The role of tazarotene-induced gene 1 in carcinogenesis:is it a tumor suppressor gene or an oncogene?

Tazarotene-induced gene 1(TIG1)is induced by a derivative of vitamin A and is known to regulate many important biological processes and control the development of cancer.TIG1 is widely expressed in various tissues;yet in many cancer tissues,it is not expressed because of the methylation of its promoter.Additionally,the expression of TIG1 in cancer cells inhibits their growth and invasion,suggesting that TIG1 acts as a tumor suppressor gene.However,in some cancers,poor prognosis is associated with TIG1 expression,indicating its protumor growth characteristics,especially in promoting the invasion of inflammatory breast cancer cells.This review comprehensively summarizes the roles of the TIG1 gene in cancer development and details the mechanisms through which TIG1 regulates cancer development,with the aim of understanding its various roles in cancer development.

Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies

FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.

Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats

AIM To evaluate the relationship between expression of ras, p53, bcl 2 gene products, and hepatocarcinogenesis since endotoxemia produced from lipopolysaccharide admi nistration and/or the hypophagocytic state of splenectomy significantly accelerated hepatocarcinogenesis induced by thioacetamide. 〖WTH4〗METHODS〓〖WTXFZ〗The hepatocarcinoma model was induced by oral intake of 0 03% thioacetamide for six months. During the induction of hepatocarcinoma model, rats were additionally treated with splenectomy and/or lipopolysaccharide administration. The techniques of flow cytometry, immunohistochemistry and immunoelectronmicroscopy were applied to quantitative analysis of the expression of oncogene proteins. RESULTS In this model system, overexpression of ras p21 protein mainly occurred on precancerous cell population or in early stage of hepatocyte transformation. And the levels of ras p21 declined when nuclear DNA aneuploid increased. Expression of bcl 2 protein slowly and steadily rose with more hepatocytes staying in S+G2M phases as the hepatocarcinoma became more malignant. P53 was moderately expressed during the hepatocarcinogenesis. There was no statistical correlation between endotoxemia levels and the changes of ras, p53 and bcl 2 gene products. CONCLUSION Over expression of oncogene ras p21 was likely to be a precursor of the premalignant hepatocytes and it might be responsible for the initiation of hepatocarcinogenesis. Bcl 2 protein expression is proportional to the severity of the malignancies. P53 may be a key pathway on the transformation and development of hepatocarcinoma. This study confirmed the hypothesis that there are multiple genes and multiple steps involved in hepatocarcinogenesis. Expressions of oncogene proteins reflected the properties of the premalignant and malignant cells, but not directly related to endotoxemia statistically.[JP]

Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis

IM To study the alterations of tumor suppressor gene p16 and Rb in the carcinogenesis of the stomach. METHODS Different mucosal biopsies were endoscopically obtained, all samples were immediately fixed with 10% buffered formalin, embedded with paraffin and sectioned serielly. Alterations of p16 and Rb protein in 12 cases of superficial gastritis, 15 atrophic gastritis, 20 atypical hyperplasia and 40 cancerous tissues were detected by the immunohistochemical method (ABC). RESULTS Different degrees of nuclear immunostaining of p16 and Rb occurred on gastric epithelium in different stages of lesions. With the lesions progressing, the positive immunostaining rate of p16 protein had a decreasing tendency (833%→733%→300%→275%), and on the other hand, that of Rb protein had an increasing tendency (250%→467%→600%→675%). A negative correlationship was found between these two parameters in the gastric cancer. Of 40 cases of gastric cancer, a negative relationship was observed in 20 cases. In comparison with both positive (9 cases) and both negative tissues (11 cases), there was a significant difference (500%,225%,275%) (P<005).CONCLUSION Abnormal expression of p16 and Rb plays an important role in gastric carcinogenesis.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM: To investigate SBA2 expression in CRC cell lines and surgical specimens of CRC and autologous healthy mucosa. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples. Normalization of the results was achieved by simultaneous amplification of beta-actin as an internal control. RESULTS: In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and beta-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts P【0.01). SBA2 expression was significantly (0.01】P 【 0.05) correlated with the grade of differentiation in CRC, with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in non-metastasis samples 0.01】P【0.05). CONCLUSION: SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.

