Anti-tumor effects of LncRNA-CR594175 silenceing on hepatocellular carcinoma cell line HepG2 in vivo and in vitro

Background Hepatocellular carcinoma (HCC) is one of the cancers of highest incidence and mortality worldwide. Methods High-throughput screening was applied to find the most varied expressed LncRNA in primary hepatocellular carcinoma and explore the interaction network of it. Results We found that lncRNA-CR594175 was the lncRNA of most varied expression, which was lower in adjacent noncancerous tissues than in primary HCC, and was lower in primary HCC than in its metastases. Knockdown of CR594175 inhibited the proliferation and invasion of HCC cells HepG2. The mechanism study revealed that CR594175, as a RNA sponge, broke the negative regulation of CTNNB1 (Catenin, beta-1) by hsa-miR142-3p, and once CR594175 was silenced, the highly expressed hsa-miR142-3p regained its regulation on CTNNB1 and inhibited the proliferation and invasion through Wnt signaling pathway. Conclusions Our present study demonstrates for the first time that CR594175 silencing suppressed proliferation and invasion of HCC cells HepG2 in vivo and in vitro by restoring the negative regulation of CTNNB1 by hsa-miR142-3p, laying a solid theoretic base for using lncRNA-CR594175 as genetic therapy target for HCC and offering a reasonable explanation for inactivation of miRNA in difference tumors or tumor of different stages.

HCC cell lines HepG2, obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in RPMI-1640 (Invitrogen, CA, USA) supplemented with 10% FBS (Fetal bovine calf serum, Invitrogen). 293TN cells, purchased from ATCC (MD, USA), were maintained in DMEM (Dulbecco minimum essential medium, Invitrogen) supplemented with 10% FBS. All these adherent cells were passaged by 0.25% trypsin digestion (Invitrogen) and incubated in an atmosphere of 5% CO 2 at 37 °C.
Assessment of CR594175, hsa-miR142-3p, CTNNB1 protein and Wnt pathway related proteins in HCC tumors and its metastatic tissues HCC, the metastatic tissues and para-carcinoma tissues from 24 patients (diagnosed in the First Affiliated Hospital of Zhengzhou University and detailed patient information was shown in Tab.1) were collected, followed by total RNA extraction and real-time PCR for measurement of CR594175 and hsa-miR142-3p and total protein extraction and western blotting for CTNNB1 and Wnt pathway related proteins (E-cadherin, C-myc, CyclinD1 and MMP-9).

Lentivirus packaging
A siRNA sequence complementarily binding to CR594175 was chosen. The target sequences of siRNA (5'-GAATCCTCGGAGACAGCAG-3') are homologous to CR594175, respectively. The oligonucleotide templates of these shRNAs were chemically synthesized and cloned into the linear pSIH1-H1-copGFP shRNA Vector (System Biosciences, CA, USA) which was obtained through digestion by BamH I and EcoR I(Takara, Dalian, China) and purification by agarose gel electrophoresis. An invalid siRNA sequence (5'-AATCGTCGAGGGCCAGACA-3') was used as a NC (negative control). Sequencing was used to confirm the vectors constructed (pSIH1-shRNA-CR594175 and pSIH1-NC). The CDS sequence of human CTNNB1 (NM_001904.3) was amplified by using the primers 5'-GGAATTCGCCACCATGGCTACTCAAGCTGATTTG-3' and 5'-CGGGATCC TTACAGGTCAGTATCAAACC-3', which contain an EcoRI cutting site and Kozak sequence and a BamhI cutting site, respectively, with the cDNA prepared by reverse transcription of RNA isolated from 293TN cells. The PCR product was digested and cloned into pcDH1-CMV lentiviral expressing vector; the recombinant vector was named pcDH1-CTNNB1. The products of the vectors were confirmed by DNA sequencing. Endotoxin free DNA was prepared in all cases.
2 μg of each pSIH1-shRNA-CR594175 vector or pSIH1-NC and 10 μg pPACK Packaging Plasmid Mix (System Biosciences) were co-transfected using Lipofectamine 2000(Invitrogen) in accordance with the manufacturer's protocol. The medium was replaced with DMEM plus 1% FBS. Forty eight hours later, the supernatant was harvested and then cleared by centrifugation at 5000×g at 4 °C for 5 minutes, and passed through a 0.45 µm PVDF membrane (Millipore, MI, USA). The titer of virus was determined by gradient dilution. The packaged lentiviruses were named as Lv-shRNA-CR594175 and Lv-NC. Recombinant lentivirus Lv-CTNNB1 and Lv-miR142-3p was packaged by the same way.

