LOC101060264 Silencing Suppresses Invasion and Metastasis of Human Colon Cancer.

BACKGROUND We explored the regulatory effects of long noncoding RNA (lncRNA) LOC101060264 silencing mediated by shRNA on invasion and metastasis of human colon cancer. MATERIAL AND METHODS Initially, 2 shRNA plasmids for LOC101060264 silencing - shRNA1 and shRNA2 - were introduced into LoVo cells. Following transfection, the expressions of LOC101060264, E-cadherin, and vimentin were determined. Next, to explore the regulatory effects of LOC101060264 silencing on cell growth, cell cycle, invasion, and migration abilities of LoVo cells, we performed MTT, flow cytometry, Transwell assay, and scratch assay, respectively. Furthermore, in nude mice with xenografted tumors, the tumor volume and weight were measured, and the expressions of PCNA, E-cadherin, vimentin, and MMP-9 in tumor tissues were determined by immunohistochemistry. RESULTS The level of E-cadherin increased and the level of vimentin decreased after LOC101060264 silencing mediated by shRNA1 and shRNA2 in LoVo cells. Silencing LOC101060264 repressed the migration, invasion, and proliferation of LoVo cells in vitro and inhibited tumor growth in nude mice in vivo. We also studied the expression of these proteins and found reduced expression of PCNA, vimentin, and MMP-9 protein, and found enhanced expression of E-cadherin protein. Moreover, the inhibitory effect of shRNA2 on the above cell behaviors was stronger than that of shRNA1. CONCLUSIONS In summary, LOC101060264 silencing decreased LoVo cell invasiveness via suppressing ETM and attenuated tumor metastasis, which provides a novel therapeutic target for patients with colon cancer.

Background

Colon cancer is one of the commonest cancers worldwide, with higher incidence and mortality in men than in women [1]. Conspicuous changes have been demonstrated in incidence and death rates in Asia and Africa [2]. The main symptoms of colon cancer patients are anemia, rectal bleeding, and abdominal pain, which are affected by the size and location of tumors [3]. In exploring therapeutic targets for colon cancer treatment, the molecular mechanisms that mediate colon cancer formation and progression have attracted considerable attention.

Long noncoding RNAs (lncRNAs) play critical roles in various biological processes [4]. lncRNAs, as a chief class of regulatory molecules, are involved in diverse disorders [5]. A study conducted by Li et al. found that lncRNAs are essential in tumorigenesis and metastasis of cancer [6]. lncRNAs are involved in colon cancer progression and play a vital role in colon cancer cell biological processes [7]. Kong et al. reported that lncRNA LINC01133 inhibits epithelial-mesenchymal transition (EMT) and metastasis in colon cancer through downregulation of transforming growth factor-β (TGF-β) [8]. EMT is a cellular process related to metastasis and invasion, with presence of mesenchymal properties and loss of epithelial features [9]. The EMT process plays a critical role in tumor invasion and metastasis. The loss of intercellular adhesion molecules (i.e., E-cadherin), downregulation of epithelial markers (e.g., cytokeratin), and upregulation of mesenchymal markers (e.g., vimentin) are characteristics of EMT [10]. In tumor invasion, the E-cadherin high/vimentin-positive expression indicates the leader cancer cells guide the follower cells at their rear. Carcinogenesis is a complex and multistage process involving cell proliferation (proliferating cell nuclear antigen, PCNA), EMT, and metastasis (matrix metallopeptidase 9, MMP-9) [11].

Short hairpin RNA (shRNA)-mediated RNA interference has been applied for gene silencing, and holds great promise for the treatment of cancer and other genetic disorders [12,13]. To block hepatic metastases of colon cancer cells, Bao et al. used an shRNA against Rac1 delivered by activatable cell-penetrating peptide [14]. A shRNA was used to knockdown ferrochelatase (FECH) in human Caco-2 colon cancer cells, and results showed that silence of FECH was protective against colon cancer tumorigenesis [15]. In the present study we silenced the expression of LOC101060264 by shRNA interference, and investigated the roles of LOC101060264 silencing in EMT process of LoVo cells, so as to find a potential therapeutic target for colon cancer.

