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. 2022 Mar 21;13(6):1808-1819.
doi: 10.7150/jca.65071. eCollection 2022.

Silencing LINC00491 Inhibits Pancreatic Cancer Progression through MiR-188-5p-induced Inhibition of ZFP91

Zhi Fang  1   2 , Bin Ruan  1   2 , Min Zhong  1   2 , Jianping Xiong  1   2 , Yanxia Jiang  3 , Zhiwang Song  1   2
Affiliations

Silencing LINC00491 Inhibits Pancreatic Cancer Progression through MiR-188-5p-induced Inhibition of ZFP91

Zhi Fang et al. J Cancer. .

Abstract

Background: Pancreatic cancer is recognized as one of the most malignant tumors with poor prognosis. Recently, long noncoding RNAs (lncRNAs) are considered as a potential prognostic biomarker of PC. However, the concrete biological effect of lncRNAs in PC remains unmasked. Herein, we explored the mechanism of LINC00491 in PC. Methods: Quantitative real-time PCR (qRT-PCR) was administrated to detected the expression of LINC00491 in PC tissues and cell lines. Loss-of-function experiment in vitro and in vivo was carried out to figure out the biological effects of LINC00491 on PC carcinogenesis. Luciferase reporter assay, subcellular fractionation, western blotting, pull-down assay and RNA immunoprecipitation assay were further used to explore the mechanism of PC tumorigenesis of LINC00491. Results: An increase of LINC00491 was detected in PC cell lines and tissues. Silencing LINC00491 in vitro and in vivo subsequently hindered cell migration, invasion, proliferation and tumor growth, respectively. Further research confirmed a negative and a positive connection of LINC00491 with microRNA 188-5p (miR-188-5p) level and Zinc finger protein 91 (ZFP91), a target of miR-188-5p, respectively. qRT-PCR and western blotting found that miR-188-5p was upregulated while LINC00491 was downregulated, concomitant with ZFP91 decreasing in PC cells or node mouse tumors, which could be significantly restored by inhibiting miR-188-5p. Besides, overexpression of miR-188-5p partially restored the inhibitory effect of LINC00491 diminution on proliferation, migration, and invasion of PC cells. Conclusion: LINC00491 promotes PC proliferation, migration, invasion via the miR-188-5p/ZFP91 axis, suggesting LINC00491 could be a new therapeutic target for PC.

Keywords: LINC00491; ZFP91; carcinogenesis; miR-188-5p; pancreatic cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
High expression of LINC00491 in PC cell lines and tissues was associated with poor prognosis. A. qRT-PCR showed a remarkable increase of LINC00491 in PC tissues. B. qRT-PCR detected the expression of LINC00491 in PC cell lines. *p<0.05, **p<0.01, ***p<0.001. At least 3 independent experiments were repeated and the results were shown by mean ± SD.
Figure 2
Figure 2
LINC00491 promoted cell migration, invasion and proliferation of PC in vitro. A. qRT-PCR was performed to validate transfection efficiency of LINC00491 siRNA in SW1990 and MIAPaCa-2. B. CCK-8 assay detected the cell proliferation viability of SW1990 and MIAPaCa-2. C. Colony formation assays showed the difference of cell proliferation in PC cells with LINC00491 silencing. D-E. Transwell assays detected the cell migration and invasion capability of SW1990 and MIAPaCa-2. *p<0.05, **p<0.01, ***p<0.001. All experiments were performed independently at least for 3 times and the results were presented by mean ± SD.
Figure 3
Figure 3
Reciprocal interaction of LINC00491 and miR-188-5p. A. The subcellular localization of LINC00491 in SW1990 and MIAPaCa-2 by subcellular fractionation assay. B-C. qRT-PCR showed the relative expression of miR-188-5p when down-regulating or up-regulating the expression of LINC00491. D-E. qRT-PCR quantified LINC00491 followed by increasing or decreasing the expression of miR-188-5p. F. The level of miR-188-5p in PC tissues. G. The remarkable negative correlation between LINC00491 with miR-188-5p in PC tissues. H. qRT-PCR revealed the expression of miR-188-5P in PC cell lines. *p<0.05, **p<0.01, ***p<0.001. At least 3 replicate experiments were performed and final results were presented by mean ± SD.
Figure 4
Figure 4
LINC00491 was a sponge for miR-188-5p. A. The sequence of miR-188-5p, wt-LINC00491 and mut-LINC00491. B-C. miR-188-5p was a target of LINC00491 validated by RIP assay and luciferase reporter assay revealed. D. qRT-PCR was conducted to evaluate the relative expression of LINC00491 after pull-down assays. *p<0.05, **p<0.01, ***p<0.001. At least 3 replicate experiments were analyzed and the results were presented by mean ± SD.
Figure 5
Figure 5
The reciprocal interaction of LINC00491 and miR-188-5p in PC cells migration, invasion and proliferation. A-B. CCK-8 assay and colony formation assay were conducted to evaluate the proliferation abilities of SW1990 and MIAPaCa-2 transfected with si-Con, si-LINC00491, miR-188-5p inhibitor, si-LINC00491 and miR-188-5p inhibitor, respectively. C-D. Transwell assays indicated the ability of migration and invasion in SW1990 and MIAPaCa-2 under the corresponding treatment. *p<0.05, **p<0.01, ***p<0.001. Each experiment was repeated in 3 independent trials and mean ± SD was used to descripted the results.
Figure 6
Figure 6
LINC00491 could up-regulate the expression of ZFP91 through binding and inhibit miR-188-5p. A. ZFP91 relative mRNA expression levels in SW1990 and MIAPaCa-2 among four different transfections. B. ZFP91 relative protein expression levels in the two PC cell lines under different treatment. *p<0.05, **p<0.01, ***p<0.001. A minimum of 3 replicates were conducted and the results were shown as mean ± SD.
Figure 7
Figure 7
Down-regulation of LINC00491 suppressed PC tumor growth and reduced the expression of ZFP91. A. The pictures of the nude mice subcutaneous tumors. B-C. The tumor weights and volumes of sh-LINC00491 nude mice group compared to sh-Con mice group. D. The relative protein expression of ZFP91 in subcutaneous tumors. E. The expression of LINC00491 in subcutaneous tumors. F. The expression level of miR-188-5p in subcutaneous tumors. *p<0.05, **p<0.01, ***p<0.001. 3 independent trials in each experiment were needed and the data were presented by mean ± SD.
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