Genetic intervention through a lentiviral approach
Cells were divided into four groups: a control group, Lv-NC group (infected with Lv-NC), Lv-shRNA-CR594175 group (infected with Lv-shRNA-CR594175) and Lv-CTNNB1 group (infected with Lv-CTNNB1). HepG2 in logarithmic phase were seeded to 6-well plates at 5 × 105 cells/well. One day later, viral solution was added at an MOI of 10. The infection efficiency was evaluated by observing and analyzing the fluorescent mark 72 hours after infection. And total RNA and protein were isolated from the cells and subjected to realtime PCR and western blotting for CR594175 and CTNNB1 protein, respectively.
We used Targetscan (http://www.targetscan.org/) to predict whether a hsa-miR142-3p binding site exists within the 3′-UTR of human CTNNB1 mRNA. The results showed that a seven-base hsa-miR142-3p seed sequence is present in the 3′-UTR of CTNNB1 mRNA. The same tool was used to predict the binding sites of hsa-miR142-3p on CR594175.A suspension of 293TN cells in logarithmic phase growth was prepared and the number of viable cells counted using a hemocytometer in conjunction with trypan blue staining. The cells were seeded into six-well plates at a concentration of 2×10 5 cells per well and maintained in Dulbecco's Modified Eagle's medium supplemented with 10 % fetal calf serum at 37℃ for 24 hours in a 5 % CO2 atmosphere. The transfection of plasmid DNA and RNA was performed using Lipofectamine 2000 (Invitrogen, USA). Transfection of cells with pRL-TK(100 ng) served as a reference for luciferase detection. Luciferase activity was measured using the dual luciferase reporter assay system (Promega, USA) 48 hours after transfection. The experiment to observe the effect of CR594175 depletion on the inhibition of luciferase by hsa-miR142-3p mimics was carried out in HepG2 cells, the plasmid transfection and luciferase activity assay the same as used in validation of target site.

Cellular proliferation assay
HepG2 cells were divided into seven groups: a control group, Lv-NC group, Lv-shRNA-CR594175 group, Lv-miR142-3p group, Lv-shRNA-CR594175 and Lv-miR142-3p group, Lv- supplemented with 10% FBS was used as chemoattractant. Cells that migrated and invaded the underside of the membrane were fixed in 4% paraformaldehyde. The invading cells were stained by Calcein-AM, and the number was then determined by fluorescence and reported as relative fluorescence units (RFUs). The grouping and cell treatment were the same as those for cell viability assay. Seventy-two hours after lentiviral infection, cells were trypsinized and the viable cells were counted by using trypan blue staining, and seeded into the upper chamber of transwell at a density of 5 × 10 5 cells/well, and incubated under normal conditions for 48 hours, and then stained and counted. The grouping was the same as in the proliferation assay.

Effect of CR594175 silencing on expression of β-catenin and downstream functional proteins of Wnt pathway
HepG2 cells were divided into three groups: a control group, Lv-NC group and Lv-shRNA-CR594175. Cells in logarithmic phase were seeded to 6-well plates at 5×105 cells/well.
One day later, viral solution was added and the infection efficiency was evaluated by observing and analyzing the fluorescent mark 72 hours after infection. Protein were isolated and subjected to western blotting for CTNNB1, E-cadherin, C-myc, CyclinD1 and MMP-9 protein, respectively.

Detection of protein contents in cell or tissues
The total protein was extracted from the cells using M-PER mammalian protein extraction reagent (Pierce, IL, USA) or from tissues using T-PER tissue protein extraction reagent (1:250) and β-actin (1:1000) (Santa Cruz, USA), followed the secondary HRP-conjugated anti-mouse/rabbit antibody (Abcam, CA, USA). After washing, the bands were detected by chemiluminescence and imaged with X-ray films. β-actin was used as an endogenous reference for normalization.

