Long non-coding antisense RNA KRT7-AS is activated in gastric cancers and supports cancer cell progression by increasing KRT7 expression

Abstract

Alterations in long non-coding RNAs (lncRNAs) are associated with human carcinogenesis. One group of lncRNAs, which are antisense in orientation to coding mRNAs (ASs), have been recently described in cancers but are poorly understood. We sought to identify ASs involved in human gastric cancer (GC) and to elucidate their mechanisms of action in carcinogenesis. We performed massively parallel RNA sequencing in GCs and matched normal tissues, as well as in GC-derived and normal gastric epithelial cell lines. One AS, designated Homo sapiens keratin 7 (KRT7-AS), was selected due to its marked upregulation and concordant expression with its cognate sense counterpart, KRT7, in GC tissues and cell lines. KRT7-AS formed an RNA-RNA hybrid with KRT7 and controlled KRT7 expression at both the mRNA and the post-transcriptional levels. Moreover, forced overexpression of the KRT7-overlapping region (OL) of KRT7-AS (but not its non-KRT7-OL portions) increased keratin 7 protein levels in cells. Finally, forced overexpression of full-length KRT7-AS or OL KRT7-AS (but not its non-KRT7-OL regions) promoted GC cell proliferation and migration. We conclude that lncRNA KRT7-AS promotes GC, at least in part, by increasing KRT7 expression.

Figure 1. Identification of an overlapping antisense transcript at the KRT7 gene locus

A. 5 gastric cancer-derived cell lines were tested: SNU5, NINC87, AGS MKN28 and KATOIII. KRT7-AS RNA expression was elevated in MKN28 and KATOIII. Expression level was normalized to HFE145 normal gastric epithelial cells.* P< .05; ** P< .01. B. Upper chart of this panel shows genome organization of the KRT7 gene at locus 12q13.13. The chart shows the start and end positions of these genes as indicated on the UCSC site (Genome Bioinformatics). Black arrows indicate transcription direction, and blue blocks are exons. The schema is in scale. “+”at both sides of strand represents the positive strand; “-“represents the negative strand. Lower chart of this panel is a schematic representation of RP3-416H24.1 (ensemble gene transcript ENST00000546686, on the minus DNA strand) and KRT7 mRNA (RefSeq gene NM_005556 on the positive DNA strand). The schema is not drawn to scale. Blocks with colors (green, blue and red) represent exons. Red block in KRT7-AS transcript represents overlapping region (OL). Red block in KRT7 mRNA represents the seventh exon and also OL region. “E” followed by number connotes exon and serial number. Block filed with grey color represents intronic portion of KRT7-AS. Black arrows represent direction of transcription. Dotted filed lines between colored blocks represent introns in KRT7. Primer sites were indicated as follows: a, KRT7-AS-specific RT-primer/KRT7-AS qPCR reverse primer; ①KRT7-AS qPCR forward primer; ②KRT7-AS PCR reverse primer; ③KRT7-AS PCR forward primer;④KRT7 mRNA qPCR forward primer; ⑤KRT7 mRNA qPCR reverse primer. “+”at both sides of strand represents the positive strand; “-“represents the negative strand. C. Antisense-specific RT-PCR screen for KRT7-AS. Antisense-specific RT-PCR detected expression of KRT7-AS RNA from the antisense strand in KATOIII and MKN28 cells. M, Gene ruler expression ladder (SM1551, Life technology); scale is illustrated at left. The cellular origin of the cDNA templates are: Lane 1, no template; Lane 2, genomic DNA PCR control (Applied Biosystems, Foster City, California, USA), Lane 3, KATOIII; Lane 4, MKN28;. cDNAs were reverse-transcribed using KRT7-AS-specific RT-primer. PCR products were amplified using KRT7-AS PCR forward/reverse primer pairs. Sites are indicated in Figure 1C as “a”,②,③.

Figure 2. Expression pattern of KRT7-AS and KRT7 mRNAs in cell lines and tissues

A. 5 GC-derived cell lines were measured: SNU5, NCI-N87, AGS, MKN28 and KATOIII. KRT7 mRNA was highly expressed in MKN28 and KATOIII human gastric cancer cells. Expression levels were normalized to HFE145 normal gastric epithelial cells. **, P< .01. B. Expression of KRT7-AS in 31 gastric cancer tissues. Expression levels are shown as log₂-fold change vs. matching normal adjacent tissue. C. Expression of KRT7 mRNA in 31 gastric cancer tissues. Expression levels are shown as log₂-fold change vs. matching normal adjacent tissue. D. Correlation between KRT7-AS and KRT7 mRNA expression in gastric cancer tissues.

