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, 8 (17), 28226-28236

MiR-let-7a Inhibits Cell Proliferation, Migration, and Invasion by Down-Regulating PKM2 in Cervical Cancer

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MiR-let-7a Inhibits Cell Proliferation, Migration, and Invasion by Down-Regulating PKM2 in Cervical Cancer

Man Guo et al. Oncotarget.

Abstract

In recent decades, miRNA has been reported as a crucial modulator in some biology progressions. This work aims to assess the expression and role of miR-let-7a and pyruvate kinase muscle isozyme M2 (PKM2) in CC tissues and cell lines. Here, we identified that miR-let-7a expression was decreased in CC tissues, and SiHa and HeLa cells (all P < 0.001), however, PKM2 expression was increased in these samples. Statistically, miR-let-7a was inversely associated with PKM2 mRNA or protein (p = 0.013, p = 0.015, respectively). In-vitro assays revealed that ectopic miR-let-7a expression repressed SiHa and HeLa cell proliferation, migration and invasion, and enhanced SiHa and HeLa cell apoptosis. Furthermore, luciferase reporter assays revealed the 3'-UTR of PKM2 was identified a target of miR-let-7a, by which miR-let-7a affected the expression of PKM2 in SiHa and HeLa cells. Besides, PKM2 plasmids partially abrogated the inhibitory effects of miR-let-7a, while si-PKM2 enhanced the inhibitory effects of miR-let-7a. In vivo, miR-let-7a mimics indeed repressed tumor growth in mice xenograft model. In conclusion, our results demonstrated that miR-let-7a inhibits cell proliferation, migration and invasion by down-regulation of PKM2 in cervical cancer. miR-let-7a/PKM2 pathway may be a useful therapeutic target for CC patients.

Keywords: CC; PKM2; miR-let-7a.

Conflict of interest statement

CONFLICTS OF INTEREST

None.

