| Objective Many clustered protocadherin genes(PCDHs)within chromosome 5q31 are frequently down-regulated in colorectal cancer(CRC)due to the hypermethylation of this region,and some of them have been identified as tumor suppressors.Moreover,mutations in the Kras gene have also been shown to be associated with the development and prognosis of colorectal cancer.The aim of this study was to screen for down-regulated PCDH which is significative to occurrence of colorectal cancer,and investigate the expression of down-regulated PCDH,PCDHGA7,in colorectal cancer with different Kras genes and its relationship with clinicopathological factors and clinical prognosis..Methods(1)A variety of PCDHs,which were significantly down-regulated in colorectal cancer,were screened by analyzing the transcriptome sequencing(RNA-seq)data of the Cancer Genome Atlas(TCGA).(2)Determining a new member of ?-PCDH subfamily in the screened down-regulated PCDH,PCDHGA7,which is associated with decreased overall survival in patients with KRAS-wild-type CRCs by analyzing the genomic transcriptome(RNA-seq)data of TCGA.(3)We collected 20 pairs of colorectal cancer and corresponding adjacent cancerous tissues,and then extracted the RNA of the sample.The m RNA expression level of PCDHGA7 was detected by reverse transcription PCR(RT-PCR)and quantitative real-time PCR(q PCR).(4)20 pairs of frozen tissues were made into paraffin sections and immunohistochemical staining was performed.The staining results were photographed,and the expression of PCDHGA7 protein was evaluated according to the positive rate of antibody expression.(5)To further expand the sample size,230 cases of colorectal cancer FFPE specimens were collected and their DNA was extracted.The amplification of KRAS gene was performed by PCR.Then carry out nucleic acid electrophoresis band verification on the amplified samples to recover and purify the KRAS gene band.Finally,the purified sample was subjected to Sanger sequencing of KRAS gene mutation,and the samples of KRAS-WT were selected therefrom.(6)The selected KRAS-WT samples were made into tissue microarray(TMA),then immunohistochemical staining,and the protein expression of PCDHGA7 was quantitatively analyzed using software.Then the results were divided into PCDHGA7 high expression and low expression group based on the median expression of PCDHGA7.At last,the correlation between the two groups and clinical pathological features and prognosis was analyzed.Results(1)In the TCGA cohort,the expression of many PCDHs spanning the alpha,beta and gamma clusters was altered in colorectal cancers,with 19 of 53 showing significantly lower than normal tissues,but 3 of 53 showing significantly higher.Of note,the most significantly down-regulated genes(p-value < 0.01)belong to the beta and gamma families.(2)In the TCGA cohort,low expression of PCDHGA7 in KRAS-WT colorectal cancer patients was associated with the decrease in overall survival(P = 0.0225).(3)Compared with the corresponding adjacent tissues,the expression of PCDHGA7 in cancer tissues was significantly decreased(P < 0.0001).(4)By grouping individuals based on PCDHGA7 expression,we found that patients whose tumors had high PCDHGA7 expression had a significantly higher risk of tumor recurrence after surgery(p = 0.048)and they presented more frequently at advanced tumor stages(p = 0.030).(5)After overall survival analysis,patients with low PCDHGA7 expression in their tumors had significantly shorter overall survival than those with high PCDHGA7 expression(P = 0.0321).Conclusion(1)Multiple clustered PCDHs were down-regulated in CRC by TCGA dataset analysis.(2)PCDHGA7 is associated with overall survival in TCGA patients of CRC with wild-type KRAS.(3)PCDHGA7 expression is significantly down-regulated in CRC in the sample analysis of our hospital(4)Low PCDHGA7 expression is correlated with poor prognostic features in CRC with wild-type KRAS. |
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