Material and Methods

Cell culture

The LoVo cells was purchased from the Shandong University (Jinan, Shandong, China). LoVo cells were preserved in nitrogen before culture. For cell culture, LoVo cells were rapidly placed in a water bath at 37°C for 5 min, centrifuged at 403×g for 10 min, and cultured with Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium containing 10% fetal calf serum (FCS) at 5% CO2 and 37°C. Cells in exponential phase were used for experiments.

Cell transfection and grouping

The shRNA oligonucleotide template was synthesized with the interference plasmid p-super-EGFP1 vector. Two shRNA plasmids were synthesized by Shanghai Invitrogen Biotechnology Co. (Shanghai, China). LOC101060264 shRNA plasmids were constructed and identified by double-enzyme digestion and sequencing. After plasmids were stably transfected into tumor cells, LOC101060264-shRNA plasmids LOC101060264 shRNA1 and LOC101060264 shRNA2 were generated, and their sequences were 5′-UUUUCUACCAGGUCGGUAC-3′ and 5′-AAUUCUUAAAUUGGGCUGG-3′, respectively [16]. Before transfection, LoVo cells were randomly assigned into a control group (without transfection), a shRNA-NC group (transfected with LOC101060264 shRNA-NC plasmid), a shRNA1 group (transfected with LOC101060264 shRNA1 plasmid), or a shRNA2 group (transfected with LOC101060264 shRNA2 plasmid). Lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA, USA) was used for transfection of plasmid into LoVo cells. After transfection for 4 h, the culture solution was brought to 2 mL with serum-free medium, and then incubated in the incubator for 48 h.

Immunofluorescence

The cells (1×106 cells/mL) spread across the treated cover glass. First, after different treatments as required, the cells were fixed with 4% paraformaldehyde. Next, 0.2% Triton X-100 was used for membrane rupture. Subsequently, cells were sealed with 5% bovine serum albumin (BSA) and incubated at 37°C for 30 min. Then, cells were incubated with corresponding primary antibodies E-cadherin (1: 100; ab15148; Abcam, Inc., shanghai, China) and vimentin (1: 100; ab92547; Abcam, Inc., Shanghai, China) at 37°C for 30 min, and at 4°C overnight. After cells were rinsed with phosphate buffer saline (PBS), we added Texas red-labeled goat anti-rabbit IgG H&L (1: 200, ab6719, Abcam) secondary antibody and FITC-labeled goat anti-rabbit IgG H&L secondary antibody (1: 50, ab6717, Abcam) for incubation at 37°C for 30 min. After rinsing with PBS, cells were stained with 4′,6-Diamidino-2-Phenylindole (DAPI) and mounted with glycerol. Fluorescence image acquisition was performed using a Nikon A1R confocal imaging system (Nikon, Tokyo, Japan). Then, cells were visualized and images were captured under an inverted laser confocal microscope (FV1200; New Discovery Technology (China) Co., Shanghai, China). Image J software was used to analyze the positive expression.

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay

The cell concentration was adjusted to 2.5×104 cells/mL, and 200 μL of each well was seeded in a 96-well culture plate. The cells in the control, shRNA-NC, shRNA1, and shRNA2 groups were adhered to the wall and grew well. A control well and 6 parallel wells were used for each group. At a wavelength of 492 nm, the optical density (OD) value of each well was measured.

