Downregulation of coding transmembrane protein 35 gene inhibits cell proliferation, migration and cell cycle arrest in osteosarcoma cells

Huang,Yinjun; Zhao,Shichang; Zhang,Yadong; Zhang,Changqing; Li,Xiaolin

doi:10.3892/etm.2016.3381

Downregulation of coding transmembrane protein 35 gene inhibits cell proliferation, migration and cell cycle arrest in osteosarcoma cells

Authors:
- Yinjun Huang
- Shichang Zhao
- Yadong Zhang
- Changqing Zhang
- Xiaolin Li
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Affiliations: Department of Orthopedic Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, P.R. China
Published online on: May 23, 2016 https://doi.org/10.3892/etm.2016.3381
Pages: 581-588
Copyright: © Huang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Osteosarcoma (OSA) is the most common primary tumor of the bone. Resistance to chemotherapy and the fast rapid development of metastatic lesions are major issues responsible for treatment failure and poor survival rates in OSA patients. Tetraspanins comprise a family of transmembrane receptor glycoproteins that affect tumor cell migration through tetraspanin‑integrin interaction. The present study focused on a four‑pass transmembrane protein gene, transmembrane protein 35 (TMEM35) gene, and examined its role in the growth, migration and cell cycle progression of OSA cells. In addition, the study discussed whether the TMEM35 gene, which encodes the TMEM35 protein, may be a potential therapeutic target for OSA. In the current study, reverse transcription‑quantitative polymerase chain reaction was performed to examine TMEM35 expression in OSA and matched healthy tissues. Small interfering RNAs (siRNAs) were transfected into SaOS2 and U2OS cells to knockdown the TMEM35 expression. Soft‑agar colony formation assay was performed to evaluate cell growth, and cell cycle progression was analyzed by flow cytometry. Wound‑healing and Boyden chamber assays were also performed to investigate cell invasion and migration by the SaOS2 and U2OS cells. TMEM35 protein was analyzed in a functional protein interaction networks database (STRING database) to predict the functional interaction partner proteins of TMEM35. The results indicated that TMEM35 was abnormally expressed in OSA tissues. Of the 37 examined patients, TMEM35 expression was significantly increased in the OSA tissues of 24 patients (64.86%; P<0.05), when compared with the expression in normal tissues. Furthermore, TMEM35 knockdown following transfection with siRNAs inhibited the colony formation ability of SaOS2 and U2OS cells in soft agar. Flow cytometric analysis also revealed that TMEM35 knockdown by RNA interference may result in G1 phase arrest and a decreased cell population at the S phase. TMEM35 knockdown inhibited cell migration in SaOS2 and U2OS cells in wound‑healing assays. In conclusion, TMEM35, a member of the tetraspanin family, serves an important role in the growth of OSA cells.

