The Partial Role of KLF4 and KLF5 in Gastrointestinal Tumors

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Hindawi Gastroenterology Research and Practice Volume 2021, Article ID 2425356, 13 pages https://doi.org/10.1155/2021/2425356

Review Article The Partial Role of KLF4 and KLF5 in Gastrointestinal Tumors

Jun-Chen Li,1 Qiu-Han Chen,2 Rui Jian,3 Jiang-Rong Zhou,1 Yun Xu,4 Fang Lu,1 Jun-Qiao Li,5 and Hao Zhang 1

1Department of Gastroenterology, The Second Aﬃliated Hospital of Chongqing Medical University, Chongqing 400010, China 2Blood Puriﬁcation Center, University-Town Hospital of Chongqing Medical University, Chongqing 400000, China 3Department of Gastroenterology, People’s Hospital of Chongqing Banan District, Chongqing 401320, China 4Department of Gastroenterology, Chongqing Sixth People’s Hospital, Chongqing 400060, China 5Department of Gastroenterology, Chongqing North-Kuanren General Hospital, Chongqing 401147, China

Correspondence should be addressed to Hao Zhang; [email protected]

Received 30 April 2021; Revised 8 July 2021; Accepted 12 July 2021; Published 28 July 2021

Academic Editor: Fausto Rosa

Copyright © 2021 Jun-Chen Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. KLF4 and KLF5 are members of the KLF transcription factor family, which play an important role in many gastrointestinal tumors. To gain a deeper insight into its function and role, bioinformatics was used to analyze the function and role of KLF4 and KLF5 in gastrointestinal tumors. Methods. Data were collected from several online databases. Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN database analysis, Kaplan-Meier Plotter analysis, LOGpc system, the Pathology Atlas, and the STRING website were used to analyze the data. We download relevant data from TCGA and then perform GO enrichment and KEGG enrichment analysis. The effects of KLF5 on gastric cancer cell proliferation were measured by CCK-8 assay. The effect of KLF5 on the expression of CyclinD1 and MMP9 was detected by Western blot. Results. KLF4 and KLF5 were differentially expressed in normal and tumor tissues of the gastrointestinal tract, and their differential expression is related to several genes or pathways. KEGG analysis showed that KLF5 was coexpressed with endocytosis-related genes. KLF5 promotes the proliferation of gastric cancer cells and the expression of metastasis-related molecules. Conclusion. KLF4 and KLF5 are of great significance for developing gastrointestinal tumors and can be used as therapeutic targets.

1. Introduction factor family members can regulate their homeostasis and pathophysiology. These transcription factors control multi- Malignant tumors are common diseases that significantly ple processes and are essential for the normal functioning threaten human health, and transcription factors play an of the digestive system [2]. important role in tumor occurrence and development. Krüppel-like factor 4 (KLF4) belongs to the SP/KLF fam- Krüppel-like factors (KLFs) and specific proteins (SPs) ily, a transcription factor with a zinc finger structure [8]. belong to the family of transcription factors, containing con- KLF4 is a DNA-binding transcriptional regulator highly served zinc finger domains that bind to target DNA expressed in the skin and gastrointestinal tract epithelial cells, sequences [1]. The KLF/SP family is expressed in various tis- especially in cell differentiation regions [9]. However, KLF4 is sues and has different tissue-specific activities and functions downregulated in many patients with epithelial cancer, [2]. These genes not only regulate physiological processes including esophageal cancer [10], gastric cancer [11], colo- such as embryogenesis, growth, development, differentiation, rectal cancer [12], and bladder cancer [13], leading to cell and proliferation but also regulate the pathogenesis of many proliferation. For example, the absence of KLF4 in the mouse diseases, including inflammatory diseases and cancers [3–6]. esophagus can increase proliferation of epithelial cells [14]. A previous study showed that the loss of KLF/SP transcrip- KLF4 is often deleted in gastrointestinal tumors [15], which tion factors is associated with many human diseases and can- can cause tumor growth of gastric cancer and colorectal cers [7]. In the digestive system, the KLF/SP transcription cancer [11]. 2 Gastroenterology Research and Practice

