Identification of HPV16 E6 infection-induced lncRNA SNHG1
The pEGFP-N1-E6 plasmids were transfected into the A549 and HCC827 cell line, and HPV16 E6 empty vectors transfections served as controls. According to the NCBI database, SNHG1 exists as several splice variant transcripts. Primers were designed according to the NCBI sequence to amplify the full-length transcript of SNHG1. QPCR analysis of A549-HPV16 E6 cells revealed that the expression of SNHG1 resulted in several transcripts of different lengths. Among these transcripts, the full length 1021 nt transcript was the most abundant in A549-HPV16 E6 cells based on TA clone sequencing analysis. Therefore, the function of the 1021 nt transcript was investigated in this study.
To identify lncRNAs associated with HPV infection, we analyzed lncRNAs expression difference among A549, A549-HPV16 E6 empty vector and A549-HPV16 E6 groups by qRT-PCR analysis. We chose some lncRNAs those were reported to play important roles in lung cancer development. As shown in Figure 1A, the expression of lncRNAs related to HPV16 E6 infection were screened. We found SNHG1 expression was higher in A549-HPV16 E6 group than that in other groups. By qRT-PCR analysis A549-HPV16 E6 and HCC827-HPV16 E6 group exhibited the highest expression of SNHG1 compared with control groups (Figure 1B). We next quantified SNHG1 expression in the cytoplasmic and nuclear fractions of RNA from cells infected with HPV16 E6. SNHG1 expression increased in both the cytoplasmic and nuclear fractions of cells upon HPV16 E6 infection, and the upregulation of SNHG1 among cytoplasmic RNA was more marked than that among nuclear RNA, indicating that both nuclear and cytoplasmic SNHG1 might play roles in HPV16 E6 infection (Figure 1C).
HPV16 E6 could promote the angiogenesis by SNHG1 in lung cancer cells
As shown in Fig 2A and 2B, by qRT-PCR assay the expression of SNHG1 was knocked down or overexpressed obviously in A549-HPV16 E6 and HCC827-HPV16 E6 cells by transfecting siRNA, pcDNA3.1-SNHG1, and control sequences. The sequences of siRNA were in supplementary Table 2. We found that tube formation in HPV16 E6-vector group was more than that in HPV16 E6 empty vector group and SNHG1 overexpression could enhance the effect of HPV16 E6 on tube formation. However, knocking down SNHG1 could inhibit the effect (Fig 2C).
Cytoplasm SNHG1 directly interacted with EGFR to regulate the angiogenesis
Furthermore the mechanistic insights into SNHG1 was explored. Because SNHG1 was located in the nucleus and cytoplasm simultaneously, we attempted to explore the possibility that SNHG1 functions by physically interacting with proteins. A biotinylated SNHG1 RNA pull-down assay and subsequent mass spectrometry (MS) analysis of the differentially displayed bands revealed that EGFR was the main protein bound to SNHG1. Many studies has shown EGFR can participate cancer angiogenesis[13]. Furthermore using immunoblotting assay we studied the relation between SNHG1 and EGFR from A549-HPV16 E6 (Fig 3A) and HCC827-HPV16 E6 (Fig 3D) cells total protein. RNA immunoprecipitation (UV-RIP) assays with a EGFR-specific antibody confirmed the direct interaction between SNHG1 and EGFR (Fig 3B and 3E). Then, native RIP was performed to confirm binding between SHNG1 and EGFR (Fig 3C and 3F). The interaction of SNHG1 with EGFR to influence tube formation was evaluated using EGFR knocking down in A549-HPV16 E6 and HCC827-HPV16 E6 cells respectively. As shown in Fig 3G and 3H, EGFR knocking down inhibited tube formation of A549-HPV16 E6 or HCC827-HPV16 E6 cells and the inhibition of tube formation in the SNHG1-knockdown A549-HPV16 E6 or HCC827-HPV16 E6 cells was enhanced by knocking down EGFR. The tube formation in the SNHG1-overexpression A549-HPV16 E6 or HCC827-HPV16 E6 cells was rescued by EGFR siRNA.
Angiogenesis effects of SNHG1 depended on EGFR signal paths
STAT3 and NF-κB pathways are modulated by EGFR[14, 15]. We wanted to determine whether HPV16 E6 regulated EGFR downstream signaling pathways by SNHG1. In our study western blot analysis showed HPV16 E6 could promote p-EGFR, and p-STAT3 and p-IκBa expression. SNHG1 overexpression increased the above effect while SNHG1 knockdown reduced the effect (Fig 4A and 4B). To detected the effect of NF-κB pathway on tube formation of A549-HPV16 E6 and HCC827-HPV16 E6 cells we added NF-κB inhibitor (BAY) in HPV16 E6-siNC and HPV16 E6-siSNHG1 groups respectively. Tube formation assays showed BAY can inhibit tube formation of lung cells (Fig 4C). NF-κB pathway can influence the tube formation of lung cells.
IL-6 was a target gene of SNHG1/EGFR signaling downstream and SNHG1 influenced the expression of IL-6 by EGFR/NF-κB
By qRT-PCR we performed a screening in SNHG1 knock down A549-HPV16 E6 cells to identify the downstream targets responsible for the biological function of SNHG1. IL-6 and VEGF-D expression were downregulated following SNHG1 knockdown (Fig 5A). IL-6, which is shown to be a target gene of EGFR/NF-κB pathway and promoted angiogenesis in numerous tumors[16], was also significantly decreased in SNHG1 knockdown cells. IL-6 expression in the cultivated media from SNHG1-deficient A549-HPV16 E6 and HCC827-HPV16 E6 cells in the presence of BAY was largely reduced compared with control cells (Fig 5B and 5C). Above experiments suggested that IL-6 was a target gene of SNHG1/EGFR/NF-κB signaling.
IL-6 influenced the angiogenesis by EGFR/STAT3 pathway
IL-6 signaling has been reported to play an important role in VEGF dependent tumor angiogenesis[17]. Therefore, we next examined its role in HPV16 E6 and SNHG1 mediated angiogenesis. As shown in Fig 6A and 6B, p-STAT3 and VEGF-D expression were upregulated in HPV16 E6-A549 and HPV16 E6-HCC827 cells compared with HPV16 E6 empty vector cells, furthermore IL-6 treatment could enhance the effect. VEGF-D expression in cells media from SNHG1-deficient A549 and HCC827 cells were rescued by the treatment with IL-6. STAT3 pathway can influence tube formation of lung cells. To detected the effect of STAT3 pathway on tube formation of A549-HPV16 E6 and HCC827-HPV16 E6 cells we added STAT3 inhibitor (stattic) in HPV16 E6-siNC and HPV16 E6-siSNHG1 cells respectively. Tube formation assays showed stattic could inhibit tube formation of lung cells (Fig 6C).
VEGF-D expression was regulated by STAT3 pathway
As shown in Fig 7A and 7B, VEGF-D, which was previously shown to be a target gene of STAT3 pathway and promoted angiogenesis in tumors[18], was also significantly decreased in A549-HPV16 E6 and HCC827-HPV16 E6 cells with the presence of stattic compared with A549-HPV16 E6 and HCC827-HPV16 E6 cells. VEGF-D expression decreased in SNHG1 knockdown cells and the effect could be strengthed by stattic.