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1	Hashimoto K, Hatori M, Hosaka M, Watanabe M, Hasegawa T and Kokubun S: Osteosarcoma arising from giant cell tumor of bone ten years after primary surgery: A case report and review of the literature. Tohoku J Exp Med. 208:157–162. 2006. View Article : Google Scholar : PubMed/NCBI
2	Hayashi K, Zhao M, Yamauchi K, Yamamoto N, Tsuchiya H, Tomita K, Kishimoto H, Bouvet M and Hoffman RM: Systemic targeting of primary bone tumor and lung metastasis of high-grade osteosarcoma in nude mice with a tumor-selective strain of Salmonella typhimurium. Cell Cycle. 8:870–875. 2009. View Article : Google Scholar : PubMed/NCBI
3	Takeuchi A, Yamamoto N, Shirai T, Nishida H, Hayashi K, Watanabe K, Miwa S and Tsuchiya H: Successful correction of tibial bone deformity through multiple surgical procedures, liquid nitrogen-pretreated bone tumor autograft, three-dimensional external fixation, and internal fixation in a patient with primary osteosarcoma: A case report. BMC Surg. 15:1242015. View Article : Google Scholar : PubMed/NCBI
4	Stiller CA: International patterns of cancer incidence in adolescents. 33:631–645. 2007.
5	Zhang Y, Zhang L, Zhang G, Li S, Duan J, Cheng J, Ding G, Zhou C, Zhang J, Luo P, et al: Osteosarcoma metastasis: Prospective role of ezrin. Tumour Biol. 35:5055–5059. 2014. View Article : Google Scholar : PubMed/NCBI
6	García-López MA, Barreiro O, García-Diez A, Sánchez-Madrid F and Peñas PF: Role of tetraspanins CD9 and CD151 in primary melanocyte motility. J Invest Dermatol. 125:1001–1009. 2005. View Article : Google Scholar : PubMed/NCBI
7	Gartlan KH, Belz GT, Tarrant JM, Minigo G, Katsara M, Sheng KC, Sofi M, van Spriel AB, Apostolopoulos V, Plebanski M, et al: A complementary role for the tetraspanins CD37 and Tssc6 in cellular immunity. J Immunol. 185:3158–3166. 2010. View Article : Google Scholar : PubMed/NCBI
8	Goschnick MW and Jackson DE: Tetraspanins - structural and signalling scaffolds that regulate platelet function. Mini Rev Med Chem. 7:1248–1254. 2007. View Article : Google Scholar : PubMed/NCBI
9	Gourgues M, Clergeot PH, Veneault C, Cots J, Sibuet S, Brunet-Simon A, Levis C, Langin T and Lebrun MH: A new class of tetraspanins in fungi. Biochem Biophys Res Commun. 297:1197–1204. 2002. View Article : Google Scholar : PubMed/NCBI
10	Jiang X, Zhang J and Huang Y: Tetraspanins in cell migration. Cell Adh Migr. 9:406–415. 2015. View Article : Google Scholar : PubMed/NCBI
11	Jones EL, Demaria MC and Wright MD: Tetraspanins in cellular immunity. Biochem Soc Trans. 39:506–511. 2011. View Article : Google Scholar : PubMed/NCBI
12	Köberle M, Kaesler S, Kempf W, Wölbing F and Biedermann T: Tetraspanins in mast cells. Front Immunol. 3:1062012. View Article : Google Scholar : PubMed/NCBI
13	Krementsov DN, Weng J, Lambelé M, Roy NH and Thali M: Tetraspanins regulate cell-to-cell transmission of HIV-1. Retrovirology. 6:642009. View Article : Google Scholar : PubMed/NCBI
14	Perron JC and Bixby JL: Tetraspanins expressed in the embryonic chick nervous system. FEBS Lett. 461:86–90. 1999. View Article : Google Scholar : PubMed/NCBI
15	Higashiyama M, Taki T, Ieki Y, Adachi M, Huang CL, Koh T, Kodama K, Doi O and Miyake M: Reduced motility related protein-1 (MRP-1/CD9) gene expression as a factor of poor prognosis in non-small cell lung cancer. Cancer Res. 55:6040–6044. 1995.PubMed/NCBI
16	Zheng R, Yano S, Zhang H, Nakataki E, Tachibana I, Kawase I, Hayashi S and Sone S: CD9 overexpression suppressed the liver metastasis and malignant ascites via inhibition of proliferation and motility of small-cell lung cancer cells in NK cell-depleted SCID mice. Oncol Res. 15:365–372. 2005.PubMed/NCBI
17	Saito Y, Tachibana I, Takeda Y, Yamane H, He P, Suzuki M, Minami S, Kijima T, Yoshida M, Kumagai T, et al: Absence of CD9 enhances adhesion-dependent morphologic differentiation, survival and matrix metalloproteinase-2 production in small cell lung cancer cells. Cancer Res. 66:9557–9565. 2006. View Article : Google Scholar : PubMed/NCBI
18	Park JJ, Jin YB, Lee YJ, Lee JS, Lee YS, Ko YG and Lee M: KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer. BMC Cancer. 12:812012. View Article : Google Scholar : PubMed/NCBI
