Twist2 promotes kidney cancer cell proliferation and invasion by regulating ITGA6 and CD44 expression in the ECM-receptor interaction pathway

Abstract

Twist2 is a member of the basic helix-loop-helix (bHLH) family and plays a critical role in tumorigenesis. Growing evidence has proven that Twist2 is involved in tumor progression; however, the role of Twist2 in human kidney cancer and its underlying mechanisms remain unclear. Real-time polymerase chain reaction and Western blot analysis were used to detect the expression of Twist2 in kidney cancer cells and tissues. Cell proliferation, cell cycle, apoptosis, migration, and invasion assay were analyzed using the Cell Count Kit-8, flow cytometry, wound healing, and Transwell analysis, respectively. In this study, we showed that Twist2 was upregulated in human kidney cancer tissues compared with normal kidney tissues. Twist2 promoted cell proliferation, inhibited cell apoptosis, and augmented cell migration and invasion in human kidney-cancer-derived cells in vitro. Twist2 also promoted tumor growth in vivo. Moreover, we found that the knockdown of Twist2 decreased the levels of ITGA6 and CD44 expression. This result indicates that Twist2 may promote migration and invasion of kidney cancer cells by regulating ITGA6 and CD44 expression. Therefore, our data demonstrated that Twist2 is involved in kidney cancer progression. The identification of the role of Twist2 in the migration and invasion of kidney cancer provides a potential appropriate treatment for human kidney cancer.

Keywords: CD44; ITGA6; Twist2; kidney cancer.

Figure 1

Twist2 is upregulated in kidney cancer cell lines and tissues. Notes: (A, B) The expression of Twist2 in the normal human kidney cell line and four human kidney cancer cell lines was assessed by RT-PCR and Western blot. (C) mRNA expression of Twist2 in 21 paired primary kidney cancer tissues (Tumor) and their corresponding normal tissues (Normal) assessed by RT-PCR. (D) Expression of Twist2 in four primary kidney cancer tissues and their corresponding normal tissues measured by Western blot. *P<0.01, **P<0.001 compared with corresponding normal controls. Abbreviations: N, normal tissues; T, tumor tissues; RT-PCR, real-time polymerase chain reaction.

Figure 2

Effect of Twist2 on kidney cancer cell proliferation. Notes: (A) Successful knockdown of Twist2 was confirmed by Western blot at 48 hours after infection with shTwist2 or shNC control lentivirus. (B) 786-0 cell proliferation was detected using CKK-8 assay at 0, 12, 24, 48, and 72 hours. (C) Successful overexpression of Twist2 was confirmed by Western blot at 48 hours after infection with pWPXL-Twist2 or black pWPXL (NC) control lentivirus. (D) ACHN cell proliferation was detected using CCK-8 assay at 0, 12, 24, 48, and 72 hours. *P<0.001 compared with shNC or NC. Abbreviations: shNC, recombinant lentivirus pLKO.1-EGFP-scramble shRNA; NC, black pWPXL; CCK, Cell Counting Kit; shTwist2, recombinant lentivirus pLKO.1-EGFP-Twist2.

Figure 3

Effect of Twist2 on kidney cancer cell cycle and apoptosis. Notes: (A, B) 786-0 and ACHN cell cycle profiles were analyzed using flow cytometry. (C, D) 786-0 and ACHN cells were stained with annexin V-fluorescein, and apoptotic rate was analyzed using flow cytometry. *P<0.001 compared with shNC or NC. Abbreviations: shNC, recombinant lentivirus pLKO.1-EGFP-scramble shRNA; NC, black pWPXL; shTwist2, recombinant lentivirus pLKO.1-EGFP-Twist2.

Figure 3

Effect of Twist2 on kidney cancer cell cycle and apoptosis. Notes: (A, B) 786-0 and ACHN cell cycle profiles were analyzed using flow cytometry. (C, D) 786-0 and ACHN cells were stained with annexin V-fluorescein, and apoptotic rate was analyzed using flow cytometry. *P<0.001 compared with shNC or NC. Abbreviations: shNC, recombinant lentivirus pLKO.1-EGFP-scramble shRNA; NC, black pWPXL; shTwist2, recombinant lentivirus pLKO.1-EGFP-Twist2.

Figure 4

Effect of Twist2 on kidney cancer cell migration and invasion. Notes: (A, B) The migration of 786-0 and ACHN cells was performed in in vitro scratch wound healing assay, and photographs were taken 0, 24, and 48 hours after the wound was made. (C, D) The invasion of 786-0 and ACHN cells was performed by Transwell assay, and photographs were taken at 48 hours after incubation in Matrigel precoated Transwell chamber (× 200). *P<0.001 compared with shNC or NC. Abbreviations: shNC, recombinant lentivirus pLKO.1-EGFP-scramble shRNA; NC, black pWPXL; shTwist2, recombinant lentivirus pLKO.1-EGFP-Twist2.

Figure 5

Effect of Twist2 on the expression of ITGA6 and CD44. Notes: The mRNA and protein levels of ITGA6 and CD44 were detected by RT-PCR and Western blot in 786-0 (A, B) and ACHN cells (C, D). *P<0.001 compared with shNC or NC. Abbreviations: RT-PCR, real-time polymerase chain reaction; shNC, recombinant lentivirus pLKO.1-EGFP-scramble shRNA; NC, black pWPXL; shTwist2, recombinant lentivirus pLKO.1-EGFP-Twist2.

Figure 6

Effect of Twist2 on tumor growth in vivo. Notes: (A) 786-0 cells transfected with shNC or shTwist2 were subcutaneously injected in athymic nude mice. Tumor growth was shown 46 days after injection (n=6). (B) Tumor weight in shTwist2-treated mice was significantly decreased compared with that in control and shNC mice. (C) shTwist2 decreased tumor growth in mice compared with that in control and shNC mice. (D, E) Expression of Twist2 and Ki67 was detected by Western blot at 46 days after shNC or shTwist2 injection. Scale bars: 10 mm. *P<0.001 compared with shNC. Abbreviations: shNC, recombinant lentivirus pLKO.1-EGFP-scramble shRNA; NC, black pWPXL; shTwist2, recombinant lentivirus pLKO.1-EGFP-Twist2.