shRNA‑mediated knockdown of KNTC1 inhibits non-small-cell lung cancer through regulating PSMB8

Abstract

In view of the important roles played by Kinetochore proteins in mitosis, we believed that they may contribute to the development and progression of human cancers, which has been reported recently elsewhere. Kinetochore-associated 1 (KNTC1) participates in the segregation of sister chromatids during mitosis, the effects of which on non-small-cell lung cancer (NSCLC) remain unclear. Here, we sought to identify the biological significance of KNTC1 in NSCLC. KNTC1 protein expression in NSCLC tissues was investigated by immunohistochemistry. Lentivirus delivered short hairpin RNA (shRNA) was utilized to establish KNTC1 silence NSCLC cell lines. The effects of KNTC1 depletion on NSCLC cell proliferation, migration, apoptosis, and tumor formation were analyzed by MTT assay, wound-healing assay, transwell assay, flow cytometry assay, and in nude mouse models in vivo. After KNTC1 reduction, NSCLC cell viability, proliferation, migration, and invasion were restrained. A xenograft tumor model was also provided to demonstrate the inhibited tumorigenesis in NSCLC. In addition, the downstream mechanism analysis indicated that KNTC1 depletion was positively associated with PSMB8. The findings of the present study suggested that KNTC1 may have a pivotal role in mediating NSCLC progression and may act as a novel therapeutic target for NSCLC.

Fig. 1. KNTC1 was upregulated in NSCLC tissues.

A The expression levels of KNTC1 in NSCLC tumor tissues and para-carcinoma tissues were determined by immunohistochemical staining. B Kaplan-Meier survival analysis was performed to reveal the relationship between KNTC1 expression and prognosis of NSCLC patients. C The transfection efficiencies of shKNTC1 in NSCLC cell lines were evaluated by observing the fluorescence of GFP. Magnification times: ×200. D The KNTC1 mRNA expression in NSCLC cell lines after transfection was analyzed by qRT-PCR. E The expression of KNTC1 protein in NSCLC cell lines after transfection was detected by western blot. ***P < 0.001.

Fig. 2. KNTC1 knockdown inhibited NSCLC development in vitro.

A The cell proliferation rate was evaluated in NSCLC cell lines after transfection by MTT assay. B The effects of KNTC1 knockdown on cell cycle were determined by flow cytometry. C The effects of KNTC1 knockdown on cell apoptosis were examined by flow cytometry. D Human apoptosis antibody array was utilized to illustrate the regulation of the expression of apoptosis-related proteins by KNTC1 knockdown in A549 cells transfected with or without KNTC1. Protein expression was presented in grayscale and visualized by R studio. E The migration rate of cells was detected in NSCLC cell lines after transfection by transwell assay. Magnification times: ×200. F The migration rate of cells was detected in NSCLC cell lines after transfection by wound-healing assay. G The interaction between KNTC1 and AKT pathway was verified via the IPA analysis. H The expression of some cancer-associated factors was detected by western blot. Results were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. Exploration the underlying mechanism of KNTC1 regulating NSCLC.

A The heatmap of DEGs identified by RNA-sequencing of NCI-H1299 cells treated with shCtrl (n = 3) or shKNTC1 (n = 3). B The enrichment of the DEGs in canonical signaling pathways was analyzed by the IPA analysis. C, D The expression of several most significantly differentially expressed genes identified by qPCR (C) and western blot (D) in NCI-H1299 with shKNTC1. E MTT assay was performed to detect the levels of cell proliferation of NCI-H1299 cells after transfecting S100A10, BAG2, RRP9, DPP3, ZNF655 and PSMB8 corresponding lentiviruses. F The potential linkage of KNTC1, PSMB8 and Ephrin Receptor Signaling, Huntington’s Disease Signaling and Actin Cytoskeleton Signaling was illustrated via the IPA analysis. G The expression levels of PSMB8 in NSCLC tumor tissues and para-carcinoma tissues were determined by immunohistochemical staining. **P < 0.01.

Fig. 4. Knockdown of KNTC1 rescued the promotion effects on NSCLC by PSMB8 overexpression.

A Celigo cell counting assay was employed to show the synergistic effects of KNTC1 and PSMB8 on A549 cell proliferation. B The flow cytometry was performed to show the synergistic effects of KNTC1 and PSMB8 on A549 cell apoptosis. C Colony formation assay was used to evaluate the ability of A549 cells to form colonies in PSMB8, shPSMB8 and shKNTC1 + PSMB8 groups. D The migration rate of cells was detected in PSMB8, shPSMB8 and shKNTC1 + PSMB8 groups by wound-healing assay. E The migration rate of cells was detected in PSMB8, shPSMB8, and shKNTC1 + PSMB8 groups by transwell assay. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. KNTC1 knockdown inhibited NSCLC tumor growth in vivo.

A A nude mice model of KNTC1 knockdown was constructed. B The fluorescence intensity was obtained through injection of d-Luciferase before sacrificing the mice. C The volume of tumors was tested from feeding to sacrifice. D The weight of tumors was measured after sacrificing mice. E The photograph of tumors was taken after removing tumors. F The value of Ki-67 was detected by IHC in tumor sections. **P < 0.01, ***P < 0.001.