19	Jee B, Jin K, Hahn JH, Song HG and Lee H: Metastasis-suppressor KAI1/CD82 induces homotypic aggregation of human prostate cancer cells through Src-dependent pathway. Exp Mol Med. 35:30–37. 2003. View Article : Google Scholar : PubMed/NCBI
20	Lee HA, Park I, Byun HJ, Jeoung D, Kim YM and Lee H: Metastasis suppressor KAI1/CD82 attenuates the matrix adhesion of human prostate cancer cells by suppressing fibronectin expression and β1 integrin activation. Cell Physiol Biochem. 27:575–586. 2011. View Article : Google Scholar : PubMed/NCBI
21	Radford KJ, Thorne RF and Hersey P: CD63 associates with transmembrane 4 superfamily members, CD9 and CD81 and with beta 1 integrins in human melanoma. Biochem Biophys Res Commun. 222:13–18. 1996. View Article : Google Scholar : PubMed/NCBI
22	Si Z and Hersey P: Expression of the neuroglandular antigen and analogues in melanoma. CD9 expression appears inversely related to metastatic potential of melanoma. Int J Cancer. 54:37–43. 1993. View Article : Google Scholar : PubMed/NCBI
23	Sauer T and Suciu V: The role of preoperative axillary lymph node fine needle aspiration in locoregional staging of breast cancer. Ann Pathol. 32:e24–e28. 2012. View Article : Google Scholar : PubMed/NCBI
24	Tran PV, Georgieff MK and Engeland WC: Sodium depletion increases sympathetic neurite outgrowth and expression of a novel TMEM35 gene-derived protein (TUF1) in the rat adrenal zona glomerulosa. Endocrinology. 151:4852–4860. 2010. View Article : Google Scholar : PubMed/NCBI
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
26	Fromigué O, Hamidouche Z and Marie PJ: Blockade of the RhoA-JNK-c-Jun-MMP2 cascade by atorvastatin reduces osteosarcoma cell invasion. J Biol Chem. 283:30549–30556. 2008. View Article : Google Scholar : PubMed/NCBI
27	Kuhn M, Szklarczyk D, Franceschini A, Campillos M, von Mering C, Jensen LJ, Beyer A and Bork P: STITCH 2: An interaction network database for small molecules and proteins. Nucleic Acids Res. 38:D552–D556. 2010. View Article : Google Scholar : PubMed/NCBI
28	Hemler ME: Tetraspanin functions and associated microdomains. Nat Rev Mol Cell Biol. 6:801–811. 2005. View Article : Google Scholar : PubMed/NCBI
29	Whitelegge JP: Integral membrane proteins and bilayer proteomics. Anal Chem. 85:2558–2568. 2013. View Article : Google Scholar : PubMed/NCBI
30	Cobbold C, Monaco AP, Sivaprasadarao A and Ponnambalam S: Aberrant trafficking of transmembrane proteins in human disease. Trends Cell Biol. 13:639–647. 2003. View Article : Google Scholar : PubMed/NCBI
31	Pieck KI: More than 50% of drugs target membrane proteins. http://www.irbbarcelona.org/en/news/more-than-50-of-drugs-target-membrane-proteins (In Italian)Accessed. December 30–2014
32	Ke AW, Shi GM, Zhou J, Huang XY, Shi YH, Ding ZB, Wang XY, Devbhandari RP and Fan J: CD151 amplifies signaling by integrin α6β1 to PI3K and induces the epithelial-mesenchymal transition in HCC cells. Gastroenterology. 140:1629–1641, e15. 2011. View Article : Google Scholar : PubMed/NCBI
33	Sadej R, Romanska H, Baldwin G, Gkirtzimanaki K, Novitskaya V, Filer AD, Krcova Z, Kusinska R, Ehrmann J, Buckley CD, et al: CD151 regulates tumorigenesis by modulating the communication between tumor cells and endothelium. Mol Cancer Res. 7:787–798. 2009. View Article : Google Scholar : PubMed/NCBI
34	Ke AW, Shi GM, Zhou J, Wu FZ, Ding ZB, Hu MY, Xu Y, Song ZJ, Wang ZJ, Wu JC, et al: Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma. Hepatology. 49:491–503. 2009. View Article : Google Scholar : PubMed/NCBI
35	Roland AV and Moenter SM: Regulation of gonadotropin-releasing hormone neurons by glucose. Trends Endocrinol Metab. 22:443–449. 2011. View Article : Google Scholar : PubMed/NCBI
36	Mantyh PW, Koltzenburg M, Mendell LM, Tive L and Shelton DL: Antagonism of nerve growth factor-TrkA signaling and the relief of pain. Anesthesiology. 115:189–204. 2011. View Article : Google Scholar : PubMed/NCBI
37	Binder DK and Scharfman HE: Brain-derived neurotrophic factor. Growth Factors. 22:123–131. 2004. View Article : Google Scholar : PubMed/NCBI
38	Nilsson SF and Bill A: Vasoactive intestinal polypeptide (VIP): Effects in the eye and on regional blood flows. Acta Physiol Scand. 121:385–392. 1984. View Article : Google Scholar : PubMed/NCBI
39	Rosenfeld CR, White RE, Roy T and Cox BE: Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation. Am J Physiol Heart Circ Physiol. 279:H319–H312. 2000.PubMed/NCBI