Flow cytometry

Following transfection for 48 h, the cells were treated with freezing PBS and then centrifuged at 179×g for 5 min to discard the supernatant. Next, the cells were adjusted to a cell concentration of approximately 1×105 cells/mL, fixed with pre-cooled 70% ethanol, and placed at −20°C overnight. After washing twice with PBS to remove the supernatant, cells were added to 100 μL RNase A and treated in a water bath at 37°C in dark for 30 min. Finally, cells were stained with 400 μL propidium iodide (PI) (Sigma-Aldrich Co., St. Louis, MO, USA) for 4 min at 30°C in the dark, and the cell cycle distribution was measured by flow cytometry (NovoCyte; New Discovery Technology (China) Co., Shanghai, China).

Transwell assay

The diluted Matrigel was placed on the upper layer of a Transwell chamber (Beijing Lebo Biotechnology Co., Beijing, China). After solidification, 2×104 cells were inoculated in each well. Serum complete medium was added to the lower layer for 48 h. Next, the chamber was repeatedly washed with PBS and fixed with 95% alcohol for 5 min. Then, it was repeatedly rinsed with PBS and stained with 0.1% crystal violet solution (Biochannel, Nanjing, China). Finally, the cells migrated to the lower layer and were counted under an inverted microscope.

Scratch test

Cells in exponential phase of each group were detached, counted, and seeded in a 6-well plate at a density of 2×105 cells/well, with 3 parallel wells. When the cell confluence reached about 95%, the scratch test was performed with medium substituted by RPMI-1640 medium containing 5% FCS. A straight line was scratched using the tip of a 10-uL pipette gun head, and cells were washed with PBS 3 times. The culture medium was added to cells and for further incubation at 37°C for 24–48 h. Then, the cell migration was visualized under an inverted microscope at the scheduled time points 0 h and 24 h, then photographed and recorded. The healing rate of the scratch area was analyzed and calculated as: scratch area=(scratch area at 0 h–scratch area at each time point)/scratch area at 0 h×100%. Cell migration ability was determined.

Tumorigenicity assay in nude mice

Forty BALB/C male nude mice aged 6±2 weeks and weighing 25±2 g were supplied by the Animal Center of Shandong University (Jinan, Shandong, China). Nude mice were randomly allocated into a control group (n=10), a shRNA-NC group (n=10), a shRNA1 group (n=10), or a shRNA2 group (n=10). LoVo cells in exponential phase following transfection were obtained. We subcutaneously inoculated 0.2 mL cell suspension into mice in the back of the neck or in the axilla. After inoculation, the tumor growth was observed at regular weekly intervals. The diameter of subcutaneous nodules >0.5 cm was defined as the presence of tumor formation [17]. After tumors were successfully formed in nude mice, the tumor volume was measured daily and recorded to draw the tumor growth curve of each group. After the experiment, pentobarbital sodium was injected into the abdominal cavity of each mouse. All the nude mice were anesthetized by 3% pentobarbital sodium (50 mg/kg) and killed through cervical vertebra acetabular method. The tumor tissues were excised and weighed, and the maximum diameter (L) and the minimum diameter (W) of the transplanted tumors were measured. The volume of the transplanted tumor was calculated as V=W2×L×0.5. Animal experiments were performed strictly following the NIH guidelines (NIH Pub. No. 85-23, revised 1996) and were approved by the Second Hospital of Shandong University (2018-10072).

Immunohistochemistry

Briefly, after dewaxing with xylene, dehydration in graded ethanol, 4-μm-thick sections of renal tissue were placed in citrate buffer solution for antigen extraction, then 3% H2O2 was used to block the antigen for 10 min. The cancer tissue was incubated with the following antibodies: Rabbit anti-mouse PCNA antibody (1: 100, ab15497, Abcam, UK), Rabbit anti-mouse E-cadherin antibody (1: 100, ab239883, Abcam, UK), Rabbit anti-mouse vimentin antibody (1: 100, ab193555, Abcam, UK), and Rabbit anti-mouse MMP-9 antibody (1: 200, ab38898, Abcam, UK). After washing 3 times with PBS, goat anti-rabbit IgG H&L antibodies (1: 100, ab150077, Abcam, UK) were added and incubated for 45 min, and the sections were counterstained with hematoxylin after visualizing with a Diaminobenzidine (DAB) kit. Image J software was used to analyze the positive expression.

