| Research BackgroundAs a common malignant tumor in biliary system,Gallbladder cancer(GBC)is characterized by high malignancy and poor prognosis.At present,early radical cholecystectomy is the only effective way to improve long-term prognosis of GBC patients.However,the lack of typical early clinical symptoms and specific diagnostic indicators,together with certain limitations of imageological diagnosis,results in early diagnosis difficulty and low surgical resection rate.Therefore,there is a desperate expectation to find the key gene of GBC and apply it into targeting therapy and then to improve the prognosis and survival rate of patients.The Warburg effect is a characteristic performance of tumor metabolism,manifested as efficient aerobic glycolysis and inefficient ATP production in many solid tumors.During aerobic glycolysis,LDHA metabolizes pyruvate to lactic acid,which provides needed energy for the rapid growth of tumor cells and abundant biosynthesis.LDHA is observed to increase in many tumors,including breast,colorectal,and pancreatic cancer,and is associated with poor prognosis.LDHA silencing leads to the reduction of lactate level,cell viability,and cellular invasiveness.Studies in vitro and in vivo have shown that LDHA exhibits its carcinogenic activity,at least partially,by regulating cell glycolysis metabolism.Thus LDHA can possibly be used as a cancer therapy target.However,the expression level of LDHA in GBC and its involvement in energy metabolism have been seldom reported.MicroRNA(miRNA)plays a crucial role in post-transcriptional gene regulation.It regulates a variety of biological behaviors such as cell proliferation,invasion,migration and apoptosis by controlling multiple target genes.Aberrant expression of miRNA is correlated with the occurrence and progression of tumors.Research reports indicate that miRNA may inhibit or promote the expression of related genes through different signaling pathways of GBC patients and may provide new strategies for the diagnosis and treatment of GBC as oncogenes or tumor suppressor genes.In conclusion,the first part of this study analyzed the expression of LDHA at the tissue level of GBC and analyzed the correlation between the expression of LDHA and clinical features such as age,gender,tumor size,pathological type,TNM stage and survival time.The second part first observed the proliferation,invasion,tumorigenicity and apoptosis of GBC cells by inhibiting the expression or activity of LDHA at GBC cell level.The impact on Warburg effect was then examined by measuring glucose uptake,lactate and ATP levels.At last,the influence of LDHA silencing on the metastatic capacity of GBC cells in vivo was observed by the nude mouse tumor-bearing experiment.In the third part,miR-30d-5p,a target gene of LDHA,was comprehensively screened and identified by means of analyte informatics.The effects of miR-30d-5p targeting regulation of LDHA on the biological behavior of GBC were investigated at GBC tissue and cell level.Part I: Correlation Analysis between Expression of LDHA inGallbladder Cancer Tissues and Its Clinic Features Objective:To study the expression and localization of LDHA in GBC tissues and to analyze the relationship between the expression of LDHA and the clinical features of GBC patients,so as to provide a clinical basis for the further study of GBC.Methods:Approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University,a total of 171 paraffin tissue samples(28 GBC samples,43 adjacent cancer samples)and 7 frozen tissue samples were collected.Paraffin specimens were stained with HE staining and immunohistochemical method to verify the expression of LDHA.The frozen samples were used to observe the localization of LDHA in GBC cells by immunofluorescence technique.The difference in protein expression level of LDHA was verified by Western blot.LDHA expression and follow-up data including patient age,gender,tumor size,pathological type,pathological grade,TNM stage and survival were analyzed with statistical methods.Result:1.LDHA expression was mainly located in the cell membrane and cytoplasm.GBC tissue LDHA expression was significantly higher than the corresponding para-carcinoma tissue and the difference was statistically significant(P <0.0001).2.LDHA was highly expressed in GBC tissues at the protein level.Compared with adjacent tissues,the difference was statistically significant(P = 0.0257).3.Compared with TNM stage,the expression of LDHA in TNM III-IV patients was higher than that in TNM I-II patients,the difference was statistically significant(P <0.0001);LDHA was highly expressed in patients with metastasis,and compared with non-metastatic group,the difference was statistically significant(P = 0.0011).The survival time of patients with low expression of LDHA was much longer than that of patients with high expression of LDHA,and the difference was statistically significant(P <0.0001).4.LDHA expression,tumor TNM staging and metastatic state are independent risk factors for GBC patients.Conclusion: LDHA is highly expressed in GBC tissue and localized in cell membrane and cytoplasm.The expression level