Study Of Expression And Function Role Of Long Non-coding RNA PANK2 In Colorectal Cancer

Background and objective:Colorectal cancer(CRC)is a common malignant tumor of the digestive system.Its incidence rate ranks third in the world and mortality ranks fourth.In China,colorectal cancer has become the third leading cause of malignant tumor digestive system.Early colorectal cancer is concealed and it is easy to be misdiagnosed and missed.Once diagnosed,the main treatments are endoscopic resection,surgery,neoadjuvant chemotherapy,adjuvant chemotherapy,radiotherapy and targeted therapy.The purpose of tumor treatment is to kill tumor cells as much as possible,reduce the number of tumor cells and thus improve the overall survival(OS)and progression-free survival(PFS)to improve the prognosis of patients.However,the current status of colorectal cancer treatment is not optimistic and there are characteristics of high recurrence rate,high metastasis rate and high drug resistance rate.Finding molecular markers that is related to the prognosis of colorectal cancer,as well as diagnostic and therapeutic targets based on molecular markers for tumor discovery will have important clinical guiding value and basic theoretical significance.Although many studies have found that many factors play a role in the pathogenesis of colorectal cancer including protein-coding genes and non-coding genes,the occurrence and evolution of colorectal cancer is an intricate process of control and there are still many unknown.There are still many unknown factors that require further research.Long non-coding RNA(LncRNA)is a class of non coding RNA longer than 200 nucleotides.LncRNA is widely distributed in human genes.Although LncRNA does not have the potential to encode proteins,LncRNA can participate in the formation of complex gene expression regulatory networks and regulate various biological processes.Recent studies have shown that LncRNA can play an important role in stem cell growth,differentiation and apoptosis.In addition,LncRNA is involved in the regulation of multiple molecular pathways,causing changes in gene expression,ultimately regulating cell proliferation,apoptosis and cell migration.LncRNA plays an important role in biological processes such as cell cycle regulation,epigenetic regulation,transcriptional regulation and post-transcriptional regulation.It plays an important role,especially in the occurrence and development of malignant tumors.Current researchs suggest that LncRNA can play an important role in the development of colorectal cancer.The long-chain non-coding RNA-ENST00000464452 is one of splice variants of the long-chain non-coding RNA PANK2(pantothenate kinase 2),located at Chromosome chr 20:4151129-4153322(+),with 2194 bp in length and an exon.There are very few reports on LncRNA PANK2 and only relevant bioinformatics databases suggest that LncRNA PANK2 can be expressed in the large intestine,esophagus,gallbladder,lung,ovary and thyroid.LncRNA PANK2 can be abnormally expressed in colon adenocarcinoma,gastric cancer and lung adenocarcinoma and is closely related to DNA methylation level.It suggested that LncRNA PANK2 may play an important role in the development of tumors.Therefore,this study is the first comprehensive analysis of the expression and biological effects of LncRNA PANK2 in colorectal cancer and preliminary explored its molecular mechanism of tumor regulation.The study consists of three parts.Part I:the expression of LncRNA PANK2 in colorectal cancer different tissues and clinicopathological correlation analysis.Part II:the regulation of LncRNA PANK2 expression on the proliferation,migration,invasion and apotosis of colorectal cancer cell lines.Part III:LncRNA PANK2 high expression competitively binds to microRNA19a-3p to upregulate SOX4 expression and promotes CRC cell metastasis.Part I Abnormal expression of LncRNA PANK2 in colorectal cancer tissues and the correlation with clinicopathologiesObjective:To investigate the correlation between the expression change of the LncRNA PANK2 and the clinicopathological characteristics in cancer tissues,margin tissues and peripheral lymph nodes of colorectal cancer patients.Methods:1.Retrospective analysis of clinical datas of 39 patients with colorectal cancerundergoing surgical resection.Cancer tissues,marginal tissues and