| Background:Colorectal cancer is one of the most common gastrointestinal cancers.It is characterized by high morbidity,high metastasis rate,and high mortality rate.Although the advancement of modern medicine and the continuous innovation of diagnostic techniques have greatly improved the prognosis of patients with colorectal cancer,the recurrence rate and mortality of colorectal cancer are still high,resulting from recurrence and metastasis of colorectal cancer.Therefore,screening tumor markers at the molecular level is of great significance for the prevention,diagnosis,treatment,and prognosis of colorectal cancer.Colorectal cancer is affected by many factors at the cellular and molecular level,and therefore it is often associated with a series of pathological processes,including solid tumors,invasion,and metastasis.In recent years,studies have found that hypoxia is one of the factors leading to the occurrence and development of malignant tumors.Hypoxia-inducible factor-la(HIF-la)is the only transcription factor discovered to date that has biological activity in a specific hypoxic state.Uncontrolled growth of tumor cells leads to abnormal hyperplasia of tumor tissue,which will cause severe hypoxia in the local tissues.At the same time,the balance between energy supply and energy consumption is destroyed.The above process induces the expression of HIF-1α,followed by transcription of downstream target genes,so blood,oxygen and energy increased and tumor cells are adaptable to the hypoxic microenvironment.Therefore,HIF-la plays an important role in the development and progression of malignacies.It is involved in the regulation of tumor development at various stages through a series of complex signal transduction pathways.It has been confirmed that HIF-la is overexpressed in many malignant tumors of various origins.The correlation between HIF-la and the malignant progression of tumors has also become an important and hot topic in the medical research field in recent years.AMPK-related protein kinase 5(AMPK5),also known as NUAK1,is an important signaling molecule downstream of Akt.Studies have shown that when activated ARK5 promotes phosphorylation of the tumor suppressor ATM,which in turn induces the phosphorylation of its downstream gene p53,which loses its biological activity and ultimately leads to abnormal human gene expression.At present,studies have confirmed that abnormal expression of ARK5 has been detected in many humanmalignant tumors.In recent years,the abnormal expression of HIF-la and ARK5 in tumor tissues has gradually become of great importance to medical workers.Both of them have abnormal expression in a variety of tumor tissues and are closely related to the growth,invasion and metastasis of tumors.They have certain value in the diagnosis and prognosis of some tumors.However,the value of HIF-la and ARK5 expression in colorectal cancer and related molecular mechanisms are not yet fully cleared and needs further research.Objective:This study was designed to detect the expression and significance of HIF-la and ARK5 in colorectal cancer tissues,analyze the effect of HIF-1α gene silencing on the proliferation,apoptosis,invasion and migration of ARC5 cells,and the expression of ARK5.The effect of HIF-1α on the growth of transplanted tumors in nude mice was designed to understand the mechanism by which HIF-1α promotes the development and progression of colorectal cancer through regulating ARK5,and to provide a reliable basis for the clinical diagnosis and targeted treatment of colorectal cancer.Method:1、Sixty patients with colorectal cancer who underwent surgery in the Department of General Surgery of People’s Hospital of Wuhan University from September 2014 to September 2015 were collected.The expressions of HIF-la and ARK5 mRNA and protein in these tissues were detected by real-time fluorescence quantitative PCR,Western blot,and immunohistochemistry,and the expression of HIF-la and ARK5 were analyzed in patients with colorectal cancer,including age,gender,and TNM staging.The relationship between the degree of pathological differentiation,lymph node metastasis,and liver metastasis was analyzed.The correlation between the expression of HIF-la and ARK5 in colorectal cancer was analyzed.2.Human colon cancer SW480 cells were cultured with normoxia and 100 pmol/L CoC12(hypoxia),and the expression of HIF-la and ARK5 mRNA in the cells was detected.SW480 was randomly divided into 3 groups.control group,cells were treated with 100 μmol/L CoCl2 to prepare hypoxia models;si-control group:cells were transfected with scramble SiRNA sequence.;siHIF-1α group,cells were transfected with siHIF-la.After that,100 pmol/L of CoC12 was added to the cells to detect the expression of HIF-1α and ARK5 mRNA in the above cells.CCK-8 assay was used to detect the proliferation activity of the cells in each group.Flow cytometry was used to detect cell apoptosis.Transwell assay was used to detect cell invasion ability.Cell scratch assay was used to detect cell migration ability.3.Human colon cancer cell line SW480 was used to inoculate the right scapular of the nude mice and a nude mouse model of colon cancer SW480 cell xenografts was established to observe the tumorigenesis.Nude mice were randomly divided into 3 groups,control group:cells were routinely cultured,without any treatment,then inoculated subcutaneously in nude mice;si-control group:cells were transfected with si-control plasmid,and