Krüppel-like factor 5 (KLF5), also known as BTEB2 and 2.6. STRING. The STRING (https://string-db.org/) database IKLF, belongs to the KLF family [16]. KLF5 can regulate cell is a protein interaction (PPI network) database. STRING col- proliferation, cell cycle, apoptosis, and differentiation [9]. In lects, scores, and integrates all publicly available sources of addition, KLF5 may play an important role in various protein interaction information and supplements these tumors, including breast cancer, prostate cancer, bladder sources by calculating predictions [28]. STRING allows users cancer, skin cancer, colon cancer, and esophageal cancer to visualize the subset as an interactive network and can also [13, 17–19]. Studies have shown that there is a relationship perform gene set enrichment analysis such as Gene Ontology between KLF5 and the occurrence and development of intes- and KEGG. tinal tumors. KLF5 is related to the mutation of APC and RAS genes. Both mutations are the two most common types 2.7. The Pathology Atlas. The Pathology Atlas contains of mutations in human colorectal cancer [20, 21]. In gastric mRNA and protein expression data from 17 different forms cancer, the poorly differentiated subtype of gastric cancer of human cancer. The resulting 5 million IHC cancer tissue shows high expression of KLF5 [22], and patients with high images are presented here [29]. expression of KLF5 have a poor prognosis [23]. In gastrointestinal tumors, changes in the expression 2.8. Cell Proliferation Assay and Western Blot. Cell prolifera- levels of KLF4 and KLF5 have an impact on tumor develop- tion rates were assessed by Cell Counting Kit 8 assay accord- ment [2]. Bioinformatics was used for analysis to understand ing to the supplier’s instructions. The effects of KLF5 on the function and roles of KLF4 and KLF5 in gastrointestinal gastric cancer cell proliferation were measured by CCK-8 tumors. assay. Total proteins were collected from gastric cancer cells using RIPA lysis and extraction buffer, and the concentration 2. Methods of proteins was measured using Bradford protein concentration assay kit. The effect of KLF5 on the expression of 2.1. GEPIA (Gene Expression Profiling Interactive Analysis). CyclinD1 and MMP9 was detected by Western blot (Supple- The data of GEPIA comes from RNA sequencing expression mental 1). data of 9,736 tumors and 8,587 normal samples of TCGA and GTEx projects. GEPIA uses standard processing pipelines to 2.9. Statistical Analysis. Data were presented as the mean ± analyze data and is a newly developed interactive Web server. SD t ff , and the -test was used to compare the two groups (using It can perform tumor/normal di erential expression analysis, Prism 5 statistical software). P value less than 0.05 was con- analysis based on cancer type or pathological stage, patient sidered statistically significant. survival analysis, etc. [24].

2.2. UALCAN. UALCAN is a comprehensive and interactive 3. Results Web resource for analyzing cancer OMICS data. UALCAN 3.1. KLF4 and KLF5 mRNA Expression Levels in provides users with easy access to published cancer OMICS Gastrointestinal Tumors. GEPIA was used to compare the data (TCGA and MET500), patient survival analysis, gene expression levels of KLF4 and KLF5 between tumor tissues promoter methylation analysis, gene correlation analysis, and normal tissues of the digestive tract (Figures 1(a)– etc. [25]. 1(d)). KLF4 was lowly expressed in gastrointestinal tumors, and the low expression in COAD and READ was statistically 2.3. KMplot (the Kaplan-Meier Plotter). The Kaplan-Meier significant (P <0:05) (Figure 1(c)). KLF5 is highly expressed Plotter (http://www.kmplot.com/analysis/) is capable of in gastrointestinal tumors, and the low expression in COAD, accessing the effect of 54675 genes on survival in 21 cancer READ, and STAD was statistically significant (P <0:05) types. Sources of the system database include GEO, EGA, (Figure 1(d)). and TCGA. This tool can be applied to perform survival curve analysis [26]. 3.2. Prognostic Analysis of KLF4 and KLF5 Expression and Patient Survival. To assess the correlation between KLF4 2.4. LOGpc System (Long-Term Outcome and Gene and KLF5 and clinical disease, UALCAN, GEPIA, and Expression Profiling Database of Pan-Cancers). The LOGpc Kaplan-Meier Plotter were used for prognostic survival anal- system encompasses 209 expression datasets and provides 13 ysis. As shown in Figures 1(e)–1(h), the high expression of types of survival terms for 31310 patients of 27 distinct malig- KLF4 in COAD, ESCA, and STAD showed a significant pos- nancies (http://bioinfo.henu.edu.cn/CRC/CRCList.jsp). This itive correlation with the survival rate of patients. As shown tool can be applied to perform survival curve analysis of in Figures 1(i), 1(j), and 1(l), the survival rate of patients ESCA [27]. was poor with high expression of KLF5 in COAD, ESCA, and STAD. Although there was no significant difference 2.5. TCGA (the Cancer Genome Atlas Database). The relevant (P >0:05), we observed that the high expression of KLF5 data of rectal adenocarcinoma were downloaded from the was negatively correlated with the survival rate of patients TCGA database (https://portal.gdc.cancer.gov/), and then, according to the trend. Unlike above, the high expression of the R package was used for GO enrichment analysis and KLF5 in READ was significantly positively correlated with KEGG enrichment analysis. the survival rate of patients (P <0:05) (Figure 1(k)). Gastroenterology Research and Practice 3

700 COAD ESCA READ STAD 1200 COAD ESCA READ STAD KLF4 KLF5

600 1000 P < 0.05 P > 0.05 P < 0.05 P > 0.05 P < 0.05 P > 0.05 P < 0.05 P < 0.05

500 800

400 600 300

400 200 Transcripts per million (TPM) per million Transcripts (TPM) per million Transcripts 200 100