peripheral lymph nodes specimens of colorectal cancer groups were collected.2.Real-time quantitative PCR was used to detect the expression level of LncRNA PANK2 in cancer tissues,margin tissues and peripheral lymph nodes.3.The relationships between the expression levels of LncRNA PANK2 and clinicalpathological features of patients were analysed.Results:1.A total of 39 patients were enrolled,with an age range of 42-88 years,mean 65.90±10.34 years old,no lymph nodes metastasis in 24 cases and lymph nodes metastasis in 15 cases.There were no statistical differences in age,gender,tumor location,tumor volume,the depth of tumor invasion,degree of tumor differentiation between patients with lymph nodes metastasis and no lymph nodes metastasis.2.The expression of LncRNA PANK2 in different tissues was compared.The expression of LncRNA PANK2 in margin tissues(0.97±0.46),cancer tissues(1.25±0.53),lymph nodes(1.70±0.52).The expression of LncRNA PANK2 in cacers was higher than margin tissues(P<0.05).The expression of lymph nodes was significantly higher than that of cancer tissues(P<0.001)and margin tissues(P<0.001).3.The expression of LncRNA PANK2 in margin tissues,cancer tissues,lymphnodes of patients with lymph node metastasis was significantly higher than that of patients without lymph nodes metastasis(P=0.008、0.003、0.015).Conclusion:Overexpression of LncRNA PANK2 is closely related to metastasis of colorectal cancer.Part II Effect of regulation of LncRNA PANK2 expression on proliferation,migration,invasion and apoptosis of colorectal cancer cell lines Objective:To investigate the effects of down-regulation of LncRNA PANK2 expression onproliferation,migration,invasion and apotosis of colorectal cancer cell lines.Methods:1.We constructed lentiviral vector which downregulated LncRNA PANK2 expression and transfected it into HCT 116 cells to obtain HCT 116 cells with silenced expression of LncRNA PANK2.2.MTT assay was used to detect the effect of silencing LncRNA PANK2 on the proliferation of HCT 116 cells.3.FACS apoptosis assay was used to detect the effect of silencing LncRNA PANK2 on the apoptosis of HCT 116 cells.4.Transwell assay was used to detect the effect of silencing LncRNA PANK2 on the migration ability of HCT 116 cells.5.Cell invasion assay was used to detect the effect of silencing LncRNA PANK2 on the invasion ability of HCT 116 cells.Results:1.The LVpFU-LncRNA PANK2 vector was successfully constructed and the sequencing and BLAST results showed complete agreement.RT-qPCR results showed that the expression level of LncRNA PANK2 in HCT 116 cells transfected with LncRNA PANK2 KD2 group was significantly down-regulated compared with the control group(P<0.05).HCT116 cell line expressing silencing of LncRNA PANK2 was obtained.2.The results of MTT showed that compared with the control group,the growth of HCT 116 cells(OD 490 and OD 490/fold)in the transfected LncRNA PANK2 KD2 group was significantly inhibited(P<0.05)and this inhibition was observed.As time advanced,such phenomenon was more significant.3.FACS apoptosis test results showed that compared with the control group(3.47±0.17%),the apoptotic cell rate of LncRNA PANK2 KD2 group(4.05±0.11%)was significantly increased(P<0.05),indicating silencing LncRNA PANK2 cells increased the ability of apotosis.4.Transwell assays showed that compared with the control group(1256±8.23),the LncRNA PANK2 KD2 group metastasis cell number(9±1.76)decreased significantly(P<0.05),indicating that the silencing of LncRNA PANK2 cells were significantly inhibited.5.The results of cell invasion assays showed that compared with the control group(248±4.44),the number of invasive cells in the LncRNA PANK2 KD2 group(76±11.61)were significantly decreased(P<0.05),Compared with the control group(1.00±0.02),the relative multiple of invasion cells(0.31±0.05)in the LncRNA PANK2 KD2 group decreased significantly(P<0.05),indicating that the invasion of silencing LncRNA PANK2 cells were significantly inhibited.Conclusion:Silencing the expression of LncRNA PANK2 in vitro can effectively inhibit the proliferation,migration,invasion and increase apotosis rate of colorectal cancer cells.Part III:LncRNA PANK2 high expression competitively binds to microRNA19a-3p to upregulate SOX4 expression and promotes CRC cell metastasis.Objective:The microRNA-19a-3p was