Research of Biological Functions and Potential Molecular Mechanisms of TTC9 Gene in Lung Adenocarcinoma

Introduction

Lung adenocarcinoma (LUAD) is one of the most commonly diagnosed subtypes of lung cancer, and one of the deadliest cancers. Tetratricopeptide repeat domain 9A (TTC9) is upregulated and has played an oncogenic role in some malignant tumors. However, the expression and role of TTC9 has not yet been elucidated in LUAD. Researching the biological functions and potential molecular mechanism of the TTC9 gene in LUAD is important for finding targets for LUAD and for the development of therapies.

Methods

LUAD tissues and non-tumor tissues were confirmed by experienced pathologists. All cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% streptomycin at 37 °C with 5% CO₂.

To analyze the expression profile of TTC9 mRNA, transcriptome sequencing data from the Cancer Genome Atlas database were mined, and genetic and epigenetic analyses, correlation analysis of differentially expressed genes with TTC9, tumor microenvironment analysis and gene set enrichment analysis were performed, respectively. To further elucidate the biological functions and molecular mechanisms of TTC9 in LUAD, lentivirus design and transfection assays, qPCR and protein blotting assays, cell counting assays, wound healing assays, transwell migration assays, cell invasion assays, apoptosis and cell cycle assays, and animal experiments were performed.

Results

In the pan-cancer expression profiles, the mRNA expression of TTC9 was found to be significantly up-regulated in a variety of malignant tumors, including LUAD. These results confirmed the overexpression of TTC9 in lung adenocarcinoma. TCGA-LUAD data were further integrated to analyze the clinical significance of TTC9 mRNA expression in patients with LUAD. The results revealed that high TTC9 mRNA expression was significantly correlated with female, lymph node invasion, residual tumor and outcome status. High TTC9 mRNA expression was an independent risk factor for unfavorable prognosis in patients with LUAD. The relationship between TTC9 mRNA expression and DNA methylation levels was investigated by data analysis of the TCGA and CCLE databases.

Data mining through TCGA and CCLE databases revealed that TTC9 mRNA expression was negatively correlated with DNA methylation at both tissue and cellular levels. DEGs co-expressed with TTC9 in LUAD were identified. the most significant genes positively correlated with TTC9 were TSPAN15, SYTL2 and GPR110. They were significantly upregulated in primary LUAD tumor tissues and play a key role in the regulation of cell development, growth, invasion, metastasis and cytokinesis. In addition, TTC9 might play a role in regulating humoral and cellular immune responses, which in turn affected the tumor microenvironment of LUAD. The results of these bioinformatics analyses suggest that TTC9 may be a promising biomarker in LUAD.

Fig. 1 The possible mechanism of TTC9 gene regulation in LUAD.

To explore the biological function of TTC9 gene, the lentivirus interference technology was used to silence the expression of TTC9 gene in LUAD cell line. The results of CCK-8 and mice xenograft model experiments showed that silencing TTC9 gene significantly inhibited the proliferation of LUAD cells in vitro and in vivo. Knockdown of TTC9 promoted cell apoptosis, inhibited cell migration and invasion, and induced cell cycle arrest in G2 phase.

Fig. 2 Relationship between TTC9 expression, immune infiltration and survival in patients with LUAD.

Furthermore, Western blot results confirmed that inhibition of TTC9 resulted in the dephosphorylation of p38 MAPK signaling pathway associated with the initiation and progression of biological processes in human cancers and decreased the expression of p-p38 and p38. Knockdown of TTC9 increased the expression of BAX, Caspase-3 and E-cadherin, while decreased the expression of N-cadherin. These findings revealed that inhibition of TTC9 could promote cell apoptosis, inhibit the proliferation, invasion and metastasis of LUAD cells through p38 MAPK signaling pathway. Thus, these results highlight the candidate eligibility of TTC9 as an effective target for therapeutic intervention in LUAD.

Fig. 3 Effect of TTC9 on signaling pathway-related proteins and tumorigenicity of LUAD cells.

Summary

This article investigates the expression profile, biological functions and potential molecular mechanisms of the TTC9 gene in LUAD. TTC9 expression was significantly overexpressed in LUAD tissues compared with that in normal lung tissues and was closely correlated with gender, lymph node metastasis, and survival status. Knockdown of TTC9 promoted PC9 cell apoptosis and inhibited cell proliferation, migration and invasion, leading to cell cycle arrest in G2 phase. Moreover, inhibition of TTC9 suppressed the tumorigenicity of PC9 cells in nude mice. TTC9 might serve as oncogene in LUAD through cancer-related signaling pathways including p38 MAPK pathway. The expression of TTC9 gene might be modulated by DNA copy number variant and DNA methylation. Therefore, TTC9 may be a novel therapeutic target for the treatment of LUAD.

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Reference:

Huang, X.; et al. Knockdown of TTC9 inhibits the proliferation, migration and invasion, but induces the apoptosis of lung adenocarcinoma cells. Heliyon, 2022, 8(11): e11254.

Transfected stable cell lines	Premade virus particles	Clones
Panoply™ Human TTC9 Knockdown Stable Cell Line	Human TTC9 adenoviral particles	Rabbit TTC9 ORF clone (XM_008248540.1)	human TTC9 ORF clone (NM_015351.1)	Mouse Ttc9 ORF clone (NM_001033149.2)	Rat Ttc9 ORF Clone (NM_001134731.1)
Panoply™ Human TTC9 Over-expressing Stable Cell Line	Human TTC9 (NM_015351) lentivirus particles	Human TTC9 cDNA Clone (NM_015351.1)	Rat Ttc9 cDNA Clone (NM_001134731.1)	TTC9 miRNA 3'UTR clone

* For research use only. Not intended for any clinical use.

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