Silencing TCAB1 suppresses proliferation of hepatocellular carcinoma cells by inducing apoptosis

Objectives: Telomerase Cajal body protein 1 (TCAB1) is closely related to the occurrence, development and prognosis of tumors, and may a ﬀ ect the sensitivity of tumor radiotherapy. There are no reports about the e ﬀ ect of TCAB1 gene expression on proliferation and apoptosis of HEPG2 cells. We plan to investigate whether silencing TCAB1 using siRNA is helpful for the diagnosis and treat-ment of hepatocellular carcinoma. Methods: Three siRNA sequences (siTCAB1-1, siTCAB1-2, siTCAB1-3) targeting TCAB1 gene and one negative sequence (NC) were designed, synthesized and then transfected into HEPG2 cells, separately. The expressions of TCAB1 and telomerase reverse transcriptase (TERT) in mRNA and protein level were detected by RT-qPCR and Western blot assays. Moreover, proliferation and apoptosis of HEPG2 cells were measured by MTT assay, and ﬂ ow cytometry, respectively. Results: RT-qPCR and Western blot data both showed that TCAB1 expression in the siTCAB1 group was signi ﬁ cantly lower than that in the blank control and NC groups (p < 0.05). However, TERT expression was not signi ﬁ cantly di ﬀ erent among those groups (p > 0.05). MTT result showed that HEPG2 cells proliferation in the siTCAB1 group was lower than that in the blank control and NC groups (p < 0.05). The apoptotic rate in the siTCAB1 group was signi ﬁ cantly increased compared with the blank control and NC groups (p < 0.01). Conclusions: Silencing TCAB1 can inhibit proliferation and promote apoptosis of HEPG2 cells, providing a potential therapeutic method for diagnosis and treatment of hepato-cellular carcinoma.


Introduction
Hepatocellular carcinoma is a primary hepatic carcinoma which is known to be a highly fatal tumor, ranking the third of all cancer-induced deaths with respect to incidence rate.The annual number of newly diagnosed hepatocellular carcinomas in the world reaches as high as 0.7 million and the annual deaths are as high as 0.6 million [1,2].The high death rate of hepatocellular carcinoma is mainly caused by early diagnosis troubles and most patients are diagnosed in late stages.Existing operation and chemical treatments are not sufficient to increase the survival rate.Therefore, it is crucial to develop new therapeutic strategies for hepatocellular carcinoma [3,4].The occurrence of hepatocellular carcinoma involves a complicated dynamic process controlled by genetic and apparent epigenetic codes [5,6].Mastering the molecular genetic pathogenesis of hepatocellular carcinoma is extremely important for developing new therapies.Although quite a few reports on genetic mechanisms of the liver are available, the mechanisms related to hepatocellular carcinoma still remain unknown.Therefore, it is necessary to further explore the causes and pathogenesis of hepatocellular carcinoma.
Telomere is a section with a special linear structure at the end of heterochromatin, and telomerase is a kind of ribonucleoprotein enzyme with telomeric repeats [7,8].Telomerase is formed by TERT, telomerase RNA gene (TERC) and some proteins related to telomere enzyme activity and assembly [9,10].TCAB1 (also known as WRAP53 or WDR79) is a key core component of telomerase protein discovered in 2009, which mainly exists in Cajal body [11], can transport telomerase from the Cajal body to the position of telomere synthesis at the end of telomere, and thereby ensure the normal activation mechanism to maintain the telomere length [12].TCAB1 belongs to WD40 family homologue.WD40 family is closely related to cell apoptosis, cell cycle progression, protease degradation and RNA metabolism [13].TCAB1 may also participate in biological behaviors such as cell apoptosis and cell cycle regulation.TCAB1 gene is located on chromosome 17p13 and overlaps with p53 gene in a "head-to-head" manner.TCAB1 gene is the antisense transcription gene of p53, and TCAB1 mRNA can target the 5′ untranslated region of p53 mRNA.The formation of TCAB1/p53 RNA complex can stabilize p53 mRNA and promote p53 protein synthesis.Therefore, it is speculated that the induction of TCAB1 expression may increase the sensitivity of p53 dependent apoptotic cells [14].TCAB1 can also regulate telomerase signal transduction, and its expression is closely related to telomere formation [15].TCAB1 protein can promote proliferation of progenitor cells and tumor cells by increasing length of telomeres at the ends of chromosomes, thus promoting the survival of a variety of cancer cells [16].The latest research confirmed that TCAB1 also participated in DNA damage response [17], and has the function of promoting the repair of damaged DNA.Therefore, TCAB1 can not only regulate p53 and affect telomere synthesis, but also promote the damaged DNA repair, which plays an important role in maintaining gene stability and preventing diseases.
According to available studies, silencing TCAB1 can inhibit proliferation of lung cancer, head and neck cancer [18,19].Nevertheless, the regulation mechanism of the TCAB1 gene in hepatocellular carcinoma is unknown.In this study, TCAB1 in HEPG2 cells was silenced by using si-RNA mediation technology and its influences on hepatoma carcinoma cells were evaluated.

