LncRNA KCNQ1OT1 regulates microRNA-9-LMX1A expression and inhibits gastric cancer cell progression

LncRNA KCNQ1OT1 is downregulated in human GC tissues

It is possible that miR-9 upregulation in GC (see our previous study [7]) could be due to downregulation of certain LncRNAs. As they could function as the ceRNAs of miR-9. To explore this possibility, the human LncRNA database, LncBase (Predicted v.2), was consulted to identify possible miR-9-targeting LncRNAs. These potential LncRNAs were further verified by other LncRNAs/miR databases, including StarBase and miRbase. The bioinformatic analyses identified that one LncRNA, KCNQ1OT1, putatively targeted miR-9. Its expression in GC tissues was tested next. Total RNA was extracted from fresh GC tissues and the adjacent normal epithelial tissues from twelve (12) primary GC patients [7]. KCNQ1OT1 was determined by qPCR assay, using the previously-described primers [23].

The results show that KCNQ1OT1 levels are significantly downregulated in cancer tissues (“Can”) (Figure 1A), as compared to its levels in the surrounding epithelial (“Epi”) tissues (Figure 1A). Therefore, in GC cancer tissues KCNQ1OT1 downregulation correlated with miR-9 upregulation (see the previous results from same set of tissue samples [7]).

Figure 1. LncRNAKCNQ1OT1 is downregulated in human GC tissues. Expression of LncRNAKCNQ1OT1 (A) in twelve (12) different human gastric cancer (GC) tissues (“Can”) and matched surrounding normal epithelial tissues (“Epi”) was shown, results were normalized to U6 RNA. All values were expressed as the mean ± standard deviation (Same for all Figures). *P <0.05 vs. “Epi” tissues.

Forced overexpression of LncRNA KCNQ1OT1 induces miR-9 depletion and LMX1A upregulation in AGS cells

In order to test the potential effect of LncRNAKCNQ1OT1 on miR-9-LMX1A expression, a lentiviral GV248 construct encoding KCNQ1OT1 promoter (“LV-KCNQ1OT1”) was transfected to AGS cells. Following selection by puromycin, two stable cell lines (namely “sL1/sL2”) were established. Testing KCNQ1OT1 expression, by qPCR assays, demonstrated that mature KCNQ1OT1 levels increased over 8 to 10 folds in the stable cells (Figure 2A). Significantly, with KCNQ1OT1 overexpression miR-9 levels were dramatically downregulated (over 60–70%, Figure 2B), resulting in increased LMX1A 3′-UTR luciferase activity (Figure 2C). Moreover, LMX1A mRNA (Figure 2D) and protein (Figure 2E and 2F) levels were significantly increased in AGS cells with LV-KCNQ1OT1. Therefore, KCNQ1OT1 overexpression downregulated miR-9, leading to upregulation of its target LMX1A in AGS cells. The lentiviral control vector (“Vec”) had no significant effect on KCNQ1OT1-miR-9-LMX1A expression in AGS cells (Figure 2A–2F).

Figure 2. Forced overexpression of LncRNAKCNQ1OT1 induces miR-9 depletion, LMX1A upregulation and AGS cell inhibition. AGS cells were infected with KCNQ1OT1-expressing lentivirus (“LV-KCNQ1OT1”) or scramble control vector lentivirus (“Vec”) for 24h, following puromycin selection two stable lines (“sL1/sL-2”) with LV-KCNQ1OT1 established, expression of KCNQ1OT1 (A), miR-9 (B) and LMX1A mRNA (D) were tested by qPCR assays; The LMX1A 3’-UTR luciferase activity was shown (C); Expression of the listed proteins in total cell lysates were tested by Western blotting assay (E, results quantified in F); Cells were further cultured for the indicated time periods, cell survival and proliferation were tested by CCK-8 assay (G) and EdU staining (H), respectively; Cell migration and invasion were tested by “Transwell” (I) and “Matrigel Transwell” assay (J), respectively; Cell apoptosis was tested by Western blotting (testing apoptosis-associated proteins, K), Annexin V FACS (L) and TUNEL staining assay (M). The exact same number of viable cells of different genetic treatment were plated initially (at 0h) for the functional assays (Same for all following Figures). “Ctrl” stands for the parental control cells (Same for all Figures). For each assay, n=5. *P <0.05 vs. “Vec” cells. Experiments in this figure were repeated five times, and similar results were obtained. Bar=100 μm (H–J).

Forced overexpression of LncRNAKCNQ1OT1 inhibits AGS cell progression in vitro

LMX1A regulates a number of physiological and pathological processes [8]. It is considered as a tumor suppressor [8, 9, 11]. LMX1A downregulation is detected in GC and many other cancers [9, 10]. Our previous study has shown that LMX1A overexpression can inhibit AGS cell progression in vitro. We therefore analyzed the functional consequences of KCNQ1OT1 overexpression.