Statistical analysis

SPSS 21.0 statistics software (IBM Corporation, Armonk, NY, USA) was used for data analysis. Measurement data are presented by mean±standard deviation. Multiple groups were compared by analysis of variance (ANOVA). p<0.05.

Results

E-cadherin increased and vimentin decreased after LOC101060264 silencing in LoVo cells

The levels of E-cadherin and vimentin in transfected LoVo cells were detected by immunofluorescence assay. As shown in Figure 1, E-cadherin was mainly expressed in the nucleus and cytoplasm, and vimentin was mainly expressed in the cytoplasm and a small amount in the nucleus in the control group. After transfection, the expressions of E-cadherin and vimentin were affected to varying degrees. After transfection of the shRNA-NC sequence, there was no obvious change in the expressions of E-cadherin and vimentin. After transfection of shRNA1 and shRNA2 sequences, the expression of E-cadherin increased and that of vimentin decreased. The changes in expression of E-cadherin and vimentin in LoVo cells transfected with shRNA2 sequence were more obvious. Immunohistochemical results (Figure 2) showed that LOC101060264 silencing upregulated E-cadherin and downregulated vimentin in LoVo cells, further confirming the immunofluorescence results.

Figure 1

Immunofluorescence assay determines positive protein expression and shows elevated E-cadherin and reduced vimentin expression after LOC101060264 silencing. (A) Expression of E-cadherin in nucleus and cytoplasm; (B) Expression of vimentin in nucleus and cytoplasm; (C) Positive rate of immunofluorescence of E-cadherin and vimentin in different groups. * p<0.05, compared with the control group and the shRNA-NC group; # p<0.05, compared with the shRNA2 group.

Figure 2

Immunohistochemical assay determines positive protein expression and shows elevated E-cadherin and reduced vimentin expression after LOC101060264 silencing. * p<0.05, compared with the control group and the shRNA-NC group; # p<0.05, compared with the shRNA2 group.

Suppression of LOC101060264 inhibits LoVo cell proliferation and arrests the cell cycle progression of LoVo cells

Cell viability was observed by MTT assay at 48 h, 72 h, and 96 h. Compared with the control and shRNA-NC groups, the viability of LoVo cells was notably decreased at 48 h, 72 h, and 96 h in the shRNA1 and shRNA2 groups (p<0.05). Compared with the shRNA1 group, the viability of LoVo cells was significantly decreased at 48 h, 72 h, and 96 h in the shRNA2 group (p<0.05), while cell growth exhibited no marked difference between the control group and the shRNA-NC group (p>0.05) (Figure 3A). These findings signify that silencing LOC101060264 expression by shRNA interference could inhibit the proliferation of human colon cancer LoVo cells.

Figure 3

MMT assay for the viability of LoVo cells and shows silencing LOC101060264 inhibits cell viability (A). The results of flow cytometry for LoVo cell cycle show that suppression of LOC101060264 arrest cell cycle progression (B, C). * p<0.05 compared with the control group and the shRNA-NC group; # p<0.05 compared with the shRNA2 group.

To analyze the role of LOC101060264 in cell cycle progression in the LoVo cell line, flow cytometry was performed. As shown in Figure 3B, 3C, the percentage of LoVo cells in G0/G1 phase in the shRNA1 and shRNA2 groups was notably higher than that of the control and shRNA-NC groups (p<0.05), while the percentage of cells in S and G2/M phases were significantly lower than those of the control and shRNA-NC groups (p<0.05). In contrast to the shRNA1 group, the percentage of LoVo cells in G0/G1 phase was significantly higher in the shRNA2 group (p<0.05), and the percentage of cells in S and G2/M phases was markedly reduced (p<0.05). Cell cycle in the control group was similar to that in the shRNA-NC group (p>0.05). The results show that inhibition of LOC101060264 can arrest LoVo cells in the G0/G1 phase.

