| Objective: Colorectal cancer is one of the common human malignancies.At present,the treatment of colorectal cancer is mainly based on the location,stage and cancer progression of the cancer.Early cancer mainly depends on surgery or supplemented with preoperative or postoperative chemoradiotherapy,endocrine therapy,etc.However,when the cancer enters the middle and late stages,especially after severe metastasis,it cannot be achieved by surgical treatment.Traditional treatment methods mainly use chemotherapeutics and radiation therapy to control the spread and deterioration of cancer.In recent years,targeted gene therapy has become a new hot spot in tumor treatment,and it has also provided many new directions for the treatment of colorectal cancer,which has improved the quality of life of patients with colorectal cancer in all aspects.In many years of research,it has been found that the most important metastatic site of colorectal cancer is the liver.Within six months of the diagnosis of primary colorectal cancer,about 25%-30% of patients have liver metastases.Liver metastasis of colorectal cancer is a very complicated biological process,and its mechanism is not very obvious.Liver metastasis is the main cause of colorectal cancer occurrence and poor prognosis.Micro RNA(mi RNA),as a class of endogenous small RNAs with a length of 20-24 nucleotides,has a variety of important regulatory functions in cells.Mi RNA mutations and ectopic expression are closely related to a variety of human cancers.Mi RNAs can inhibit the development of tumors or promote the development of tumors,and at the same time can inhibit the expression of a variety of tumor-related genes.A large number of studies have shown that mi RNAs play an important role in tumorigenesis and development,including malignant biological behaviors that affect tumor cell proliferation,apoptosis,and metastasis.Micro RNA(mi RNA),as a non-coding short-stranded RNA,regulates the expression of multiple target genes at the post-transcriptional level and involves a series of regulation of cell development processes.mi RNA has become a hot spot in life science research in recent years,and many studies have shown that it is closely related to the occurrence of malignant tumors.Micro RNA-196a-5p(mi R-196a-5p)has been reported to be involved in the metastasis of some cancers.Mi RNA-196 a has recently discovered biological target molecules that are closely related to the development of breast cancer and cervical cancer.However,the mechanism of mi R-196a-5p colorectal cancer liver metastasis has not been clearly reported.NF-κB protein is a transcription factor that binds to target genes to regulate its expression.Recent studies have reported that activation of the NF-κB pathway is associated with metastasis in different types of cancer.A study showed that STX2 forms a forward signal loop with NF-κB.In addition,it has been clarified that inactivation of the NF-κB signaling pathway inhibits the invasion,metastasis and EMT of colorectal cancer cells.In this study,we explored the effects of mi R-196a-5p and its potential target IκBα on colorectal cancer cell(CRC)migration,invasion,and epithelial-mesenchymal transition(EMT).Mi R-196b-5p is involved in expression in a variety of cancers.In colorectal cancer,mi R-196b-5p has also been shown to function as an oncogene.ING(positive growth inhibitory gene)includes 5 family members,of which ING5 is located at 2q37.3 of the human chromosome,which contains 8 exons and 7 introns.The ING5 protein from amino to carboxyl terminus contains Leucine zipper-like,newly conserved regions,nuclear localization sequences,and plant homology domains.Its different regions perform different functions.Many studies have found that the expression of ING protein is down-regulated or deleted during the development of malignant tumors,and the expression of ING in cancer cells is involved in the regulation of apoptosis.Many in vitro and in vivo experiments have also found that ING protein regulates apoptosis,but its competitive inhibitory effect with the downstream gene ING5 has not been clearly reported.The purpose of this study was to investigate whether mi R-196b-5p competes with ING5 