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Objective:To observe the expression of CD36 in hepatocellular carcinoma tissues and cell lines, and to investigate the effects of CD36 on the proliferation and migration abilities of human hepatocellular carcinoma cell lines and human hepatocellular carcinoma cell xenograft models in nude mice.Methods:Differences in the expression levels of CD36 transcripts in 371 hepatocellular carcinoma and paracancerous tissues were analyzed based on information from The Cancer Genome Atlas (TCGA) database. Cancer tissues and corresponding paracancerous tissues of 48 hepatocellular carcinoma patients who were diagnosed and underwent surgical treatment at the Affiliated Hospital of Yangzhou University from January 2019 to February 2021 were prospectively collected, and the levels of CD36 mRNA in the tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method. Western blotting was used to detect CD36 protein levels in human hepatocellular carcinoma cell lines Huh7 and HCCLM3 and human normal liver cell line LO2. Plasmids containing CD36 interfering sequences and empty plasmids were transfected into Huh7 cells or HCCLM3 cells for sh-CD36 group and control group, respectively. The CCK-8 assay was used to detect the proliferation ability (expressed as absorbance value) of cells in each group at 0, 12, 24, 36, 48 and 60 h of culture, and the scratch healing assay and Transwell assay were used to detect the migration ability of cells in each group. The Huh7 cells of sh-CD36 group or control group were injected into the axillary subcutis of BALB/c nude mice, with 4 mice in each group, to construct nude mice models of human hepatocellular carcinoma xenografts; the long and short diameters of tumor were measured weekly after 1 week of inoculation, and the tumor volume was calculated. The nude mice were put to death after 5 weeks of inoculation, and the tumor specimens were collected and weighed; the tumor cell morphology was observed under the microscope, and the expressions of CD36 and Ki-67 proteins in the tumor tissues was detected by immunohistochemistry (IHC).Results:Analysis of the data from the TCGA database showed that the level of CD36 transcripts was higher in hepatocellular carcinoma tissues compared with that in paracancerous tissues (4.2±1.8 vs. 3.2±1.5, t = 2.28, P = 0.035). Tissues detection using qRT-PCR in 48 patients with hepatocellular carcinoma showed that the relative expression of CD36 mRNA in hepatocellular carcinoma tissues was higher than that in paracancerous tissues (0.76±0.26 vs. 0.48±0.23, t = 3.52, P < 0.001). Western blotting assay showed that CD36 protein level in Huh7 and HCCLM3 cells was higher than that in LO2 cells, which were (1.42±0.11) times and (1.68±0.16) times higher than LO2 cells, respectively (both P < 0.001). At the mRNA and protein levels, the CD36 of Huh7 and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group (both P < 0.001). CCK-8 assay showed that the proliferative ability of Huh7 cells and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group after 36 and 24 h of culture (both P < 0.01). Scratch healing assay showed that the scratch healing rates of Huh7 cells [(12±3)% vs. (30±5)%, t = 4.01, P < 0.001] and HCCLM3 cells [(15±4)% vs. (29±5)%, t = 4.16, P < 0.001] in the sh-CD36 group were lower than those in the corresponding control group at 48 h of culture; Transwell assay showed that the number of Huh7 cells [(46±6) cells/field of view vs. (88± 6) cells/field of view, t = 5.56, P < 0.001] and HCCLM3 cells [(42±5) cells/field of view vs. (82±7) cells/field of view, t = 5.34, P < 0.001] penetrating into the membrane in 24 h in the sh-CD36 group was less than that in the corresponding control group. Five weeks after subcutaneous injection, the tumor volume [(682±268) mm 3vs. (1 375±512) mm 3, t = 4.73, P = 0.006] and tumor mass [(432±95) mg/mouse vs. (871±109) mg/mouse, t = 6.57, P < 0.001] of nude mice injected with Huh7 cells of the sh-CD36 group were lower than those of nude mice injected with Huh7 cells of the control group; under the microscope, the density of tumor cells in transplanted tumor specimens of nude mice injected with Huh7 cells of the sh-CD36 group was lower than that in nude mice injected with Huh7 cells of the control group, and the expression levels of both CD36 and Ki-67 proteins were also low. Conclusions:CD36 expression is up-regulated in cancer tissues of hepatocellular carcinoma patients and human hepatocellular carcinoma cell lines Huh7 and HCCLM3, and it may associate with cell proliferation and migration of hepatocellular carcinoma. Knockdown of CD36 expression significantly inhibits the proliferation and migration abilities of hepatocellular carcinoma cells in vitro, and inhibits the tumors of human hepatocellular carcinoma cell xenograft models in nude mice.
