CRISPR/Cas9 is an efficient, accurate, and optimizable genome-editing tool. Here, we present a modified CRISPR/Cas9 genome-editing protocol for single nucleotide mutation in adherent cell lines. The protocol was adapted to focus on ease of use and efficiency. The protocol here describes how to generate a single nucleotide mutation in cultured 22Rv1 cells. We have also used the protocol in other adherent cell types. Thus, the protocol can be applied to assessing the effect of non-coding single nucleotide polymorphisms (SNPs) in a variety of cell types. For complete details on the use and execution of this protocol, please refer to Gao et al. (2018).
Objective To investigate the role of PI3K/Akt signal pathway in albumin-induced transforming growth factor beta 1 (TGF-β1) expression and proliferation in renal proximal tubule cells (HK-2).Methods HK-2 cells were cultured in vitro and allocated to three groups: control, albumin(BSA) and BSA+ Ly294002 (PI3K/Akt specific inhibitor).Cell proliferation was assessed by MTT method.TGF-β1 mRNA expression was determined by RT-PCR and protein outputs from TGF-β1, phosphorylated PI3K (p-PI3K) and Akt (p-Akt) was measured by Western blotting at 12, 24 and 48 h of culture.Results MTT revealed that higher dosage of BSA (30 mg/ml) overload induced significant proliferation in HK-2 cells (P< 0.05).Compared with control group, remarkable increase or up-regnlation was shown in expression of TGF-β 1 mRNA (0.472±0.025 vs 0.233±0.021, P<0.05), protein output from TGF-β1 (296±20.1 vs 100±13.2, P<0.05), p-PI3K (P<0.05) and p-Akt (P<0.05), with BSA induction at 12.Addition of Ly294002 was demonstrated to inhibit HK-2 proliferation at 12 hours and protein expressions in TGF-β1, p-PI3K and p-Akt at 48 h (P<0.05).Conclusion Albumin may induce TGF-β1 production through the activation of PI3-K/Akt signal pathway in renal tubular epithelial cells.
Key words:
Serum albumin; Transforming growth factor beta 1; Signal transduction; Renal tubular epithelial cells; Cell culture
Tumor metastasis, the main characteristic and important indication of malignant tumor, is also the primary cause of death for most cancer patients. The tumor metastasis is a multistep process involving various factors, multiple interactions of many genes as well as the microenvironment. To investigate the mechanism of tumor metastasis will help us understand the essences of metastasis process, therefore exploring molecular targets for clinical diagnosis and treatment of cancer.
Key words:
Tumor metastasis; Mechanism; Epithelial-mesenchymal transitions; Cancer stem cell
Abstract HBV infection is recognized as a serious global health problem, and hepatitis B virus infection is a complicated chronic disease leading to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). New biochemical serum markers could be used to advance the diagnosis and prognosis of HBV-associated liver diseases during the progression of chronic hepatitis B into cirrhosis and HCC. We determined whether the 4210 Da and 1866 Da polypeptides are serum metabolite biomarkers of hepatopathy with hepatitis B virus. A total of 570 subjects were divided into five groups: healthy controls, those with natural clearance, and patients with CHB, LC, and HCC. The 1866 Da and 4210 Da polypeptides were measured by Clin-ToF II MALDI-TOF–MS. There were significant differences in 4210 Da and 1866 Da levels among the five groups ( P < 0.001). For the differential diagnosis of CHB from normal liver, the areas under the receiver operating characteristic (ROC) curve of 4210 Da and 1866 Da and their combination via logistic regression were 0.961, 0.849 and 0.967. For the differential diagnosis of LC from CHB, the areas under the ROC curve were 0.695, 0.841 and 0.826. For the differential diagnosis of HCC from CHB, the areas under the ROC curve were 0.744, 0.710 and 0.761, respectively. For the differential diagnosis of HCC from LC, the areas under the ROC curve of 4210 Da and 1866 Da were 0.580 and 0.654. The positive rate of 1866 Da was 45.5% and 69.0% in AFP-negative HCC patients and that of 4210 Da was 60.6% 58.6% in AFP-negative HCC patients of the study HCC vs. CHB and HCC vs. LC. The 4210 Da and 1866 Da polypeptide levels were positively correlated with HBV DNA levels ( P < 0.001, r = 0.269; P < 0.001, r = 0.285). The 4210 Da and 1866 Da polypeptides had good diagnostic value for the occurrence and progression of HBV-related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma and could serve to accurately guide treatment management and predict clinical outcomes.
The present study aimed to develop and validate a new nomogram for predicting the incidence of hepatocellular carcinoma (HCC) among chronic hepatitis B (CHB) patients receiving antiviral therapy from real-world data.The nomogram was established based on a real-world retrospective study of 764 patients with HBV from October 2008 to July 2020. A predictive model for the incidence of HCC was developed by multivariable Cox regression, and a nomogram was constructed. The predictive accuracy and discriminative ability of the nomogram were assessed by the concordance index (C-index), calibration curves, and decision curve analysis (DCA). Risk group stratification was performed to assess the predictive capacity of the nomogram. The nomogram was compared to three current commonly used predictive models.A total of 764 patients with HBV were recruited for this study. Age, family history of HCC, alcohol consumption, and Aspartate aminotransferase-to-Platelet Ratio Index (APRI) were all independent risk predictors of HCC in CHB patients. The constructed nomogram had good discrimination with a C-index of 0.811. The calibration curve and DCA also proved the reliability and accuracy of the nomogram. Three risk groups (low, moderate, and high) with significantly different prognoses were identified (p < 0.001). The model's performance was significantly better than that of other risk models.The nomogram was superior in predicting HCC risk among CHB patients who received antiviral treatment. The model can be utilized in clinical practice to aid decision-making on the strategy of long-term HCC surveillance, especially for moderate- and high-risk patients.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
