Upregulation of LINC00265 is associated with poor clinical outcomes among patients with GC
To determine the specific role of LINC00265 in GC, the expression profile of this lncRNA was examined in 32 pairs of GS tissue samples and adjacent normal tissue samples. LINC00265 was found to be overexpressed in the GC tissue samples relative to the adjacent normal gastric tissues, as revealed by reverse-transcription quantitative PCR (RT-qPCR; Figure 1A, P < 0.05). Additionally, the expression of LINC00265 was quantified in a panel of GC cell lines (KATO-III, SGC-7901, BGC-823, HGC-27, AGS, NCI-N87, SNU-1, and SNU-16) and in normal gastric cells (GES cells). The results showed that LINC00265 expression was higher in the eight tested GC cell lines than in GES cells (Figure 1B, P < 0.05). Notably, patients with GC overexpressing LINC00265 showed shorter overall survival and disease-free survival (Figure 1C, P = 0.0354; Figure 1D, P=0.0120) than the patients with GC underexpressing LINC00265. These results implied that LINC00265 may be closely associated with the pathogenesis of GC.
A reduction in LINC00265 expression inhibits the malignant characteristics of GC cells in vitro
Having detected the aberrant upregulation of LINC00265 in GC, we next attempted to determine the functions of LINC00265 in GC progression. NCI-N87 and KATO III cells were chosen for subsequent experiments and were transfected with either a small interfering RNA [siRNA] against LINC00265 (si-LINC00265) or a negative control siRNA (si-NC). LINC00265 was successfully knocked down in NCI-N87 and KATO III cells after transfection of si-LINC00265 (Figure 2A, P < 0.05). A Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the influence of si-LINC00265 on GC cell proliferation. The si-LINC00265 transfection obviously reduced the proliferative ability of NCI-N87 and KATO III cells compared with that in the si-NC group (Figure 2B, P < 0.05). Then, colony formation assay was conducted to test whether si-LINC00265 introduction increases GC cell apoptosis. As expected, the number of si-LINC00265 group was lower among NCI-N87 and KATO III cells after transfection with si-LINC00265 (Figure 2C, P < 0.05). In addition, Transwell migration assays revealed that the LINC00265 knockdown notably reduced the migration (Figure 2D, P < 0.05) of NCI-N87 and KATO III cells. In general, these findings suggested that the LINC00265 downregulation slowed the malignant progression of GC in vitro.
LINC00265 acts as a competing endogenous RNA (ceRNA) on miR-144-3p in GC cells
To investigate the molecular events involved in LINC00265–mediated GC progression, a nuclear/cytoplasmic fractionation assay was conducted to determine the distribution of LINC00265 inside GC cells. The data indicated that LINC00265 is mainly located in the cytoplasm of GC cells (Figure 3A); this finding suggested that this lncRNA may serve as a ceRNA for some miRNA(s) [28]. Herein, the candidate miRNAs that could be inactivated by LINC00265 were predicted via starBase 3.0. The results indicated that LINC00265 contains one conserved binding site for miR-144-3p (Figure 3B). Luciferase reporter was performed to further characterize the relation between LINC00265 and miR-144-3p in GC cells. MiR-144-3p agomir (agomiR-144-3p) transfection-mediated upregulation of miR-144-3p (Figure 3C, P < 0.05) noticeably decreased the luciferase activity of the LINC00265-WT plasmid (the plasmid expressing LINC00265 containing the wild-type binding site for miR-144-3p; P < 0.05); however, the luciferase activity of LINC00265-MUT (the plasmid expressing LINC00265 containing a mutant binding site for miR-144-3p) was unaffected in both NCI-N87 and KATO III cells when miR-144-3p was overexpressed, as evidenced by the luciferase reporter assay (Figure 3D). This finding suggested that miR-144-3p is a target of LINC00265 in GC cells. MiR-144-3p expression was subsequently measured in the 40 pairs of GC tissue samples and adjacent normal gastric tissues by RT-qPCR. MiR-144-3p was found to be significantly underexpressed in the GC tissue samples (Figure 3E, P < 0.05). In addition, the expression of miR-144-3p was evaluated in LINC00265–deficient NCI-N87 and KATO III cells. The LINC00265 knockdown remarkably upregulated miR-144-3p in NCI-N87 and KATO III cells (Figure 3F, P < 0.05). Overall, these results meant that LINC00265 functions as a ceRNA (molecular sponge) for miR-144-3p in GC cells.
MiR-144-3p acts as a tumor-suppressive miRNA in GC cells
To examine the manner in which miR-144-3p regulates the malignancy of GC, the NCI-N87 and KATO III cell lines were transfected with either agomiR-144-3p or agomir-NC and, then, subjected to functional experiments. Transfection with agomiR-144-3p appreciably decreased proliferation (Figure 4A, P < 0.05) of NCI-N87 and KATO III cells. Transwell migration assays showed that, when endogenous miR-144-3p was overexpressed, NCI-N87 and KATO III cells had weaker migratory (Figure 4C, P < 0.05). In summary, these data suggested that miR-144-3p may suppress GC progression.
