Effects of RNA interference-mediated gene silencing of SIAH2 on human hepatocellular carcinoma cell line HepG2 invitro*

, -, -, , -

(1.Department of Gastroenterology, The First Affiliated Hospital of Gannan Medical University;2.Department of Medical Services, The First Affiliated Hospital of Gannan Medical University, Ganzhou Jiangxi 341000)

Effects of RNA interference-mediated gene silencing of SIAH2 on human hepatocellular carcinoma cell line HepG2invitro*

LIUYao1,HEXing-bo2,HUANGWen-feng1,ZHOUYun1,HUANGCai-bin1

(1.DepartmentofGastroenterology,TheFirstAffiliatedHospitalofGannanMedicalUniversity;2.DepartmentofMedicalServices,TheFirstAffiliatedHospitalofGannanMedicalUniversity,GanzhouJiangxi341000)

Objective:To investigate the relations between SIAH2 and biological processes of hepatocellular carcinoma.Methods:Expressions of SIAH2 were detected in hepatocellular carcinoma tissues and adjacent non-tumorous liver tissues by immunohistochemistry. SIAH2 specific shRNA expression plasmids were constructed and transfected into HepG2 cells. The expressions of SIAH2 were examined by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western blot. Cell proliferation was measured by MTS. Flow cytometry assay was used to test cell apoptosis and cell cycle. The ability of invasion was evaluated by transwell. Results:Overexpressions of SIAH2 were detected in hepatocellular carcinoma tissues. Expression of SIAH2 in HepG2 cells was significantly down-regulated by SIAH2 specific small hairpin RNA. The mean actual absorbance in HepG2-S3 group was shown by MTS assay to be significantly lower than that in HepG2-neo (P<0.05), HepG2-NC(P<0.05) and untransfected HepG2 cells(P<0.05). In addition,knock down of SIAH2 in HepG2 cells induced cell cycle arrest and apoptosis, and resulted in significant inhibition of cell invasion.Conclusion:SIAH2 plays oncogenic role in hepatocellular carcinoma by promoting cell proliferation and invasion, and can be exploited as a target for hepatocellular carcinoma treatment.

Hepatocellular carcinoma cell line HepG2;Seven in absentia homologs 2;Small hairpin RNA

0 Introduction

Hepatocellular carcinoma (HCC) ranks as the fifth and seventh most common cancer in men and women, respectively,and the third most common cause of cancer-related mortality worldwide[1].Despite advances in the detection and therapeutic strategies of HCC over the past few years,the mortality rate and overall prognosis has not been improved for cases with the presence of advanced or terminal stage disease at diagnosis[2].Thus the identification of new biological markers that have a sufficient sensitivity and specificity for the early diagnosis of HCC is necessary.

A RING finger E3 ubiquitin ligase, seven in absentia homolog (SIAH) mediates ubiquitination and degradation of substrates that are important in several biological processes[3].Structurally, the SIAH family presents a divergent N-terminal domain, a highly conserved catalytic RING domain, two zinc finger domains, and a substrate-binding domain[4].In humans, SIAH has two isoforms, SIAH1 and SIAH2, which can exert distinct functions in cellular processes, including cell cycle control, DNA damage response, tumorigenesis, and metastasis[5].Several studies have shown an oncogenic role for SIAH proteins, especially SIAH2, in breast[6],prostate cancer[7],and lung cancer[8].On the contrary, SIAH proteins, especially SIAH1, have been found to act as a tumor suppressor in gastric tumors[9].This could be explained as a consequence of the specificity of each subunit to degrade different substrates.

Mainly due to the oncogenic role of SIAH2 in some types of cancer, the aim of the study is to document SIAH2 expression pattern in human hepatocellular carcinoma samples compared to adjacent non-tumorous liver tissues from the same patients, and to illustrate the role and relevant molecular mechanism of SIAH2 in the development of HCC cellsinvitro.

