LINC00958 regulated miR-627-5p/YBX2 axis to facilitate cell proliferation and migration in oral squamous cell carcinoma

Abstract

Oral squamous cell carcinoma (OSCC), the subtype of head and neck cancers, is notorious for its high incidence and death rate. The role of long non-coding RNAs (lncRNAs) is discovered to be significant for the canceration and cancer progression. Long intergenic non-protein coding RNA 958 (LINC00958) is discovered as a carcinogene in multiple cancers, such as gastric cancer, pancreatic cancer, and glioma, but there has been no report about how LINC00958 functions in OSCC. The objective of our study is to unfold function and mechanism investigation on LINC00958 in OSCC. First, TCGA database showed the upregulation and prognostic significance of LINC00958 in head and neck squamous carcinoma. Then, we discovered in OSCC clinical samples that LINC00958 presented high expression and predicted poor prognosis. Also, LINC00958 was elevated in OSCC cells. In vitro gain- and loss-function experiments proved that LINC00958 facilitated cell growth, retarded apoptosis, accelerated migration, and epithelial-to-mesenchymal transition (EMT) in OSCC. Mechanistically, we confirmed the cytoplasmic expression of LINC00958 in OSCC cells, and revealed that LINC00958 sequestered miR-627-5p to upregulate YBX2 expression. Rescue assays indicated that LINC00958 regulated OSCC cell proliferation, motility and EMT through YBX2. Together, we showed that LINC00958 promoted OSCC progression through miR-627-5p/YBX2 axis, indicating LINC00958 as a new prognostic marker, and provided new perspectives for molecular targeted treatment for OSCC.

Keywords: LINC00958; OSCC; YBX2; miR-627-5p.

Figure 1.

Upregulation and Clinical Significance of LINC00958 in OSCC. (a) TCGA data showed the upregulation of LINC00958 in head and neck squamous carcinoma (HNSC). (b) TCGA database showed that LINC00958 upregulation indicated poor prognosis in HNSC. (c) RT-qPCR analyses showed the upregulation of LINC00958 in OSCC tissues. (d) Kaplan-Meier analysis and log-rank test showed that high LINC00958 expression predicted poor prognosis in OSCC. (e) RT-qPCR analyses confirmed the upregulation of LINC00958 in OSCC cell lines. *P < .05; **P < .01.

Figure 2.

LINC00958 Aggravated Cell Proliferation and Attenuated Apoptosis in OSCC. (a) RT-qPCR results of LINC00958 silencing by shLINC00958#1 or shLINC00958#2 in SCC15 cells and its overexpression by pcDNA3.1/LINC00958 in Fadu cells. (b) CCK-8 results of cell proliferation in response to LINC00958 knockdown or overexpression. (c) Colony formation results of cell proliferation in response to LINC00958 knockdown or overexpression. (d) The ratio of EdU positive cells upon LINC00958 knockdown or overexpression. (e) Caspase-3 activity in OSCC cells upon LINC00958 knockdown or overexpression. **P < .01; ***P < .001.

Figure 3.

LINC00958 Facilitated Cell Migration and EMT in OSCC. (a) Cell migration upon LINC00958 knockdown or overexpression was assessed by transwell migration assay. (b–c) The mRNA and protein levels of E-cadherin and N-cadherin upon LINC00958 knockdown or overexpression were detected by RT-qPCR and western blot analyses. (d) IF results of the expression of E-cadherin and N-cadherin upon LINC00958 knockdown or overexpression. **P < .01.

Figure 4.

LINC00958 Targeted miR-627-5p in OSCC. (a–b) Subcellular fractionation and FISH assay showed the cytoplasmic localization of LINC00958 in OSCC cells. (c) RT-qPCR results showed that only miR-627-5p, instead of miR-3174, was downregulated in OSCC tissues. (d) Spearman’s correlation curve showed the negative correlation between LINC00958 and miR-627-5p in OSCC samples. (e) RT-qPCR results of the downregulation of miR-627-5p in OSCC cell lines. (f) The binding sites between LINC00958 and miR-627-5p and the mutant sites. (g) RT-qPCR results confirmed the overexpression of miR-627-5p by miR-627-5p mimic in SCC15 cells and the knockdown of miR-627-5p by miR-627-5p inhibitor in Fadu cells. (h) Luciferase reporter assay confirmed that miR-627-5p interacted with LINC00958. (i) RIP assay confirmed the interplay between miR-627-5p and LINC00958. *P < .05; **P < .01; ***P < .001.

Figure 5.

LINC00958/miR-627-5p Targeted YBX2 in OSCC. (a) The Venn pattern showed that 7 genes (PHLPP2, FCHSD2, RANBP9, EBAG9, SKIDA1, UBN2, and YBX2) were commonly predicted as the targets for miR-627-5p in four bioinformatics databases (microT, miRmap, PITA, and PicTar). (b) RT-qPCR results showed that among the seven candidate mRNAs, only YBX2 could be reduced or induced by overexpression or knockdown of miR-627-5p in OSCC cells. (c) Western blot analyses confirmed that protein levels of YBX2 could be reduced or induced by overexpression or knockdown of miR-627-5p in OSCC cells. (d) RT-qPCR and western blot analyses showed that mRNA and protein levels of YBX2 could be induced or reduced by overexpression or knockdown of LINC00958 in OSCC cells. (e) The binding sequences between YBX2 and miR-627-5p and the mutant sequences. (f) Luciferase reporter assays confirmed that miR-627-5p targeted YBX2 mRNA. (g) Luciferase reporter assay proved that LINC00958/miR-627-5p targeted YBX2 mRNA. (h) RT-qPCR results of the upregulation of YBX2 in OSCC tissues and cell lines. (i) Spearman’s correlation curve showed that YBX2 was positively correlated with LINC00958 and negatively correlated with miR-627-5p. *P < .05; ***P < .01.

Figure 6.

LINC00958 Regulated Cell Growth, Motility, and EMT through YBX2 in OSCC. (a) The silencing of YBX2 by shYBX2#1 or shYBX2#2 in Fadu cells and the overexpression of YBX2 by pcDNA3.1/YBX2 in SCC15 cells were confirmed by RT-qPCR analyses. (b–c) SCC15 cells were transfected with shNC+pcDNA3.1, shLINC00958#1+ pcDNA3.1, or shLINC00958#1+ pcDNA3.1/YBX2. Fadu cells were transfected with pcDNA3.1+ shNC, pcDNA3.1/LINC00958+ shNC, or pcDNA3.1/LINC00958+ shYBX2#1 for subsequent experiments. CCK-8 and colony formation results of SCC15 and Fadu cells in each group. (d) Caspase-activity of SCC15 and Fadu cells in each group was evaluated to assess cell apoptosis level. (e) Transwell migration results of SCC15 and Fadu cells in each group. (f–g) RT-qPCR and western blot analysis of E-cadherin and N-cadherin levels of SCC15 and Fadu cells in each group. *P < .05; **P < .01; ***P < .01.