The Mechanism Study On The Phosphofructokinase Related MiRNA In Tumor Glycolysis

Background and objectiveCancer is one of the most malignant diseases in the world. Among all cancers, lung cancer is the most leading cause of death and the second leading cause of death for men and women, respectively. Cancer cells obtain energy mainly through glycolysis rather than oxidative compared with the normal cells, this phenomenon now termed as Warburg effect. Phosphofructokinase(PFK) is the most critical rate-limiting enzyme involved in glycolysis, which catalyzes the conversion of fructose 6-phosphate to fructose 1, 6-bisphosphate. Micro RNAs(miRNAs) are a class of endogenous, non-coding RNAs that perform their functions at the posttranscriptional level through targeting 3′ untranslated region(3′ UTR) of m RNAs. Increasing evidences demonstrate that miRNAs play important roles in cancer progression by promoting cancer cell proliferation or apoptosis. Although many miRNAs involved in glycolysis, miRNA that regulates glycolysis by targeting PFK remains largely unknown. Our research was designed to identify the role and the regulatory mechanism of PFK-related miRNA in glycolysis in lung cancer cells. This work will offer the evidence for the molecular targeted agent development in lung cancer treatment.Methods(1) We consulted and analyzed references to screen out the main isoform of PFK, the liver type(PFKL).(2) We performed the bioinformatic tools to predict that PFKL is the potential target of miR-128.(3) According to the synthesis principle of miRNA design, miR-128 mimics, miR-128 inhibitor, and the corresponding negative control were synthesized. To increase or decrease the miRNA level in cells, we used the Lipofectamine 2000 to transfect miR-128 mimics or inhibitor into lung cancer cells according to the product manual. Quantitative real time PCR(qRT-PCR) was used for the miR-128 expression 24 hr post-transfection.(4) qRT-PCR and Western blot were performed to analyze the change of PFKL m RNA level and protein expression after miR-128 mimics and inhibitor were being transfected in lung cancer cells, respectively. Cotransfection lung cancer cells with miR-128 and pc DNA-PFKL(PFKL overexpression plasmid) could rescue the PFKL m RNA level downregulated by miR-128.(5) To further confirm that PFKL is the direct target of miR-128, the Dual Luciferase Report Assay was used.(6) MTT and crystal violet staining were used to analyze cell proliferation and cell colony formation after transfection of RNAs. Fluorospectrophotometry Assay was used to analyze the intracellular ATP content after transfection of RNAs. To validate the influences that RNAs made on glycolysis, Spectrophotometry Assay was used to analyze the glucose uptake and lactate production in lung cancer cells.(7) qRT-PCR was used to analyse the expression of miR-128 and PFKL m RNA level after inhibiting the AKT signaling pathway by GSK690693.(8) To validate the mechanism that underlying miR-128 regulates glycolysis, Western blot were used to analyze the total and the phosphorylated AKT expression after transfected lung cancer cells with miR-128 and miR-128-in, respectively.Results(1) PFKL m RNA level and miR-128 expression is relative high in NCI-H460 and NCI-H1299 lung cancer cell line, respectively. The expression of miR-128 is inversely correlation with PFKL m RNA level.(2) We found that PFKL was the direct target of miR-128. The Dual Luciferase Report Assay further validates the target relationship between them. Overexpression of miR-128 could significantly reduce PFKL m RNA level and protein expression, and miR-128 knockdown could increase PFKL m RNA level and protein expression.(3) Both upregulation of miR-128 and PFKL knockdown could significantly inhibit the proliferation and colony formation of NCI-H460; whereas, miR-128 knockdown could promote the proliferation and colony formation of NCI-H1299.(4) PFKL was significantly high-expressed and miR-128 expression was markedly low-expressed in clinical lung cancer samples compared with the normal samples, respectively. Notably, PFKL expression was negatively correlated with miR-128 expression in lung cancer samples.(5) Both miR-128 overexpression and PFKL knockdown could increase intracellular ATP content, and decrease glucose uptake and lactate production in NCI-H460. Conversely, both miR-128 knockdown and PFKL overexpression could decrease intracellular ATP content, and increase glucose uptake and lactate production in NCI-H1299.(6) MiR-128 expression and PFKL mRNA level were upregulated and downregulated respectively after inhibiting the AKT signaling pathway.(7) MiR-128 overexpression observably decreased the phosphorylated but not the total AKT expression, whereas miR-128 knockdown made no effects both on the total and the phosphorylated AKT expression.Conclusions This study elucidated the regulatory mechanism between miR-128 and PFKL. Furthermore, we validated the role that miRNA plays on regulating the key rate-limiting enzyme involved in glycolysis. Meanwhile, our results suggested that miR-128 regulates glycolysis through targeting 3′ UTR of PFKL, thus affects the intracellular ATP content, glucose uptake and lactate production. These data demonstrated that miRNA plays an important role in glycolysis; and also, miR-128 may acts as a new target for lung cancer therapy.