The test results of the glucose uptake and lactate production test kit showed that the NC group and sh HK2+MTSS1 group had significantly higher glucose uptake and lactate production compared with the HK2 knockdown group alone.The results show that knocking down HK2 can significantly reduce the inhibitory effect of MTSS1 on the glycolysis of breast cancer cells.The difference was statistically significant(P<0.05).10.The results of the scratch healing experiment and the Transwell migration experiment show that knocking out HK2 can significantly reduce the inhibition of tumor migration caused by high expression of MTSS1,and further using Transwell to detect cell invasion ability and found that knocking out HK2 can significantly reduce the inhibition of tumor migration caused by high expression of MTSS1 inhibition of invasion.The difference was statistically significant(P<0.05).11.The experimental results of the nude mouse breast cancer metastasis model showed that the breast cancer cells had obvious metastasis in the lungs,but the metastasis in the liver was not obvious.HE results showed that compared with the high-expression MTSS1 group alone,the combined glycolysis inhibitor 3-BP group could significantly reduce the inhibition of metastasis caused by high-expression MTSS1.12.The results of co-immunoprecipitation showed that MTSS1 and HK2 protein had obvious binding.13.The results of mitochondrial isolation showed that high expression of MTSS1 can reduce HK2 targeted by mitochondria,suggesting that MTSS1 may inhibit aerobic glycolysis by reducing the level of mitochondrial-bound HK2.ConclusionsMTSS1 regulates breast cancer aerobic glycolysis and metastasis by affecting HK2 kinase activity;MTSS1 inhibits breast cancer aerobic glycolysis by competitively binding to mitochondrial targeted HK2;inhibiting aerobic glycolysis can significantly reduce the effects of MTSS1 on breast cancer Inhibition of cell migration and invasion and breast cancer metastasis.