| Endometrial cancer is one of the most commonly diagnosed malignancy of the female reproductive system, the incidence of which is rising in recent years, due to the early onset of symptoms, most patients can be diagnosed and treated at early period, the overall prognosis after surgery is comparatively good, but still some patients occurred early recurrence or tumor metastasis, which become the main reason for poor prognosis among these patients. Many endometrial cancer related molecular markers such as PTEN/ P53/ Ki67/ K-ras has been found, but there are still many limitations at its clinical application.Human kallikrein belong to the serine protease family, constituted of KLK1-15, KLK family members has been found involved in tumor proliferation, invasion, drug resistance and other aspects of tumor function. KLK4 is a frequently researched molecule of the KLK family, which is unique in that KLK4’s expression mainly located in the nucleus, while other KLK members located in the cytoplasm, it has been found KLK4 is highly expressed in prostate cancer, ovarian cancer, breast cancer, promote the proliferation of prostate cancer cells and invasion and metastasis of ovarian cancer, researches show that KLK4 can regulate the expression of cell cycle proteins, involved in the tumor cells epithelial to mesenchymal transition. KLK4 was reported as a hormone regulated gene and overexpressed in endometrial cancer, but the exact function and mechanism is unknown.Endometrial cancer is a typical hormone-dependent tumor, we consider KLK4 may also exist certain functions in endometrial cancer. In this research, we discussed the relationship between the expression of KLK4 and the clinicopathological parameters in endometrial cancer, explored its function and possible mechanism.Part1 Expression of KLK4 and its clinical significance in endometrial carcinoma Objective to investigate expression of human kallikrein4(KLK4) in endometrial cancer and its relationship with prognosis, providing new thoughts for improving the prognosis of endometrial cancer.Methods Immunohistochemical methods were used to detect the expression of KLK4 in 81 endometrial carcinoma, 30 normal endometrium and 30 atypical endometrium hyperplasia, staining scores were correlated with clinicopathological information and follow-up data to explore its relationship with prognosis.Results KLK4 is predominantly localized to the nucleus of glandular epithelium cells, the high expression rate of KLK4 in normal endometrium, atypical endometrial hyperplasia and endometrial cancer were 30%, 53%, 67%, the difference was statistically significant(P=0.002); high expression of KLK4 is positively correlated with histological grade(P=0.021) and deep muscular invasion(P=0.031), survival analysis showed that high KLK4 expression was significantly associated with decreased progression-free survival(P=0.032).Conclusion High expression of KLK4 in endometrial carcinoma is related to cancer cell differentiation and local invasion, it may be used as a marker to predict early recurrence of endometrial cancer.Part 2 Expression and regulation of KLK4 in endometrial cancer cell linesObjective To investigate the expression and regulation of KLK4 in endometrial cancer cell linesMethods Real-time PCR and western blot was used to detect KLK4 m RNA and protein expression in Ishikawa, HEC-1B and RL95-2 cells. Ishikawa cells was cultured in estrogen-stripped medium and were added to 10-10 mol / L, 10-8mol / L and 10-6mol / L of 17-β estrogen, KLK4 m RNA and protein was detected after 24 h, 48 h, and 72 h. Then ishikawa cells was treated with 10-8mol / L estrogen in combination with 10-9mol / L, 10-7mol / L and 10-5mol /L ICI182,780 to observe the effects of estrogen receptor antagonists on KLK4 expression.Results KLK4 is expressed in different endometrial cancer cells lines at different extent, estrogen can promote KLK4 expression in ishikawa cells, the conceration of 10-8mol / L for 48 h has the most obvious effect. ICI182,780 can block the effect of estrogen’s upregulation effect on KLK4, the concentration of 10-7mol / L has the most obvious effect of downregulation.Conclusion Estrogen can increase KLK4 expression in endometrial cancer cells, while estrogen receptor antagonists could block such effect, suggesting that estrogen regulate KLK4 expression of endometrial cancer cells through estrogen receptor.Part 3 The effect of KLK4’s over-expression on the function of endometrial cancer cells Objective To investigate possible roles of KLK4 in endometrial cancerMethods pcDNA3.1(+)-KLK4 plasmids were transfected into RL95-2 cells and continuously screened by G418 to establish cell lines can stably overexpress KLK4. MTT assay was applied to evaluate proliferate ability of RL95-2 cells, the migration and invasion ability in vitro was evaluated by scratch test and transwell invasion assay.Results KLK4 was successfully constructed into pc DNA3.1(+) and established cell lines can stably overexpress KLK4. MTT assys showed overexpression of KLK4 cannot promote RL95-2 cell’s proliferation, but can significantly promote its ability of migration and invasion in vitro.Conclusion Overexpression of KLK4 can promote RL95-2 cells’ s invasion and metastasis in vitro, but cannot promote its proliferation.Part 4 A preliminary study on the mechanism of RL95-2 cell’s invasion after overexpression of KLK4Objective To investigate the possible mechanisms of RL95-2 cell’s invasion after overexpression of KLK4Methods Real-time PCR, western blot and immunofluorescence was performed to detect e-cadherin m RNA and protein expression. E-cadherin over-expression plasmids were transiently transfected into KLK4 overexpressing cells to observe its invasive ability in vitro.Results In contrast to control group and blank group, e-cadherin expression level was significantly downregulated after overexpression of KLK4, transiently transfect pc DNA3.1(+)-e-cadherin into KLK4 overexpressing cells caused a decline of the cell’s invasion ability.Conclusion KLK4 maybe a inhibitor of e-cadherin and may promote RL95-2 cell’s invasion by inhibiting the expression of e-cadherin. |
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