Animal xenografts
Nude mice were purchased from Shanghai SLAC Laboratory Animal Co.,Ltd (Shanghai, China) and housed at the animal experiment center of Zhengzhou University, where the implantation experiment was performed. All the protocols were previously approved by the Zhengzhou University Animal Ethics Committee. HepG2 cells (1×10 6 ) were suspended in 200 μl medium, and injected subcutaneously into the flank regions of 48 female athymic nude mice. Two weeks after inoculation, visible subcutaneous tumors were detected, and the tumors were measured approximately 2.5 mm in diameter 3 weeks after inoculation.
All animals were randomly divided into 3 groups (8 mice per group): the Model group, the NC group, and the CR594175-silencing group. For the intervention groups, each animal received 30 μl recombinant lentivirus (1×10 8 IFU) twice a week since the second week for 4 weeks, while the model group received the same volume of saline instead. Tumor diameter was measured weekly since the second week, and the data was used to plot the tumor growth curves. The formula for calculating the tumor volume was: V=ab2/2, a and b are the long and short diameters of the tumor, respectively.

Statistical analysis
All data is expressed as mean ± SD, and analyzed by one way ANOVA. Least Significant Difference (LSD) was used for multiple comparisons between any two means. P-values < 0.05 were considered statistically significant. All statistical analysis was performed using SPSS 13.0 software.

Assessment of CR594175, hsa-miR142-3p and CTNNB1 protein in tissues from HCC and metastatic tissues
The data of CTNNB1 mRNA and protein levels demonstrates that in comparison with paracarcinoma tissues, CTNNB1 protein was increased in HCC and its metastasis (p <0.01), and more in HCC metastasis than in primary HCC (p<0.05); but there were no obvious differences between the mRNA levels in three groups of tissues (p >0.05). These results suggest that the high expression of CTNNB1 is due to inactivation of post-transcriptional regulation. The expression of CR594175 and hsa-miR142-3p in the adjacent tissues, HCC and their metastasis were positively correlated with snail protein levels and was higher in the HCC and their metastasis than that in the adjacent tissues (p <0.05) (Fig1-A). We also detected the downstream functional proteins of Wnt pathway E-cadherin, C-myc, CyclinD1 and MMP-9 levels in HCC and their metastasis as compared with the adjacent tissue (p<0.01), that being more significant in HCC metastasis than in primary HCC (p<0.05). A reverse trend was observed in E-cadherin to the three proteins mentioned above in these tissues (Fig1-B).

Effect of silencing CR594175 and expression CTNNB1 via lentiviral approach HCC cells
Recombinant lentiviruses, Lv-NC,Lv-shRNA-CR594175 and Lv-CTNNB1, were used to infect HepG2. GFP(Green fluorescent protein) was detected in most of the cells 72 hours after infection, and the proportion of GFP-expressing cells suggested that the gene delivery efficiency was higher than 95% in the HepG2 (Fig.2A). CR594175 was significantly decreased by Lv-shRNA-CR594175 (p<0.05), and no change in cells infected with Lv-CTNNB1(p 0.05); CTNNB1 protein level was significantly increased by Lv-CTNNB1 and decreased by Lv-shRNA-CR594175 (p<0.05) (Fig.2B). These findings suggest that CR594175 silencing down-regulated CTNNB1 expression in HepG2, overexpression of CTNNB1 had no obvious effect on CR594175, so there is an obvious upstream and downstream relation between CR594175, hsa-miR142-3p and CTNNB1.

Luciferase experiments
Our bioinformatics analysis identified an 8-base hsa-miR142-3p seed sequence in the 3′-UTR of CTNNB1 mRNA. We therefore constructed luciferase reporter vectors to verify whether this site represents a valid hsa-miR142-3p target. Reporter vectors were generated that contained the wild-type CTNNB1 3′-UTR or a variant in which the hsa-miR142-3p target site within the 3′-UTR had been mutated. Both reporter constructs expressed luciferase at a high level. However, the miR142-3p mimic significantly inhibited luciferase activity in cells transfected with the reporter vector encoding the wild type 3′-UTR (42.15±3.98 vs. 8.07±0.88; p < 0.01), while the miR142-3p inhibitor significantly increased luciferase activity in these cells (43.02±2.02 vs. 52.81±9.04; p < 0.05).
Conversely, in cells transfected with the reporter vector encoding the mutated hsa-miR142-3p target site, neither the miR142-3p mimic nor the miR142-3p inhibitor had any observable effect on luciferase activity (p >0.05). Co-transfection of miR142-3p-NC (nontargeting control) had no effect on the luciferase activity of either of the vectors (p > 0.05). These results verified the presence of a hsa-miR142-3p target site in the 3′-UTR of CTNNB1 mRNA and demonstrated that binding of hsa-miR142-3p to this target site downregulates CTNNB1 expression (Fig. 3A). Interestingly, miR142-3p mimics lost its inhibition on the activity of luciferase expressed by wild-type (wt) luciferase reporter vector in HepG2, and regained the inhibition after CR594175 silencing (Fig. 3B). Taken together, these data suggest that CR594175 overexpression impaired negative regulation of CTNNB1 by hsa-miR142-3p.