Results showed that AGS cells with LV-KCNQ1OT1 presented with reduced cell viability (vs. control cells, Figure 2G). Furthermore, KCNQ1OT1 overexpression inhibited EdU incorporation (Figure 2H) in AGS cells, indicating proliferation inhibition. “Transwell” and “Matrigel Transwell” assay results, in Figure 2I and 2J, demonstrated that ectopic overexpression of KCNQ1OT1 potently inhibited AGS cell migration and invasion in vitro. Additionally, apoptosis activation was detected in KCNQ1OT1-overexpressed AGS cells, evidenced by cleavages of caspase-3, caspase-9 and poly adenosine diphosphate (ADP) ribose (PARP) (Figure 2K), as well as increased Annexin V staining (Figure 2L) and nuclear TUNEL ratio (Figure 2M). The lentiviral control vector (“Vec”) had no significant effect on the function of AGS cells (Figure 2G–2M).

Forced overexpression of LncRNAKCNQ1OT1 induces miR-9 reduction and LMX1A upregulation in primary human GC cells

Next, we tested whether LncRNAKCNQ1OT1 exerted similar functions in primary human GC cells. As described previously [7], the primary human GC cells, derived from two independent patients (namely “GC-1/GC-2”), were infected with LV-KCNQ1OT1, which resulted in 5-6 folds increase of KCNQ1OT1 expression (Figure 3A). On the contrary, miR-9 levels were significantly decreased (Figure 3B), with LMX1A mRNA (Figure 3C) and protein (Figure 3D) levels increased in KCNQ1OT1-overexpressed GC cells. LMX1B protein expression was unaffected by KCNQ1OT1 overexpression (Figure 3D).

Figure 3. Forced overexpression of LncRNAKCNQ1OT1 induces miR-9 reduction and LMX1A upregulation in primary human GC cells. The primary human GC cells (“GC-1/GC-2”) were infected with LncRNAKCNQ1OT1-expressing lentivirus (“LV-KCNQ1OT1”) or scramble control vector lentivirus (“Vec”) for 72h, expression of KCNQ1OT1 (A), miR-9 (B) and LMX1A mRNA (C) were tested by qPCR assay; LMX1A and LMX1B protein expression in total cell lysates was tested by Western blotting assay (D, LMX1A protein results were quantified); Cells were further cultured for the indicated time periods, cell proliferation and viability were tested by EdU staining assay (E) and CCK-8 (F), respectively, with cell migration and invasion tested by “Transwell” and “Matrigel Transwell” assay (G and H); Cell apoptosis was quantified via the TUNEL staining assay (I) and Annexin V-FACS assay (J). For each assay, n=5. *P <0.05 vs. “Vec” cells. Experiments in this figure were repeated three times, and similar results were obtained. Bar=100 μm (E and G).

Functional studies show that KCNQ1OT1 overexpression by LV-KCNQ1OT1 inhibited EdU incorporation (Figure 3E) and cell viability (Figure 3F) in primary human GC cells. Furthermore, LV-KCNQ1OT1-expressing primary GC cells presented with decreased cell migration and invasion (vs. Vector control cells, Figure 3G, 3H). Contrarily, the increased TUNEL staining (Figure 3I, vs control cells) and Annexin V ratio (Figure 3J) were detected in LV-KCNQ1OT1-expressing primary GC cells, indicating apoptosis activation. Therefore, ectopic KCNQ1OT1 overexpression inhibited viability, proliferation, migration and invasion, but inducing apoptosis activation in primary GC cells.

LncRNA KCNQ1OT1 siRNA induces miR-9 upregulation and LMX1A downregulation, promoting GC cell progression in vitro

Next, the siRNA strategy was employed to knockdown KCNQ1OT1. Two siRNAs, targeting non-overlapping sequence of KCNQ1OT1 (namely “seq1/seq2” [24]), were transfected to AGS cells. Analyzing KCNQ1OT1 expression by qPCR confirmed that the applied siRNAs resulted in over 60-70% reduction of KCNQ1OT1 expression in AGS cells (Figure 4A). Significantly, KCNQ1OT1 siRNA induced miR-9 upregulation (Figure 4B) and LMX1A mRNA/protein downregulation (Figure 4C). Importantly, KCNQ1OT1-silenced cells presented with increased cell viability (Figure 4D) and EdU staining (Figure 4E), as compared to cells with scramble control siRNA (“si-C”) (Figure 4D, 4E). AGS cell migration and invasion, tested by “Transwell” assay (Figure 4F) and “Matrigel Transwell” assay (Figure 4G), were augmented by KCNQ1OT1 siRNAs as well.