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Objective:To explore the association of miRNA-146a (miR-146a), miRNA-196a2 (miR-196a2), and miRNA-499 (miR-499) single nucleotide polymorphisms with genetic susceptibility in hepatocellular carcinoma.Methods:A case-control study was designed. A total of 175 patients (hepatocellular carcinoma group) in Affiliated Hospital of Yangzhou University from April 2015 to March 2019 and 302 healthy people undergoing physical examination during the same period (the control group) were selected. The genotype distribution of miR-146a, miR-196a2 and miR-499 in the peripheral blood of the two groups were detected by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Logistic regression model was used to analyze the association of 3 genotypes of miRNA, genotypes of hepatitis virus infectors with genetic susceptibility in hepatocellular carcinoma. The relationship between miR-146a gene polymorphism and demography factor as well as the clinical characteristics was also analyzed by using Spearman correlation analysis.Results:In hepatocellular carcinoma group, miR-146a single nucleotide polymorphism of CC, CG, GG site genotypes had 52 (29.7%) cases, 86 (49.1%) cases, 37 (21.1%) cases, respectively; in the control group, the corresponding genotypes had 137 (45.4%) cases, 135 (44.7%) cases and 30 (9.9%) cases, respectively, and the difference in genotype distribution of both groups was statistically significant ( χ2 = 17.23, P < 0.05). There were no statistical differences in genotype distribution of miR-196a2 and miR-499 ( χ2 = 0.51, P = 0.776; χ2 = 0.05, P = 0.976). Single factor logistic regression analysis showed that in the co-dominant model of miR-146a genotype, genotypes of CG ( OR = 1.96, 95% CI 1.13-3.41, P = 0.017) and GG ( OR = 3.30, 95% CI 1.85-5.89, P<0.01) had elevated risk of hepatocellular carcinoma compared with CC genotype. In the dominant model, the risk of hepatocellular carcinoma in CG+GG genotypes was increased compared with that in CC genotype ( OR=1.97, 95% CI 1.33-2.93, P = 0.001); in the recessive model, the risk of hepatocellular carcinoma in GG genotype was increased compared with that in CG+ GG genotype ( OR=2.43, 95% CI 1.44-4.11, P = 0.001). Single factor logistic regression analysis showed that there was no significant difference in the risk of hepatocellular carcinoma in the co-dominant, dominant and recessive models between miR-196a2 and miR-499 genotypes (all P > 0.05). For hepatocellular carcinoma patients with positive hepatitis B virus (HBV), CG genotype had a 2.02-fold (95% CI 1.06-5.07) risk of hepatocellular carcinoma compared with CC genotype, and GG genotype had a 3.12-fold (95% CI 1.66-10.07) risk of hepatocellular carcinoma compared with CC genotype; CG+GG genotype had a 1.91-fold (95% CI 1.85-3.38) compared with CC genotype, GG genotype had a 1.54-fold (95% CI 1.15-6.08) compared with CG+GG genotype. The increasing risk of hepatocellular carcinoma by miR-146a gene polymorphisms was not found in hepatocellular carcinoma patients with hepatitis C virus (HCV) infection or without HBV and HCV infection. Spearman correlation analysis showed that miR-146a gene polymorphisms was not related with age, gender, smoking, drinking, family history of cancer, alanine transaminase and aspartate aminotransferase (all P>0.05). Conclusions:GG and CG genotypes of miR-146a increase the risk of genetic susceptibility in hepatocellular carcinoma, especially for patients with HBV infection. miR-196a2 and miR-499 single nucleotide polymorphisms don't increase the risk of hepatocellular carcinoma.
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Objective To explore the protective effect of astilbin in hepatic ischemia-reperfusion injury (HIRI).Methods SD rats were divided into Sham group (control group),HIRI group (ischemia-reperfusion group),astilbe (low dose group,middle dose group,high dose group),and estabilished the model of rat HIRI.After liver were reperfused with blood (in 4 h,8 h,16 h),collecting the specimens of blood and liver tissues.Detection of serum alanine aminotransferase (ALT),aspertate aminotransferase (AST);Then observed the changes of liver cell microstructure;Western blot analysised the expression of HMGB1,TLR4,NF-kB,TNF-α in liver tissue.Results The serum ALT levels of Sham group in 4 h,8 h,16 h were (58.11 ±4.81) U/L,(57.12 ± 5.33) U/L,(57.63 ±4.54) U/L,the serum ALT levels of HIRI group in 4 h,8 h,16 h were (540.38 ± 21.41) U/L,(831.21 ± 20.11) U/L,(191.95 ± 15.35) U/L.Compared with Sham group,the serum ALT levels of HIRI group were significantly increased(P ＜ 0.01).Compared with HIRI group,The serum ALT levels of three dose groups in 4 h,8 h,16 h were significantly declined,including high dose group lower the most obvious (The serum ALT levels of high dose group in 4 h,8 h,16 h were (223.75 ± 10.53) U/L,(412.14 ±23.59) U/L,(205.25 ± 15.48) U/L (P ＜0.01).The results of light microscope indicated that drug groups significantly reduce the liver cell damage.The results of Western blot displayed that High dose group of HMGB1,TLR4 protein expression in 4 h,8 h,16 h drop significantly than HIRI group(P ＜0.05).High dose group of NFkB,TNF-α protein expression in postoperative 8 h,16 h decrease significantly than HIRI group (P ＜ 0.05),but in postoperative 8 h,there was no statistically significant difference compared with group HIRI (P＞0.05).Conclusion Astilbe pretreatment can reduce HIRI and its mechanism may be associated with downregulating the axis of HMGB1/TLR4/NF-kB/TNF-α,proceed to the next inhibiting the inflammatory response.
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This paper discusses the problems, clinical application and limitations of argon-helium cryosurgical scalpel and liquid nitrogen cryosurgical scalpel. The feasibility of self-absorption liquid-CO2 cryosurgical scalpel is analyzed. The result shows that self-absorption liquid-CO2 cryosurgical scalpel can be applied to cryosurgery.