Relationship between Fas/ FasL expression and apoptosis of colon adenocarcinoma cell lines

INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Adenovirus-mediated expression of pig α(1,3) galactosyltransferase reconstructs Gal α(1,3) Gal epitope on the surface of human tumor cells

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

虎杖苷调节Akt/MDM2/p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响

目的探讨虎杖苷(PD)调节蛋白激酶B/原癌基因MDM2/抑癌基因p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响。方法以人胆囊癌细胞株(GBC-SD)为研究对象,体外培养人胆囊癌细胞株(GBC-SD),使用浓度为10~160 mmol/L的虎杖苷处理细胞24、48、72 h,采用CCK-8法检测细胞的增殖能力,确定最佳实验浓度。将GBC-SD细胞分为对照组(Control组)、虎杖苷低、中、高浓度组(PD-L组、PD-M组、PD-H组)、虎杖苷+Akt激活剂组(PD+SC79组),Transwell小室法评价细胞的迁移能力,Hoechst染色观察细胞的凋亡,流式细胞术检测细胞周期与细胞凋亡,Western blot检测Akt、MDM2、p53磷酸化水平,建立荷瘤小鼠模型评价虎杖苷对胆囊癌肿瘤生长的影响。结果浓度为10~160 mmol/L的虎杖苷处理细胞24 h,可显著抑制GBC-SD细胞的增殖活性,选择10、20、40 mmol/L的虎杖苷进行后续实验;与Control组比较,PD-L组、PD-M组、PD-H组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著降低,G0/G1期细胞比例、P-p53蛋白表达显著升高,且呈浓度依赖性(P<0.05);与PD-H组比较,PD+SC79组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著升高,G0/G1期细胞比例、P-p53蛋白表达显著降低(P<0.05);虎杖苷干预治疗后,小鼠移植瘤的生长速度显著降低(P<0.05)。结论虎杖苷可以通过调节Akt/MDM2/p53信号通路使细胞周期阻滞,抑制胆囊癌细胞增殖、迁移。

颅脑外伤患者血清IL-33、sST2、TNF-α、HMGB1水平与病情严重程度及短期预后的关系

目的探讨颅脑外伤患者血清白细胞介素(IL)-33、可溶性生长刺激表达基因2蛋白(sST2)、肿瘤坏死因子-α(TNF-α)、高迁移率族蛋白B1(HMGB1)与病情严重程度及短期预后的关系。方法回顾性分析2022年1月至2024年1月渭南市中心医院收治的142例颅脑外伤患者的临床资料。根据格拉斯哥昏迷量表(GCS)评估病情严重程度分为轻度组(13~15分)(n=47)、中度组(9~12分)(n=59)、重度(3~8分)(n=36)。根据格拉斯哥预后量表(GOS)评估28 d预后分为预后良好组(4~5分)(n=115)、预后不良组(1~3分)(n=27)。收集患者资料,包括性别、年龄、体重指数、吸烟、饮酒、高血压、糖尿病、外伤原因、IL-33、sST2、TNF-α、HMGB1。比较不同病情严重程度、不同预后患者的临床资料;采用Spearman秩相关分析检验颅脑外伤患者病情严重程度与IL-33、sST2、TNF-α、HMGB1水平的相关性;通过受试者操作特征(ROC)分析IL-33、sST2、TNF-α、HMGB1预测颅脑外伤患者预后不良的价值。结果重度组IL-33、sST2、TNF-α、HMGB1水平分别为(40.69±9.64)pg/mL、(7.75±2.14)ng/mL、(252.43±41.37)ng/L、(11.42±3.27)μg/L,中度组IL-33、sST2、TNF-α、HMGB1水平分别为(30.47±7.22)pg/mL、(6.45±1.37)ng/mL、(181.29±22.75)ng/L、(8.05±2.13)μg/L,均高于轻度组[(21.53±5.84)pg/mL、(5.61±1.05)ng/mL、(140.36±16.41)ng/L、(4.87±1.42)μg/L],差异均有统计学意义(P<0.05)。IL-33、sST2、TNF-α、HMGB1水平与颅脑外伤患者病情严重程度呈正相关(r=0.561、0.518、0.593、0.537,P<0.05)。预后不良组IL-33、sST2、TNF-α、HMGB1水平分别为(74.08±20.37)pg/mL、(10.56±2.54)ng/mL、(286.31±42.81)ng/L、(13.45±3.07)μg/L,均高于预后良好组[(22.64±6.19)pg/mL、(6.95±1.73)ng/mL、(153.76±31.45)ng/L、(5.86±1.49)μg/L],差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,IL-33、sST2、TNF-α、HMGB1预测颅脑外伤患者预后不良的曲线下面积为0.891、0.846、0.905、0.928,均有P<0.05。结论IL-33、sST2、TNF-α、HMGB1表达水平随颅脑外伤患者病情严重程度升高而升高,且与其短期不良预后有关。

