Crispr-Cas9-based long non-coding RNA interference and activation identified that the aberrant expression of Myc-regulated ST8SIA6 antisense RNA 1 promotes tumorigenesis and metastasis in hepatocellular carcinoma

Abstract

Objective: Long non-coding RNAs (lncRNAs) participate in the formation, progression, and metastasis of cancer. This study aimed to explore the roles of the lncRNA ST8SIA6 antisense RNA 1 (ST8SIA6-AS1) in tumorigenesis and elucidate the underlying molecular mechanism of its upregulation in hepatocellular carcinoma (HCC).

Material and methods: A total of 56 in-house pairs of HCC tissues were examined, and ST8SIA6-AS1 levels were determined through real-time polymerase chain reaction (PCR). The biological behavior of ST8SIA6-AS1 by Crispr-Cas9-based gene repression and activation was determined in vitro and in vivo. The binding sites and biological behavior of Myc proto-oncogene and forkhead box A on chromatin were investigated through luciferase reporter assays, chromatin immunoprecipitation-quantitative PCR, and co-immunoprecipitation (co-IP) assays. The regulatory mechanisms of ST8SIA6-AS1 expression were analyzed with encyclopedia of DNA elements and gene expression profiling interactive analysis.

Results: The expression of ST8SIA6-AS1 significantly increased in multiple HCC cell lines and the 56 in-house pairs of HCC tissues (P = 0.0018). Functionally, high-efficiency Crispr-Cas9-based knockdown of ST8SIA6-AS1 revealed that ST8SIA6-AS1 knockdown attenuated the proliferation, migration, and infiltration of HCC cells and considerably reduced the growth rate of subcutaneous and orthotopic HCC tumors. Conversely, ST8SIA6-AS1 overexpression considerably improved the oncogenic characteristics of the HCC cells. Furthermore, ST8SIA6-AS1 upregulation was regulated by the direct binding of transcription factor Myc to the -260 bp to+155 bp and +1003 bp to +1312 bp regions of the ST8SIA6-AS1 transcription start site, which is a segment with high level of H3K27 acetylation. Myc knockdown or treatment with the BET bromodomain inhibitor JQ-1 considerably reduced ST8SIA6-AS1 RNA expression in the HCC cells.

Conclusion: Our study has established the oncogenic role of ST8SIA6-AS1 and elucidated the Myc-dependent upregulation mechanism of ST8SIA6-AS1 in HCC, providing a profound theoretical molecular basis for the carcinogenic function of ST8SIA6-AS1 in HCC.

Keywords: Hepatocellular carcinoma; Long non-coding RNA; Myc proto-oncogene; ST8SIA6 antisense RNA 1; Tumorigenesis.

Figure 1:

Expression of ST8SIA6-AS1 was considerably upregulated in HCC and HCC cell lines. (a) The relative expression of ST8SIA6-AS1 in 369 HCC samples compared with 160 normal samples obtained from the GEPIA database. (b) The ST8SIA6-AS1median expression of tumor (red) and normal samples (green) in Bodymap (from the GEPIA database). (c) The ST8SIA6-AS1 expression profile across all tumor samples and corresponding normal tissues obtained from the GEPIA database. (d) The expression levels of ST8SIA6-AS1 in HCC cell lines. (e) The relative expression of ST8SIA6-AS1 in 56 in-house pairs of HCC tissues. (f) Pairwise analysis of the expression of ST8SIA6-AS1 in 56 in-house pairs of HCC tissues. Error bars are shown as mean ± SD, and ANOVA-test was used. (The red column represents the up-regulated expression of ST8SIA6-AS1 in HCC compared to the paracancer sample, while the green column represents the down-regulated expression of ST8SIA6-AS1 in HCC compared to the paracancer sample.) The asterisk indicates that the data have statistical differences, *P < 0.05, **P < 0.01, and ***P < 0.001. (ST8SIA6-AS1: ST8SIA6 antisense RNA 1, TPM: Transcripts per million, NS: no significance, LIHC: Liver hepatocellular carcinoma, HCC: Hepatocellular carcinoma, GEPIA: Gene expression profiling interactive analysis, SD: Standard deviation, ANOVA: Analysis of variance, ACC: Adrenocortical carcinoma, BLCA: Bladder Urothelial Carcinoma, BRCA: Breast invasive carcinoma, CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma, CHOL: Cholangiocarcinoma, COAD: Colon adenocarcinoma, DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma, ESCA: Esophageal carcinoma, GBM: Glioblastoma multiforme, HNSC: Head and Neck squamous cell carcinoma, KICH: Kidney Chromophobe, KIRC: Kidney renal clear cell carcinoma, KIRP: Kidney renal papillary cell carcinoma, LAML: Acute Myeloid Leukemia, LGG: Brain Lower Grade Glioma, LIHC: Liver hepatocellular carcinoma, LUAD: Lung adenocarcinoma, LUSC: Lung squamous cell carcinoma, OV: Ovarian serous cystadenocarcinoma, PAAD: Pancreatic adenocarcinoma, PCPG: Pheochromocytoma and Paraganglioma, PRAD: Prostate adenocarcinoma, READ: Rectum adenocarcinoma, SARC: Sarcoma, SKCM: Skin cutaneous melanoma, STAD: Stomach adenocarcinoma, STES: Stomach and esophageal carcinoma, TGCT: Testicular germ cell tumors, THCA: Thyroid carcinoma, THYM: Thymoma, UCEC: Uterine corpus endometrial carcinoma,UCS: Uterine carcinosarcoma).

