AC245100.4 is up-regulated in prostate cancer tissues and cells.
AC245100.4 is transcribed from the positive strand of a genomic DNA sequence located on chromosome 1q21.1 (from 143699511 to 143717173) with length of 1555 bp. In this study, the expression of AC245100.4 was higher in prostate cancer tissues compared with normal prostate tissues from TCGA data (Fig. 1A). To prove whether the lncRNA is associated with prostate cancer, we detected the expression of AC245100.4 in common tumor cells. The results showed that AC245100.4 has relatively high expression in PCa, especially in PC3 and DU145 cells (Fig. 1B&C), and it was mainly located in cytoplasm (Fig. 1D). These results suggested that AC245100.4 may be closely related to prostate cancer and may play a major biological role in the cytoplasm.
AC245100.4 regulates the proliferation, migration and apoptosis of PCa cells in vitro.
It has now become widely accepted that lncRNAs function as crucial regulators at each step during genetic information processing in the living cell25, 26. In this study, we down-regulated the expression of AC245100.4 in PC3 and DU145 cell, then detect the biological function of the cells. The results showed that the proliferation and migration of above cells were inhibited through interference of AC245100.4 (Fig. 2A-D). Furthermore, we found that the percentage of apoptosis was increased via knockdown of AC245100.4 (Fig. 2E&F). Meantime, the protein expression of Bcl-2 was decreased and the expression of activated caspase-3 was increased compared with control cells (Fig. 2G). These results suggested that AC245100.4 was closely related to prostate cancer and plays an important biological role as an oncogene.
AC245100.4 regulates the proliferation and migration PCa cells in vivo.
To explore the effect of AC245100.4 on tumor proliferation and metastasis in vivo, DU145 cells with sh-AC245100.4 or sh-ctrl were inoculated into male nude mice subcutaneously. The smaller subcutaneous tumors were found in the sh-AC245100.4 group were significantly compared with the sh-ctrl group (Fig. 3A&B). consistently, the mean volume and weight of tumors were substantially lower in the group of sh-AC245100.4 (Fig. 3C&D). Tumors formed from sh-AC245100.4 DU145 cells suggested the weak positivity for Ki-67 than tumors from the sh-ctrl (Fig. 3E). Moreover, the metastatic nodules formed in sh-AC245100.4 were less than in sh-ctrl group (Fig. 3F). These observations were consistent with the results in vitro. Taken together, these findings suggested that the interference of AC245100.4 inhibited tumor proliferation and metastasis of PCa cells in vivo.
AC245100.4 regulates the expression of NR4A3.
To date, the specific function of lncRNAs in the regulation of tumor progression is not well known. Thus, to explore the precise mechanism of lncRNAs in PCa progression could increase the understanding of the specific mechanisms of PCa tumorigenesis. In this research, the second-generation sequencing (Next generation sequencing, NGS) was used to explore the mechanism of AC245100.4. There were 117 up-regulated genes and 72 down-regulated genes in the condition of AC245100.4 silence (Fig. 4A and 4B). GO functional classification of differential genes shows that differential genes mainly exist in cell process, biological regulation, biological process regulation and other molecular functions (Fig. 4C&D). KEGG pathway analysis showed that differential genes were mainly enriched in cell communication, signal transduction, molecular folding and classification degradation, cancer, metabolism, immunity and other pathways (Fig. 4E&F). Among 189 different genes, we further performed RT-qPCR to detect the relative mRNA expression of several genes related to tumor progression via the knockdown of AC245100.4 (Znf177, APIP, GDI2, YOD1, NR4A3). Interestingly, NR4A3 as a significantly up-regulated gene caught our attention (Supplement.1). Then, we found suggested that the knockdown of AC245100.4 could increase the expression of NR4A3 (Fig. 4G&H). Moreover, the same results could be found in xenograft mouse model by immumo-histochemical staining (Fig. 4I). Anyway, the expression of NR4A3 was upregulated via the silence of AC245100.4. However, the correlation between NR4A3 and AC245100.4 and the role of NR4A3 on progression of PCa are still a fog.
Overexpression of NR4A3 inhibits proliferation and induces apoptosis in PCa.
