| BackgroundCervical cancer is a common gynecological malignancy,and its incidence has been rising in recent years,and the incidence is very high in Asian populations.In our country,the occurrence of cervical cancer is also very common,and it has increasingly become a serious problem that threatens women’s life and health.The main reason for the death of cervical cancer is that some patients have reached the advanced stage at the time of diagnosis.In the course of treatment,they have resisted in radiotherapy and chemotherapy,and even metastasis.Therefore,it is urgent to find a new treatment method.The utilization of glucose by tumor cells is preferentially selected by aerobic glycolysis,namely the Warburg effect,which leads to an increase in the consumption of glucose,resulting in a large amount of lactic acid.In addition,abnormal utilization of glutamine by tumor cells also leads to the increasing production of lactic acid.These energy metabolism pathways all lead to an increase in intracellular lactic acid levels.To avoid acidosis,tumor cells excrete lactic acid into the microenvironment through membrane proteins and cause microenvironmental acidification,which will promote the development of many malignant biological behaviors of cancer,including tissue invasion,metastasis,neovascularization and the response to hypoxia.In addition,lactic acid-induced acidification of the tumor microenvironment(pH 6.0—6.5)can inhibit the host’s local anti-cancer immunity.There is an increasing evidence that targeted tumor reprogramming of energy metabolism can provide an effective treatment for cancer treatment.The Monocarboxylate Transporters(MCTs)family includes plasma membrane proteins involved in the transport of lactic acid and other monocarboxylates.The extracellular flow of lactic acid/H+ is generally thought to be mediated by MCT4.The expression of MCT4 is associated with high glycolysis of cells,and the high expression of MCT4 in tumors is associated with poor prognosis of many types of cancers,including cervical cancer.More and more studies have shown that inhibiting the expression or function of MCTs can inhibit the growth of cancers,which provides new therapeutic ideas for cancers.Currently,most small molecule inhibitors targeting MCTs can only specifically target MCT1 or MCT2(eg,AZD3965,AR-C155858),but not specifically inhibit MCT4,and a specific treatment for MCT4 that mediates lactate efflux is still lacking.Objective 1.To detect the protein level of MCT4 in normal cervical epithelium and cervical cancer cells,then select appropriate siRNA concentration and the duration time of silencing MCT4 gene.The expression level of MCT4 was tested by western blot;2.To explore the effects of silencing MCT4 gene on the biological behaviors of cervical cancer cells Hela and Siha,such as secreting lactic acid,proliferation,invasion,migration and apoptosis.Materials and methods Experimental method(1)To detect the MCT4 protein expression level in normal cervical epithelium and cervical cancer cells:The western blot was used to detect the expression of MCT4 in Hcer-Epic,Hela and Siha and whether the expression of MCT4 was different.(2)Screening the appropriate concentration of siRNA and the duration time of silencing MCT4 gene:Two specific siRNA sequences,siMCT4#1 and siMCT4#2,were transfected with HeLa and Siha with the concentration of 50 nM,100 nM,150 nM,200 nM,and 300 nM,respectively,and siRNAs with better silencing effect were selected as suitable concentrations.Then,the siRNA was transfected with Hela and Siha with this concentration,respectively.Proteins were extracted at the time of 24 h,48h,72 h and 96 h respectively.The expression of MCT4 was detected by western blot.(3)The effect of silencing MCT4 on the biological behaviors of cervical cancer cells:Hela and Siha were divided into 4 groups: blank control group(Ctrl),negative control group(siNC),experimental group 1(siMCT4#1)and experimental group 2(siMCT4#2).When the MCT4 gene was silenced with the suitable concentration,the MCT4 protein levels in the Hela and Siha groups were detected by western blot.After the siRNA was transfected for 96 hours,the lactate concentration in the culture medium was measured by the lactic acid kit.The proliferation ability of the cells was detected by CCK8 experiments,the migration ability of the two cell lines was detected by the scratch test,the invasion ability of the two cell lines was detected by the Transwell invasion test,and the apoptosis numbers of the cells after transfection was detected by flow cytometry.Results 1.The expression of MCT4 in three cell lines showed that the expression of MCT4 protein in Hela and Siha was significantly higher than that of normal cervical epithelial cells,Hcer-Epic,while expression of MCT4 in Siha was slightly higher than that in Hela.2.Western blot results showed that in Hela,Siha,with the increase of transfection concentration,the gray value of MCT4 gradually decreased,but there is no significant difference between the 200 nM group and 300 nM group;When transfected Hela,Siha with siMCT4#1,siMCT4# 2 at the concentration of 200 nM,with the increase of transfection time,the gray value of MCT4 gradually decreased.3.When MCT4 gene was specifically silenced at the concentration of 200 nM,the MCT4 protein levels in Hela and Siha was decreased.After the MCT4 gene was specifically silenced in Hela and Siha,the lactic acid secretion of both two cell lines decreased,and the proliferation,migration and invasion of both two cell lines also decreased,while the apoptosis numbers in two cell lines both increased.Conclusions 1.The expression of MCT4 protein in cervical cancer cells,Hela and Siha,was significantly higher than that in normal cervical epithelial cells--Hcer-Epic;2.Silencing MCT4 gene with siRNA was concentration-dependent and time-dependent in a certain range.The silencing effect of 200 nM siRNA was best,and there was no significant difference with 300 nM.With the prolongation of transfection time,the silencing effect was obvious and the silencing effect could persist 96 hours.3.siRNA can down-regulate the expression of MCT4 protein by silencing MCT4 gene in Hela and Siha,thereby inhibiting the secretion of lactic acid in two cell lines,inhibiting the proliferation,migration and invasive capacity of two cell lines,and promoting Hela,Siha Apoptosis. |
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