1 Materials and Methods

1.1 Immunohistochemistry and evaluation Paraffin-embedded 50 specimens of hepatocellular carcinoma tissues and adjacent non-tumorous liver tissues from same patients were obtained from the First Affiliated Hospital of Gannan Medical University. Clinicopathological data including sex, age, tumor size, TNM stage, and the presence of cirrhosis were recorded from the institute database. Mouse-anti-human polyclonal SIAH2 antibody (Abcam, USA) was used for immunohistochemistry assay, which was performed following the protocol of Universal SP kit (Zhongshan Goldenbridge Biotechnology, Peking, China). All stained tissues were reviewed and scored by two pathological doctors independently and blindly. Positive staining of SIAH2 protein presents brown in cytoplasm, partly in cytomembrane. Semi-quantitative counting method was used to determine positive staining described as following: selected 10 visual fields under high power lens (×400) randomly, counted the numbers of positive cells in 100 cells per field, calculated the average positive rate. Intensity of staining was scored as 0 (Cells without brown staining), 1 (with mild brown staining), 2 (with moderate brown staining), and 3 (with intense brown staining). Positive cells percentage was scored as 0 (0%), 1 (1%～25%), 2 (26%～50%), 3 (51%～75%), and 4 (76%～100%). The final positive scores=positive score × staining intensity score. Final score of each case was the result of the positive score multiplied by the intensity score. Score more than 2 was considered as positive.

1.2 ShRNAs synthesis and plasmids construction Single shRNA strands were 5′-GATCC-N′19-TTCAAGAGA-N19-TTTTTTA-3′(sense) and 5′-AGCTTAAAAAA-N19-TCTCTTGAA-N′19-G-3′(antisense). N19 was the sense sequence of SIAH2 target oligonucleotides, N′19 was antisense sequence of SIAH2 target oligonucleotides. Three different template oligonucleotides targeting SIAH2 (GeneBank, NM_005067.5) were as follow: SIAH2-S1 sense:5′-ACGCCCACAAGAGCATTAC-3′;SIAH2-S2 sense:5′-TGTTCCCTGACCCTGCACC-3′;SIAH2-S3 sense:5′-AGATAATGGGAACCTTG GA-3′.As a negative control, one scrambled sequence 5′-GCAGATAGGTAGG CGTTAT-3′ was designed. These sequences were submitted to BLAST against human genome sequence to ensure that only SIAH2 gene was targeted. Enzymes including sites of BamHⅠand HindⅢ were constructed into extreme of oligonucleotides fragment. All single shRNA strands were cloned into pGenesil-1. The four shRNAs inserted vectors were named as pSIAH2-S1,pSIAH2-S2, pSIAH2-S3 and pSIAH2-NC respectively.

1.3 Cell lines and culture The human hepatocellular carcinoma cell line HepG2 (with high level of SIAH2 expression measured in our preliminary study) was purchased from American Type Culture Collection (ATCC). The cells were cultured in RPMI1640 medium (GIBCO,USA) supplemented with 10% fetal bovine serum (GIBCO,USA), 100 U·mL-1penicillin, and 100 g·mL-1streptomycin at 37 ℃ with 5% CO2. The cells in the logarithmic phase of growth were used in all experiments described below.

1.4 Cell transfection Cell transient transfection was performed following the protocol of Lipofectamine 2000 (Invitrogen,USA). The transfection efficiency was observed by fluorescent microscope.The untransfected cells,empty vector (pGenesil-1) transfected cells (HepG2-neo), and nonspecific shRNA (pSIAH2-NC) transfected cells (HepG2-NC) were used as controls. Cells were transient transfected with pSIAH2-S1, pSIAH2-S2 and pSIAH2-S3. Names of the transfected cells were HepG2-S1, HepG2-S2, HepG2-S3, respectively. Successful knockdown of SIAH2 was analyzed by Western blot analysis and PCR.

1.5 PCR Total RNA of all transfected cells were extracted with the Trizol at 48 h after transfection. First-strand cDNA was generated using 2 μg total RNA via MMLV-reverse transcriptase using random primers. A final reaction of 20 μL was used to determine the mRNA level by PCR. The specific primers were as follow: SIAH2 5’-GATCCACATGAACGCACG-3’(forward) and 5’-GAGGGAAGCCACGTGTAAAC-3’ (reverse); GAPDH 5’-AGAAGGCTGGGGCTCATTTG-3’(forward) and 5’-AGGGGCCATCCACAGTCTTC-3’(reverse). Thermal cycling was initiated with a denaturation step at 94 ℃ for 2 min followed by 35 cycles of denaturing at 94 ℃ for 30 s, annealing at 48 ℃ for 30 s and extension at 72 ℃ for 30 s, and followed by the final extension at 72 ℃ for 10 min.