0 0 T ( n = 92) T ( n = 92) T ( n = 275) T ( n = 182) T ( n = 408) T ( n = 275) T ( n = 182) T ( n = 408) N ( n = 349) N ( n = 286) N ( n = 318) N ( n = 211) N ( n = 349) N ( n = 286) N ( n = 318) N ( n = 211) (a) (b) ⁎ 10 P P P P < 0.05 P > 0.05 P < 0.05 P > 0.05 10 < 0.05⁎ > 0.05 < 0.05 P < 0.05 ⁎ ⁎ ⁎ 8 8 6 6 4 4 2 2 KLF4 KLF5 0 0 COAD ESCA READ STAD COAD ESCA READ STAD T = 275, T = 182, T = 92, T = 408, T = 275, T = 182, T = 92, T = 408, N = 349 N = 286 N = 318 N = 211 N = 349 N = 286 N = 318 N = 211 (c) (d)

1 COAD Disease free survival 1.0 Log rank P = 0.0076 ESCA HR (high) = 0.52 n P (HR) = 0.0087 0.75 High ( = 71) n (high) = 91 0.8 n (low) = 91

0.50 0.6 Low (n = 208) 0.4 0.25 Percent survival Percent Survival probability Survival 0.2 P < 0.05 0 P < 0.001 0.0 0 1000 2000 3000 4000 0 20 40 60 80 100 120 Days Months

Low KLF4 TPM High KLF4 TPM (e) (f)

Figure 1: Continued. 4 Gastroenterology Research and Practice

Disease free survival 1.0 Log rank P = 0.13 1.0 HR = 0.72 (0.6 – 0.86) HR (high) = 0.48 Log rank P = 0.00028 P (HR) = 0.14 STAD 0.8 n (high) = 46 0.8 n (low) = 46

0.6 0.6 High (n = 600) 0.4 0.4 READ Probability n Percent survival Percent Low ( = 275) 0.2 0.2 P = 0.14 P < 0.001 0.0 0.0 0 20 40 60 80 100 120 0 50 100 150 Months Time (months)

Low KLF4 TPM Expression High KLF4 TPM Low High (g) (h)

Overall survival 1.0 1.0 COAD Log rank P = 0.054 HR (high) = 0.61 ESCA P (HR) = 0.055 n (high) = 135 0.8 0.8 n (low) = 135

0.6 0.6

0.4 0.4

Probability HR = 0.64518

Percent survival Percent 95% Cl, 0.4 – 1.0407 0.2 0.2 P = 0.055 P = 0.072 0.0 0.0 0 50 100 150 0 20 40 60 Months Months

Low KLF5 TPM High KLF5 TPM (i) (j)

Overall survival Disease free survival 1.0 Log rank P = 0.026 1.0 Log rank P = 0.71 HR (high) = 0.33 HR (high) = 1.1 P (HR) = 0.034 STAD P (HR) = 0.7 n (high) = 46 n (high) = 192 0.8 n (low) = 46 0.8 n (low) = 192

0.6 0.6

0.4 0.4

Percent survival Percent READ survival Percent 0.2 0.2 P < 0.05 P > 0.05 0.0 0.0 0 20406080 100 120 0 20406080 100 120 Months Months

Low KLF5 TPM Low KLF5 TPM High KLF5 TPM High KLF5 TPM (k) (l)

Figure 1: The expression and survival curve of KLF4 or KLF5 in gastrointestinal tumors. (a–d) Expression of KLF4 or KLF5 in gastrointestinal tumors. KLF4 was lowly expressed in COAD, ESCA, READ, and STAD tumors and had statistical significance in COAD and READ (P <0:05). KLF5 was highly expressed in COAD, ESCA, READ, and STAD tumors and was statistically significant in COAD, READ, and STAD (P <0:05). (e–h) The survival curve of KLF4 in gastrointestinal tumors. Patients with high expression of KLF4 had a good prognosis in COAD, ESCA, and STAD (P <0:05). (i–l) The survival curve of KLF5 in gastrointestinal tumors. Patients with high KLF5 expression had a good prognosis in READ (P <0:05). T means tumor and N means normal. 3.3. Expression of KLF4 and KLF5 Based on Drinking reduced (P <0:05) (Figure 2(b)), while that of KLF5 was not Frequency or Helicobacter pylori Infection. UALCAN was significantly different in STAD with Helicobacter pylori used to compare the effects of different drinking frequencies infection (P >0:05) (Figure 2(d)). for tumor patients on the expression levels of KLF4 and KLF5 in ESCA. In tumor patients with a history of drinking 5 days 3.4. The Methylation Status of KLF4 and KLF5 Promoters in a week, the expression level of KLF4 in ESCA was signifi- Gastrointestinal Tumors. UALCAN was adopted to analyze cantly reduced (P <0:01) (Figure 2(a)), whereas KLF5 did the changes in the methylation levels of KLF4 and KLF5 pro- not show a significant difference (P >0:05) (Figure 2(c)). moters between normal and tumor tissues of the gastrointes- Then, the relationship between KLF4 and KLF5 expression tinal tract. We found that there was no significant change in levels and Helicobacter pylori infection in gastric cancer the promoter methylation level of KLF4 in COAD and ESCA was explored. We found that the expression level of KLF4 (P >0:05) (Figures 2(e) and 2(f)), while the promoter meth- in STAD with Helicobacter pylori infection was significantly ylation level of KLF4 was significantly reduced in READ and Gastroenterology Research and Practice 5