Suppression of LOC101060264 inhibits LoVo cell migration and invasion

Cell invasion ability and migration ability were evaluated by scratch test and Transwell assay, respectively. Cell invasion was measured by the numbers of cells penetrating Matrigel (Figure 4A) and cell migration was measured by cell migration distance (Figure 4B). In the control, shRNA-NC, shRNA1, and shRNA2 groups, the number of LoVo cells that permeated the Matrigel to the back of the Transwell membrane was 82.11±9.52, 86.52±8.11, 64.54±6.98, and 47.13±5.28, respectively. Invasion of cells in the shRNA1 and shRNA2 groups was significantly greater than that in the control and shRNA-NC groups (p<0.05). The number of invading cells was decreased evidently by shRNA2 in comparison to shRNA1 (p<0.05), but no significant difference was observed between cells without transfection and cells transfected with shRNA-NC (p>0.05). At 24 h after scratching, the healing rate of the scratch area in the shRNA1 and shRNA2 groups was notably slower than that in the control and shRNA-NC groups (p<0.05). In contrast to the shRNA1 group, the healing rate of scratch area in the shRNA2 group was significantly reduced (p<0.05). These findings indicate that silencing LOC101060264 inhibits the invasion and migration of human colon cancer cell line LoVo.

Figure 4

Transwell assay and scratch test showed that LOC101060264 inhibits LoVo cell invasion and migration. (A) Transwell assay for cell invasion showed LoVo cell invasion was inhibited in the shRNA1 and shRNA2 groups; (B) Scratch test shows that LOC101060264 inhibits LoVo cell migration. * p<0.05, compared with the control group and the shRNA-NC group; # p<0.05, compared with the shRNA1 group.

Downregulation of LOC101060264 inhibits tumor growth in nude mice

Tumor volumes and growth curves of transplanted tumors were observed after inoculation in nude mice (Figure 5A, 5B). After 28 days, all the nude mice were killed and tumors were weighed. The weights of transplanted tumors in each group gradually increased in a time-dependent manner. The nude mice in the shRNA1 group and the shRNA2 group had smaller volume than in the control group and the shRNA-NC group at each time point (p<0.05). Compared with the shRNA1 group, the tumor volume of nude mice in the shRNA2 group decreased significantly at all time points (p<0.05), but there was no evident difference in the control group and the shRNA-NC group (p>0.05). At the end of the experiment on day 28, the weights of subcutaneous transplanted tumors in the nude mice without treatment and injected with shRNA-NC were (2.16±0.36) g and (2.12±0.23) g, respectively, which were evidently greater than that of shRNA1(0.75±0.08 g) and shRNA2 (0.47±0.05 g) (p<0.05). Compared with the shRNA1 group, the weight of subcutaneous transplanted tumors in the shRNA2 group was evidently lower (p<0.05). These findings reveal that downregulation of LOC101060264 suppresses tumor growth in nude mice.

Figure 5

The tumor volume is decreased in nude mice when LOC101060264 is downregulated. (A) Transplanted tumors in nude mice were smaller in the shRNA1 group and the shRNA2 group; (B) Tumor volume was decreased in the shRNA1 group and the shRNA2 group; * p<0.05, compared with the control group and the shRNA-NC group; # p<0.05, compared with the shRNA1 group.

E-cadherin increased and vimentin, PCNA, and MMP-9 decreased after LOC101060264 silencing in tumor tissues

The expressions of E-cadherin, PCNA, vimentin, and MMP-9 in transfected LoVo cells were detected by immunohistochemical assay (Figure 6). After different transfections, the expressions of above proteins were affected to varying degrees. After transfection of shRNA-NC sequence, there was no obvious change in the expressions of these proteins. After transfection of shRNA1 and shRNA2 sequences, the expression of E-cadherin increased, and that of vimentin, PCNA, and MMP-9 decreased (p<0.05). The changes in expression of these proteins in LoVo cells transfected with shRNA2 sequence were more obvious (p<0.05).

