Abstract:

Objective To investigate the targeting relationships involving miR-331-3p，BCL6 and ITGA9， and to examine their effects on the proliferation， migration， invasion， and immune cell infiltration levels of adenoid cystic carcinoma （ACC） cells. Methods A dual-luciferase reporter assay was employed to verify the targeting relationships among miR-331-3p， BCL6， and ITGA9. The miR-331-3p mimic and cells transfected with pcDNA3.1-BCL6 plasmid were established using salivary adenoid cystic carcinoma-83 （SACC-83） tumor cells. Real-time quantitative PCR was used to detect the mRNA expression levels of miR-331-3p， BCL6， and ITGA9. Western blotting was performed to detect the protein relative expression levels of BCL6 and ITGA9. Cell proliferation was assessed using the CCK-8 assay. Cell migration and invasion were evaluated using scratch wound healing and Transwell assays， respectively. Furthermore， transfection models were established in ACC metastasis （ACC-M） cells using empty vector plasmids， ITGA9 and BCL6 overexpression plasmids， miRNA NC mimic， and miR-331-3p mimic. Western blotting was performed to detect the protein relative expression levels of BCL6 and ITGA9. Real-time quantitative PCR was used to detect the relative expression levels of chemokine genes secreted by tumor cells （CXCL9， CXCL10， and CXCL11）. Results The dual luciferase assay results indicated that miR-331-3p could specifically bind to BCL6. BCL6 could specifically bind to ITGA9. In SACC-83 cells， the relative expression levels of miR-331-3p were 1.01±0.16， 0.67±0.05， and 0.35±0.12 in the mimic+NC group， mimic+BCL6 group， and mimic NC+BCL6 group， respectively. The BCL6 mRNA relative expression levels were 1.01±0.21， 2.06±0.27， and 4.48±0.36， respectively， while the ITGA9 mRNA relative expression levels were 1.00±0.04， 0.55±0.07， and 0.35±0.17， respectively， with statistically significant differences （F=22.28， P=0.002； F=116.65， P<0.001； F=26.72， P=0.001）. The expression levels of miR-331-3p and ITGA9 decreased progressively， whereas BCL6 expression increased progressively in the above three groups （all P<0.05）. The ITGA9 protein relative expression levels in SACC-83 cells were 0.96±0.03， 0.87±0.02， and 0.67±0.05 in the mimic+NC group， mimic+BCL6 group， and mimic NC+BCL6 group， respectively. The BCL6 protein relative expression levels were 0.92±0.01， 1.13±0.02， and 1.72±0.02， respectively， with statistically significant differences （F=61.87， P<0.001； F=7.18， P=0.026）. The ITGA9 protein relative expression decreased progressively， while BCL6 protein relative expression increased progressively in the above three groups （all P<0.05）. The proliferation activities of SACC-83 cells were 100.00%±6.05%， 82.03%±2.47%， and 53.18%±5.47% in the mimic+NC group， mimic+BCL6 group， and mimic NC+BCL6 group， respectively. The apoptosis rates in these groups were 6.21%±0.23%， 15.31%±0.74%， and 27.47%±1.09%， respectively； migration rates were 54.17%±9.15%， 34.37%±5.84%， and 11.17%±5.25%， respectively； invasion rates were 52.33%±8.74%， 36.67%±4.73%， and 14.00%±2.00%， respectively， with statistically significant differences （F=69.15， P<0.001； F=572.93， P<0.001； F=28.66， P=0.001； F=32.56， P=0.001）. The proliferation activity， migration rate， and invasion rate of SACC-83 cells decreased progressively， while the apoptosis rate increased progressively in the above three groups （all P<0.05）. After ACC-M cells were transfected with the overexpression vectors of ITGA9 and miR-331-3p mimic， the mRNA relative expression levels of CXCL9， CXCL10， and CXCL11 all decreased （all P<0.05）； after transfection with BCL6 vectors respectively， the mRNA relative expression levels of CXCL9， CXCL10， and CXCL11 all increased （all P<0.05）. Conclusions miR-331-3p promotes ITGA9 expression by inhibiting BCL6. This regulation enhances the proliferation， migration， and invasion capabilities of ACC cells； reduces the infiltration of cytotoxic lymphocytes within the tumor； and thereby promotes tumor progression.

Key words: Carcinoma, adenoid cystic, MicroRNAs, Immune infiltration, Signaling axis