Expression of KLF4 in ESCA based on drinking frequency Expression of KLF5 in ESCA based on drinking frequency 400 1000 P > 0.05 300 P < 0.01 750

200 500

100 250

0 0 Transcripts per million Transcripts Transcripts per million Transcripts

–100 –250 ( n = 6) ( n = 6) ( n = 2) ( n = 6) ( n = 6) ( n = 6) ( n = 2) ( n = 6) Normal Normal ( n = 11) ( n = 34) ( n = 11) ( n = 48) ( n = 11) ( n = 34) ( n = 11) ( n = 48) 1 day/week 1 day/week 0 days/week 2 days/week 3 days/week 4 days/week 5 days/week 7 days/week 0 days/week 2 days/week 3 days/week 4 days/week 5 days/week 7 days/week (a) (b)

Expression of KLF4 in STAD based on H.pylori infection status Expression of KLF5 in STAD based on H.pylori infection status 300 P < 0.05 600 P > 0.05 250 500 200 400 150 300 100 200 50 100 Transcripts per million Transcripts 0 per million Transcripts 0 –50 –100 Normal With H.pylori Without H.pylori Not available Normal With H.pylori Without H.pylori Not available (n = 34) infection infection (n = 153) (n = 34) infection infection (n = 153) (n = 20) (n = 157) (n = 20) (n = 157) (c) (d)

Promoter methylation level of Promoter methylation level of Promoter methylation level of KLF4 in COAD KLF4 in ESCA KLF4 in READ 0.06 0.05 0.05 0.05 0.045 0.04 0.045 0.04 0.04 0.035 0.03 0.035 0.03 Beta value Beta Beta value Beta Beta value Beta 0.03 0.02 0.025 0.025 0.02 P > 0.05 P > 0.05 P < 0.05 0.02 0.01 0.015 Normal Tumor Normal Tumor Normal Tumor (n = 37) (n = 313) (n = 16) (n = 185) (n = 7) (n = 98) (e) (f) (g)

Promoter methylation level of Promoter methylation level of Promoter methylation level of KLF4 in STAD KLF5 in COAD KLF5 in ESCA 0.35 0.06 0.055 0.3 0.055 0.05 0.25 0.05 0.045 0.2 0.045 0.04 0.15 0.04 0.035 Beta value Beta value Beta value 0.1 0.035 0.03 0.05 0.03 0.025 P < 0.05 P > 0.05 P > 0.05 0 0.025 0.02 Normal Tumor Normal Tumor Normal Tumor (n = 2) (n = 395) (n = 37) (n = 313) (n = 16) (n = 185) (h) (i) (j)

Figure 2: Continued. 6 Gastroenterology Research and Practice

Promoter methylation level of Promoter methylation level of KLF5 in READ KLF5 in STAD 0.05 0.06

0.045 0.05 0.04 0.04 0.035 0.03 Beta value Beta Beta value Beta 0.03

0.025 0.02 P < 0.05 P < 0.05 0.02 0.01 Normal Tumor Normal Tumor (n = 7) (n = 98) (n = 2) (n = 395) (k) (l)

Figure 2: In ESCA and STAD, KLF4 expression is related to drinking frequency or Helicobacter pylori infection. The promoters of KLF4 and KLF5 genes are hypomethylated in READ and STAD. (a–d) Expression of KLF4 and KLF5 based on drinking frequency or Helicobacter pylori infection. (e–l) The methylation status of KLF4 and KLF5 promoters in gastrointestinal tumors.

ELK1 CEBPG

SPI1 ESR2

CEBPA CEBPB KDM6A HDAC7 HDAC1 JUN

HDAC5 ESR1 YAP1 KAT5 GSK3B

SP1 RARA KLF5 CREBBP WWTR1 SETD7 NCOR1 CREBBP HDAC2 CDH1 EP300 EP300 PAX9 CTNNB1 HDAC1 KLF4 KDM6B TP53 NCOR2 RXRA FBXW7 YAP1

SMURF2 NFKB1 HDAC2 VHL UBC SUMO1 AURKA

WWP1 SKP1 CUL1

ACTA2 HUWE1 TCEB2 KLF5 KLF4 CUL2

(a) (b)

Figure 3: STRING interaction network of KLF4/KLF5 gene interacting proteins. (a) STRING interaction network of interacting proteins of KLF4 gene. (b) STRING interaction network of interacting proteins of KLF5 gene.