Figure 6

Immunohistochemical assay determines positive protein expression and shows elevated E-cadherin and reduced vimentin, PCNA, and MMP-9 expression after LOC101060264 silencing. (A) Positive expression of immunohistochemistry shows that the expression of E-cadherin was increased and vimentin, PCNA, and MMP-9 were decreased most in cells transfected with shRNA sequence. (B) Number of positive expressions of immunohistochemistry. * p<0.05, compared with the control group and the shRNA-NC group; # p<0.05, compared with the shRNA1 group.

Discussion

Colon cancer is one of the commonest cancers in China, with higher incidence rates in men and in developed regions [18]. Of crucial importance, dysregulated lncRNAs are reported to be closely associated with proliferation, metastasis, invasion, and apoptosis in colon cancer, with clinical significance [19]. We focused our attention on the role of LOC101060264 in cellular behaviors of colon cancer LoVo cells. Our results demonstrate that silencing of LOC101060264 inhibits colon cancer cell proliferation, invasion, and migration in vitro, as well as tumor growth in vivo.

Our results showed that E-cadherin expression in the colon cancer cell line LoVo is increased and vimentin is decreased after shRNA-mediated LOC101060264 silencing, supporting that LOC101060264 silencing can inhibit the EMT process. In vivo experimentation further demonstrated that downregulated LOC101060264 inhibited tumor growth in nude mice with a decrease in EMT. Partially in line with our study, a previous study noted that E-cadherin expression was enhanced but vimentin was reduced by the suppression of sphingosine kinase 1 (SphK1) in colon cancer cells, indicative of an inhibitory role of SphK1 on EMT [20]. E-cadherin and vimentin are of pivotal importance as markers of the EMT process, and the process strongly affects the development of invasion and metastasis in colon cancer [21]. Loss of E-cadherin has been associated with the acquisition of invasive ability, which is related to high tumor grade and poor prognosis [22]. EMT is associated with the metastasis of colon cancer and increases tumor cell invasiveness, and is of great importance in tumor progression [23,24]. Thus, our findings show that LOC101060264 silencing alleviated colon cancer progression and is a promising colon cancer treatment through inhibiting EMT.

Through MTT assay, scratch test, and Transwell assay, we verified that downregulated LOC101060264 inhibited the proliferation, invasion, and migration of the LoVo human colon cancer cell line. This finding was similar to a previous study which showed the downregulation of lncRNA taurine-upregulated gene 1 (TUG1) inhibited cell proliferation and invasion in clear cell renal cell carcinoma and bladder cancer [25,26]. Furthermore, Liu et al. found that lncRNA loc285194 suppressed the growth of tumor cells in osteosarcoma [27]. In addition, dysregulated lncRNAs were documented to act as tumor promoters or suppressors in colon cancer. Li et al. found that downregulated lncRNA XLOC-010588 inhibits colon cancer cell invasion and migration [28]. Consistent with our study, a previous study has indicated that shRNA against CD44 functions as an inhibitor in the colon cancer cell proliferation, invasion, and migration, and is promotes cell apoptosis [29]. Epigenetic gene silencing of tumor suppressors plays a critical effect on the tumorigenesis of colon cancer.

Conclusions

In conclusion, our data demonstrate that silencing LOC101060264 inhibits proliferation, migration, and invasion of the human colon cancer cell line LoVo, and prevents tumor growth with a decrease of EMT in nude mice. These findings show that LOC101060264 is involved in the initiation and progression of colon cancer, and is an attractive therapeutic target for colon cancer. Further research is needed to elucidate the underlying mechanism of LOC101060264 in colon cancer, as well as the signaling pathways involved.