Effects Of Calcium Channel Protein STIM1&Orai1 On The Proliferation And Invasion Of Human Liver Cancer

Objective: Detect the expression differences of protein STIM1&Orai1 in liver cells and between different liver cancer cell lines. Explore the effect of STIM1&Orai1 on biological functions of proliferation and invasion of hepatocarcinoma cells.Method: 1.Use RT-PCR to get gene STIM1 and Orai1, construct the recombinant vectors PLV-C1-STIM1-flag and PLV-C1-Orai1-flag,which express proteins fused protein STIM1 and Orai1,and contruct the recombinant vectors PLV-C1-STIM1 and PLV-C1-Orai1, which only express proteins STIM1 and Orai1.2.Use the combinant vectors PLV-C1-STIM1-flag,PLV-C1-Orai1-flag,PLV-C1-STIM1 and PLV-C1-Orai1, to package lentivirus and detect the viral titer. Use PLV-C1-STIM1-flag and PLV-C1-Orai1-flag viruses coinfect Hep G2 cells.Use Western-blot to detect the expression of STIM1 or Orai1.Use CCD to detect the free calcium in coinfect line and normal cells. Use Immunofluorescence to position STIM1 and Orai1 in Hep G2 cells. 3.Cell culturing; Use Western-blot to compare the expression of protein STIM1&Orai1 in liver cells and between different liver cancer cell lines.4.Use MTT to detect the differences of the cell growth curve.Use the flow cytometry instrument to detect the differences of cell cycle.Teocin inducing-culture the cells for 2d, then use the flow cytometry instrument to detect the apoptosis rate.Use “Transwell” method detect the cell invasion and migration ability.Result: 1.Successfully constructed four recombinant vectors PLV-C1-STIM1-flag 、PLV-C1-Orai1-flag,PLV-C1-STIM1-flag and PLV-C1-Orai1-flag. 2.Use two recombinant vectors lentivirus coinfect, get the coinfect cell,used western-blot, havedetected the overexpression of protein STIM1&Orai1.STIM1 mainly distributed in the cytoplasm, Orai1 protein was mainly distributed in the cell membrane.Used CCD,have detected the calcium level of coinfect cell increased obviously.3.The expression of calcium channel protein STIM1 / Orai1 in hepatoma cell lines increased than the normal liver cell. 4.Used MTT have got the differences of the cell growth curve,the growth rate of coinfect cell significally faster.The test of cell cycle showed : The G1&G2 phase of coinfect cell significantly shortened,S-phase cells ratio and proliferation index have increased. 5.The apoptosis rate has no obvious change.The overexpression of STIM1&Orai1 have not affected the cell invasion and migration ability of Hep G2 cell.Conclusion: 1.The expression of calcium channel protein STIM1 / Orai1 in hepatoma cell lines increased than the normal liver cell. 2.STIM1&Orai1 can promote the proliferation of Hep G2 cell, but no significantly affect on the ability against the mitochondrial pathways of apoptosis.Overexpress the STIM1&Orai1 have no obvious effect on the invasion and migration ability of Hep G2 cell.