1.6 Western blot At 72 h after transfection, cells in different treatment groups were collected and lysed, and the protein concentration was detected by BCA protein assay kit. Supernatants were loaded on a 12%SDS-PAGE gel, and they were then wet transferred onto PVDF membranes. The membranes were incubated with monoclonal rabbit anti-human SIAH2 antibody (SantaCruz Biotechnology Inc, USA 1∶1 000) in TBST and 5% nonfat dry milk overnight at 4 ℃. After the overnight incubation with the primary antibodies, membranes were washed and incubated with HRP-conjugated goat anti-rabbit secondary antibody (SantaCruz Biotechnology Inc, USA) in TBST for 2 h. The bands were visualized with ECL Plus and exposed to X-ray film.

1.7 Cell proliferation assay For the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega),HepG2-S3 cells were seeded into 96-well plates at a density of 1×104cells per well, and the untransfected cells, HepG2-neo and HepG2-NC were used as controls. Five duplicate wells were set up for each group. At 24, 48, and 72 h, 20 μL of MTS reagent was added to each well, incubated for 4 h at 37 ℃,and the results were analyzed by a plate reader at 490 nm. Each assay was performed in triplicate. The inhibitory rate of cell proliferation was calculated using the following formula: Inhibitory rate (%)= (1-A490of treated group)/(A490of control group) ×100%.

1.8 Flow cytometry assay Apoptosis analysis: At 48 h after cells were transfected in 6-well plates (1×105cells per well), cells in different treatment groups were gently trypsinized and washed with cold PBS twice. At least 20,000 cells were resuspended in 500 μL 1×binding buffer stained with 5 μL 7-AAD and 1 μL AnnexinV-PE. Then, rates of apoptosis were analyzed by flow cytometry.

Cell cycle analysis: At 48 h after transfection, cells indifferent treatment groups were digested by trypsin (0.25%) and fixed in 70% ethanol for 1 h at room temperature. After centrifugation, the cell pellet was resuspended in PBS (pH 7.4) containing 100 μL Rnase A(1 mg·mL-1), and 400 μL PI (50 μg·mL-1) at room temperature for 30 min. Then cell cycle was determined by flow cytometry using a FACS can flow cytometer at 488 nm.

1.9Invitrocell invasion assay Invasion assay was performed using Transwell chambers. Briefly, the 8-μm pore size filters were coated with 100 μL of 1 mg·mL-1Matrigel (BD Biosciences, Bedford, MA). 500 μL RPMI1640 medium containing 10% FBS was added to the lower chambers. After transfection for 48 h, cells were harvested and homogeneous single cell suspensions (2×105cells/well) were added to the upper chambers. The invasion lasted for 24 h at 37 ℃ in a CO2incubator. After that, noninvasive cells on the upper surface of the filters were carefully scraped off with a cotton swab, and cells migrated through the filters were fixed and stained with 0.1% crystal violet for 10 min at room temperature, and finally, examined and photographed by microscopy (×200). Quantification of migrated cells was performed.

1.10 Statistic analysis Statistical analyses were performed with SPSS 17.0 software package. Quantitative results were given as mean±SD. Difference between two experimental groups was evaluated by the students’ttest and differences among groups were analyzed using One-Way ANOVA and SNK-qtest.Pvalues less than 0.05 were considered as statistically significant.

2 Results

2.1 Overexpressions of SIAH2 in HCC tissues We examined SIAH2 expression in HCC tissue by Immunohistochemistry. As shown in Figure 1, SIAH2 staining was weak in the adjacent non-tumorous liver tissues but was obviously strong in HCC tissues. More importantly, we found that SIAH2 staining intensity was correlated with the clinicopathological characteristics of HCC (Table 1). SIAH2 expression was positively correlated with the tumor TNM stage.

HCC tissues showed higher density staining of SIAH2 (A: DAB staining, ×100; B: DAB staining, ×400), but adjacent non-tumorous liver tissues showed lower staining (C: DAB staining, ×100; D: DAB staining, ×400).

Figure 1 Immunohistochemistry analysis of SIAH2 expression in HCC tissues and adjacent non-tumorous liver tissues.

