Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells

AIM:To investigate the effects of osteopontin(OPN)gene expression knockdown on colon cancer Lovo cells in vitro.METHODS:Four candidate small interfering RNA(siRNA)constructs targeting the OPN gene and a scrambled control sequence(NC-siRNA)were synthesized and inserted into a pGPU6/GFP/Neo expression vector.After confirmation by restriction enzyme digestion and DNA sequencing,the recombinant plasmids were subsequently transfected into a human colon cancer cell line(Lovo)using a liposome transfection method.Stably transfected cells were maintained with G418 selection and referred to as Lovo-OPN-1,-2,-3,-4,and Lovo-NC cells.Knockdown efficiency of each of the four siRNA constructs was determined by realtime reverse transcription polymerase chain reaction assays and western blotting,and the construct with the most effective silencing was used for subsequent experiments.Cell proliferation,adhesion,and Matrigel invasion assays were performed to analyze the effects of OPN knockdown in stably transfected Lovo cells.The levels of four angiogenic factors,namely vascular endothelial growth factor(VEGF),matrix metalloproteinase(MMP)-2,MMP-9 and urokinase plasminogen activator were detected by enzyme-linked immunosorbent assays(ELISA).RESULTS:Recombinant vectors containing OPNspecific and scrambled siRNA sequences were successfully constructed and stably transfected into Lovo cells.Compared with the control Lovo and Lovo-NC cells,the levels of OPN mRNA and protein expression in LovoOPN-1,-2,-3,and-4 were significantly reduced(all P<0.05),with the most efficient reduction observed in Lovo-OPN-4 cells(P<0.05).Relative to untransfected Lovo cells,OPN mRNA expression levels in Lovo-NC and Lovo-OPN-4 cells were 1.008±0.067 and 0.160±0.023,respectively.The relative OPN protein expression levels in Lovo,Lovo-NC,and Lovo-OPN-4 cells were 3.024±0.211,2.974±0.630,and 0.121±0.008,respectively.Moreover,transfection with the scrambled sequence had no effect on the expression of OPN.After24,48,72,and 96 h of cultivation,absorption values at 450 nm to assess proliferation of Lovo-OPN-4 cells were 0.210±0.017,0.247±0.024,0.314±0.037,and 0.359±0.043,respectively,which were significantly lower than those of Lovo(0.244±0.031,0.313±0.024,0.513±0.048 and 0.783±0.051)and LovoNC cells(0.241±0.029,0.309±0.022,0.563±0.023,and 0.735±0.067)(all P<0.05).The absorption values at 595 nm,which were measured in a cell adhesion assay,showed that adhesion of Lovo-OPN-4 cells(0.215±0.036)was significantly decreased compared to Lovo(0.490±0.037)and Lovo-NC cells(0.462±0.043)(P<0.05).The number of invasive Lovo-OPN-4 cells(16.1±1.9)was also significantly decreased compared to Lovo(49.9±5.4)and Lovo-NC cells(48.8±4.5)(P<0.05).ELISA assays showed significant reductions in Lovo-OPN-4 cells compared to Lovo and Lovo-NC cells with regard to the expression of VEGF(1687.85±167.84 ng/L vs 2348.54±143.80 ng/L and 2284.39±138.62 ng/L,respectively),MMP-2(2966.07±177.36μg/L vs 4084.74±349.54μg/L and 4011.41±424.48μg/L,respectively),MMP-9(3782.89±300.64μg/L vs5062.90±303.02μg/L and 4986.38±300.75μg/L,respectively)and uPA(1152.69±120.79μg/L vs1380.90±147.25μg/L and 1449.80±189.92μg/L,respectively)(all P<0.05).CONCLUSION:Knockdown of OPN gene expression suppresses colon cancer cell growth,adherence,invasion,and expression of angiogenic factors.

NQO1 C609T polymorphism correlated to colon cancer risk in farmers from western region of Inner Mongolia