Cell culture and transfection
The frozen HEPG2 cells was dissolved in a 37 °C water bath and then transferred to a centrifuge tube to collect cells.Subsequently RPMI 1,640 medium with 10 % bovine albumin was added into the tube for cell resuspension.The cells were cultured in an incubator (SANYO, Shanghai, China) at 37 °C and 5 % CO 2 to the logarithmic phase.Medium in the incubator was replaced over time according to cell growth state [20].At 1 d before the transfection, HEPG2 cells were seeded in a 6-well plate at the rate of 5 × 10 5 cells per well and then cultured in an incubator.According to protocol of the Lipofectamine 2,000 transfection reagent, siTCAB1-1, siTCAB1-2, siTCAB1-3 and NC were transfected to HEPG2 cells at the concentration of 90 nmol/L when the adherent convergence of cells was about 70 %.Three repeats were set up and non-transfected conventionally cultured HEPG2 cells were named as the blank control group.All cells were incubated for 5 h at 37 °C and 5 % CO 2 and then cultured in RPMI 1640 medium with 10 % FBS for 48 h.All cultured cells were applied in the following experiments.

RT-qPCR assay for detecting expressions of TCAB1 and TERT mRNA
Total RNA from HEPG2 cells in different groups was extracted conventionally by using the Trizol reagent and was quantified.RNA transcription was then performed according to the instructions of the VAZYME transcription kit, using the following program: 15 min at 42 °C, 15 min at 70 °C, and stored at 4 °C.The cDNA obtained from the transcription was diluted 6 times with ddH 2 O and stored for later use.Primer sequences were designed by Primer Premier 5.0 according to TCAB1 and TERT sequences obtained from NCBI (Table 2).Amplification was performed according to the instructions of the SYBR Green kit using a CFX96 Touch Real-Time PCR Detection System (ABI, Shanghai, China).The reaction conditions comprised 50 °C 2 min, 95 °C 10 min; 95 °C 30 s and 60 °C 30 s, run for 40 cycles.The data were analyzed in 2 −△△Ct method.Each experimental setup gave three parallel datasets and each dataset was analyzed three times.
Table : The segment information of the interference sequences.

Primer sequences
Western blot assay for detecting expressions of TCAB1 and TERT The supernate in each well was removed following 48 h siRNA, and 120 μL pre-cooled RIPA cell lysis buffer (1 mLRIPA + 10 μL PMSF (10 mM) + 10 μL phosphatase inhibitors) was added into each well and incubated on ice for 30 min to extract the total proteins.Protein concentration in each group was tested and analyzed to assure equivalent proteins for SDS-PAGE electrophoresis, before sample loading.Total protein loading in each sample is 40 μg.After electrophoresis, proteins were transferred onto the PVDF membranes, and incubated with TCAB1 (1:1,000 diluted), TERT (1:1,000 diluted) and GAPDH (1:1,000 diluted) primary antibodies overnight at 4 °C.The blots were washed with PBS several times and then incubated with HRP-conjugated second antibody (1:50,000 diluted) for 1 h at room temperature.Next, the blots were washed with PBS, and then exposed to ECL luminescent solution.The images were recorded and the grey values were scanned to analyze expressions of target proteins.Experimental analyses were repeated three times.