to inhibit malignant biological behavior in colorectal cancer cells.In summary,mi R-196a-5p plays a key role in the migration,invasion,and epithelial-mesenchymal transition(EMT)of colorectal cancer cells through targeted binding to IκBα;It inhibits the proliferation,migration and invasion of colorectal cancer cells,and promotes malignant biological behaviors such as colorectal cancer cell apoptosis;Mi R-196b-5p promotes the proliferation,migration and invasion of colorectal cancer cells,and inhibits apoptosis,while ING5 inhibits the proliferation,migration and invasion of colorectal cancer cells,and promotes apoptosis.In addition,mi R-196b-5p affects the biological behavior of colorectal cancer cells through targeted binding with ING5,which provides a new target for the treatment of colorectal cancer.This study provides new ideas for targeted treatment of colorectal cancer.Methods:1.Culture of colorectal cancer cells SW480,HCT116,lovo and human embryonic kidney cells HEK293 T.2.Double luciferase gene report3.Real-time PCR was used to detect the expression of mi R-196a-5p,mi R-196b-5p,and ING5 in colorectal cancer tissues and colorectal cancer cells.4.CCK8 method to detect cell proliferation.5.The distribution of mi R-196a-5p was detected by immunohistochemistry.6.Immunofluorescence assay to detect protein expression and distribution.7.Transwell method was used to detect cell migration and invasion.8.Cell scratch test method to detect cell migration.9.Flow cytometry to detect apoptosis and cell cycle10.Western blot was used to detect protein expression.11.Nude mouse xenograft model to observe the tumor growth and nude mice survival time.Results:1.Mi R-196a-5p is highly expressed in colorectal cancer cell lines SW480,SW620,HCT116,Caco-2 and Lo Vo cell lines.2.Mi R-196a-5p promotes the proliferation of colorectal cancer cells.3.Mi R-196a-5p promotes migration and invasion of colorectal cancer cells.4.Mi R-196a-5p promotes liver metastasis in colorectal cancer.5.Mi R-196a-5p promotes epithelial-mesenchymal transition(EMT)in colorectal cancer cells.6.Inhibition of NF-κB signaling pathway can inhibit epithelial-mesenchymal transition(EMT)in colorectal cancer cell lines.7.Mi R-196a-5p promotes epithelial-mesenchymal transition(EMT)and colorectal cancer cell line migration and invasion through targeted binding to IκBα.8.Mi R-196b-5p is highly expressed in colorectal cancer cells(SW480 and HCT116).9.Mi R-196b-5p promotes the proliferation,migration and invasion of colorectal cancer cells and inhibits apoptosis.10.ING5 inhibits the proliferation,migration and invasion of colorectal cancer cells,and promotes the apoptosis of colorectal cancer cells.11.ING5 is a target gene of mi R-196b-5p,mi R-196b-5p can inhibit the expression of ING5.12.ING5 knockdown simultaneously inhibited the expression of mi R-196b-5p in colorectal cancer cells,inhibited the proliferation,migration and invasion of colorectal cancer cells and promoted apoptosis.13.mi R-196b-5p plays a role in promoting tumor growth in vivo xenograft models.Conclusions:1.Mi R-196a-5p is highly expressed in colorectal cancer cells SW480,SW620,HCT116,Caco-2 and Lo Vo.2.Mi R-196a-5p promotes the proliferation,migration,invasion,EMT and liver metastasis of rectal cancer cells.3.IκBα is the target gene of mi R-196a-5p.4.Mi R-196a-5p promotes the migration,invasion,and EMT of colorectal cancer cells by targeting 3’UTR of IκBα m RNA.5.Mi R-196b-5p is highly expressed in colorectal cancer cells(SW480 and HCT116),while ING5 is low.6.Mi R-196b-5p can promote the proliferation,migration and invasion of colorectal cancer cells,and inhibit apoptosis.7.ING5 can inhibit the proliferation,migration and invasion of colorectal cancer cells and promote apoptosis.8.ING5 is the target gene of mi R-196b-5p.Mi R-196b-5p promotes the proliferation,migration and invasion of colorectal cancer cells by targeting the 3’UTR that binds to the ING5 m RNA,and inhibits apoptosis.9.Double knockdown of mi R-196b-5p and ING5 inhibited the proliferation,migration,and invasion of colorectal cancer cells(SW480 and HCT116),and significantly inhibited the growth of tumors in vivo. |
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