Figure 4. LncRNA KCNQ1OT1 siRNA induces miR-9 upregulation and LMX1A downregulation, promoting GC cell progression in vitro. AGS cells (A–G) or primary human GC cells (“GC-1/GC-2”) (H–L) were transfected with 500 nM of KCNQ1OT1 siRNA (“seq1/seq2”, two rounds, total 48h) or the scramble control siRNA (“si-C”, two rounds, total 48h), expression of KCNQ1OT1 (A and H), miR-9 (B and I) and LMX1A mRNA (C and J) were tested by qPCR assays; LMX1A and LMX1B protein expression was tested by Western blotting assay (C and K, LMX1A protein results were quantified); Cells were further cultured for the indicated time periods, cell viability (D) and proliferation (E and L) were tested by the listed assays; Cell migration and invasion were tested by “Transwell” (F) and “Matrigel Transwell” (G) assays respectively. For each assay, n=5. *P <0.05 vs. “si-C” cells. Experiments in this figure were repeated three times, and similar results were obtained. Bar=100 μm (E–G).

In primary human GC cells (“GC-1/GC-2”), transfection with the KCNQ1OT1 siRNA (“seq2”) similarly resulted in KCNQ1OT1 downregulation (Figure 4H), miR-9 upregulation (Figure 4I) and LMX1A downregulation (Figure 4J, 4K). Cell proliferation, tested by an EdU staining assay, was enhanced as well (Figure 4L). These results further confirmed a tumor-suppressive function by KCNQ1OT1, and KCNQ1OT1 siRNA promoted GC cell progression in vitro.

In LMX1A-knockout AGS cells altering KCNQ1OT1 expression fails to change cell viability and proliferation

To verify that LMX1A is one target of KCNQ1OT1, we utilized the CRISPR/Cas9 gene-editing method to knockout LMX1A (see our previous study [7]). Two stable LMX1A knockout (“Cas9-LMX1A-ko”) AGS cell lines were established (namely “S1/S2”) [7]. Results in Figure 5A confirmed LMX1A protein depletion in the stable cells, with LMX1B unaffected (Figure 5A). LV-KCNQ1OT1 (see Figure 2) or KCNQ1OT1 siRNA (“seq2”, see Figure 3) were tranduced to the Cas9-LMX1A-ko cells, both significantly altered KCNQ1OT1 expression (Figure 5B). Significantly, neither LV-KCNQ1OT1 nor KCNQ1OT1 siRNA altered viability (Figure 5C) and proliferation (EdU ratio, Figure 5D) in Cas9-LMX1A-ko AGS cells. Thus, in LMX1A-knockout AGS cells KCNQ1OT1 did not change cell viability and proliferation, indicating that LMX1A is one target of KCNQ1OT1.

Figure 5. In LMX1A-knockout AGS cells altering KCNQ1OT1 expression fails to change cell viability and proliferation. AGS cells were transfected with the lentiCRISPR/Cas9 LMX1A knockout constructs containing non-overlapping sgRNA sequences (“S1/S2”), following FACS sorting and puromycin selection two stable lines were obtained (“Cas9-LMX1A-ko”). LMX1A and LMX1B protein expression was tested (A, LMX1A protein results were quantified). LV-KCNQ1OT1 or KCNQ1OT1 siRNA (500 nM) were transduced to the Cas9-LMX1A-ko AGS cells (“S1/S2”) for 72h, KCNQ1OT1 expression (B), cell viability (C) and proliferation (D) were shown. (E) The proposed signaling cascade of this study. For each assay, n=5. *P <0.05 vs. “Ctrl” cells. Experiments in this figure were repeated three times, and similar results were obtained.

Chemicals and reagents

Puromycin, neomycin and polybrene were obtained from Sigma-Aldrich (St. Louis, Mo). All antibodies were purchased from Abcam (Cambridge, MA). RNA reagents, Lipofectamine 2000 and other transcription reagents were obtained from Invitrogen (Shanghai, China). All sequences and plasmids were provided by Genechem (Shanghai, China).

Cell culture

AGS cells were cultured as previously described [7]. The primary human GC cells, derived from two independent written informed-consent GC patients (“GC-1” and “GC-2”), were cultured in the described medium [31]. The protocols were approved by the Ethics Board of Minhang Hospital, Fudan University (Ethics number: 2015-052, Principle Investigator, Dr. Feng Li), in according to Declaration of Helsinki.