MTT analysis of cell proliferation capacity
The cells in the blank control, NC, and siTCAB1 groups were separately transfected with plasmids as above-mentioned method, seeded in a 96-well plate (200 μL cell suspension/well) and cultured for 48 h.And then, 20 μL MTT reagent was added into each well and incubated at 37 °C for 4 h.The medium was removed and 150 μL DMSO was added and oscillated for 10 min.The absorbance at OD568 nm was measured with Microplate Reader (Thermo Fisher Scientific, Shanghai, China).

Cell apoptosis test based on flow cytometry
The cells in the blank control, NC, and siTCAB1 groups were separately transfected with plasmids as above-mentioned method, and then collected following 48 h culture.The cells were centrifuged for 5 min at 1,200 rpm and the supernates were removed.The cells were washed with PBS twice and then centrifuged for 5 min at 1,200 rpm.The following experimental procedures were as the instructions of the Apoptosis kit.Flow cytometry (Beckman Coulter, Shanghai, China) was used for detection.

Statistical analysis
All experiments were repeated at least three times.The data are given as mean ± SD.The comparisons were analyzed by Student's t-test for pairwise comparisons.p value less than 0.05 was considered statistically significant.All statistical analyses were done using SPSS version 19.0.

Transfection efficiency of HEPG2 cells
The levels of TCAB1 mRNA in different groups of cells at 48 h after transfection were tested by RT-qPCR.The quantitative results showed that the expression levels of TCAB1 in all three siTCAB1-1, siTCAB1-2 and siTCAB1-3 groups were lower than that in the blank control and NC groups (p<0.05).Notably, TCAB1 expression in the siTCAB1-3 group was extremely low (p<0.01)(Figure 1).Therefore, siTCAB1-3 was chosen as the experimental group (siTCAB1 group) to interfere target in the following experiments.

The influence of silencing TCAB1 on TCAB1 and TERT by RT-qPCR in HEPG2 cells
The mRNA expression levels of TCAB1 and TERT in the blank control, NC and siTCAB1 groups were tested by RT-qPCR at 48 h after transfection (Figure 2).Compared with the blank control, mRNA level of TCAB1 in the NC group changed minimally (p>0.05), while it was strongly downregulated in the siTCAB1 transfected cells (p<0.05).However, TERT mRNA expression remained unchanged in all groups (p>0.05).Therefore, we can speculate that silencing TCAB1 may not influence TERT gene expression.

Effects of silencing TCAB1 on expressions of TCAB1 and TERT proteins in HEPG2 cells
The protein expression levels of TCAB1 and TERT in the blank control, NC and siTCAB1 groups were analyzed by Western blot at 48 h after transfection (Figure 3).Compared with the blank control, expression of TCAB1 in the NC group changed minimally (p>0.05).However, its expression in the siTCAB1 group declined significantly (p<0.05).TERT expression remained basically constant in all groups (p>0.05).It can be speculated that silencing TCAB1 in HEPG2 cells will not influence TERT protein expression.

Effects of silencing TCAB1 on proliferation of HEPG2 cells
The proliferation of HEPG2 cells at 48 h after silencing TCAB1 was tested by MTT assay.According to data analysis (Figure 4), the cell proliferation rate was 76.73 % in siTCAB1 group and 99.44 % in NC group.The cell proliferation rate in siTCAB1 group was lower than that of the blank control and NC groups (p<0.05).There was no significant difference between the NC and the blank control groups (p>0.05).These data indicate that silencing TCAB1 may weaken proliferation of HEPG2 cells.

Effects of silencing TCAB1 on apoptosis of HEPG2 cells
The analysis of apoptosis of HEPG2 cells in the blank control, NC and siTCAB1 groups at 48 h after transfection were performed by flow cytometry.Apoptosis rate of HEPG2 cells in NC group was similar with that in the blank control group (p>0.05),Compared with NC group, the early apoptosis rate of HEPG2 cells in siTCAB1 group increased by 9.02 % ± 0.85 % and the late apoptosis rate increased by 1.41 % ± 0.42 % (Figure 5).The apoptosis rate of HEPG2 cells in siTCAB1 group increased significantly than that of the blank control and NC groups (p<0.01),but was fluctuated more significantly in   early stages of apoptosis.The data analysis showed that down-regulation of TCAB1 expression could promote the apoptosis of HEPG2 cells.