then inoculated subcutaneously in nude mice;siHIF-Group la:Cells were transfected with siHIF-1α and subsequently inoculated subcutaneously in nude mice.The length(L)and width(W)of the transplanted tumors in each group were measured with a vernier caliper every 5 days.The transplanted tumor volume was calculated.On the 20th day,the nude mice were sacrificed and the complete transplanted tumor tissues were collected and weighed.Western blot was used to detect the expression of ARK5 protein in the transplanted tumor tissue.Results:1.Compared with adjacent tissues,the expression of ARK5 and HIF-1α mRNA and protein in colorectal cancer tissues were significantly increased(P<0.05).The size and shape of normal colorectal tissue cells are basically the same.There is only one nucleus in the cell,and there is a certain ratio between the nucleus and the cytoplasm.Compared with normal colorectal cancer cells,colorectal cancer cells increase in size and shape,and some of them increase in size into tumor giant cells.Nuclei increase in size and shape,showing dual-nuclear,giant nucleus,multinuclear,and heteromorphism.Nuclear,etc.,the ratio between the cytoplasm and the nucleus is also different than normal colorectal cells.The HIF-la and ARK5 proteins are mainly expressed in the cytoplasm and nucleus.In 60 colon cancer tissues,the positive expression rates of HIF-1α and ARK5 proteins were 76.7%(46/60)and 71.7%(43/60),respectively.Univariate analysis showed that HIF-1α and ARK5 protein expression and tumor stage(73.9%and 74.4%,respectively),pathological differentiation(78.3%and 81.4%,respectively),lymph node metastasis(82.6%and 79.1%,respectively)and liver cancer metastasis(32.6%and 30.2%,respectively).Linear correlation analysis evaluated the relationship between HIF-1α and ARK5 expression and showed a correlation between the two(r = 0.776,P = 0.001).2.Compared with the normoxic group,the expression of ARK5 and HIF-1αmRNA in the hypoxic group was significantly higher(P<0.05).Compared with the si-control group,the expression of ARK5 and HIF-la mRNA in the siHIF-la group was significantly decreased(P<0.05).The above results indicate that the HIF-la gene silencing colorectal cancer cell line was successfully constructed and can be used for subsequent experiments.At the same time,the expression of ARK5mRNA decreased after HIF-la knockdown,suggesting that ARK5 may be a downstream factor of HIF-1α forits expression is regulated by HIF-la.There was no significant difference in proliferation between the control group and the si-control group(P>0.05).Compared with the si-control group,the proliferation of the siHIF-1α group was significantly decreased(P<0.05).There was no significant difference in apoptosis rate between control group and si-control group(P>0.05).Compared with si-control group,the apoptosis rate of siHIF-1α group was significantly higher(P<0.05).There was no significant difference in the number of invading cells between the control group and the si-control group(P>0.05).Compared with the si-control group,the number of invading cells in the siHIF-la group was significantly decreased(P<0.05).There was no significant difference in the cell migration rate between the control group and the si-control group(P>0.05).Compared with the si-control group,the cell migration rate of the siHIF-la group was significantly lower(P<0.05).3、When human colon cancer SW480 cells were inoculated into the right scapular of the nude mouse on the 8th day,the transplanted tumor with the size of soybean was visible in this site.In this experiment,all the 30 nude mice were tumorigenic.After nude mice became tumorigenic,each group of nude mice was normal in eating and drinking water,and there was no abnormality in urine and stool.The nude mice in each group had good mental status,normal activities,and rapid response.In addition,the color of the nude mice in each group was normal and the xenografts did not rupture.Compared with the control group,the tumor volume in the si-control group had no significant difference at any time point(P>0.05).Compared with the si-control group,the transplant tumor volume of the siHIF-la group significantly decreased at various time points(P<0.05)..No statistical difference was found in the weight of tumors between the control and si-control group(P>0.05),while compared with the si-control group,the weight of tumors in siHIF-la group was significantly reduced(P<0.05).No significant difference was found in ARK5 expression of the transplanted tumor tissue between the control and si-control group(P>0.05),while compared with the si-controlgroup,the ARK5 protein in siHIF-la group was significantly reduced(P<0.05).Conclusion:1.This study found that the expression levels of HIF-la and ARK5 were abnormally up-regulated in colorectal cancer tissues and correlated with TNM stage,pathological differentiation,lymph node metastasis,and hepatic metastasis,and there was a positive correlation between HIF-1α and ARK5.2.Hypoxic environment can increase the expression of HIF-1αmRNA and ARK5 mRNA in human colorectal cancer SW480 cells.HIF-la promotes the proliferation,invasion and migration and inhibited apoptosis of SW480 cells through upregulating ARK5。3、Silencing HIF-1α effectively inhibits the growth of transplanted colon cancer cells in nude mice.The mechanism may be related to the down-regulation of ARK5 expression after HIF-la inhibition. |
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