Upregulation Of HOXA10 Protein Predicts Poor Prognosis In Patients With Colorectal Cancer

Colorectal cancer(CRC)is one of the most common and deadly tumor types in both men and women in the world.Decades ago,colorectal cancer was also a malignant tumor with a low incidence.However,at present,it has become the highest incidence of gastrointestinal cancer,and accounts for about 10% of cancer-related deaths in Western countries.The "rising" of colorectal cancer incidence in developed countries can be attributed to an aging population,healthless modern diet,and increased risk factors such as smoking,low exercise levels,and obesity.Despite the widespread adoption of effective preventive screening measures and numerous major advances in comprehensive treatment options in recent years,colorectal cancer remains one of the most serious malignancies that threaten human health.One of the important causes of this situation is drug resistance.Despite advances in cytotoxicity and targeted therapies,the current long-term management of resistance to treatment remains one of the biggest challenges for incurable metastatic cancer.Because drug resistance allows tumor cells to accumulate means of evading various treatments and eventually lead to the death of the host.The homeobox(HOX)gene is an important regulator of embryonic morphogenesis and differentiation.The HOX gene family plays a key role in development,differentiation,apoptosis,angiogenesis,locomotion,and receptor signaling.Because of this,changes in the expression of these genes may have important implications for tumorigenesis and tumor progression as well as cancer diagnosis and treatment.Many studies have shown that different homeobox genes may be differentially expressed in some organs as tumor suppressor or tumor promoter.In terms of its oncogene properties,homeobox genes are usually expressed during the embryonic stage,reactivated in the tumor,and down-regulated in normal differentiated adult tissues.In contrast,some homeobox genes are expressed in normal differentiated adult tissues but are down-regulated in tumors.Interestingly,homeobox genes may have both tumor-promoting and tumor-suppressing properties,depending on the particular organ or cell lineage of the expression.Some long or short non-coding RNAs are also involved in regulating the transcription or expression of homeobox genes.Therefore,it is important to understand the abnormal expression patterns of homeobox genes in specific cancer types or cell lineages and their carcinogenic and invasive mechanisms for certain types of cancer.Many studies have shown that HOX gene expression contributes to the development of CRC.The HOXA10 gene,a member of the HOX family,is also an regulator of embryonic morphogenesis and differentiation,but it is not evident in several types of cancer.It has been reported that the deregulation of HOXA10 is related to the progression of endometrial cancer,but HOXA10 can also induce the expression of p53 in breast cancer cells and reduce its invasiveness.At the same time,HOXA10 is also a transcription factor involved in the proliferation of hematopoietic stem cells and progenitor cells.Its overexpression is associated with the development of cancer and the poor prognosis of patients with acute myeloid leukemia and solid cancer.It is still unknown whether the characteristics of this oncogene and tumor suppressor gene are actually present in colorectal cancer.It is not clear what role HOXA10 plays in the development of colorectal cancer.Therefore,studying the expression status and function mechanism of HOXA10 in colorectal cancer may have extremely positive significance in clinical practice.This study attempts to clarify the clinical significance of HOXA10 protein expression in colorectal cancer and explain its relationship with prognosis and prognosis of colorectal cancer.Part ? The expression and its Clinical characteristics of protein HOXA10 in colorectal cancerObjective:1.Description HOXA10 expression status of colorectal cancer,and to investigate its significance in the occurrence of colorectal cancer.2.Observing the relationship between the expression of HOXA10 and PTEN in colorectal cancer and explaining the predictive value of HOXA10 in the prognosis of colorectal cancer.Methods:1.Independent studies on the expression of HOXA10 protein in colorectal cancer from the Oncomine database was retrieved.2.Follow-up CRC patients from February 2005 to February 2010,who underwent radical excision followed by 5-fluorouracil(5-FU)-based adjuvant chemotherapy during February 2005 to February 2010 in Xuzhou Central Hospital.The endpoint of follow-up was disease progression.3.Immunohistochemistry was performed on pairs of cancerous and normal