Effects Of Recombinant IGF-1α On The Proliferation Of Human Endometrial Carcinoma Cell Ishikawa

Endometrial cancer is the fourth most common cancer in women, following breast, lung, and colorectal cancer, following breast, lung, and colorectal cancer. In 2008, there were 40,100 new cases diagnosed and 7470 disease-related deaths. Women have a 2.6% lifetime risk of developing this malignancy. Although usually detected in early, more favorable stages, cancers identified at late stages are associated with high levels of morbidity and mortality. The incidence of the disease has increased gradually each year and endometrial cancer mortality has more than doubled from 1987 to 1998. Moreover, while the incidence and mortality rates from several other cancers have plateaued or decreased in the last decade, rates for endometrial cancer continue to rise. The reasons underlying this increase are multifactorial, but the research shows that the IGFs is likely a contributing factor in endometrial carcinoma.Although the high rate of early diagnosis of endometrial cancer, advanced clinical stage of morbidity and mortality are still not optimistic. Because the risk factors of endometrial cancer are multiple, not yet fully understood, so there is no good means of prevention and measures. In recent years, more research show support that insulin-like growth factor system is realated with tumorigenesis, and possibly through autocrine or IGFs paracrine mode of participation in the process of endometrial hyperplasia and even endometrial carcinoma development and metastasis. study of IGFs will help to further understand and improve the theory of the etiology of endometrial cancer, and promotes endometrial cancer early prevention and reversal treatment of Insulin-like growth factor system (insulin-like growth factor system, IGFs) is a complex molecular networks, including two ligands (IGF-1 and IGF-2), two receptors (IGF-1R, IGF-2R) , 6 high-affinity binding proteins (IGFBP1-6) and IGFBP proteases, which IGF-1, IGF-2 and higher concentrations of IGFBP3 in the loop. A large number of cancer epidemiology display the two factors of IGF-1,IGFBP3 and the ratio between them is closely related with cancer. Insulin-like growth factor-1 (insulin-like growth factor-1, IGF-1) is synthesized by the liver, gene located on chromosome 12q22, the full 153 amino acids, the main chain of 70 amino acids (activity units), and the structure is similar to insulin and is modulated by insulin and growth hormone (GH), promoting the body with growth, anabolic, neurological and physiological activity of bone regeneration, and angiogenesis, tumorigenesis, and promote tumor cell proliferation and also play an important role in metastasis.1. IGF-1 in different types of endometrial carcinomaThis study used immunohistochemistry to detect the differences expression of IGF-1 in normal endometrium, endometrial hyperplasia and endometrial cancer tissue, our research found found that IGF-1 were positive expression in different phases of normal endometrium, but no over-expression, but the difference was not significant (p> 0.05), IGF-1 was also positive expression in different types of endometrial hyperplasia ,also no over-expression, but the difference was not significant (p> 0.05), whereas in endometrial cancer, IGF-1 expression and over-expression were both significantly higher than that in normal endometrium and endometrial hyperplasia ,there is significantly difference (p<0.05), it suggested IGF-1 may be involved in endometrial carcinogenesis, whereas IGF-1 in the histological grade and pathological expression and excessive period of no significant difference in expression. However, IGF-1's over-expression increased in the lower histological grading of endometrial carcinoma, it hints that IGF-1 may be prompted to have an important role in typeⅠendometrial cancer , maybe related to endometrial cancer risk factors such as obesity hyperinsulinemia and diabetes-related signal transduction pathways. According to research results suggest that down-regulation of expression of IGF-1 in patients with lifestyle intervention or approach to clinical prevention of maybe decline the possibility of endometrial carcinoma.2. the successful construction and expression of IGF-1αrecombinant proteinIn this study, we use multiplex PCR cloning of IGF-1αgene, using restriction enzyme digestion and connection methods ,connect to the pMD-18 cloning vector, DNA sequencing results showed that the construction is successfully; we select pET28a expression vector, transformed into E. coli ,induced to nickel chelate affinity chromatography purified fusion protein up to 90%, and the expression of recombinant protein IGF-1α, was highly purified protein, in this study fwe synthesized ull 153aa of IGF-1α, maybe retain the active part of IGF-1α.In this study, multiplex PCR, cloned by the IGF-1αgene, and successfully constructed pET28a-IGF-1αplasmid, to achieve the recombinant protein in Escherichia coli. the SDS-PAGE electrophoresis analysis showed that the exogenous protein is IGF-1αgene expression products, and by nickel chelate affinity chromatography, the highly pure protein with activity.This study used a prokaryotic expression system of E. coli. E. coli is the earliest and most widely used in gene expression technology compared with other systems, has the characerastic of efficient expression and anti-pollution ability, promotes protein expression, downstream protein purification and so on. pET28a (+) prokaryotic expression vector containing the T7 RNA polymerase specificity can be recognized by strong T7 promoter. Foreign genes placed under the control of T7 promoter by IPTG induced expression of foreign proteins efficiently. The experiment using metal chelate chromatography, purified recombinant protein IGF-1α, the chromatographic method has the following advantages, first of all ,it has higher protein binding capacity, and can be regenerated hundreds of times, but does not affect its chromatographic properties, the third is cheap. Fourth, as most of the protein is missing metal affinity, metal chelate chromatography can recover a large number of recombinant proteins, in view of the above advantages, metal chelate chromatography proved to be technologies for better purification compared with other chromatographic technical means.The purity of the recombinant protein is more than 90%, and can meet the requirements of downstream experiments to study the IGF-1 in the development of endometrial adenocarcinoma experimental basis in the role.3. the role of recombinant protein IGF-1αin proliferation of endometrial cancer cell Ishikawa cells .Further in vitro studies used recombinant protein IGF-1 on endometrial cancer cell line Ishikawa cell cycle, we observed by flow cytometry, IGF-1 by affecting the Ishikawa endometrial cancer cell lines in cell cycle and promote cells from the G0/G1→S advance , to achieve the proliferation role and the role was found to be dose-dependent. When IGF-1 concentration is 10ng/ml, 25ng/ml, 50ng/ml, after cultured 24h, the Gl phase cells decreased significantly, S phase cells was significantly higher. the cell proliferation index (PI) and S-phase fraction (SPF) were significantly increased , promoting the cell proliferation and accelerating the cell cycle. and the same concentration of recombinant protein IGF-1αeffect on cancer cells after different period, the cell proliferation index(PI ) and S-phase fraction (SPF) were also significantly increased.Conclusion, IGF-1 played an important role in the development of endometrial cancer , IGF-1 in the carcinogenesis mechanism may be as a mitrogen and related to other oncogenes and tumor suppressor genes and signal pathways involving the G1 to S cell transformation, promeoting the cell proliferation. the IGF family promote cell proliferation in the process of endometrial cancer, theses findings help increase the understanding of tumor etiology and pathogenesis, and possibly for cancer prevention, reversal and new forms of treatment.