CBX4 upregulation was correlated with poor prognosis of GC and enhances the malignant characteristics of gastric cancer
Again, to determine the specific role of CBX4 in GC, the expression profile of this lncRNA was examined in 40 pairs of GS tissue samples and adjacent normal tissue samples. CBX4 was found to be overexpressed in the GC tissue samples relative to the adjacent normal gastric tissues, as revealed by reverse-transcription quantitative PCR (RT-qPCR; Figure 5A, P < 0.05). Additionally, the expression of CBX4 was quantified in a panel of GC cell lines (KATO-III, SGC-7901, BGC-823, HGC-27, AGS, NCI-N87, SNU-1, and SNU-16) and in normal gastric cells (GES cells). The results showed that LINC00265 expression was higher in the eight tested GC cell lines than in GES cells (Figure 5B, P < 0.05). A cohort of 140 pair tissues samples were performed IHC assay, the IHC result showed that CBX4 was mainly expressed in the cytoplasm. Positive immmunoreactivity was depicted in 90.7% (127/140) of cancerous tissues and 27.1% (38/140) of noncancerous tissues. Elevated expression of CBX4 in cancer was found in 72.9% (102/140) of cases (Fig. 5C). Notably, patients with GC overexpressing CBX4 showed shorter overall survival (Fig. 5D, P = 0.0184) than the patients with GC underexpressing CBX4. To further reveal the influence of CBX4 on GC cells, CBX4 was successfully knocked down in NCI-N87 and KATO III cells after transfection of si-CBX4 (Figure 6A&B, P < 0.05). A Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the influence of si- CBX4 on GC cell proliferation. The si-CBX4 transfection obviously reduced the proliferative ability of NCI-N87 and KATO III cells compared with that in the si-NC group (Figure 6C, P < 0.05). Then, colony formation assay was conducted to test whether si-CBX4 introduction increases GC cell apoptosis. As expected, the number of si-CBX4 group was lower among NCI-N87 and KATO III cells after transfection with si-CBX4 (Figure 6D, P < 0.05). In addition, Transwell migration assays revealed that the CBX4 knockdown notably reduced the migration (Figure 6E, P < 0.05) of NCI-N87 and KATO III cells. These results indicated that CBX4 may be closely associated with the pathogenesis of GC and could enhance the ability of proliferation and metastasis of gastric cancer cells.
CBX4 mRNA is a direct target of miR-144-3p in GC cells
By means of target prediction tools, including starBase 3.0 and TargetScan, CBX4 was predicted as a potential target gene of miR-144-3p (Figure 7A). To validate this prediction, a luciferase reporter assay was performed on NCI-N87 and KATO III cells after cotransfection with either agomiR-144-3p or agomir-NC and either plasmid CBX4-WT (a plasmid expressing luciferase mRNA containing the CBX4 3′-UTR harboring a wild-type binding site for miR-144-3p) or plasmid CBX4-MUT (a plasmid expressing luciferase mRNA containing the CBX4 3′-UTR harboring a mutated binding site for miR-144-3p). The ectopic expression of miR-144-3p significantly reduced the luciferase activity of CBX4-WT in NCI-N87 and KATO III cells (P < 0.05). By contrast, mutation of the binding site abrogated this phenomenon (Figure 7B). Next, the expression levels of CBX4 in miR-144-3p-overexpressing NCI-N87 and KATO III cells were determined to investigate whether CBX4 expression can be inhibited by miR-144-3p in GC. As expected, the protein levels (Figure7C, P < 0.05) and mRNA (Figure 7D, P < 0.05) of CBX4 in NCI-N87 and KATO III cells diminished in response to the agomiR-144-3p transfection. Collectively, the above findings identified CBX4 mRNA as a direct target of miR-144-3p in GC cells.
LINC00265 enhances the malignant characteristics of GC cells in vitro through the miR-144-3p/CBX4 axis
To test whether the oncogenic effects of LINC00265 on the malignancy of GC cells were mediated by its influence on the miR-144-3p/CBX4 pathway, rescue experiments were performed on LINC00265-deficient NCI-N87 and KATO III cells via transfection with miR-144-3p antagomir (antagomiR-144-3p). MiR-144-3p expression was found to be efficiently upregulated in NCI-N87 and KATO III cells after the co-transfection with si-LINC00265 and antagomiR-NC. But the miR-144-3p expression levels were relatively downregulated after co-transfection with si-LINC00265 and antagomiR-144-3p (Figure 7F, P < 0.05). On the contrary, protein expression levels of CBX4 were downregulated both in NCI-N87 and KATO III cells after the co-transfection with si-LINC00265 and antagomiR-NC, and regulated relatively co-transfection with si-LINC00265 and antagomiR-144-3p (Figure 7G, P < 0.05). These data revealed that the LINC00265 knockdown reduced the malignancy of GC cells in vitro by decreasing the sponging of miR-144-3p by LINC00265 and, thereby, reducing CBX4 expression.
The LINC00265 knockdown inhibits the in vivo tumor growth of GC cells
Xenograft tumors were induced to test whether there is a similar influence of LINC00265 on tumor growth in vivo as in the above experiments in vitro. NCI-N87 cells transfected with either si-LINC00265 or si-NC were subcutaneously injected into a flank of nude mice. At 28 days postinoculation, the tumor growth curve indicated that the growth of tumor xenografts was much slower in the si-LINC00265 group than in the si-NC group (Figure 8A and 8B, P < 0.05). At the end of this experiment, all the mice were euthanized and the tumor xenografts were excised and weighed. The weight of the tumor xenografts derived from si-LINC00265–transfected NCI-N87 cells was obviously lower (Figure 8C, P < 0.05). Additionally, LINC00265 expression was still low in the tumor xenografts from the si-LINC00265 group (Figure 8D, P < 0.05). The expression of miR-144-3p was higher (Figure 8E, P < 0.05), whereas the protein expression of CBX4 was lower (Figure 8F, P < 0.05), in the si-LINC00265 group than in the si-NC group. These results indicated that the downregulation of LINC00265 retarded the in vivo tumor growth of GC cells by decreasing the output of the miR-144-3p–CBX4 axis.