Table 1 Correlation of SIAH2 expression with clinical and pathological of hepatocellular carcinoma

CharacteristicsnSIAH2Positive(%) χ2 PValueGender0．421>0．05 Male4235(83．3) Female86(75．0)Diameter/cm7．950>0．05 >52116(76．2) ≤52923(79．3)AFP/ng·mL-11．823>0．05 >252822(78．6) ≤252217(77．3)HBsAg1．576>0．05 Negative2619(73．1) Positive2418(75．0)Cirrhosis2．035>0．05 No2618(69．2) Yes2413(54．2)TNMstage6．871<0．01 Ⅰ、168(50．0) Ⅲ、Ⅳ3431(91．2)

2.2 Down-regulation of SIAH2 expressions by shRNA RT-PCR and Western blot were performed to analyze the mRNA and protein expression levels of SIAH2 in HepG2 cells at 72 h after transfection. As shown in Figure 2, compared to untransfected HepG2 cells and HepG2-neo, HepG2-NC cells, the levels of SIAH2 mRNA and protein were significantly down-regulated in HepG2-S1, HepG2-S2, HepG2-S3 cells, especially in HepG2-S3 cells. According to these results, HepG2-S3 cells that showed the highest inhibitory rate of SIAH2 were used for further assay described below. No significant change in SIAH2 mRNA and protein expression was found among untransfected HepG2 cells and HepG2-neo, HepG2-NC cells.

2.3 Effects of SIAH2 silencing on cell proliferation According to Table 2, the proliferation of HepG2-S3 cells was obviously inhibited from the first day, when compared with control cells. The proliferation inhibition rate of HepG2-S3 cells were (36.3±2.32)%,(55.2±4.2)% and (51.1±1.24)% at 24, 48 and 72 h, respectively (P<0.05) compared to control group at each time point. There were no differences among untransfected HepG2 cells, HepG2-neo, and HepG2-NC cells. Thus, knockdown of SIAH2 by shRNA could inhibit the growth of HepG2 cells.

The best inhibitory effects of SIAH2 were identified in HepG2-S3 cells by RT-PCR (upper) and Western blot (lower), which were both performed for three times independently. Bar graphs show the relative expression levels of SIAH2 mRNA (upper) and protein (lower). *P<0.05 versus control groups.

Figure 2 Down-regulation of SIAH2 by siah2-shRNA in HepG2 cells

Table 2 Suppression of proliferation by SIAH2-shRNA in HepG2 cells measured by MTS assay.

*P<0.05 vs other groups.

2.4 Effects of SIAH2 silencing on cell cycle and apoptosis Cell cycle analysis by FCM revealed that SIAH2 shRNA could induce changes in cell cycle of HepG2 cells (Figure 3). There were no significant differences (P>0.05) in the percentages of cells at each phase among untransfected HepG2 cells, HepG2-neo and HepG2-NC cells. Compared to untransfected HepG2 (44.62±0.23)%, HepG2-neo (46.87±0.12)% and HepG2-NC cells (45.29±0.35)% respectively, there were significant differences (P<0.05) in the percentage of cells in G0/G1 phase in HepG2-S3 group (49.68±0.25)%. Similarly, there was a significant difference (P<0.05) in the percentage of cells in S phase in HepG2-S3 group (22.94±0.66)% versus untransfected HepG2 (25.66±0.16)%, HepG2-neo group (25.71±0.87)% and HepG2-NC group (25.92±0.37)%, respectively. The results indicated that silencing SIAH2 gene could increase the percentage of cells at G0/G1 phase and decrease the percentage of cells at S phase, showing that HepG2 cells were blocked in G0/G1 stage after inhibiting SIAH2 gene by pSIAH2-S3 shRNA.

As shown in Figure 4, cell apoptosis rate (LR) in HepG2-S3 cells was markedly increased to (39.58±1.08)%, higher than (0.75±2.31)% for HepG2,(0.84±1.95)% for HepG2-NC,and (0.90±1.86)% for HepG2-NC cells (P<0.05). The results of apoptosis assay indicated that the inhibitory effect of cell growth might be due to the enhancement of apoptosis by SIAH2 shRNA.

A: HepG2; B: HepG2-neo; C: HepG2-NC; D: HepG2-S3. The % cells within different cells stages were analyzed by flow cytometry. Data were representative of three experiments. Data were expressed as mean±S.D. *P<0.05 vs. other groups.

Figure 3 The cell cycle of HepG2 was measured by flow cytometry

A: HepG2; B: HepG2-neo; C: HepG2-NC; D: HepG2-S3. The % cells within normal and apoptosis were analyzed by flow cytometry. Data were representative of three experiments. Data were expressed as meanS.D. *,P<0.05 vs. other groups.

