The Relationship Between HMLH1Gene Methylation And Adriamycin Resistance Of Osteosarcoma And Its Clinical Significance

Chapter I Research on the role of hMLHl gene promoter methylation in adriamycin resistance of osteosarcoma cellObjective To investigate whether hMLH1gene promoter methylation correlate with adriamycin resistance of osteosarcoma MG63/ADR cell; To investigate whether demethylation drug5-Aza-dc correlate with hMLH1gene promoter methylation、gene expression and adriamycin resistance of osteosarcoma MG63/ADR cell. Methods Methylation specific PCR was used to test hMLHl methylation status of adriamycin resistance of osteosarcoma MG63/ADR cell and its parental cell MG63. MTT method and flow cytometry was used to test adriamycin resistance index of MG63/ADR cell、the cytotoxicity of5-Aza-dc on MG63/ADR、IC50and adriamycin resistance reversal index of MG63/ADR cell48h after MG63/ADR cell dealed with different concentration of5-Aza-dc. Methylation specific PCR, real-time fluorescent quantitative PCR and Western blot method was used to test the influence of hMLHl methylation status、mRNA and protein expression caused by5-Aza-dc. Results Methylation specific PCR showed that hMLH1of MG63cell was unmethylated, hMLHl of MG63/ADR was partly methylated.The IC50of adriamycin in MG63cells and MG63/ADR cells were2.28±0.12μM、11.18±0.11μM, MG63/ADR cells’resistance index was4.90. MTT method and flow cytometry showed that after48h’s intervened by5-Aza-dc, non-toxic dose and low-toxic dose of MG63/ADR cell were50μM and100μM. non-toxic dose and low-toxic dose reduced IC50of adriamycin to6.18±0.13μM、4.42±0.12μM; adriamycin resistance reversal index werel.81、2.53. Methylation specific PCR, real-time fluorescent quantitative PCR and Western blot method demonstrated that hMLH1methylation was well removed by non-toxic and low-toxic dose5-Aza-dc, the expression of hMLH1mRNA and its protein were increased. Conclusin MG63was hMLH1unmethylated cell, MG63/ADR was hMLH1partly methylated cell. hMLHl promoter methylation may be involved in the adriamycin resistance of osteosarcoma. Non-toxic and low-toxic dose5-Aza-dc could well remove the hMLHl methylation、 increased expression of hMLHl and had no inhibition of proliferation of cells, for these they can partly reverse the adriamycin resistance of osteosarcoma.Figture13, Table8, Reference41. Chapter Ⅱ The relationship between hMLHl gene methylation status and its protein expression in osteosarcoma tissue and linical pathology and prognosisObjective To test the hMLHl gene methylation status in the osteosarcoma tissue which haven’t accepted any anti-tumor treatment, To discus the relationship between hMLHl gene methylation in osteosarcoma tissues and clinical pathology and prognosis of patients with osteosarcoma. Methods Phenol/chloroform method was used to extract FFPE osteosar-coma tissue DNA of50osteosarcoma patients. Nested methylation specific PCR was used to test hMLHl methylation status. Immunohistochemical method was used to detect hMLHl protein expression. The relationship between hMLHl methylation and patients’ clinicopathological features was analysed by X2test. Univariate Kaplan-Meier and Log-rank test were used for hMLHl methylation and overall survival analysis and disease-free survival analysis. The relationship between hMLHl methylation and its protein expression analysed by Spearman rank correlation analysis. Results46osteosarcoma tissue samples was amplificated successfully by nested methylation specific PCR, in which20cases with the hMLHl gene methylation, accounting for44.48%. hMLHl gene methylation wasn’t associated with age、sex、pathological types、Enneking staging and grading, just correlated with5disease-free survival. Univariate Kaplan-Meier and Log-rank test showed that overall survival curves of c and hMLH1gene unmethylation patients were separated, survival time of hMLH1gene unmethylation patients were better than hMLH1gene unme-thylation patients patients, but there was no statistical significance. Conclusions hMLH1gene methylation is a common event in osteosar-coma, it is associated with5disease-free survival of osteosarcoma, but is not correlate with overall survival of osteosarcoma. hMLH1gene hyme-thylation is expected to becaome a prediction of recurrence and a bio-marker for osteosarcoma patients. HMLH1gene promoter methylation is an important mechanism of its function inactivation and negatively correlated with protein expression. Figture7, Table2, Reference14.