analyzed and verified as the target gene of LncRNA PANK2.At the same time,the protein target SOX4 was finally targeted by this signal axis and the mechanism of biological function effects were predicted.Methods:1.Bioinformatics Technology Screening LncRNA PANK2 Candidate Target Genes and Validation.2.Combining gene comparison,location analysis,and biological information prediction,it is speculated that SOX4 is the final protein effect target.3.qRT-PCR test and database analysis were used to probe the correlation between SOX4 and colorectal cancer development.4.Construct a knock down cell lines of SOX4 and explore the biological activities of SOX4 through MTT experiments,FACS apoptosis detection,scratch detection,invasion detection and related signal pathway proteins detection.Results:1.Bioinformatics screens microRNA-19a-3p as LncRNA PANK2 binding target gene.2.Fluorescence reporter system detection combined with RT-PCR to detect knockdown and over-expression of LncRNA PANK2 found that microRNA-19a-3p expression was negatively regulated and was significantly different in different tissues of colorectal cancer.These results suggested that microRNA-19a-3p was the directly regulating target gene of LncRNA PANK2.3.Using TargetScan and miRTarBase for database target prediction,SOX4 is the final effector protein target.4.Based on the prediction results,we analyzed the correlation between SOX4 expression and prognosis in different tissues of colorectal cancer.At the same time,Pearson analysis revealed that SOX4 was significantly negatively correlated with microRNA-19a-3p.5.The SOX4 knockdown cell lines were successfully constructed.The cell vatality detection revealed that the growth curve of HCT 116 was reduced with the down-regulation of SOX4.At the same time,the apoptosis was significantly increased in the flow cytometry test and the migration capacity of the knockdown cell lines were decreased in the migration ability test and the invasion capacity were decreased in the invasion ability test.Finally,Western-Blot detected that after down-regulation of SOX4,the protein c-Myc that binds to SOX4 was significantly down-regulated,and at the same time,migration-related effector proteins were significantly changed.All the above results suggest that reducing the expression of SOX4 weakened the biological activity of colorectal cancer cells.6.LncRNA PANK2 high expression competitively binds to microRNA19a-3p to upregulate SOX4 expression and promotes CRC cell metastasis.Conclusion:Silencing the expression of SOX4 in vitro can effectively inhibit the proliferation,migration,invasion and increase apotosis rate of colorectal cancer cells.Based on the results of previous genome-wide sequencing,a novel LncRNA PANK2 was screened as a significantly different gene in colorectal cancer.This study conducted an in-depth study of this gene.This study is divided into three parts.Firstly,the expression of LncRNA PANK2 was compared in the cancer tissues,margin tissues and lymph tissues of colorectal cancer patients.The results showed that the expression of LncRNA PANK2 in colorectal cancer tissues was significantly higher than that of margin tissues.The expression in lymph nodes was significantly higher than in cancer tissues and margin tissues.The cancer tissues,margin tissues,and lymph nodes tissues in patients with lymph nodes metastasis were significantly higher than those without lymph nodes metastasis.These results suggested that the overexpression of LncRNA PANK2 was closely related to the metastasis of colorectal cancer.Secondly,we explored the biological activity of the LncRNA PANK2 in vitro using basic cell biology methods and found that silencing LncRNA PANK2 in vitro can effectively inhibit the proliferation,migration and invasion of colorectal cancer cells,and promote its apoptotic capacity.Finally,in combination with bioinformatics,we further explored the molecular sponges and potential protein targets that LncRNA PANK2 may adsorb,and found that LncRNA PANK2 can competitively bind to microRNA19a-3p to upregulate SOX4 expression and promote CRC cell metastasis.In summary,LncRNA PANK2 may become a biological marker for early warning of colorectal cancer metastasis and a new genetic target for clinical treatment,which can better provide basis for clinical treatment and medical reference.