Figure 4 The apoptotic rate of HepG2 cells was detected by flow cytometry

2.5 Effects of SIAH2 silencing on invasion ability Theinvitrocell invasion assay was performed and the number of invading cells was counted. As displayed in Figure 5, HepG2-S3 group showed significantly decreased invasiveness, compared to untransfected HepG2 cells (P<0.05) and HepG2-neo(P<0.05), HepG2-NC cells(P<0.05), respectively. These results demonstrated that transfection with SIAH2 shRNA could reduce the invasion of HepG2 cells.

Cell invasive ability was assessed by Matrigel invasion assay.

A: HepG2; B: HepG2-neo; C: HepG2-NC;D:HepG2-S3 (Hematoxylin staining, ×400). Each bar represented the cell numbers adherent on lower membrane. *P<0.05 versus control groups.

Figure 5 Inhibition of invasion by SIAH2 shRNA in HepG2 cells

3 Discussion

HCC is a common and aggressive malignant tumor worldwide with dismal outcomes. Epidemiological studies indicate that risk factors for HCC include chronic viral hepatitis, cirrhosis, heavy alcoholism, non-alcoholic fatty liver disease, and certain inherited metabolic conditions, such as hemochromatosis and alpha-1-antitrypsin deficiency[10].This disease is characterized by highly recurrent rate after curative resection and resistance to chemotherapy[11].Therefore, how to effectively inhibit the proliferative and metastatic biological behavior of HCC is a key problem to improve the outcome.

Genomics and molecular biology studies indicated that the genetic abnormality of some genes, including potential oncogenes and tumor suppressor genes, could be involved in HCC development. The function of these genes mainly depends on the level of their proteins influenced by post-translational modifications such as ubiquitination, phosphorylation and acetylation[11]. However, the molecular mechanisms that contributes to post-translational regulation of these genes in HCC remains to be largely uncovered[12].

The ubiquitin-proteasome pathway (UPP) is a common cellular process for protein degradation in eukaryotes and involves in the regulation of cellular process including cell cycle, transcription, apoptosis, cell adhesion, angiogenesis, and tumor growth[13].This pathway consists of a series of enzymatic reactions involving an ubiquitin-activating protein (E1), an ubiquitin-conjugating protein (E2) and an ubiquitin ligase (E3)[14].E3 ligases play a central role in the recognition, and hence, targeting of substrates for ubiquitination. Accordingly,considerable efforts have been devoted to understand mechanisms underlying E3 function[15].

SIAH proteins are RING finger E3 ubiquitin ligases, which mediate proteasomal protein degradation by poly-ubiquitination. Two SIAH proteins have been identified in humans, SIAH1 and SIAH2, which can exert distinct functions in cellular processes including cell cycle control, DNA damage response, tumorigenesis and metastasis. Several SIAH substrates have been described to date, including the hypoxia-regulating family of prolyl hydroxylases (PHDs), PML, TRAF2, PPAR, AKAP121, HDAC3, DCC, HIPK2 and DYRK2[8].SIAH1 and SIAH2 have been implicated in interacting with and in modulating the stability of different substrates to play a different role in the tumorigenesis control[16-18].SIAH proteins (especially SIAH1) have been found to act as a tumor suppressor in breast cancer[19],gastric tumors[20].On the contrary, Several groups have shown an oncogenic role for SIAH proteins, especially SIAH2, in breast[6]and prostate[3，7]，although the molecular mechanisms have not yet been fully elucidated.

In this study, we found that SIAH2 expression was significantly higher in HCC tissues. These data suggest that SIAH2 overexpression is correlated with the progression of HCC. To elucidate the role of SIAH2 in the tumorigenesis of HCC, we designed and synthesized three specific shRNAs against SIAH2 gene to investigate the effects of SIAH2 inhibition on HepG2 cells. RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation and an excellent strategy for specific gene silencing. ShRNA is formed by hairpin structures and stretches of double-stranded RNA, which will be cleaved by the ribonuclease dicer to produce mature miRNA inside the targeted cells. Recently the vector-based approach of shRNA interference has been developed in order to achieve stable, long-term, and highly specific suppression of gene expression in mammalian cells. These shRNA expression vectors have many advantages: they can be stably introduced into cells and persistently effective, either as selectable plasmids or as retroviruses[21].

Results of RT-PCR and Western blot showed specific SIAH2-shRNAs could effectively knockdown endogenous expression of SIAH2 in HepG2 cells. As a consequence of SIAH2 gene knockdown, the proliferation and invasion of HepG2 cells were obviously inhibited, but the apoptosis rate was significantly increased. FCM results showed cell cycle changes. These results showed inhibition of SIAH2 could suppress the growth and metastatic potential of HepG2 cellsinvitro, which suggested that SIAH2 might contribute to the growth and metastasis of HCC. Next, we will investigate the effect of SIAH2-shRNA on the growth of HepG2 hepatocellular carcinoma xenografts in nude mice,invivo.