Previous research has found that heterogeneously expressed NR4A3 could decrease the growth of non-small cell lung cancer27. In addition, lncRNA LINC00467, as an oncogenic lncRNA, it was a highly expressed in Hepatocellular carcinoma by repressing NR4A328. Therefore, whether AC245100.4 can regulate the expression of NR4A3, and the relationship between NR4A3 and the occurrence and development of prostate cancer, which still need to be explored. TCGA data shows that NR4A3 was lower expressed in prostate cancer tissues than its in normal prostate tissues (Fig. 5A). Also, the same result was found in prostate cancer cells and normal prostate cells (Fig. 5B). Therefore, the above results indicate that NR4A3 may be associated with prostate cancer. Given that NR4A3 was lower expressed in prostate cancer cells, we over-expressed NR4A3 in PC3 and DU145 cell lines (Supplement.2). At the same time, the proliferation of above cells was decreased compared with the control cells (Fig. 5C&D). Importantly, the apoptosis rates were increased in the PC3 and DU145 cells by overexpression of NR4A3 (Fig. 5E&F). Meantime, the Bcl-2 protein was down-regulated and the activated caspase-3 protein was up-regulated in above cells (Fig. 5G).
To study the role of NR4A3 on tumor growth and metastasis in vivo, DU145 cells with NR4A3 plasmid or control vector were inoculated into nude mice. Five weeks later, the volume and weight of tumors were substantially smaller in the NR4A3 group then its in the control group (Fig. 5H, I&J). The formed tumors in NR4A3 overexpression group had lower positivity for Ki-67 compared with the control group (Fig. 5K). Taken together, these results demonstrated NR4A3 may play the role of tumor suppressor in PCa.
Knockdown of AC245100.4 increases the expression of NR4A3 by regulating the phosphorylation status of STAT3.
Previous studies have shown that NR4A3 was transcriptionally silenced by STAT3 in the gastric cancer29. Meantime, several lncRNAs have been revealed to regulate phosphorylation of STAT3 in a direct binding manner or other manners27, 30. Therefore, we suspected that AC245100.4 increased phosphorylation of STAT3 by direct binding, thus silenced NR4A3. As shown in the results, the protein level of p-STAT3 was decreased by knockdown of AC245100.4, while there was no change in total STAT3 (Fig. 6A). Also, the relative expression of STAT3 had no change via the knockdown of AC245100.4 (Fig. 6B). IL6 is believed to mediate STAT3 tyrosine phosphorylation and cryptotanshinone is a STAT3 inhibitor31. To further determine whether STAT3 could influence the expression of AC245100.4, PC3 and DU145 cells were stimulated with IL6 or cryptotanshinone. However, we didn’t observe the change of the expression of AC245100.4 after with IL-6 or cryptotanshinone treatment (Fig. 6C&D). These findings indicated that STAT3 is the downstream of AC245100.4. Further RIP assays suggested that AC245100.4 was directly bound to STAT3 (Fig. 6E). To investigate whether the knockdown of AC245100.4 could influence the enrichment of p-STAT3 in the promoter of NR4A3. As shown in Fig. 6F, ChIP assays suggested that the knockdown of AC245100.4 prominently suppressed the enrichment of p-STAT3 in the promoter of NR4A3. In a word, AC245100.4 regulating the expression of NR4A3 was accomplished by directly bound to STAT3 and inducing its phosphorylation status.
Silence of NR4A3 attenuates the tumor suppressive roles of AC245100.4 knockdown in PCa.
To investigate whether the tumor-promoting role of AC245100.4 was accomplished by NR4A3 in PCa, we designed three interfering segments of NR4A3 and selected the segment with the best interference efficiency for subsequent experiments (Fig. 7A&B). We co-transfected interfering fragments of NR4A3 and AC245100.4 in PC3 and DU145 cells. Next, we found that the silence of NR4A3 reversed the inhibition of cell viability caused by AC245100.4 knockdown (Fig. 7C&D). Besides, the increasing protein level of NR4A3 was verified in the group of the silence of AC245100.4, however, the results were reversed by treating with IL6 (Fig. 7E). In brief, these results proved convincingly that NR4A3 drops off the tumor promoting role of AC245100.4 in PCa and that NR4A3 may act as a potential tumor suppressor in PCa.