Discussion
Abnormal expression level of TCAB1 was found in multiple types of cancer cells [21,22].The expression of WRAP53 is increased in esophageal squamous cell carcinoma (ESCC), and WRAP53 overexpression is correlated with tumor progression.WRAP53 could be a useful biomarker for ESCC [23].The expression of TCAB1 is up-regulated in the development of nasopharyngeal carcinoma induced by Epstein-Barr virus (EBV), and TCAB1 is involved in stimulating telomerase activity and regulating the DNA damage response within the context of EBV infection [24].WDR79 is overexpressed in non-small cell lung cancer (NSCLC) tissues and cells and it might, therefore play an important role in the tumorigenesis of NSCLC [25].A notable overexpression of TCAB1 was observed in head and neck carcinoma clinical specimens as well as in carcinoma cell lines.TCAB1 might facilitate the occurrence and development of head and neck carcinomas [19].It suggests that TCAB1 may be involved in the occurrence and development of various tumors.TCAB1 can be used as a marker for the early diagnosis of tumors.According to RT-qPCR and Western blot data, silencing TCAB1 in HEPG2 cells down-regulates TCAB1 gene expression without affecting TERT expression.Our results are consistent with the reports of telomerase activity observed after TCAB1 mutations [26].In the induced pluripotent stem cells (iPSCs) from a form of dyskeratosis congenita caused by TCAB1 mutations, telomerase catalytic activity is unperturbed, but the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs [26].Silencing TCAB1 by siRNA targeting technology may not stop telomerase activity in tumor cells, but it may shorten telomeres by interrupting telomerase and telomere in some way [9,11,27].The results showed that silencing TCAB1 will not affect the activity of TERT, and the possible mechanism is that telomerase is not localized from Cajal body to the position of telomere synthesis at the end of telomere in HEPG2 cells.
Cell proliferation is closely linked to the occurrence of tumors.According to MTT analysis on proliferation of HEPG2 cells after silencing TCAB1, inhibiting expression of TCAB1 can affect proliferation and splitting speeds of cells.Rao and coauthors [23] found that siRNA targeting WRAP53 sequence was synthesized and transfected into ESCC cell line EC109.The down-regulation of WRAP53 expression inhibited the proliferation of ESCC cell line EC109.To investigate the impact of TCAB1 depletion in lung adenocarcinoma cells, A549 cells were transfected with TCAB1 siRNA and images were captured using an inverted microscope.The results exhibited an evident weakness in the reproductive capacity of TCAB1-depleted cells.MTT assay revealed decreased activity of cellular enzymes in TCAB1-depleted cells.These results indicated that TCAB1 siRNA effectively inhibits the proliferation of A549 cells [28].Wrap53 expression was upregulated in colorectal cancer tissue specimens and cell lines.Knockdown of Wrap53 expression significantly increased the percentage of cells in the G1 phase but reduced the percentage of cells in the S phase (p<0.05), and suppressed tumor cell proliferation and invasion capacity in vitro [29].Our results are consistent with the reports of the observed inhibition of cell proliferation caused by interference with TCAB1 expression.These findings indicate that TCAB1 plays a key role in cell proliferation and may be a potential target for treatment of hepatocellular carcinoma.
Cell apoptosis is a natural law of life and plays an important supporting role in controlling the dynamic equilibrium of cell quantity in the body.Cancer cells can proliferate infinitely, because their apoptosis mechanism is inhibited.If the maturity and differentiation of tumor cells can be inhibited, tumor cells may lose immortality and induce cell apoptosis, thus realizing the goal of curing cancer [30].In this study, apoptosis rate of HEPG2 cells in siTCAB1 group at 48 h after transfection was significantly greater than those in the blank control and NC groups (p<0.05).This strongly suggests that silencing TCAB1 can activate apoptotic signals to induce and accelerate cell apoptosis.Rao and coworkers [23] found that siRNA targeting WRAP53 sequence was transfected into ESCC cell line EC109.The downregulation of WRAP53 expression increased the expression of Bax protein and the apoptosis rate of EC109.SiRNA lentivirus targeting WDR79 gene was transfected into A549 cells and H1299 cells.The results showed that the early apoptosis rate of H1299 cells increased by 10 % and the late apoptosis rate increased by 4 %.The apoptosis rate of A549 cells also increased by 15 %.It indicated that WDR79 interference caused NSCLC cell apoptosis [25].In the study of clinical tissues of nasopharyngeal carcinoma, and it was found that TCAB1 was significantly overexpressed in EBV positive specimens.After TCAB1 knockdown, depletion of TCAB1 leaded to both cell cycle arrest and apoptosis, and inhibited the activation of ataxia telangiectasia and Rad3 related protein induced by EBV, resulting in accumulation of DNA damage [24].WRAP53 knockdown induced colorectal cancer cell line apoptosis in vitro [29].Our results were consistent with the previous studies, implied that silencing TCAB1 suppresses proliferation of hepatocellular carcinoma cells by inducing apoptosis.
To sum up, silencing TCAB1 will inhibit proliferation and accelerate early apoptosis of HEPG2 cells, thus providing a way for us to realize the goal of treating hepatocellular carcinoma.In future, TCAB1 might be useful as a prognostic biomarker or a potential target for the diagnosis and therapy of hepatocellular carcinoma.However, the mechanism that TCAB1 uses to promote the occurrence of hepatocellular carcinoma still remains unknown and needs to be explored further.
Research funding: This study was supported by the Major science and technology innovation projects in Xinxiang City (ZG1403).Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.Competing interests: Authors state no competing of interest.Informed consent: Not applicable.Ethical approval: Not applicable.

