Up-regulation of UXT and down-regulation of MEG3 were observed in breast cancer.
To investigate the expression of UXT and MEG3, we collected breast tumor tissues and their adjacent tissues from 13 cases of patients and conducted the following experiments such as immunohistochemistry, qRT-PCR and western blot analysis. As shown in Fig. 1A, the positive expression of UXT in tumor tissues was significantly stronger than the adjacent tissues. The data of qRT-PCR and western blot analysis also displayed that UXT was highly expressed while MEG3 was low expressed in breast tumor tissues compared with adjacent normal tissues (Fig. 1B-1D). Moreover, we further examined the levels of UXT and MEG3 in various breast cancer cell lines including ZR75 and MCF7, as well as normal breast cell MCF10A. As expected, higher level of UXT and lower level of MEG3 were also observed in breast cancer cell lines, compared with that of control (Fig. 1E-1G).
Knockdown of UXT or overexpression of MEG3 inhibited proliferation, promoted apoptosis and delayed migration and invasion of MCF7 and ZR75 cells.
To explore the biological effects of UXT and MEG3 in the tumorigenesis of breast cancer, we down-regulated UXT expression by using siRNA duplexes and up-regulated MEG3 expression through introducing pcDNA 3.1-MEG3 into ZR75 and MCF7 cells, following by testing the alteration of proliferation, apoptosis, migration and invasion. As shown in Fig. 2A, the level of MEG3 both in ZR75 and MCF7 cells was remarkably increased after transfection of pcDNA 3.1-MEG3. As expected, the mRNA and protein levels of UXT were also knocked down after transfection of its specific siRNA (Fig. 2B and 2C). Along with the increasing level of MEG3 or decreasing level of UXT, CCK-8 assay presented the inhibited cell viability of ZR75 and MCF7 cells (Fig. 2D), and the increased apoptotic cells (Fig. 2E). Furthermore, both migratory and invasive cells were remarkably reduced in plasmid pcDNA3.1 MEG3 group (Fig. 2G). Likewise, UXT knockdown by siRNA also inhibited the capacity of migration and invasion of MCF7 and ZR75 cells (Fig. 2H). Therefore, these data indicated that there is a negative correlation between UXT and MEG3 in regulating the progression of breast cancer.
UXT bound with DNMT3b to promote MEG3 methylation, in turn down-regulating MEG3 expression.
To assess the methylation of MEG3 promoter, methylation-specific PCR was conducted, and we found that the bright band of 160 kb was only present in the methylated group of positive, MCF7 and ZR75 cells, whereas there was no same band existing in corresponding un-methylated group and both group of negative cells (Fig. 3A), suggesting that the hypermethylation of MEG3 promoter in breast cancer cells. It has been demonstrated that the methylation of MEG3 is regulated by DNA (cytosine-5)-methyltransferase 3b (DNMT3b) . Thus, DNMT3b level in breast cancer was detected in clinical specimens (13 cases) and cell lines. The results showed that DNMT3b was highly expressed in breast tumor tissues and cell lines (Fig. 3B-3D). Knockdown of DNMT3b increased the level of MEG3 (Fig. 3E-3G). Moreover, the data of co-immunoprecipitation identified the interaction between UXT and DNMT3b (Fig. 3H). Since silencing of UXT obviously reduced the level of DNMT3b (Fig. 3H), while knockdown of DNMT3b did not alter the expression of UXT (Fig. 3I), indicating that DNMT3b might be a downstream effector in UXT-mediated breast cancer tumorigenesis. Furthermore, the elevated level of MEG3 induced by UXT knockdown was dramatically reversed by DNMT3b overexpression (Fig. 3J). In sum up, these data implied that the negative regulation of UXT on MEG3 expression was depended on DNMT3b-mediated methylation modification.
MEG3 bound with p53 to regulate its transcriptional activity.
To explore the downstream mechanism of MEG3, we detected the expression of p53 in normal and breast cancer cells and found that p53 was down-regulated in breast cancer cells (Fig. 4A). Moreover, overexpression of MEG3 also induced the increased level of p53 (Fig. 4B). Thus, we hypothesized whether MEG3 could bind to p53 and regulate its transcription. As MEG3 contains three conserved motifs, named as M1, M2 and M3, several relevant MEG3 deletion mutants was generated for further confirmation. Luciferase reporter assay was performed to test the mutant for the ability to activate p53-mediated transcription activity and the results showed that each deletion mutants failed to stimulate the transcriptional activity of p53 (Fig. 4C), indicating that full length of MEG3 is crucial for p53 transcription. Furthermore, RNA immunoprecipitation (RIP) assay suggested that MEG3 could directly bind to p53 (Fig. 4D). Knockdown of p53 diminished the effects of MEG3 overexpression on apoptosis, migration, invasion, which was further verified by the detection of apoptosis-related proteins including Bax, Bcl-2, p21 and p53 (Fig. 4E-4G). All above, it is suggested that p53 was involved MEG3-mediated apoptosis, migration and invasion of MCF7 and ZR75 cells.
UXT negatively regulated the MEG3/p53 axis in a DNMT3b-dependent manner to promote cancer growth in vivo.
To identify whether the regulation of UXT on MEG3/p53 axis in a DNMT3b-dependent manner is fit in vivo, the stable cells of UXT knockdown combined with DNMT3b knockdown or overexpression were constructed and then subcutaneously injected into the right frank of nude mice to confirm the influence of UXT and DNMT3b on tumor growth. As shown in Fig. 5A and 5B, the inhibition of UXT knockdown on tumor growth was further strengthened by DNMT3b knockdown but weakened by DNMT3b overexpression. Similarly, increasing levels of MEG3 and p53 were presented by the knockdown of both UXT and DNMT3b while the up-regulation of MEG3 and p53 induced by UXT knockdown was almost abolished by DNMT3b overexpression (Fig. 5C and 5D). Whereas, the data of DNMT3b using qRT-PCR exerted the opposite trend (Fig. 5E). As expected, immunohistochemical staining of p53 and DNMT3b further validated the conclusion (Fig. 5F). Thus, UXT negatively regulated the MEG3/p53 axis in a DNMT3b-dependent manner to promote the growth of nude mice xenograft.