Human tissues

As described previously [7], fresh GC cancer tissues along with matched surrounding normal gastric epithelial tissues from twelve (12) primary informed-consent GC patients (all male, 41–67 years old, with no prior chemotherapy and radiotherapy) were acquired. Tissues were washed, minced, and homogenized before further analysis.

qPCR assay

For each treatment, aliquots of 100 ng total RNAs were reverse transcribed into cDNA with Reverse Transcriptase M-MLV (Promega, Madison, WI). The quantitative reverse transcriptase PCR (qPCR) assay was performed by the previously-described protocol [7], using ^ΔΔCt method for target mRNA quantification and GAPDH as the internal control. KCNQ1OT1 and miR-9 expression was normalized to U6 RNA. Primers for miR-9, LMX1A, GAPDH and U6 were reported previously [7]. The primers for KCNQ1OT1, forward, 5′-CTTTGCAGCAACCTCCTTGT; reverse, 5′-TGGGGTGAGGGATCTGAA, were also reported early [32].

Forced KCNQ1OT1 overexpression

The PCR primer sets, 5′-GGGGTACCCCAGGTGACAAGGTGCAGGCGC and reverse primer: 5′-ACAGAGTTCCTCGTTGGGAGCTTGAAGATCTTC [32], were applied to amplify the upstream KCNQ1OT1 promoter region from GC cells, inserted into the hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector (GV248, Genechem). The construct, along with the lentiviral packaging constructs [7], were transfected to HEK-293 cells to generate KCNQ1OT1 expression lentivirus (“LV-KCNQ1OT1”). The lentivirus was filtered, enriched and added to GC cells. When necessary, stable cells were selected by puromycin (5.0 μg/mL) for a total of 4-5 passages. Control cells were infected with control lentivirus with empty vector.

KCNQ1OT1 siRNA

GC cells were seeded at 50% confluence. Two different siRNAs (designed and verified by RIBOBIO, Guangzhou, China [24]) against non-overlapping sequence of KCNQ1OT1 were individually transfected (siRNA concentration at 500 nM) by Lipofectamine 2000 reagent for 24h, repeated for a second round for another 24h (total 48h). KCNQ1OT1 knockdown was confirmed by qPCR assay. Control cells were transfected with the scramble non-sense control siRNA (“si-C”).

Luciferase reporter assay

As reported [7], the human LMX1A 3’-UTR with the putative binding sites of miR-9 (see the sites at [7]) was amplified by PCR and then inserted into the firefly luciferase reporter vector, pGL4.13 (luc2/SV40) (Promega), at the XbaI site and downstream from the stop codon of the luciferase gene. AGS cells were plated at 1 × 10⁵ cells per well into six-well plates (60% confluence), transfected with pGL4.13 construct along with the Renilla luciferase reporter vector and pRL-SV40 (Promega). Following the indicated genetic treatments, LMX1A 3’-UTR luciferase activity value was tested, and results were normalized to that in the control cells.

Cell viability assay

Cells were seeded onto the 96-well plates (3 × 10³ cells/ well) [7]. Cell viability was determined via the Cell Counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan), with optical density (OD) values measured at 570 nm.

In vitro cell migration and invasion assays

As described [33], GC cells were seeded on “Transwell” upper chamber (at 3 × 4000 cells per chamber, BD Biosciences). The complete medium (with 10% FBS) was added to the lower compartments. After 24h the migrated cells on the lower surface were stained. Matrigel (Sigma) was added in the chamber surface when analyzing cell invasion. Five repeated views in each condition were included to calculate the average number of migrated/invasive cells.

EdU assay

EdU (5-ethynyl-20-deoxyuridine) Apollo-567 Kit (RIBOBIO, Shanghai, China) was employed to test cell proliferation. Briefly, cells were seeded onto the twelve-well plates. Following treatment, EdU and DAPI dyes were added, and cells were incubated for additional 8h. Cell nuclei were then visualized under a fluorescent microscope. For each condition at least 500 cells in five random views were included to calculate EdU ratio (EdU/DAPI×100%).

Apoptosis assays

Testing cell apoptosis, by the Annexin V FACS and TUNEL staining assay, was described in detail in our previous study [7].

LMX1A knockout

The two different lentiCRISPR-GFP-puro LMX1A knockout constructs (see our previous study [7]) were independently transfected to AGS cells by Lipofectamine 2000. GFP-positive cells were FACS-sorted, cultured for another three weeks. Stable cells were achieved by puromycin selection, named as “Cas9-LMX1A-ko” cells.

Western blotting

Total cellular lysates were resolved by SDS-PAGE (10%) gels, transferred to a PVDF membrane. The latter was incubated with blocking buffer and following primary and secondary antibodies. ECL method was applied to detect the immuno-reactive bands.

Statistical analyses

The GraphPad Prism software was employed for statistical analyses. All values were expressed as the mean ± standard deviation (SD). Repeated-measures analysis of variance (RMANOVA) followed by Dunnett’s post hoc test for multiple comparisons were applied to evaluate statistical significance. All differences were considered significant at P < 0.05. To determine significance between two treatment groups, the two-tailed t tests were carried out.