tissues to detect the expression of HOXA10 and phosphatase and tensin homology(PTEN).The correlation between HOXA10 expression and 5-year DFS in colorectal cancer was analyzed.Result:1.Seven independent studies from the Oncomine database showed that HOXA10 is highly expressed in CRC tissue compared to the normal control group.2.A total of 85 patients with CRC,who underwent radical excision followed by 5-fluorouracil(5-FU)-based adjuvant chemotherapy during February 2005 to February 2010 in Xuzhou Central Hospital were enrolled.The median follow-up time was 97 months(6 to 120 months),during which 70 patients relapsed within 5 years after surgery.3.Immunohistochemistry showed that 58 cases(68.2%)showed positive expression of HOXA10 protein in tumor tissues and negative expression in paired adjacent normal tissues.And the up-regulation of HOXA10 protein was synchronized with the down-regulation of PTEN.Although not related to clinical pathological features,there was a significant correlation between up-regulation of HOXA10 and reduction of 5-year disease-free survival(DFS).The Cox proportional hazards model further revealed that,in addition to lymph node metastasis,HOXA10 expression is an independent factor in DFS prediction in CRC patients.Conclusion:1.Compared with normal tissues,HOXA10 protein is overexpressed in most colorectal cancer.2.The up-regulation of HOXA10 protein was synchronized with the down-regulation of PTEN,and its up-regulation was associated with a reduction of 5-year disease-free survival(DFS).3.HOXA10 may be a potential biomarker in colon cancer,and its overexpression indicates a poor prognosis.4.HOXA10 may affect the prognosis of colorectal cancer by down-regulating PTEN levels.Part ? HOXA10 affects proliferation,apoptosis and chemosensitivity of colorectal cancer in vivo and in vitroObjective:1.To study the effect of HOXA10 knockdown on the biological behavior of colorectal cancer Lo Vo and HT29 cell lines.2.To study the effect of HOXA10 knockdown on the 5-FU chemosensitivity of colorectal cancer Lo Vo and HT29 cell lines.Methods:1.Lentivirus-mediated RNA interference was used to knock down HOXA10 expression in Lo Vo and HT-29 cell lines,then cells' proliferation,apoptosis and tumor growth in vivo were detected.2.Cells in each group were intervened with a gradient of 5-FU,and cell proliferation,apoptosis,and colony formation were detected.3.The si RNA-HOXA10 transfected cells was planted on the right lower limb of a nude mouse and the growth of the colorectal cancer implanted tumor was compared with whether 5-FU was used or not.Result:1.HOXA10 knockdown reduces HOXA10 protein expression.2.HOXA10 knockout can inhibit proliferation,induce apoptosis and increase the cytotoxicity of 5-FU in vitro.3.HOXA10 knockout enhances 5-FU cytotoxicity in nude mice.4.Conclusion:1.Lentiviral plasmid HOXA10 si RNA transfected Lo Vo and HT29 cell lines can significantly interfere with the expression of HOXA10.2.HOXA10 knockdown reduced the proliferation of colorectal cancer cell lines Lo Vo and HT29,and increased their apoptosis.3.HOXA10 knockdown significantly increase sensitivity to 5-FU chemotherapy in vitro and in vivo.Part ? MiR-135 a affects 5-Fu Chemosensitivity by Regulating HOXA10Objective:1.Find the upstream regulatory factor miRNA of HOXA10.2.Validate the above miRNA function and clarify its mechanism of action.Method:1.Bioinformatics predicts miRNA binding to HOXA10.2.Luciferase assay validates HOXA10's targets for predicting miRNAs.3.Synthesize the above-identified miRNAs and intervene in Lovo and HT29 cells,and observe their proliferation.4.5-FU intervenes in the above cells to detect their IC50.5.Over-express HOXA10 cells and observe their proliferation.Result:1.Using mi Rcode and Target Scan website to analyze the upstream regulation of HOXA10 miRNA,preliminary screening mi R-135 a and miR-135 b complementary binding with HOXA10 non-coding region.2.Luciferase experiments confirmed that HOXA10 is the target of mi R-135 a and mi R-135 b.3.Realtime PCR confirmed that the expression of mi R-135 a was significantly down-regulated in colon cancer Lo Vo and HT29 cells compared with human colon epithelial cells HCo Epi C.4.Lo Vo and HT29 cells transfected with the mi R-135 a plasmid were inhibited from proliferation and the IC50 of 5-Fu was reduced.5.MiR-135 a mimic can induce apoptosis in Lo Vo and HT29 cells,and overexpression of HOXA10 can reverse the effect of mi R-135 a to a certain extent.Conclusion:1.MiR-135 a is an upstream regulator of HOXA10.2.Over-expression of mi R-135 a can inhibit the proliferation of Lo Vo and HT29 cells,induce their apoptosis,and increase their sensitivity to 5-Fu.3.MiR-135 a may act by inhibiting HOXA10 overexpression.