LncRNA HCP5 enhances the proliferation and migration of cervical cancer via miR-216a-5p/CDC42 axis

Abstract

To investigate the important roles of the cancer-promoting long non-coding RNAs (lncRNAs) in cervical cancer, the up-regulated lncRNAs and prognostic analysis were identified through Lnc2Cancer and Lncar. LncRNA-regulated miRNA and miRNA-target mRNA were analyzed based on starBase v2.0 and miTarbase to predict the lncRNA-miRNA-mRNA ceRNA network. Based on the above findings, the abnormally expressed histocompatibility leukocyte antigen complex P5 (HCP5) was identified in 31 cervical cancer patients through RT-qPCR. The stable cell lines were constructed to explore the effect of HCP5 on the promotion of cervical cancer and the regulatory role on the expression of miR-216a-5p and CDC42. Cell Counting Kit-8 (CCK8) assay, cell clone formation, and transwell assay were used to examine proliferation and migration ability of cervical cancer cells. The results displayed that the overexpression of HCP5 promoted cervical cancer cell proliferation and migration in vitro, and the elevated HCP5 can also promote tumor growth in vivo. Besides, RT-qPCR and western blot assay revealed that elevated HCP5 suppressed miR-216a-5p expression and then up-regulated the expression of CDC42. In contrast, knocking down HCP5 resulted in increased expression of miR-216a-5p and then downregulated the expression of CDC42. Rescue experiments also demonstrated that miR-216a-5p could in part intercept in promotion impact caused by HCP5 on cervical cancer cells. Above all, HCP5, as an oncogene, can promote proliferation and migration ability of cervical cancer via the regulation of the miR-216a-5p/CDC42 axis.

Keywords: HCP5, miR-216a-5p, CDC42; cervical cancer; proliferation, migration.

Figure 1

HCP5 was upregulated in cervical cancer. (a) Venn diagram of LncRNAs upregulated and LncRNA associated with poor prognosis in patients with cervical cancer. (b, c) The roles of 4 hub LncRNAs in 46 malignant tumors based on the lncar database. (d) The expression levels of HCP5 in the cancerous tissues and adjacent tissues from 31 cervical cancer specimens. ns, not significant; *P < 0.05, **P < 0.01, ***P<0.001.

Figure 2

HCP5 enhances the proliferation, migration, and clone formation of cervical cancer cells. (a) The effect of HCP5 knockdown and overexpression in SiHa and HeLa cells by the transduction of the corresponding lentivirus. (b) CCK8 assay was used to assess the proliferative capacity of SiHa and HeLa cells at 24h, 48h, and 72h after knockdown or overexpression of HCP5. (c, d) Transwell assay was perfomed to detect the migration ability of SiHa and HeLa cells with knockdown or overexpression of HCP5 after being seeded in the upper chamber of Transwell for 24h. (e, f) Clone formation images of cells in SiHa and HeLa with knockdown or overexpression of HCP5. lv-shcon represents control of knockdown model of HCP5, lv-shhcp5 represents the knockdown model of HCP5, lv-con represents control of overexpression model of HCP5, lv-hcp5 represents overexpression model of HCP5. All the experiments were performed three times in duplicate. The data were presented as mean ± SD. **P<0.01, ***P<0.001, ****P<0.0001.

Figure 3

Expression of miR-216a-5p and CDC42 is regulated by HCP5 in SiHa and HeLa cells. (a, b and c, d) The RT-qPCR assay was used to analyze the relative gene expression of miR-216a-5p and CDC42 in SiHa and HeLa cells after overexpression or knockdown of HCP5. (e, f) Western Blot assay was performed to assess the expression of CDC42 protein in the established cells. All the experiments were performed three times in duplicate. The data were presented as mean ± SD. **P<0.01, ***P<0.001, ****P<0.0001.

Figure 4

The effect of HCP5 on proliferation and migration by targeting CDC42 in SiHa and HeLa cells was partially negatively regulated by miR-216a-5p. CCK8 assay using the established cells treated with miR-216a-5p inhibitor (a) or mimic (b). (c, d) Cell migration was evaluated by transwell assay in different group cells. Western blot assay analyzed the protein expression of CDC42 in SiHa and HeLa cells with knockdown or overexpression of HCP5 after being treated with miR-216a-5p inhibitor (e) or mimic (f). All the experiments were performed three times in duplicate. The data was presented as mean ± SD, *P < 0.05, **P < 0.01, ***P<0.001.

Figure 5

HCP5 promoted tumorigenesis of SiHa cells in vivo. In the in vivo experimental analysis, 5x10⁶ SiHa cells of different conditions were inoculated into the right posterior subcutaneous of nude mice. (a, b) Image of Xenograft tumors generated by SiHa cells and the corresponding tumor volume and tumor weight. (c, d) Representative images of IHC staining for H&E and immunohistochemical images showing the protein expression level of Ki67 in the tumor tissue samples. Four nude mice were included in each group of experiments. The data were presented as mean ± SD. *P<0.05, **P<0.01.