Long non-coding RNA LINC00472 inhibits oral squamous cell carcinoma via miR-4311/GNG7 axis

Abstract

Emerging studies indicate that long non-coding RNAs play important roles in oral squamous cell carcinoma (OSCC). However, the function of the majority of long non-coding RNAs is still unclear. Recently, LINC00472 has been reported to play crucial roles in multiple cancers. However, the role of LINC00472 in oral squamous cell carcinoma (OSCC) is still not clear. This study found that LncRNA LINC00472 was significantly down-regulated in several squamous cell carcinoma cancer tissues and OSCC cell lines. Over-expression of LINC00472 in OSCC cells inhibited OSCC progression and alleviated OSCC immune responses. Additionally, we confirmed that LINC00472 functioned as an hsa-miR-4311 sponge and regulated the expression of GNG7 (guanine nucleotide-binding protein, gamma 7). Also, we found that LINC00472 over-expression could suppress xenograft tumor growth in vivo. Our study provides evidence that LINC00472 plays an essential role in inhibiting oral squamous cell carcinoma progression and affecting immune responses by directly binding to hsa-miR-4311 to regulate the expression of GNG7 positively.

Keywords: GNG7; LINC00472; OSCC; hsa-miR-4311.

Graphical abstract

Figure 1.

LncRNA LINC00472 was down-regulated in several squamous cell carcinomas and OSCC cell lines (a) Transcripts per million (TPM) of LINC00472 detected in CESC, HNSC, and LUSC patients. CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; HNSC, head, and neck squamous cell carcinoma; LUSC, lung squamous cell carcinoma. T represented tumor tissues, and N represented normal tissues. (b) Expression of LINC00472 in normal human oral keratinocytes NHOK cell and 6 human OSCC cancer cell lines, CAL27, FADU, HN12, HSU3, SCC9, and SCC25. Error bars represent standard deviations, n = 3; ***p < 0.001.

Figure 2.

LINC00472 inhibited OSCC progression and affected immune responses in vitro (a) Expression level of LINC00472 after over-expression of LINC00472 quantified using qPCR. (b) Cell proliferation of FADU cells after over-expression of LINC00472 evaluated using MTT assay. (c) The migration ability of FADU cells after over-expression of LINC00472 was measured by transwell experiments (without Matrigel). (d) Quantification result of (C). (e) The invasion ability of FADU cells after over-expression of LINC00472 was measured by transwell experiments (with Matrigel). (f) Quantification result of (E). (g) The expression level of CXCL9 and CXCL10 after over-expression of LINC00472 was quantified using qPCR. (h) CXCL9 and CXCL10 levels after over-expression of LINC00472 were detected using Western blot. Scale bar = 50 μm. Error bars represent standard deviations, n = 3; **p < 0.01, ***p < 0.001.

Figure 3.

LINC00472functioned as the sponge of hsa-miR-4311 (a) hsa-miR-4311 was up-regulated in FADU cells. (b) The relative expression level of hsa-miR-4311 after over-expression of LINC00472 was quantified using qPCR. (c) Dual-luciferase assay in FADU cells transfected with either with control or miR-4311 mimics in LINC00472. (d) The correlation of LINC00472 and hsa-miR-4311 was confirmed using RNA immune-precipitation assay with the anti-AGO2 antibody. (e) Cell proliferation of FADU cells after the inhibition of hsa-miR-4311 evaluated using MTT assay. (f) The migration ability of FADU cells after the inhibition of hsa-miR-4311 was measured by transwell experiments (without Matrigel). (g) Quantification result of (F). (h) The invasion ability of FADU cells after the inhibition of hsa-miR-4311 was measured by transwell experiments (with Matrigel). (i) Quantification result of (H). (j) The expression level of CXCL9 and CXCL10 after the inhibition of hsa-miR-4311 was quantified using qPCR. (k) CXCL9 and CXCL10 levels after the inhibition of hsa-miR-4311 were detected using Western blot. Scale bar = 50 μm. Error bars represent standard deviations, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

LINC00472 inhibited OSCC progression and affected immune response by regulating the expression of GNG7 (a) Dual-luciferase assay in FADU cells transfected with either with control or miR-4311 mimics in GNG7. (b) Expression of GNG7 in normal human oral keratinocytes NHOK cell and 6 human OSCC cancer cell lines, CAL27, FADU, HN12, HSU3, SCC9, and SCC25. (c) GNG7 level after over-expression of LINC00472 or down-regulation of hsa-miR-4311; GNG7 level after down-regulation of LINC00472 or up-regulation of hsa-miR-4311. (d) Cell proliferation of FADU cells after up-regulation of hsa-miR-4311, GNG7, or hsa-miR-4311+ GNG7 evaluated using MTT assay. (e) The migration ability of FADU cells after up-regulation of hsa-miR-4311, GNG7, or hsa-miR-4311+ GNG7 measured by transwell experiments (without Matrigel). (f) The invasion ability of FADU cells after up-regulation of hsa-miR-4311, GNG7, or hsa-miR-4311+ GNG7 measured by transwell experiments (with Matrigel). (g) Quantification result of (E). (h) Quantification result of (F). (i) The expression level of CXCL9 and CXCL10 after up-regulation of hsa-miR-4311, GNG7, or hsa-miR-4311+ GNG7 were quantified using qPCR. (j) CXCL9 and CXCL10 levels after up-regulation of hsa-miR-4311, GNG7, or hsa-miR-4311+ GNG7 quantified using Western blot. Scale bar = 50 μm. Error bars represent standard deviations, n = 3; **p < 0.01, ***p < 0.001.

Figure 5.

LINC00472 alleviated oral squamous cell carcinoma and inhibited tumor metastasis in vivo (a) Control or LINC00472-overexpressing FADU cells were injected into the right rear flank of nude mice. The representative pictures of the xenografted tumors. (b) Quantification result of the tumor size of (A). (c) GNG7 level in the OSCC tumor tissues detected by WB. (d) The levels of proliferation markers, PCNA, and ki-67 in the OSCC tumor tissues detected by WB. (e) Ratio of ki-67 positive cells detected by IHC staining. (f) HE staining of metastatic nodules in the lungs of the control or LINC00472-overexpressing group. Scale bar = 20 μm. Error bars represent standard deviations, n = 6; **p < 0.01.