Application of Western Blot Analysis in Studying the Proliferation of NM23B Gene Cells

NM23 is a tumor metastasis suppressor gene, which has regulatory functions on cell proliferation, differentiation and ontogeny. At present, 10 subtypes of NM23 have been found, and the more thorough research is NM23A and NM23B. Western blot analysis shows that NM23B has hepatocyte proliferation effect on SD rats cultured in vitro. Western blot analysis, also known as western blot analysis, is a method of detecting a certain protein in a complex sample based on the specific binding of antigen and antibody.

Because of the high resolution of SDS-PAGE and the high specificity and sensitivity of solid-phase immunoassay, Western blotting can detect medium-sized target proteins as low as 1-5ng (as low as 10-100pg), and has now become a protein analysis. One of the conventional techniques, and also an experimental method commonly used in molecular biology, biochemistry and immunogenetics, Medicilon can provide Western blotting experimental technology services. Western blotting is often used to identify a certain protein, and can perform qualitative and semi-quantitative analysis of the protein.

The mechanism of action of nm23 protein

The nm23 protein is highly homologous to the awd protein and nucleoside diphosphate kinase (ndpk) of Drosophila:

(1) The awd protein of Drosophila

The amino acid sequence of human nm23 protein and awd protein is 77% to 78% identical. The awd protein plays an important regulatory role in the embryonic development of Drosophila. For example, mutations or decreased expression of the awd gene will lead to many tissues and organs of Drosophila. Abnormal differentiation and death of wing disc cells. Based on this, it is speculated that nm23 protein may be necessary for normal tissue development. If nm23 protein is inactivated or reduced, it will lead to a disordered state that is conducive to abnormal differentiation and tumor metastasis.

(2) Nucleoside diphosphate kinase (ndpk)

Nucleoside diphosphate kinase (ndpk) is a family of multifunctional homogenous enzymes ubiquitous in cells, mainly distributed in the cytoplasm and plasma membrane, and its function involves the process of cellular activities that depend on ntp (nucleoside triphosphate). Ndpk catalyzes this reaction to affect microtubule polymerization, spindle formation and regulate cell movement. On the one hand, its abnormalities cause cell chromosomal aneuploidy aberrations to destroy the normal morphological structure of cells and promote tumorigenesis. On the other hand, by affecting the changes in the cytoskeleton response to cell signals, it causes cell movement and participates in infiltration and metastasis. The nm23 protein shows ndpk activity, so it is likely to play a role in regulating cell signal transduction, cell differentiation and other processes through the same or similar pathway as ndpk.

NM23 promotes cell proliferation

To observe the effect of NM23B in the proliferation of rat hepatocytes cultured in vitro, the method is to construct NM23B overexpression vector, interference vector and its negative control vector, respectively transfect SD rat BRL-3A hepatocytes, and set up a blank control group (CON) , Interference NC group (siRNA-NC), interference group (siRNA), overexpression group (OE), overexpression NC group (OE-NC), the mRNA changes of Cyclin D1, NM23B and PCNA in each group of cells were analyzed by quantitative PCR .

Western blot analysis was used to compare the changes of Cyclin D1, NM23B and PCNA protein in each group of cells, flow cytometry was used to detect the cell cycle ratio of each group, and cell proliferation was detected by the cell proliferation-toxicity test (CCK 8) method. turn out

(1) The mRNA and protein expression of Cyclin D1, NM23B and PCNA in the overexpression group OE group was significantly higher than that in the CON group (P<0.05).

(2) On the other hand, the mRNA and protein expression of Cyclin D1, NM23B and PCNA in the interference group siRNA group was significantly lower than that in the CON group (P<0.05), and="" the="" interference="" nc="" group="" oe-nc="" had="" no="" statistics="" compared="" with="" con="" academic="" difference="" p="">0.05);

(3) The ratio of G0/G1 phase cells in the OE group was significantly lower than that in the CON group (P<0.05), and the ratio of G0/G1 phase cells in the siRNA group was significantly higher than that in the CON group (P<0.05). At the same time, the proportion of S-phase cells in the OE group was significantly higher than that in the CON group (P<0.05), and the proportion of S-phase cells in the siRNA group was significantly lower than that in the CON group (P<0.05). the="" sirna-nc="" group="" and="" oe-nc="" were="" similar="" to="" con="" group.="" ratio="" was="" not="" statistically="" different="" p="">0.05).

CCK-8 detection of cell proliferation showed that the proliferation rate of the OE group was significantly faster than that of the CON group (P<0.05), the proliferation rate of the siRNA group was significantly slower than that of the CON group (P<0.05), the="" sirna-nc="" group="" and="" oe-nc="" compared="" with="" con="" there="" was="" no="" statistical="" difference="" between="" two="" groups="" p="">0.05).

Therefore, overexpression of NM23B can enhance the expression of Cyclin D1 and PCNA, shorten the cell cycle, thereby promoting the proliferation of hepatocytes; interfering with the expression of NM23B can weaken the expression of Cyclin D1 and PCNA, prolong the cell cycle, thereby inhibiting the proliferation of hepatocytes. Confirmed the role of NM23B in the proliferation of SD rat hepatocytes cultured in vitro, laying a theoretical foundation for subsequent in vivo experiments.

The relationship between nm23 and tumors

The nm23 gene has the effect of inhibiting tumor metastasis, which has been confirmed in in vitro transfection experiments and animal experiments. In the study of cell cloning of melanoma, it was found that nm23 significantly inhibited the carcinogenesis of melanoma cells, including inhibiting cell viability, inhibiting tumor occurrence, inhibiting tumor metastasis, and changing the response of melanoma. In recent years, clinical examinations have found that the expression of nm23 is negatively correlated with the invasion and metastasis of various tumors, and has an important impact on the prognosis of patients.

Studies have shown that the tumor suppressor effect of nm23 gene is tissue specific: in some tumors, such as breast cancer, liver cancer, malignant melanoma, ovarian cancer, etc., nm23 protein expression is negatively correlated with tumor metastasis and poor clinical prognosis, and its gene is missing It is closely related to tumor metastasis, showing its characteristics as a tumor metastasis suppressor gene; in other tumors, such as lung cancer, prostate cancer, bladder cancer, neuroblastoma, bone tumors, endometrial cancer, etc., the expression of nm23 gene Levels and deletions seem to have nothing to do with tumor metastasis, but they are related to tumor differentiation, proliferation and progression; even in some tumors, such as neuroblastic carcinoma, when it deteriorates or metastasizes, the expression of nm23 increases instead, and it seems that its expression level increases. Related to the proliferation of cancer cells. These results indicate that nm23 gene not only functions as a tumor metastasis suppressor gene, but also has more other biological functions.

Therefore, the nm23 gene not only has the lease of cell proliferation, but also has the function of suppressing tumor metastasis. However, the nm23 gene does not have the function of suppressing cancer metastasis in all types of tumors. Studies have shown that its role has tissue differences. The mechanism of this difference is not clear, indicating that a comprehensive understanding of the nm23 gene is far from complete. , More in-depth research is needed.

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