Figures

Figure 1
Figure 1. Expression of miR-let-7a in human CC tissues and cell lines
(A) Relative miR-let-7a messenger RNA-expression levels in representative three samples and cell lines, including SiHa and HeLa were detected by RT-PCR analysis. The average expression was normalized to U6 expression. Each bar represents the mean of three independent experiments. (B) Real-time PCR analysis of miR-let-7a expression in 35 cases of cancer and paired normal tissues. The average expression was normalized to U6 expression. All data are represented as mean ± SEM of at least three replicate experiments unless otherwise noted. *denotes significance at P < 0.001 relative to normal cervical cancer tissues or NEEC by student t-test.
Figure 2
Figure 2. Expression of PKM2 in CC tissues and cell lines, and its correlation with the expression miR-let-7a
(A) RT-PCR analysis of PKM2 mRNA expression in 35 cases of cancer tissues (C.T.), paired normal tissues (N.T.), and cell lines including SiHa and HeLa. Quantification analysis was defined as the relative density of PKM2 mRNA to GAPDH. GAPDH was used as an internal control. Results shown are the mean ± SEM of repeated independent experiments. (B) The expression of PKM2 protein was examined in 35 cases of CC tissues, paired normal tissues, and cell lines including SiHa and HeLa using western blot. The average PKM2 expression was normalized to β-actin expression. All data are represented as mean ± SEM of at least three replicate experiments unless otherwise noted. *denotes significance at P < 0.001 relative to normal cervical cancer tissues or NEEC by student t-test. (C) Representative immunostaining of PKM2 in non-neoplastic human cervical tissues and cervical cancer tissues, including cervical cancer grade 1, grade 2 and grade 3. (D) According to Pearson′s correlation analysis, the expression of PKM2 mRNA or protein in 35 cases of cervical cancer tissues was inversely associated with the expression of miR-let-7a (p = 0.013, p = 0.015, respectively).
Figure 3
Figure 3. miR-let-7a upregulation inhibited SiHa and HeLa cell proliferation, migration and invasion
(A) Validation of miR-let-7a expression levels after transfection with miR-let-7a mimics, miR-let-7a inhibitor, or the relative controls by RT-PCR analysis. CCK-8 assays revealed that up-regulation of miR-let-7a inhibited cell proliferation of SiHa and HeLa cells. *denotes significance at P < 0.01 relative to NC miRNAs by ANOVA. (BC) Transwell assay was conducted to identify the role of miR-let-7a in migration and invasion, and up-regulation of miR-let-7a inhibited SiHa and HeLa cell migration and invasion. Representative micrographs and quantification of crystal violet-stained cell colonies were > 0.1 mm. Cells that penetrated the membrane were photographed at 100× magnification. Each bar represents the mean of three independent experiments. *denotes significance at P < 0.01 relative to NC miRNAs by student t-test.
Figure 4
Figure 4. Effects of miR-let-7a on cell apoptosis in SiHa and HeLa cells
SiHa (A) and HeLa (B) cells were transfected with miR-let-7a mimics, miR-let-7a inhibitor, or the relative controls as indicated for 48 h, and then cells were stained with Annexin V-FITC/PI, and analyzed by flow cytometry as described in methods. The statistic data were presented as mean ± SEM from three independent experiments. Representative images for cell apoptosis stained with Annexin V-FITC/PI.
Figure 5
Figure 5. miR-let-7a suppressed PKM2 expression by directly targeting the PKM2 3′-UTR and altered levels of proteins related to proliferation in SiHa and HeLa cells
(A) PKM2 protein expression in SiHa cells transfected with miR-let-7a or the miR-let-7a inhibitor was detected by Western blotting analysis. β-actin served as the loading control. Luciferase reporter assay of SiHa cells with the pGL3-PKM2-3′-UTR-wt or pGL3-PKM2-3′-UTR-mut were co-transfected with and miR-let-7a mimics, miR-let-7a-in or NC with increasing amounts (50 nM) of oligonucleotides. β-actin served as the loading control. *denotes significance at P < 0.01 relative to NC miRNAs by student t-test. (B) PKM2 protein expression in HeLa cells transfected with miR-let-7a or the miR-let-7a inhibitor was detected by Western blotting analysis. β-actin served as the loading control. Luciferase reporter assay of HeLa cells with the pGL3-PKM2-3′-UTR-wt or pGL3-PKM2-3′-UTR-mut were co-transfected with and miR-let-7a mimics, miR-let-7a-in or NC with increasing amounts (50 nM) of oligonucleotides. β-actin served as the loading control. *denotes significance at P < 0.01 relative to NC miRNAs by student t-test.
Figure 6
Figure 6. Effects of overexpression or inhibition of PKM2 on miR-let-7a-inhibited cell proliferation, migration and invasion
(A) The proliferation capacity of miR-let-7a-overexpressing SiHa and HeLa cells was partially improved when cells were transfected with PKM2 plasmids in comparison with miR-NC. (B, C) The migration and invasion of miR-let-7a-overexpressing SiHa and HeLa cells were effectively improved when cells were transfected with PKM2 plasmids. *P < 0.001, vs. vector. (D) The proliferation capacity of miR-let-7a-overexpressing SiHa and HeLa cells was partially inhibited when cells were transfected with si-PKM2 compared with si-control. (E, F) The migration and invasion of miR-let-7a-overexpressing SiHa and HeLa cells were effectively improved when cells were transfected with si-PKM2. *P < 0.001, vs. si-control.
Figure 7
Figure 7. miR-let-7a affected the growth of SiHa-engrafted tumor
SiHa cells stably transfected with miR-let-7a-expressing plasmids or empty vector were subcutaneously injected into nude mice (3 nude mice for experiment group, and 3 nude mice for control group) and tumor volumes were measured every week. Tumors were resected and weighed at 4 weeks after cell injection. Each injection contained 40 ng of miR-let-7a mimic in 10 μl saline solution. Mice were stitched and reanimated under warm light. Control mice were injected with mimic control. (A) The expression of miR-let-7a was detected using qRT-PCR assay. (B) The expression of PKM mRNA was detected using qRT-PCR assay. (C) We identified that miR-let-7a mimics could repress the SiHa-engrafted tumor growth when compared with control-induced SiHa-engrafted tumors. The weight of miR-let-7a-treated tumors was obviously lower compared with control-treated SiHa-engrafted solid tumor mass. (D) The expression of PKM2 protein detected using western blot. *P < 0.001, v.s. control.

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References

    1. Liu Z, Zhou H, Wang W, Fu YX, Zhu M. A novel dendritic cell targeting HPV16 E7 synthetic vaccine in combination with PD-L1 blockade elicits therapeutic antitumor immunity in mice. Oncoimmunology. 2016;5:e1147641. - PMC - PubMed
    1. Li XL, Liu XX, Cao GS, Ju DD, Jiang H. Narrowing Resection of Parametrial Tissues Is Feasible in Low-Risk Cases of Stage IA2-IB1 Cervical Cancer. J Cancer. 2016;7:1481–6. - PMC - PubMed
    1. Sun L, Liu M, Sun GC, Yang X, Qian Q, Feng S, Mackey LV, Coy DH. Notch Signaling Activation in Cervical Cancer Cells Induces Cell Growth Arrest with the Involvement of the Nuclear Receptor NR4A2. J Cancer. 2016;7:1388–95. - PMC - PubMed
    1. Luo CL, Liu YQ, Wang P, Song CH, Wang KJ, Dai LP, Zhang JY, Ye H. The effect of quercetin nanoparticle on cervical cancer progression by inducing apoptosis, autophagy and anti-proliferation via JAK2 suppression. Biomed Pharmacother. 2016;82:595–605. - PubMed
    1. Su K, Wang CF, Zhang Y, Cai YJ, Zhang YY, Zhao Q. The inhibitory effects of carnosic acid on cervical cancer cells growth by promoting apoptosis via ROS-regulated signaling pathway. Biomed Pharmacother. 2016;82:180–91. - PubMed

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