The Role And Mechanism Of NHE1in The Invasion And Metastasis Of MHCC97-H Cell Line

Hepatocellular carcinoma (HCC), the fifth most common malignancy in theworld, is one of the most common malignant tumor, and the second leadingcause cancer related death in China. With the great difficulties in early diagnosisand treatment, the prognosis of this highly malignant tumor is often not good,posing a great threat to public health. Although great progress has been madedue to development of combined therapy mainly with surgical resection in re-cent years, the prognosis of those, who received surgical resection and a varietyof adjuvant therapy, remains poor. There are many reasons for this, while themain reason may be difficulty of early diagnosis. Clinically, most patients, oftendiagnosed at advanced stage, have a great difficulty in treatment, and the post-operative tumor recurrence and metastasis is common. According to some re-searches, the majority of patients with hepatocellular carcinoma died of metas-tasis-related complications. Therefore, studying the molecular mechanism ofmetastasis of liver cancer and proposing appropriate treatment measures have avery important clinical significance. Sodium hydrogen exchanger1(Na+/H+exchanger1, NHE1) is one of ionexchange protein on the cell membrane, whose main function is to regulate theintracellular pH value, and regulate the cytoskeleton by the binding to F-actin.Over-expression or high activity of NHE1are observed in many tumors, and it isthought to be involved in tumor cell proliferation, apoptosis, differentiation, in-vasion and metastasis, etc. However, the role of NHE1in HCC remains unclear.ObjectiveTo design and synthesize specific NHE1-siRNA, and explore the role ofNHE1in invasive and metastatic process of hepatoma cell line MHCC97-H.MethodsThree specific NHE1gene small interfering RNA (siRNA) was transfectedinto hepatoma cell line of MHCC97-H, using liposome. The mRNA and proteinexpression of NHE1was confirmed by real time PCR and Western blot. Intra-cellular and extracellular pH changes in each group were also detected. One ofthe most efficient NHE1-siRNA was used for subsequent experiments, in whichit was transfected into MHCC97-H. Immunofluorescence staining, scanningelectron microscopy, migration and invasion assays, western blot, gelatin zymo-graphy experiments were used to detect changes of related index in each group.Results1. The siRNA specifically against NHE1gene was successfully transfectedinto the hepatoma cell MHCC97-H. The NHE1-siRNA with the sequence (sensestrand:5’-GCCCUGUUAAUCAUUCCGUTT-3’, antisense strand:5’-ACGGAAUGAUUAACAGGGCTT-3’) was found to have the highest inhibition efficiencyby real time PCR, western blot and intracellular and extracellular pH value re-sults. This specific NHE1-siRNA was used for further experiment. 2. After specific of inhibition of NHE1, fluorescence and scanning electronmicroscopy showed that pseudopodia retraction and loss of polarity inMHCC97-H cells transfected with NHE1-siRNA and disorder of intracellularactin network, the reduced pseudopodia retraction formation were also observed.Migration assay results suggested that cell migration were reduced after the spe-cific inhibition of NHE1.3. The number of cells penetrated the basement membrane of the transwellchamber were significantly reduced in MHCC97-H cells transfected with spe-cific siRNA of NHE1. Western blot and gelatin zymography demostrated de-creased expression and activity of MMP-2and MMP-9in NHE1-siRNA group.It can be inferred that NHE1may cause the acidification of the extracellular en-vironment, thus increase the expression and activity of MMP-2and MMP-9andpromotes invasion and metastasis of MHCC97-H.ConclusionNHE1may regulate cell movement by modulating the cytoskeleton and themorphological changes of hepatoma cells; On the other hand, increase the ex-pression and activity of MMP-2and MMP-9by adjusting the intracellular andextracellular pH value. The common role of these two processes promote inva-sion and metastasis of liver cancer cells, indicating a potential target of NHE1inHCC and that the inhibition of NHE1can inhibit this process.