of LDHA in GBC was correlated with tumor TNM stage,metastasis and survival prognosis.Part II: The Effect of LDHA on the Biological Behavior ofGallbladder Cancer Cells Objectives:To verify the proliferation,invasion and apoptosis of GBC cells in vitro at the cellular level after the inhibition of LDHA expression or activity;to verify the effect of LDHA silencing on GBC cell metastasis in vivo.Methods:The GBC cell line used in this study was confirmed by Western blot to verify LDHA expression in GBC cells after LDHA knockdown and FX-11 treatment.After LDHA knockdown,the proliferation of GBC cells was detected by CCK-8 assay and EdU staining.The invasive ability of GBC cells was detected by invasion assay.The glucose uptake,lactate and ATP levels were measured by colorimetric assay.After treatment with FX-11,the activity of GBC cells was detected by LDHA kit.The proliferation of GBC cells was detected by CCK-8.The invasion ability of GBC cells was detected by invasion assay.The ability of tumorigenesis of GBC cells was detected by cloning experiments and the apoptosis of GBC cells by flow cytometry.The effect of LDHA knockdown on metastasis ability of GBC cells was observed by nude mouse tumorigenicity assay.The tumor volume and weight change were recorded,and the tumor growth curve was drawn.Transplanted tumors were verified by HE staining and immunohistochemistry.Result:1.LDHA was highly expressed in two GBC cell lines,NOZ and GBC-SD.The LDHA protein expression level of GBC cell line transfected with LDHA siRNA was significantly decreased in a concentration-dependent gradient.The knockdown efficiency of 60 nmol/L LDHA siRNA was the highest and the difference was statistically significant(P <0.05).2.After LDHA knockdown,GBC cells decreased grape intake and levels of lactate and ATP were reduced;meanwhile,the proliferation and invasion ability weakened.3.After inhibiting the activity of LDHA,FX-11 attenuated the proliferation,invasion and clonality,and increased the early apoptosis.4.After LDHA knock-out,GBC cells formed tumors in nude mice in vivo in a relatively late time,the tumor size was small and the growth rate was slow.The difference was statistically significant(P <0.05).The pathological features of GBC were found by HE staining and immunohistochemistry suggested that the expression of LDHA was low.The difference was statistically significant(P <0.05).Conclusion:LDHA was highly expressed in GBC cells.After inhibiting the expression or activity of LDHA,ATP,glucose uptake and lactate level decreased,while GBC cell proliferation decreased significantly,invasion ability decreased significantly,and early apoptosis rate increased.The vivo tumorigenicity experiments confirmed that inhibition of LDHA expression in GBC cells decreased in vivo metastasis and significantly increased survival time.Part III: Screening and Identifying the LDHA TargetingmiR-30d-5pObjective:To screen and identify miRNA that can target LDHA using the GEO database;to verify their impact on GBC biological behavior.Methods:To screen the miRNA that can target LDHA using the GEO database;to detect the expression of miR-30d-5p in GBC and adjacent tissues;to analyze the correlation between the expression of miR-30d-5p and clinical features include TNM staging,metastasis and prognosis.The correlation between miR-30d-5p and LDHA was analyzed.LDHA mRNA expression was verified at RNA level by overexpression of miR-30d-5p,and LDHA expression was verified at the protein level.Dual luciferase reporter gene confirmed that LDHA is a miR-30d-5p target gene.After overexpression of miR-30d-5p,CCK-8 was used to detect the proliferation of cells,the ability of tumorigenesis was confirmed by cloning experiments and the invasion ability was verified by invasion experiments.Result:1.The miR-30d-5p targeted to LDHA was screened,which was significantly down-regulated in GBC tissues,the difference was statistically significant(P <0.005).2.The expression of miR-30d-5p was correlated with tumor TNM stage(P = 0.0066)and tumor metastasis(P = 0.0036).The patients with low expression of miR-30d-5p had short survival time and the difference was statistically significant(Log-rank test,P = 0.0328).3.The expression level of LDHA mRNA significantly decreased after miR-30d-5p overexpression and the difference was statistically significant(P <0.05);the level of LDHA protein significantly decreased and the difference was statistically significant(P <0.05);Dual luciferase reporter gene confirmed that LDHA is a miR-30d-5p target gene.4.After overexpression of miR-30d-5p,the proliferation of GBC cells,the ability of cloning tumorigenesis,and the invasion abilityweakened.Compared with the control group,the difference was statistically significant(P <0.05).Conclusion:The low expression of miR-30d-5p in GBC tissue is correlated with LDHA expression,TNM stage,metastasis and survival time.miR-30d-5p may target the expression of LDHA by binding to LDHA mRNA.After miR-30d-5p was overexpressed,the proliferation,invasion and tumorigenicity of GBC cells significantly decreased. |
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