LINC01232 exerts oncogenic activities in pancreatic adenocarcinoma via regulation of TM9SF2

Abstract

Pancreatic adenocarcinoma (PAAD), one of the most prevailing malignant tumors in digestive system, is identified as one of the main culprits of cancer-associated mortality. Despite long intergenic non-protein coding RNA 1232 (LINC01232) is found to be upregulated in TCGA PAAD tissues and associated with poor prognosis, the potential of LINC01232 in PAAD progression still needs more explorations. In this study, LINC01232 was chosen to be the research object in PAAD cellular processes. Functionally, loss-of function assays were carried out and the experimental results indicated that suppression of LINC01232 hindered the deterioration of PAAD by affecting cell proliferation and migration. Furthermore, relationship between LINC01232 and its nearby gene transmembrane 9 superfamily member 2 (TM9SF2) was investigated. The same expression pattern of TM9SF2 in TCGA PAAD samples was observed. It was found that upregulation of LINC01232 could be a crucial factor for the dysregulation of TM9SF2. Mechanistically, LINC01232 recruited EIF4A3 to boost TM9SF2 mRNA stability. Besides, our findings demonstrated that the transcriptional activation of LINC01232 and TM9SF2 was mediated by SP1. Therefore, we concluded that LINC01232 executed carcinogenic properties in PAAD progression via regulation of TM9SF2. In conclusion, this study was the first to unveil the role and molecular mechanism of LINC01232, suggesting LINC01232 as a promising molecular target for pancreatic cancer treatment.

Fig. 1. LINC01232 expression is upregulated in PAAD samples and associated with poor patients’ prognosis.

a TCGA data of LINC01232 expression in PAAD and normal samples. b TCGA survival analysis of PAAD patients with high or low LINC01232 expression. c LINC01232 expression was detected in 40 pairs of PAAD tissues and adjacent normal tissues. d qRT-PCR assay was implemented to estimate the expression level of LINC01232 in normal cell line (HPDE6-C7) and PAAD cells (CAPAN-1, BxPC-3, JF305, PANC-1, and SW1990). *P < 0.05, **P < 0.01

Fig. 2. Silencing of LINC01232 hampered cell proliferation and promoted cell apoptosis in PAAD.

a The efficiency of LINC01232 knockdown in PANC-1 and SW1990 cells was verified by qRT-PCR. b, c Cell proliferative ability was assessed by MTT and colony formation assays after LINC01232 knockdown. d, e TUNEL-staining assay and flow cytometry analysis were adopted to determine the apoptosis of PANC-1 and SW1990 cells after indicated transfection. *P < 0.05, **P < 0.01

Fig. 3. Knockdown LINC01232 led to the repression of cell migration, invasion and EMT in PAAD.

a–c Transwell assays were carried out to detect cell migration and invasion in LINC01232-downregulated PAAD cells. d The levels of EMT-related proteins was measured by western blotting. e Immunofluorescence assay was applied to further confirm the levels of E-cadherin and N-cadherin in PANC-1 and SW1990 cells. f The phenotype of PANC-1 and SW1990 cells transfected with different plasmids was observed. **P < 0.01

Fig. 4. LINC01232 positively regulated the expression of TM9SF2.

a Upregulation of TM9SF2 expression in TCGA PAAD tissues. b TCGA correlation analysis of LINC01232 and TM9SF2. c TM9SF2 expression and its correlation with LINC01232 in 40 PAAD tissues. d TM9SF2 expression in normal cell line and PAAD cell lines. e mRNA and protein level of TM9SF2 were examined in PAAD cells transfected with LINC01232-specific shRNA and control shRNA. f, g Subcellular fractionation and FISH assay were carried out to determine the nuclear-cytoplasmic fractionation of LINC01232 in PAAD cells. h Ago2-RIP experiment was utilized to estimate the capability of LINC01232 to combine with RISC complex. *P < 0.05, **P < 0.01

Fig. 5. LINC01232 recruited EIF4A3 to stabilize TM9SF2.

a Pull-down silver staining and the peak map for mass spectrometry analysis were used to demonstrate the interaction between EIF4A3 and LINC01232. b, c RIP assay was applied to validate the binding of EIF4A3 to LINC01232 and TM9SF2. d The effect of LINC01232 knockdown on the binding of EIF4A3 to TM9SF2 was assessed by RIP assay. e, f After Actinomycin D treatment, the mRNA stability of TM9SF2 in indicated cells was determined by qRT-PCR. *P < 0.05

Fig. 6. LINC01232 and TM9SF2 synergistically promoted PAAD progression.

a, b Cell proliferation in cells transfected with sh-NC, sh-LINC01232, or co-transfected with sh-LINC01232 and TM9SF2 expression vector was measured by the MTT and colony formation assays. c Cell apoptosis rate was evaluated by caspase-3 activity assay. d, e Cell migratory and invasive capacities were determined by transwell assays. f EMT process was estimated by western blotting. g Tumors derived from cells transfected with different plasmids. h, i Tumor volume and tumor weight in different groups were calculated and shown. *P < 0.05, **P < 0.01

Fig. 7. SP1 acted as a transcriptional activator of LINC01232 and TM9SF2 in PAAD cells.

a After transcription factors were respectively overexpressed, we observed that upregulation of SP1 led to the most significant increase of TM9SF2 and LINC01232 expression. b The levels of TM9SF2 and LINC01232 in response to inhibition of SP1 were detected by qRT-PCR. c ChIP assay revealed the binding capability of SP1 to LINC01232 and TM9SF2 promoters. d DNA motif of SP1. e, f Specific binding sites of SP1 for LINC01232 and TM9SF2 promoters were validated by luciferase reporter gene assays. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

Graphical abstract illustrated the function and molecular mechanism of LINC01232 in PAAD progression