Study Of NHE1 MRNA Expressions In Hepatocellular Carcinoma Tissue And Paraneoplastic Tissue And Construction Of PIRES2-AcGFP1-NHE1 Eukaryotic Expression Vector

The Na+/H+ exchanger (NHE) is a ubiquitous protein in mammalian cells. It is an integral membrane protein which exchanges one intracellular H+ ion for an extracellular Na+ ion, thereby protection cells from intracellular acidification. Among the NHE, the most wide distribution isoform is NHE1. In addition, NHE1 participates in regulation of intracellular pH (pHi) values and cell volume, ion transport, cell proliferation, differentiation and apoptosis, tumor metastasis and invasion, and so on.Recently, a new theory that an increasing attention has been paid on the regulating the tumor cell intracellular pH values has been proposed, and NHE1 is on the focus of this theory. Many researches have proved that the expression of NHE1 was significantly high in diverse kinds of tumor cells such as lung cancer, breast cancer, melanoma, gastric cancer, etc. The tumor cells have the advantage of anaerobic glycolysis for the increase of oxygen and nutrients can not keep pace with the development of tumor. And then much lactic acid and H+ ion will be produced, but in situ tumor spectroscopic studies utilizing the 31P isotope have reported that tumor cells have alkaline intracellular pH values (pHi=7.12-7.65)and acidic interstitial extracellular pH values(pHe=6.2-6.9). Apparently, the tumor extracellular environment is more acidic than the intracellular environment, creating a reversed pH gradient across the cell membrane that increases as the tumor progresses. The development and maintenance of this gradient is directly due to the ability of the tumor to secrete H+ ion, and this secretion mainly depends on the Na~+/H~+ exchanger1 (NHE1).Based on this point, this article respectively researched the expression and significance of NHE1 mRNA and protein in hepatocellular carcinoma (HCC) tissue and paraneoplastic tissue in part one and two, and constructed pIRES2-AcGFP1-NHE1 eukaryotic expression vector in part three in order to investigate the regulatory mechanism in hepatocellular carcinoma.Part 1Objective: To research expression and significance of Na+/H+ exchanger1 mRNA in hepatocellular carcinoma tissue and paraneoplastic tissue.METHODS: Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), we detected the expressions of NHE1 mRNA in the tissues of 34 HCC, 34 paraneoplastic tumor samples and 21 Non-HCC liver tissue samples from the patients.RESULTS: NHE1 mRNA expressed in all the HCC tissues, and the relative expression quantity in HCC and paraneoplastic tissues are respecively 2.892±0.882 and 0.802±0.206. The relative expression quantity detected in 21 cases of normal liver tissue and other liver disease specimen was 0.872±0.186. Perform group t-test with the relative expression quantity of NHE1 detected in HCC tissue and paraneoplastic tissue, the two have significant difference (t=13.458, P<0.001); perform group t-test with the expression quantity detected in paraneoplastic tissue and the liver tissue for control, the two have significant difference:(t=10.320, P<0.001); perform group t-test with the expression quantity of NHE1 mRNA detected in para-carcinoma tissue and the control liver tissue, the two have no statistical significance(t=1.276, P>0.10). Perform paired t-test with the expression quantity of NHE1 mRNA detected in the HCC tissue and paraneoplastic tissue of the same patient, the two have significant difference (t=13.168, P<0.001).Conclusion: NHE1 mRNA may play an important role for the development of HCC.Part 2Objective: To study the expression of Na+/H+ exchanger1 protein in primary human hepatocelluar carcinoma and the relationship with invasion and matastasis, and also its clinical significance.Methods: Immunohistochemical SP method was used to detect the expression of NHE1 protein in HCC and paraneoplastic tissues. The association between the expression of NHE1 protein and the clinicopathologic parameters of patients was analyzed.Results: NHE1 protein expression levels and positive rates in human HCC were significantly higher than that in paraneoplastic tissues. NHE1 protein expression was significantly correlated with histological grade, the clinical TNM stage and portal tumor thrombus, but was not related with patients'age, size of tumor, number of tumors, serum AFP level.Conclusion: NHE1 protein is upregulated in human hepatocellular carcinoma tissues. The higher expression of NHE1 protein was closely related to the degree of malignancy of HCC, disease progression, invasion and metastasis of HCC.Part 3Objective: To construct pIRES2-AcGFP1-NHE1 eukaryotic expression vector.Methods: RT-PCR was applied to amplify human cDNA from HepG2 cells with a pair of specific sequence primers carrying a restriction enzyme site Xho I or sal I on each 5'end. NHE1 cDNA was inserted into T vector for sequencing, and then insterted into pIRES2-AcGFP1 vector. We tested the whole sequence of pIRES2-AcGFP1-NHE1 recombined eukaryotic expression vector.Results: The NHE1 gene was successfully cloned and its sequence was analyzed, which basically accorded with the genebank. And then the cloned NHE1 gene was inserted into clone vector pIRES2-AcGFP1. We successfully constructed the recombination expression plasmids pIRES2-AcGFP1-NHE1, which were identified by PCR and enzyme digestion. The sequence of pIRES2-AcGFP1-NHE1 is identical with the genbank.Conclusion: The pIRES2-AcGFP1-NHE1 eukaryotic expression vector was successfully constructed. It is indicated that the expression of GFP in the eukaryotic cells which were transinfected by a recombination plasmid could act as a sign of NHE1 expression in eukaryotic cells. This vector can pave the way for the further research on the regulatory mechanism of NHE1 on the development, metastasis and invasion of hepatocellular carcinoma.