STAD (P <0:05) (Figures 2(g) and 2(h)). The changes in the TCEB2, HUWE1, CUL1, SKP1, VHL, YAP1, SPI1, SETD7, promoter methylation level of KLF5 in tumors were further CDH1, and PAX9 (Supplemental 2.3). The protein molecules analyzed, and no signiﬁcant change was found in the pro- (top 25) that interact with KLF5 include HDAC2, YAP1, moter methylation level of KLF5 in COAD and ESCA EP300, CEBPA, CTNNB1, NCOR1, JUN, SUMO1, ACTA2, (P >0:05) (Figures 2(i) and 2(j)). However, in READ and WWTR1, RARA, NCOR2, HDAC1, CREBBP, ESR2, RXRA, STAD, the methylation level of KLF5 promoter was signiﬁ- ESR1, FBXW7, UBC, SMURF2, GSK3B, WWP1, NFKB1, cantly reduced (P <0:05) (Figures 2(k) and 2(l)). CEBPB, and CEBPG (Supplemental 2.4).

3.5. PPI Network Analysis. To identify protein molecules that 3.6. Gene Analysis of Coexpression with KLF4 or KLF5 in interact with KLF4 and KLF5, the STRING database was Rectal Adenocarcinoma (READ). We used UALCAN to study used to generate a PPI network (Figure 3). The protein mol- which genes in rectal adenocarcinoma are related to the ecules (top 25) that interact with KLF4 include HDAC7, SP1, expression of KLF4 or KLF5. As shown in Figures 4(a) and HDAC2, CTBP1, HDAC1, KDM6A, CREBBP, ELK1, 4(b), the genes positively or negatively related to KLF4 KDM6B, EP300, HDAC5, KAT5, TP53, AURKA, CUL2, expression were analyzed, and the top 25 genes were listed. Gastroenterology Research and Practice 7

Expression pattern of input genes in rectum adenocarcinoma Expression pattern of input genes in rectum adenocarcinoma

KLF4 KLF4 BCAS1 FOXQ1 GPR120 MYL6B PTGER4 PDRG1 NR3C2 RTKN ST6GALNAC1 PQBP1 SIPA1L2 COMMD7 ST3GAL4 RPS21 CASP7 USE1 PAPSS2 NUTF2 MUC4 WWOX AKAP5 ROMO1 CHST5 RPS11 RAB27A DRAP1 B3GALT5 CTNNBL1 B3GNT6 DYNLRB1 CBFA2T3 CDK5 SLC22A23 ANXA9 SPDEF BCL11A ST6GALNAC6 SNRPD2 NR4A2 C11orf73 FCGBP CARS2 GNA14 ETNK2 TPSG1 LY6G6E PLCL2 GNAS Normal Tumor Normal Tumor Log2 (TPM + 1) Log2 (TPM + 1) 0 5 10 15 0 5 10 15 (a) (b)

Expression pattern of input genes in rectum adenocarcinoma Expression pattern of input genes in rectum adenocarcinoma

KLF5 KLF5 FBXL3 C19orf60 DIS3 SCO2 VPS36 FAM128A ERCC5 DGCR6 RB1 DGCR6L UBL3 TNFSF12-TNFSF13 ELF1 STRA13 CD164 MIF RNF6 MRPL12 UTP14C UBE2S PDCD6IP MRPL41 AKAP11 MTP18 RAB5A YIF1A SPHAR NDUFS8 COG6 C19orf20 ZMYM2 RPL23AP82 UFM1 LSM10 FNDC3A ENDOG PIBF1 TUBA1A SH3BGRL2 TRAPPC5 COG3 PGAM4 TPP2 RPL18A MLL5 ERI3 RYBP GPX1 Normal Tumor Normal Tumor

Log2 (TPM + 1) Log2 (TPM + 1)

0 5 10 15 0 5 10 15 (c) (d)

Figure 4: Genes related to KLF4/KLF5 in READ. (a) Genes positively correlated with KLF4 in READ. (b) Genes negatively correlated with KLF4 in READ. (c) Genes positively correlated with KLF5 in READ. (d) Genes negatively correlated with KLF5 in READ.

Then, the genes that were positively or negatively correlated The top 20 enriched GO terms in the cellular component with KLF5 expression were analyzed, and the top 25 genes (CC) category include endosome membrane, cell-cell junc- were listed accordingly (Figures 4(c) and 4(d)). tion, cell leading edge, early endosome, trans-Golgi network, tight junction, apical junction complex, and bicellular tight 3.7. GO Analysis and KEGG Enrichment Analysis of Genes junction (Figures 5(c) and 5(d)). The cell connections Coexpressed with KLF5. Taking KLF5 as an example, the involved in these categories were necessary for tumor related pathway of KLF5 coexpressed genes in rectal adeno- metastasis. carcinoma was analyzed. The data on genes coexpressed with The data of coexpressed genes with KLF5 were down- KLF5 in rectal adenocarcinoma were downloaded and loaded from TCGA, and then KEGG analysis was applied applied GO analysis on this data (Figure 5). The top 20 (Figures 5(e) and 5(f)). Genes coexpressed with KLF5 were enriched GO terms are shown in Figures 5(a) and 5(b), enriched in endocytosis. Related literature has also shown including phospholipid metabolism process, Golgi vesicle that proper inhibition of endocytosis can sensitize tumor transport, glycerolipid metabolism process, glycerophospho- immunotherapy. Genes coexpressed with KLF5 were also lipid metabolism process, glycoprotein biosynthesis process, abundant in the RAS pathway. Combined with relevant liter- glycosylation, and cell junction assembly, in the biological ature, the analysis illustrated that the expression of KLF5 was process (BP) category. More genes were involved in phos- related to the regulation of these pathways. pholipid metabolism among these categories, and the metabolism was of great signiﬁcance for tumor growth and 3.8. KLF5 Promotes Proliferation of MGC-803 Cells. To verify metastasis. the eﬀect of KLF5 on MGC-803 cell proliferation, we tested 8 Gastroenterology Research and Practice