Many studies have focused on SIAH2 primary functions by targeting selected substrates, such as DYRK2, PHD and HIPK2, for proteasomal degradation by poly-ubiquitination to participate in the development of cancer. Our research found that SIAH2 might increase the stability of paternally expressed gene 10 (PEG10) by mono-ubiquitination. PEG10 is an imprinted gene with an active paternal allele but silent maternal allele. The PEG10 gene is activated in a great majority of hepatocellular carcinomas, although its expression was absent in normal liver cells. A functional role for PEG10 in the growth-promoting activities has also been demonstrated in HCC cells. CO-IP and confocal showed that cytosol SIAH2 co-localizes and binds to PEG10, inhibits the degradation of PEG10, to promote the tumor progression.

In summary, here we presentinvitroevidence that SIAH2 promotes HepG2 cells growth and invasion. SIAH2 plays an important role in the regulation of apoptosis and cell cycle. Based on these findings, intervention with SIAH2 expression may provide a therapeutic approach in HCC development and metastasis.

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體外研究RNA干擾介導(dǎo)的SIAH2基因沉默對(duì)人肝癌細(xì)胞HepG2的影響

劉 瑤1，賀興波2，黃文峰1，周 云1，黃才斌1

(贛南醫(yī)學(xué)院第一附屬醫(yī)院 1.消化內(nèi)科;2.醫(yī)務(wù)科，江西 贛州 341000)

目的：研究發(fā)現(xiàn)泛素連接酶SIAH2在多種腫瘤中高表達(dá)，為了研究SIAH2和肝癌發(fā)病之間的相關(guān)性，利用RNA干擾技術(shù)觀察泛素連接酶SIAH2基因表達(dá)抑制對(duì)人肝癌細(xì)胞株HepG2細(xì)胞周期和凋亡的影響。方法：采用免疫組化檢測(cè)50例HCC和相應(yīng)癌旁組織中SIAH2蛋白的表達(dá)。構(gòu)建特異性靶向SIAH2的重組干擾質(zhì)粒轉(zhuǎn)染人肝癌細(xì)胞HepG2，采用RT-PCR和Western blotting 技術(shù)檢測(cè)重組質(zhì)粒對(duì)SIAH2 mRNA和蛋白表達(dá)的影響，MTS比色法測(cè)定重組質(zhì)粒轉(zhuǎn)染對(duì)細(xì)胞體外增殖能力的影響，流式細(xì)胞術(shù)檢測(cè)重組質(zhì)粒轉(zhuǎn)染后細(xì)胞周期和凋亡的變化。Transwell 實(shí)驗(yàn)檢測(cè)細(xì)胞體外侵襲能力變化。結(jié)果：SIAH2在肝癌組織中表達(dá)明顯升高。靶向SIAH2的干擾RNA能顯著抑制SIAH2 mRNA和蛋白的表達(dá)水平；與HepG2-neo組、 HepG2-NC組和未轉(zhuǎn)染HepG2組相比，SIAH2干擾組(HepG2-S3)細(xì)胞增殖速度明顯減慢，細(xì)胞明顯阻滯于G1期，細(xì)胞凋亡率明顯升高，體外侵襲能力減弱。結(jié)論：SIAH2通過促進(jìn)肝癌細(xì)胞增殖和侵襲在肝癌發(fā)病中發(fā)揮癌基因作用，上述研究為以SIAH2為靶點(diǎn)的肝癌基因治療提供實(shí)驗(yàn)依據(jù)。

肝癌細(xì)胞HepG2;SIAH2;小干擾RNA

2016-07-18)(責(zé)任編輯：敖慧斌)

HUANG Cai-bin,male, M.D.,Professor,Department of Gastroenterology.E-mail: hcbgmu@163.com

R730.231 Document code：A Article ID：1001-5779(2016)06-0831-08

10.3969/j.issn.1001-5779.2016.06.001

*Funding: National Natural Science Foundation of China (Grant No. 81160257), The Science and Technology Project of

Ganzhou City (Grant No. GZ2015ZSF072)，The Science and Technology Project from the Health and Family Planning Commission of Jiangxi Province (Grant No. 20161096).