SPP1、DEC1、C1QTNF6蛋白与口腔鳞状细胞癌患者临床病理指标及预后的关系

目的:探讨口腔鳞状细胞癌患者血清重组人分泌型磷蛋白1(SPP1)、软骨分化的表达基因1(DEC1)和补体C1q/肿瘤坏死因子相关蛋白6(C1QTNF6)的表达水平与其临床病理指标及预后的关系。方法:免疫组织化学染色法和电化学发光免疫分析法检测88例口腔鳞状细胞癌患者癌组织和血清中SPP1、DEC1和C1QTNF6蛋白的表达水平;Pearson相关性分析和Kaplan-Meier生存分析法分析患者血清SPP1、DEC1和C1QTNF6蛋白表达水平与其临床病理指标的相关性和对患者预后的影响。多因素Cox回归法分析影响口腔鳞状细胞癌患者预后的危险因素。结果:免疫组织化学染色结果显示口腔鳞状细胞癌患者癌组织中SPP1、DEC1和C1QTNF6蛋白的阳性表达率较癌旁组织分别增加了1.94、2.98和2.35倍(P<0.05或P<0.01);电化学发光免疫分析法结果显示口腔鳞状细胞癌患者血清SPP1、DEC1和C1QTNF6蛋白表达水平较正常人分别上调了8.61、6.20和4.03倍(P<0.05或P<0.01);Pearson相关性分析结果显示患者血清SPP1、DEC1和C1QTNF6蛋白表达水平与患者发生多灶嗜神经侵犯、肿瘤浸润程度、淋巴结转移、较高的TNM分期呈正相关(P<0.05);Kaplan-Meier生存分析结果显示,血清SPP1、DEC1和C1QTNF6蛋白高表达水平的口腔鳞状细胞癌患者总生存率低;多因素Cox回归分析结果表明多灶嗜神经侵犯、肿瘤高浸润度、淋巴结转移和高的TNM分期是影响预后的危险因素(P<0.05)。结论:口腔鳞状细胞癌患者癌组织和血清中SPP1、DEC1和C1QTNF6蛋白表达水平升高,且与患者发生多灶嗜神经侵犯、肿瘤浸润和淋巴结转移、较高的TNM分期呈正相关,而与患者预后呈负相关。

A preliminary study on ras protein expression in human esophageal cancer and precancerous lesions

INTRODUCTIONThe esophageal carcinoma is a common malignanttumor in Linzhou City (Linxian) of Henan Provincein northern China.Although the etiology andnatural history of csophageal carcinoma are notclear,a substantial amount of evidence has beenprovided to suggest that the development of humanesophageal squamous cell carcinomas (SCC) is amultistage progressive process.An

Current status of gene therapy in gastroenterology

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Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

