Effects of p16 on the Cell Cycle and Fatty Acid Metabolism of Gallbladder Cancer Cells
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摘要:
目的 通过临床水平和细胞水平验证p16的表达模式，并进一步探索p16对人胆囊癌（gallbladder cancer，GBC）细胞周期及脂肪酸代谢的影响。 方法 收集胆囊结石伴胆囊炎标本30例，GBC标本30例。采用荧光定量PCR检测各GBC样本及胆囊炎样本的p16mRNA的水平、采用免疫组织化学染色检测各GBC样本及胆囊炎样本的p16蛋白表达水平。利用肿瘤细胞的CCK8检测稳转细胞株的增殖能力、采用流式细胞技术检测肿瘤细胞的凋亡和分裂周期。QPCR检测低表达p16癌细胞中过氧化物酶体相关基因；线粒体氧化相关基因;脂肪酸合成相关基因的表达情况。 结果 p16mRNA及p16蛋白在GBC中的表达显著高于慢性胆囊炎（P < 0.05）。低表达p16之后，肿瘤细胞凋亡率降低，G0/G1期降低，S期升高，细胞分裂加快，差异极其显著（P < 0.0001 ）。低表达p16之后，GBC中的大部分相关基因显著上调，差异具有统计学意义（P < 0.05）。结论 p16在GBC中显著高表达。p16能够抑制GBC细胞周期的进展。p16可能通过调节脂肪酸代谢的途径影响GBC细胞的发生发展。 Abstract:Objective To verify the expression pattern of p16 at the clinical and cellular levels, and further explore the effects of p16 on the cell cycle and fatty acid metabolism of human gallbladder cancer （GBC） cells. Methods Thirty specimens of gallstones with cholecystitis and thirty GBC specimens were collected. Quantitative fluorescence PCR （QPCR） was used to detect p16 mRNA levels in GBC samples and cholecystitis samples. Immunohistochemical staining was performed to detect p16 protein expression levels in GBC samples and cholecystitis samples. CCK8 assay was used to detect the proliferative capacity of stably transfected cell lines. Flow cytometry was employed to detect apoptosis and cell cycle progression in tumor cells. QPCR was used to detect the expression of peroxisome-related genes, mitochondrial oxidation-related genes, and fatty acid synthesis-related genes in cancer cells with low p16 expression. Results The expression of p16 mRNA and p16 protein in GBC was significantly higher than that in chronic cholecystitis （P < 0.05）. After p16 downregulation, the apoptosis rate of tumor cells decreased, the proportion of cells in G0/G1 phase decreased, the S phase proportion increased, and cell division accelerated, with extremely significant differences （P < 0.0001 ）. After p16 downregulation, most related genes in GBC were significantly upregulated, with statistically significant differences （P < 0.05）.Conclusions p16 is significantly highly expressed in GBC. p16 can inhibit the progression of GBC cell cycle. p16 may affect the occurrence and development of GBC cells through regulating the pathway of fatty acid metabolism. -
Key words:
- p16 /
- Gallbladder cancer /
- Cell cycle /
- Fatty acid metabolism /
- Tumor
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图 1 QPCR检测临床样本中p16的表达
*P < 0.05
Figure 1. QPCR detection of p16 expression in clinical samples
图 2 IHC检测临床样本中p16的表达
A：免疫组化染色后图像（200×，标尺：50 μm）；B：p16在GBC和胆囊炎组织中的阳性表达率；*P < 0.05。
Figure 2. IHC detection of p16 expression in clinical samples
图 3 QPCR检测p16在细胞中的表达
****P < 0.0001。
Figure 3. QPCR detection of p16 expression in cells
图 5 干扰效率筛选及细胞转染
A：3条干扰链的p16相关水平；B：sh-NC和sh-p16的荧光检测结果；**P < 0.01，***P < 0.001。
Figure 5. Interference efficiency screening and cell transfection
图 6 稳转细胞株WB及QPCR验证
A：NOZ-sh-NC和NOZ-sh-p16的免疫印迹图；B：NOZ-sh-NC和NOZ-sh-p16的p16相关水平；*P < 0.05。
Figure 6. WB and qPCR validation of stable transfected cell lines
图 7 NOZ细胞增殖及细胞凋亡周期检测
A：NOZ-sh-NC和NOZ-sh-p16的细胞凋亡检测；B：NOZ-sh-NC和NOZ-sh-p16的细胞周期检测；C：sh-NC和sh-p16在96 h里，在450 nm波长下吸光度值的变化；D：NOZ-sh-NC和NOZ-sh-p16在细胞周期中，凋亡率、细胞周期各阶段、CDK4和CDK6的状况；*P < 0.05，**P < 0.01，****P < 0.0001。
Figure 7. Detection of NOZ cell proliferation and apoptosis cycle
图 8 NOZ细胞脂肪酸代谢相关基因检测
*P < 0.05，**P < 0.01，ns P > 0.05。
Figure 8. Detection of fatty acid metabolism related genes in NOZ cells
表 1 引物及序列
Table 1. The sequences of the primers
基因 正向引物序列（5′-3′） 反向引物序列（5′-3′） GAPDH CCTTCCGTGTTCCTACCCC GCCCAAGATGCCCTTCAGT CDK4 CGGCCTGTGTCTATGGTCG CAGTCGCCTCAGTAAAGCCA CDK6 TCTGATTACCTGCTCCGCGA CCTCCTCTTCCCTCCTCGAA ACOX1 TTACACACATCCTGGACGGC AATTGAGGCCCACAGGTTCC EHHADH TCAACGCGATCAGGTAACGG TCAACGCGATCAGGTAACGG PPARα CCCTGTCTGCTCTGTGGACT GAGCTCCAAGCTGTGGTGACA CPT1a GACAGCTACGCCAAATCTCTAC GGACATGACGTACTCCCAAAG ACADVL GCGATGACAGCAAAGACGAT GACGGGTATGGGAACACCTG ACACA GACTGGGTAGAGCGATTGATG CTGACAGAGGACTGATGTGATG ACSL1 TTTTGTTAAGTGCTGGTGCC ACCACCACTACCCATGACAG FASN CTGAAGGACCTGTCTAGGTTTG CGGAGTGAATCTGGGTTGAT P16 TTCCTGGACACGCTGGT ACCTTCCGCGGCATCTAT 
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表 2 干扰靶点
Table 2. Interference target
编号 Target序列信息 靶点区域 CDKN2A-sh1 AGTAACCATGCCCGCATAGCT 3′UTR区 CDKN2A-sh2 CCTCGGGAAACTTAGATCATC 3′UTR区 CDKN2A-sh3 CACTACCGTAAATGTCCATTT 3′UTR区
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