Figure 2:

ST8SIA6-AS1 can promote the proliferation of HCC cells in vitro. (a) Schematic illustration of Crispr-Cas9-based transcriptional silencing and activation of lncRNA ST8SIA6-AS1. This figure was independently created by the authors of this article. (b and c) CC9i efficiently downregulated the expression of ST8SIA6-AS1 in MHCC-97H and HCCLM3. (d and e) the growth curve of the HCC cell lines. (f) Experimental scheme for Crispr-Cas9-based cell proliferation competition assay (This figure was independently created by the author using Adobe Illustrator CC 2018). The abundance of sgRNAs in the HCC cell lines was detected at P0 (cell passage number), P3, and P6 through Real-time PCR. (g and h) The abundance of each sgRNA was detected through qPCR in the HCC cell lines at P0, P3, and P6. (i) Colony-forming assay was performed on MHCC-97H. The colonies were counted after 2 weeks of culture. (j) The distribution of the cell cycle phase in MHCC-97H was detected through flow cytometry. (k) CC9a efficiently upregulated the expression of ST8SIA6-AS1 in two of three sgRNAs. (l) The proliferation of SMMC-7721 was detected with a growth curve. (m) The overexpression of ST8SIA6-AS1 increased the colony-forming capacity of SMMC-7721. Error bars, mean ± S.E.M. data were analyzed with ANOVA test. The asterisk indicates that the data have statistical differences, *P < 0.05, **P < 0.01, and ***P < 0.001. (ST8SIA6-AS1: ST8SIA6 antisense RNA 1, NS: No significance, PI: Propidium iodide, CC9i: Crispr-Cas9-based gene interference, NC: Negative control, HCC: Hepatocellular carcinoma, lncRNA: Long noncoding RNAs, sgRNAs: Single-guide RNAs, PCR: polymerase chain reaction, qPCR: Quantitative polymerase chain reaction, CC9a: Crispr-Cas9-based gene activation, S.E.M.: Standard error of the mean, ANOVA: Analysis of variance.)

Figure 3:

ST8SIA6-AS1 promotes the migration and invasion of HCC cells in vitro. (a) Compared with the control group (CC9i-NC), the knockdown of ST8SIA6-AS1 (CC9i-1 and CC9i-3) decreased the migration capacity of HCCLM3. (b) Compared with the control group (CC9a-NC) the overexpression of ST8SIA6-AS1 (CC9a-2 and CC9a-3) enhanced the migration ability of SMMC-7721. (c-e) Cell migration and invasion capacity of HCCLM3, MHC-97H (c and d), and SMMC-7721 (e) were measured by Transwell migration and Matrigel invasion assays. The asterisk indicates that the data have statistical differences, *P < 0.05, **P < 0.01, and ***P < 0.001. (ST8SIA6-AS1: ST8SIA6 antisense RNA 1, HCC: Hepatocellular carcinoma, NC: Negative control, CC9a: Crispr-Cas9-based gene activation, CC9i: Crispr-Cas9-based gene interference.)

