Influence of osteopontin short hairpin RNA on the proliferation and invasion of human renal cancer cells

Abstract

The influence of short hairpin RNA (shRNA)-mediated osteopontin (OPN) gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated. Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids, which were transferred into the cultured ACHN cells by Lipofectamine™ 2000. The cells transfected by shRNA expression vectors (ACHN/OPN) were visualized under an inverted microscope and screened by G418. Untreated cells (ACHN) and cells transfected by mock vectors (ACHN/Vect) were used as control groups. The expression levels of OPN mRNA and protein were detected by real-time PCR and Western blot respectively. The cell cycle and ratios of apoptotic cells were assessed by flow cytometry. MTT method was used for drawing the growth curve and observing cell proliferation in vitro. The abilities of migration and invasion in three groups were measured by Transwell chamber test. The expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in three groups were examined by Western blot. Our results showed that the recombinant plasmid could be successfully transferred into ACHN cells by LipofectamineTM 2000. Compared with untreated cells, the expression levels of OPN mRNA and protein in ACHN/OPN cells were decreased by 59.68% and 76.42%, respectively (P<0.05), ACHN/OPN cells were blocked in S phase and apoptotic ratio increased significantly (P<0.05), however, no significant differences were found between ACHN/Vect and ACHN. Recombinant plasmid significantly attenuated expression levels of MMP-2 and MMP-9 proteins and suppressed the proliferation, migration, and invasion of ACHN cells. This study suggested that OPN may play an important role in the growth and invasion of human renal cancer ACHN cells, and these processes are correlated with the activations of MMP-2 and MMP-9. Our data provided preliminary experimental evidence for the feasibility of RNA interference technology in gene therapy of human renal cancer.

Keywords

osteopontin / short hairpin RNA / RNA interference / human renal cancer cells

References

[1] RittlingS.R., ChambersA.F.. Role of oesteopontin in tumor progression. Br J Cancer, 2004, 90(10): 1877-1881

[2] WaiP.Y., KuoP.C.. The role of osteopontin in tumor metastasis. J Surg Res, 2004, 121(2): 228-241

[3] RudlandP.S., Platt-HigginsA., El-TananiM., et al.. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res, 2002, 62(12): 3417-3427

[4] ShimadaY., WatanabeG., KawamuraJ., et al.. Clinical significance of osteopontin in esophageal squamous cell carcinoma: comparison with common tumor markers. Oncology, 2005, 68(2–3): 285-292

[5] CastellanoG., MalaponteG., MazzarinoM.C., et al.. Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression. Clin Cancer Res, 2008, 14(22): 7470-7480

[6] PhilipS., KunduG.C.. Osteopontin induces nuclear factor kappa B-mediated promatrix metalloproteinase-2 activation through I kappa B alpha /IKK signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways. J Biol Chem, 2003, 278(16): 14 487-14 497

[7] RangaswamiH., BulbuleA., KunduG.C.. Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/I kappa B alpha kinase-dependent nuclear factor kappa B-mediated promatrix metalloproteinase-9 activation. J Biol Chem, 2004, 279(37): 38 921-38 935

[8] DesaiB., MaT., ZhuJ., et al.. Characterization of the expression of variant and standard CD44 in prostate cancer cells: identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface. J Cell Biochem, 2009, 108(1): 272-284

[9] LiptonA., Colombo-BerraA., BukowskiR.M., et al.. Skeletal complications in patients with bone metastases from renal cell carcinoma and therapeutic benefits of zoledronic acid. Clin Cancer Res, 2004, 10(18Pt2): 6397S-6403S

[10] WeberK.L., DoucetM., PriceJ.E.. Renal cell carcinoma bone metastasis: epidermal growth factor receptor targeting. Clin Orthop Relat Res, 2003, 415(Suppl): S86-S94

[11] WeberK., DoucetM., KominskyS.. Renal cell carcinoma bone metastasis—elucidating the molecular targets. Cancer Metastasis Rev, 2007, 26(3–4): 691-704

[12] ButlerW.T.. The nature and significance of osteopontin. Connect Tissue Res, 1989, 23(2–3): 123-136

[13] DenhardtD.T., GuoX.. Osteopontin: a protein with diverse functions. FASEB J, 1993, 7(15): 1475-1482

[14] PrinceC.W.. Secondary structure predictions for rat osteopontin. Connect Tissue Res, 1989, 21(1–4): 15-20

[15] TuckA.B., ArsenaultD.M., O’MalleyF.P., et al.. Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene, 1999, 18(29): 4237-4236

[16] KhodavirdiA.C., SongZ., YangS., et al.. Increased expression of osteopontin contributes to the progression of prostate cancer. Cancer Res, 2006, 66(2): 883-888

[17] PiyathilakeC.J., FrostA.R., ManneU., et al.. Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung. Clin Cancer Res, 2002, 8(3): 734-744

[18] WuY., DenhardtD.T., RittlingS.R.. Osteopontin is required for full expression of the transformed phenotype by the ras oncogene. Br J Cancer, 2000, 83(2): 156-163

[19] NaqviA.R., IslanM.N., ChoudhuryN.R., et al.. The fascinating world of RNA interference. Int J Biol Sci, 2009, 5(2): 97-117

[20] BrummelkampT.R., BernardsR., AgamiR.. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, 296(5567): 550-553

[21] WadhwaR., KaulS.C., MiyagishiM., et al.. Know-how of RNA interference and its applications in research and therapy. Mutat Res, 2004, 567(1): 71-84

[22] PaulC.P., GoodP.D., WinerI., et al.. Effective expression of small interfering RNA in human cells. Nat Biotechnol, 2002, 20(5): 505-508

[23] TuckA.B., HotaC., WilsonS.M., et al.. Osteopontin induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways. Oncogene, 2003, 22(8): 1198-1205

[24] TuckA.B., ElliottB.E., HotaC., et al.. Chambers, Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met). J Cell Biochem, 2000, 78(3): 465-475

[25] LinY.H., HuangC.J., ChaoJ.R., et al.. Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor. Mol Cell Biol, 2000, 20(8): 2734-2742

[26] ZhuB., SuzukiK., GoldbergH.A., et al.. Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin. J Cell Physiol, 2004, 198(1): 155-167

[27] DasR., MahabeleshwarG.H., KunduG.C., et al.. Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells. J Biol Chem, 2003, 278(3): 28 593-28 606

[28] KatsoR., OkkenhaugK., AhmadiK., et al.. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol, 2001, 17: 615-675

[29] SamolovB., SteenB., SeregardS., et al.. Delayed inflammation-associated corneal neovascularization in MMP-2-deficient mice. Exp Eye Res, 2005, 80(2): 159-166

[30] ScorilasA., KaramerisA., AmogiannakiN., et al.. Overexpression of matrix-metalloproteinase-9 in human breast cancer: a potential favorable indicator in node-negative patients. Br J Cancer, 2001, 84(11): 1488-1496

[31] HrabecE., StrekM., NowakD., et al.. Elevated level of circulating matrix metalloproteinase-9 in patients with lung cancer. Respir Med, 2001, 95(1): 1-4

[32] SakamotoY., MafuneK., MoriM., et al.. Overexpression of MMP-9 correlates with growth of small hepatocellular carcinoma. Int J Oncol, 2000, 17(2): 237-243