DNA ploidy and c-Kitmutation in gastrointestinal stromal tumors

AIM: To investigate the prognostic significance of c-Kitgen emutation and DNA ploidy in gastointestinal stromal tumors (GISTs).METHODS: A total of 55 cases of GISTs were studied for the expression of c-Kit by immunohistochemistry, and the c-Kit gene mutations in exons 9, 11, 13, and 17 were detected by polymerase chain reaction-single strand confirmation polymarphism (PCR-SSCP) and denaturing high performance liquid chromatography (D-HPLC) techniques. DNA ploidy was determined by flow cytometry.RESULTS: Of the 55 cases of GISTs, 53 cases (96.4%) expressed c-Kit protein. The c-Kit gene mutations of exons 11 and 9 were found in 30 (54.5%) and 7 cases (12.7%),respectively. No mutations were found in exons 13 and 17.DNA aneuploidy was seen in 10 cases (18.2%). The c-Kit mutation positive GISTs were larger in size than the negative GISTs. The aneuploidy tumors were statistically associated with large size, high mitotic counts, high risk groups, high cellularity and severe nuclear atypia, and epithelioid type.There was a tendency that c-Kit mutations were more frequently found in aneuploidy GISTs.CONCLUSION: DNA aneuploidy and c-Kit mutations can be considered as prognostic factors in GISTs.

ZNF217 expression correlates with the biological behavior of human ovarian cancer cells

The aim of the study was to investigate the correlation of zinc-finger protein 217 （ZNF217） gone ex- pression with the biological behavior of human ovarian cancer HO-8910 cells. Methods： The expression of ZNF217 in ovarian carcinoma cell line：s was detected by RT-PCR and Western blot, respectively. The biological behaviors of the transfectants were investigated by MTT, in vitro Boyden chamber and in vivo invasion assay, respectively. Results： RT-PCR and Western blotting revealed that transfection of ZNF217 into the HO-8910 cells significantly increased their proliferation along with mark- edly enhanced in vitro and in vivo invasion and metastatic abilities. MTT assay showed that the proliferation ability of pEGFP- N1-ZNF217/HO-8910 cells was significantly higher than that of pEGFP-N1/HO-8910 cells and HO-8910 cells （P 〈 0.001）. The Boyden chamber assay showed that the numbers of migrating pEGFP-N1-ZNF217/HO-8910, pEGFP-N1/HO-8910 and HO-8910 cells were （141.25 ± 13.91） cells/200 x field, （82.50 ± 11.73） cells/200 × field and （81.75 ± 12.12） cells/200 x field, respectively, with a significant difference between them （F = 29.274, P 〈 0.001）. The nude mouse experiment showed that the in vivo tumor formation ability of pEGFP-N1-ZNF217/HO-8910 cells was significantly higher than that of pEGFP-N1/HO-8910 cells （P 〈 0.001）. Conclusion： Based on these clinical and laboratory observations, we conclude that ZNF217 may contribute to ovarian cancer invasion and metastasis, and associated with worse clinical outcomes. We evaluated ZNF217＇s role as a biomarker of ovarian carcinogenesis and tumor progression in patient samples and explored possible molecular mechanisms in promoting tumor growth and invasion.

Potential effect of hepatitis C Virus non-structural protein 4B on liver carcinogenesis

Objective：To investigate the effect of hepatitis C virus non-structural protein 4B（HCV NS4B） on c-Myc, P53, ras gene expression＂ and apoptosis in hepatic cells and study the possible role that NS4B played in the carcinogenesis of heparoma. Methods： The recombinant plasmid（PCXN2-NS4B, PCXN2-P53） and the empty, vector were transfected or co-transfected into Chang liver cells with liposome. Screening was performed with G418. Plasmid mRNA was detected by RT-PCR. The pro rein expressions of c-Myc and ras genes were analyzed by immunocytochemistry. The expressions of wild-type P53 （wtp53） gene were detected by in situ hybridization. TUNEL（flow cytometry） was used for assessing the rate of apoptosis. Results：No expression of c-Myc gene was found in PCXN2 group. The expression of c-Myc gene in NS4B group was 21.3% ＋ 1.2%. The ex pression of ras gene in PCXN2 group was lower than that in NS4B group. Compared with PCXN2 group, the expression of P53 mRNA was not promoted or inhibited in NS4B group. But the expression of P53 mRNA in NS4B-P53 group was lower than that in P53 group. In PCXN2, NS4B, P53 and NS4B-P53 group, the rates of apoptosis were 17.02% ± 1.24%, 11.94% ± 2.24%, 25.84% ± 3.49% and 18.34% ± 1.55% respectively. Conclusion ：HCV NS4B induces the expression of c-Myc and ras gene. HCV NS4B may play a role in the inhibition of cell death through P53-dependent manner. Results from this study suggested that HCV NS4B might contribute to the viral carcinogenesis.

