RNAi-mediated Silencing Of Aquaporin-3 Inhibits The Proliferation And Invasion Of XWLC-05 Human Lung Cancer Cells In Vitro And In Xenograft Tumor Of Athymic Mice

Objective:To construct the siRNA expression vector targeting AQP3 and to evaluate its ability to down-regulate the expression of AQP3 in XWLC-05 human lung cancer cells, furthermore, to evaluate the effects of AQP3 silence on the proliferation and invasion of XWLC-05 human lung cancer cells vivo and in vitro.Methods:We searched the gene sequence of human AQP3 in the Genbank, selected the optimal target sequence,and then according to the characters of pSilencer 4.1-CMV neo siRNA expression vector,designed the homologue siRNA synthetic templates with hairpin,two siRNA expression vectors, pAQP3-siRNA1and pAQP3-siRNA2 targeting AQP3, were constructed using pSilencer4.1-CMV vector. After recombinant plasmid pAQP3-siRNA1 and pAQP3-siRNA2 were transfected into XWLC-05 lung cancer cells 48h, morphologic changes of cells were observed, the levels of mRNA and protein expressions in XWLC-05 human lung cancer cells were determined by RT-PCR and Western blot analysis respectively. At different time points(at 24h,48h and 72h) after transfection, the cell cultures were measured for cell proliferation levels using MTS assay, phase distribution and apoptosis of tumor cell cycle was analyzed by flow cytometry(FCM) after transfection and non-transfection; cell invasivenesses was tested by Matrigel method. At 48h after transfection, activities of MMP-2,MM-9 were measured by gelatin zymography, to investigate the conceivable mechanism. Through these to evaluate the influences of AQP3 silence on the proliferation and invasion of XWLC-05 human lung cancer cells. At last, subcutaneous xenograft tumors in athymic mice were induced by inoculation of XWLC-05. The general conditions of the athymic mice, Body Weight Changes after the experiments, blood cells analysis when the experiments ended, tumorigenicity rate; growth of the xenograft tumors were observed after inoculation, measured the diameters of tumor every 3 days and worded out the gross tumor volume, the growth curves were drawn, histopathology of the xenograft tumor was done, and the expression of AQP3 in the xenograft tumor was observed by immunohistochemistry. Through these to evaluate the influences of AQP3 silence on the xenograft tumors in athymic mice of XWLC-05 human lung cancer cells.Results:Two siRNA expression vectors targeting AQP3 were constructed successfully, named pAQP3-siRNA1 and pAQP3-siRNA2 respectively. After 48h transfection, cell density of experiment group decreased, rate of propagation step down and vacuoles emerged in cytoplasm, but there were no these changes in the other two groups, level of AQP3-mRNA was decreased significantly in AQP3-siRNA1 XWLC-05 cells(65% of that in control group) compared with AQP3-siRNA2 XWLC-05 cells(79% of that in control group) (P<0.05).The level of protein expression of AQP3 in AQP3-siRNA1 XWLC-05 cells was decreased significantly (53% of that in control group) compared with AQP3-siRNA2 XWLC-05 cells(73% of that in control group) (P< 0.05).Similar knockdown of AQP3 expression at both mRNA and protein levels were induced by pAQP3-siRNA. The results of MTS assay indicated that there were no obvious inhibition of the proliferation level of XWLC-05 cells at 24h after transfection, but the proliferation level were obviously decreased at 48h and 72h after transfection with pAQP3-siRNA1(P<0.05); compared with the other two groups, percentage of G1 phase cells in experimental group increased significantly(P<0.05), and formed obvious apoptotic peak in experimental group;compared with control group, the number of invasive and migratory cells declined after transfection in experimental group, and compared with 48h,72h, there were less cells of invasive and migratory declined at 24h after transfection(P<0.05), but there was no significant difference between 48h and 72h after transfection(P>0.05); the OD values of eluant in experimental group were significantly decreased compared with the other two groups(P<0.05); the results showed that the activities of MMP-2 were significantly decreased in AQP3-siRNAl group compared with the other two groups(P<0.05). The animal models were set up successfully. All athymic mice were alive when the experiment ended, the body weights of athymic mice increased with different degree, but there was no significant difference between the two groups(P>0.05). It's higher on red blood cell count in experiment group than control group (P< 0.05), and there no significant difference between the two groups on white blood cell count and platelet count(P>0.05).The macroscopic xenograft tumor emerged 7-10 days after inoculation, there were 15 tumors emerged in experiment group, the rate is 75%; 16 tumors emerged in control group, the rate is 80%, and there no significant difference between the two groups(P>0.05); the growth curve of control group is steep, range of ascensus is big, and the growth curve of experiment group is gently. The xenograft tumors were significantly decreased in the AQP3-siRNA group compared with the control group. Histopathology:the tumor cells in the control group formed cancer nest, structures as crypt or glandular cavity formed, inequality of size, more atypia, cell nucleus got bigger, nuclear chromatin is anachromasis, nuclear membrane increased thickness, more pathological karyokinesis; in experiment group, the number of tumor cell decreased, lamellar necrosis, less karyokinesis, more karyopyknosis, karyorrhexis and caryorrhexis, fibroplasias in interstitium and phlogocyte infiltration. Immunohistochemisty: AQP3 resides in plasmalemma and cytolymph, positive staining is brown, intensity of AQP3 is lower in experiment group than in control group.Conelusion:AQP3 specific siRNA can significantly and specifically down-regulate the expression of AQP3 in XWLC-05 cells.It can efficiently inhibit the proliferation and invasion of XWLC-05 human lung cancer cell in vitro. The mechanism maybe is regulating multiplication cycle of tumor cells and inducing cell apoptosis to inhibit the proliferation of tumor cells, and reduction of AQP3 gene expression affected the activities of MMP-2, lead to depressions of infiltrating and metastasis of XWLC-05 cells. It also can inhibit growth of xenograft tumor in athymic mice, accelerate necrosis and apoptosis of tumor cells.