P. adjust P. adjust Phospholipid metabolic process Bicellular tight junction assembly 0.02 Golgi vesicle transport Tight junction assembly 0.02 Glycerolipid metabolic process Post–Golgi vesicle–mediated transport Glycerophospholipid metabolic process Golgi vesicle transport Glycoprotein biosynthetic process Tight juction organization Glycosylation 0.03 Apical junction assembly 0.03 Cell junction assembly Phospholipid metabolic process Protein localization to plasma membrance Glycerophospholipid metabolic process Post–Golgi vesicle–mediated transport Cell–cell junction assembly Cell–cell junction assembly Glycosylation 0.04 0.04 Cell–cell junction organization Endosome organization Cell junction assembly Cytosolic transport Positive regulation of exocytosis Bicellular tight junction assembly Tight junction assembly Golgi to plasma membrane transport 0.05 0.05 Glycerolipid metabolic process Tight juction organization Protein localization to plasma membrance Apical junction assembly Cell–cell junction organization Endosome organization Cytosolic transport Positive regulation of exocytosis Microtubule nucleation 0.06 Golgi to plasma membrane transport 0.06 Glycoprotein biosynthetic process Microtubule nucleation 0 5 10 15 0.02 0.03 0.04 0.05 0.06 0.07 Gene ratio BP BP

Count 4 12 8 16 (a) (b)

P. adjust P. adjust Endosome membrane Tight junction Cell–cell junction Apical junction complex 0.04 0.04 Cell leading edge Bicellular tight junction Early endosome Golgi cisterna Trans–Golgi network Golgi stack Tight junction Endosome membrane Apical junction complex Trans–Golgi network 0.08 WASH complex 0.08 Bicellular tight junction Golgi cisterna membrane Golgi stack SWI/SNF complex Golgi cisterna Tethering complex Rufe Cell–cell junction Golgi cisterna membrane Rufe 0.12 Tethering complex 0.12 Endoplasmic reticulum quality control compartment WASH complex Early endosome SWI/SNF complex Cell leading edge Endoplasmic reticulum quality control compartment COPI–coated vesicle COPI–coated vesicle 0 5 10 15 0.020.010.03 0.04 0.05 0.06 Gene ratio CC CC

Count 4 12 8 16 (c) (d)

P. adjust P. adjust Endocytosis 0.2 Endocytosis 0.2 Viral carcinogenesis Viral carcinogenesis Ras signaling pathway Lysine degradation Protein processing in endoplasmic reticulum Phosphatidylinositol signaling system Tight junction Type II diabetes mellitus Phosphatidylinositol signaling system Protein processing in endoplasmic reticulum Gastric cancer Tight junction 0.4 Hepatitis C Fc gamma R–mediated phagocytosis Lysine degradation Legionellosis Fc gamma R–mediated phagocytosis Ras signaling pathway 0.4 Glycerophospholipid metabolism Glycerophospholipid metabolism Choline metabolism in cancer Choline metabolism in cancer Type II diabetes mellitus Glycosphingolipid biosynthesis – lacto and neolacto series 0.6 Legionellosis Gastric cancer 0.6 Melanoma Mucin type O–glycan biosynthesis Platinum drug resistance Hepatitis C Melanoma Bacterial invasion of epithelial cells Platinum drug resistance Inositol phosphate metabolism Bacterial invasion of epithelial cells 0.8 Glycosphingolipid biosynthesis – lacto and neolacto series 0.8 Inositol phosphate metabolism Mucin type O–glycan biosynthesis 0 5 10 0.03 0.06 0.09 Gene ratio KEGG KEGG

Count 2.5 10.0 5.0 12.5 7.5 (e) (f)

Figure 5: GO enrichment analysis of genes coexpressed with KLF5 in rectal adenocarcinoma. (a, b) Bar chart or bubble chart in biological process (BP) category. (c, d) Bar chart or bubble chart in cell component (CC) category. (e, f) KEGG enrichment analysis of genes coexpressed with KLF5 in rectal adenocarcinoma. Gastroenterology Research and Practice 9