Effect of CR594175 knock down on proliferation and invasion of HepG2 cells
As suggested by cell proliferation assays, CR594175 silencing effectively inhibited the proliferation of HepG2 cells 72 hours after infection (p<0.01),while CTNNB1 overexpression could promote the proliferation of HepG2 cells (p < 0.05), and CTNNB1 overexpression could reverse the proliferation suppression caused by CR594175 silencing (p<0.05).Overexpression of miR142-3p had no obvious effect on the proliferation of HepG2, but it showed a strong inhibitory activity of proliferation in the case of CR594175 silenced (Fig. 4A). Therefore, it was suggested that CR594175 silence inhibited cell proliferation by suppressing the expression of CTNNB1, which was reversed by exogenous expression of CTNNB1.Furthermore, cell invasion assay results demonstrated that CR594175 silencing inhibited invasion in HepG2 cells (p < 0.01), and CTNNB1 overexpression could reverse the invasion inhibition caused by CR594175 silencing (P < 0.05). Overexpression of miR142-3p had no obvious effect on invasion of HepG2, but it showed a strong inhibitory activity of invasion in the case of CR594175 silenced (Fig. 4B).
Because exogenous CTNNB1 has no wild promoter, we deduced that some transcription factors may bind to CTNNB1 promoter and activate its transcription, which may mediate the regulation of CTNNB1 by CR594175. In vivo experiment showed that 4 consecutive weeks of CR594175 silencing significantly decreased tumor volume. After administration for five weeks, the tumor volume of the model group was 701.21±54.13 mm 3 , the NC control group was 672.34±49.06 mm 3 and the CR594175 silencing group was 212.31±57.71 mm 3 . The tumor inhibition rates in the NC group and CR594175 silencing group were 4.12% and 69.73%, respectively, with a statistically significant difference between the CR594175 silencing group and the other two groups (p <0.01) (Fig. 4C).

Wnt pathway
We assessed E-cadherin, C-myc, CyclinD1 and MMP-9 in the ASLNC02525-depleted HepG2 cells. The results showed that C-myc, CyclinD1 and MMP-9 were decreased and E-cadherin was increased by CR594175 silencing (p 0.01 vs Lv-shRNA-CR594175 group) but not the Lv-NC (p 0.05 vs Lv-NC group) (Fig. 5). That indicated CR594175 silencing could inhibited the classic Wnt pathway by increasing the expression of CTNNB1 protein (Fig. 5).

