The effect of LncRNA NNT-AS1 targeting miR-199a-5p on the proliferation, migration and invasion of oral squamous cell carcinoma cells

doi:10.11958/20251271

Abstract

Abstract:

Objective To investigate the effect of long non-coding RNA (LncRNA) NNT-AS1 on proliferation, migration and invasion of oral squamous cell carcinoma (OSCC) cells by regulating microRNA (miR)-199a-5p. Methods CAL-27 cells were assigned into the control group, the sh-NC group, the sh-NNT-AS1 group, the sh-NNT-AS1+miR-In-NC group and the sh-NNT-AS1+miR-199a-5p-In group. The proliferation, colony formation, migration and invasion abilities of CAL-27 cells were detected by EdU method, clone formation, scratch healing and Transwell experiments. The expressions of LncRNA NNT-AS1 and miR-199a-5p in human keratinocyte cell line HaCaT, human OSCC cell line CAL-27 and each group of cells were detected by qRT-PCR experiment. Western blot experiments were performed to detect the expressions of epithelial-mesenchymal transition (EMT)-related proteins [E-cadherin, N-cadherin, Vimentin, and epidermal growth factor receptor (EGFR)]. The targeting relationship between LncRNA NNT-AS1 and miR-199a-5p was verified by dual luciferase reporter gene assay. Results Compared with HaCaT cells, the level of LncRNA NNT-AS1 was elevated in CAL-27 cells, while the level of miR-199a-5p was decreased (P<0.05). Compared with the control group and the sh-NC group, the EdU positive rate, colony formation rate, scratch healing rate, cell invasion number, expression levels of LncRNA NNT-AS1, N-cadherin, Vimentin and EGFR proteins were decreased in the sh-NNT-AS1 group, while the expression levels of miR-199a-5p and E-cadherin protein were increased (P<0.05). Compared with the sh-NNT-AS1 group and the sh-NNT-AS1+miR-In-NC group, the EdU positive rate, colony formation rate, scratch healing rate, cell invasion number, N-cadherin, Vimentin and EGFR protein expression levels were increased in the sh-NNT-AS1+miR-199a-5p-In group, while the expression levels of miR-199a-5p and E-cadherin protein were decreased (P<0.05). Compared with the miR-NC group, the relative luciferase activity of NNT-AS1-WT cells transfected with miR-199a-5p mimic was decreased (P<0.05). Conclusion LncRNA NNT-AS1 is highly expressed in OSCC. It can promote the proliferation, migration, invasion and EMT process of OSCC cells by negatively regulating miR-199a-5p.

Key words: squamous cell carcinoma of head and neck, mouth neoplasms, RNA, long noncoding, long non-coding RNA NNT-AS1, microRNA-199a-5p

CLC Number:

R739.85

Figures/Tables 11

References 22

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基因名称	引物序列（5'→3'）	产物大小/bp
LncRNA NNT-AS1	上游：ACGTGCAGACAACATCTACCT	168
LncRNA NNT-AS1	下游：TACAACACCTTCCCGCAT	168
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	189
GAPDH	下游：CTGGCCCGGAAGACATCTG	189
miR-199a-5p	上游：CGCGCCCAGTGTTCAGACTAC	124
miR-199a-5p	下游：AGTGCAGGGTCCGAGGTATT	124
U6	上游：CTCGCTTCGGCAGCACA	116
U6	下游：AACGCTTCACGAATTTGCGT	116

基因名称	引物序列（5'→3'）	产物大小/bp
LncRNA NNT-AS1	上游：ACGTGCAGACAACATCTACCT	168
LncRNA NNT-AS1	下游：TACAACACCTTCCCGCAT	168
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	189
GAPDH	下游：CTGGCCCGGAAGACATCTG	189
miR-199a-5p	上游：CGCGCCCAGTGTTCAGACTAC	124
miR-199a-5p	下游：AGTGCAGGGTCCGAGGTATT	124
U6	上游：CTCGCTTCGGCAGCACA	116
U6	下游：AACGCTTCACGAATTTGCGT	116

组别	EdU阳性率/%	集落形成率/%	划痕愈合率/%	细胞侵袭数量/个
对照组	41.95±5.81	36.67±4.33	82.15±10.36	123.67±14.82
sh-NC组	40.26±5.37	34.83±4.17	79.72±9.19	119.83±12.38
sh-NNT-AS1组	17.42±2.23^ab	16.50±2.50^ab	38.41±4.54^ab	57.67±6.23^ab
sh-NNT-AS1+miR-In-NC组	18.35±2.19	15.17±1.83	36.67±4.83	55.33±5.96
sh-NNT-AS1+miR-199a-5p-In组	39.73±5.54^cd	31.33±4.67^cd	80.53±9.61^cd	106.50±12.14^cd
F	45.474^＊＊	47.048^＊＊	51.442^＊＊	56.876^＊＊

组别	EdU阳性率/%	集落形成率/%	划痕愈合率/%	细胞侵袭数量/个
对照组	41.95±5.81	36.67±4.33	82.15±10.36	123.67±14.82
sh-NC组	40.26±5.37	34.83±4.17	79.72±9.19	119.83±12.38
sh-NNT-AS1组	17.42±2.23^ab	16.50±2.50^ab	38.41±4.54^ab	57.67±6.23^ab
sh-NNT-AS1+miR-In-NC组	18.35±2.19	15.17±1.83	36.67±4.83	55.33±5.96
sh-NNT-AS1+miR-199a-5p-In组	39.73±5.54^cd	31.33±4.67^cd	80.53±9.61^cd	106.50±12.14^cd
F	45.474^＊＊	47.048^＊＊	51.442^＊＊	56.876^＊＊

组别	LncRNA NNT-AS1	miR-199a-5p
对照组	1.00±0.00	1.00±0.00
sh-NC组	1.02±0.13	0.98±0.11
sh-NNT-AS1组	0.42±0.07^ab	3.05±0.43^ab
sh-NNT-AS1+miR-In-NC组	0.45±0.08	2.89±0.38
sh-NNT-AS1+miR-199a-5p-In组	0.44±0.08	1.12±0.14^cd
F	85.647^＊＊	94.011^＊＊