The expression and role of hypoxia-induced long noncoding RNA LINC00970 in salivary adenoid cystic carcinoma cells

doi:10.3877/cma.j.issn.1674-1366.2023.03.009

Abstract

Abstract:

Objective

To investigate the relationship and function between the expression of long noncoding RNA (lncRNA) LINC00970 and hypoxia in salivary adenoid cystic carcinoma (SACC) cells.

Methods

After 48 hours of hypoxia (1% O₂) induction, lncRNA chip was used to detect the lncRNA induced by hypoxia in SACC-83 cells and verified by qRT-PCR. Clinical samples were used to analyze the expression and prognostic impact of LINC00970 in SACC tissues. After transfection of SACC-LM and SACC-83 cells with LINC00970 shRNA plasmids, the cell proliferation, clonal formation ability, cell invasion and migration ability, and the expression of E-cadherin and N-cadherin were detected by CCK-8, plate cloning, Transwell assay and Western blot, respectively. Experiments such as fluorescence quantitative PCR, Western blot and Transwell were repeated three times to get the average value of the sample, and SPSS 20.0 software was used for statistical analysis. The difference between the relative expression of LINC00970 between SACC tissue cells and normal salivary gland tissue was analyzed by paired t-test. The independent t-test was used to compare the influence of silent LINC00970 on the proliferation, cloning, invasion and migration ability of SACC-83 and SACC-LM cells, as well as the effect of LINC00970 silence on EMT-related proteins. The KaplanMeier method was used to calculate the correlation between the expression of LINC00970 and the total survival time of SACC patients, and to draw the survival curve. P<0.05 was taken as statistically significant for the difference.

Results

The results of lncRNA microarray and qRT-PCR showed that LINC00970 was the most highly expressed lncRNA in SACC-83 cells in response to hypoxia (15.13 ± 0.57 vs 1.08 ± 0.06, t = 24.56, P<0.001) . Analysis of clinical sample data showed that the expression of LINC00970 in SACC tissues was significantly higher than that in normal salivary gland tissues (0.49 ± 0.41 vs 0.08 ± 0.16, t = 2.53, P<0.001) , and the survival rate of patients with high expression of LINC00970 was significantly lower than that of patients with low expression of LINC00970 (HR = 0.42, P = 0.03) . After the knockdown of LINC00970 in SACC cells, the invasion and migration ability promoted by hypoxia were inhibited, and the expression of interstitial marker N-cadherin was down-regulated after increased hypoxia, while the expression of epithelial marker E-cadherin was up-regulated after hypoxia-induced decrease.

Conclusions

Hypoxia can induce the expression of LINC00970 in SACC cells, with its high expression closely related to the poor prognosis of SACC patients. LINC00970 may promote cell invasion and migration by regulating EMT.

Key words: Carcinoma, adenoid cystic, Salivary, Cell hypoxia, RNA, long noncoding, Epithelial-mesenchymal transition

Yunying Zhu, Xiaolin Gao, Yanping Ge, Zhangsong Wang, Zhaoyu Lin, Jinsong Li, Donghui Wu. The expression and role of hypoxia-induced long noncoding RNA LINC00970 in salivary adenoid cystic carcinoma cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2023, 17(03): 210-217.

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References 23

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基因名称	上游引物	下游引物
LINC00970	TTGGATTCCCTGCTGGTTTC	TCCTTCACAGCGTCAAATGG
ADAMTSL4-AS1	AGCAAGGAACTGCAGACTTG	TGTGCTTTTGGGACTGCATG
HOTTIP	TTTTTGGGGCGGTGTTTACC	AAACCGAAGAGGCTCAAAGC
LINC00658	AATCACCAGCACCACTTTGC	AGGGTCTCTCATGATGTTGCAG
MESTIT1	ACAACACAGGCCAGAAACAC	ACATGTTCCATGGCCTTCAG
LINC01149	AACAATTCACCCACCTTGGC	AGACTATCGATGCTGGGAAAGG
RPS6KA2-AS1	CAACACAGGTGAAATGGCTCAG	AGGAAGAAACCGCAAGCAAC
LINC00896	ACCTTGCCTGCTGAGACATAG	AAACCCAACACGATGACTGC
LIFR-AS1	TGGCCAAGTTTGCAGTCATC	GGCTGCAGTGAGCTGTAATTG
LINC00351	AACTCCGGACACATTTGAGC	AATGAAGCTGCAGACCCTTG

基因名称	上游引物	下游引物
LINC00970	TTGGATTCCCTGCTGGTTTC	TCCTTCACAGCGTCAAATGG
ADAMTSL4-AS1	AGCAAGGAACTGCAGACTTG	TGTGCTTTTGGGACTGCATG
HOTTIP	TTTTTGGGGCGGTGTTTACC	AAACCGAAGAGGCTCAAAGC
LINC00658	AATCACCAGCACCACTTTGC	AGGGTCTCTCATGATGTTGCAG
MESTIT1	ACAACACAGGCCAGAAACAC	ACATGTTCCATGGCCTTCAG
LINC01149	AACAATTCACCCACCTTGGC	AGACTATCGATGCTGGGAAAGG
RPS6KA2-AS1	CAACACAGGTGAAATGGCTCAG	AGGAAGAAACCGCAAGCAAC
LINC00896	ACCTTGCCTGCTGAGACATAG	AAACCCAACACGATGACTGC
LIFR-AS1	TGGCCAAGTTTGCAGTCATC	GGCTGCAGTGAGCTGTAATTG
LINC00351	AACTCCGGACACATTTGAGC	AATGAAGCTGCAGACCCTTG

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