Discussion
The invasion and metastasis of cancer refers to cancer cells break away from the primary focus and transfer to discrete tissues and proliferate into cancer of the same nature, i.e. the diffusion and growth of cells from primary tumor to remote organs [13]. This process depends on the interaction between cancer cells and internal environment promoting their survival, growth, and angiogenesis, as well as invasion and metastasis [14]; therefore, inhibiting proliferation and invasion of tumor cell is the key factor to inhibit tumor metastasis.
LncRNAs, endogenous RNAs longer than 200 nucleotides discovered recently and attracting much interest, have been found to have functions associated with various biological processes via epigenetic modification. Increasingly major studies suggest tangible involvement of lncRNA in cancer. Analysis of chromatin reveals that there are over 1000 lncRNAs in mammals, which are less conservative than the coding RNAs. As an important type of regulators, lncRNAs exert their functions through a variety of ways.
Although they were firstly regarded as by-products by RNA polymerase II, or transcriptional noise, recent studies have shown that lncRNAs are associated with multiple biological processes such as chromosome silencing, chromatin modification and transcriptional regulation [15,16]. Abnormal lncRNA expression is found in diversified diseases, and lncRNA expression profiles in a variety of tumors are different form normal cells. The proportion of lncRNAs in the total transcripts of genome is far larger than that of encoding RNAs. And lncRNAs play crucial roles in the regulatory network though their interaction with DNA, RNA and proteins. In addition to gene expression regulation, lncRNAs are closely related to evolution of species, embryonic development, metabolism and tumorigenesis. The evidence on involvement of lncRNAs in diseases including cancers will provide basis and target for diagnosis and treatment of diseases. Sun Shu-han et al.
We screened for differential lncRNAs in several pairs of selected HCC and adjacent tissue by using lncRNA chips. The reason that CR594175 caught our attention was that its expression was not only increased in HCC than in para-carcinoma tissues, but also was increased more in HCC metastases than in primary HCC, indicating that CR594175 would be associated with the process of HCC. We knocked down CR594175 in HepG2 cells, and found that the proliferation and invasion were reduced, as well as downstream protein of Wnt pathway CMYC, CyclinD1 and MMP-9,while E-cadherin is the opposite,so we believe elevated CR594175 levels contribute to metastases of HCC through Wnt pathway. To figure out how CR594175 promotes Wnt pathway in HCC, we analyzed key proteins involved in invasion and migration in HCC cells with CR594175 silencing and control cells, and found that the expression of CTNNB1 was consistent with CR594175, which was confirmed in primary HCC, metastatic HCC and adjacent tissue. Interestingly, our RIP-Seq experiment showed that CR594175 did not bind to CTNNB1 directly (Data not shown). We next quantified the transcription and protein levels of CTNNB1 in HCC with CR594175 silencing and found that CTNNB1 expression was abnormal at the post-transcription level, suggesting an inactivation of post-regulation of CTNNB1 due to high expression of CR594175, which led to Overactivation of Wnt pathway in HCC. As a typical posttranscription regulating factor, miRNA naturally became our pointcut to investigate the relation between CR594175 and CTNNB1. Bioinformatics suggests that there is a 7-base seeding region of hsa-miR142-3p on CTNNB1's 3'UTR and 2 seeding regions on the 600base CR594175. As a result, we speculated that elevated CR594175 bound to hsa-miR142-3p as miRNA sponge and disabled the negative regulation of CTNNB1 by hsa-miR142-3p, so CTNNB1 expression was increased and resulted in proliferation and invasion of HCC cells.
The interaction between lncRNAs and miRNAs has an important influence on the onset and development of cancer [17,18]. MiRNAs are able to regulate lncRNAs in a targeted way: a study has shown that miR-21 targets lncRNA GAS5 in addition to protein coding genes [19]. LncRNAs can also affect the onset and development of cancer by regulating expression of miRNAs. According to existing studies, lncRNAs regulate miRNAs through three ways: (1) to competitively combine to 3'-UTR of mRNAs with miRNAs, so as to inhibit negative regulation by miRNAs-Pang et al. found that an anti-sense RNA complementary to BACE1 can complement to BACE1 mRNA, competitively inhibiting the negative regulation of BACE1 by miRNA [20]; (2) to regulate target genes by forming pre-miRNAs after RNA splicing and producing specific miRNAs; and (3) to act as endogenous miRNA sponge to suppress miRNA function, so as to affect malignant biological behavior of cancer cells [21].
The most important finding of this study is CR594175 silencing could restore the negative regulation of CTNNB1 by hsa-miR142-3p to inhibit cancer, directly based on following facts: (1) hsa-miR142-3p negatively regulated CTNNB1 by binding to its 3'UTR, which was found in HCC with CR594175 silencing but not with those with high CR594175 expression levels; (2)CR594175 silencing inhibited proliferation and invasion of HCC cells, which was reversed by overexpression of CTNNB1; (3) Overexpression of hsa-miR142-3p had no observable effect on proliferation and invasion of HCC cells, but inhibited that when CR594175 was depleted. Considering CTNNB1 overexpressed by the lentiviral system has no wild 3'UTR, it would not be affected by miRNA. So we think that there is a CR594175/hsa-miR142-3p/CTNNB1 pathway regulating metastasis of HCC.

Conclusion
The study demonstrates that CR594175 plays a key role in the process of HCC metastasis, and offers a possible explanation for that hsa-miR142-3p loses its basic function of resisting HCC tumors. In the long run, lncRNA will not only be a direct target for gene    Hsa-miR142-3p binds to CTNNB1 3'UTR, which is interfered by CR594175. A)293 cells were transfected with pGL3-wt-CTNNB1 or pGL3-mt-CTNNB1 in the presence or absence of miR142-3p-mimic or inhibitor and subjected to luciferase activity silencing group, data are expressed as the means ± SD.