Figure 3. Regulatory function of KRT7-AS overexpression to KRT7

A. Overexpression of KRT7-AS in MKN28 and KATOIII cells after vector transfection. B. Western blot illustrates increased keratin 7 protein levels in both MKN28 and KATOIII cells after forced KRT7-AS overexpression. C. Unchanged KRT7 mRNA levels in MKN28 and KATOIII cells after forced KRT7-AS overexpression. Expression was normalized to empty vector-transfected cells.

Figure 4. Regulatory function of KRT7 mRNA downregulation to KRT7-AS and the rescue effect of KRT7-AS overexpression in KRT7 mRNA-downregulated cells

A. Target sites of two siRNAs to KRT7 mRNA. Black triangles represent target sites of two different siRNAs specific to KRT7 mRNA which all avoid the region overlapping with KRT7-AS. B. No change of KRT7-AS transcript was detected after KRT7 mRNA downregulation. Expression levels were normalized to control-siRNA transfected cells. C. Left panel: Overexpression of KRT7-AS led to a three-fold upregulation of KRT7 mRNA in KRT7 mRNA-downregulation MKN28 cells. Right panel: Overexpression of KRT7-AS led to a two-fold upregulation of KRT7 mRNA in KRT7 mRNA-downregulated KATOIII cells. Expression levels were normalized to control-siRNA and empty vector transfected cells. *, P< .05; **, P< .01. D. Western blot illustrates increased keratin 7 protein levels in KRT7 mRNA-dowregulated MKN28 and KATOIII cells after forced KRT7-AS overexpression. E. Overexpression efficiency of KRT7-AS vector in the KRT7 mRNA-downregulated MKN28 and KATOIII cells. Expression levels were normalized to control-siRNA and empty vector transfected cells. **, P< .01.

Figure 5. KRT7-AS and KRT7 mRNA form a duplex RNA-RNA structure at their mutually overlapping region, which protects KRT7 mRNA from degradation

A. Upper chart is a schematic representation of the PCR amplification region for overlapping (OL) and non-overlapping (non-OL) regions of KRT7-AS. Lower gel images illustrate OL and non-OL RT-PCR products in various samples. Total RNA was extracted and purified, single-stranded RNA was digested with increasing amounts of RNase A+T (indicated by the black wedge and multiple “++++”) and the remaining double-stranded RNA was subjected to RT-PCR to amplify the overlapping or non-overlapping regions of KRT7-AS. B. Stability of KRT7 mRNA over time was measured by qPCR relative to time 0 after blocking new RNA synthesis with α-amanitin (50 mM) (indicated with black arrow). MKN28 cells were transfected with KRT7-AS-containing vector or empty vector for 24 h, then further exposed to 50 mM α-amanitin for 6, 12 or 24 h. Cells were harvested, and the stability of the KRT7 mRNA was analyzed by qPCR. 18S RNA, a product of RNA polymerase I, was used as a control and was unchanged after α-amanitin treatment.

Figure 6. The OL region of KRT7-AS only increases KRT7 protein level, but has little effect on KRT7 mRNA level

A. Schema of KRT7-AS full-length (FL) vector, overlapping (OL) vector, and non-overlapping (non-OL) vector construction. B. Keratin 7 protein level increased in MKN28 cells after OL- and FL-vector transfection, but no change occurred in non-OL vector-transfected MKN28 cells. C. Keratin7 protein level increased in KATOIII cells after OL- and FL-vector transfection, but no change occurred in non-OL vector-transfected KATOIII cells. D. KRT7 mRNA levels did not change substantially in KRT7-AS OL-, FL- or non-OL vector-transfected MKN28 cells and KATOIII cells. Expression was normalized to empty vector-transfected cells.

Figure 7. Overexpression KRT7-AS OL and FL promotes proliferation and migration of GC cells

A. Cell proliferation assays in MKN28 and KATOIII cells. In MKN28 cells, KRT7-AS OL- and FL-overexpression induced significantly higher growth rates at day 5, P< .05. In KATOIII cells, no significant change was found. B. Cell cycle analyses in MKN28 and KATOIII cells. Relative to empty vector-transfected cells, KRT7-AS OL- and FL-overexpression induced a significant increase in cells at S+G1/M phase. *, P < .05; **, P< .01. C. Scratch assay results in MKN28 cells. Significantly more cells migrated in KRT7-AS OL- and FL- overexpressing cells than in empty vector-transfected cells or non-OL-overexpressing cells; P< .05.

Figure 8. Rescue effect on proliferation by FL KRT7-AS overexpression in KRT7 siRNA–treated cells

Cells were treated with either control siRNA + empty vector, KRT7 siRNA + empty vector, or KRT7 siRNA + FL KRT7-AS vector in cell proliferation assays. A. Growth rate in MKN28 cells. B. Growth rate in KATOIII cells.