BTN3A3 Regulates Glucolytic Metabolism And Growth Metastasis Of Cervical Cancer

Part 1 Expression and clinical significance of BTN3A3 protein in cervical cancerObjective:To investigate the expression and clinical significance of BTN3A3 protein in human cervical cancer.Methods:Immunohistochemistry was used to detect the expression of BTN3A3 protein in 55 normal cervical tissues and 69 cervical cancer tissues(including 34 squamous cell carcinoma,30 adenocarcinoma and 5 adenosquamous carcinoma).The positive expression rate of BTN3A3 protein in cervical cancer patients with different clinicopathological characteristics was compared,and the relationship between BTN3 A3 protein positive expression and pathological characteristics was analyzed.Results:The positive expression rate of BTN3A3 protein in cervical cancer tissues was higher than that in normal cervical tissues[18.2%(10/55)](P<0.05),and the high expression rate of BTN3A3 protein in patients with stage Ⅲ and vascular cancer thrombus[88.9%(16/18),71.9%(23/32)](all P<0.05).Conclusions:BTN3A3 protein is highly expressed in cervical cancer tissues,which is related to the stage of patients and vascular tumor thrombus,but has no obvious relationship with age,histological type,differentiation degree and HPV infection.Part 2 Effect of regulating BTN3A3 gene expression on biological behavior of cervical cancer cell line in vitroObjective:The siRNA of BTN3A3 gene was synthesized and transfected into Hela cells to observe the effect of knocking down the gene on the physiological function of cervical cancer cells.Methods:According to the mRNA sequence of BTN3A3 gene,the siRNA fragment was designed and synthesized and transfected into Hela cells by liposome method.western blot and real-time quantitative PCR were used to detect the gene interference effect,and then the growth curve,clone formation,scratch test and Transwell test were used to detect the changes of cell growth,proliferation,migration and invasion ability.Results:The silencing efficiency of BTN3A3 gene detected by RT-PCR was about 68%.Further cell function experiments showed that the growth rate of cells in the interference group,namely si BTN3A3 group,lagged behind that in NC group from the second day(P<0.05).Compared with the control group(258 ±24.64),the number of cell clones in si BTN3A3 group(120.3 ± 13.37)decreased significantly(P<0.05).In the cell scratch experiment,the change of migration area in the interference group was only about 50%of that in the control group;The number of cells passing through matrix glue and membrane in interference group was(200.3 ± 10.65),which was less than that in control group(137.7 ±10.27)(P<0.05).Conclusions:BTN3A3 can promote the growth,proliferation,migration and invasion of cervical cancer HeLa cells.Part 3 BTN3 A3 affects the physiological function of cervical cancer by regulating glycolytic pathwayObjective:1.Screening proteins that may interact with BTN3A3 protein;2.The interaction between BTN3 A3 and LDHA,the key enzyme of glycolysis,was verified,and BTN3A3 affected the physiological function of cervical cancer cells through glycolytic pathway.Methods:1.Design primers according to BTN3A3 gene and amplify the full length of BTN3A3 gene.Use Xhol I and Hind III to double-digest the gene amplification product and Myc empty vector;The recombinant plasmid MycBTN3A3 was constructed by connecting the double-digested btn3a3 and Myc vector with T4-ligase,and transfected HeLa cells with Myc empty vector respectively.After 48 hours,the cells were collected,and a small number of samples were identified by Western blot.After the plasmid transfection was successful,the remaining samples were enriched with Myc beads.Finally,the same amount of 2× SDS was added,and the samples were run with electrophoresis protein gel,and the protein gel was developed by silver staining.2.BTN3A3 and LDHA with different tags were co-transferred in HeLa cells,and the interaction between BTN3A3 and the selected LDHA protein was verified by immunoprecipitation method.3.The localization of BTN3A3 and LDHA in cells was observed by immunofluorescence.4.After interfering with BTN3A3 gene expression in HeLa cells,glucose uptake,lactic acid and APT levels were detected by using related detection kits.Results:Myc-BTN3A3 was successfully expressed in transfected 293T cells,and the sequencing comparison was correct.After cutting off the differential bands on the silver-dyed protein gel,mass spectrometry was performed to screen out hundreds of proteins that may interact with BTN3A3,including LDHA.In immunoprecipitation experiment,Myc beads were used to aggregate BTN3A3 protein with Myc tag,and Flag antibody detection showed that LDHA protein with Flag tag was also precipitated.Further immunofluorescence experiments showed that the red fluorescence of BTN3A3 and the green fluorescence of LDHA were both in cytoplasm.After interfering with the expression of BTN3A3,glucose uptake in the interfering group(1.143 ± 0.1269)was lower than that in the control group(1.577 ± 0.07046)(P<0.05),while ATP and lactic acid production in the interfering group were 601.6± 18.48 and 5.247± 0.1827 respectively,which were also significantly lower than those in the control group(786.5± 8.181,7.51± 0.4258)Conclusions:1.There is an interaction between BTN3A3 and LDHA protein;2.BTN3A3 affects glucose uptake,lactic acid and ATP production in HeLa cells.3.BTN3A3 may affect the physiological function of cervical cancer cells by promoting glycolytic pathway.