Figure 1 :
Figure 1: The expression level of TCAB1 in HEPG2 cells transfected with siTCAB1-1, siTCAB1-2, siTCAB1-3 and NC sequences by RT-qPCR.After transfection, HEPG2 cells were continuously cultured for 48 h.RT-qPCR was used to detect the mRNA expression level of TCAB1 in HEPG2 cells of each group.GAPDH was taken as the internal reference.Data were presented as the mean ± SD (n=3).Student's t-test.*p<0.05,** p<0.01 vs. the blank control and NC groups.

Figure 2 :
Figure 2: The expression level of TERT and TCAB1 in HEPG2 cells transfected with siTCAB1 by RT-qPCR.HEPG2 cells were transfected with siTCAB1 and then cultured for 48 h.RT-qPCR was used to detect the mRNA expression levels of TERT and TCAB1 in HEPG2 cells of each group.GAPDH was taken as the internal reference.Data were presented as the mean ± SD (n=3).Student's t-test.*p<0.05 vs. the blank control and NC groups.

Figure 3 :
Figure 3: The expression level of TERT and TCAB1 in HEPG2 cells transfected with siTCAB1 by Western blot.(A) HEPG2 cells were transfected with siTCAB1 and then cultured for 48 h.TCAB1 and TERT expression at protein levels were examined with Western blot in each group.GAPDH was taken as the internal reference.(B) The protein levels of TCAB1 and TERT were quantified by densitometry.The grey values were scanned to analyze the levels of TCAB1 and TERT proteins relative to GAPDH in each group.Statistical analysis was performed by Student's t-test.*p<0.05 vs. the blank control and NC groups.

Figure 4 :
Figure 4: The proliferation capacity of HEPG2 cells transfected with siTCAB1 by MTT analysis.HEPG2 cells were transfected with siTCAB1 and cultured for 48 h.And then, 20 μL MTT reagent was added into each well and incubated at 37 °C for 4 h.The absorbance at OD568 nm was measured with a Microplate Reader in each group.Statistical analysis was performed by Student's t-test.*p<0.05 vs. the blank control and NC groups.

Figure 5 :
Figure 5: The apoptosis of HEPG2 cells transfected with siTCAB1 was detected by flow cytometry.(A) Representative charts showing flow cytometry analysis of apoptosis.Flow cytometry was used to detect apoptosis of HEPG2 cells in each group.HEPG2 cells were transfected with siTCAB1 and cultured for 48 h, then stained with classical Annexin V/7-AAD.The percentage of apoptotic cells was analyzed via flow cytometry in each group.(B) Bar chart showing apoptosis rate of HEPG2 cells transfected with siTCAB1 in each group.The percentage of apoptotic cells were presented as the mean ± SD (n=3).Student's t-test.**p<0.01 vs. the blank control and NC groups.

Table  :
Primer sequences used for RT-qPCR.