Figure 4:

ST8SIA6-AS1 knockdown significantly reduces the growth of liver tumors in vivo. (a and b). The tumor volume of subcutaneous xenografts in nude mice injected with the HCCLM3 cells. (c) ST8SIA6-AS1 expression level was determined in the tumors of the control and ST8SIA6-AS1 knockdown groups at the end of the experiment. NC-T1, NC-T2, and NC-T3 refer to three randomly selected tumor tissues in the control group. CC9i-T1, CC9i-T2, and CC9i-T3 refer to three randomly selected tumor tissues in the ST8SIA6-AS1 knockdown group. (d) Ki67 immunohistochemical staining in xenograft tumor sections. (e) The knockdown of ST8SIA6-AS1 expression decelerated the growth of liver tumors in situ (tumor and sections of liver tissue). CC9i-NC: Control group; CC9i-3: Experimental group 3. Error bars, mean ± s.e.m. Data were analyzed with ANOVA. The asterisk indicates that the data have statistical differences, *P < 0.05, **P < 0.01, and ***P < 0.001. (ST8SIA6-AS1: ST8SIA6 antisense RNA 1, NC: Negative control, CC9i: Crispr-Cas9-based gene interference, S.E.M.: Standard error of the mean, ANOVA: Analysis of variance.)

Figure 5:

Myc regulates the expression of ST8SIA6-AS1 by binding to its promoter region. (a) Genome browser tracks showing the Myc, FOXA1, and POLR2A binding sites in the ST8SIA6-AS1 gene locus. Data from the ENCODE database. (b) Overexpression of Myc and FOXA1 in SMMC-7721 significantly increased the RNA level of ST8SIA6-AS1. (c) MHCC-97H cells were transfected with negative control Small interference RNA (Scramble) or siRNA targeting c-Myc and FOXA1, immunoblotting analysis was performed. Asterisk, non-specific band. (d and e) SiRNA were used to knock down Myc and FOXA1 in the MHCC-97H and HCCLM3 cells, and the expression level of ST8SIA6-AS1 was detected through qPCR. (f) MHCC-97H and HCCLM3 cells with or without JQ-1 treatment for 24 h and ST8SIA6-AS1 were detected through qPCR. (g) The luciferase reporter plasmid of the binding site was co-transfected with the Myc or FOXA1-expressing plasmid in 293T. Luciferase activity was detected 24 h after transfection. Vector: Luciferase reporter vector control, BS: Plasmid with binding site region (−260 bp to +1489 bp region of the ST8SIA6-AS1 TSS). (h and i). ChIP-qPCR were performed to detect the binding of Myc or FOXA1 on the ST8SIA6-AS1 promoter region. (j and k) co-IP experiments were performed in 293T cells, and antibodies against HA and Flag tags were used. Protein levels were detected through Western blotting. Data were analyzed with Student’s t-test and ANOVA. The asterisk indicates that the data have statistical differences, *P < 0.05, **P < 0.01, and ***P < 0.001. (ST8SIA6-AS1: ST8SIA6 antisense RNA 1, TFBSs: Transcription factor binding sites, cCREs: Candidate cis-regulatory elements, OE: Overexpression, FOXA1: Forkhead box A1, qPCR: Quantitative polymerase chain reaction, BS1: Binding site 1, BS2: binding site 2, TSS: transcription start site, co-IP: Co-immunoprecipitation, ChIP-qPCR: Chromatin immunoprecipitation-quantitative polymerase chain reaction, siRNA: Small interfering RNA, HA-c-Myc: HA-tagged c-Myc, Flag-FOXA1: Flag-tagged FOXA1, #: Non-specific bands. Error bars: Mean ± Standard error of the mean, NS: No Significance, ANOVA: Analysis of variance, ENCODE: Encylopedia of DNA Elements.).

Figure 6:

Co-expression relationship between ST8SIA6-AS1 ang Myc target genes. (a) The scatter plot shows the correlation analysis between ST8SIA6-AS1 and 240 Myc target genes in liver tumors and GETx liver tissues (data from GEPIA). The two dotted lines refer to R = 0.1 and P = 0. (b) Representative scatter plots of co-expression between ST8SIA6-AS1 and Myc target genes. (c) Schematic of ST8SIA6-AS1 upregulation in the HCC cells. The transformation of HCC cells caused the upregulation of Myc, promoting the binding of c-Myc proteins to the promoter region, transcriptional activation of ST8SIA6-AS1, and the proliferation, migration, and invasion ability of the HCC cells. (ST8SIA6-AS1: ST8SIA6 antisense RNA 1, TPM: Transcripts per million, HCC: Hepatocellular carcinoma, GEPIA: Gene expression profiling interactive analysis.)