| Background:Non-small-cell lung cancer (NSCLC) is the leading malignant tumor worldwide as far as the harm to human health is concerned. The mechanism of NSCLC is intensive involved with aberrant gene promoter methylation (abbreviate to methylation). PR domain zinc finger protein (PRDM) is a tumor suppressor gene family, which possesses histone protein methyltransferase activity and can suppress target gene transcription, and involved in regulation of several life proceedings include differentiation and organ development. PRDM family catch more and more attention in recent years, studies have demonstrated that aberrant methylation of PRDM gene extensively participate in the mechanism of tumors, whereas is rare reported in lung cancer study area.Objective:To investigate whether aberrant methylation of PRDM family members is involved in the mechanism of NSCLC; and to discover the methylation profile of PRDM family in NSCLC.Methods:1Clinical samples study Seventy five NSCLC patients including52lung squamous cell carcinoma patients and23lung adenocarcinoma patients were enrolled for research. Primary tumor tissues (abbreviate to tumor tissue), adjacent nontumorous normal tissues(abbreviate to adjacent tissue) and distant lung tissues were collected from each of the subjects, then mRNA expression of PRDM genes was detected by reverse transcription polymerase chain reaction (RT-PCR) in order to discover the family members with absent or decreased mRNA expression in tumor tissues (for ease of description, we call them temporarily lung cancer associated PRDM, and abbreviate as LCA-PRDM). Sequently, methylation status of the LCA-PRDM were analyzed by methylation-specific PCR (MSP) for all of the tumor tissues, adjacent tissues and distant lung tissues, correspondingly, Western-blot and immunohisto- chemistry were adopted to analyze the protein expression. Finally, the influence of LCA-PRDM methylation on mRNA and protein expression as well as the correlation of methylation with clinicopathological characteristics of patients was analyzed.2NSCLC cell lines study Human lung squamous cell carcinoma HTB-182cells, human lung adenocarcinoma A549cells and immortalized human bronchial epithelia HBE cells were cultured in vitro. Each of the cell line was treated by5-aza-2’-deoxycitydine with graded concentration (0μmol/L as control,1μmol/L,5μmol/L and10μmol/L), and the cell growth curve were detected based on MTT assay. Cell were harvested and the methylation status of PRDM2, PRDM5and PRDM16were analyzed by MSP, correspondingly, the mRNA and protein of PRDM2, PRDM5, PRDM16were analyzed by RT-PCR and Western-blot respectively.3Tumor xenograft study Human lung squamous cell carcinoma SK-MES-1cells were cultured in vitro and the methylation status of PRDM5was analyzed by MSP. Sequence, cells were treated with5-aza-2’-deoxycitydine in different concentration for demethylation and the cell growth curve were detected based on MTT assay. Twenty two male BALB/c nude mice were divided into control group and5-aza-2’-deoxycitydine treatment group randomly with11mice in each group, and then subcutaneously inoculated with SK-MES-1cells. When the short diameter of the tumor xenograft reached0.5cm, the mice in5-aza-2’-deoxycitydine treatment group were administered5-aza-2’-deoxycitydine three times per week by intraperitoneal injection up to four weeks later, and the mice in control group were treated with PBS three times per week by intraperitoneal injection, and the tumor xenografts were measured every other two days. When the mice were sacrificed, the tumors were dissected for weigh, the methylation status, and the mRNA and protein expression of PRDM5for tumor xenografts were analyzed by MSP, RT-PCR and Western-blot respectively.Results:1Clinical samples study (1)For lung squamous cell carcinoma and lung adenocarcinoma patients, the mRNA of PRDM1, PRDM2, PRDM5, PRDM6, PRDM10, PRDM14, PRDM15, PRDM16were detected from the tumor tissues, adjacent tissues and distant lung tissues of both. The expression of PRDM2mRNA, PRDM5mRNA and PRDM16mRNA were decreased/absent in tumor tissues compared with in distant lung tissues.