Objective： To investigate the relationship between NAD（P）H：quinone oxidoreductase 1 （NQOI） C609T polymorphism and colon cancer risk in farmers from western region of Inner Mongolia. Methods： Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was performed to analyze NQO1 C609T polymorphism from 160 healthy controls and 76 colon cancer patients. Results： Among the colon cancer patients, the incidence of NQOI T allele （53.29%） was significantly higher than it in control group （33.75%, P〈0.001）. The individuals with NQO1 T allele had higher risk [2.239 （95% CI： 1.510-3.321） times] to develop colon cancer than individuals with NQO1 C allele. The incidence of NQO1 （TFI-） （34.21%） in colon cancer patients was higher than that in control group （15.62%, P〈0.001）. Odds ratios （OR） analysis suggested that NQO1 （T/F） and NQOI （T/C） genotype carriers had 3.813 （95% CI： 1.836-7.920） times and 2.080 （1.026-4.219） times risk compared with wild-type NQO1 （C/C） gene carriers in developing colon cancer. Individuals with NQO1 （T/I＇） genotype had 2.541 （95% CI： 0.990-6.552） times, 3.713 （95% CI： 1.542-8.935） times, and 3.471 （95% CI： 1.356-8.886） times risk than individuals with NQOI （T/C） or NQOI （C/C） genotype in well- differentiated, moderately-differentiated, and poorly-differentiated colon cancer patients, respectively. Conclusions： NQO1 gene C609T could be one of risk factors of colon cancer in farmers from western region of Inner Mongolia,

Cytochrome P450 2E1 RsaI/PstI and DraI Polymorphisms Are Risk Factors for Lung Cancer in Mongolian and Han Population in Inner Mongolia

Objective： To explore the relationship between cytochrome P450 2E1 （CYP2E1） RsaI/PstI and DraI polymorphism and lung cancer susceptibility in Mongolian and Han population in Inner Mongolia of China. Methods： CYP2E1 RsaI/PstI and DraI polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in 64 lung cancer patients, 150 healthy Mongolian and 150 healthy Han individuals. The distribution of genotype and allele frequencies of CYP2E1 RsaI/PstI and DraI polymorphisms were studied. Results： The risk of lung cancer was increased in individuals with CYP2E1 （cl/cl） and CYP2E1 （DD） with OR values of 2.431 （95%CI=1.082-5.460） and 2.778 （95%CI=1.358-5.683） respectively （P0.05）. When CYP2E1 RsaI/PstI and DraI polymorphisms were combined, the risk of lung cancer was reduced in individuals with CYP2E1 （cl/c2＋c2/c2 and DD＋CC） with OR values of 0.233 （95%CI=0.088-0.615, P0.05）. In smokers, the susceptibility to lung cancer was higher in the individuals with CYP2E1 （c1/c1） and CYP2E1 （DD） than in the individuals with c2 and C allele （P0.05, OR=2.643 and 4.308 respectively）. There was no significant difference in distribution of CYP2E1 genotype frequency between healthy Mongolian, Han population and lung cancer patients, healthy controls in Inner Mongolia. Conclusion： CYP2E1 （c1/c1） and CYP2E1 （DD） are predisposing factors of lung cancer in population in Inner Mongolia. CYP2E1 （c2﹢C） co-mutation may decrease the risk of lung cancer. Smoking exerts synergetic effect with CYP2E1 （c1/c1） and CYP2E1 （DD） on the occurrence of lung cancer.

Association of rs1137101 with hypertension and type 2 diabetes mellitus of Mongolian and Han Chinese

BACKGROUND Hypertension(HTN)and type 2 diabetes mellitus(T2DM)are often coincident,and each condition is considered a risk factor for the other.Both occur frequently in the Inner Mongolia region of China.The reasons for differences in risk between Han and Mongolian ethnic groups are not known.The LEPR gene and its polymorphism,rs1137101(Gln223Arg),are both considered risk factors for HTN and T2DM,but any role of rs1137101 in the occurrence of HTN+T2DM remains unclear for Mongolian and Han populations in the Inner Mongolia region.AIM To investigate the relationship between rs1137101 and the occurrence of HTN with T2DM in Mongolian and Han populations in Inner Mongolia METHODS A total of 2652 subjects of Han and Mongolian ethnic origins were enrolled in the current study,including 908 healthy controls,1061 HTN patients and 683 HTN patients with T2DM.RESULTS The association between the rs1137101 polymorphism and HTN with T2DM was analyzed,and differences between Han and Mongolian individuals assessed.There was a significant correlation between rs1137101 and HTN(co-dominant,dominant,over-dominant and log-additive models)and HTN+T2DM(co-dominant,dominant,over-dominant and log-additive models)after adjustment for sex and age in individuals of Mongolian origin.rs1137101 was significantly associated with HTN(co-dominant,recessive and log-additive models)and HTN+T2DM(codominant,dominant,over-dominant and log-additive models)in the Han Chinese population.CONCLUSION Mongolian and Han subjects from Inner Mongolia with HTN who had rs1137101 were protected against the development of T2DM.Allele A has the opposite impact on the occurrence of HTN in Mongolian and Han Chinese populations.