Effects Of Lactic Acid Dehydrogenase - A Expression On Proliferation And Apoptosis Of Cholangiocarcinoma Cell Line Hucct - 1 In

Intrahepatic cholangiocarcinoma (ICC) is a devastating malignancy that arises from the epithelial lining of the intrahepatic bile ducts, with worldwide data demonstrating an increase in the incidence and mortality. Due to the difficulty in detecting at the early stage and the current disappointing chemotherapy strategies, the prognosis of patients with advanced cholangiocarcinoma is very poor; therefore, new insights into cancer cell-specific biological pathways are urgently needed to promote development of rationally targeted therapeutics.Even under aerobic conditions, cancer cells preferentially undergo glycolysis followed by fermentation, this phenomenon is known as aerobic glycolysis (or the Warburg effect). Lactic dehydrogenase A (LDHA),the NADH-dependent enzyme that catalyzes the conversion of pyruvate to lactate, is a key glycolytic enzyme. Inhibition of LDH-A should allow a blockade of the aerobic glycolysis of tumor cell. Attenuation of LDH-A expression stimulated the OXPHOS (oxidative phosphorylation) capacity, and severely diminished the tumorigeniciry of neu-initiated mammary tumor cells.Previous studies showed that LDH-A was strongly expressed in gastric, pulmonary, gynaecologic and breast malignancies associated with poor prognosis. However, expression and the effects of LDH-A in ICC has not been described up to now. Therefore, we first choosed a ICC cell line overexpress LDHA, then constructed three short hairpin RNAs (shRNA) targeting LDHA, and transfected into cholangiocarcinoma cell lines hucctl, validated the interference effect of these shRNAs by quantitative real time PCR and Western blot analysis. Transfected the shRNA with the highest inhibited effect into hucctl analyzed cell proliferation and cell apoptosis by MTT assay and annexin V-7-AAD apoptosis detection assay respectively.In the present study, we found: 1. Compared to human normal biliary epithelial cell line HiBEPic,LDHA was overexpressed in ICC cell line Hucct-1and QBC939;2. Three short hairpin RNAs (shRNA) targeting LDHA were combined to pGPU6/GFP/Neo vector, and transfected into cholangiocarcinoma cell lines hucct1.Validated the interference effect of these shRNAs by quantitative real time PCR and Western blot analysis. siLDH-A3has the highest inhibited effect, the expression of ldh-a mRNA was inhibited to53%±11%, compared to the negative control group (p<0.05); the expression of LDHA was decreased to29%±2%, compared to the negative control group (p<0.05);3. Reduction of LDH-A by RNAi (RNA interference) inhibited cell growth in the liquid culture and induced cell apoptosis. MTT assay shows that hucctl LDH-A knocked-down cell lines(RNAi group) proliferation rates are slower than negative control hucctl cell lines(NC group), the absorbance value of transfected cells in96h decrease markedly compared to the negative control cells (0.95±0.04Vs0.73±0.04)(p<0.05);the apoptosis rates of RNAi (20.47%±3.35%) increased obviously compared to that of NC cells(9.0%±1.08%).4. Furthermore, we found that inhibition of LDH-A elevated the cytoplasmic ROS levels. In other words, overexpression of LDH-A in cholangiocarcinoma might be associated with ROS downregulation.Conclusion:Taken together, these results obtained in vitro suggest that LDH-A is a potential therapeutic target for ICC.