(2) In lung squamous cell carcinoma patients, the frequency of PRDM2methylation for tumor tissues, adjacent tissues and distant lung tissues was67.3%,50.0%and17.3%, the frequency of PRDM5methylation for tumor tissues, adjacent tissues and distant lung tissues was73.1%,44.2%and21.1%, and the frequency of PRDM16methylation for tumor tissues, adjacent tissues and distant lung tissues was80.8%,40.4%and21.2%. For lung adenocarcinoma patients, the frequency of PRDM2methylation for tumor tissues, adjacent tissues and distal lung tissues was78.3%,34.8%and21.7%, the frequency of PRDM5methylation for tumor tissues, adjacent tissues and distant lung tissues was82.6%,47.8%and17.4%, and the frequency of PRDM16methylation for tumor tissues, adjacent tissues and distant lung tissues was82.6%,52.2%and30.4%. The methylation frequencies of PRDM2, PRDM5and PRDM16for tumor tissues were significantly higher than for distal lung tissues, and the frequencies for adjacent tissues were between tumor tissues and distant lung tissues.(3) For both the lung squamous cell carcinoma patients and lung adenocarcinoma patients, the protein expression of PRDM2, PRDM5and PRDM16in adjacent tissues was higher than in tumor tissues and the expression in distant lung tissues were higher than in adjacent tissues.(4) The mRNA and protein of PRDM2, PRDM5and PRDM16had not been detected from tissues with totally-methylated gene status. For lung squamous cell carcinoma and lung adenocarcinoma, the expression of mRNA and protein in tumor tissues, adjacent tissue and distant lung tissue about PRDM2, PRDM5and PRDM16were compared between partly-methylated and non-methylated gene statuses, the results demonstrated that partly-methylated gene status presented lower expression of mRNA and protein than non-methylated gene status.(5) As regard the correlation analysis of PRDM2, PRDM5, PRDM16methylation with clinicopathological characteristics of patients, the result demonstrated that in lung squamous cell carcinoma patients, methylation of PRDM2and PRDM16were correlated with smoke status (smokers with higher methylation frequency) and methylation of PRDM5was correlated with tumor differentiation (poor differentiation tumor presented higher methylation frequency).2NSCLC cell lines study (1)5-aza-2’-deoxycitydine treatment had displayed growth depressive effect on all of the three cell lines including A549, HTB-182and HBE, whereas the effect was more intensive to A549and HTB-182than to HBE.(2) The PRDM2, PRDM5and PRDM16gene were all slightly methylated in control HBE cells;5-aza-2’-deoxycitydine treatment presented no obviously influence on the methylation status of the three PRDMs as well as expression levels of mRNA and protein.(3) For A549and HTB-182, the PRDM2, PRDM5and PRDM16genes were all highly methylated in control cells,5-aza-2’-deoxy-citydine treatment concentration-dependent reversed the high methylation status of the three PRDMs, and upregulated the expression levels of mRNA and protein, furthermore, HTB-182cell was more sensitive respond to5-aza-2’-deoxy-citydine treatment than A549cell.3Tumor xenograft study (1) The PRDM5gene were methylated in SK-MES-1.(2)5-aza-2’-deoxycitydine treatment had displayed growth depressive effect on SK-MES-1.(3) Tumor xenografts were formed in all of the22BALB/c nude mice in five to nine days after subcutaneously inoculated with SK-MES-1cells. The growth speed of tumor xenografts in5-aza-2’-deoxycitydine treated nude mice was significant slower than on control nude mice, and the final weight of tumor was significantly less in nude mice treated by5-aza-2’-deoxycitydine (0.86±0.42g) than in control nude mice(2.03±0.63g).(4) PRDM5methylation was detected in tumor xenografts from8of the11control nude mice, but only from3of the11nude mice treated by5-aza-2’-deoxycitydine.(5) The relative expression intensity of PRDM5mRNA and protein were0.52±0.10and0.52±0.18in tumor xenografts from5-aza-2’-deoxycitydine treated nude mice, whereas were0.16±0.11and0.15±0.10in tumor xenografts from control nude mice. The expression levels of PRDM5mRNA and protein in5-aza-2’-deoxycitydine treated nude mice were significant higher than in control nude mice. Conclusions:(1) Expression of PRDM2, PRDM5and PRDM16is absent or decreased in non-small-cell lung cancer tissue.(2) Gene methylation is the primary cause of absent or decreased PRDM2, PRDM5, PRDM16expression in non-small-cell lung cancer tissue.(3) Smoking is probably the important cause for PRDM2and PRDM5methylation in non-small-cell lung cancer tissue.(4) Demethylation drugs could suppress the growth of non-small-cell lung cancer, and the mechanism is probably involved with alteration the methylation status of genes including PRDM2, PRDM5and PRDM16. |
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