从GEO数据库筛选结肠癌差异关键基因及验证

目的通过基因表达综合(GEO)数据库分析筛选出结肠癌(CC)组织差异表达基因,同时通过体外实验进行验证,挖掘潜在的结肠癌差异关键基因。方法从GEO数据库获取人CC数据集GSE10950和GSE74602,利用GEO2R和韦恩(Venn)图在线分析工具筛选CC和正常结肠组织的差异表达基因(DEGs)。通过DAVID在线工具对差异表达基因进行基因本体论(GO)和京都基因与基因组百科全书(KEGG)通路富集分析,然后利用Cytoscape软件构建蛋白质相互作用(PPI)网络,并选出核心基因。使用GEPIA数据库进行生存分析,将与预后显著相关的核心基因视为关键基因。用细胞转染及CCK8法进一步验证关键基因的功能。结果从2个数据集中获得了515个DEGs,其中223个表达上调和292个表达下调。GO富集分析显示上调DEGs参与细胞周期负调控、转录调控等生物学过程,KEGG信号通路分析上调DEGs主要富集于细胞周期和DNA复制等信号通路。PPI网络筛选出33个核心基因。经UCLCAN分析发现,CCNB1、CCNA2、CDC20、CDKN3、DLGAP5、HMMR和NCAPG高表达的CC患者生存期较短(P<0.05)。通过GEPIA数据库验证,CC患者中7个基因的表达水平升高,差异有统计学意义(P<0.05)。KEGG通路分析表明CCNB1、CDC20和CCNA2在细胞周期中高度富集。敲低CCNB1、CDC20和CCNA2表达可显著抑制结肠癌细胞增殖。结论筛选出可能参与结肠癌发展的7个关键基因,其中CCNB1、CDC20和CCNA2可能成为CC的诊断分子标志物和治疗靶点。

DOK3基因表达下调对原代结肠癌细胞生物学行为的影响及其可能机制

目的 分析对接蛋白3(DOK3)基因表达下调对原代结肠癌细胞增殖、侵袭/迁移能力的影响,并基于DOK3与G蛋白偶联受体84(GPR84)的相互作用探讨可能的作用机制。方法 培养原代结肠癌细胞HCT16,将其分为对照组、小干扰RNA(siRNA)-NC组、siRNA-DOK3组进行实验。分别将siRNA-NC、siRNA-DOK3转染至siRNA-NC组、siRNA-DOK3组,而仅将培养液加入对照组。培养24 h后通过实时荧光定量PCR评估转染效果,再使用CCK-8法、Transwell实验分别检测各组细胞的增殖情况、侵袭/迁移能力,采用蛋白免疫共沉淀实验检测HCT16细胞中DOK3与GPR84之间的相互作用关系。结果 与对照组比较,siRNA-DOK3组HCT16细胞中DOK3 mRNA表达量降低(P<0.05),而siRNA-NC组HCT16细胞中DOK3mRNA表达量差异无统计学意义(P>0.05)。培养48 h、72 h后,siRNA-DOK3组HCT16细胞的增殖活力较对照组增加(P<0.05)。与对照组相比,siRNA-DOK3组HCT16细胞的迁移和侵袭数量增加(P<0.05),而siRNA-NC组HCT16细胞的迁移和侵袭数量差异无统计学意义(P>0.05)。蛋白免疫共沉淀实验结果显示,在HCT16细胞中GPR84与DOK3相互结合。结论 DOK3基因可能作为抑癌基因,参与了结肠癌的发生与发展,且其可能通过与GPR84结合发挥抑癌作用。