1.5

1.0 con KLF5 shcon shKLF5 KLF5 MGC-803 0.5 MGC–803 GAPDH OD value (450 nm) value OD 0.0 0 244872 Time (h)

con+shcon KLF5 shKLF5

(a) (b) con KLF5 shcon shKLF5 CyclinD1

MMP9 MGC-803

a-Tubulin Normal tissue Primary tumor

Figure 6: (a) MGC-803 cells were transfected with an overexpressing KLF5 plasmid to detect the expression levels of KLF5 protein. (b) CCK- 8 detects the effect of KLF5 on cell proliferation. (c) MGC-803 cells were transfected with an overexpressing KLF5 plasmid and shKLF5 plasmid to detect the expression levels of CyclinD1 and MMP9 proteins. (d) Immunohistochemical analysis of KLF5 expression in stomach cancer and normal stomach tissue. Data derived from the Pathology Atlas database. whether shKLF5 inhibition can reduce the viability of MGC- tent with the previous reports, the results of GEPIA bio- 803 cells. shKLF5 resulted in significantly decreased cell via- informatics analysis exhibited that KLF4 showed low bility than the control group. Then, we tested whether overexpression in gastrointestinal tumors. In addition, patients expression of KLF5 promoted the viability of MGC-803 with high expression of KLF4 had a better survival rate. cells, and overexpression of KLF5 increased cell viability A previous study has shown that overexpression of compared to the control group (Figure 6(b)). KLF4 will lead to a decrease in the expression of N-cadherin, MMP2, and MMP9 [34]. On the contrary, the loss 3.9. KLF5 Regulates the Expression of Proliferating-Associated of KLF4 weakened the inhibition of N-cadherin, MMP2, CyclinD1 and Metastasis-Associated MMP9 Proteins. To elu- and MMP9, leading to increased tumor cell invasion cidate the mechanism by which KLF5 promotes proliferation and migration, thereby affecting survival and prognosis and migration in gastric cancer, we next studied the expres- [35]. KLF5, in contrast to KLF4, showed high expression sion levels of CyclinD1 and MMP9 proteins. We used in gastrointestinal tumors. High expression of KLF5 can MGC-803 cells, and CyclinD1 and MMP9 proteins showed promote cancer progression, such as the increase in a significant increase after overexpression of KLF5. Then, KLF5 can promote breast cell proliferation and tumori- the inhibition of shKLF5 was tested. After treatment with genesis [36]. Related literature indicates that patients with shKLF5, CyclinD1 and MMP9 proteins were significantly high expression of KLF5 have poor survival rates. How- decreased (Figure 6(c)). ever, our results indicated that patients with high expression of KLF5 in rectal adenocarcinoma have a higher 4. Discussion survival rate. It may be caused by the difference in the sample size of the data. KLF4 and KLF5 are important members of the KLF family P53 exerts its function and effect by regulating other [7]. Due to their abundant expressions in gastrointestinal genes [37]. When P53 is wild-type, KLF5 promotes the pro- epithelial crypt cells were first identified as an intestinal- liferation of esophageal keratinocytes. Conversely, when rich KLF (IKLF) [14, 30]. KLF4 and KLF5 have received P53 is mutant, KLF5 inhibits cell proliferation [38]. However, extensive attention as a key transcription factor and potential whether P53 can affect the level of KLF5 protein remains to drug target [19, 31]. be elucidated. The presence of P53 can ensure the expression In tumors, the deletion of a certain gene can lead to of KLF4 protein, and the tumor suppressor effect of KLF4 changes in the corresponding tumor suppressor function, will be exerted [39]. A previous study has shown that KLF4 thereby promoting tumors [32]. KLF4 exhibits cancer acts as a tumor suppressor in colorectal cancer and KLF4 suppressive effects in gastrointestinal tumors, and its plays an important role in γ-ray-induced DNA damage absence often leads to tumor deterioration [33]. Consis- repair [40]. 10 Gastroenterology Research and Practice

The direct contact of alcohol with the mucous mem- a key molecule that affects the function of KLF5 and tran- branes of the digestive tract can induce many changes in scriptional regulation that controls lipid metabolism [55]. metabolism and function [41]. Excessive drinking not only The SUMOylation of KLF4 can regulate the transcription can cause duodenal erosion, upper jejunal bleeding, and and proliferation of vascular smooth muscle cells [56]. mucosal damage but also can regulate the intestinal mucosal By regulating the pathway of KLF4 and KLF5 interacting immune system [42]. Alcohol-induced mucosal damage proteins, the expression of KLF4 and KLF5 can be increases the risk of esophageal cancer [41]. We found that regulated. alcohol can affect the expression of KLF4 in esophageal can- KLF4 plays an important role in stem cell renewal and cer, and there is no significant difference in the effect of KLF5 reprogramming [57]. An important advantage of pluripotent expression. Among patients with esophageal cancer, those stem cells (PSCs) is that they have the potential to proliferate who had a history of drinking 5 days a week had lower indefinitely. However, this feature may also be a double- KLF4 expression. Oxidative stress of alcohol can trigger edged sword. The inherent tumorigenic properties, immuno- chronic inflammation and carcinogenesis by forming reac- genicity, and heterogeneity of stem cells are practical prob- tive oxygen species [43]. Therefore, it was speculated that lems that limit the use of stem cells [58]. PSC KLF4 alcohol might affect the expression of KLF4 by causing cer- negatively regulates the epithelial cell-to-mesenchymal tran- tain metabolic changes. sition of gastrointestinal cancer by interacting with the Helicobacter pylori is a Gram-negative bacterium, which Notch, TGF-β, and Wnt signaling pathways [15]. is the leading cause of chronic gastritis and peptic ulcer dis- KLF5 was used as an example to analyze its coexpressed ease [44]. Helicobacter pylori’s main virulence factor CagA genes in colon adenocarcinoma. The abnormal lipid metabo- can induce the occurrence of gastric cancer [45]. Meanwhile, lism of cells has also received extensive attention and studied CagA can upregulate miR-155 to inhibit the expression of as potential pathogenesis of various tumors [59]. GO analysis KLF4 and promote the malignant transformation of normal showed that genes coexpressed with KLF5 are primarily epithelial cells [46]. It was revealed that gastric cancer involved in phospholipid metabolism in the biological pro- patients infected by Helicobacter pylori had significantly cess (BP) category. It has been reported that certain changes lower KLF4 expression than noninfected patients. Therefore, in lipid metabolism can promote tumorigenesis [60]. Helicobacter pylori can play a role in promoting cancer by Changes in lipid metabolism can also activate important inhibiting the KLF4 expression. oncogenic signaling pathways, including the Hippo/YAP Abnormal DNA methylation is relatively common in and Wnt/β-catenin pathways [61]. Interventions on key cancer, and most gene methylation abnormalities are prone metabolites in KLF5 or its coexpressed genes and designing to tumors [47]. In gastric cancer, abnormal DNA methyla- drug targets may be able to treat tumors. tion in the gene promoter region can lead to the inactivation GO analysis revealed that genes coexpressed with KLF5 of tumor suppressor genes and other cancer-related genes are involved in cell junctions in the category of cell compo- and is an epigenetic marker in gastric cancer [48]. In colorec- nents. The cell connection is very important for tumor tal cancer, DNA methylation can be used as a biomarker and metastasis. If the tight connection of the cell is weakened, it evaluate its prognosis [49]. Bioinformatics analysis showed will cause abnormal cell migration, thereby spreading the that in gastric cancer and rectal adenocarcinoma, the pro- cancer cells [62]. Perhaps by regulating the expression of moter region of KLF4 and KLF5 was significantly hypo- KLF5, it can reduce tumor metastasis and play an anticancer methylated. It was speculated that in gastric cancer and role. rectal adenocarcinoma, abnormal methylation in the pro- KEGG analysis showed that genes coexpressed with moter region of KLF4 and KLF5 is involved in the progres- KLF5 were enriched in endocytosis. The endocytosis sion or suppression of KLF4 and KLF5. mechanism regulates the interaction between cells and PPI plays an important role in various cellular pathways, the environment by controlling the lipid and protein com- allowing us to explore the function of proteins by under- position of the plasma membrane [63]. Endocytosis plays standing protein interaction partners [50]. The STRING a vital role in cancer cell signal transduction, cancer cell database was used to analyze the proteins that interacted with invasion, and metastasis [64, 65]. It has been demon- KLF4 and KLF5. The pathways involved in the protein inter- strated that inhibition of endocytosis can effectively action between KLF4 and KLF5 are the Notch signaling path- enhance the therapeutic effect of monoclonal antibodies way, general transcription pathway, and SUMO E3 ligase against tumors and make tumor immunotherapy sensitive pathway. According to previous reports, the Notch signaling [66]. The coexpressed genes of KLF5 are enriched in can enhance the expression of KLF4 and KLF5 and regulate endocytosis; thus, inhibiting or promoting the expression the proliferation of conjunctival epithelium and goblet cell of these genes may regulate the expression of KLF5. Genes differentiation [51]. In intestinal tumors and colorectal can- coexpressed with KLF5 are also abundant in the RAS cer cells, the Notch signaling can inhibit the expression of pathway. RAS activation inhibits TGF-β-induced KLF5 KLF4 and reduce proliferation and tumor formation [52]. acetylation [67], and the deacetylation state of KLF5 may In bladder cancer, Notch-1 regulates the proliferation and be an important mechanism, by which KLF5 and HDAC differentiation of cancer cells by inhibiting the expression of promote cell proliferation and tumor growth [68]. There- KLF4 [53]. SUMOylation is a posttranslational modification fore, by regulating the expression level of KLF5 or its that promotes nuclear localization of KLF5 by inhibiting coexpressed genes, the development of cancer can be NES activity [54]. It has been reported that SUMOylation is inhibited. Gastroenterology Research and Practice 11

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